U.S. patent application number 12/325776 was filed with the patent office on 2009-06-11 for compositions comprising vascular and myocyte progenitor cells and methods of their use.
Invention is credited to Piero ANVERSA, Jan Kajstura, Annarosa Leri.
Application Number | 20090148421 12/325776 |
Document ID | / |
Family ID | 40651427 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090148421 |
Kind Code |
A1 |
ANVERSA; Piero ; et
al. |
June 11, 2009 |
COMPOSITIONS COMPRISING VASCULAR AND MYOCYTE PROGENITOR CELLS AND
METHODS OF THEIR USE
Abstract
The invention provides compositions of adult cardiac vascular
progenitor cells (VPCs) and adult cardiac myocyte progenitor cells
(MPCs) useful for the treatment of various cardiac conditions. The
invention also encompasses methods of generating a biological
bypass, repairing damaged myocardium, and treating or preventing
hypertensive cardiomyopathy and heart failure with the compositions
of the invention. Methods of isolating the cardiac progenitor cells
are also disclosed.
Inventors: |
ANVERSA; Piero; (Boston,
MA) ; Leri; Annarosa; (Boston, MA) ; Kajstura;
Jan; (Brookline, MA) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Family ID: |
40651427 |
Appl. No.: |
12/325776 |
Filed: |
December 1, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60991515 |
Nov 30, 2007 |
|
|
|
Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
C12N 5/0692 20130101;
A61P 9/10 20180101; A61K 35/12 20130101; A61P 9/00 20180101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/34 20060101
A61K035/34; A61P 9/00 20060101 A61P009/00 |
Claims
1. A pharmaceutical composition comprising adult vascular
progenitor cells and a pharmaceutically acceptable carrier, wherein
the vascular progenitor cells are lineage negative, c-kit positive,
and flk1 positive.
2. The pharmaceutical composition of claim 1, wherein said vascular
progenitor cells are isolated from human myocardium.
3. The pharmaceutical composition of claim 1, wherein said vascular
progenitor cells differentiate predominantly into endothelial cells
or smooth muscle cells in vitro.
4. The pharmaceutical composition of claim 1, wherein the
concentration of vascular progenitor cells is about
1.times.10.sup.5 cells/ml to about 1.times.10.sup.7 cells/ml.
5. The pharmaceutical composition of claim 1 further comprising
adult myocyte progenitor cells, wherein the myocyte progenitor
cells are lineage negative, c-kit positive, and flk1 negative.
6. The pharmaceutical composition of claim 5, wherein said myocyte
progenitor cells are isolated from human myocardium.
7. The pharmaceutical composition of claim 5, wherein said myocyte
progenitor cells differentiate predominantly into cardiomyocytes in
vitro.
8. The pharmaceutical composition of claim 5, wherein the
concentration of myocyte progenitor cells is about 1.times.10.sup.5
cells/ml to about 1.times.10.sup.7 cells/ml.
9. The pharmaceutical composition of claim 5, wherein the ratio of
vascular progenitor cells to myocyte progenitor cells is about
1:1.
10. A method for generating a biological bypass in a subject in
need thereof comprising: obtaining myocardial tissue from the
subject; extracting vascular progenitor cells from said myocardial
tissue; expanding said vascular progenitor cells in culture; and
administering said vascular progenitor cells to a stenotic or
occluded artery in the subject's heart, wherein the vascular
progenitor cells differentiate into endothelial cells and/or smooth
muscle cells, thereby forming coronary vessels that reestablish
blood flow to the myocardium.
11. The method of claim 10, wherein the coronary vessels include
coronary arteries, arterioles, and capillaries.
12. The method of claim 11, wherein the coronary vessels have
diameters ranging from about 6 .mu.m to about 2 mm.
13. The method of claim 11, wherein the coronary arteries are at
least 500 .mu.m in diameter.
14. The method of claim 10, wherein the vascular progenitor cells
are administered by a catheter.
15. A method for restoring structural and functional integrity to
damaged myocardium in a subject in need thereof comprising:
obtaining myocardial tissue from the subject; extracting vascular
progenitor cells from said myocardial tissue; expanding said
vascular progenitor cells in culture; and administering said
vascular progenitor cells to the damaged myocardium, wherein the
vascular progenitor cells differentiate into endothelial cells and
smooth muscles cells forming functional coronary vessels, thereby
increasing blood flow to the damaged myocardium.
16. The method of claim 15 further comprising extracting myocyte
progenitor cells from said myocardial tissue, expanding said
myocyte progenitor cells in culture; and administering said myocyte
progenitor cells to the damaged myocardium, wherein the myocyte
progenitor cells differentiate into cardiomyocytes forming
functional myocardium, thereby increasing contractile function.
17. The method of claim 16, wherein the myocyte progenitor cells
are administered simultaneously with the vascular progenitor
cells.
18. The method of claim 16, wherein the myocyte progenitor cells
are administered at a specified time interval after vascular
progenitor cells.
19. The method of claim 18, wherein the myocyte progenitor cells
are administered after said vascular progenitor cells have
generated functional coronary vessels.
20. The method of claim 16, wherein the vascular progenitor cells
and myocyte progenitor cells are administered by a catheter.
21. The method of claim 20, wherein the vascular progenitor cells
and myocyte progenitor cells are administered to the border zone of
the damaged myocardium.
22. The method of claim 16, wherein the damaged myocardium is an
infarct.
23. The method of claim 22, wherein the vascular progenitor cells
and myocyte progenitor cells are administered to the middle of the
infarct.
24. A method for treating or preventing hypertensive cardiomyopathy
in a subject in need thereof comprising: administering the
pharmaceutical composition of claim 5 to the subject's heart,
wherein the vascular progenitor cells and myocyte progenitor cells
engraft in said subject's heart, thereby repopulating diminished
progenitor cell niches or forming new progenitor cell niches.
25. The method of claim 24, wherein the vascular progenitor cells
and myocyte progenitor cells are autologous.
26. The method of claim 24, wherein the probability of the subject
having heart failure is reduced following administration of the
pharmaceutical composition.
27. A method for treating or preventing heart failure in a subject
in need thereof comprising: obtaining myocardial tissue from the
subject; extracting vascular progenitor cells from said myocardial
tissue; expanding said vascular progenitor cells in culture; and
administering said vascular progenitor cells to the subject's
heart, wherein the vascular progenitor cells differentiate into
endothelial cells and smooth muscles cells forming functional
coronary vessels, thereby increasing cardiac function.
28. The method of claim 27 further comprising extracting myocyte
progenitor cells from said myocardial tissue, expanding said
myocyte progenitor cells in culture; and administering said myocyte
progenitor cells to the damaged myocardium, wherein the myocyte
progenitor cells differentiate into cardiomyocytes forming
functional myocardium, thereby increasing cardiac function.
29. The method of claim 28, wherein the myocyte progenitor cells
are administered simultaneously with the vascular progenitor
cells.
30. The method of claim 28, wherein the myocyte progenitor cells
are administered at a specified time interval after vascular
progenitor cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/991,515, filed Nov. 30, 2007, which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
cardiology, and more particularly relates to compositions of
cardiac progenitor cells or cardiac stem cells and methods of using
the compositions for repairing damaged myocardium and/or generating
a biological coronary bypass.
BACKGROUND OF THE INVENTION
[0003] Acute and chronic post-infarction ischemic heart failure in
humans is characterized by myocardial regeneration which is limited
to the myocyte compartment of the surviving myocardium (259-270).
Additionally, small areas of spontaneous myocardial regeneration
which invade the infarct shortly after the ischemic event have been
identified (267). In addition to the loss in muscle mass, the
coronary vasculature remains defective and the extent and
regulation of myocardial perfusion are severely impaired (271-281).
Alterations in the balance between oxygen demand and supply have
been viewed for a long time as critical determinants of the
evolution of the ischemic myopathy (282). Pathology of the coronary
circulation together with humoral, mechanical and biochemical
factors sustain the ischemic myopathy and condition its unfavorable
progression to terminal failure (283-288).
[0004] Despite advances in understanding the etiology of coronary
artery disease (CAD) together with early diagnosis of pre-clinical
atherosclerotic lesions and treatment of conventional risk factors,
cardiovascular disease continues to be the leading cause of death
in the industrialized world (289). Coronary atherosclerosis is the
result of the evolving and complex interplay of endothelial injury,
inflammatory mediators and the accumulation of oxidized lipids
within the arterial wall (290-292). The presence of
pro-inflammatory and anti-inflammatory cytokines mediates the
cross-talk between the injured endothelial cells and the
constituents of the vessel wall which condition the progression of
the atherosclerotic plaque (292-296). The site of coronary artery
stenosis is characterized by a large fibrous cap, a small lipid
core and calcification; vessel pathology typically shows inward
growth and narrowing of the lumen. Conversely, non-constrictive
coronary atherosclerosis manifests itself with a lipid deposition
and a thin fibrous cap without a change in vessel luminal diameter
(291). However, it is the latter which is commonly involved in the
initiation of an acute coronary syndrome triggered by thrombosis
secondary to plaque rupture or erosion (297-299). More than 50% of
these events occur in the proximal portion of the epicardial
coronary arteries (300) resulting in sudden death, myocardial
infarction or ischemic cardiomyopathy.
SUMMARY OF THE INVENTION
[0005] One objective of the invention is to interfere with the
evolution of coronary artery disease by regenerating the various
portions of the coronary circulation together with cardiomyocytes
through the delivery of resident cardiac progenitor cells (PCs)
capable of differentiating into vascular endothelial cells (ECs),
smooth muscle cells (SMCs) and cardiomyocytes. The inventors have
surprisingly discovered two subsets of cardiac PCs: vascular
progenitor cells and myocyte progenitor cells. Vascular progenitor
cells are c-kit positive and flk1 positive and predominantly
differentiate into ECs and SMCs. Myocyte progenitor cells are c-kit
positive and flk1 negative and predominantly differentiate into
cardiomyocytes.
[0006] The present invention provides compositions, including
pharmaceutical compositions, of adult cardiac progenitor cells
useful for the treatment of various cardiac conditions. In one
embodiment of the invention, the pharmaceutical composition
comprises adult vascular progenitor cells and a pharmaceutically
acceptable carrier, wherein the vascular progenitor cells are
lineage negative, c-kit positive, and flk1 positive. In another
embodiment, the pharmaceutical composition further comprises adult
myocyte progenitor cells, wherein the myocyte progenitor cells are
lineage negative, c-kit positive, and flk1 negative. The vascular
progenitor cells and myocyte progenitor cells may be isolated from
human myocardium or myocardial vessels. In some embodiments, the
ratio of vascular progenitor cells to myocyte progenitor cells in
the composition can be varied to optimize cell therapy treatment
for a particular condition or a particular patient. In a preferred
embodiment, the ratio of vascular progenitor cells to myocyte
progenitor cells is about 1:1.
[0007] The present invention also provides a method for generating
a biological bypass in a subject in need thereof. In one
embodiment, the method comprises obtaining myocardial tissue from
the subject; extracting vascular progenitor cells from said
myocardial tissue; expanding said vascular progenitor cells in
culture; and administering said vascular progenitor cells to a
stenotic or occluded artery in the subject's heart, wherein the
vascular progenitor cells differentiate into endothelial cells
and/or smooth muscle cells, thereby forming coronary vessels that
reestablish blood flow to the myocardium. The coronary vessels may
include coronary arteries, arterioles, and capillaries with
diameters ranging from about 6 .mu.m to about 2 mm.
[0008] The present invention also encompasses a method for
restoring structural and functional integrity to damaged myocardium
in a subject in need thereof. In one embodiment, the method
comprises obtaining myocardial tissue from the subject; extracting
vascular progenitor cells from said myocardial tissue; expanding
said vascular progenitor cells in culture; and administering said
vascular progenitor cells to the damaged myocardium, wherein the
vascular progenitor cells differentiate into endothelial cells and
smooth muscles cells forming functional coronary vessels, thereby
increasing blood flow to the damaged myocardium. In another
embodiment, the method further comprises extracting myocyte
progenitor cells from said myocardial tissue, expanding said
myocyte progenitor cells in culture; and administering said myocyte
progenitor cells to the damaged myocardium, wherein the myocyte
progenitor cells differentiate into cardiomyocytes forming
functional myocardium, thereby increasing contractile function. The
myocyte progenitor cells may be administered simultaneously with
the vascular progenitor cells or after a particular time
interval.
[0009] The present invention also provides a method for treating or
preventing hypertensive cardiomyopathy in a subject in need thereof
comprising administering a pharmaceutical composition comprising
vascular progenitor cells and myocyte progenitor cells to the
subject's heart, wherein the vascular progenitor cells and myocyte
progenitor cells engraft in said subject's heart, thereby
repopulating diminished progenitor cell niches or forming new
progenitor cell niches. In one embodiment, the vascular progenitor
cells and myocyte progenitor cells are autologous. In another
embodiment, the probability of the subject having heart failure is
reduced following administration of the pharmaceutical
composition.
[0010] The present invention includes a method for treating or
preventing heart failure in a subject in need thereof. In one
embodiment, the method comprises obtaining myocardial tissue from
the subject; extracting vascular progenitor cells from said
myocardial tissue; expanding said vascular progenitor cells in
culture; and administering said vascular progenitor cells to the
subject's heart, wherein the vascular progenitor cells
differentiate into endothelial cells and smooth muscles cells
forming functional coronary vessels, thereby increasing cardiac
function. In another embodiment, the method further comprises
extracting myocyte progenitor cells from said myocardial tissue,
expanding said myocyte progenitor cells in culture; and
administering said myocyte progenitor cells to the damaged
myocardium, wherein the myocyte progenitor cells differentiate into
cardiomyocytes forming functional myocardium, thereby increasing
cardiac function.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Pro-epicardium. (A, B) Embryo at E9 showing the
localization of c-kit-positive EGFP-positive progenitor cells (PCs)
(B: green). Area in the rectangle is shown at higher magnification
in C and D. Arrows delimit the pro-epicardium. EGFP-positive PCs
express c-kit (C, white) and flk1 (D, magenta). PCs positive for
c-kit and flk1 are indicated by asterisks.
[0012] FIG. 2. Progenitor Cells in c-kit-EGFP mice. Expression of
flk1 (upper panels, magenta), c-kit (upper panels, white) and SCF
(lower panels, yellow) in EGFP-positive PCs (lower panels, green)
in the primitive streak (A), cardiac crescent (B), heart tube (C),
looping heart (D) and four-chambered heart (E). Arrows:
c-kit-positive flk1-positive PCs. Arrowheads: c-kit-positive
flk1-negative PCs.
[0013] FIG. 3. A. Formation of vessels. Regeneration of vessels by
proliferation of resident differentiated cells or recruitment of
circulating progenitors. B. Cardiac niches. Vascular niches contain
quiescent VPCs that following activation leave the niche area and
give rise to transient amplifying cells (TACs) differentiating into
SMCs, ECs and adventitial cells. Similarly, myocardial niches
contain quiescent MPCs that following activation leave the niche
area and give rise to TACs differentiating into myocytes.
[0014] FIG. 4. Vascular niches. C-kit-positive (A, C, E, G: green,
arrows) flk1-positive (B, D, F, H: white, arrows) VPCs in the
endothelium (A, B: not stained; E, F, I: vWF, yellow), SMC layer
(A, B: .alpha.-SM-actin, red) and adventitia (A, B: not stained; C,
D: collagen, blue) of coronary arterioles and tangentially
sectioned capillaries (E-I) in the mouse (A, C, E) and rat (G)
heart. Connexin 43 (magenta, arrowheads) is present between
c-kit-flk1-positive cells and ECs (G-I). GATA4 positive cells (E:
white; I: red). *VPCs positive for c-kit and flk1 only (I). A
continuous basal lamina (yellow) that defines the vascular niche is
not present (C; inset). Some of the cells adjacent to vessel (A, B)
are c-kit-positive flk1-negative. The functional role of these
cells is identified only by their differentiation potential in
vitro, as discussed in the detailed description.
[0015] FIG. 5. Myocardial niches. Some MPCs (A-D: c-kit, green)
express GATA4 (B, D: yellow, arrowheads) or Ets1 (C: white,
arrowheads). *MPCs positive for c-kit and negative for GATA4. Small
developing myocytes are also apparent (B, double arrows). (B:
.alpha.-sarcomeric actin, .alpha.-SA, red; double arrows). (E) A
continuous basal lamina (white) that defines the myocardial niche
is not present. A single PC that expresses c-kit and flk1 is also
present (D: VPC, open arrow).
[0016] FIG. 6. Clonogenic VPCs and MPCs. Clones generated by
deposition of single VPCs and MPCs. VPCs express c-kit and flk1
(A), and MPCs c-kit only (B). Clonogenic VPCs (C) differentiate
mostly into ECs (vWF: yellow) and SMCs (.alpha.-SMA: green) and
MPCs (D) mostly into myocytes (.alpha.-SA: red). E: Differentiation
pattern of VPCs and MPCs. Results are mean.+-.SD. *Difference
between VPCs and MPCs.
[0017] FIG. 7. EPCs and myocardial regeneration. Human EPCs
injected in the infarcted immunodeficient mouse heart formed human
(Alu sequences, green dots in nuclei) coronary vessels (left panel:
.alpha.-SM-actin, yellow; middle panel: vWF, white) and new
myocytes (right panel: .alpha.-SA, red; arrows). *Spared
myocytes
[0018] FIG. 8. Mechanisms of asymmetric division of stem cells. (A)
Cell polarization involves the establishment of distinct membrane
domains, apical and basolateral, through the formation of adherens
junctions. (B) The orientation of the mitotic spindle depends on
polarity proteins which determine the localization of the cell fate
determinants. (C) The distribution of the cell fate determinants
Numb and .alpha.-adaptin conditions the pattern of stem cell
division. The uniform localization of these endocytic proteins at
the two poles of the dividing stem cell results in the generation
of two daughter cells with identical fate. But the non-uniform
localization of these endocytic proteins at one pole only of the
dividing stem cell results in the generation of two daughter cells
with different fate.
[0019] FIG. 9. MPC division. A-C: Myocardial niche with 7 MPCs
(c-kit, green); 1 symmetrically dividing MPC (A: phospho-H3,
yellow, arrow) shows .alpha.-adaptin (B: magenta, arrow) and
internalized Notch (C: white, arrow) at both poles. D-F: Myocardial
niche with 7 MPCs; 1 asymmetrically dividing MPC (D: phospho-H3,
yellow, arrow) shows Numb (E: magenta, arrow) and Notch (F: white,
arrow) at one pole of the cell.
[0020] FIG. 10. Immortal strand and silent sister theories. (A)
During mitosis, the oldest template (blue) and the newer DNA
strands (red) randomly segregate in the daughter cells (blue and
red) or the oldest template DNA strands co-segregate in the
daughter stem cell (blue). (B) Chromatid-specific segregation of
chromosomes during mitosis results in the silencing of stem-related
genes in the daughter early committed cell (ECC). Copies of the
parental genes are indicated by 1 and 2. With asymmetric division,
the stem cell (SC) inherits active (ready to be transcribed)
self-renewal genes (capital letters) while the ECC inherits
repressed self-renewal genes (lower case letters). With symmetric
division, two cells identical to the mother SC are formed. If these
cells receive appropriate signals from the niche environment, they
will restore the gene expression typical of SCs and acquire the SC
phenotype (adapted from ref. 129).
[0021] FIG. 11. Transcriptional profile of BMPCs and MPCs. Freshly
isolated BMPCs and MPCs were compared. The spectrum of transcripts
was similar in the two cell populations with the exception of mRNA
for proteins specific of myeloid cells, mast cells and macrophages
which were expressed in BMPCs and mRNA for genes involved in
proliferation and differentiation of cells of mesodermal and
ectodermal origin which were expressed in MPCs. Genes and
fold-increases are indicated.
[0022] FIG. 12. Notch signaling in MPCs. A: As shown by
immunoprecipitation (IP), the intracellular domain of Notch1 and
RBP-Jk form a complex in MPCs. SN: supernatant used as negative
control. B, C: RBP-Jk binding to the promoter of Nkx2.5. B:
Gel-shift assay: arrows indicate the position of the RBPJk-shifted
and supershifted bands. Co, unlabeled self-oligonucleotide; NS Co,
unlabeled non-specific oligonucleotide; Ab: RBP-Jk antibodies;
Nkx2.5: oligonucleotide only. C: ChIP: arrows indicate the position
of the PCR product representing the Nkx2.5 promoter. DNA templates
were obtained from a protein-DNA complex immunoprecipitated with
RBP-Jk-specific antibody (Nkx2.5) or IgG only (IgG). Hes1 promoter
was used as positive control and MEF2C promoter as negative
control. Input: Genomic DNA without immunoprecipitation. CTRL:
control with no template. D, E: Mouse MPCs following the activation
of the Notch receptor express the Notch intracellular domain (D,
NICD: green) in their nuclei. NICD-positive MPCs express Nkx2.5 (E:
magenta). F: Treatment with .gamma.-secretase inhibitor which
blocks the Notch signaling, markedly attenuates the nuclear
accumulation of NICD and expression of Nkx2.5. G, H: In infarcted
mice, numerous small myocytes are positive for NICD (G: green) and
BrdU (H: white). I, J: Newly formed vessels in infarcted heart:
SMCs (.alpha.-SMA: red) and ECs (vWF: yellow) express NICD (I:
green) and are positive for BrdU (J: white). K: The Notch ligand
Delta-4 accumulates in the wall of regenerated vessels.
[0023] FIG. 13. Tail-cuff measurements of blood pressure in
sham-operated mice and mice subjected to renal artery clipping (4
weeks). Renal artery stenosis is associated with a marked increase
in arterial blood pressure (A) and multiple foci of replacement
fibrosis (B).
[0024] FIG. 14. Vascular and myocardial niches. A: Large section of
a dog coronary artery. Area in the rectangle is shown at a higher
magnification in the lower panel. One c-kit positive (green) and
flk1-positive (white) cell is present in the intimal layer (not
stained) of the vessel. The expression of connexin 43 (Cnx 43,
magenta) at the interface between the VPCs and endothelial cells is
shown in the inset. B, C: Transverse sections of human coronary
arterioles (SMCs, .alpha.-SMA; red). Clusters of several c-kit
positive (green) and flk1-positive (white) VPCs are present in the
adventitia (not stained). D: Myocardial niche containing several
c-kit positive (green) cells. These cells are flk1 negative; they
correspond to MPCs. The expression of connexin 43 (magenta) is
shown at the interface between two MPCs, between a MPC and a
myocyte (.alpha.-SA, red), and between a MPC and a fibroblast
(procollagen, yellow) is illustrated in the insets.
[0025] FIG. 15. Phenotypic characterization of VPCs and MPCs.
Freshly isolated dog VPCs and MPCs were expanded in vitro (P3-P4)
and analyzed by FACS. A: VPCs were negative for hematopoietic
markers and .alpha.-SA and expresses at very low levels desmin,
CD31, von Willebrand factor (vWf) and TGF-.beta.1 receptor. B: MPCs
were negative for hematopoietic markers, CD31, von Willebrand
factor (vWf) and TGF-.beta.1 receptor and expressed at low levels
.alpha.-SA and desmin. C: Cytospin preparation of freshly sorted
VPC shows the expression of c-kit (green) and flk1 (red),
confirming the FACS data.
[0026] FIG. 16. VPCs and MPCs are self-renewing, clonogenic and
multipotent. A: Clones derived from a single VPC and MPC obtained
from a canine coronary artery (VPC clone) and myocardium (MPC
clone) are shown by phase-contrast microscopy. B: The dog VPC clone
is positive for c-kit (green), flk1 (red) and both c-kit and flk1
(yellow). C: The dog MPC clone is positive for c-kit (green) but is
negative for flk1. D, E: In differentiating medium, VPCs (D)
differentiate mostly into SMCs (.alpha.-SMA, green) and ECs (vWf,
yellow), while MPCs (E) differentiate predominantly into myocytes
(.alpha.-SA, red).
[0027] FIG. 17. Engraftment of VPCs into the vessel wall of
coronary arteries. A and B: DiI-labeled VPCs (red) were placed on
the endothelial surface of calcein-labeled dog coronary artery
(green) and examined by two-photon microscopy. After 15 hours, the
appearance of green fluorescence in the two DiI-labeled VPCs
(arrow) indicates the transfer of calcein through functional gap
junctions. C and D: DiI-labeled VPCs (C, red) were co-cultured with
a segment of human right coronary artery loaded with calcein (D,
green). Several DiI-labeled VPCs show yellow-green fluorescence
(arrowheads) which indicates the transfer of calcein through
functional gap junctions. Blue fluorescence corresponds to
collagen. E: VPCs were co-cultured with a segment of a human
coronary artery and their translocation within the vessel wall was
followed for 8 hours by two-photon microscopy. This panel
corresponds to the superimposition of two images of the same field
taken one hour apart. The first corresponds to the position of VPCs
in the vessel wall at 5 hours (red dots) and the second at 6 hours
(white dots). Yellow arrows indicate the direction of migration.
Blue fluorescence corresponds to collagen.
[0028] FIG. 18 shows the ensuing reactive hyperemia after (left to
right) release (R) 5, 10, 15 and 30 second occlusion of the
coronary artery. There is a large hyperemic response which will be
used to assess the degree of coronary flow reserve.
[0029] FIG. 19 shows that injection of VPCs resulted in formation
of large coronary arteries, 1.5 mm in diameter which stain for A:
.alpha.-smooth muscle actin (Red), B: green fluorescent protein
(green), C: a marker for primate DNA (white, Alu) and, D: the merge
of A & B. E, F & G: indicates staining of smooth muscle
actin, green fluorescent protein and the merge of E & F,
respectively on small coronary microvessels, less than 100 .mu.m in
diameter. Thus, the injection of VPCs resulted in formation of
large coronary arteries and arterioles.
[0030] FIG. 20. A. During occlusion of the LAD using the hydraulic
occluder, injection of contrast did not appear in the distal LAD
circulation in 2 of the dogs (i.e. there was little collateral
blood flow), whereas there was a substantial and obvious appearance
of contrast in the distal circulation in the one dog receiving VPCs
(evidence of a newly developed circulation) as shown above in A by
the dark arrows. B. Ligation resulted in apical wall thinning and
paradoxical motion (see arrows). Thus, we have created a model of a
large infarct accompanied by heart failure.
[0031] FIG. 21. A. Injection of HGF or IGF results in an increase
in contractile function in the ischemic zone following 4 hours of
total occlusion of the LAD (301). In addition, evidence of newly
generated cardiac myocytes and an increase in ejection fraction,
stroke volume (figure from Linke et al (301)) and shortening (panel
A) that was proportional to the regeneration of the myocardium was
observed. B. In another set of experiments (panel B) designed to
use an adeno-associated virus containing VEGF to grow new blood
vessels in the area of an infarction (small LAD infarct as
proposed), an increase in segment function and segment work in
segments that were either paradoxical to begin with or in those
that had reduced shortening (310) was observed. This was associated
with an increase in the number of cardiomyocytes in the area of the
infarct.
DETAILED DESCRIPTION OF THE INVENTION
[0032] As used herein, "autologous" refers to something that is
derived or transferred from the same individual's body (i.e.,
autologous blood donation; an autologous bone marrow
transplant).
[0033] As used herein, "allogeneic" refers to something that is
genetically different although belonging to or obtained from the
same species (e.g., allogeneic tissue grafts or organ
transplants).
[0034] As used herein, "stem cells" are used interchangeably with
"progenitor cells" and refer to cells that have the ability to
renew themselves through mitosis as well as differentiate into
various specialized cell types. The stem cells used in the
invention are somatic stem cells, such as bone marrow or cardiac
stem cells or progenitor cells. "Vascular progenitor cells" or VPCs
are a subset of adult cardiac stem cells that are c-kit positive
and flk1 (e.g. VEGFR-2) positive, which generate predominantly
endothelial cells and smooth muscle cells. "Myocyte progenitor
cells" or MPCs are a subset of adult cardiac stem cells that are
c-kit positive and flk1 negative, which generate cardiomyocytes
predominantly.
[0035] As used herein, "adult" stem cells refers to stem cells that
are not embryonic in origin nor derived from embryos or fetal
tissue.
[0036] Stem cells employed in the invention are advantageously
selected to be lineage negative. The term "lineage negative" is
known to one skilled in the art as meaning the cell does not
express antigens characteristic of specific cell lineages. And, it
is advantageous that the lineage negative stem cells are selected
to be c-kit positive. The term "c-kit" is known to one skilled in
the art as being a receptor which is known to be present on the
surface of stem cells, and which is routinely utilized in the
process of identifying and separating stem cells from other
surrounding cells.
[0037] As used herein, the term "cytokine" is used interchangeably
with "growth factor" and refers to peptides or proteins that bind
receptors on cell surfaces and initiate signaling cascades thus
influencing cellular processes. The terms "cytokine" and "growth
factor" encompass functional variants of the native cytokine or
growth factor. A functional variant of the cytokine or growth
factor would retain the ability to activate its corresponding
receptor. Variants can include amino acid substitutions,
insertions, deletions, alternative splice variants, or fragments of
the native protein. The term "variant" with respect to a
polypeptide refers to an amino acid sequence that is altered by one
or more amino acids with respect to a reference sequence. The
variant can have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties, e.g.,
replacement of leucine with isoleucine. Alternatively, a variant
can have "nonconservative" changes, e.g., replacement of a glycine
with a tryptophan. Analogous minor variations can also include
amino acid deletion or insertion, or both. Guidance in determining
which amino acid residues can be substituted, inserted, or deleted
without eliminating biological activity can be found using computer
programs well known in the art, for example, DNASTAR software.
[0038] As used herein "damaged myocardium" refers to myocardial
cells which have been exposed to ischemic conditions. These
ischemic conditions may be caused by a myocardial infarction, or
other cardiovascular disease or related complaint. The lack of
oxygen causes the death of the cells in the surrounding area,
leaving an infarct, which will eventually scar.
[0039] As used herein, "patient" or "subject" may encompass any
vertebrate including but not limited to humans, mammals, reptiles,
amphibians and fish. However, advantageously, the patient or
subject is a mammal such as a human, or a mammal such as a
domesticated mammal, e.g., dog, cat, horse, and the like, or
production mammal, e.g., cow, sheep, pig, and the like.
[0040] The pharmaceutical compositions of the present invention may
be used as therapeutic agents--i.e. in therapy applications. As
herein, the terms "treatment" and "therapy" include curative
effects, alleviation effects, and prophylactic effects. In certain
embodiments, a therapeutically effective dose of progenitor cells
is applied, delivered, or administered to the heart or implanted
into the heart. An effective dose or amount is an amount sufficient
to effect a beneficial or desired clinical result. Said dose could
be administered in one or more administrations.
[0041] Mention is made of the following related pending patent
applications:
[0042] U.S. Application Publication No. 2003/0054973, filed Jun. 5,
2002, which is herein incorporated by reference in its entirety,
discloses methods, compositions, and kits for repairing damaged
myocardium and/or myocardial cells including the administration
cytokines.
