U.S. patent application number 12/091405 was filed with the patent office on 2012-01-19 for use of tumor necrosis factor-alpha receptor p75 for treatment of ischemia-induced neovascularization.
This patent application is currently assigned to CARITAS ST. ELIZABETH MEDICAL CENTER OF BOSTON INC. Invention is credited to David A. Goukassian, Douglas W. Losordo.
Application Number | 20120014879 12/091405 |
Document ID | / |
Family ID | 37968413 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120014879 |
Kind Code |
A1 |
Losordo; Douglas W. ; et
al. |
January 19, 2012 |
USE OF TUMOR NECROSIS FACTOR-alpha RECEPTOR p75 FOR TREATMENT OF
ISCHEMIA-INDUCED NEOVASCULARIZATION
Abstract
Improvements on the basic method used for BEAMing increase
sensitivity and increase the signal-to-noise ratio. The
improvements have permitted the determination of intrinsic error
rates of various DNA polymerases and have permitted the detection
of rare and subtle mutations in DNA isolated from plasma of cancer
patients.
Inventors: |
Losordo; Douglas W.;
(Chicago, IL) ; Goukassian; David A.; (West
Newton, MA) |
Assignee: |
CARITAS ST. ELIZABETH MEDICAL
CENTER OF BOSTON INC
BOSTON
MA
|
Family ID: |
37968413 |
Appl. No.: |
12/091405 |
Filed: |
October 24, 2006 |
PCT Filed: |
October 24, 2006 |
PCT NO: |
PCT/US06/41545 |
371 Date: |
July 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60729235 |
Oct 24, 2005 |
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|
Current U.S.
Class: |
424/9.2 ;
424/85.1; 424/93.21; 424/94.4; 435/320.1; 435/325; 435/455;
435/6.1; 435/6.13; 435/7.21; 514/13.3; 514/44R; 514/7.6; 514/7.7;
514/8.1; 514/8.2; 514/8.5; 514/8.9; 514/9.1; 514/9.5; 514/9.6 |
Current CPC
Class: |
G01R 31/3277 20130101;
G01R 31/50 20200101; H02H 3/33 20130101; G01R 31/52 20200101; H02H
1/0015 20130101; A61P 9/10 20180101; C12Q 1/6858 20130101; C12Q
1/6858 20130101; C12Q 2565/537 20130101; C12Q 2563/155 20130101;
C12Q 2527/125 20130101 |
Class at
Publication: |
424/9.2 ;
514/44.R; 435/455; 424/93.21; 514/8.1; 514/9.5; 514/9.1; 514/7.6;
514/13.3; 514/9.6; 514/8.9; 514/8.2; 424/85.1; 514/8.5; 514/7.7;
424/94.4; 435/320.1; 435/325; 435/7.21; 435/6.13; 435/6.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C12N 15/85 20060101 C12N015/85; A61K 38/18 20060101
A61K038/18; A61K 38/19 20060101 A61K038/19; A61P 9/10 20060101
A61P009/10; A61K 38/44 20060101 A61K038/44; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; G01N 33/566 20060101
G01N033/566; C12Q 1/68 20060101 C12Q001/68; A61K 48/00 20060101
A61K048/00; A61K 38/30 20060101 A61K038/30 |
Claims
1. A method of treating, reducing the severity of, or preventing
ischemia in a subject having or at risk of developing ischemia, the
method comprising, a) contacting a cell of the subject with a
nucleic acid molecule encoding a p75/TNFR2 polypeptide or a
fragment thereof; and b) expressing the p75/TNFR2 polypeptide in
the cell, wherein the method treats or prevents ischemia in the
subject.
2. A method of enhancing angiogenesis in a tissue before, during,
or after an ischemic event, the method comprising a) contacting a
cell with a nucleic acid molecule encoding a p75/TNFR2 polypeptide
or a fragment thereof; and b) expressing the p75/TNFR2 polypeptide
in the cell, wherein the method enhances angiogenesis in the
tissue.
3. The method of claim 1, further comprising administering the cell
to the subject, wherein the method enhances angiogenesis.
4. The method of claim 1, wherein the method reduces apoptosis in
the subject.
5. The method of claim 1, wherein the method enhances the local
release of angiogenic growth factors and cytokines in the
tissue.
6. The method of claim 1, further comprising the step of
administering to the subject an angiogenic factor selected from the
group consisting of: vascular endothelial growth factor (VEGF),
hepatocyte growth factor (HGF), basic fibroblast growth factor
(bFGF), angiopoietin 1, angiopoietin 2 and monocyte chemotactic
protein-1 (MCP-1).
7. The method of claim 1, further comprising the step of
administering to the subject an endothelial cell mitogen selected
from the group consisting of acidic and basic fibroblast growth
factors, vascular endothelial growth factor, epidermal growth
factor, transforming growth factor a and 13, platelet-derived
endothelial growth factor, platelet-derived growth factor, tumor
necrosis factor a, hepatocyte growth factor, insulin like growth
factor, erythropoietin, colony stimulating factor, macrophage-CSF,
granulocyte/macrophage CSF and nitric oxide synthase.