[0043] U.S. Application Publication No. 2006/0239983, filed Feb.
16, 2006, which is herein incorporated by reference in its
entirety, discloses methods, compositions, and kits for repairing
damaged myocardium and/or myocardial cells including the
administration of cytokines and/or adult stem cells as well as
methods and compositions for the development of large arteries and
vessels. The application also discloses methods and media for the
growth, expansion, and activation of human cardiac stem cells.
[0044] In development, the cardiogenic mesoderm contains two
populations of progenitor cells (PCs), which are destined to
generate pre-cardiomyocytes and pre-endocardial cells while
coronary vessels are formed independently (1-5). Endothelial and
smooth muscle precursors migrate from the pro-epicardium and
differentiate into sinusoidal vesicles that create capillary
channels (6-8). When the closed vessel network is established and
connections with the aorta are made, smooth muscle precursors
migrate to segments of the endothelial channels and coronary
arteries are formed (8-11). Five classes of PCs have been
implicated in cardiac development: endocardial, myocardial,
endothelial, smooth muscle and peri-vascular connective tissue cell
progenitors-precursors. The contracting heart is avascular for
several days (2, 7), strengthening the notion that the origin of
the coronary vasculature is distinct from the muscle mass. The
inventors have identified in the developing mouse heart that
c-kit-positive-flk1-positive PCs together with c-kit-positive
flk1-negative PCs are present in the pro-epicardium from which the
coronary circulation is formed and in the primitive myocardium from
which cardiomyocytes originate (FIGS. 1 and 2). Thus, c-kit is
present in two PC classes differentiating into vascular cells and
cardiomyocytes. To provide evidence in favor of the interaction of
c-kit, flk1 and the c-kit ligand, stem cell factor (SCF), in
cardiac development their colocalization was established in embryos
from c-kit-EGFP mice; flk1, c-kit and SCF were concurrently
expressed in the primitive streak, cardiac crescent, primitive
heart tube, looping heart and four-chambered heart (FIG. 2). A
second subset of c-kit-positive flk1-negative cells was also
found.
[0045] The identification that several PCs regulate cardiac
development together with observations of myocyte and vessel
formation in the adult (12-17), has led to the recognition that the
heart is a dynamic organ regulated by a stem cell compartment
(18-20). Moreover, this intrinsic cellular system promotes partial
cardiac repair following injury (21-23). Although several cardiac
PC classes have been described (21, 22, 24-32), the inventors have
discovered c-kit-positive flk1-negative myocyte progenitor cells
(MPCs) appear to represent the most potent cell for myocardial
regeneration (18-22). The inventors have focused on the functional
characterization of MPCs and their ability to form a myocyte
progeny that reaches the adult phenotype in rodent, dogs and humans
(16, 21-23, 33). These cells acquire the electrical, mechanical and
calcium transient properties of mature myocytes (21, 23, 33). Also,
MPCs give rise to coronary arterioles and capillary structures.
Myocyte regeneration is impressive but vessel growth is not as
striking as myocyte growth (21-23, 33). This differential response
is consistent with in vitro results in which MPCs differentiate
predominantly into myocytes and to a lesser extent into endothelial
cells (ECs) and smooth muscle cells (SMCs) (21, 33). Thus, MPCs
acquire predominantly a cardiomyogenic fate but possess also a
restricted ability to form ECs and SMCs. Accordingly, the present
invention provides isolated myocyte progenitor cells, wherein the
myocyte progenitor cells are c-kit positive and flk1 negative. In
one embodiment, the myocyte progenitor cells differentiate
predominantly into cardiomyocytes, that is at least 80%, at least
85%, at least 90%, or at least 95% of the cells generated from
myocyte progenitor cells are cardiomyocytes.
[0046] The notion that PCs in the adult heart generate de novo
coronary vessels is at variance with the traditional view of
coronary vessel biology. It is generally believed that, in contrast
to active vessel growth in the embryonic and neonatal heart, the
adult coronary vasculature is quiescent (34). A certain degree of
expansion of the vascular bed is considered possible only after
tissue injury (35, 36). This process can be mediated by three
mechanisms (FIG. 3A): (a) angiogenesis that corresponds to the
sprouting of mature ECs from pre-existing vessels in response to
angiogenic growth factors (36); (b) vasculogenesis that corresponds
to sites of active neovascularization mediated by recruitment of
circulating endothelial progenitor cells (EPCs) from the bone
marrow (37-40); and (c) adaptive arteriogenesis or collateral
vessel formation that corresponds to the development of large
vessels from pre-existing arteriolar anastomosis (41). This process
is mediated by shear-stress which upregulates angiogenic and
inflammatory factors (34, 35). Thus, at the site of
vascularization, ECs are assumed to originate from adjacent
pre-existing blood vessels or from recruited EPCs. SMCs are derived
from a pool of circulating progenitors or, in analogy to
atherogenesis, from mature cells in the media (41-43).
[0047] The contribution of resident vascular PCs to vasculogenesis
is a relatively new concept in vascular biology. Among the most
likely candidates, a population of Sca-1 positive PCs located in
the adventitia of the mouse aorta may represent a novel vascular
primitive cell (44-47). Multipotent, self-renewing cells with
characteristics similar to embryonic mesangioblasts have been
isolated from the embryonic aorta (48-50) and PCs with vasculogenic
potential have been identified in the human thoracic aorta (51).
Importantly, in the presence of tissue ischemia, various organs
contribute to the release of a pool of circulating PCs distinct
from the bone marrow which have powerful vasculogenic properties
(52). Collectively, this novel information suggests that
undifferentiated cells may reside in the vessel wall and play a
relevant role in vessel homeostasis and regeneration.
[0048] Therefore, in analogy to cardiomyogenesis that is promoted
by activation, proliferation and differentiation of resident MPCs,
the inventors suggested that the physiological turnover of vascular
ECs and SMCs and vasculogenesis following injury are regulated by
the commitment of resident c-kit-positive flk1-positive vascular
progenitor cells (VPCs). The inventors have found that
c-kit-flk1-positive PCs from the adult mouse heart generate single
cell clones and these clonogenic cells differentiate predominantly
into ECs and SMCs and to a much smaller extent into myocytes (FIG.
6). Thus, c-kit-flk1-positive PCs are nested in vascular niches and
possess the fundamental properties of stem cells: they are
self-renewing, clonogenic and multipotent. They appear to
constitute a novel class of adult VPC distinct from MPCs. MPCs are
located in myocardial niches, do not express flk1 and differentiate
predominantly into cardiomyocytes. Thus, the present invention also
provides isolated vascular progenitor cells, wherein the vascular
progenitor cells are c-kit positive and flk1 (e.g. VEGFR-2)
positive. In one embodiment, the vascular progenitor cells
differentiate predominantly into ECs and SMCs, that is at least
80%, at least 85%, at least 90%, or at least 95% of the cells
generated from vascular progenitor cells are ECs and SMCs.
[0049] Given the regenerative capacity of these new classes of
cardiac PCs and their propensity for generating particular cardiac
lineages, MPCs and VPCs are particularly useful in generating new
myocardial tissue and vessels, respectively. Accordingly, the
present invention provides a method for restoring structural and
functional integrity to damaged myocardium in a subject in need
thereof. Restoration of structural and functional integrity
preferably requires the generation of new functional myocardium
comprised of new cardiomyocytes as well as new myocardial vessels
comprised of new endothelial and smooth muscle cells. In one
embodiment, the method comprises obtaining myocardial tissue from
the subject; extracting vascular progenitor cells from said
myocardial tissue; expanding said vascular progenitor cells in
culture; and administering said vascular progenitor cells to the
damaged myocardium, wherein the vascular progenitor cells
differentiate into endothelial cells and smooth muscles cells
forming functional coronary vessels, thereby increasing blood flow
to the damaged myocardium. Preferably, the vascular progenitor
cells are c-kit positive and flk1 positive.
[0050] In another embodiment, the method further comprising
extracting myocyte progenitor cells from said myocardial tissue,
expanding said myocyte progenitor cells in culture; and
administering said myocyte progenitor cells to the damaged
myocardium, wherein the myocyte progenitor cells differentiate into
cardiomyocytes forming functional myocardium, thereby increasing
contractile function. Preferably, the myocyte progenitor cells are
c-kit positive and flk1 negative.
[0051] Administration of VPCs and/or MPCs are used to restore
structural and functional integrity to damaged myocardium and or
damaged myocardial vessels resulting from cardiovascular diseases,
including, but not limited to, atherosclerosis, ischemia,
hypertension, restenosis, angina pectoris, rheumatic heart disease,
congenital cardiovascular defects and arterial inflammation and
other diseases of the arteries, arterioles and capillaries or
related complaint. In some embodiments, the subject is suffering
from a myocardial infarction and the damaged myocardium is an
infarct. The vascular progenitor cells and myocyte progenitor cells
may be administered to a border zone of the damaged myocardium
(e.g. infarct) and/or they may be administered to the middle of the
infarct.
[0052] The present invention also provides a method of generating a
biological bypass in a subject in need thereof. This method can be
used in conjunction with surgical procedures, such as stenting and
angioplasty, or is preferably used in place of such surgical
procedures. In one embodiment, the method comprises obtaining
myocardial tissue from the subject; extracting vascular progenitor
cells from said myocardial tissue; expanding said vascular
progenitor cells in culture; and administering said vascular
progenitor cells to a stenotic or occluded artery in the subject's
heart, wherein the vascular progenitor cells differentiate into
endothelial cells and/or smooth muscle cells, thereby forming
coronary vessels that reestablish blood flow to the myocardium.
[0053] The coronary vessels that may be formed include coronary
arteries, arterioles, and capillaries. The formed coronary vessels
may have diameters ranging from about 6 .mu.m to about 2 mm. In one
embodiment, the coronary vessel has a diameter of over 100 .mu.m.
In a further embodiment, the formed coronary vessel has a diameter
of at least 125, at least 150, at least 175, at least 200, at least
225, at least 250, at least 275, at least 300, at least 325, at
least 350, at least 375, at least 400, at least 425, at least 450,
or at least 475 .mu.m. In a preferred embodiment, formed coronary
arteries have a diameter of at least 500 .mu.m. In yet another
embodiment of the present invention, the formed coronary vessel
provides a biological bypass around an area in need of therapy or
repair, including around an occlusion or blockage, such that blood
flow, blood pressure, and circulation are restored or improved.
Improvements or enhancements in blood flow and cardiac function or
contractility can be assessed using standard techniques known to
those skilled in the art of cardiology, including, but not limited
to, hemodynamic analysis and echocardiography.
[0054] The present invention also encompasses methods of treating
or preventing hypertensive cardiomyopathy. Hypertensive
cardiomyopathy is a weakening of the heart muscle or a change in
heart muscle structure caused by prolonged high blood pressure,
which can lead to heart failure.
[0055] The evolution of hypertensive cardiomyopathy may be
conditioned by the formation of dysfunctional vascular and
myocardial niches and loss of functionally-competent VPCs and MPCs.
Thus, it may be possible to interfere with the etiology of
hypertensive cardiomyopathy by repopulating dysfunctional niches
and the PC pool with functionally-competent VPCs and MPCs or by
creating new vascular and myocardial niches. The newly repopulated
niches can provide a sufficient number of VPCs and MPCs to
regenerate an unlimited number of coronary vessels and myocytes so
that the heart can have the capacity to correct anatomical changes
produced by pathologic loads and sustain pump function
indefinitely.
[0056] VPC and MPC niches possess distinct structural and
functional properties (FIG. 3B). The inventors have shown that VPCs
are nested within the endothelium, the media and the adventitia of
different classes of coronary vessels of the mouse and rat heart
(FIG. 4). Gap and adherens junctions made by connexins and
cadherins are present between VPCs, and between VPCs and ECs, SMCs
and fibroblasts. VPCs in the intima and media appear as single
cells or in groups of 2-4 cells. Larger pockets of VPCs are found
predominantly in close proximity to large-intermediate arteries and
resistance arterioles. Cell clusters consist of undifferentiated
VPCs and lineage committed cells (LCC), i.e., ECs and SMCs. Larger
clusters in the adventitia are similar but occasionally show one or
two cells committed to the myocyte fate. Collectively, these
structural properties are consistent with vascular niches.
[0057] Vascular niches are distinct from myocardial niches for
their localization along the territory of the coronary vessels and
cellular composition. In myocardial niches, MPCs are intimately
associated with cells predominantly committed to the myocyte
lineage although EC and SMC progenitor-precursors may be found
(FIG. 5). In myocardial niches, myocytes and fibroblasts but not
ECs operate as supporting cells (53). Thus, vascular and myocardial
niches function in tandem to sustain heart homeostasis.
[0058] In one embodiment of the invention, the method for treating
or preventing hypertensive cardiomyopathy in a subject in need
thereof comprises administering vascular progenitor cells and
myocyte progenitor cells to the subject's heart, wherein the
vascular progenitor cells and myocyte progenitor cells engraft in
said subject's heart, thereby repopulating diminished progenitor
cell niches or forming new progenitor cell niches. In preferred
embodiments, the vascular progenitor cells and myocyte progenitor
cells are autologous.
[0059] Following administration of the progenitor cells to the
subject's heart, the vascular progenitor cells engraft within
established vascular niches or form new vascular niches within the
walls of coronary vessels. Similarly, following their
administration, myocyte progenitor cells engraft within established
myocyte niches or form new myocyte niches within the myocardium,
particularly in the subject's atria or myocardial apex.
Repopulation of the progenitor cell niches within a subject's heart
restores the regenerative capacity of the subject's heart and can
reduce the symptoms or occurrence of cardiovascular disease or
heart failure. Thus, the present invention also provides a method
for restoring regenerative capacity to a subject's heart by
administering VPCs and/or MPCs to the subject's heart. In one
embodiment, the probability of the subject having heart failure is
reduced following administration of the progenitor cells.
[0060] The present invention also includes a method for treating or
preventing heart failure in a subject in need thereof. Heart
failure may be the result of diminished functional capacity of
resident VPCs and MPCs or a depletion of functional VPCs and MPCs
within their respective niches in the heart. In one embodiment of
the invention, the method comprises obtaining myocardial tissue
from the subject; extracting vascular progenitor cells from said
myocardial tissue; expanding said vascular progenitor cells in
culture; and administering said vascular progenitor cells to the
subject's heart, wherein the vascular progenitor cells
differentiate into endothelial cells and smooth muscles cells
forming functional coronary vessels, thereby increasing cardiac
function.
[0061] In another embodiment, the method further comprises
extracting myocyte progenitor cells from said myocardial tissue,
expanding said myocyte progenitor cells in culture; and
administering said myocyte progenitor cells to the damaged
myocardium, wherein the myocyte progenitor cells differentiate into
cardiomyocytes forming functional myocardium, thereby increasing
cardiac function. Increased cardiac function may be reflected as
increased exercise capacity, increased cardiac ejection volume,
decreased left ventricular end diastolic pressure, decreased
pulmonary capillary wedge pressure, increased cardiac output,
increased cardiac index, lowered pulmonary artery pressures,
decreased left ventricular end systolic and diastolic dimensions,
decreased left and right ventricular wall stress, and decreased
wall tension. In some embodiments, multiple administrations of VPCs
and/or MPCs may be made to the subject's heart. For example, VPCs
and/or MPCs may be administered in two or more, three or more, four
or more, five or more, or six or more injections. Injections may be
made at the base of the heart, the apex, or the mid-region. In one
embodiment, two injections of VPCs and/or MPCs are made at each of
the apex, mid-region, and base.
[0062] Preferably, one or more symptoms of heart failure are
reduced or alleviated following administration of VPCs and/or MPCs.
Symptoms of heart failure include, but are not limited to, fatigue,
weakness, rapid or irregular heartbeat, dyspnea, persistent cough
or wheezing, edema in the legs and feet, and swelling of the
abdomen.
[0063] Progenitor cells may be isolated from tissue specimens (e.g.
myocardium or myocardial vessels) obtained from a subject or
patient. By way of example, myocardial tissue specimens may be
minced and placed in appropriate culture medium. Cardiac progenitor
cells growing out from the tissue specimens can be observed in
approximately 1-2 weeks after initial culture. At approximately 4
weeks after the initial culture, the expanded progenitor cells may
be collected by centrifugation. Other methods of isolating adult
cardiac progenitor cells from a subject are known in the art and
can be employed to obtain suitable progenitor cells for use in the
methods of the invention. U.S. Patent Application Publication No.
2006/0239983, filed Feb. 16, 2006, which is herein incorporated by
reference in its entirety, describes media appropriate for
culturing and expanding adult progenitor cells, particularly human
cardiac progenitor cells. However, one of ordinary skill in the art
would be able to determine the necessary components and modify
commonly used cell culture media to be employed in culturing the
isolated cardiac progenitor cells of the invention.
[0064] It is preferable that the cardiac progenitor cells of the
invention are lineage negative. Lineage negative progenitor cells
can be isolated by various means, including but not limited to,
removing lineage positive cells by contacting the progenitor cell
population with antibodies against lineage markers and subsequently
isolating the antibody-bound cells by using an anti-immunoglobulin
antibody conjugated to magnetic beads and a biomagnet.
Alternatively, the antibody-bound lineage positive stem cells may
be retained on a column containing beads conjugated to
anti-immunoglobulin antibodies. The cells not bound to the
immunomagnetic beads represent the lineage negative progenitor cell
fraction and may be isolated. For instance, cells expressing
markers of the cardiac lineage (e.g. markers of vascular cell or
cardiomyocyte commitment) may be removed from cardiac progenitor
cell populations to isolate lineage negative cardiac progenitor
cells. Markers of the vascular lineage include, but are not limited
to, GATA6 (SMC transcription factor), Ets1 (EC transcription
factor), Tie-2 (angiopoietin receptors), VE-cadherin (cell adhesion
molecule), CD62E/E-selectin (cell adhesion molecule),
alpha-SM-actin (.alpha.-SMA, contractile protein), CD31 (PECAM-1),
vWF (carrier of factor VIII), Bandeiraera simplicifolia and Ulex
europaeus lectins (EC surface glycoprotein-binding molecules).
Markers of the myocyte lineage include, but are not limited to,
GATA4 (cardiac transcription factor), Nkx2.5 and MEF2C (myocyte
transcription factors), and alpha-sarcomeric actin (.alpha.-SA,
contractile protein).
[0065] In a preferred embodiment of the invention, the lineage
negative progenitor cells express the stem cell surface marker,
c-kit, which is the receptor for stem cell factor. Positive
selection methods for isolating a population of lineage negative
progenitor cells expressing c-kit are well known to the skilled
artisan. Examples of possible methods include, but are not limited
to, various types of cell sorting, such as fluorescence activated
cell sorting (FACS) and magnetic cell sorting as well as modified
forms of affinity chromatography. In a preferred embodiment, the
lineage negative progenitor cells are c-kit positive.
[0066] Vascular progenitor cells are isolated by selecting cells
expressing the VEGFR2 receptor, flk1, from the c-kit positive
progenitor cell population, isolated as described above. Thus,
vascular progenitor cells are lineage negative, c-kit positive, and
flk1 positive. Similarly, myocyte progenitor cells are isolated
from the c-kit progenitor cell population by selecting cells that
do no express flk1. Therefore, myocyte progenitor cells are lineage
negative, c-kit positive, and flk1 negative. Similar methods for
isolating c-kit positive progenitor cells may be employed to select
cells that express or do not express the flk1 receptor (e.g.
immunobeads, cell sorting, affinity chromatography, etc.).
[0067] Isolated lineage negative, c-kit positive progenitor cells
(e.g. VPCs and MPCs) may be plated individually, for instance in
single wells of a cell culture plate, and expanded to obtain clones
from individual progenitor cells. In some embodiments, cardiac
progenitor cells that are c-kit positive and flk1 positive are
plated individually to obtain pure cultures of vascular progenitor
cells. In other embodiments, cardiac progenitor cells that are
c-kit positive and flk1 negative are plated individually to obtain
pure cultures of myocyte progenitor cells.
[0068] In certain embodiments of the invention, the vascular
progenitor cells or myocyte progenitor cells are activated prior to
administration to a subject. Activation of the progenitor cells may
be accomplished by exposing the progenitor cells to one or more
cytokines. Suitable concentrations of the one or more cytokines for
activating the progenitor cells include a concentration of about
0.1 to about 500 ng/ml, about 10 to about 500 ng/ml, about 20 to
about 400 ng/ml, about 30 to about 300 ng/ml, about 50 to about 200
ng/ml, or about 80 to about 150 ng/ml. In one embodiment, the
concentration of one or more cytokines is about 25, about 50, about
75, about 100, about 125, about 150, about 175, about 200, about
225, about 250, about 275, about 300, about 325, about 350, about
375, about 400, about 425, about 450, about 475, or about 500
ng/ml. In some embodiments, the vascular progenitor cells or
myocyte progenitor cells are activated by contact with hepatocyte
growth factor (HGF), insulin-like growth factor-1 (IGF-1), or a
variant thereof.
[0069] HGF positively influences stem cell migration and homing
through the activation of the c-Met receptor (Kollet et al. (2003)
J. Clin. Invest. 112: 160-169; Linke et al. (2005) Proc. Natl.
Acad. Sci. USA 102: 8966-8971; Rosu-Myles et al. (2005) J. Cell.
Sci. 118: 4343-4352; Urbanek et al. (2005) Circ. Res. 97: 663-673).
Similarly, IGF-1 and its corresponding receptor (IGF-1R) induce
cardiac stem cell division, upregulate telomerase activity, hinder
replicative senescence and preserve the pool of
functionally-competent cardiac stem cells in the heart (Kajstura et
al. (2001) Diabetes 50: 1414-1424; Torella et al. (2004) Circ. Res.
94: 514-524; Davis et al. (2006) Proc. Natl. Acad. Sci. USA 103:
8155-8160). In some embodiments, the vascular progenitor cells or
myocyte progenitor cells are contacted with HGF and IGF-1.
[0070] Some other non-limiting examples of cytokines that are
suitable for the activation of the vascular progenitor cells or
myocyte progenitor cells include Activin A, Bone Morphogenic
Protein 2, Bone Morphogenic Protein 4, Bone Morphogenic Protein 6,
Cardiotrophin-1, Fibroblast Growth Factor 1, Fibroblast Growth
Factor 4, Flt3 Ligand, Glial-Derived Neurotrophic Factor, Heparin,
Insulin-like Growth Factor-II, Insulin-Like Growth Factor Binding
Protein-3, Insulin-Like Growth Factor Binding Protein-5,
Interleukin-3, Interleukin-6, Interleukin-8, Leukemia Inhibitory
Factor, Midkine, Platelet-Derived Growth Factor AA,
Platelet-Derived Growth Factor BB, Progesterone, Putrescine, Stem
Cell Factor, Stromal-Derived Factor-1, Thrombopoietin, Transforming
Growth Factor-.alpha., Transforming Growth Factor-.beta.1,
Transforming Growth Factor-.beta.2, Transforming Growth
Factor-.beta.3, Vascular Endothelial Growth Factor, Wnt1, Wnt3a,
and Wnt5a, as described in Kanemura et al. (2005) Cell Transplant.
14:673-682; Kaplan et al. (2005) Nature 438:750-751; Xu et al.
(2005) Methods Mol. Med. 121:189-202; Quinn et al. (2005) Methods
Mol. Med. 121:125-148; Almeida et al. (2005) J Biol Chem.
280:41342-41351; Barnabe-Heider et al. (2005) Neuron 48:253-265;
Madlambayan et al. (2005) Exp Hematol 33:1229-1239; Kamanga-Sollo
et al. (2005) Exp Cell Res 311:167-176; Heese et al. (2005)
Neuro-oncol. 7:476-484; He et al. (2005) Am J Physiol.
289:H968-H972; Beattie et al. (2005) Stem Cells 23:489-495; Sekiya
et al. (2005) Cell Tissue Res 320:269-276; Weidt (2004) Stem Cells
22:890-896; Encabo et al (2004) Stem Cells 22:725-740; and
Buytaeri-Hoefen et al. (2004) Stem Cells 22:669-674, the entire
text of each of which is incorporated herein by reference.
[0071] Functional variants of the above-mentioned cytokines can
also be employed in the invention. Functional cytokine variants
would retain the ability to bind and activate their corresponding
receptors. Variants can include amino acid substitutions,
insertions, deletions, alternative splice variants, or fragments of
the native protein. For example, NK1 and NK2 are natural splice
variants of HGF, which are able to bind to the c-MET receptor.
These types of naturally occurring splice variants as well as
engineered variants of the cytokine proteins that retain function
can be employed to activate the progenitor cells of the
invention.
[0072] The present invention involves administering a
therapeutically effective dose or amount of progenitor cells to a
subject's heart. An effective dose is an amount sufficient to
effect a beneficial or desired clinical result. Said dose could be
administered in one or more administrations. In some embodiments,
at least three effective doses are administered to the subject's
heart. In other embodiments, at least five effective doses are
administered to the subject's heart. Each administration of
progenitor cells may comprise a single type of progenitor cell
(e.g. VPC or MPC) or may contain a mixture of VPCs and MPCs. In one
embodiment, VPCs and/or MPCs are administered to a border zone of
the damaged myocardium. More than one administration of the
progenitor cells may be administered to the border zone, for
instance, two or more, three or more, four or more, or five or more
administrations may be applied to the border zone of the damaged
myocardium.
[0073] In some embodiments, it may be beneficial to alter the
number of MPCs and VPCs to optimize the ratio that governs flow and
function to increase segment function and to fully restore
contractile function of the anterior wall of the heart. Thus, in
one embodiment, the MPCs are administered simultaneously with VPCs.
The ratio of VPCs to MPCs may be adjusted to obtain more
endothelial cells, smooth muscle cells, and myocardial vessels or
more cardiomyocytes and myocardium. For example, suitable ratios of
VPCs to MPCs include, but are not limited to, 1:20; 1:10; 1:5, 1:2;
1:1:2:1, 5:1; 10:1, and 20:1. In a preferred embodiment, the ratio
of VPCs to MPCs is 1:1. In other embodiments, MPCs are administered
at a particular time interval after VPCs. These embodiments allow
for the development of coronary circulation by differentiation of
the injected VPCs to support the differentiation, growth and
function of later injected MPCs. In one embodiment, MPCs are
administered after VPCs have generated functional coronary vessels.
Examples of suitable time intervals include, but are not limited
to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6
months, 9 months, 12 months, 18 months or 24 months.
[0074] An effective dose of progenitor cells may be from about
2.times.10.sup.4 to about 2.times.10.sup.7, more preferably about
1.times.10.sup.5 to about 6.times.10.sup.6, or most preferably
about 2.times.10.sup.6. As illustrated in the examples, about
2.times.10.sup.6 to about 1.times.10.sup.7 progenitor cells are
used to effect generation of new myocardium and new myocardial
vessels in a canine model. Although there would be a size
difference between the heart of a canine and the heart of a human,
it is likely that this range of progenitor cells would be
sufficient in a human as well. However, the precise determination
of what would be considered an effective dose may be based on
factors individual to each patient, including their size, age, size
of donor heart, type of repopulating progenitor cells (e.g. VPCs,
or MPCs), and amount of time after myocardial damage. One skilled
in the art, specifically a physician or cardiologist, would be able
to determine the number of progenitor cells that would constitute
an effective dose without undue experimentation.
[0075] The progenitor cells (e.g. stem cells) may be administered
to the heart by injection. The injection is preferably
intramyocardial. As one skilled in the art would be aware, this is
the preferred method of delivery for progenitor cells as the heart
is a functioning muscle. Injection by this route ensures that the
injected material will not be lost due to the contracting movements
of the heart.
[0076] In another embodiment, the progenitor cells are administered
by injection transendocardially or trans-epicardially. In another
embodiment of the invention, the progenitor cells are administered
using a catheter-based approach to deliver the trans-endocardial
injection. The use of a catheter precludes more invasive methods of
delivery wherein the opening of the chest cavity would be
necessitated. As one skilled in the art would appreciate, optimum
time of recovery would be allowed by the more minimally invasive
procedure. A catheter approach involves the use of such techniques
as the NOGA catheter or similar systems. The NOGA catheter system
facilitates guided administration by providing electromechanic
mapping of the area of interest, as well as a retractable needle
that can be used to deliver targeted injections or to bathe a
targeted area with a therapeutic. Any of the embodiments of the
present invention can be administered through the use of such a
system to deliver injections or provide a therapeutic. One of skill
in the art will recognize alternate systems that also provide the
ability to provide targeted treatment through the integration of
imaging and a catheter delivery system that can be used with the
present invention. Information regarding the use of NOGA and
similar systems can be found in, for example, Sherman (2003) Basic
Appl. Myol. 13: 11-14; Patel et al. (2005) The Journal of Thoracic
and Cardiovascular Surgery 130:1631-38; and Perrin et al. (2003)
Circulation 107: 2294-2302; the text of each of which are
incorporated herein in their entirety.
[0077] In still another embodiment, the progenitor cells may be
administered to a subject's heart by an intracoronary route. This
route obviates the need to open the chest cavity to deliver the
cells directly to the heart. One of skill in the art will recognize
other useful methods of delivery or implantation which can be
utilized with the present invention, including those described in
Dawn et al. (2005) Proc. Natl. Acad. Sci. USA 102, 3766-3771, the
contents of which are incorporated herein in their entirety.
[0078] The present invention also encompasses a pharmaceutical
composition comprising adult vascular progenitor cells and a
pharmaceutically acceptable carrier, wherein the vascular
progenitor cells are lineage negative, c-kit positive, and flk1
positive. The vascular progenitor cells may be isolated from human
myocardium. In one embodiment, the vascular progenitor cells are
isolated from the subject to whom they will be administered (i.e.
the vascular progenitor cells are autologous). The vascular
progenitor cells preferably differentiate predominantly (e.g.
greater than 80%) into endothelial cells or smooth muscle cells in
vitro.
[0079] In another embodiment, the pharmaceutical composition
further comprises adult myocyte progenitor cells, wherein the
myocyte progenitor cells are lineage negative, c-kit positive, and
flk1 negative. The myocyte progenitor cells may be isolated from
human myocardium. In one embodiment, the myocyte progenitor cells
are isolated from the subject to whom they will be administered
(i.e. the myocyte progenitor cells are autologous). The myocyte
progenitor cells preferably differentiate predominantly (e.g.
greater than 80%) into cardiomyocytes in vitro.
[0080] The pharmaceutical composition may comprise a concentration
of vascular progenitor cells and/or myocyte progenitor cells from
about 2.times.10.sup.4 to about 2.times.10.sup.7, more preferably
about 1.times.10.sup.5 to about 6.times.10.sup.6, or most
preferably about 2.times.10.sup.6. In one embodiment, the
pharmaceutical composition comprises a concentration of vascular
progenitor cells and/or myocyte progenitor cells from about
1.times.10.sup.5 cells/ml to about 1.times.10.sup.7 cells/ml. In
some embodiments, the pharmaceutical composition may comprise
vascular progenitor cells and myocyte progenitor cells in a
particular ratio. This ratio may be adjusted to generate more
vascular tissue (i.e. a higher number of VPCs compared to MPCs) or
more myocardium (i.e. a higher number of MPCs compared to VPCs).
The ratio of VPCs to MPCs in the pharmaceutical composition may be
1:20; 1:10; 1:5, 1:2; 1:1:2:1, 5:1; 10:1, and 20:1. In a preferred
embodiment, the ratio of VPCs to MPCs is 1:1.
[0081] The invention also comprehends methods for preparing
compositions, such as pharmaceutical compositions, including VPCs
and/or MPCs as described herein, for instance, for use in inventive
methods for treating or preventing cardiovascular diseases, such as
myocardial infarction, hypertensive cardiomyopathy, and heart
failure. In one embodiment, the pharmaceutical composition
comprises vascular progenitor cells and a pharmaceutically
acceptable carrier, wherein said vascular progenitor cells are
c-kit positive and flk1 positive. In another embodiment, the
pharmaceutical composition comprises myocyte progenitor cells and a
pharmaceutically acceptable carrier, wherein said myocyte
progenitor cells are c-kit positive and flk1 negative. In still
another embodiment, the pharmaceutical composition comprises
vascular progenitor cells, myocyte progenitor cells and a
pharmaceutically acceptable carrier, wherein said vascular
progenitor cells are c-kit positive and flk1 positive and said
myocyte progenitor cells are c-kit positive and flk1 negative.
[0082] In an additionally preferred aspect, the pharmaceutical
compositions of the present invention are delivered via injection.
These routes for administration (delivery) include, but are not
limited to, subcutaneous or parenteral including intravenous,
intraarterial (e.g. intracoronary), intramuscular, intraperitoneal,
intramyocardial, transendocardial, trans-epicardial, intranasal
administration as well as intrathecal, and infusion techniques.
Accordingly, the pharmaceutical composition is preferably in a form
that is suitable for injection.
[0083] When administering a therapeutic of the present invention
parenterally, it will generally be formulated in a unit dosage
injectable form (solution, suspension, emulsion). The
pharmaceutical formulations suitable for injection include sterile
aqueous solutions or dispersions and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
The carrier can be a solvent or dispersing medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils.
[0084] Proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion, and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, may also be used as solvent
systems for compound compositions.
[0085] Additionally, various additives which enhance the stability,
sterility, and isotonicity of the compositions, including
antimicrobial preservatives, antioxidants, chelating agents, and
buffers, can be added. Prevention of the action of microorganisms
can be ensured by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, and the
like. In many cases, it will be desirable to include isotonic
agents, for example, sugars, sodium chloride, and the like.
Prolonged absorption of the injectable pharmaceutical form can be
brought about by the use of agents delaying absorption, for
example, aluminum monostearate and gelatin. According to the
present invention, however, any vehicle, diluent, or additive used
would have to be compatible with the progenitor cells.
[0086] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the present
invention in the required amount of the appropriate solvent with
various amounts of the other ingredients, as desired.
[0087] The pharmaceutical compositions of the present invention,
e.g., comprising a therapeutic dose of progenitor cells (e.g. VPC
and/or MPCs), can be administered to the patient in an injectable
formulation containing any compatible carrier, such as various
vehicles, adjuvants, additives, and diluents. Examples of
compositions comprising a therapeutic of the invention include
liquid preparations for parenteral, subcutaneous, intradermal,
intramuscular, intracoronarial, intramyocardial or intravenous
administration (e.g., injectable administration), such as sterile
suspensions or emulsions. Such compositions may be in admixture
with a suitable carrier, diluent, or excipient such as sterile
water, physiological saline, glucose or the like. The compositions
can also be lyophilized. The compositions can contain auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, gelling or viscosity enhancing additives, preservatives,
flavoring agents, colors, and the like, depending upon the route of
administration and the preparation desired. Standard texts, such as
"REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985,
incorporated herein by reference, may be consulted to prepare
suitable preparations, without undue experimentation.
[0088] The compositions can be isotonic, i.e., they can have the
same osmotic pressure as blood and lacrimal fluid. The desired
isotonicity of the compositions of this invention may be
accomplished using sodium chloride, or other pharmaceutically
acceptable agents such as dextrose, boric acid, sodium tartrate,
propylene glycol or other inorganic or organic solutes. Sodium
chloride is preferred particularly for buffers containing sodium
ions.
[0089] Viscosity of the compositions may be maintained at the
selected level using a pharmaceutically acceptable thickening
agent. Methylcellulose is preferred because it is readily and
economically available and is easy to work with. Other suitable
thickening agents include, for example, xanthan gum, carboxymethyl
cellulose, hydroxypropyl cellulose, carbomer, and the like. The
preferred concentration of the thickener will depend upon the agent
selected. The important point is to use an amount which will
achieve the selected viscosity. Viscous compositions are normally
prepared from solutions by the addition of such thickening
agents.
[0090] A pharmaceutically acceptable preservative can be employed
to increase the shelf-life of the compositions. Benzyl alcohol may
be suitable, although a variety of preservatives including, for
example, parabens, thimerosal, chlorobutanol, or benzalkonium
chloride may also be employed. A suitable concentration of the
preservative will be from 0.02% to 2% based on the total weight
although there may be appreciable variation depending upon the
agent selected.
[0091] Those skilled in the art will recognize that the components
of the compositions should be selected to be chemically inert with
respect to the active compound. This will present no problem to
those skilled in chemical and pharmaceutical principles, or
problems can be readily avoided by reference to standard texts or
by simple experiments (not involving undue experimentation), from
this disclosure and the documents cited herein.
[0092] The inventive compositions of this invention are prepared by
mixing the ingredients following generally accepted procedures. For
example, isolated progenitor cells can be resuspended in an
appropriate pharmaceutically acceptable carrier and the mixture
adjusted to the final concentration and viscosity by the addition
of water or thickening agent and possibly a buffer to control pH or
an additional solute to control tonicity. Generally the pH may be
from about 3 to 7.5. Compositions can be administered in dosages
and by techniques well known to those skilled in the medical and
veterinary arts taking into consideration such factors as the age,
sex, weight, and condition of the particular patient, and the
composition form used for administration (e.g., liquid). Dosages
for humans or other mammals can be determined without undue
experimentation by the skilled artisan, from this disclosure, the
documents cited herein, and the knowledge in the art.
[0093] Suitable regimes for initial administration and further
doses or for sequential administrations also are variable, may
include an initial administration followed by subsequent
administrations; but nonetheless, may be ascertained by the skilled
artisan, from this disclosure, the documents cited herein, and the
knowledge in the art.
[0094] The present invention also includes kits for restoring
structural and functional integrity to damaged myocardium or
generating a biological bypass. In one embodiment, the kit
comprises a pharmaceutical composition, instructions for
administering the pharmaceutical composition, and optionally an
administration device, wherein the pharmaceutical composition
comprises vascular progenitor cells. In another embodiment, the kit
comprises a pharmaceutical composition, instructions for
administering the pharmaceutical composition, and optionally an
administration device, wherein the pharmaceutical composition
comprises myocyte progenitor cells. In still another embodiment,
the kit comprises a pharmaceutical composition, instructions for
administering the pharmaceutical composition, and optionally an
administration device, wherein the pharmaceutical composition
comprises vascular progenitor cells and myocyte progenitor cells.
The vascular progenitor cells and myocyte progenitor cells may be
in the same pharmaceutical composition or they may be in separate
pharmaceutical compositions packaged in different containers within
the kit. Administration devices that may optionally be included in
the kit include a catheter, syringe, or any other appropriate
administration device.
[0095] This invention is further illustrated by the following
additional examples that should not be construed as limiting. The
contents of all references, patents and published patent
applications cited throughout this application, as well as the
Figures, are incorporated herein by reference in their
entirety.
EXAMPLES
Example 1
Identification of VPCs and Vascular Niches in the Mouse Heart
[0096] The objective of the experiments outlined in this example is
to identify and characterize vascular niches containing vascular
progenitor cells within the vasculature of murine hearts.
Homozygous c-kit-EGFP mice at 4 months are used for this study
(182). They were generated through microinjection of FVB/NJ 0.5 dpc
zygotes with clone 2 of the c-kit-EGFP construct. Founder animals
were genotyped by PCR and backcrossed to stabilize the transgene.
High expressing lines were characterized by PCR and
immunohistochemistry for colocalization of endogenous c-kit and
EGFP; testes, heart and bone marrow were examined. Although EGFP is
under the control of the c-kit promoter, mice do not develop a
dilated myopathy. Cardiac function and anatomy were measured in a
group of 28 male homozygous mice at 6-11 months of age. No
significant differences between wild-type and c-kit-EGFP mice were
observed in left ventricular (LV) anatomy or LV hemodynamics.
A. In Vivo Studies
[0097] Vascular niches. The heart is arrested in diastole and fixed
by perfusion of the coronary vasculature with 10% buffered formalin
(23, 66-68). The left (LV) and right (RV) ventricle are separated
and five slices each, perpendicular to the origin of the left and
right coronary artery, from the base to the apex are obtained.
Serial sections, 4 .mu.m thick, are collected, 200 .mu.m apart, for
a total of approximately 30 sections per ventricle. These sections
include the entire thickness of the LV or RV wall so that the left
and right coronary arteries are sampled from their origin near the
aorta to the level of resistance arterioles and capillaries. The
various segments of the coronary arteries are identified by
staining smooth muscle cells (.alpha.-SMA, calponin), endothelial
cells (PECAM-1, vWF) and adventitial fibroblasts (procollagen).
Vascular progenitor cells (VPCs) are recognized with antibodies
specific for c-kit and flk1. Transcription factors specific for
vascular (GATA6, Ets1, VEZF1) and myocyte (Nkx2.5, MEF2C) lineages
are detected by a mixture of antibodies. Additionally, the presence
of junctional proteins (connexin 43, 45, 40, 37; VE-, N-, R-,
T-cadherin) between VPCs and between VPCs and endothelial cells
(ECs), smooth muscle cells (SMCs) and fibroblasts is assessed.
These analyses are all performed by confocal microscopy (13, 16,
21-23, 33, 53, 183).
[0098] Size, number and cellular composition of vascular niches.
The methodology employed in this study has been previously
described (See ref. 53). Briefly, in each vascular niche, multiple
parameters are measured: number and diameter of primitive and
committed cells; long and short diameter of cell clusters. The
volume of each niche is calculated assuming a shape which will vary
from an ellipsoid to spherical configuration. The number of niches,
VPCs and committed cells are expressed per length of coronary
arteries, arterioles and capillaries (148, 183).
[0099] BrdU pulse-chase assays. The protocol below is based on the
assumption that the cell cycle lasts 24 hours and S phase 8 hours.
Three assays are performed to identify 1) slowly-cycling VPCs; 2)
transition of VPCs from the stem cell compartment to the amplifying
cell pool; and 3) EC and SMC lifespan and their turnover rate.
[0100] Short-term pulse-chase assay. Mice are injected with BrdU 3
times in 8 hours (length of S phase) and sacrificed .about.15 min
after the last injection (Pulse) or 1, 3 and 7 days later (Chase).
If VPCs control cell turnover in the vessel wall, the following
results are anticipated: Pulse: All BrdU-labeled cells are expected
to be bright. BrdU-bright cells are cycling cells that have
incorporated BrdU and correspond to VPCs and amplifying cells.
Differentiated ECs and SMCs are not expected to be BrdU-positive at
this time point. Chase: Following 1, 3 and 7 days of chasing,
BrdU-bright and BrdU-dim cells may be found. Because of the short
chasing period, VPCs are predicted to be bright. VPCs are
hypothesized to divide rarely and asymmetrically. The daughter stem
cell will be BrdU-bright and the daughter committed cell will
undergo rounds of division and simultaneously differentiate, i.e.,
amplifying cells. Due to the low turnover rate of ECs and SMCs in
the vessel wall (184-188), a few BrdU-dim ECs and SMCs may be
present and most likely constitute the progeny of VPCs.
Alternatively, BrdU-dim ECs and SMCs may derive from already
committed amplifying cells that incorporated BrdU during pulse. The
number of BrdU-dim ECs and SMCs should increase with time of
chasing.
[0101] Long-term pulse-chase assay. Mice are injected with BrdU 3
times/day for 4 days and sacrificed .about.15 minutes after the
last injection (Pulse) or 4 and 8 weeks later (Chase). Pulse:
BrdU-labeled VPCs, ECs and SMCs will consist mostly of BrdU-bright
cells because cycling cells will continue to incorporate BrdU
preventing its dilution. The number of BrdU-labeled cells provide
information on the cumulative growth rate within the vessel wall in
4 days. Chase: Because of the long period of chasing, BrdU-bright
cells detected at 4 and 8 weeks correspond to slowly or rarely
cycling cells that were in S phase during pulse and did not divide
during chase. If these cells express c-kit and flk1, they
correspond to long-term label retaining cells, i.e. VPCs. BrdU-dim
cells are interpreted as cells that underwent label dilution during
chase, i.e., the progeny of VPCs that incorporated BrdU during
pulse or the progeny of BrdU-labeled amplifying cells.
BrdU-negative cells are considered cells that have lost the label
as a result of multiple (>10) rounds of divisions (189) with
chase or were not cycling during pulse.
[0102] Very long-term pulse-chase assay. Mice are injected with
BrdU 3 times/day for 4 days and sacrificed 6 months later. Pulse:
see above. Chase: Over 6 months, amplifying cells labeled during
pulse should have undergone a number of divisions leading to
complete loss of BrdU. In comparison with 8 weeks of chasing (see
above), the number of BrdU-negative ECs and SMCs should increase
and the number of BrdU-positive ECs and SMCs should decrease. Thus,
it is reasonable to assume that all BrdU-labeled cells at 6 months
are the progeny of BrdU-bright VPCs that incorporated BrdU during
pulse and have undergone rare division during the 6 month chasing.
This protocol provides evidence in favor of a progenitor-product
relationship between VPCs and ECs and SMCs.
[0103] In all cases, bright and dim BrdU-positive VPCs are counted.
Levels of fluorescence greater than 4,000 and lower than 2,000
units (pixel.times.average intensity) are considered representative
of bright and dim cells, respectively (53). VPCs with intermediate
levels of fluorescence, >2,000 but <4,000, are excluded to
score long-term label retaining lineage-negative VPCs. Under this
condition, the autofluorescence of the section together with the
signal generated by the irrelevant antibody, employed as a negative
control for BrdU staining, is <10 units. Labeling >50 units
is included. BrdU-negative VPCs are also counted. An identical
approach is utilized to evaluate the fraction of EC and SMC nuclei
labeled by BrdU, bright and dim. However, EC and SMC nuclei with
intermediate fluorescence intensity (>2,000 and <4,000) are
included in the analysis.
[0104] EC and SMC Lifespan. EC and SMC lifespan are determined by
the equations developed for hierarchically structured cell
populations (190). This methodology is described in detail in ref.
53.
B. In Vitro Studies
[0105] VPCs. VPCs are harvested by enzymatic dissociation and
characterized by FACS. VPCs are deposited in individual wells of
Terasaki plates (21, 22, 33). Myocyte progenitor cells (MPCs) are
similarly isolated and used for comparison.
[0106] FACS (MoFlo, Dako). VPCs are incubated with 1-5 .mu.g/100
.mu.l primary antibody against c-kit and flk1 and markers listed
below. Primary antibodies are directly conjugated with FITC or Cy5
(33). Bone marrow lineages: CD2 (T cells, Natural Killer cells),
CD3 (T cells), CD8 (T cells), CD11b/Mac-1 (neutrophils), CD11c
(neutrophils), CD14 (monocytes), CD16 (neutrophils, monocytes),
CD19 (B cells), CD20 (B cells), CD24 (B cells), CD41 (hematopoietic
cells), CD34 (HSCs, EPCs), CD45 (leukocytes, mast cells), CD133
(HSCs, EPCs), TER119 (erythrocytes); Vascular lineage: GATA6 (SMC
transcription factor), Ets1 (EC transcription factor), Tie-2
(angiopoietin receptors), VE-cadherin (cell adhesion molecule),
CD62E/E-selectin (cell adhesion molecule), alpha-SM-actin
(.alpha.-SMA, contractile protein), CD31 (PECAM-1), vWF (carrier of
factor VIII), Bandeiraera simplicifolia and Ulex europaeus lectins
(EC surface glycoprotein-binding molecules); Myocyte lineage: GATA4
(cardiac transcription factor), Nkx2.5 and MEF2C (myocyte
transcription factors), alpha-sarcomeric actin (.alpha.-SA,
contractile protein).
[0107] Clonogenicity. VPCs are seeded in single wells (21, 22, 33).
Cloning efficiency (number of clones/number of seeded cells) are
determined and clones are expanded in F12 medium. For subcloning,
cells from a clone are plated in single wells and the formation of
clones analyzed. Population doubling time is calculated by linear
regression of log 2 values of cell number. BrdU (1 .mu.g/ml) is
added for one week to measure the fraction of cycling and
non-cycling cells. In view of the long labeling period, BrdU
positive and negative cells are considered cycling and non-cycling
VPCs, respectively. Ki67 labeling provides the number of cycling
cells at the time of observation (21, 22, 33, 191). To determine
the self-renewal potential of the founder cell, the number of
lineage negative (LinNEG) VPCs are counted within the clone. LinNEG
cells are cells negative for markers of vascular cell commitment.
Identical analysis is performed in each subclone.
[0108] VPC differentiation. Clonogenic VPCs are grown in
differentiating medium (DM; 10.sup.-8 M dexamethasone). The
fraction of cells committed to SMCs (GATA6, TGF.beta.1 receptor,
(.alpha.-SMA, calponin), ECs (Ets1, VEZF1, CD31, vWF, VE-cadherin)
and myocytes (Nkx2.5, MEF2C, .alpha.-SA, .alpha.-actinin, troponin
I, troponin T, cardiac MHC, connexin 43, N-cadherin) is studied by
FACS and immunocytochemistry (33). MPCs are used for comparison.
Cell differentiation is confirmed by real-time RT-PCR.
[0109] Functional competence of differentiated progeny. For SMC
differentiation, cells are grown in collagen IV-coated dishes in DM
supplemented with 1 ng/ml human recombinant TGF-.beta.1 (192).
Cells with electrophysiological properties of adult SMCs are
defined. For EC differentiation, cells are seeded in
methylcellulose plates (Methocult) with 100 ng/mL VEGF. Colonies
taking up DiI-Ac-LDL and binding lectin are defined (193, 194).
[0110] Calcein dye transfer assay. SMCs, ECs and fibroblasts are
loaded with calcein-AM and VPCs are labeled with DiI (53). Labeled
VPCs are cultured in the presence of SMCs, ECs and fibroblasts for
2 hours. This approach is followed for the detection of functional
gap junctions between VPCs and putative supporting cells. Since
calcein does not transfer spontaneously between cells, the presence
of green fluorescence in VPCs will be indicative of the transfer of
calcein through functional gap junctions to VPCs. This analysis is
done by two-photon microscopy. Then, the same preparations are
fixed, stained for connexins, and examined by confocal microscopy
to confirm in the same cells that calcein transfer is mediated by
functional gap junctions (53). In additional experiments, the gap
junction blocker heptanol (53) is added to the cells prior to their
co-culture.
[0111] Vessel culture. Coronary arteries are isolated to test the
ability of clonogenic VPCs to engraft. Coronary arteries are loaded
with calcein (green), cultured and placed on the stage of a
two-photon microscope enclosed in a chamber at constant
temperature, 37.degree. C., and CO2 concentration, 5%. A suspension
of .about.10,000 DiI-labeled VPCs (red) is allowed to come in
contact with the vessel lumen or adventitia. The transfer of
calcein (green) to DiI-labeled cells (red) is recorded continuously
for 48 hours by two-photon microscopy. The appearance of green
fluorescence in the DiI-labeled VPCs (red) documents the formation
of functional gap junctions. To determine efficiency of
engraftment, VPCs positive for DiI and calcein are counted in the
vessel wall together with the sites of engraftment with ECs, SMCs,
fibroblasts, myofibroblasts and pericytes (195, 196). The ability
of VPCs to invade the vessel wall from the lumen or adventitia and
establish adherens and gap junctions with ECs, SMCs and adventitial
cells is determined by two photon microscopy and calcein transfer
assay in living vessels. Two-photon microscopy allows us to
document the speed of migration of VPCs. Presence of cadherins and
connexins between VPCs and vascular cells are evaluated after
fixation by confocal microscopy. ECs, SMCs, fibroblasts and
pericytes are identified respectively by vWF, calponin, procollagen
and NG2-proteoglycan (33, 195, 196). VPC commitment is determined
by expression of transcription factors and cytoplasmic
proteins.
C. Characterization of VPC and MPC Niches
[0112] The niche microenvironment controls the number of progenitor
cells (i.e. stem cells) and their progeny by influencing the
pattern of division. Progenitor cells self-renew by symmetric
division, which generates two daughter stem cells, or by asymmetric
division, which generates one daughter cell that is identical to
the mother cell and a second daughter cell which has a separate
fate (71-80). The non-stem cell sister is a short-lived committed
PC that proliferates and simultaneously differentiates, i.e., the
amplifying cell (18, 19, 81, 82). Upon maturation, the amplifying
cell cannot divide further; it has reached terminal differentiation
and growth arrest. The terminally differentiated cell may retain
only the ability to increase in size undergoing hypertrophy (15,
18-20). Asymmetric cell replication can occur by three mechanisms
(FIG. 8): (a) Generation of cell polarity which is determined by
basal contacts (basal lamina) and lateral interactions (neighboring
cells) (83-88); (b) Orientation of the mitotic spindle which is
controlled by spindle-polarizing factors (89-94); and (c)
Segregation of cell fate determinants into one of the daughter
cells (95-99).
[0113] The lack of epithelial organization in the heart together
with the absence of a well-defined basal lamina surrounding the
vascular and myocardial niches (FIGS. 4 and 5) makes it impossible
to define an apical-basal axis and recognize the polarity of VPCs
and MPCs. Similarly, the cleavage plane that dictates the
orientation of the mitotic spindle is strictly related to the
apical-basal polarity (84, 86-88, 90, 92). Thus, cell fate
determinants are employed to characterize the pattern of VPC and
MPC division as shown in our data (FIG. 9).
[0114] The inhomogeneous intracellular segregation of selective
proteins in daughter cells at the time of mitosis may constitute
the intrinsic determinant of VPC and MPC fate. Genes, including
Numb, .alpha.-adaptin and members of the Notch pathway, interact to
enable progenitor cells (i.e. stem cells) to produce differently
destined sibling cells (80, 97-99). Notch signaling mediates
numerous developmental cell decisions. Although some controversy
exists (100), Notch preserves the pool of neural stem cells in the
prenatal and adult brain (101-107). In the hematopoietic system,
Notch often leads to transcriptional suppression of
lineage-specific genes, restricting the number of cells that adopt
a specific fate (108, 109). Numb can segregate to one of the two
daughter cells or be equally distributed in the cytoplasm of both
daughter cells (80, 97-99). Numb is expressed during mitosis, from
late prophase to telophase, and in the early stages of life of the
new daughter cell (110). Numb localizes to endocytic vesicles and
binds to the endocytic protein .alpha.-adaptin inducing the
internalization and inactivation of the Notch receptor (111).
Therefore, asymmetric partitioning of gene products at mitosis
conditions cell fate (FIG. 8): cells that receive Numb become
unresponsive to Notch while Numb-negative cells retain their
responsiveness to Notch and adopt the phenotype associated with
Notch activation (80, 86, 112). Signaling through the Notch
receptor can occur only between closely adjacent stem cells and
supporting cells. Notch ligands are transmembrane proteins. Upon
ligand binding, the Notch receptor is cleaved so that its
intracellular domain is translocated to the nucleus where it forms
complexes with transcription factors of the recombinant DNA binding
protein (RBP) family (113-115). This pathway may be operative in
the heart and regulate sternness or commitment of VPCs and MPCs
(116).
[0115] A relevant aspect of asymmetric division of adult somatic
stem cells is cosegregation of the template DNA strands ("oldest")
in the "mother" cell (117, 118). Template DNA strands are
duplicated during S phase when the new DNA strands are formed.
During mitosis of non stem cells, original template and synthesized
DNA strands are randomly segregated in the daughter cells (119).
However, it has been suggested that adult somatic stem cells are
capable of cosegregating the original template DNA strands
("oldest") in consecutive divisions (120) so that the daughter cell
that inherits the "oldest" template DNA retains stem cell features
while the daughter cell that acquires the "newer" DNA strands
enters the transit amplifying pool (FIG. 10).
[0116] The role of VPCs in vascular cell turnover and growth, and
the role of MPCs in cardiomyogenesis may be dictated by differences
in their intrinsic properties or signals in the separate niche
microenvironment, or both. Possibly, the inherent features of stem
cell classes can be determined by characterizing their
transcriptional profile with oligonucleotide microarray techniques
(133-137). These methods provide the opportunity to compare gene
expression between distinct progenitor cell types and possibly
recognize genes or clusters of genes involved in self-renewal,
multipotentiality and lineage specification (137-1139). The ability
of VPCs and MPCs to self-renew and undergo asymmetric division may
be linked to a common genetic signature of these cell classes. This
shared core of genes may correspond to those highly expressed in
all uncommitted cells: Oct4, Nanog and Sox2 (140-142). A signature
set of VPC and MPC genes may also exist and help to define the
different function of these cells in cardiac homeostasis. We have
begun to use this approach for understanding the ability of bone
marrow progenitor cells (BMPCs) to generate functional myocardium
after infarction (66-68, 143-145). Progenitor cells resident in the
bone marrow and the heart share a core of "stemness" genes but each
progenitor cell appears to express tissue-restricted genes that may
determine the efficiency of differentiation into specific lineages
(FIG. 11).
[0117] Our data suggest that the Notch pathway is involved in the
lineage specification of MPCs to myocytes. New myocytes, however,
retain a poorly differentiated dividing phenotype (amplifying
cells) and contribute to the expansion of the cardiac cell pool.
Notch signaling may be equally relevant to the differentiation of
VPCs and the generation of amplifying ECs and SMCs. In the search
for the molecular control of MPC commitment, we have found that a
perfect consensus site for the Notch effector protein, RBP-Jk, is
present in the promoter region of Nkx2.5. This suggests that Nkx2.5
is a novel target gene of Notch. This possibility is supported by
several initial studies which included immunohistochemistry,
band-shift assay, chromatin immunoprecipitation and
beta-galactosidase reporter assay. Notch inhibition in vivo
attenuates cardiomyogenesis after infarction. In the acutely
infarcted heart, there is a consistent localization of the active
form of Notch in EC and SMC nuclei of newly formed coronary vessels
(FIG. 12). The Notch ligand Delta 4 accumulates in the wall of
these developing vascular structures, suggesting that this ligand
receptor interaction in VPCs may promote vasculogenesis.
Example 2
Restoration of Vascular and Myocyte Niches may Reverse Heart
Failure
[0118] Heart failure may be a stem cell disease. The alteration in
coronary perfusion and muscle contractile behavior of the
decompensated heart may result from depletion of functional VPCs
and MPCs which become unable to form a number of vascular cells and
cardiomyocytes required to counteract the abnormal hemodynamic
load. Although multiple variables including defects in hormonal
regulation, calcium metabolism, contractile regulatory proteins,
and complex signal transduction pathways with upregulation or
downregulation of a variety of gene products have been recognized,
the initial triggering event of heart failure remains obscure (157,
236, 237).
[0119] Pressure loading induces concentric ventricular hypertrophy,
in which wall thickness increases without chamber enlargement (148,
238-240). In its compensated form, mural thickening is the result
of an increase in myocyte diameter and/or myocyte number in the
absence of tissue injury (238-242). These events lead to an
increase in ventricular mass-to-chamber volume ratio that
normalizes the abnormal elevation in systolic stress. These
adaptations constitute the anatomical counterpart of compensated
concentric hypertrophy and are typically present in patients with
aortic stenosis (15) or systemic hypertension (243) with modest
ventricular dysfunction (243-245). However, the long-term effects
of increased pressure loads result in expansion in cavitary
diameter and relative wall thinning, altering the balance between
ventricular mass and chamber volume, on the one hand, and
afterload, on the other (246-248). These factors define concentric
hypertrophy in its decompensated stage in which circumferential
systolic and diastolic stress are increased. Multiple foci of
myocardial damage represented by areas of replacement fibrosis
across the ventricular wall become apparent and chamber volume
expands with time (246). Chronic ventricular dilation is the
critical determinant of the initiation of ventricular dysfunction
and its progression to terminal failure (148). Systemic
hypertension is one of the major causes of heart failure in humans
(249).
[0120] To address the question of whether VPCs and MPCs can be used
to alleviate hypertension-induced heart failure, we use two-kidney
one-clip renal hypertension in which the increase in systolic blood
pressure occurs gradually and worsens with time (250-253). The
two-kidney one clip renal hypertension model mimics acquired
systemic hypertension in humans (246-248, 254-257). Initially, the
increase in systemic blood pressure is paralleled by a
corresponding increase in the myocyte and vascular compartment;
concentric hypertrophy is apparent and the pressure load is
sustained by the expansion in the muscle mass (246, 247, 250-257).
Chronically, myocardial damage develops, the chamber dilates, the
thickness of the wall decreases and ventricular failure supervenes
(246, 247, 257). Structurally, myocyte death, vascular rarefaction
and collagen accumulation precede the decline in systemic blood
pressure and the onset of ventricular decompensation; and the
severity of tissue injury is strictly connected to the extent of
functional impairment.
[0121] The evolution of hypertensive cardiomyopathy may be
conditioned by the formation of dysfunctional vascular and
myocardial niches and loss of functionally-competent VPCs and MPCs.
Because VPCs and MPCs possess the inherent ability to regenerate an
unlimited number of coronary vessels and myocytes, the heart should
have the capacity to correct the anatomical changes produced by
pathologic loads and sustain pump function indefinitely.
[0122] It may be possible to interfere with the etiology of
hypertensive cardiomyopathy by repopulating dysfunctional niches
and the progenitor cell pool with functionally-competent VPCs and
MPCs or by creating new vascular and myocardial niches. Since
EGFP-positive VPCs and MPCs are administered, the newly formed
niche structures are easily identified and characterized. By
definition, a niche has to contain at least one undifferentiated
stem cell. In the hypertensive cardiomyopathic heart, old niches
may host the new VPCs and MPCs, but putative new niches may be
created as well. The presence of groups of engrafted EGFP-positive
VPCs and MPCs together with EGFP-negative recipient progenitor
cells connected by adherens junctions and gap junctions allows us
to define expanded niches while pockets of EGFP-positive VPCs and
MPCs only will reflect the generation of putative new niches.