8-10. (canceled)
11. The method of claim 3, wherein the cell is delivered directly
to an ischemic tissue or is delivered systemically.
12. (canceled)
13. The method of claim 1, wherein the nucleic acid molecule is
present in a vector.
14-15. (canceled)
16. A method of enhancing p75/TNFR2 expression in a cell, the
method comprising a) contacting a cell with a nucleic acid molecule
encoding a p75/TNFR2 polypeptide or a fragment thereof; and b)
expressing the p75/TNFR2 polypeptide in the cell.
17-21. (canceled)
22. The method of claim 20, wherein the cell is an endothelial
progenitor cell or bone marrow derived cell.
23. An expression vector comprising a nucleic acid molecule
encoding a mammalian p75/TNFR2 polypeptide or a fragment thereof
operably linked to a promoter sufficient to direct expression of
the p75 TNFR2 receptor polypeptide in a cell.
24-26. (canceled)
27. The vector of claim 26, wherein the vector is selected from the
group consisting of adenoviral vectors, adeno-associated viral
vectors, retroviral vectors, lentiviral vectors, alphaviral
vectors, and herpes virus vectors.
28-30. (canceled)
31. A host cell comprising the vector of claim 23.
32-34. (canceled)
35. The host cell of claim 31, wherein the cell is an endothelial
progenitor cell or a bone marrow-derived cell.
36. (canceled)
37. The host cell of claim 31, wherein the cell is in vitro or in
vivo.
38. A pharmaceutical composition comprising an effective amount of
an expression vector encoding a human p75/TNFR2 polypeptide or a
fragment thereof in a pharmaceutically acceptable excipient,
wherein the p75/TNFR2 polypeptide is operably linked to a promoter
sufficient to drive expression of the p75/TNFR2 polypeptide in a
mammalian cell.
39. (canceled)
40. The pharmaceutical composition of claim 38, wherein the
promoter is sufficient to drive expression in an endothelial
progenitor cell or a bone marrow-derived cell.
41. A kit for the treatment or prevention of ischemia, the kit
comprising an effective amount of an expression vector encoding a
human p75/TNFR2 polypeptide or a fragment thereof in a
pharmaceutically acceptable excipient, wherein the p75/TNFR2
polypeptide is operably linked to a promoter sufficient to drive
expression of the p75/TNFR2 polypeptide in a mammalian cell.
42. (canceled)
43. A method of monitoring a subject being treated for ischemia,
the method comprising a) administering a treatment that enhances
the expression of a p75/TNFR2 polypeptide in a cell of the subject;
and b) measuring apoptosis or an increase in angiogenesis in a
tissue of the subject relative to a reference, wherein a decrease
in apoptosis or an increase in angiogenesis indicates a reduced
severity of ischemia in the subject.
44-50. (canceled)
51. A method for identifying a candidate compound a candidate
compound useful for the treatment of ischemia, the method
comprising the steps of: (a) contacting a cell expressing p75/TNFR2
polypeptide with a candidate compound; and (b) detecting an
increase in the level or the biological activity of a p75/TNFR2
polypeptide or nucleic acid molecule in the cell relative to a
reference, wherein an increase in the level or the biological
activity of p75/TNFR2 polypeptide identifies a candidate compound
useful for the treatment of ischemia.
52. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Aging is associated with an increased risk of
atherosclerotic disease of the coronary and peripheral arteries. In
either vascular system, the extent of ischemic damage and degree of
subsequent functional recovery after arterial obliteration largely
depends on the development of new collateral blood vessels. Aging
is associated with impaired angiogenesis in murine and rabbit limb
ischemia models. Aging is also accompanied by a steady decline in
immune functions, such as defects in signaling pathways and altered
expression of cytokines, such as interferon-gamma (IFN.gamma.) and
vascular endothelial growth factor (VEGF). Angiogenesis is
associated with perivascular inflammation and monocyte/macrophage
accumulation.
[0002] Tumor necrosis factor alpha (TNF-.alpha.), a
macrophage/monocyte-derived pluripotent mediator, can function as
an angiogenic factor in one system and as an anti-angiogenic factor
in another. These mutually exclusive effects have been attributed
to TNF-.alpha. concentration and duration of exposure; that is, low
concentrations and short exposure is angiogenic, whereas high
concentrations and prolonged exposure is anti-angiogenic.
TNF-.alpha. has been reported to induce the expression of many
important immune- and angiogenesis-related genes through two
different TNF-.alpha. receptors: TNF-.alpha.R1 (p55) and
TNF-.alpha.R2 (p75). In various vascular endothelial cells,
TNF-.alpha. increased the expression of the well-known angiogenic
factors VEGF, basic fibroblast growth factor (bFGF), and
interleukin-6 (IL-6). The role of the two distinct TNF-.alpha.
receptors in mediating these responses are still unclear.
[0003] Given that angiogenesis is impaired in elderly individuals,
this dysfunction may be related to alterations in TNF-.alpha.
receptor expression. Methods of enhancing blood flow in the elderly
are required to treat or prevent ischemia.