Specific Methods
[0123] Hypertension. Renal hypertension is produced in female mice
at 4 months of age. Under anesthesia, a silver clip with an
aperture of 70 .mu.m is placed on the left renal artery while
leaving the controlateral artery untouched (246, 247, 254). Blood
pressure increases in 10-15 days after renal artery clipping and
further with time. Sham-operated animals are used as control.
Arterial blood pressure is measured by the tail-cuff method every
15 days. Systolic blood pressure greater that 150 mmHg is
indicative of hypertension (FIG. 13).
[0124] Echocardiography. Echocardiography is performed every two
weeks in un-anesthetized mice using a Sequoia 256c (Acuson)
equipped with a 13-MHz linear transducer (15L8) (23, 33, 145).
[0125] Cell implantation. VPCs and MPCs are isolated from
.beta.-actin-EGFP male transgenic mice as described in Example 1
and injected in the myocardium of female hypertensive mice
following the recognition of ventricular dysfunction established by
echocardiography. This is expected to appear .about.3 months after
the onset of hypertension and to deteriorate further at 6 months.
Six injections, two at the base two at the mid-region and two near
the apex are performed. Each injection consists of 10,000 VPCs,
10,000 MPCs or 5,000 VPCs and 5,000 MPCs for a total of 60,000
cells in each case. Mice injected with PBS and untreated
sham-operated mice are used as controls. Short-term studies at 1, 3
and 7 days after cell implantation evaluate homing and engraftment
of donor VPCs and MPCs (145). The distribution of EGFP-positive
male VPCs and MPCs in new niches and pre-existing niches is also
established. At 1 and 2 months, the progeny of the injected cells
is evaluated. Additionally, the morphometric approach discussed in
Example 1 will be employed to define number, size and composition
of VPC and MPC niches.
[0126] Ventricular performance. At sacrifice, animals are
anesthetized and the right carotid artery cannulated with a
microtip pressure transducer (Millar SPR-671). A 3 lead ECG is also
obtained. A four channel 100 kHz 16-bit recorder with built-in
isolated ECG amplifier (iWorks IX-214) is used. The catheter is
advanced into the LV for the evaluation of LV pressures and dP/dt
(23, 33, 145). The heart is fixed by perfusion as described in
Example 1.
[0127] Integration of regenerated myocardium with recipient
myocardium. Calcium transient in newly formed EGFP-positive
myocytes and resident pre-existing myocytes is determined by an ex
vivo preparation and two-photon microscopy (33, 145). For cell
physiology see refs. 33 and 145.
[0128] Coronary blood flow. This parameter is obtained with
non-radioactive microspheres (see ref. 183).
[0129] Size, number and cellular composition of vascular and
myocardial niches is determined as described in Example 1.
Additionally, sections are stained with GFP and Y chromosome to
identify the implanted cells and their progeny. Senescent VPCs and
MPCs are identified by the expression of p53 and p16 and by
measuring the length of telomeres by Q-FISH (16, 175, 258).
Apoptotic VPCs and MPCs within the niches are determined by TdT
assay.
[0130] PCR for GFP and Y-chromosome DNA. These protocols confirm
the morphological data.
[0131] Cell fusion. Cell fusion between EGFP-positive donor cells
and recipient cells are determined by FISH assay for
sex-chromosomes (14, 33, 66, 68, 145). Also, VPCs and MPCs are
infected with a lentivirus carrying Cre recombinase and injected in
hypertensive 1oxP mice (33).
[0132] Data analysis. Hypertension is induced in female mice which
will be divided in several subgroups: 2 times of treatment (3 month
and 6 months), 4 modalities of treatment (VPCs, MPCs, VPCs and
MPCs, and vehicle) and 5 time points at which mice are sacrificed.
Each subgroup consists of 10 mice, for a total of 400 mice
(2.times.4.times.5.times.10=400). Fifty sham-operated normotensive
mice are used as controls. One thousand male .beta.-actin-EGFP
transgenic mice will be required to obtain the VPCs and MPCs to be
implanted.
[0133] The results of this series of experiments are expected to
show that the implanted VPCs and MPCs will engraft into the mouse
heart both in established niches as well as newly established
niches. Mice receiving progenitor cells (VPCs, MPCs, or VPCs and
MPCs) will exhibit reduced symptoms of hypertensive cardiomyopathy
and heart failure as compared to mice receiving vehicle only. Mice
that receive both VPCs and MPCs are expected to show the greatest
recovery from symptoms.
Example 3
Identification and Characterization of VPCs and MPCs in Dogs
[0134] There are several aims of this Example: (a) To demonstrate
that the normal canine heart contains a population of lineage
negative c-kit-positive flk1-positive cells, i.e., VPCs, which are
located in the intima, media and adventitia of the coronary
vasculature including the capillary network; (b) To demonstrate
that VPCs are located in vascular niches present in the various
segments of the dog coronary circulation; (c) To demonstrate that
VPCs can be isolated and expanded from adult dog epicardial
coronary arteries and small samples of atrial and ventricular
myocardium; (d) To demonstrate that VPCs possess the properties of
stem cells and differentiate into ECs and SMCs and only to a
limited extent into myocytes; (e) To demonstrate that the canine
heart contains a population of lineage negative c-kit-positive
flk1-negative cells, i.e., MPCs, which are located in myocardial
niches; (f) To demonstrate that MPCs can be isolated and expanded
from small samples of atrial and ventricular myocardium; (g) To
demonstrate that MPCs possess the properties of stem cells and
differentiate into myocytes and only to a limited extent into ECs
and SMCs; and (h) To demonstrate that the molecular signature of
VPCs differs from that of MPCs.
[0135] To achieve these objectives, samples of dog coronary
arteries and myocardium are studied. Following the documentation
that VPCs and MPCs are present in the canine heart, the question is
whether VPCs and MPCs reside in the coronary circulation and the
myocardium or translocate from the bone marrow to the vessel wall.
Stem cells possess critical properties that can be determined to
establish the origin of VPCs and MPCs. Stem cells are stored in
niches (370-374) and stem cell quiescence, activation, growth and
differentiation are all modulated within the niche structure
(375-377). For the documentation of niches within an organ, stem
cells have to be found, the anchoring of stem cells to the
supporting cells identified and the existence of a
progenitor-product relationship established (374, 376, 378).
[0136] VPCs and MPCs are expected to be connected to supporting
cells by junctional and adhesion proteins represented by connexins
and cadherins (379, 381). Connexins are gap junction channel
proteins that mediate passage of small molecules involved in
cell-to-cell communication (381, 382). Cadherins are
calcium-dependent transmembrane adhesion molecules, which have a
dual function; they anchor stem cells to their microenvironment and
promote cross talk between stem cells and between stem cells and
supporting cells (380).
[0137] Additionally, the clonal efficiency of VPCs and MPCs is
established together with their ability to form a differentiated
progeny. This information is critical biologically and clinically.
Stem cells have to be self-renewing, clonogenic and multipotent in
order to be classified as stem cells (301-304, 311-313).
[0138] Finally, the transcriptional profile of VPCs and MPCs is
determined to establish shared and distinct genotypic properties
among these two populations of primitive cells (383, 386). By
comparing global gene expression patterns, we can identify distinct
genes or clusters of genes involved in self-renewal,
multipotentiality and lineage specification (387, 389). Changes in
gene expression is correlated with the phenotype (proteins) and
functional state (differentiation) of the cells. Moreover,
microarray technologies monitor the expression of thousands of
genes offering a comprehensive view of the molecular signature of
stem cells with separate roles in vivo (383-386).
[0139] The baseline gene expression of undifferentiated VPCs and
MPCs is determined first. Moreover, the ability of both VPCs and
MPCs to self-renew and undergo asymmetric division may be linked to
a common genetic identity of the two cell categories. This shared
core of genes may correspond to Oct4, Nanog and Sox2 which modulate
self-renewal and multipotentiality (390-396). Also, previously
unidentified genes may be recognized. During differentiation, the
loss of PC multipotentiality may correlate with alterations in gene
expression that regulate vascular and cardiomyocyte genomic
identity. The analysis of gene expression profiles includes VPC and
MPC clones grown in non-differentiating medium. Clonogenic VPCs and
MPCs from individual clones are cultured in "generic"
differentiating medium and in "predominantly" EC-producing,
SMC-producing or myocyte-producing medium for a short (2 days),
intermediate (10 days) and long (21 days) period. The gene
expression profile of cells kept in generic medium is compared with
that of cells exposed to specific media to determine: 1. Genes
involved in unipotent and multipotent specification; 2. Genes
involved in early and late commitment and terminal differentiation;
and 3. Genes involved in the acquisition of functional competence.
Expression profiles are examined by clustering analysis and this
approach may identify putative differentiation pathways of VPCs
into ECs and SMCs, and MPC into myocytes.
[0140] Our results have shown that the dog heart possesses a
compartment of undifferentiated cells characterized by the
expression of the stem cell antigen c-kit; canine c-kit-positive
progenitor cells (PCs) are self-renewing, clonogenic and
multipotent (301), which are the fundamental properties of stem
cells (302, 303). Additionally, this population of c-kit-positive
resident PCs can be activated after infarction to invade the
damaged tissue and promote the formation of new myocardium which
consists mostly of cardiomyocytes and to a limited extent of
coronary vessels. In an effort to address the dramatic problem of
coronary artery disease (CAD), data have been obtained in dogs and
humans supporting the notion that the cardiac c-kit-positive PC
pool is not homogeneous but consists of primitive cells distributed
separately in the coronary vasculature and in the muscle
compartment (FIG. 14).
[0141] The expression of the VEGFR2/flk1 or kinase domain receptor
(KDR) represents the earliest marker of angioblast precursors
(305-309) and allows us to recognize and sort separately
c-kit-positive PCs with powerful vasculogenic (flk1-positive) and
cardiomyogenic (flk1-negative) potential. Our data (FIG. 15)
indicate that flk1-positive c-kit-positive PCs are negative for
markers of hematopoietic cell lineages including CD34, CD41, CD45,
CD133 and a cocktail of antibodies against lineage markers of bone
marrow derived cells: CD2 (T cells and natural killer cells), CD3
(T cells), CD8 (T cells), CD14 (monocytes), CD16
(neutrophils/monocytes), CD19 (B cells), CD20 (B cells), CD24 (B
cells), CD56 (natural killer cells), CD66b (granulocytes) and
glycophorin A (red blood cells). Also, only a small fraction of
flk1-positive c-kit-positive cells is positive for the EC adhesion
protein CD31 and the SMC TGF-.beta.1 receptor protein; the myocyte
contractile protein .alpha.-sarcomeric actin is undetectable.
Therefore, the dog heart contains a pool of lineage negative
flk1-positive c-kit-positive cells that possess the phenotypic
properties of multipotent precursors.
[0142] The possibility to sort cardiac PCs which express or do not
express flk1 in dogs has posed the critical question whether these
two categories of cells are self-renewing, clonogenic and
multipotent (301-304, 311-316). The acquisition of this information
is critical for the definition of these cell classes and
characterization of their differentiation behavior. To address this
problem, PCs were sorted and individual cells were deposited in
single wells of Terasaki plates (301-304, 311-313). After
.about.3-4 weeks multicellular clones were obtained. Clonogenic
flk1-positive c-kit-positive PCs exposed to differentiating medium
acquired mostly the EC and SMC lineage and in minimal proportion
the myogenic phenotype. Conversely, clonogenic flk1-negative
c-kit-positive PCs generated predominantly myocytes and to a
limited extent SMCs and ECs (FIG. 16). These results at the single
cell level strongly suggest that the PC pool contains two distinct
classes of PCs: coronary vascular progenitor cells (VPCs) and
myocyte progenitor cells (MPCs). Additionally, these observations
point to the presence of an unsuspected VPC located within the wall
of coronary vessels distinct from the MPC distributed within the
myocardium.
[0143] In an additional series of experiments, we have utilized a
simplified in vitro preparation to collect information on the
migratory behavior and homing properties of VPCs to determine
whether a self-renewing, clonogenic and multipotent VPC stored in
vascular niches is present in the dog heart and whether it
possesses unique phenotypic and functional properties in vitro
(FIG. 17). Segments of large coronary arteries isolated from the
dog heart and from explanted and discarded human hearts were used.
The results of these experiments show: (a) that VPCs have the
ability to move from the lumen of intact large coronary arteries
into the vessel wall and home to the intima, media and adventitia
forming gap junctions and adherens junction with resident ECs, SMCs
and adventitial cells; and (b) that VPCs implanted in proximity of
the adventitia of intact large coronary arteries or large coronary
arteries with endothelial-medial damage and/or adventitial damage
home to the adventitia, form connection with fibroblasts, pericytes
and myofibroblast-like cells in the adventitia and then migrate to
the media and intima establishing junctional complexes with
resident cells.
Specific Methods
[0144] Specimens for histology and immunocytochemistry. Samples of
large coronary arteries together with specimens of myocardium
containing intermediate- and small-sized coronary arteries and
capillary profiles are examined to detect VPC niches and their
distribution within the different portions of the coronary
circulation and their localization in the vessel wall. Samples of
myocardium from the atria and ventricles are analyzed for the
recognition of putative myocardial niches and their distribution in
the heart. Vascular niches are compared to MPC niches to establish
differences in the phenotypic properties of VPCs, MPCs and
supporting cells.
[0145] Samples for in vitro studies. Five specimens of canine
coronary arteries and myocardium are collected. The in vitro
characteristics of VPCs are compared with those of MPCs. VPCs and
MPCs are harvested by enzymatic dissociation (304) and single cell
suspension characterized by FACS to determine their surface
phenotype. Sorted VPCs and MPCs are deposited in individual wells
of Terasaki plates.
[0146] FACS. VPCs and MPCs are suspended at a concentration of
.about.100,000 cells/ml PBS. Aliquots of 100 .mu.l of cell
suspensions are incubated for 20 minutes at 4.degree. C. with 1-5
.mu.g/100 .mu.l of primary antibody against c-kit and KDR and
markers listed below. Primary antibodies are directly conjugated
with FITC or Cy5 for FACS analysis (MoFlo, Dako; 304). Antigens
studied are as follows: Bone marrow lineages=CD2 (T cells, Natural
Killer cells), CD3 (T cells), CD8 (T cells), CD14 (monocytes), CD16
(neutrophils, monocytes), CD19 (B cells), CD20 (B cells), CD24 (B
cells), CD34 (HSCs, EPCs), CD45 (leukocytes, mast cells), CD133
(HSCs, EPCs), glycophorin A (erythrocytes); Vascular lineage=GATA6
(SMC transcription factor), Ets1 (EC transcription factor), Tie-2
(angiopoietin receptors), VE-cadherin (cell adhesion molecule),
CD62E/E-selectin (cell adhesion molecule), alpha-SM-actin
(contractile protein), CD31 (PECAM-1), vWF (carrier of factor
VIII); Myocyte lineage=GATA4 (cardiac transcription factor), Nkx2.5
and MEF2C (myocyte transcription factors), alpha-sarcomeric-actin
(contractile protein). The specificity of the antibodies against
bone marrow epitopes is tested on cells obtained from canine
blood.
[0147] Clonogenicity and growth properties. Cloning efficiency
(number of developed clones/number of seeded single cells) is
determined (304). For subcloning, cells from a clone are plated in
single wells and analyzed. Clonogenic cells are counted daily and
population doubling time is calculated by linear regression
analysis of log 2 values of cell number (402). To determine the
fraction of cycling and non-cycling cells, BrdU (1 .mu.g/ml) is
added one week after plating in a restricted number of wells. In
view of the long labeling period, BrdU positive and negative cells
are considered cycling and non-cycling, respectively. Ki67 labeling
provides the number of cycling cells at the time of observation
(301, 304, 311).
[0148] VPC and MPC differentiation and immunocytochemistry.
Clonogenic cells are grown in differentiating medium (DM, 10-8 M
dexamethasone). The number and the relative fractions of cells
committed to the SMC, EC and myocyte lineage is studied by FACS and
immunocytochemistry (304). Antibodies for SMCs (GATA-6, TGF.beta.1
receptor, alpha-SM-actin, SM-heavy chain 22), ECs (Ets1, CD31, vWF,
VE-cadherin) and myocytes (Nkx2.5, MEF2C, .alpha.-sarcomeric-actin,
.alpha.-cardiac-actinin, troponin I, troponin T, cardiac myosin
heavy chain, connexin 43, N-cadherin) are employed. For in vitro
immunocytochemistry and later for in vivo immunohistochemistry, all
antibodies are conjugated with quantum dots to exclude
autofluorescence artifacts see (266, 270, 304).
[0149] Functional competence of VPCs. For SMC differentiation,
cells are grown in collagen IV-coated dishes in DM supplemented
with 1 ng/ml human recombinant TGF.beta.1 (403). Cells with
electrophysiological properties of adult SMCs are defined. For EC
differentiation, cells are seeded in methylcellulose plates with
100 ng/mL VEGF. Colonies taking up Dil-Ac-LDL and binding lectin
are defined (404).
[0150] RNA extraction and array hybridization. Clonogenic VPCs and
MPCs and BMPCs are resuspended in Trizol. RNA is extracted and
.about.1 .mu.g of total RNA is converted to biotin-labeled cRNA
using Gene Chip One-Cycle Target labeling kit and hybridized on the
human genome array at the Affymetrix Facility of Albert Einstein
College of Medicine. A minimum of 3-5 independent hybridizations is
performed in triplicates for each condition (385, 405, 406). The
MIAME (minimal information about a microarray experiment)
guidelines are followed for data presentation (407). The Affymetrix
software MicroArray Suite 5.0 (MAS 5.0) is used to generate
absolute expression estimates (absent/present call) from the raw
data. Software default thresholds are employed to determine the
present (P) or absent (A) calls (.alpha.1=0.04, .alpha.2=0.06, and
.tau.=0.015). The data obtained from MAS 5.0 are then normalized
and further analyzed in the Gene-Spring software version 6.2.
Per-chip normalization is done as follows: values below 0.01 are
set to 0.01, and then each measurement is divided by the 50th
percentile of all measurements in that sample. Per-gene
normalization is done as follows: each gene is divided by the
median of its measurements in all samples. If the median of the raw
values is less than 10, each measurement for that gene is divided
by 10. Genes are considered to be differentially expressed in VPCs
and MPCs only when three criteria are met: (a) Difference in
expression is at least twofold; (b) The gene is identified by MAS
5.0 as present in two out of three replicates or present or
marginal in all three replicates in the group with the highest
expression level; and (c) Difference in expression is significant
(P<0.05 in an unpaired t-test with Welch's correction).
Classification of genes into functional clusters is done by
collecting annotations and keywords with the Onto-Express Tool
Affymetrix Net Affx, and the Simplified Gene Ontology Tool included
in GeneSpring 6.2 Software (408). Microarray data are confirmed by
real-time RT-PCR.
[0151] Real-time RT-PCR and Western blotting. RNA is isolated using
Trizol from clonogenic VPCs and MPCs and BMPCs freshly isolated
from the sternum of patients undergoing open heart surgery; 1 .mu.g
of RNA is employed for reverse transcription (RT) into cDNA using
SuperScript III cDNA synthesis kit (304). RNA is incubated with
5'-phosphorylated oligo(dT)20 primer. Real time RT-PCR analysis is
performed on 7300 Real Time PCR System and run in duplicate using
1/20th of the cDNA per reaction. Gene sequences for primer design
are obtained from the NCBI database. Primers are chosen (Primer3
software), and their specificity is tested with electronic PCR
using human genome and human transcript database. Cycling
conditions are established according to the designed primers. Data
are analyzed using the Automatic Baseline of the Sequence Detection
software, and the threshold is fixed at 0.05 manually for cycle
threshold (Ct) determination. PCR efficiency is evaluated using a
standard curve of five serial dilution points; quantified values
are normalized against the input determined by the housekeeping
gene GAPDH or beta-actin. Real-time RT-PCR products are run on 2%
agarose/1.times.TBE gel. Amplified fragments are cut out, DNA is
extracted and amplified and sequences are determined in sense and
antisense directions. The expression of selected genes is confirmed
at the protein level (301, 304, 311).
Example 4
Implantation of VPCs Generates a Biological Bypass in the Presence
of a Stenotic or Occluded Coronary Artery
[0152] The tight coupling of coronary blood flow to cardiac or
myocyte function allows for increases in cardiac output to support
normal physiologic function for instance in exercise or pregnancy
(331, 342). However, due to the tight coupling and little potential
overlap in mechanisms, the heart is left in the precarious position
where a limitation in blood flow or loss of cardiac myocytes,
together or separately, may have substantial consequences for
cardiac pump function. During a limitation in coronary blood flow,
due to stenosis or total occlusion of a blood vessel, there is an
exponential relationship between flow and function (343) and the
limited delivery of oxygen and substrates to contracting myocytes
will limit contraction and may result in ischemic cell death.
Unlike apoptotic cell death where the myocyte is essentially
reabsorbed (344, 345, 346), ischemic cell death results in
replacement fibrosis and scarring. If substantial, the scaring will
lead to altered cardiac diastolic compliance and function. This can
alter cardiac diastolic function (wall tension), and the time and
distance over which oxygen diffusion primarily occurs in the heart.
However, following myocardial ischemia, blood vessels are lost
altering the architecture of the coronary circulation and the
delivery of oxygen to support oxidative metabolism in cardiac
myocytes. Myocyte contractile or systolic function is reduced or
eliminated due to lack of oxygen and substrate, myocytes die
leaving scar tissue which alters diastolic function and the
diffusion distance for oxygen to the remaining myocytes. Thus
remodeling of the heart occurs and this adds to the cardiac
dysfunction.
[0153] The intimate relationship between the coronary circulation
and the cardiac myocytes it perfuses supports the contention that
there is an optimum relationship between the two and that both
structures are needed to support cardiac performance. Classical
therapeutic approaches to the treatment of myocardial ischemia have
centered on drugs including clot busters or surgical interventions
such as stenting and bypass surgery, for instance, to restore
coronary blood flow. These have been largely effective in restoring
cardiac function in an ischemic zone especially if the intervention
is performed within a few hours after ischemia (347, 348). However
if the ischemia lasts for a longer period of time, there may be
limited benefit to restoring blood flow since the cardiac myocytes
are already dead or have already begun an irreversible process
leading to cell death (344). Exciting and still developing
therapies address this limitation of cell death by attempting to
replace cardiac myocytes in the ischemic zone using cell based
approaches (267, 349, 350). In addition if the ischemia is
maintained so that the area of the infarct remodels, the
architecture of the heart changes so that blood vessels disappear
(rarefaction). At that time, opening of or bypassing large arteries
alone will be ineffective in restoring blood flow and function in
the area of the ischemia since the small vessels including
capillaries have been reabsorbed. At some point in time,
reperfusion will be ineffective since blood flow will not get to
the surviving myocytes and myocyte loss (mass) leads to reduced
inotropic state. Thus, it is also necessary to grow a functional
coronary vasculature, including large vessels and capillaries
during the development of cell based therapies.
[0154] Currently attention is being paid to the ability to reverse
remodel the chronically ischemic heart via methods to recruit or
use stem cells. A recent study by Bearzi et al (304) has shown that
injection of human cardiac stem cells labeled with GFP, which were
expanded in vitro, at the time of infarction, results in the
generation of labeled cardiac myocytes (84% of the cells) and blood
vessel cells (8% of the cells). These cells were functionally
integrated into the mouse heart and showed contractile function up
to 2 weeks after injection. Furthermore, the restructured heart
contained a myocyte to capillary ratio of 8/1 with a diffusion
distance for oxygen of approximately 18 .mu.m. This ratio is
smaller than the 1/3 normally seen in the heart and may indicate an
oxygen supply reserve in the newly regenerated heart. On the other
hand, the size of the newly developed myocytes ranged from 100 to
1900 .mu.m.sup.3 somewhat smaller than the average mouse
cardiomyocyte, 25000 .mu.m.sup.3, and this has to be factored into
evaluation of the requirements of oxygen in those cells (the oxygen
cost of growth and contraction) which is unknown. This study
provides strong evidence that these therapies using cardiac derived
adult stem cells grow both myocytes and capillaries and are
potentially useful.
[0155] We have developed approaches to recruit cardiac stem cells
to treat myocardial ischemia in the dog heart. There are a number
of advantages to this approach including: a) the ability to study
the dog in the conscious state free from the complications of
anesthesia and recent surgery (353); b) the ability to measure
regional and global cardiac function more precisely; c) the ability
to design and carry out experiments in a longitudinal design where
cardiac function is measured repeatedly over time in the same
animal; d) the availability of classic well described models from
the literature; and d) the ability to record cardiac function on
line in real time and to accurately assess the evaluation the
impact of loading conditions on contractility.
[0156] The objective of this experiment is to determine whether in
the presence of a stenotic or occluded coronary artery
catheter-delivered or locally injected VPCs generate a biological
bypass which reestablishes blood flow to the distal myocardium
restoring in part regional ventricular function.
[0157] Reactive hyperemia is used to assess the extent of the
coronary stenosis (ranging from small to critical to producing an
infarct). The ensuing hyperemia uses up the coronary reserve and
results in characteristic 500% increase in blood flow (FIG. 18)
(336-338). During maximum coronary dilation, relying on oxidative
metabolism and having increased oxygen extraction from 80 to 85%
the oxygen supply to the contracting myocytes also increases
approximately 5 fold. Thus the geometry of the coronary
circulation, the limitation of oxidative metabolism, an almost
maximum oxygen extraction, and the ability to increase coronary
blood flow almost 5 fold, account for increased oxygen delivery in
the heart.
[0158] Cardiac myocytes contract at rates ranging from 40 to
approximately 180 b/min in man (and in the conscious dogs that we
use). A measure of cardiac contractility, for instance LV dP/dt,
can increase about 5 fold in the conscious dog during maximum
exercise (339, 340). Together with the tachycardia, this results in
an approximate 5 fold increase in cardiac output or slightly
greater in cardiac work due to the increase in arterial pressure
which occurs during exercise (341). Thus heart rate can increase
about 4 fold, contractility approximately 5 fold and cardiac work
5-6 fold during maximum cardiac performance. From this it should be
obvious that the coronary circulation is able to accommodate a 5
fold increase in oxygen delivery and the heart is able to undertake
about a 5 fold increase in cardiac performance, indicating that
over a wide range of cardiac function, there is tight coupling
between oxygen delivery and oxygen demand. In fact, when this tight
coupling is altered due to altered blood flow regulation or a
mismatch between oxygen delivery and consumption, myocyte
dysfunction or death ensues resulting in cardiac pump dysfunction.
If this dysfunction is large or long lasting heart failure may
occur.
[0159] Three different protocols are employed in this series of
experiments. They are as follows:
Protocol 1.
[0160] Rationale: If a coronary stenosis limits blood to a portion
of the heart resulting in myocardial ischemia and dysfunction then
restoration of blood flow will restore contractile function distal
to the stenosis. Thus a therapy designed to selectively create a
biological bypass would be unique and effective. The goal of this
experiment is to determine if the injection of VPCs into the heart
results in the formation of functional blood vessels and results in
amelioration of the ischemia due to chronic coronary stenosis.
[0161] Specific methods: The dog is trained to lie on the
laboratory table and then echocardiography is performed with
special attention to the anterior wall. Dogs are instrumented for
the measurement of cardiac function (segment crystals/contraction)
and with a flow transducer, hydraulic occluder and critical
stenosis on the distal LAD. At surgery the stenosis is adjusted to
eliminate the reactive hyperemia following a 15 second coronary
artery occlusion (a critical stenosis). The left atrial appendage
is harvested from which VPCs are isolated and propagated. The chest
is closed in layers and the dog allowed to recover. Ten days after
surgery, hemodynamics are recorded, the reactive hyperemia
examined, and echocardiography performed. This is repeated each
week for 2-4 weeks (until the autologous canine stem cells
proliferate, estimates are that 10.times.10.sup.6/ml cells per
animal are used). Once sufficient cells are available, hemodynamics
(non radioactive microspheres injected through the left atrium to
measure collateral flow) are recorded and then the dog anesthetized
with sodium pentobarbital. The dog is taken to the fluoroscopy
laboratory and the LAD catheterized for the injection of contrast
(without and with occlusion of the LAD) to visualize the collateral
circulation. Following this, a specially designed balloon-needle
catheter (414) is advanced into the area of the stenosis. The
needle is advanced from the catheter and injections of 250 .mu.l of
the VPC cell suspension is given 2-5 times. In some of the dogs the
cell suspension is mixed with contrast media (414) to visualize the
sites of injection. The catheter is removed and the dog is allowed
to recover. The dog is studied each week for 4 weeks and
microspheres injected with and without occlusion to measure
collateral flow. Prior to sacrifice, the cardiac catheterization is
repeated to visualize the collateral circulation. At that time, one
half of the dogs are anesthetized and the heart perfusion fixed. In
the other half, tissue is collected for in vitro studies of
microvessel reactivity.
[0162] The injection of VPCs results in growth of GFP containing
blood vessels, visualization of a well developed collateral
circulation at catheterization, and increased deposition of
microspheres in the ischemic zone distal to the stenosis. In
isolated perfused coronary microvessels, there may be increased
flow induced dilation (indicating functional endothelium) and
increased agonist induced dilation (indicating enhanced smooth
muscle and endothelial function). There may be a relationship
between dilation in vitro and the site and amount of GFP staining.
There may be an increase in segment contractile function in the
ischemic zone that is proportional to the increase in blood
flow.
[0163] We have currently studied 6 dogs with a coronary stenosis
and injection of vascular stem cells which were labeled with GFP
prior to injection into the ischemic region of the dog heart. In
these animals, we clearly identified coronary blood vessels labeled
with GFP (FIG. 19).
[0164] The LAD coronary artery was catheterized and the coronary
circulation visualized in three dogs using a fluoroscope. Those
dogs were instrumented with a flow transducer, an occluder and a
critical stenosis. Injection of contrast in all the dogs indicated
the presence of a significant stenosis. One dog had injections of
GFP labeled VPCs during the initial surgery (3 weeks prior to
study). Most importantly, during occlusion of the LAD using the
hydraulic occluder, injection of contrast did not appear in the
distal LAD circulation in 2 of the dogs (i.e. there was little
collateral blood flow), whereas there was a substantial and obvious
appearance of contrast in the distal circulation in the one dog
receiving VPCs (evidence of a newly developed circulation) as shown
above in FIG. 20A. Hearts were perfusion fixed and tissue sections
prepared to find evidence of GFP labeled small blood vessels.
[0165] In one dog we created a large infarct with the goal of
severely compromising cardiac function to induce heart failure. By
ligating the proximal LAD at the first diagonal branch, we reduced
ejection fraction from 72% to 29% (FIG. 20B).
Protocol 2.
[0166] Rationale: The primary goal of acute cardiac
catheterization/stenting and thrombolytics to treat myocardial
infarction is to restore blood flow to the area of the infarction,
particularly the border zone and thereby reduce or reverse the
ischemic damage and contractile dysfunction. Thus, current
therapies already are designed to selectively increase blood flow
to the ischemic myocardium. There are no therapies designed to
increase blood flow after a chronic infarction since the remodeling
of the infarct includes disappearance of blood vessels. The goal of
this study is to determine if the injections of VPCs into the
border and ischemic zone of a distal (small infarct) LAD occlusion
results in increased blood flow to the infarct and enhances
contractile function. These studies use a small infarction so that
the complications of heart failure are not present.
[0167] Specific methods: Surgery is performed using general
anesthesia and sterile technique for the measurement of cardiac
function (including segment function) and with an occluder and flow
transducer on the LAD. The left atrial appendage is harvested for
the collection of VPCs and MPCs. The dog is allowed to recover for
10 days. Hemodynamic studies, echocardiography and cardiac
catheterization is performed and then the dog anesthetized, the
distal LAD permanently occluded and the dog allowed 2-4 weeks to
recover. After 2-4 weeks the LAD is catheterized to visualize blood
flow to the area of the infarct and microspheres injected, then
under general anesthesia using an echo-guided spinal needle or
Mercator catheter, injections of VPCs (250 .mu.l) are made into the
middle of the infarct and then into the border zone (3-5
injections). The dog is allowed to recover and then hemodynamic
recordings made each week for 4 weeks. At that time the dogs are
anesthetized, cardiac catheterization performed, and microspheres
injected and the heart perfusion fixed, or collected for in vitro
studies of coronary microvessels.
[0168] The results are expected to show an increase in microsphere
measured blood flow to the border zone of the infarct and a small
increase in segment function (local contraction). There may be
evidence of GFP containing blood vessels and in vitro these may
have enhanced flow and agonist induced dilation.
Protocol 3.
[0169] Rationale: Myocardial infarction in man is often associated
with the development of heart failure due to reduced inotropic
state (myocyte loss). Furthermore, once a large infarct is
established and heart failure evident, the heart failure progresses
due to increased wall stress and subsequent remodeling. There are
no current therapies which are targeted to the sequence of events
leading to the progression of ischemic heart failure. The use of
VPCs would be a unique approach to restoring blood flow to a large
ischemic area of the LAD. The goal of these experiments is to
determine the effectiveness of injection of VPCs as a therapy in
the presence of a large established infarction.
[0170] Specific methods: In these studies we create a large
infarct, sufficient to reduce ejection fraction acutely and
permanently by occluding the proximal LAD. Dogs are trained to lie
on the laboratory table and echocardiography performed. Dogs are
instrumented for the measurement of cardiac function and segment
function in the potentially ischemic zone. A flow transducer and
hydraulic occluder are placed on the proximal LAD just distal to
the first diagonal branch. The left atrial appendage is harvested.
The dog is allowed to recover. After recovery and echocardiography,
microspheres are injected to measure blood flow and then the dog
anesthetized for permanent occlusion of the proximal LAD. The dog
is allowed to recover and the size of the infarct determined by
echo, following microsphere injection and hemodynamic measures. The
infarct is visualized using fluoroscopy. After 2-4 weeks the dog is
anesthetized and using an echo guided spinal needle or Mercator
catheter, VPCs injected into the middle of the infarct and around
the border zone. The dog is allowed 2-4 weeks during which
hemodynamics and echocardiography is performed. At the terminal
experiment, cardiac catheterization is performed to visualize blood
flow to the infarct.
[0171] The results are expected to show some increase in GFP
labeled blood vessels, in microsphere measured blood flow, and
visualized blood flow to the ischemic region and some increase in
cardiac function at best proportional to the increase in blood
flow.
Example 5
Implantation of MPCs Replaces the Scar with Functionally Competent
Myocardium in the Presence of a Stenotic or Occluded Coronary
Artery
[0172] The objective of this series of experiments is to determine
whether in the presence of a stenotic or occluded coronary artery
catheter-delivered or locally injected MPCs replace the scar with
functionally competent myocardium restoring in part regional
ventricular function.
[0173] Three different protocols are employed in this series of
experiments. They are as follows:
Protocol 1.
[0174] Rationale: The inability of reperfusion therapy to increase
contractile function distal to a chronic coronary artery stenosis
is due to the lack of functioning cardiac myocytes and not to the
ability to increase blood flow to the ischemic zone. The goal of
this experiment is to determine if the injection of MPCs into the
heart results in the formation of functional cardiac myocytes and
results in amelioration of the ischemia due to chronic coronary
stenosis.
[0175] Specific methods: The dog is trained to lie on the
laboratory table and then echocardiography is performed with
special attention to the anterior wall. Dogs are instrumented for
the measurement of global and segmental cardiac function and with a
flow transducer, hydraulic occluder and critical stenosis on the
distal LAD. At surgery the stenosis is adjusted to eliminate the
reactive hyperemia following a 15 second coronary artery occlusion
(a critical stenosis). The left atrial appendage is harvested from
which MPCs are isolated and propagated. The chest is closed in
layers and the dog allowed to recover. Ten days after surgery
hemodynamics are recorded, the reactive hyperemia examined, and
echocardiography performed. This is repeated each week for 2-4
weeks (until the autologous canine stem cells proliferate,
estimates are that 10.times.10.sup.6/ml cells per animal are used).
Once sufficient cells are available, hemodynamics (non radioactive
microspheres injected through the left atrium to measure collateral
flow) are recorded and then the dog anesthetized with sodium
pentobarbital. The dog is taken to the fluoroscopy laboratory and
the LAD catheterized to visualize the collateral circulation and to
inject MPCs using a specially designed balloon-needle catheter
(414). The needle is advanced from the catheter and injections of
250 .mu.l of the MPC cell suspension is given 2-5 times. In some of
the dogs, the cell suspension is mixed with contrast media (414) to
visualize the sites of injection. The catheter is removed and the
dog allowed to recover. The dog is studied each week for 4 weeks
and microspheres injected to measure collateral flow. At that time
the cardiac catheterization is repeated to visualize collateral
blood flow and one half the dogs is anesthetized and the heart
perfusion fixed. In the other half, tissue is collected for in
vitro studies of microvessel reactivity.
[0176] The results of these experiments are expected to show that
the injection of MPCs results in a partially developed collateral
circulation (fluoroscope) growth of GFP containing cardiac myocytes
and few GFP labeled blood vessels. There may be minimal increased
deposition of microspheres in the ischemic zone distal to the
stenosis. There may be a relationship between increased cardiac
function and the site and number of GFP staining MPCs.
[0177] We have previously published (301) that injection of HGF or
IGF results in an increase in contractile function in the ischemic
zone following 4 hours of total occlusion of the LAD. In addition,
we found evidence of newly generated cardiac myocytes and an
increase in ejection fraction, stroke volume (301) and shortening
(right FIG. 21, panel A) that was proportional to the regeneration
of the myocardium.
[0178] In another set of experiments (FIG. 21, panel B) designed to
use an adeno-associated virus containing VEGF to grow new blood
vessels in the area of an infarction (small LAD infarct as
proposed), we also found an increase in segment function and
segment work in segments that were either paradoxical to begin with
or in those that had reduced shortening (310). This was associated
with an increase in the number of cardiomyocytes in the area of the
infarct.
Protocol 2.
[0179] Rationale: It is not the ability to increase blood flow
during reperfusion injury to the border zone of a small infarct
that limits recovery, rather it is the lack of functioning
myocytes. This is important since a small infarct may enlarge or
extend with time if wall stress increases resulting in additional
ischemic damage. The goal of this study is to determine if the
injections of MPCs into the border and ischemic zone of a distal
(small infarct) LAD occlusion results in increased blood flow and
contractile function. These studies use a small infarction so that
complications of heart failure are not present.
[0180] Specific methods: Surgery is performed using general
anesthesia and sterile technique for the measurement of cardiac
function (including segment function) and with an occluder and flow
transducer on the LAD. The left atrial appendage is harvested for
the collection of MPCs. The dog is allowed to recover for 10 days.
Hemodynamics studies and echocardiography are performed and then
the dog anesthetized, the distal LAD permanently occluded and the
dog allowed 2-4 weeks to recover. Hemodynamic recordings are made
after 2-4 weeks, and then under general anesthesia, cardiac
catheterization performed, and catheter injections of MPCs (250
.mu.l) using an echo-guided spinal needle or Mercator are made into
the middle of the infarct and then into the border zone (3-5
injections). The dog is allowed to recover and then hemodynamic
recordings made each week for 4 weeks. At that time microspheres
are injected, the dogs are anesthetized for cardiac
catheterization, and the heart perfusion fixed or collected for in
vitro studies of coronary microvessels.
[0181] The results are expected to show an increase in local
contraction in the border zone of the infarct. There may be no
increase in microsphere measured blood flow to the border zone of
the infarct but a demonstrable increase in segment function (local
contraction). It is also expected that there will be evidence of
GFP containing MPCs.
Protocol 3.
[0182] Rationale: Myocardial infarction in man is often associated
with the development of heart failure due to reduced inotropic
state (myocyte loss). Furthermore, once a large infarct is
established and heart failure evident, the heart failure progresses
due to increase wall stress and subsequent remodeling. There are no
current therapies which are targeted to the sequence of events
leading to the progression of ischemic heart failure. The use of
MPCs would be a unique focused approach to restoring contractile
function to a large ischemic area perfused by the LAD. The goal of
these experiments is to determine the effectiveness of injection of
MPCs as a therapy in the presence of a large established
infarction.
[0183] Specific methods: In these studies we create a large
infarct, sufficient to reduce ejection fraction acutely and
chronically by occluding the proximal LAD. Dogs are trained to lie
on the laboratory table and echocardiography performed. Dogs are
instrumented for the measurement of cardiac function and segment
function in the potentially ischemic zone. A flow transducer and
hydraulic occluder are placed on the proximal LAD just distal to
the first diagonal branch. The left atrial appendage is harvested.
The dog is allowed to recover. After recovery and echocardiography,
microspheres are injected to measure blood flow and then the dog
anesthetized for cardiac catheterization and induction of a
permanent occlusion of the proximal LAD. The dog is allowed to
recover and the size of the infarct determined by echo, following a
second microsphere injection and hemodynamic measures. After 2-4
weeks, the dog is anesthetized, cardiac catheterization performed
and using an echo guided spinal needle or Mercator catheter, MPCs
are injected into the middle of the infarct and around the border
zone. The dog is allowed 2-4 weeks recovery during which
hemodynamics and echocardiography are performed.
[0184] There may be some increase in GFP-MPCs and perhaps some
increase in cardiac function proportional to the increase in MPCs.
There may be little increase in blood flow using microspheres or
contrast media.
Example 6
Implanted VPCs and MPCs Generate a Biological Bypass and
Functionally Competent Myocardium in the Presence of a Stenotic or
Occluded Coronary Artery
[0185] To address the use of strategies to recruit stem cells into
the ischemic dog heart, we have performed two studies in dogs with
anterior wall infarction following permanent occlusion of the LAD
coronary artery. In the first study (301), 6 hours after permanent
occlusion, a time point during which there is no recovery of
function during reperfusion (347, 348), through an echo guided
spinal needle we injected a combination of hepatocyte growth factor
(HGF) and insulin like growth factor (IGF), based on previous
studies (363-365), into the center of the infarct (as determined by
paradoxical motion using the echo) and into the border zone (as
identified by reduced systolic function on the echo). We then
recorded hemodynamics in the conscious dog each week for 4 weeks
and harvested the heart to identify newly formed myocytes and blood
vessels. In the conscious dog, injection of IGF and HGF caused
recovery in contractile function in both the center of the infarct
and in the border zone. This was proportional to the number of
newly formed cardiac myocytes and blood vessel cells of stem cell
origin. Importantly these cells were derived from adult resident
cardiac stem cells based on cell specific markers. Of some
significance is the fact that we found increased numbers of both
blood vessels and newly formed myocytes leading to the speculation
that both cell types are needed to re-establish the relationship
between coronary blood flow and contractile function.
[0186] We performed studies using the same model and injected an
adeno-associated virus containing the human VEGF165 gene into the
center and border zone 4 hours after permanent occlusion of the LAD
coronary artery (310). Again the dogs were studied in the conscious
state for 4 weeks before tissues were collected. In those studies,
designed to grow blood vessels using VEGF, we found expression of
human VEGF in the dog heart and its receptors in the infarct and
found recovery of segment function in both the center of the
infarct and in the border zone. Importantly, we also found
cardiomyocytes of stem cell origin. Thus using two different
strategies in the same model we found recovery of function
associated with new cardiomyocytes and new blood vessels again
supporting the concept that re-establishment of the flow/function
relationship is required for a new therapy to be effective.
Importantly, Bearzi et al. (304) also found both newly formed blood
vessels and cardiomyocytes in the mouse heart suggesting that
development of an integrated myocardium is required to restore
function in the ischemic heart.
[0187] Three studies using swine as a model for altered cardiac
function and stem cell repair during ischemia have been published.
The first by Liu et al. (366) examined the effects of autologous
bone marrow stem cell administration via a jell patch placed on the
ischemic area 60 minutes after release of a coronary artery
occlusion. The acute application of stem cell therapy resulted in
both neovascularization and new myocyte formation and
proliferation. Together, approximately 3 weeks after infarction
there was a substantial increase in systolic wall thickening,
approximately 40%, and of neovascularization near the patch,
including evidence of both coronary arteries and capillaries. These
studies using autologous bone marrow stem cells acutely after
infarction, support our conclusion that it is best to remodel the
heart creating both new blood vessels and myocytes. The same
authors (367) recently performed studies designed to examine the
effect of allogeneic bone marrow stem cells administered at the
time of infarction in the pig. That study indicated an increase in
vascular density in treated animals 4 weeks after infarction,
however, they also concluded that the contractile effect,
decreasing ejection fraction from 55 to 30% with a return to 41% in
the treated group, was not due to new myocytes but rather to
"patchy spared myocytes." Another study by Suzuki et al. (368)
puzzles over the lack of new blood vessel formation and a
flow/function relationship in hibernating myocardium transfected
with FGF-5 (fibroblast growth factor given in an adenovirus). In
that study, there was an increase in shortening in the hibernating
myocardium from 2.4 to 4.6 mm 14 days after administration of FGF
and only a small increase in blood flow. Furthermore, the authors
discuss the possibility that the lack of new blood vessel growth
may limit the functional recovery of the heart, again pointing out
that the proper coupling of flow and function may result in
development of optimum therapies. Therefore strategies which
produce both new myocytes and a corresponding circulation may be
most efficacious in the treatment and consequences of myocardial
ischemia.
[0188] The objective of the experiments described in this Example
is to determine whether in the presence of a stenotic or occluded
coronary artery catheter-delivered or locally injected VPCs and
MPCs generate a biological bypass and functionally competent
myocardium, which together restore blood flow and regional
ventricular function.
[0189] Three different protocols are employed in this series of
experiments. They are as follows:
Protocol 1.
[0190] Rationale: The myocardium normally functions with a specific
ratio of blood vessels to myocytes and the loss of that
relationship in the process of myocardial ischemia is partially
responsible for cardiac contractile dysfunction. The
reestablishment of the optimum ratio of cardiac myocytes and blood
vessels is most effective in restoring cardiac contractile
function. The goal of this experiment is to determine if the
injection of both MPCs and VPCs into the heart results in the
formation of functional cardiac myocytes and blood vessels and
results in salvage of the ischemia due to chronic coronary
stenosis.
[0191] Specific methods: The dog is trained to lie on the
laboratory table and then echocardiography is performed with
special attention to the anterior wall. Dogs are instrumented for
the measurement of cardiac function and with a flow transducer,
hydraulic occluder and critical stenosis on the distal LAD. At
surgery, the stenosis is adjusted to eliminate the reactive
hyperemia following a 15 second coronary artery occlusion (a
critical stenosis). The left atrial appendage is harvested from
which both MPCs and VPCs are isolated and propagated. The chest is
closed in layers and the dog is allowed to recover. Ten days after
surgery hemodynamics are recorded, the reactive hyperemia examined
and echocardiography performed. This is repeated each week for 2-4
weeks (until the autologous canine stem proliferate, estimates are
that 2.times.10.sup.6 cells per ml per animal are used, of
combination of VPCs and MPCs). Once sufficient cells are available,
hemodynamics (non radioactive microspheres are injected through the
left atrium to measure collateral flow) are recorded and then the
dog anesthetized with sodium pentobarbital. The dog is taken to the
fluoroscopy laboratory and the LAD catheterized to visualize the
collateral circulation and then for use of a specially designed
balloon-needle catheter (414). The needle is advanced from the
catheter and injections of 250 .mu.l of the mixture of VPC and MPCs
cell suspension is given 2-5 times each. In some of the dogs the
cell suspension is mixed with contrast media (Mercator paper) to
visualize the sites of injection. The catheter is removed and the
dog allowed to recover. The dog is studied each week for 4 weeks
and microspheres injected to measure blood flow. On the last day,
under general anesthesia cardiac catheterization is performed to
visualize the collateral circulation. At that time one half the
dogs are anesthetized and the heart perfusion fixed. In the other
half, tissue is collected for in vitro studies of microvessel
reactivity.
[0192] The injection of the mixture of MPCs and VPC may result in
growth of GFP containing myocytes and blood vessels, increased
deposition of microspheres in the ischemic zone distal to the
stenosis, and importantly increased regional contractile function.
There may be a large collateral network by fluoroscopy. In isolated
perfused coronary microvessels there may be increased flow induced
dilation (indicating functional endothelium) and increased agonist
induced dilation (indicating enhanced smooth muscle and endothelial
function). There may be a relationship between dilation in vitro
and the site and amount of GFP staining in microvessels. There may
be an increase in contractile function in the ischemic zone that is
greater than can be accounted for by the increase in blood flow,
indicating the important role played by MPCs in recovery of
contractile function in the ischemic heart. It is possible that the
ratio of enhanced blood flow (VPCs) and enhanced contraction (MPCs)
is critical to the recovery of the ischemic heart and that
adjusting the number of each VPC and MPC (an optimal ratio) may
have to be performed to optimize salvage for each particular
condition or subject. It may be possible to inject VPCs to grow
blood vessels first and then MPCs to grow myocytes once the
circulation has developed sufficiently to support MPC
differentiation, growth and function.
Protocol 2.
[0193] Rationale: The lack of restoration of blood flow to the
border zone of the ischemic heart may limit the survival of
myocytes that have survived the initial ischemia. On the other
hand, the reduced number of myocytes may doom the ischemic area due
to the reduction in inotropic state and cardiac contractile
function. Thus, solving only one of these two problems, reduced
blood flow or reduced myocyte mass, is not sufficient to restore
cardiac function. The goal of this study is to determine if the
injections of both MPCs and VPCs into the border and ischemic zone
of a distal (small infarct) LAD occlusion results in increased
blood flow and contractile function. These studies use a small
infarction so that the complications of heart failure are not
present.
[0194] Specific methods: Surgery is performed using general
anesthesia and sterile technique for the measurement of cardiac
function (including segment function) and with an occluder and flow
transducer on the LAD. The left atrial appendage is harvested for
the collection of VPCs and MPCs. The dog is allowed to recover for
10 days. Hemodynamic studies and echocardiography are performed and
then the dog anesthetized, the distal LAD permanently occluded and
the dog allowed 2-4 weeks to recover. After 2-4 weeks, under
general anesthesia the LAD is catheterized to visualize the
circulation to the infarct and then using an echo-guided spinal
needle or Mercator catheter injections of MPCs and VPCs (250 .mu.l
each) are made into the middle of the infarct and then into the
border zone (3-5 injections). The dog is allowed to recover and
then hemodynamic recordings made each week for 4 weeks. For the
last experiment, microspheres are injected and the dog anesthetized
for catheterization of the LAD and contrast injection. At that time
the heart is perfusion fixed or tissues collected for in vitro
studies of coronary microvessels.
[0195] There may be an increase in microsphere measured blood flow
and delivery of contrast (catheterization) to the border zone of
the infarct. There may be evidence of GFP containing cardiac
myocytes and blood vessels and in vitro these will have enhanced
flow and agonist induced dilation. There may be a substantial
increase in local segment function due to the growth and
proliferation of MPCs. It may be beneficial to alter the number of
MPCs and VPCs to optimize the ratio that governs flow and function
to increase segment function and to fully restore contractile
function of the anterior wall. In addition it may be possible to
inject VPCs first to support the differentiation, growth and
function of MPCs once the coronary circulation has developed.
Protocol 3
[0196] Rationale: Myocardial infarction in man is often associated
with the development of heart failure due to reduced inotropic
state and for instance large reductions in ejection fraction.
Furthermore, once a large infarct is established with loss of both
myocytes and blood vessels and heart failure evident, the heart
failure progresses due to increase wall stress and subsequent
remodeling. There are no current therapies which are targeted to
the sequence of events leading to the progression of ischemic heart
failure. The use of both MPCs and VPCs would be a unique
combinatorial approach to restoring blood flow and contractile
function to a large ischemic area perfused by the LAD. The goal of
these experiments is to determine the effectiveness of injection of
both MPCs and VPCs as a therapy in the presence of a large
established infarction.
[0197] Specific methods: In these studies, we create a large
infarct, sufficient to reduce ejection fraction acutely and
chronically by occluding the proximal LAD. Dogs are trained to lie
on the laboratory table and echocardiography performed. Dogs are
instrumented for the measurement of cardiac function and segment
function in the potentially ischemic zone. A flow transducer and
hydraulic occluder are placed on the proximal LAD just distal to
the first diagonal branch. The left atrial appendage is harvested.
The dog is allowed to recover. After recovery and echocardiography,
microspheres are injected to measure blood flow and then the dog
anesthetized for cardiac catheterization to visualize the
circulation and for permanent occlusion of the proximal LAD. The
dog is allowed to recover and the size of the infarct determined by
echo, following microsphere injection and hemodynamic measures.
After 2-4 weeks the dog is anesthetized, the cardiac
catheterization repeated and using an echo guided spinal needle of
Mercator catheter, MPCs and VPCs injected into the middle of the
infarct (initially at the same time and at an initial ratio of 1/1)
and around the border zone. The dog is allowed 2-4 weeks during
which hemodynamics and echocardiography are performed. On the final
day, the dog is anesthetized and cardiac catheterization performed
to visualize the circulation.
[0198] There may be a substantial increase in GFP labeled blood
vessels and myocytes, in microsphere measured blood flow to the
ischemic region, an increase in visualized blood flow by contrast
injection, and a marked increase in cardiac function which is
greater than would be predicted by the increase in blood flow alone
supporting the important role of MPCs. To achieve an ejection
fraction similar to that before infarction, the ratio of the
injected MPCs/VPCs may be adjusted or optimized. Alternatively or
additionally, the timing of injection of the VPCs and MPCs may be
adjusted.
[0199] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the appended claims is not to be limited by particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope thereof.
[0200] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
REFERENCES
[0201] 1. Fishman M C, Chien K R: Fashioning the vertebrate heart:
earliest embryonic decisions. Development. 124:2099-2117, 1997.
[0202] 2. Mikawa T: Cardiac lineages. In: R P Harvey, N Rosenthal,
eds: Heart Development. Academic Press, San Diego, pp. 19-33, 1999.
[0203] 3. Srivastava D, Olson E N: A genetic blueprint for cardiac
development. Nature. 407: 221-226, 2000. [0204] 4. Bruneau B G:
Transcriptional regulation of vertebrate cardiac morphogenesis.
Circ Res. 90:509-519, 2002. [0205] 5. Harvey R P: Patterning the
vertebrate heart. Nat Rev Genet. 3:544-556, 2002. [0206] 6. Mikawa
T: Early embryonic vascular development. Cardiovasc Res.
31:E34-E45, 1996. [0207] 7. Reese D E, Mikawa T, Bader D M:
Development of the coronary vessel system. Circ Res. 91:761-768,
2002. [0208] 8. Olivey H E, Compton L A, Barnett J V: Coronary
vessel development: the epicardium delivers. Trends Cardiovasc Med.
14:247-251, 2004. [0209] 9. Mikawa T, Fischman D A: Retroviral
analysis of cardiac morphogenesis: discontinuous formation of
coronary vessels. Proc Natl Acad Sci USA. 89:9504-9508, 1992.
[0210] 10. Mikawa T, Gourdie R G: Pericardial mesoderm generates a
population of coronary smooth muscle cells migrating into the heart
along with ingrowth of the epicardial organ. Dev Biol. 174:221-232,
1996. [0211] 11. Wessels A, Perez-Pomares J M: The epicardium and
epicardially derived cells (EPDCs) as cardiac stem cells. Anat Rec
A Discov Mol Cell Evol Biol. 276:43-57, 2004. [0212] 12. Anversa P,
Leri A, Beltrami C A, Guerra S, Kajstura J: Myocyte death and
growth in the failing heart. Lab Invest. 78:767-786, 1998. [0213]
13. Beltrami A P, Urbanek K, Kajstura J, Yao S-M, Finato N, Bussani
R, Nadal-Ginard B, Silvestri F, Leri A, Beltrami C A, Anversa P:
Evidence that human cardiac myocytes divide after myocardial
infarction. N Engl J Med. 344:1750-1757, 2001. [0214] 14. Quaini F,
Urbanek K, Beltrami A P, Finato N, Beltrami C A, Nadal-Ginard B,
Kajstura J, Leri A, Anversa P: Chimerism of the transplanted heart.
N Engl J Med. 346:5-15, 2002. [0215] 15. Urbanek K, Quaini F, Tasca
G, Torella D, Castaldo C, Nadal-Ginard B, Leri A, Kajstura J,
Quaini E, Anversa P: Intense myocyte formation from cardiac stem
cells in human cardiac hypertrophy. Proc Natl Acad Sci USA.
100:10440-10445, 2003. [0216] 16. Urbanek K, Torella D, Sheikh F,
De Angelis A, Nurzynska D, Silvestri F, Beltrami C A, Bussani R,
Beltrami A P, Quaini F, Bolli R, Leri A, Kajstura J, Anversa P:
Myocardial regeneration by activation of multipotent cardiac stem
cells in ischemic heart failure. Proc Natl Acad Sci USA.
102:8692-8697, 2005. [0217] 17. Anversa P, Leri A, Rota M, Hosoda
T, Bearzi C, Urbanek K, Kajstura J, Bolli R: Concise review: stem
cells, myocardial regeneration, and methodological artifacts. Stem
Cells. 25:589-601, 2007. [0218] 18. Leri A, Kajstura J, Anversa P:
Cardiac stem cells and mechanisms of myocardial regeneration.
Physiol Rev. 85:1373-1416, 2005. [0219] 19. Anversa P, Kajstura J,
Leri A, Bolli R: Life and death of cardiac stem cells: a paradigm
shift in cardiac biology. Circulation. 113:1451-1463, 2006. [0220]
20. Anversa P, Leri A, Kajstura J: Cardiac regeneration. J Am Coll
Cardiol. 47:1769-1776, 2006. [0221] 21. Beltrami A P, Barlucchi L,
Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso
E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P: Adult
cardiac stem cells are multipotent and support myocardial
regeneration. Cell. 114:763-776, 2003. [0222] 22. Linke A, Muller
P, Nurzynska D, Casarsa C, Torella D, Nascimbene A, Castaldo C,
Cascapera S, Bohm M, Quaini F, Urbanek K, Leri A, Hintze T H,
Kajstura J, Anversa P: Stem cells in the dog heart are
self-renewing, clonogenic, and multipotent and regenerate infarcted
myocardium, improving cardiac function. Proc Natl Acad Sci USA.
102: 8966-8971, 2005. [0223] 23. Urbanek K, Rota M, Cascapera S,
Bearzi C, Nascimbene A, De Angelis A, Hosoda T, Chimenti S, Baker
M, Limana F, Nurzynska D, Torella D, Rotatori F, Rastaldo R, Musso
E, Quaini F, Leri A, Kajstura J, Anversa P: Cardiac stem cells
possess growth factor-receptor systems that after activation
regenerate the infarcted myocardium, improving ventricular function
and long-term survival. Circ Res. 97:663-673, 2005. [0224] 24.
Hierlihy A M, Seale P, Lobe C G, Rudnicki M A, Megeney L A: The
post-natal heart contains a myocardial stem cell population. FEBS
Lett. 530:239-243, 2002. [0225] 25. Oh H, Bradfute S B, Gallardo T
D, Nakamura T, Gaussin V, Mishina Y, Pocius J, Michael L H,
Behringer R R, Garry D J, Schneider M D: Cardiac progenitor cells
from adult myocardium: homing, differentiation, and fusion after
infarction. Proc Natl Acad Sci USA. 100:12313-12318, 2003. [0226]
26. Matsuura K, Nagai T, Nishigaki N, Oyama T, Nishi J, Wada H,
Sano M, Toko H, Akazawa H, Sato H, Nakaya H, Kasanuki H, Komuro I:
Adult cardiac. Sca-1-positive cells differentiate into beating
cardiomyocytes. J Biol Chem. 279:11384-11391, 2004. [0227] 27.
Martin C M, Meeson A P, Robertson S M, Hawke T J, Richardson J A,
Bates S, Goetsch S C, Gallardo T D, Garry D J: Persistent
expression of the ATP-binding cassette transporter, Abcg2,
identifies cardiac SP cells in the developing and adult heart. Dev
Biol. 265:262-275, 2004. [0228] 28. Messina E, De Angelis L, Frati
G, Morrone S, Chimenti S, Fiordaliso F, Salio M, Battaglia M,
Latronico M V G, Coletta M. Vivarelli E, Frati L, Cossu G,
Giacomello A: Isolation and expansion of adult cardiac stem cells
from human and murine heart. Circ Res. 95:911-921, 2004. [0229] 29.
Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A, Colucci W
S, Liao R: CD31- but not CD31+ cardiac side populations cells
exhibit functional cardiomyogenic differentiation. Circ Res.
97:52-61, 2005. [0230] 30. Laugwitz K L, Moretti A, Lam J, Gruber
P, Chen Y, Woodard S, Lin L Z, Cai C L, Lu M M, Reth M, Platoshyn
O, Yuan J X, Evans S, Chien K R: Postnatal isl1+ cardioblasts enter
fully differentiated cardiomyocyte lineages. Nature. 433:585-587,
2005. [0231] 31. Rosenblatt-Velin N, Lepore M G, Cartoni C,
Beermann F, Pedrazzini T: FGF-2 controls the differentiation of
resident cardiac precursors into functional cardiomyocytes. J Clin
Invest. 115:1724-1733, 2005. [0232] 32. Tomita Y, Matsumura K,
Wakamatsu Y, Matsuzaki Y, Shibuya I, Kawaguchi H, Ieda M, Kanakubo
S, Shinmazaki T, Ogawa S, Osumi N, Okano H, Fukuda K: Cardiac
neural crest cells contribute to the dormant multipotent stem cell
in the mammalian heart. J Cell Biol. 170:1135-1146, 2005. [0233]
33. Bearzi C, Rota M, Hosoda T, Tillmanns J, Nascimbene A, De
Angelis A, Yasuzawa-Amano S, Trofimova I, Siggins R W, Cascapera S,
Beltrami A P, Zias E, Quaini F, Urbanek K, Michler R E, Bolli R,
Kajstura J, Leri A, Anversa P: Human cardiac stem cells. Proc Natl
Acad Sci USA. 104:14068-14073, 2007. [0234] 34. Carmeliet P:
Angiogenesis in life, disease and medicine. Nature. 438:932-936,
2005. [0235] 35. Carmeliet P, Jain R K: Angiogenesis in cancer and
other diseases. Nature. 407:249-257, 2000. [0236] 36. Carmeliet P:
Angiogenesis in health and disease. Nat Med. 9:653-660, 2003.
[0237] 37. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee
R, Li T, Witzenbichler B, Schatteman G, Isner J M: Isolation of
putative progenitor endothelial cells for angiogenesis. Science.
275:964-967, 1997. [0238] 38. Aicher A, Heeschen C, Mildner-Rihm C,
Urbich C, Ihling C, Technau-Ihling K, Zeiher A M, Dimmeler S:
Essential role of endothelial nitric oxide synthase for
mobilization of stem and progenitor cells. Nat Med. 9:1370-1376,
2003. [0239] 39. Urbich C, Dimmeler S: Endothelial progenitor
cells: characterization and role in vascular biology. Circ Res. 95:
343-353, 2004. [0240] 40. Asahara T, Kawamoto A: Endothelial
progenitor cells for postnatal vasculogenesis. Am J Physiol.
287:C572-579, 2004. [0241] 41. Carmeliet P: Mechanisms of
angiogenesis and arteriogenesis. Nat Med. 6:389-395, 2000. [0242]
42. Hirschi K K, Goodell M A: Hematopoietic, vascular and cardiac
fates of bone marrow-derived stem cells. Gene Ther. 9:648-652,
2002. [0243] 43. Deb A, Skelding K A, Wang S, Reeder M, Simper D,
Caplice N M: Integrin profile and in vivo homing of human smooth
muscle progenitor cells. Circulation. 110:2673-2677, 2004. [0244]
44. Hu Y, Zhang Z, Torsney E, Afzal A R, Davison F, Metzler B, Xu
Q: Abundant progenitor cells in the adventitia contribute to
atherosclerosis of vein grafts in ApoE-deficient mice. J Clin
Invest. 113:1258-1265, 2004. [0245] 45. Mayr U, Mayr M, Yin X,
Begum S, Tarelli E, Wait R, Xu Q: Proteomic dataset of mouse aortic
smooth muscle cells. Proteomics. 5:4546-4557, 2005. [0246] 46.
Torsney E, Mandal K, Halliday A, Jahangiri M, Xu Q:
Characterisation of progenitor cells in human atherosclerotic
vessels. Atherosclerosis. 191:259-264, 2007. [0247] 47. Mandal K,
Jahangiri M, Xu Q: Progenitor cells and vascular repair. In
Cardiovascular Regeneration and Stem Cell Therapy. Eds. A. Leri, P.
Anversa, W. H. Frishman, Blackwell Futura 2007, Malden, M A, pp.
57-66. [0248] 48. Minasi M G, Riminucci M, De Angelis L, Borello U,
Berarducci B, Innocenzi A, Caprioli A, Sirarella D, Baiocchi M, De
Maria R, Boratto R, Jaffredo T, Broccoli V, Bianco P, Cossu G: The
meso-angioblast: a multipotent, self-renewing cell that originates
from the dorsal aorta and differentiates into most mesodermal
tissues. Development. 129:2773-2783, 2002. [0249] 49. Sampaolesi M,
Blot S, D'Antona G, Granger N, Tonlorenzi R, Innocenzi A, Mognol P,
Thibaud J L, Galvez B G, Barthelemy I, Perani L, Mantero S,
Guttinger M, Pansarasa O, Rinaldi C, Cusella De Angelis M G,
Torrente Y, Bordignon C, Bottinelli R, Cossu G: Mesoangioblast stem
cells ameliorate muscle function in dystrophic dogs. Nature.
444:574-579, 2006. [0250] 50. Dellavalle A, Sampaolesi M,
Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, Innocenzi A,
Galvez B G, Messina G, Morosetti R, Lis S, Belicchi M, Peretti G,
Chamberlain J S, Wright W E, Torrente Y, Ferrari S, Bianco P, Cossu
G: Pericytes of human skeletal muscle are myogenic precursors
distinct from satellite cells. Nat Cell Biol. 9: 255-267, 2007.
[0251] 51. Zengin E, Chalajour F, Gehling U M, Ito W D, Treede H,
Lauke H, Weil J, Reichenspurner H, Kilic N, Ergun S: Vascular wall
resident progenitor cells: a source for postnatal vasculogenesis.
Development. 133:1543-1551, 2006. [0252] 52. Aicher A, Rentsch M,
Sasaki K, Ellwart J W, Fandrich F, Siebert R, Cooke J P, Dimmeler
S, Heeschen C: Nonbone marrow-derived circulating progenitor cells
contribute to postnatal neovascularization following tissue
ischemia. Circ Res. 100:581-589, 2007. [0253] 53. Urbanek K,
Cesselli D, Rota M, Nascimbene A, De Angelis A, Hosoda T, Bearzi C,
Boni A, Bolli R, Kajstura J, Anversa P, Leri A: Stem cell niches in
the adult mouse heart. Proc Natl Acad Sci USA. 103:9226-9231, 2006.
[0254] 54. Coultas L, Chawengsaksophak K, Rossant J: Endothelial
cells and VEGF in vascular development. Nature. 438:937-945, 2005.
[0255] 55. Huber T L, Kouskoff V, Fehling H J, Palis J, Keller G:
Haemangioblast commitment is initiated in the primitive streak of
the mouse embryo. Nature. 432:625-630, 2004. [0256] 56. Shalaby F,
Ho J, Stanford W L, Fischer K D, Schuh A C, Schwartz L, Bernstein
A, Rossant J: A requirement for Flk1 in primitive and definitive
hematopoiesis and vasculogenesis. Cell. 89:981-990, 1997. [0257]
57. Shalaby F, Rossant J, Yamaguchi T P, Gertsenstein M, Wu X F,
Breitman M L, Schuh A C: Failure of blood-island formation and
vasculogenesis in Flk-1-deficient mice. Nature. 376:62-66, 1995.
[0258] 58. Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S,
Yurugi T, Naito M, Nakao K, Nishikawa S: Flk1-positive cells
derived from embryonic stem cells serve as vascular progenitors.
Nature. 408:92-96, 2000. [0259] 59. Kattman S J, Huber T L, Keller
G M: Multipotent flk-1+ cardiovascular progenitor cells give rise
to the cardiomyocyte, endothelial, and vascular smooth muscle
lineages. Dev Cell. 11:723-732, 2006. [0260] 60. Wu S M, Fujiwara
Y, Cibulsky S M, Clapham D E, Lien C L, Schultheiss T M, Orkin S H:
Developmental origin of a bipotential myocardial and smooth muscle
cell precursor in the mammalian heart. Cell. 127:1137-1150, 2006.
[0261] 61. Moretti A, Caron L, Nakano A, Lam J T, Bernshausen A,
Chen Y, Qyang Y, Bu L, Sasaki M, Martin-Puig S, Sun Y, Evans S M,
Laugwitz K L, Chien K R: Multipotent embryonic isl1+ progenitor
cells lead to cardiac, smooth muscle, and endothelial cell
diversification. Cell. 127:1151-1165, 2006. [0262] 62. Kataoka H,
Takakura N, Nishikawa S, Tsuchida K, Kodama H, Kunisada T, Risau W,
Kita T, Nishikawa I S: Expressions of PDGF receptor alpha, c-Kit
and Flk1 genes clustering in mouse chromosome 5 define distinct
subsets of nascent mesodermal cells. Dev Growth Differ. 39:729-740,
1997. [0263] 63. Nishikawa S I, Nishikawa S, Hirashima M,
Matsuyoshi N, Kodama H: Progressive lineage analysis by cell
sorting and culture identifies FLK1+VE-cadherin+ cells at a
diverging point of endothelial and hemopoietic lineages.
Development. 125:1747-1757, 1998. [0264] 64. Bertrand J Y, Giroux
S, Golub R, Klaine M, Jalil A, Boucontet L, Godin I, Cumano A:
Characterization of purified intraembryonic hematopoietic stem
cells as a tool to define their site of origin. Proc Natl Acad Sci
USA. 102:134-139, 2005. [0265] 65. Samokhvalov I M, Samokhvalova N
I, Nishikawa S: Cell tracing shows the contribution of the yolk sac
to adult haematopoiesis. Nature. 446:1056-1061, 2007. [0266] 66.
Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson S M, Li B,
Pickel J, McKay R, Nadal-Ginard B, Bodine D M, Leri A, Anversa P:
Bone marrow cells regenerate infarcted myocardium. Nature.
410:701-705, 2001. [0267] 67. Orlic D, Kajstura J, Chimenti S,
Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine D M, Leri A,
Anversa P: Mobilized bone marrow cells repair the infarcted heart
improving function and survival. Proc Natl Acad Sci USA.
98:10344-10349, 2001. [0268] 68. Kajstura J, Rota M, Whang B,
Cascapera S, Hosoda T, Bearzi C, Nurzynska D, Kasahara H, Zias E,
Bonafe' M, Nadal-Ginard B, Torella D, Nascimbene A, Quaini F,
Urbanek K, Leri A, Anversa P: Bone marrow cells differentiate in
cardiac cell lineages after infarction independently of cell
fusion. Circ Res. 96:127-137, 2005. [0269] 69. Murasawa S, Kawamoto
A, Horii M, Nakamori S, Asahara T: Niche-dependent translineage
commitment of endothelial progenitor cells, not cell fusion in
general, into myocardial lineage cells. Arterioscler Thromb Vasc
Biol. 25:1388-1394, 2005. [0270] 70. Koyanagi M, Brandes R P,
Haendeler J, Zeiher A M, Dimmeler S: Cell-to-cell connection of
endothelial progenitor cells with cardiac myocytes by nanotubes: a
novel mechanism for cell fate changes? Circ Res. 96:1039-1041,
2005. [0271] 71. Jan Y N, Jan L Y: Asymmetric cell division.
Nature. 392:775-778, 1998. [0272] 72. Lu B, Jan L, Jan Y N: Control
of cell divisions in the nervous system: symmetry and asymmetry.
Annu Rev Neurosci. 23:531-556, 2000.
[0273] 73. Noctor S C, Martinez-Cerdeno V, Ivic L, Kriegstein A R:
Cortical neurons arise in symmetric and asymmetric division zones
and migrate through specific phases. Nat Neurosci. 7:136-144, 2004.
[0274] 74. Roegiers F, Jan Y N: Asymmetric cell division. Curr Opin
Cell Biol. 16:195-205, 2004. [0275] 75. Faubert A, Lessard J,
Sauvageau G: Are genetic determinants of asymmetric stem cell
division active in hematopoietic stem cells? Oncogene. 23:7247-55,
2004. [0276] 76. Gotz M, Huttner W B: The cell biology of
neurogenesis. Nat Rev Mol Cell Biol. 6: 777-788, 2005. [0277] 77.
Huttner W B, Kosodo Y: Symmetric versus asymmetric cell division
during neurogenesis in the developing vertebrate central nervous
system. Curr Opin Cell Biol. 17:648-657, 2005. [0278] 78. Lechler
T, Fuchs E: Asymmetric cell divisions promote stratification and
differentiation of mammalian skin. Nature. 437:275-280, 2005.
[0279] 79. Clevers H: Stem cells, asymmetric division and cancer.
Nat Genet. 37:1027-1028, 2005. [0280] 80. Verdi J M, Bashirullah A,
Goldhawk D E, Kubu C J, Jamali M, Meakin S O, Lipshitz H D:
Distinct human NUMB isoforms regulate differentiation vs.
proliferation in the neuronal lineage. Proc Natl Acad Sci USA.
96:10472-10476, 1999. [0281] 81. Braun K M, Niemann C, Jensen, U B,
Sundberg, J P, Silva-Vargas V, Watt F M: Manipulation of stem cell
proliferation and lineage commitment: visualization of
label-retaining cells in whole mounts of mouse epidermis.
Development. 130:5241-5255, 2003. [0282] 82. Tumbar T, Guasch G,
Greco V, Blanpain C, Lowry W E, Rendl M, Fuchs E: Defining the
epithelial stem cell niche in skin. Science. 303:359-363, 2004.
[0283] 83. Aaku-Saraste E, Oback B, Hellwig A, Huttner W B:
Neuroepithelial cells downregulate their plasma membrane polarity
prior to neural tube closure and neurogenesis. Mech Dev. 69:71-81,
1997. [0284] 84. Huttner W B, Brand M: Asymmetric division and
polarity of neuroepithelial cells. Curr Opin Neurobiol. 7:29-39,
1997. [0285] 85. Halfter W, Dong S, Yip Y P, Willem M, Mayer U: A
critical function of the pial basement membrane in cortical
histogenesis. J Neurosci. 22:6029-6040, 2002. [0286] 86. Malicki J:
Cell fate decisions and patterning in the vertebrate retina: the
importance of timing, asymmetry, polarity and waves. Curr Opin
Neurobiol. 14:15-21, 2004. [0287] 87. Cowan C R, Hyman A A:
Asymmetric cell division in C. elegans: cortical polarity and
spindle positioning. Annu Rev Cell Dev Biol. 20:427-453, 2004.
[0288] 88. Wodarz A: Molecular control of cell polarity and
asymmetric cell division in Drosophila neuroblasts. Curr Opin Cell
Biol. 17:475-481, 2005. [0289] 89. Strome S, Wood W B: Generation
of asymmetry and segregation of germ-line granules in early C.
elegans embryos. Cell. 35:15-25, 1983. [0290] 90. Chenn A,
McConnell S K: Cleavage orientation and the asymmetric inheritance
of Notch1 immunoreactivity in mammalian neurogenesis. Cell.
82:631-641, 1995. [0291] 91. Kaltschmidt J A, Davidson C M, Brown N
H, Brand A H: Rotation and asymmetry of the mitotic spindle direct
asymmetric cell division in the developing central nervous system.
Nat Cell Biol. 2:7-12, 2000. [0292] 92. Roegiers F,
Younger-Shepherd S, Jan L Y, Jan Y N: Two types of asymmetric
divisions in the Drosophila sensory organ precursor cell lineage.
Nat Cell Biol. 3:58-67, 2001. [0293] 93. Kusch J, Liakopoulos D,
Barral Y: Spindle asymmetry: a compass for the cell. Trends Cell
Biol. 13:562-569, 2003. [0294] 94. Grava S, Schaerer F, Faty M,
Philippsen P, Barral Y: Asymmetric recruitment of dynein to spindle
poles and microtubules promotes proper spindle orientation in
yeast. Dev Cell. 10:425-439, 2006. [0295] 95. Shen C P, Jan L Y,
Jan Y N: Miranda is required for the asymmetric localization of
Prospero during mitosis in Drosophila. Cell. 90:449-458, 1997.
[0296] 96. Shen C P, Knoblich J A, Chan Y M, Jiang M M, Jan L Y,
Jan Y N: Miranda as a multidomain adapter linking apically
localized Inscuteable and basally localized Staufen and Prospero
during asymmetric cell division in Drosophila. Genes Dev.
12:1837-1846, 1998. [0297] 97. Shen Q, Zhong W, Jan Y N, Temple S:
Asymmetric Numb distribution is critical for asymmetric cell
division of mouse cerebral cortical stem cells and neuroblasts.
Development. 129:4843-4853, 2002. [0298] 98. Shen Q, Temple S:
Creating asymmetric cell divisions by skewing endocytosis. Sci
STKE. 162:PE52, 2002. [0299] 99. Li H S, Wang D, Shen Q, Schonemann
M D, Gorski J A, Jones K R, Temple S, Jan L Y, Jan Y N:
Inactivation of Numb and Numblike in embryonic dorsal forebrain
impairs neurogenesis and disrupts cortical morphogenesis. Neuron.
40:1105-1118, 2003. [0300] 100. Androutsellis-Theotokis A, Leker R
R, Soldner F, Hoeppner D J, Ravin R, Poser S W, Rueger M A, Bae S
K, Kittappa R, McKay R D: Notch signaling regulates stem cell
numbers in vitro and in vivo. Nature. 442:823-826, 2006. [0301]
101. Wang Y, Chan S L, Miele L, Yao P J, Mackes J, Ingram D K,
Mattson M P, Furukawa K: Involvement of Notch signaling in
hippocampal synaptic plasticity. Proc Natl Acad Sci USA.
101:9458-9462, 2004. [0302] 102. Hatakeyama J, Bessho Y, Katoh K,
Ookawara S, Fujioka M, Guillemot F, Kageyama R: Hes genes regulate
size, shape and histogenesis of the nervous system by control of
the timing of neural stem cell differentiation. Development.
131:5539-5550, 2004. [0303] 103. Kageyama R, Ohtsuka T, Hatakeyama
J, Ohsawa R: Roles of bHLH genes in neural stem cell
differentiation. Exp Cell Res. 306:343-348, 2005. [0304] 104. Yu F,
Kuo C T, Jan Y N: Drosophila neuroblast asymmetric cell division:
recent advances and implications for stem cell biology. Neuron.
51:13-20, 2006. [0305] 105. Hatakeyama J, Kageyama R: Notch1
expression is spatiotemporally correlated with neurogenesis and
negatively regulated by Notch1-independent Hes genes in the
developing nervous system. Cereb Cortex. 16 (Suppl. 1):132-137,
2006. [0306] 106. Alexson T O, Hitoshi S, Coles B L, Bernstein A,
van der Kooy D: Notch signaling is required to maintain all neural
stem cell populations--irrespective of spatial or temporal niche.
Dev Neurosci. 28:34-48, 2006. [0307] 107. Hatakeyama J, Sakamoto S,
Kageyama R: Hes1 and Hes5 regulate the development of the cranial
and spinal nerve systems. Dev Neurosci. 28:92-101, 2006. [0308]
108. Lehar S M, Dooley J, Farr A G, Bevan M J: Notch ligands Delta
1 and Jagged1 transmit distinct signals to T-cell precursors.
Blood. 105:1440-1447, 2005. [0309] 109. Maillard I, Schwarz B A,
Sambandam A, Fang T, Shestova O, Xu L, Bhandoola A, Pear W S:
Notch-dependent T-lineage commitment occurs at extrathymic sites
following bone marrow. Blood. 107:3511-3519, 2006. [0310] 110. Rhyu
M S, Jan L Y, Jan Y N: Asymmetric distribution of numb protein
during division of the sensory organ precursor cell confers
distinct fates to daughter cells. Cell. 76:477-491, 1994. [0311]
111. Berdnik D, Torok T, Gonzalez-Gaitan M, and Knoblich J A: The
endocytic protein .alpha.-adaptin is required for numb-mediated
asymmetric cell division in Drosophila. Dev Cell. 3:221-231, 2002.
[0312] 112. Guo M, Jan L Y, Jan Y N: Control of daughter cell fates
during asymmetric division: interaction of Numb and Notch. Neuron.
17:27-41, 1996. [0313] 113. Zhu J, Zhang Y, Joe G J, Pompetti R,
Emerson S G: NF-Ya activates multiple hematopoietic stem cell (HSC)
regulatory genes and promotes HSC self-renewal. Proc Natl Acad Sci
USA. 102:11728-11733, 2005. [0314] 114. Vercauteren S M, Sutherland
H J: Constitutively active Notch4 promotes early human
hematopoietic progenitor cell maintenance while inhibiting
differentiation and causes lymphoid abnormalities in vivo. Blood.
104:2315-2322, 2004. [0315] 115. Gustafsson M V, Zheng X, Pereira
T, Gradin K, Jin S, Lundkvist J, Ruas J L, Poellinger L, Lendahl U,
Bondesson M: Hypoxia requires notch signaling to maintain the
undifferentiated cell state. Dev Cell. 9:575-576, 2005. [0316] 116.
Boni A, Nascimbene A, Urbanek K, Delucchi F, Gonzalez A, Siggins R,
Amano K, Yasuzawa-Amano S, Ojaimi C, Rota M, Hosoda T, Anversa P,
Kajstura J, Leri A: Notch1 receptor enhances myocyte
differentiation of cardiac progenitor cells and myocardial
regeneration after infarction. Submitted, 2007. [0317] 117. Smith G
H: Label-retaining epithelial cells in mouse mammary gland divide
asymmetrically and retain their template DNA strands. Development.
132:681-687, 2004. [0318] 118. Karpowicz P, Morshead C, Kam A,
Jervis E, Ramunas J, Cheng V, van der Kooy D: Support for the
immortal strand hypothesis: neural stem cells partition DNA
asymmetrically in vitro. J Cell Biol. 170:721-732, 2005. [0319]
119. Merok J R, Lansita J A, Tunstead J R, Sherley J L:
Cosegregation of chromosomes containing immortal DNA strands in
cells that cycle with asymmetric stem cell kinetics. Cancer Res.
62:6791-6795, 2002. [0320] 120. Cairns J: Mutation selection and
the natural history of cancer. Nature. 255:197-200, 1975. [0321]
121. Kiel M J, He S, Ashkenazi R, Gentry S N, Teta M, Kushner J A,
Jackson T L, Morrison S J: Haematopoietic stem cells do not
asymmetrically segregate chromosomes or retain BrdU. Nature. Epub
ahead of print, 2007. [0322] 122. Zhang J, Niu C, Ye L, Huang H, He
X, Tong W G, Ross J, Haug J, Johnson T, Feng J Q, Harris S,
Wiedemann L M, Mishina Y, Li L: Identification of the
haematopoietic stem cell niche and control of the niche size.
Nature. 425:836-841, 2003. [0323] 123. Armakolas A, Klar A J: Cell
type regulates selective segregation of mouse chromosome 7 DNA
strands in mitosis. Science. 311:1146-1149, 2006. [0324] 124. Watt
F M, Hogan B L: Out of Eden: stem cells and their niches. Science
287:1427-1438, 2000. [0325] 125. Potten C S, Owen G, Booth D:
Intestinal stem cells protect their genome by selective segregation
of template DNA strands. J Cell Sci. 115:2381-2388, 2002. [0326]
126. Potten C S, Hume W J, Reid P, Cairns J: The segregation of DNA
in epithelial stem cells. Cell. 15:899-906, 1978. [0327] 127. Welm
B E, Tepera S B, Venezia T, Graubert T A, Rosen J M, Goodell M A:
Sca-1(pos) cells in the mouse mammary gland represent an enriched
progenitor cell population. Dev Biol. 245:42-56, 2002. [0328] 128.
Shinin V, Gayraud-Morel B, Gomes D, Tajbakhsh S: Asymmetric
division and cosegregation of template DNA strands in adult muscle
satellite cells. Nat Cell Biol. 8:677-687, 2006. [0329] 129.
Lansdorp P M: Immortal strands? Give me a break. Cell.
129:1244-1247, 2007. [0330] 130. Kawamoto A, Murayama T, Kusano K,
IiM, Tkebuchava T, Shintani S, Iwakura A, Johnson I, von Samson P,
Hanley A, Gavin M, Curry C, Silver M, Ma H, Kearney M, Losordo D W:
Synergistic effect of bone marrow mobilization and vascular
endothelial growth factor-2 gene therapy in myocardial ischemia.
Circulation. 110:1398-1405, 2004. [0331] 131. Ii M, Nishimua H,
Iwakura A, Wecker A, Eaton E, Asahara T, Losordo D W: Endothelial
progenitor cells are rapidly recruited to myocardium and mediate
protective effect of ischemic preconditioning via "imported" nitric
oxide synthase activity. Circulation. 111:1114-1120, 2005. [0332]
132. Asai J, Takenaka H, Kusano K F, Ii M, Luedemann C, Curry C,
Eaton E, Iwakura A, Tsutsumi Y, Hamada H, Kishimoto S, Thorne T,
Kishore R, Losordo D W: Topical sonic hedgehog gene therapy
accelerates wound healing in diabetes by enhancing endothelial
progenitor cell-mediated microvascular remodeling. Circulation.
113:2413-2424, 2006. [0333] 133. Lockhart D J, Dong H, Byrne M C,
Follettie M T, Gallo M V, Chee M S, Mittmann M, Wang C, Kobayashi
M, Horton H, Brown E L: Expression monitoring by hybridization to
high-density oligonucleotide arrays. Nat Biotechnol. 14:1675-1680,
1996. [0334] 134. Li C, Wong W H: Model-based analysis of
oligonucleotide arrays: expression index computation and outlier
detection. Proc Natl Acad Sci USA. 98:31-36, 2001. [0335] 135.
Bhattacharya B, Miura T, Brandenberger R, Mejido J, Luo Y, Yang A
X, Joshi B H, Ginis I, Thies R S, Amit M, Lyons I, Condie B G,
Itskovitz-Eldor J, Rao M S, Puri R K: Gene expression in human
embryonic stem cell lines: unique molecular signature. Blood.
103:2956-2964, 2004. [0336] 136. Bruno L, Hoffmann R, McBlane F,
Brown J, Gupta R, Joshi C, Pearson S, Seidl T, Heyworth C, Enver T:
Molecular signatures of self-renewal, differentiation, and lineage
choice in multipotential hemopoietic progenitor cells in vitro. Mol
Cell Biol. 24:741-756, 2004. [0337] 137. Ivanova N B, Dimos J T,
Schaniel C, Hackney J A, Moore K A, Lemischka I R: A stem cell
molecular signature. Science. 298:601-604, 2002. [0338] 138.
Ramalho-Santos M S, Yoon Y, Matsuzaki R, Mulligan C, Melton D A:
"Stemness" transcriptional profiling of embryonic and adult stem
cells. Science. 298:597-600, 2002. [0339] 139. Sturn A, Quackenbush
J, Trajanoski Z: Genesis: cluster analysis of microarray data.
Bioinformatics. 18:207-208, 2002. [0340] 140. Kuroda T, Tada M,
Kubota H, Kimura H, Hatano S Y, Suemori H, Nakatsuji N, Tada T:
Octamer and Sox elements are required for transcriptional cis
regulation of Nanog gene expression. Mol Cell Biol. 25:2475-2485,
2005. [0341] 141. Rodda D J, Chew J L, Lim L H, Loh Y H, Wang B, Ng
H H, Robson P: Transcriptional regulation of nanog by OCT4 and
SOX2. J Biol Chem. 280:24731-24737, 2005. [0342] 142. Player A,
Wang Y, Bhattacharya B, Rao M, Puri R K, Kawasaki E S: Comparisons
between transcriptional regulation and RNA expression in human
embryonic stem cell lines. Stem Cells Dev. 15:315-323, 2006. [0343]
143. Yoon Y S, Wecker A, Heyd L, Park J S, Tkebuchava T, Kusano K,
Hanley A, Scadova H, Qin G, Cha D H, Johnson K L, Aikawa R, Asahara
T, Losordo D W: Clonally expanded novel multipotent stem cells from
human bone marrow regenerate myocardium after myocardial
infarction. J Clin Invest. 115:326-338, 2005. [0344] 144. Murasawa
S, Kawamoto A, Horii M, Nakamori S, Asahara T: Niche-dependent
translineage commitment of endothelial progenitor cells, not cell
fusion in general, into myocardial lineage cells. Arterioscler
Thromb Vasc Biol. 25:1388-1394, 2005. [0345] 145. Rota M, Kajstura
J, Hosoda T, Bearzi C, Vitale S, Esposito G, Iaffaldano G,
Padin-Ireguas M E, Gonzalez A, Rizzi R, Small N, Muraski J, Alvarez
R, Chen X, Urbanek K, Bolli R, Houser S R, Leri A, Sussman M A,
Anversa P: Bone marrow cells adopt the cardiomyogenic fate in vivo.
Proc Natl Acad Sci USA. In press, 2007. [0346] 146. Jessup M,
Brozena S: Heart failure. N Engl J Med. 348:2007-2018, 2003. [0347]
147. Courville K A, Ventura H: Hypertension and heart failure:
diagnosis and management. Curr Hypertens Rep. 8:185-190, 2006.
[0348] 148. Anversa P, Olivetti G: Cellular basis of physiological
and pathological myocardial growth. Handbook of Physiology: the
Cardiovascular System. The Heart. New York: Oxford University
Press. pp. 75-144, 2002. [0349] 149. Rakusan K: Assessment of
cardiac growth of the heart in health and disease. New York: Raven
Press. pp. 25-40, 1984. [0350] 150. Anversa P, Ricci R, Olivetti G:
Quantitative structural analysis of the myocardium during
physiologic growth and induced cardiac hypertrophy: a review. J Am
Coll Cardiol. 7:1140-1149, 1986.
[0351] 151. Anversa P, Kajstura J: Ventricular myocytes are not
terminally differentiated in the adult mammalian heart. Circ Res.
13:1-14, 1998. [0352] 152. Rakusan K: Cardiac growth maturation and
aging. Growth of the heart in health and disease. New York: Raven
Press. pp 131-64, 1984. [0353] 153. MacLellan W R, Schneider M D:
Death by design. Programmed cell death in cardiovascular biology
and disease. Circ Res. 81:137-144, 1997. [0354] 154. Anversa P,
Leri A, Beltrami C A, Guerra S, Kajstura J: Myocyte death and
growth in the failing heart. Lab Invest. 78:767-786, 1998. [0355]
155. Haunstetter A, Izumo S: Apoptosis: basic mechanisms and
implications for cardiovascular disease. Circ Res. 82:1111-1129,
1998. [0356] 156. Kang P M, Izumo S: Apoptosis and heart failure: A
critical review of literature. Circ Res. 86:1107-1113, 2000. [0357]
157. Heineke J, Molkentin J D: Regulation of cardiac hypertrophy by
intracellular signaling pathways. Mol Cell Biol. 7:589-600, 2006.
[0358] 158. Wijns W, Vatner S F, Camici P G: Hibernating
myocardium. N Engl J Med. 339:173-181, 1998. [0359] 159. Hsieh P C
H, Segers V F M, Davis M E, MacGillivray C, Gannon J, Molkentin J
D, Robbins J, Lee R T: Evidence from a genetic fate-mapping study
that stem cells refresh adult mammalian cardiomyocytes after
injury. Nature Med. 13:970-974, 2007. [0360] 160. Adams J W, Sakata
Y, Davis M G, Sah V P, Wang Y, Liggett S B, Chien K R, Brown J H,
Dorn G W: Enhanced G.crclbar.q signaling: A common pathway mediates
cardiac hypertrophy and apoptotic heart failure. Proc Natl Acad Sci
USA. 95:10140-10145, 1998. [0361] 161. Spees J L, Olson S D,
Whitney M, Prockop D J: Mitochondrial transfer between cells can
rescue aerobic respiration. Proc Natl Acad Sci USA. 103:1283-1288,
2006. [0362] 162. Moore K A, Lemischka I R: Stem cells and their
niches. Science. 311:1880-1885, 2006. [0363] 163. Yin T, Li L: The
stem cell niches in bone. J Clin Invest. 116:1195-1201, 2006.
[0364] 164. Scadden D T: The stem-cell niche as an entity of
action. Nature. 441:1075-1079, 2006. [0365] 165. Blau H M,
Brazelton T R, Weimann J M: The evolving concept of a stem cell:
entity or function? Cell. 105:829-841, 2001. [0366] 166. Spradling
A, Drummond-Barbosa D, Kai T: Stem cells find their niche. Nature
414: 98-104, 2001. [0367] 167. Lin H: The stem cell niche theory:
lessons from flies. Nat Rev Genet. 3:931-940, 2002. [0368] 168.
Song X, Zhu C H, Doan C, Xie T: Germline stem cells anchored by
adherens junctions in the Drosophila ovary niches. Science.
296:1855-1857, 2002. [0369] 169. Chen D, McKearin D: Dpp signaling
silences bam transcription directly to establish asymmetric
divisions of germline stem cells. Curr Biol. 13:1786-1791, 2003.
[0370] 170. Zhu C H, Xie T: Clonal expansion of ovarian germline
stem cells during niche formation in Drosophila. Development.
130:2579-2588, 2003. [0371] 171. Song X, Xie T:
DE-cadherin-mediated cell adhesion is essential for maintaining
somatic stem cell in the Drosophila ovary. Proc Natl Acad Sci USA.
99:14813-14818, 2002. [0372] 172. Kulessa H, Turk G, Hogan B L:
Inhibition of Bmp signaling affects growth and differentiation in
the anagen hair follicle. EMBO J. 19:6664-6674, 2000. [0373] 173.
Stappenbeck T S, Mills J C, Gordon J I: Molecular features of adult
mouse small intestinal epithelial progenitors. Proc Natl Acad Sci
USA. 100:1004-1009, 2003. [0374] 174. Fuchs E, Tumbar T, Guasch G:
Socializing with the neighbors: stem cells and their niche. Cell.
116:769-778, 2004. [0375] 175. Torella D, Rota M, Nurzynska D,
Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A,
Sussman M A, Urbanek K, Nadal-Ginard B, Kajstura J, Anversa P, Leri
A: Cardiac stem cell and myocyte aging, heart failure and IGF-1
overexpression. Circ Res. 94:514-524, 2004. [0376] 176.
Perez-Moreno M, Jamora C, Fuchs E: Sticky business: orchestrating
cellular signals at adherens junctions. Cell. 121:535-548, 2003.
[0377] 177. Goldberg G S, Valiunas V, Bring P R: Selective
permeability of gap junction channels. Biochim Biophys Acta.
1662:96-101, 2004. [0378] 178. Mitsunaga K, Araki K, Mizusaki H,
Morohashi K, Haruna K, Nakagata N, Giguere V, Yamamura K, Abe K:
Loss of PGC-specific expression of the orphan nuclear receptor
ERR-beta results in reduction of germ cell number in mouse embryos.
Mech Dev. 121: 237-246, 2004. [0379] 179. Pevny L, Placzek M: SOX
genes and neural progenitor identity. Curr Opin Neurobiol. 15:7-13,
2005. [0380] 180. Loh Y H, Wu Q, Chew J L, Vega V B, Zhang W, Chen
X, Bourque G, George J, Leong B, Liu J, Wong K Y, Sung K W, Lee C
W, Zhao X D, Chiu K P, Lipovich L, Kuznetsov V A, Robson P, Stanton
L W, Wei C L, Ruan Y, Lim B, Ng H H: The Oct4 and Nanog
transcription network regulates pluripotency in mouse embryonic
stem cells. Nat Genet. 38:431-440, 2006. [0381] 181. Ivanova N,
Dobrin R, Lu R, Kotenko J, Levorse J, DeCoste C, Xenia S, Lun Y,
Lemischka I R: Dissecting self-renewal in stem cells with RNA
interference. Nature. 444: 533-538, 2006. [0382] 182. Cairns L A,
Moroni E, Levantini E, Giorgetti A, Klinger F G, Ronzoni S,
Tatangelo L, Tiveron C, De Felici M, Dolci S, Magli M C, Giglioni
B, Ottolenghi S: Kit regulatory elements required for expression in
developing hematopoietic and germ cell lineages. Blood.
102:3954-3962, 2003. [0383] 183. Tillmanns J, Rota M, Hosoda T,
Rotatori F, DeAngelis A, Amano K, Amano S, LeCapitaine N, Esposito
G, Loredo M, Misao Y, Vitale S, Bearzi C, Urbanek K, Bolli R, Leri
A, Kajstura J, Anversa P: Formation of large coronary arteries by
cardiac stem cells: a biological bypass. Proc Natl Acad Sci USA. In
revision, 2007. [0384] 184. Thyberg J, Hedin U, Sjolund M, Palmberg
L, Bottger B A: Regulation of differentiated properties and
proliferation of arterial smooth muscle cells. Arteriosclerosis.
10:966-990, 1990. [0385] 185. Brown K E, Kindy M S, Sonenshein G E:
Expression of the c-myb proto-oncogene in bovine vascular smooth
muscle cells. J Biol Chem. 267:4625-4630, 1992. [0386] 186. Wang J,
Chen D, Walsh K: Regulation of Cdk2 activity in proliferating
versus contact-inhibited endothelial cells: The role of the p27
cyclin kinase inhibitor. Circulation. 94: I-524, 1996. [0387] 187.
Hsiao R, Sharma H W, Ramakrishnan S, Keith E, Narayanan R:
Telomerase activity in normal endothelial cells. Anticancer Res.
17:827-832, 1997. [0388] 188. Spyridopoulos I, Andres V: Control of
vascular smooth muscle and endothelial cell proliferation and its
implication in cardiovascular disease. Front Biosci. 1:d269-287,
1998. [0389] 189. Mahmud N, Devine S M, Weller K P, Parmar S,
Sturgeon C, Nelson M C, Hewett T, Hoffman R: The relative
quiescence of hematopoietic stem cells in nonhuman primates. Blood
97:3061-3068. [0390] 190. Savill N J: Mathematical models of
hierarchically structured cell populations under equilibrium with
application to the epidermis. Cell Prolif. 36:1-26, 2003. [0391]
191. Baserga R, Wiebel F: The cell cycle of mammalian cells. Int
Rev Exp Pathol. 7:1-30, 1969. [0392] 192. Chen S, Lechleicder R J:
Transforming growth factor-beta-induced differentiation of smooth
muscle from a neural crest stem cell line. Circ Res. 94:1195-1202.
[0393] 193. Urbich C, Heeschen C, Aicher A, Dernbach E, Zeiher A M,
Dimmeler S: Relevance of monocytic features for neovascularization
capacity of circulating endothelial progenitor cells. Circulation.
108:2511-2516, 2003. [0394] 194. Potente M, Urbich C, Sasaki K,
Hofmann W K, Heeschen C, Aicher A, Kollipara R, DePinho R A, Zeiher
A M, Dimmeler S: Involvement of Foxo transcription factors in
angiogenesis and postnatal neovascularization. J Clin Invest.
115:2382-2392, 2005. [0395] 195. Lamagna C, Bergers G: The bone
marrow constitutes a reservoir of pericyte progenitors. J Leukoc
Biol. 26:677-681, 2006. [0396] 196. Lindskog H, Athley E, Larsson
E, Lundin S, Hellstrom M, Lindahl P: New insights to vascular
smooth muscle cell and pericyte differentiation of mouse embryonic
stem cells in vitro. Arterioscler Thromb Vasc Biol. 26:1457-1464,
2006. [0397] 197. Berenson M L, Levine D M, Rindskopf D: Applied
statistics. Prentice Hall, Englewood Cliffs, N.J. pp. 557. [0398]
198. Souilhol C, Cormier S, Monet M, Vandormael-Pournin S, Joutel
A, Babinet C, Cohen-Tannoudji M: NAS transgenic mouse line allows
visualization of Notch pathway activity in vivo. Genesis.
44:277-286, 2006. [0399] 199. Dimmeler S, Zeiher A M, Schneider M
D: Unchain my heart: the scientific foundations of cardiac repair.
J Clin Invest. 115:572-583, 2005. [0400] 200. Rubart M, Field L J:
Cardiac regeneration: repopulating the heart. Annu Rev Physiol.
68:29-49, 2006. [0401] 201. Murry C E, Soonpaa M H, Reinecke H,
Nakajima H, Nakajima H O, Rubart M, Pasumarthi K B, Virag J I,
Bartelmez S H, Poppa V, Bradford G, Dowell J D, Willaims D A, Field
L J: Haematopoietic stem cells do not transdifferentiate into
cardiac myocytes in myocardial infarcts. Nature. 428:664-668, 2004.
[0402] 202. Nygren J M, Jovinge S, Breitbach M, Sawen P, Roll W,
Hescheler J, Taneera J, Fleishcmann B K, Jacobsen S E: Bone
marrow-derived hematopoietic cells generage cardomyocytes at a low
frequency through cell fusion, but not transdifferentaiation. Nat
Med. 10:494-501, 2004. [0403] 203. Balsam L B, Wagers A J,
Christensen J L, Kofidis T, Weissman I L, Robbins R C:
Haematopoietic stem cells adopt mature haematopoietic fates in
ischemic myocardium. Nature. 428:668-673, 2004. [0404] 204. Tosh D,
Slack J M: How cells change their phenotype. Nat Rev Mol Cell Biol.
3: 187-194, 2002. [0405] 205. Pomerantz J, Blau H M: Nuclear
reprogramming: a key to stem cell function in regenerative
medicine. Nat Cell Biol. 6:810-816, 2004. [0406] 206. Leri A,
Kajstura J, Anversa P: Identity deception: not a crime for a stem
cell. Physiology. 20:162-168, 2005. [0407] 207. Kajstura J, Leri A,
Bolli R, Anversa P: Endothelial progenitor cells:
neovascularization or more? J Mol Cell Cardiol. 40:1-8, 2006.
[0408] 208. Schroeder T, Fraser S T, Ogawa M, Nishikawa S, Oka C,
Bomkamm G V, Nishikawa S, Honjo T, Just U: Recombination signal
sequence-binding protein Jkappa alters mesodermal cell fate
decisions by suppressing cardiomyogenesis. Proc Natl Acad Sci USA.
100:4018-4023, 2003. [0409] 209. Han H, Tanigaki K, Yamamoto N,
Kuroda K, Yoshimoto M, Nakahata T, Ikuta K, Honjo T: Inducible gene
knockout of transcription factor recombination signal binding
protein-J reveals its essential role in T versus B lineage
decision. Int Immunol. 14:637-645, 2002. [0410] 210. Okada S,
Nakauchi H, Nagayoshi K, Nishikawa S, Nishikawa S, Miura Y, Suda T:
Enrichment and characterization of murine hematopoietic stem cells
that express c-kit molecule. Blood. 78:1706-1712, 1991. [0411] 211.
Orlic D, Fischer R, Nishikawa S, Nienhuis A W, Bodine D M:
Purification and characterization of heterogeneous pluripotent
hematopoietic stem cell populations expressing high levels of c-kit
receptor. Blood. 82:762-770, 1993. [0412] 212. Balazs A B, Fabian A
J, Esmon C T, Mulligan R C: Endothelial protein C receptor (CD201)
explicitly identifies hematopoietic stem cells in murine bone
marrow. Blood. 107:2317-2321, 2006. [0413] 213. Dumble M, Moore L,
Chambers S M, Geiger H, Van Zant G, Goodell M A, Donehower L A: The
impact of altered p53 dosage on hematopoietic stem cell dynamics
during aging. Blood. 109:1736-1742, 2007. [0414] 214. Kawada H,
Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Miyatake H, Muguruma Y,
Tsuboi K, Itabashi Y, Ikeda Y, Ogawa S, Okano H, Hotta T, Ando K,
Fukuda K: Nonhematopoietic mesenchymal stem cells can be mobilized
and differentiate into cardiomyocytes after myocardial infarction.
Blood. 104:3581-3587, 2004. [0415] 215: Kopan R: All good things
must come to an end: how is Notch signaling turned off? Sci STKE.
PE1, 1999. [0416] 216. Kopan R: Notch: a membrane-bound
transcription factor. J Cell Sci. 115:1095-1097, 2002. [0417] 217.
Iwatsubo T: The gamma-secretase complex: machinery for
intramembrane proteolysis. Curr Opin Neurobiol. 14:379-393, 2004.
[0418] 218. Quesenberry P J: The continuum model of marrow stem
cell regulation. Curr Opin Hematol. 13:216-221, 2006. [0419] 219.
Quesenberry P J, Colvin G A, Abedi M, Dooner G, Dooner M, Aliotta
J, Keaney P, Luo L, Demers D, Peterson A, Foster B, Greer D: The
stem cell continuum. Ann NY Acad Sci. 1044:228-235, 2005. [0420]
220. Lemischka I R: Microenvironmental regulation of hematopoietic
stem cells. Stem Cells. 15:63-68, 1997. [0421] 221. Hackney J A,
Charbord P, Brunk B P, Stoeckert C J, Lemischka I R, Moore K A: A
molecular profile of a hematopoietic stem cell niche. Proc Natl
Acad Sci USA. 99:13061-13066, 2002. [0422] 222. Juan G, Gruenwald
S, Darzynkiewicz Z: Phosphorylation of retinoblastoma
susceptibility gene protein assayed in individual lymphocytes
during their mitogenic stimulation. Exp Cell Res. 239:104-110,
1998. [0423] 223. Darzynkiewicz Z, Juan G, Traganos F: Cytometry of
cell cycle regulatory proteins. Prog Cell Cycle Res. 5:533-542,
2003. [0424] 224. Juan G, Darzynkiewicz Z: Detection of cyclins in
individual cells by flow and laser scanning cytometry. Methods Mol
Biol. 91:67-75, 1998. [0425] 225. Cheng L C, Tavazoie M, Doetsch F:
Stem cells: from epigenetics to microRNAs. Neuron. 46:363-367,
2005. [0426] 226. Ko M S, McLaren A: Epigenetics of germ cells,
stem cells, and early embryos. Dev Cell. 10:161-166, 2006. [0427]
227. Shivdasani R A: MicroRNAs: regulators of gene expression and
cell differentiation. Blood. 108:3646-3653, 2006. [0428] 228. Lee M
S, Jun D H, Hwang C I, Park S S, Kang J J, Park H S, Kim J, Kim J
H, Seo J S, Park W Y: Selection of neural differentiation-specific
genes by comparing profiles of random differentiation. Stem Cells.
24:1946-1955, 2006. [0429] 229. Fazel S, Cimini M, Chen L, Li S,
Angoulvant D, Fedak P, Verma S, Weisel R D, Keating A, Li R K:
Cardioprotective c-kit+ cells are from the bone marrow and regulate
the myocardial balance of angiogenic cytokines. J Clin Invest.
116:1865-1877, 2006. [0430] 230. Weissman I L, Anderson D J, Gage
F: Stem and progenitor cells: origins, phenotypes, lineage
commitments and transdifferentiations. Annu Rev Cell Dev Biol.
17:387-403, 2001. [0431] 231. Bhattacharya B, Miura T,
Brandenberger R, Mejido J, Luo Y, Yang A X, Joshi B H, Ginis I,
Thies R S, Amit M, Lyons I, Condie B G, Itskovitz-Eldor J, Rao M S,
Puri R K: Gene expression in human embryonic stem cell lines:
unique molecular signature. Blood. 103:2956-2964, 2004. [0432] 232.
Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk G J, Li Y,
Xu C, Fang R, Guegler K, Rao M S, Mandalam R, Lebkowski J, Stanton
L W: Transcriptome characterization elucidates signaling networks
that control human ES cell growth and differentiation. Nat
Biotechnol. 22:707-716, 2004. [0433] 233. Ginis I, Luo Y, Miura T,
Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter
M K, Itskovitz-Eldor J, Rao M S: Differences between human and
mouse embryonic stem cells. Dev Biol. 269:360-380, 2004. [0434]
234. Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P,
Stoeckert C, Aach J, Ansorge W, Ball C A, Causton H C, Gaasterland
T, Glenisson P, Holstege F C, Kim I F, Markowitz V, Matese J C,
Parkinson H, Robinson A, Sarkans U, Schulze-Kremer S, Stewart J,
Taylor R, Vilo J, Vingron M: Minimum information about a microarray
experiment (MIAME)-toward standards for microarray data. Nat Genet.
29:365-371, 2001.
[0435] 235. Draghici S, Khatri P, Bhavsar P, Shah A, Krawetz S A,
Tainsky M A: Onto-Tools, the toolkit of the modern biologist:
Onto-Express, Onto-Compare, Onto-Design and Onto-Translate. Nucleic
Acids Res. 31:3775-3781, 2003. [0436] 236. Olson E N, Schneider M
D: Sizing up the heart: development redux in disease. Genes Dev.
17:1937-1956, 2003. [0437] 237. Chien K R, Olson E N: Converging
pathways and principles in heart development and disease: CV@CSH.
Cell. 110:153-162, 2002. [0438] 238. Grossman W, Jones D, McLaurin
L P: Wall stress and patterns of hypertrophy in the human left
ventricle. J Clin Invest. 56:56-64, 1975. [0439] 239. Blake J,
Devereux R B, Herrold E M, Jason M, Fisher J, Borer J S, Laragh J
H: Relation of concentric left ventricular hypertrophy and
extracardiac target organ damage to supranormal left ventricular
performance in established essential hypertension. Am J Cardiol.
62:246-252, 1988. [0440] 240. Devereux R B, Roman M J, Palmieri V,
Okin P M, Boman K, Gerdts E, Nieminen M S, Papademetriou V,
Wachtell K, Dahlof B: Left ventricular wall stresses and wall
stress-mass-heart rate products in hypertensive patients with
electrocardiographic left ventricular hypertrophy: the LIFE study.
J Hypertens. 18:1129-1138, 2000. [0441] 241. Olivetti G, Ricci R,
Anversa P: Hyperplasia of myocyte nuclei in long-term cardiac
hypertrophy in rats. J Clin Invest. 80:1818-1821, 1987. [0442] 242.
Olivetti G, Ricci R, Lagrasta C, Manica E, Sonnenblick E H, Anversa
P: Cellular basis of wall remodeling in long-term pressure
overload-induced right ventricular hypertrophy in rats. Circ Res.
63:648-657, 1988. [0443] 243. Olivetti G, Melissari M, Balbi T,
Quaini F, Cigola E, Sonnenblick E H, Anversa P: Myocyte cellular
hypertrophy is responsible for ventricular remodeling in the
hypertrophied heart of middle aged individuals in the absence of
cardiac failure. Cardiovasc Res. 28:1199-1208, 1994. [0444] 244.
Haider A W, Larson M G, Benjamin E J, Levy D: Increased left
ventricular mass and hypertrophy are associated with increased risk
for sudden death. J Am Coll Cardiol. 32: 1454-1459, 1998. [0445]
245. Berenji K, Drazner M H, Rothermel B A, Hill J A: Does
load-induced ventricular hypertrophy progress to systolic heart
failure? Am J Physiol Heart. 289:H8-H16, 2005. [0446] 246. Capasso
J M, Palackal T, Olivetti G, Anversa P: Left ventricular failure
induced by long-term hypertension in rats. Circ Res. 66:1400-1412,
1990. [0447] 247. Anversa P, Palackal T, Sonnenblick E H, Olivetti
G, Capasso J M: Hypertensive cardiomyopathy. Myocyte nuclei
hyperplasia in the mammalian rat heart. J Clin Invest. 85:994-997,
1990. [0448] 248. Olivetti G, Melissari M, Balbi T, Quaini F,
Sonnenblick E H, Anversa P: Myocyte nuclear and possible cellular
hyperplasia contribute to ventricular remodeling in the
hypertrophic senescent heart in humans. J Am Coll Cardiol.
24:140-149, 1994. [0449] 249. Gradman A H, Alfayoumi F: From left
ventricular hypertrophy to congestive heart failure: management of
hypertensive heart disease. Prog Cardiovasc Dis. 48:326-341, 2006.
[0450] 250. Wiesel P, Mazolai L, Nussberger J, Pedrazzini T:
Two-kidney, one clip and one-kidney, one clip hypertension in mice.
Hypertension. 29:1025-1030, 1997. [0451] 251. Yang X P, Liu Y H,
Rhaleb N E, Kurihara N, Kim H E, Carretero O A: Echocardiographic
assessment of cardiac function in conscious and anesthetized mice.
Am J Physiol. 277:H1967-H1974, 1999. [0452] 252. Murat A, Pellieux
C, Brunner H R, Pedrazzini T: Calcineurin blockade prevents cardiac
mitogen-activated protein kinase activation and hypertrophy in
renovascular hypertension. J Biol Chem. 275:40867-40873, 2000.
[0453] 253. Mazzolai L, Pedrazzini T, Nicoud F, Gabbiani G, Brunner
H R, Nussberger J: Increased cardiac angiotensin II levels induce
right and left ventricular hypertrophy in normotensive mice.
Hypertension. 35:985-991, 2000. [0454] 254. Anversa P, Capasso J M:
Loss of intermediate-sized coronary arteries and capillary
proliferation after left ventricular failure in rats. Am J Physiol.
260:H1552-1560, 1991. [0455] 255. Capasso J M, Puntillo E, Halpryn
B, Olivetti G, Li P, Anversa P: Amelioration of effects of
hypertension and diabetes on myocardium by cardiac glycoside. Am J
Physiol. 262:H734-742, 1992. [0456] 256. Anversa P, Li P, Malhotra
A, Zhang X, Herman M V, Capasso J M: Effects of hypertension and
coronary constriction on cardiac function, morphology and
contractile proteins in rats. Am J Physiol. 265:H713-724, 1993.
[0457] 257. Li P, Zhang X, Capasso J M, Meggs L G, Sonnenblick E H,
Anversa P: Myocyte loss and left ventricular failure characterize
the long term effects of coronary artery narrowing or renal
hypertension in rats. Cardiovasc Res. 27:1066-1075, 1993. [0458]
258. Rota M, LeCapitaine N, Hosoda T, Boni A, De Angelis A,
Padin-Iruegas M E, Esposito G, Vitale S, Urbanek K, Casarsa C,
Giorgio M, Luscher T F, Pelicci P G, Anversa P, Leri A, Kajstura J:
Diabetes promotes cardiac stem cell aging and heart failure, which
are prevented by deletion of the p66shc gene. Circ Res. 99:42-52,
2006. [0459] 259. Beltrami C A, Di Loreto C, Finato N, Rocco M,
Artico D, Cigola E, Gambert S R, Olivetti G, Kajstura J, Anversa P.
Proliferating cell nuclear antigen (PCNA), DNA synthesis and
mitosis in myocytes following cardiac transplantation in man. J Mol
Cell Cardiol. 29: 2789-2802, 1997. [0460] 260. Kajstura J, Leri A,
Finato N, Di Loreto C, Beltrami C A, Anversa P. Myocyte
proliferation in end-stage cardiac failure in humans. Proc Natl
Acad Sci USA. 95: 8801-8805, 1998 [0461] 261. Anversa P, Leri A,
Beltrami C A, Guerra S, Kajstura J. Myocyte death and growth in the
failing heart. Lab Invest. 1998; 78:767-86. [0462] 262. Beltrami A
P, Urbanek K, Kajstura J, Yan S M, Finato N, Bussani R,
Nadal-Ginard B, Silvestri F, Leri A, Beltrami C A, Anversa P.
Evidence that human cardiac myocytes divide after myocardial
infarction. N Eng J Med. 2001; 344:1750-7. [0463] 263. Anversa P,
Nadal-Ginard B. Myocyte renewal and ventricular remodeling. Nature.
2002; 415:240-3. [0464] 264. Anversa P, Leri A, Kajstura J,
Nadal-Ginard B. Myocyte growth and cardiac repair. J Mol Cell
Cardiol. 2002; 34:91-105. [0465] 265. Anversa P, Leri A. Myocardial
regeneration. In: Zipes, Libby, Bonow, Braunwald eds. Braunwald's
Heart Disease A Textbook of Cardiovascular Medicine 7.sup.th
Edition. Elsevier Saunders, Philadelphia, Pa. 2005; 1911-1923.
[0466] 266. Leri A, Kajstura J, Anversa P. Cardiac stem cells and
mechanisms of myocardial regeneration. Physiol Rev. 2005;
85:1373-416. [0467] 267. Urbanek K, Torella D, Sheikh F, De Angelis
A, Nurzynska D, Silvestri F, Beltrami C A, Bussani R, Beltrami A P,
Quaini F, Bolli R, Leri A, Kajstura J, Anversa P. Myocardial
regeneration by activation of multipotent cardiac stem cells in
ischemic heart failure. Proc Natl Acad Sci USA. 2005; 102:8692-7.
[0468] 268. Anversa P, Kajstura J, Leri A, Bolli R. Life and death
of cardiac stem cells: a paradigm shift in cardiac biology.
Circulation. 2006; 113:1451-63. [0469] 269. Anversa P, Leri A,
Kajstura J. Cardiac regeneration. J Am Coll Cardiol. 2006;
47:1769-76. [0470] 270. Anversa P, Leri A, Rota M, Bearzi C,
Urbanek K, Kajstura J, Bolli R. Stem Cells, Myocardial regeneration
and methodological artifacts. Stem Cells. 25: 589-601, 2007. [0471]
271.13. Wicker P, Tarazi R C. Coronary blood flow in left
ventricular hypertrophy: a review of experimental data. Eur Heart
J. 1982; 3 Suppl A: 111-8. [0472] 272. Harrison D G, Barnes D H,
Hiratzka L F, Eastham C L, Kerber R E, Marcus M L. The effect of
cardiac hypertrophy on the coronary collateral circulation.
Circulation. 1985; 71:1135-45. [0473] 273. Bache R J. Effects of
hypertrophy on the coronary circulation. Prog Cardiovasc Dis. 1988:
30:403-40. [0474] 274. Karam R, Healy B P, Wicker P. Coronary
reserve is depressed in postmyocardial infarction reactive cardiac
hypertrophy. Circulation. 1990; 81:238-46. [0475] 275. Myocardial
reperfusion imaging: basic principals and clinical applications. Am
J Card Imaging. 1993; 7:11-23. [0476] 276. Giordano A, Trani C,
Lombardo A, Maseri A. Detection of hibernated myocardium using
intracoronary technetium-99m-sestamibi. Q J Nucl Med. 1997;
41:46-50. [0477] 277. Hansen C L, Rastogi A, Sangrigoli R. On
myocardial perfusion, metabolism, and viability. J Nucl Cardiol.
1998; 502-5. [0478] 278. Mari C, Strauss W H. Detection and
characterization of hibernating myocardium. Nucl Med Comun. 2002;
23:311-22. [0479] 279. Chin B B, Esposito G, Kraitchman D L.
Myocardial contractile reserve and perfusion defect severity with
rest and stress dobutamine (99m)Tc-sestamibi SPECT in canine
stunning and subendocardial infarction. J Nucl Med. 2002;
43:540-50. [0480] 280. Paeng J C, Lee D S, Cheon G J, Kim K B, Yeo
J S, Chung J K, Lee M C. Consideration of perfusion reserve in
viability assessment by myocardial TI-201 rest-redistribution
SPECT: a quantitative study with dual-isotope SPECT. J Nucl
Cardiol. 2002; 9:68-74. [0481] 281. Sciagra R, Leoncini M, Mennuti
A, Dabizzi R P, Pupi A. Classification of ischemic dysfunctional
myocardium combining perfusion quantification and contractile
reserve evaluation using nitrate-enhanced gated single photon
emission computed tomography with dobutamine test. Q J Nucl Med Mol
Imaging. 2004; 48:4-11. [0482] 282. Anversa P and Olivetti G.
Cellular basis of physiological and pathological myocardial growth.
In: Handbook of Physiology. The Cardiovascular System. The Heart.
Bethesda, Md., 2002, sect. 2 chapter 2:75-144. [0483] 283.
Lefkowitz R J, Caron M G, Stiles G L. Mechanisms of
membrane-receptor regulation. Biochemical, physiological, and
clinical insights derived from studies of the adrenergic receptors.
N Engl J Med. 1984; 310:1570-9. [0484] 284. Schrier R W, Abraham W
T. Hormones and hemodynamics in heart failure. N Engl J Med. 1999;
341:577-85. [0485] 285. I, Ojamaa K. Thyroid hormone and the
cardiovascular system. N Engl J Med 2001; 344:501-9. [0486] 286.
Weber K T. Aldosterone in congestive heart failure. N Engl J Med.
2001; 345:1689-97. [0487] 287. Jessup M, Brozena S. Heart failure.
N Engl J Med. 2003; 348:2007-18. [0488] 288. Aurigemma G P, Gaasch
W H. Clinical practice. Diastolic heart failure. N Engl J Med.
2004; 351:1097-1105. [0489] 289. www.americanheart.org/statistics
[0490] 290. Libby P, Aikawa M. Stabilization of atherosclerotic
plaques: new mechanisms and clinical targets. Nat Med. 2002;
8:1257-62. [0491] 291. Libby P, Theroux P. Pathophysiology of
coronary artery disease. Circulation. 2005; 111:3481-8. [0492] 292.
Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med.
1999; 340:115-26. [0493] 293. Hansson G K. Inflammation,
atherosclerosis, and coronary artery disease. N Engl J Med. 2005;
352:1685-95. [0494] 294. Weber C. Platelets and chemokines in
atherosclerosis: partners in crime. Circ Res. 2005; 96:612-6.
[0495] 295. Libby P, Ridker R. Inflammation and atherothrombosis
from population biology and bench research to clinical practice. J
Am Coll Cardiol. 2006; 48:A33-46. [0496] 296. Croce K, Libby P.
Intertwining of thrombosis and inflammation in atherosclerosis.
Curr Opin Hematol. 2007; 14:55-61. [0497] 297. Maseri A, Fuster V.
Is there a vulnerable plaque? Circulation. 2003; 107:2076-71.
[0498] 298. Slager C J, Wentzel J J, Gijsen F J, thury A, van der
Wal A C, Schaar J A, Serruys P W. The role of shear stress in the
destabilization of vulnerable plaques and related therapeutic
implications. Nat Clin Pract Cardiovasc Med. 2005; 2:456-64. [0499]
299. Fuster V, Moreno P R, Fayad Z A, Corti R, Badimon J J.
Atherothrombosis and high-risk plaque: part I: evolving concepts. J
Am Coll Cardiol. 2005; 46:937-54. [0500] 300. Kolodgie F D, Burke A
P, Farb A, Gold H K, Yuan J, Narula J, Finn A V, Virmani R. the
thin-cap fibroatheroma: a type of vulnerable plaque: the major
precursor lesion to acute coronary syndromes. Curr Opin Cardiol.
2001; 16:285-92. [0501] 301. Linke A, Muller P, Nurzynska D,
Casarsa C, Torella D, Nascimbene A, Castaldo C, Cascapera S, Bohm
M, Quaini F, Urbanek K, Leri A, Hintze T H, Kajstura J, Anversa P.
Stem cells in the dog heart are self-renewing, clonogenic, and
multipotent and regenerate infracted myocardium, improving cardiac
function. Proc Natl Acad Sci USA. 2005; 102:8966-8971. [0502] 302.
Melton D A, Cowen C. "Stemness": Definitions, Criteria, and
Standards. In: Lanza R, Blau H, Melton D, Moore M, Thomas E D,
Verfaillie C, Weissman I, West M. eds. Handbook of Stem Cells
Volume 2. Adult and Fetal Elsevier Academic Press. 2004;
136:xxv-xxx. [0503] 303. Verfaillie C M. "Adult" stem cells: tissue
specific or not? In: Lanza R, Blau H, Melton D, Moore M, Thomas E
D, Verfaillie C, Weissman I, West M. eds. Handbook of Stem Cells.
Volume 2. Adult and Fetal Stem Cells. Elsvier Academic Press. 2004;
137:13-20. [0504] 304. Bearzi C, Rota M, Hosoda T, Tillmanns J,
Nascimbene A, De Angelis A, Yasuzawa-Amano S, Trofimova I, Siggins
R W, Cascapera S, Beltrami A P, Zias E, Quaini F, Urbanek K,
Michler R E, Bolli R, Kajstura J, Leri A, Anversa P: Human cardiac
stem cells. Proc Natl Acad Sci USA. 104: 14068-14073, 2007. [0505]
305. Fehling H J, Lacaud G, Kubo A, Kennedy M, Robertson S, Keller
G and Kouskoff V. Tracking mesoderm induction and its specification
to the hemangioblast during embryonic stem cell differentiation.
Development. 2003; 130:4217-4227. [0506] 306. Kouskoff V, Lacaud G,
Schwantz S, Fehling H J and Keller G. Sequential development of
hematopoietic and cardiac mesoderm during embryonic stem cell
differentiation. Proc Natl Acad Sci USA. 2005; 102:13170-13175.
[0507] 307. Carmeliet P. Angiogenesis in life, disease and
medicine. Nature. 2005; 438:932-936. [0508] 308. Coultas L,
Chawengsaksophak K and Rossant J. Endothelial cells and VEGF in
vascular development. Nature. 2005; 438:937-945. [0509] 309.
Kattman S J, Huber T L, Keller G M. Multipotent Flk-1+
cardiovascular progenitor cells give rise to the cardiomyocyte,
endothelial, and vascular smooth muscle lineages. Developmental
Cell. 2006; 11:723-732. [0510] 310.52 Ferrarini M, Arsic N, Recchia
F A, Zentilin L, Zacchigna S, Xu X, Linke A, Giacca M, Hintze T H.
Adeno-associated virus-mediated transduction of VEGF165 improves
cardiac tissue viability and functional recovery after permanent
coronary occlusion in conscious dogs. Circ Res. 14; 98(7):954-61,
2006 [0511] 311. Beltrami A P, Barlucchi L, Torella D, Baker M,
Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri
A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells
are multipotent and support myocardial regeneration. Cell 114:1-20,
2003. [0512] 312. Bearzi C, Muller P, Amano K, Tang X-L, Loredo M,
Mosna F, Gatti A, Esposito G, Leri A, Kajstura J, Anversa P,
Rimoldi O, Bolli R: Identification and characterization of cardiac
stem cells in the pig heart. Circulation. 114: II-125, 2006. [0513]
313. Bolli R, Jneic H, Tang X-L, Rimoldi O, Mosna F, Loredo M,
Gatti A, Kajstura J, Leri A, Bearzi C, Abdel-Latit A, Anversa P.
Intracoronary administration of cardiac stem cells improves cardiac
function in pigs with old infarction.
Circulation. 2006; 114:II-239. [0514] 314. Reya T, Morrison S J,
Clarke M F, Weissman I L. Stem cells, cancer, and cancer stem
cells. Nature. 2001; 414:105-11. [0515] 315. Fuchs E, Raghavan S.
Getting under the skin of epidermal morphogenesis. Nat Rev Genet.
2002; 3:199-209. [0516] 316. Alonso L, Fuhs E. Stem cells of the
skin epithelium. Proc Natl Acad Sci USA. 2003; 100 Suppl:11830-5.
[0517] 317. Lapidot T, Petie I. Current understanding of stem cell
mobilization: the roles of chemokines, proteolytic enzymes,
adhesion molecules, cytokines, and stromal cells. Exp Hematol.
2002; 30:973-981. [0518] 318. Quesenberry P J, Colvin G, Abedi M.
Perspective: fundamental and clinical concepts on stem cell homing
and engraftment: a journey to niches and beyond. Exp Hematol. 2005;
33:9-19. [0519] 319. Dawn B, Stein A B, Urbanek K, Rota M, Whang B,
Rastaldo R, Torella D, Tang X L, Rezazadeh A, Kajstura J, Leri A,
Hunt G, Varma J, Prabhu S D, Anversa P, Bolli R. Cardiac stem cells
delivered intravascularly traverse the vessel barrier, regenerate
infracted myocardium, and improve cardiac function. Proc Natl Acad
Sci USA. 2005; 102:3766-3771. [0520] 320. Wei C-J, Xu X, Lo C W.
Connexins and cell signaling in development and disease. Annu Rev
Cell Dev Biol 2004; 20:811-38. [0521] 321. Doetsch F. A niche for
adult neural stem cells. Curr Opin Genet Dev. 2003; 13:543-50.
[0522] 322. Li L, Xie T. Stem cell niche: structure and function.
Annu Rev Cell Dev Biol. 2005; 21:605-31. [0523] 323. Kopp H-G,
Avecilla S T, Hooper A T, Rafii S. The bone marrow vascular niche:
home of HSC differentiation and mobilization. Physiology. 2005;
20:349-356. [0524] 324. Hayashi R, Yamato M, Sugiyaa H, Sumide T,
Yang J, Okano T, Tano Y, Nishida K. N-cadherin is expressed by
putative stem/progenitor cells and melanocytes in the human limbal
epithelial stem cell niche. Stem Cells. 2006. [Epub ahead of
print]. [0525] 325. Frisch S M, Ruoslahti E. Integrins and anoikis.
Curr Opin Cell Biol. 1997; 9:701-706. [0526] 326. Frisch S M,
Screaton R A. Anoikis mechanisms. Curr Opin Cell Biol. 2001;
13:555-562. [0527] 327. Melendez J, Turner C, Avraham H, Steinberg
S F, Schaefer E, Sussman M A. Cardiomyocyte apoptosis triggered by
RAFTK/pyk2 via Src kinase is antagonized by paxillin. J Biol Chem.
2004; 279:53516-23. [0528] 328. Reddig P J, Juliano R L. Clinging
to life: cell to matrix adhesion and cells survival. Cancer
Metastasis Rev. 2005; 24:425-439. [0529] 329. Patel, M., J. M.
Stewart, A. V. Loud, P. Anversa, J. Wang, L. Fiegel and T. H.
Hintze. Altered function and structure of the heart in dogs with
chronic elevation in plasma norepinephirne. Circulation
84:2091-2100, 1991. [0530] 330. Hintze, T. H. and Vatner, S.F.:
Comparison of effects of nifedipine and nitroglycerin on large and
small coronary arteries and cardiac function in conscious dogs.
Circ. Res. (Suppl 1) 52: 139-146, 1983 [0531] 331. Bernstein R W,
Ochoa F Y, Xu X, Forfia P, Shen W, Thompson C I and Hintze T H.
Function and production of nitric oxide in the coronary circulation
of the conscious dog during exercise. Circ Res 79:840-848, 1996
[0532] 332. Wang J, Wolin M S and Hintze T H. Chronic exercise
enhances endothelium-mediated dilation of epicardial coronary
artery in conscious dogs. Circ Res 73:829-838, 1993. [0533] 333.
Sessa W C, Pritchard K, Seyedi N, Wang J and Hintze T H. Chronic
exercise in dogs increases coronary vascular nitric oxide
production and endothelial cell nitric oxide synthase gene
expression. Circ Res 74:349-353, 1994. [0534] 334. Shen W Q, Zhang
X P, Zhao G, Wolin M S, Sessa W and Hintze T H. Nitric oxide
production and upregulation of NO synthase gene expression
contribute to vascular regulation during exercise and may be
responsible for the beneficial vascular effects of aerobic exercise
training. Med Sci Sports Exer 27:1125-1134, 1994. [0535] 335.
Stanley W C, Recchia F A, Lopashuk G D. Myocardial substrate
metabolism in the normal and failing heart. Physiol Rev
85:1093-1129, 2005. [0536] 336. Ollson R A, Gregg D E. Myocardial
reactive hyperemia in the unanesthetized dog. Am J Physiol
208:224-230, 1965 [0537] 337. Hintze, T H., Kaley, G.:
Prostaglandins and the control of blood flow in the canine
myocardium. Circ Res 40: 313-320, 1977. [0538] 338. Hintze, T. H.,
Vatner, S. F.: Reactive dilation of large coronary arteries
following brief coronary occlusion in conscious dogs. Circ. Res.
54: 50-57, 1984. [0539] 339. Vatner S F, Franklin D, Higgins C B,
Patrick T, Braunwald E. Left ventricular response to severe
exertion in untethered dogs. J Clin Invest 51:3052-3060, 1972.
[0540] 340. Vatner S F, Pagani M. Cardiovascular adjustments to
exercise: hemodynamics and mechanisms. Prog Cardiovasc Dis
19:91-108, 1976. [0541] 341. Van Citters R L, Franklin D.
Cardiovascular performance of Alaska sled dogs during exercise.
Circ Res 24:33-42, 1969. [0542] 342. Williams J G, Rincon-Skinner
T, Sun D, Wang X, Zhang S, Zhang X, Hintze T H. Role of NO in
coupling myocardial oxygen consumption and coronary vascular
dynamics during pregnancy in the dog. Am J Physiol (In Press 2007)
[0543] 343. Vatner S F, Correlation between acute reductions in
myocardial blood flow and function in conscious dogs. Circ Res
47:201-207, 1980 [0544] 344. Kajstura J, Bolli R, Sonnenblick E H,
Anversa P. Cause of death: suicide. J Molec Cardiol 40:425-437,
2006 [0545] 345. Beltrami A P, Urbanek K, Kajstura J, Yan S M,
Finato N, Bussani R, Nadal-Ginard B, Silvestri F, Leri A, Beltrami
C A, Anversa P. Evidence that human cardiac myocytes divide after
myocardial infarction. N Engl J Med. 344:1785-1787, 2001. [0546]
346. Anversa P, Leri A, Kajstura J. Cardiac regeneration. J Am Coll
Cardiol 47:1769-1776, 2006. [0547] 347. Lavallee M, Cox D, Patrick
T A, Vatner S F. Salvage of myocardial function by coronary artery
reperfusion 1, 2, and 3 hours after occlusion in conscious dogs.
Circ Res 53:235-247, 1983. [0548] 348. Gallagher K P. W(h)ither
myocardial reperfusion injury. J Thromb Thrombolysis 4:137-139,
1997. [0549] 349. Leri A, Kajstura J, Anversa P. Cardiac stem cells
and mechanisms of cardiac regeneration. Physiol Rev 85:1373-1416,
2005. [0550] 350. Nadal-Ginard, Kajstura J, Anversa P. Myocyte
death, growth, and regeneration in cardiac hypertrophy and failure.
Circ Res 92:139-150, 2003. [0551] 351. Li W, Mital S, Ojaimi C,
Csiszar A, Kaley G, Hintze T H. Premature death and age-related
cardiac dysfunction in male eNOS-knockout mice. J. Mol. Cell
Cardiol. 37(3):671-90, 2004. [0552] 352. Quinones M A, Gaassch W H,
Alexander J K. Influence of acute changes in preload, afterload,
contractile state and heart rate on ejection and isovolumic indices
of myocardial contractility in man. Circulation 53: 293-302, 1976
[0553] 353. Vatner S F. Effects of anesthesia on cardiovascular
control mechanisms. Environ Health Perspect. 26:193-206, 1978.
[0554] 354. Kajstura J, Zhang X, Liu Y, Szoke E, Cheng W, Olivetti
G, Hintze T H and Anversa P. Cellular basis of pacing-induced
dilated myopathy:myocyte cell loss and myocyte cellular
hypertrophy. Circulation 92:2306-2317, 1995. [0555] 355. Liu Y,
Cignola E, Cheng W, Kajstura J, Olivetti G, Hintze T H and Anversa
P. Myocyte nuclear mitotic division and programmed cell death
characterize the cardiac myopathy induced by rapid ventricular
pacing in dogs. Lab Invest 73:771-787, 1995. [0556] 356. Cheng W,
Sheng B, Kajstura J, Li P, Wolin M S, Sonnenblick E, Hintze T H,
Olivetti G and Anversa P. Stretch-induced programmed myocyte cell
death. J Clin Invest 96:2247-2259, 1995. [0557] 357. Leri A,
Malhotra A, Li Q, Stiegler P, Claudio P P, Giordano A, Kajstura J,
Hintze T H and Anversa P. Pacing-induced heart failure in dogs
enhances the expression of p53 and p53-dependent genes in
ventricular myocytes. Circulation 97:194-203, 1998 [0558] 358.
Setoguchi M, Leri A, Wang S, Liu Y, DeLuca A, Giordano A, Hintze T
H, Kajstura J and Anversa P. Activation of cyclins and
cyclin-dependent kinases, DNA replication and myocyte proliferation
in pacing-induced heart failure in dogs. Lab Invest 79:1545-1558,
1999. [0559] 359. Barlucci L, Leri A, Dostal D E, Fiordaliso F,
Tada H, Hintze T H, Kajstura J, Nadal-Ginard B, Anversa P. Canine
ventricular myocytes possess a renin-angiotensin system which is
upregulated with heart failure. Circ Res 2001; 88: 298-304. [0560]
360. Cesselli D, I Jakoniuk, L Barlucchi, A P Beltrami, T H Hintze,
B Nadal-Ginard. Oxidative stress-mediated cardiac cell death is a
major determinant of ventricular dysfunction and failure in dog
dilated cardiomyopathy. Circ Res. 89:279-86, 2001. [0561] 361. Leri
A, L Barlucchi, F Limana, A Deptala, Z Darzynkiewicz, T H Hintze, J
Kajstura, B Nadal-Ginard, P Anversa. Telomerase expression and
activity are coupled with myocyte proliferation a preservation of
telomeric length in the failing heart. Proc Natl Aca Sci.
98:8626-31, 2001. [0562] 362. Post H, Kajstura J, Lei B, Sessa W B,
Byrne B, Anversa P, Hintze T H, Recchia F A. Adeno-associated virus
medicated gene delivery into coronary microvessels of chronically
instrumented dogs. J Appl Physiol 95:1688-1694, 2003. [0563] 363.
Davis M E, Hsieh P C, Takashi T, Song Q, Zhang S, Kamm R D, Grodz A
J, Anversa P, Lee R T. Local myocardial insulin-like growth factor
1 (IGF-1) delivery with biotinylated peptide nanofibers improves
cell therapy for myocardial infarction. Proc Nat Acad Sci USA
103:8155-8160, 2006 [0564] 364. Torella D, Rota M, Nurzynska D,
Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A,
Sussman M A, Urbanek K, Nadal-Ginard B, Kajstura J, Leri A, Anversa
P. Cardiac stem cell and myocyte aging, heart failure, and insulin
like growth factor-1 overexpression. Circ Res 94:411-413, 2004.
[0565] 365. Dawn B, Guo Y, Rezazzadeh A, Huang Y, Stein A B, Hunt
G, Tiwari S, Varma J, Gu Y, Prabhu S D, Kajstura J, Anversa P,
Ildstad S T, Bolli R. Postinfarction cytokine therapy regenerates
cardiac tissue and improves left ventricular function. Circ Res
28:990-992, 2006. [0566] 366. Liu J, Hu Q, Wang Z, Xu C, Wang X,
Gong G, Mansoor A, Lee J, Hou M, Zeng L, Zhang J R, Jerosch-Herold
M, Guo T, Bache R J, Zhang J. Autologous stem cell transplantation
for myocardial repair. Am J Physiol 287:H501-H511, 2004. [0567]
367. Zeng L, Hu Q, Wang X, Mansoor A, Lee J, Feygin J, Zhang G,
Suntharalingam P, Boozer S, Mhashilkar A, Panetta C J, Swingen C,
Deans R, From A H L, Bache R J, Verfaille C M, Zhang J.
Bioenergetic and functional consequences of bone marrow-derived
multipotent progenitor cell transplantation in hearts with
postinfarction left ventricular remodeling. Circulation
115:1866-1875, 2007. [0568] 368. Suzuki G, Lee T C, Fallviollitta J
A, Canty J M. Adenoviral gene transfer of FGF-5 to hibernating
myocardium improves function and stimulates myocytes to hypetrophy
and reenter the cell cycle. Circ Res 96:767-775, 2005. [0569] 369.
Tillmanns J, Rota M, Hosoda T, Rotatori F, DeAngelis A, Amano K,
Amano S, LeCapitaine N, Esposito G, Loredo M, Misao Y, Vitale S,
Bearzi C, Urbanek K, Bolli R, Leri A, Kajstura J, Anversa P:
Formation of large coronary arteries by cardiac stem cells: a
biological bypass. Proc Natl Acad Sci USA. In revision, 2007.
[0570] 370. Moore K A, Lemischka I R. Stem cells and their niches.
Science. 2006; 311:1880-1885. [0571] 371. Yin T, Li L. The stem
cell niches in bone. J Clin Invest. 2006; 116:1195-1201. [0572]
372. Scadden D T. The stem-cell niche as an entity of action.
Nature. 2006; 441:1075-1079. [0573] 373. Braun K M, Niemann C,
Jensen U B, Sundberg P, Silva-Vargas V, Watt F M. Manipulation of
stem cell proliferation and lineage commitment: visualization of
label-retaining cells in whole mounts of mouse epidermis.
Development. 2003; 130:5241-5255. [0574] 374. Tumbar T, Guasch G,
Greco V, Blanpain C, Lowry W E, Rendl M, Fuchs E. Defining the
epithelial stem cell niche in skin. Science. 2004; 303:359-363.
[0575] 375. Watt F M, Hogan B L M. Out of Eden: stem cells and
their niches. Science. 2000; 287:1427-1438. [0576] 376. Spradling
A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature.
2001; 414:98-104. [0577] 377. Gotz M, Huttner W B. The cell biology
of neurogenesis. Nat Rev Mol Cell Biol. 2005; 6:777-788. [0578]
378. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors:
stem cells and their niche. Cell. 2004; 116:769-778. [0579] 379.
Urbanek K, Cesselli D, Rota M, Nascimbene A, De Angelis A, Hosoda
T, Bearzi C, Boni A, Bolli R, Kajstura J, Anversa P, Leri A. Stem
cell niches in the adult mouse heart. Proc Natl Acad Sci USA. 2006;
103:9226-31. [0580] 380. Perez-Moreno M, Jamora C, Fuchs E. Sticky
business: orchestrating cellular signals at adherens junctions.
Cell. 2003; 121:535-548. [0581] 381. Goldberg G S, Valiunas V,
Bring P R. Selective permeability of gap junction channels. Biochim
Biophys Acta. 2004; 1662:96-101. [0582] 382. Wei C-J, Xu X, Lo C W.
Connexins and cell signaling in development and disease. Annu Rev
Cell Dev Biol 2004; 20:811-38. [0583] 383. Lockhart D J, Dong H,
Byrne M C, Follettie M T, Gallo M V, Chee M S, Mittmann M, Wang C,
Kobayashi M, Horton H, Brown E L. Expression monitoring by
hybridization to high-density oligonucleotide arrays. Nat
Biotechnol. 1996; 14:1675-80. [0584] 384. Li C, Wong W H.
Model-based analysis of oligonucleotide arrays: expression index
computation and outlier detection. Proc Natl Acad Sci USA. 2001;
98:31-36. [0585] 385. Bhattacharya B, Miura T, Brandenberger R,
Mejido J, Luo Y, Yang A X, Joshi B H, Ginis I, Thies R S, Amit M,
Lyons I, Condie B G, Itskovitz-Eldor J, Rao M S, Puri R k. Gene
expression in human embryonic stem cell lines: unique molecular
signature. Blood. 2004; 103:2956-64. [0586] 386. Bruno L, Hoffmann
R, McBlane F, Brown J, Gupta R, Joshi C, Pearson S, Seidl T,
Heyworth C, Enver T. Molecular signatures of self-renewal,
differentiation, and lineage choice in multipotential hemopoietic
progenitor cells in vitro. Mol Cell Biol. 2004; 24:741-56. [0587]
387. Ivanova N B, Dimos J T, Schaniel C, Hackney J A, Moore K A,
Lemischka I R. A stem cell molecular signature. Science. 2002;
298:601-604. [0588] 388. Ramalho-Santos M S, Yoon Y, Matsuzxaki R,
Mulligan C, Melton D A. "Stemness" transcriptional profiling of
embryonic and adult stem cells. Science. 2002; 298:597-600. [0589]
389. Sturn A, Quackenbush J, Trajanoski. Genesis: cluster analysis
of microarray data. Bioinformatics. 2002; 18:207-08. [0590] 390.
Kuroda T, Tada M, Kubota H, Kimura H, Hatano S Y, Suemori H,
Nakatsuji N, Tada T. Octamer and Sox elements are required for
transcriptional cis regulation of Nanog gene expression. Mol Cell
Biol. 2005; 25:2475-85. [0591] 391. Rodda D J, Chew J L, Lim L H,
Loh Y H, Wang B, Ng H H, Robson P. Transcriptional regulation of
nanog by OCT4 and SOX2. J Biol Chem. 2005; 280:24731-7. [0592] 392.
Player A, Wang Y, Bhattacharya B, Rao M, Puri R K, Kawasaki E S.
Comparisons between transcriptional regulation and RNA expression
in human embryonic stem cell lines. Stem Cells Dev. 2006;
15:315-23. [0593] 393. Stains J P, Civitelli R. Genomic approaches
to identifying transcriptional regulators of osteoblast
differentiation.
Genome Biol. 2003; 4:222. [0594] 394. Rao R R, Stice F L. Gene
expression profiling of embryonic stem cells leads to greater
understanding of pluripotency and early developmental events. Biol
Reprod. 2004; 71:1772. [0595] 395. Terskikh A V, Miyamoto T, Chang
C, Diatchenko L, Weissman I L. Gene expression analysis of purified
hematopoietic stem cells and committed progenitors. Blood. 2003;
102:94-101. [0596] 396. Komor M, Guller S, Baldus C D, de Vos S,
Hoelzer D, Ottmann O G, Hofmann W K. Transcriptional profiling of
human hematopoiesis during in vitro lineage-specific
differentiation. Stem Cells. 2005; 23:1154-69. [0597] 397. Cheng L
C, Tavaoie M, Doetsch F. Stem cells: from epigenetics to microRNAs.
Neuron. 2005; 46:363-7. [0598] 398. Ko M S, McLaren A. Epigenetics
of germ cells, stem cells, and early embryos. Dev Cell. 2006;
10:161-6. [0599] 399. Shivdasani R A. MicroRNAs: regulators of gene
expression and cell differentiation. Blood. 2006 [Epub ahead of
print]. [0600] 400. Lee M S, Jun D H, Hwang C I, Park S S, Kang J
J, Park H S, Kim J, Kim J H, Seo J S, Park W Y. Selection of neural
differentiation-specific genes by comparing profiles of random
differentiation. Stem Cells. 2006; 24:1946-55. [0601] 401. Assmus
B, Honold J, Schachinge V, Brittenn M B, Fischer-Rasokat U, Lehmann
R, Teupe C, Pistorius K, Martin H, Abolmaai N D, Tonn T, Dimmler S,
Zeiher A M. Transcoronary transplantation of progenitor cells after
myocardial infarction. N Engl Med 355:1189-1191, 2006;
355:1274-1277, 2006 [0602] 402. Baserga R. The biology of cell
reproduction. Harvard University Press. 1985 [0603] 403. Chen S,
Lecleider R J. Transforming growth factor-beta-induced
differentiation of smooth muscle from a neural crest stem cell
line. Circ Res. 2004; 94:1195-1202. [0604] 404. Bruhl T, Heeschen
C, Aicher A, Jadidi A S, Haendeler J, Hoffmann J, Schneider M D,
Zeiher A M, Dimmeler S, Rossig L. p21Cip1 levels differentially
regulate turnover of mature endothelial cells, endothelial
progenitor cells, and in vivo neovascularization. Circ Res. 2004;
94:686-692. [0605] 405. Brandenberger R, Wei H, Zhang S, Lei S,
Murage J, Fisk G J, Li Y, Xu C, Fang R, Guegler K, Rao M S,
Mandalam R, Lebkowski J, Stanton L W. Transcriptome
characterization elucidates signaling networks that control human
ES cell growth and differentiation. Nat Biotechnol. 2004;
22:707-716. [0606] 406. Ginis I, Luo Y, Miura T, Thies S,
Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter M K,
Itskovitz-Eldor J, Rao M S. Differences between human and mouse
embryonic stem cells. Dev Biol. 2004; 269:360-380. [0607] 407.
Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P,
Stoeckert C, Aach J, Ansorge W, Ball C A, Causton H C, Gaasterland
T, Glenisson P, Holstege F C, Kim I F, Markowitz V, Matese J C,
Parkinson H, Robinson A, Sarkans U, Schulze-Kremer S, Stewart J,
Taylor R, Vilo J, Vingron M. Minimum information about a microarray
experiment (MIAME)-toward standards for microarray data. Nat Genet.
2001; 29:365-371. [0608] 408. Draghici S, Khatri P, Bhavsar P, Shah
A, Krawetz S A, Tainsky M A. Onto-Tools, the toolkit of the modern
biologist: Onto-Express, Onto-Compare, Onto-Design and
Onto-Translate. Nucleic Acids Res. 2003; 31:3775-3781. [0609] 409.
Urbanek K, Rota M, Cascapera S, Bearzi C, Nascimbene A, De Angelis
A, Hosoda T, Chimenti S, Baker M, Limana F, Nurzynska D, Torella D,
Rotatori F, Rastaldo R, Musso E, Quaini F, Leri A, Kajstura J,
Anversa P. Cardiac stem cells possess growth factor-receptor
systems that following activation regenerate the infracted
myocardium improving ventricular function and long-term survival.
Circ Res. 2005; 97:663-673. [0610] 410. Wallenstein S, Zucker C L,
Fleiss J L. Some statistical methods useful in circulation
research. Circ Res. 1980; 47:1-9. [0611] 411. Berenson M L, Levine
D M, Rindskopf D. Applied statistics. Prentice Hall, Englewood
Cliffs. Pp. 362-418, 1988. [0612] 412. Jeffery I B, Higgins D G,
Culhane A C. Comparison and evaluation of methods for generating
differentially expressed gene lists from microarray data. BMC
Bioinformatics. 2006; 7:359. [0613] 413. Hack C J. Integrated
transcriptome and proteome data: the challenges ahead. Brief Funct
Genomic Proteomic. 2004; 3:212-219. [0614] 414. Ikeno F, Lyons J,
Kaneda K, Baluom M, Benet L, Rezaee M. Novel precutaneous
adventitial drug delivery system for regional vascular treatment.
Cath. Cardiovasc Interv 63:222-230, 2004 [0615] 415. Suematsu N,
Ojaimi C, Kinugawa S, Xu X, Koller A, Recchia F A, Hintze T H.
Hyperhomocysteinemia alters cardiac substrate metabolism by
impairing NO bioavailability through oxidative stress. Circulation.
115:255-262, 2007. [0616] 416. Bernstein R D, Zhang X, Zhao G,
Forfia P R, Tuzman J, Ochoa M, Vogel T and Hintze T H. Mechanisms
of nitrate accumulation in plasma during pacing induced heart
failure in conscious dogs. Nitric oxide biology and Chemistry
1:386-396, 1998. [0617] 417. Recchia F A, McConnell P I, Bernstein
R D, Vogel T R, Xu X B and Hintze T H. Reduced nitric oxide
production and altered myocardial metabolism during the
decompensation of pacing-induced heart failure in the conscious
dog. Circ Res 83:969-979, 1998. [0618] 418. Post H, d'Agostino C,
Lionette V, Castellari M, Kang E Y, Altarejos M, X B Xu, T H
Hintze, Recchia F A. Reduced left ventricular compliance and
mechanical efficiency after prolonged inhibition of NO synthesis in
conscious dogs. J Physiol. 552:233-239, 2003. [0619] 419. Erbs S,
Linke A, Schachinger V, Assmus B, Thiele H, Diederich K W, Hoffmann
C, Dimmler S, Tonn T, Hambrecht R, Zeiher A M, Schuler G.
Restoration of microvascular function in infarct-related artery by
intracoronary transplantation of bone marrow progenitor cells in
patients with acute myocardial infarction: the Doppler substudy of
Reinfarction of Enriched Progenitor cells and Infarct Remodeling in
Acute Myocardial Infarction (REPAIR-AMI) trial. Circulation
116:366-374, 2007 [0620] 420. Stewart J M, Wang J, Zeballos G A,
Dean R, Schustek M, Ochoa M, Hintze T H. Bilaterial atriectomy
eliminates atrial peptide release during volume expansion in
conscious dogs. Circ. Res. 70:724-732, 1992. [0621] 421. Shen W Q,
Xu X, Wang J, Ochoa M, Zeballos G A, Schustek M, Stewart J M,
Hintze T H. Contribution of the ventricles and atrial appendages to
the elevation of plasma ANF during congestive heart failure in
conscious dogs. Basic Res Cardio 41:319-328, 1996. [0622] 422. Lei
B, Matsuo K, Labinsky V, Sharma N, Chandler M P, Ahn A, Hintze T H,
W C Stanley and F A Recchia. Exogeneous nitric oxide reduces
glucose transporters translocation and lactate production in
ischemic myocardium, in vivo PNAS. 102:6966-6971, 2005 [0623] 423.
Millard R W, Baig H, Vatner S F. Cardiovascular effects of
radioactive microsphere suspensions and Tween 80 solutions. Am J
Physiol 232:H331-H334, 1977 [0624] 424. Sun D, Huang A, Zhao G,
Bernstein R, Forfia P, Xu X, Koller A, Kaley G, Hintze T H. Reduced
NO-dependent arteriolar dilation during the development of
cardiomyopathy. Am. J. Physiol. Heart Circ. Physiol. 278:
H461-H468, 2000 [0625] 425. Sun D, Huang A, Mital S Kichuk M R,
Marboe C, Addonizio L J, Muhler R E, Koller A, Hintze T G, Kaley G.
Norepinephrine elicits B2 receptor mediated dilation of isolated
human coronary arterioles. Circulation. 106:550-555, 2002. [0626]
426. Lavallee M, Vatner S F. Regional myocardial blood flow and
necrosis in primates following coronary occlusion. Am J Physiol
246:H635-H639, 1984.
* * * * *
References