SUMMARY OF THE INVENTION
[0004] The invention generally provides methods and compositions
for modulating p75 receptor/TNFR2 expression for the treatment or
prevention of ischemia.
[0005] In one aspect, the invention generally features a method of
treating, reducing the severity of, or preventing ischemia in a
subject having or at risk of developing ischemia. The method
involves contacting a cell of the subject (e.g., human or
veterinary patient) with a nucleic acid molecule encoding a
p75/TNFR2 polypeptide or a fragment thereof; and expressing the
p75/TNFR2 polypeptide in the cell, where the method treats or
prevents ischemia in the subject.
[0006] In another aspect, the invention generally features a method
of enhancing angiogenesis in a tissue before, during, or after an
ischemic event. The method involves contacting a cell with a
nucleic acid molecule encoding a p75/TNFR2 polypeptide or a
fragment thereof; and expressing the p75/TNFR2 polypeptide in the
cell, where the method enhances angiogenesis in the tissue.
[0007] In yet another aspect, the invention generally features a
method of enhancing angiogenesis in a subject. The method involves
contacting a cell isolated from the subject with a nucleic acid
molecule encoding a p75/TNFR2 polypeptide or a fragment thereof;
and administering the cell to the subject, where the method
enhances angiogenesis.
[0008] In yet another aspect, the invention generally features
method of reducing apoptosis in a subject having or at risk of
developing ischemia. The method involves contacting a cell of the
subject with a nucleic acid molecule encoding a p75 TNFR2 receptor
polypeptide or a fragment thereof; and expressing the p75 TNFR2
receptor polypeptide in the cell, where the method reduces
apoptosis in the subject.
[0009] In one embodiment of the above aspects, the method enhances
the local release of angiogenic growth factors and cytokines in the
tissue. In another embodiment of the above aspects, the method
further includes the steps of administering to the subject an
angiogenic factor selected from the group consisting of: vascular
endothelial growth factor (VEGF), hepatocyte growth factor (HGF),
basic fibroblast growth factor (bFGF), angiopoietin 1, angiopoietin
2 and monocyte chemotactic protein-1 (MCP-1). In yet another
embodiment of the above aspects, the method further includes the
steps of administering to the subject an endothelial cell mitogen
selected from the group consisting of acidic and basic fibroblast
growth factors, vascular endothelial growth factor, epidermal
growth factor, transforming growth factor .alpha. and .beta.,
platelet-derived endothelial growth factor, platelet-derived growth
factor, tumor necrosis factor .alpha., hepatocyte growth factor,
insulin like growth factor, erythropoietin, colony stimulating
factor, macrophage-CSF, granulocyte/macrophage CSF and nitric oxide
synthase. In yet another embodiment of the above aspects, the cell
is in vivo or in vitro. In yet another embodiment of the above
aspects, the method further comprises the step of delivering the
cell to a subject (e.g., the cell is delivered directly to an
ischemic tissue or the cell is delivered systemically). In still
another embodiment of the above aspects, the nucleic acid molecule
is present in a vector (e.g., a viral vector). In another
embodiment of the above aspects, the nucleic acid molecule is
positioned for expression.
[0010] In another aspect, the invention generally features a method
of enhancing p75/TNFR2 expression in a cell. The method involves
contacting a cell with a nucleic acid molecule encoding a p75/TNFR2
polypeptide or a fragment thereof; and expressing the p75/TNFR2
polypeptide in the cell (e.g., a cell in vitro or in vivo).
[0011] In a related aspect, the invention generally features an
expression vector comprising a nucleic acid molecule encoding a
mammalian p75/TNFR2 polypeptide or a fragment thereof operably
linked to a promoter sufficient to direct expression of the p75
TNFR2 receptor polypeptide in a cell. In one embodiment, the
expression vector is a mammalian expression vector, such as an
expression vector suitable for expression in a human cell. In
another embodiment, the expression vector is a viral expression
vector. In yet another embodiment, the viral vector is selected
from the group consisting of adenoviral vectors, adeno-associated
viral vectors, retroviral vectors, lentiviral vectors, alphaviral
vectors, and herpes virus vectors. In another embodiment, the
mammalian p75/TNFR2 polypeptide is a murine polypeptide or a human
polypeptide. In another embodiment, the promoter drives expression
in an endothelial progenitor cell or in a bone marrow derived
cell.
[0012] In another related aspect, the invention generally features
a host cell (e.g., a mammalian cell, such as a human or murine
cell) comprising the vector of any previous aspect. In one
embodiment, the cell is an endothelial progenitor cell or is a bone
marrow-derived cell. In another embodiment, the cell is in vitro or
in vivo.
[0013] In another aspect, the invention generally features a
pharmaceutical composition comprising an effective amount of an
expression vector encoding a human p75/TNFR2 polypeptide or a
fragment thereof in a pharmaceutically acceptable excipient, where
the p75/TNFR2 polypeptide is operably linked to a promoter
sufficient to drive expression of the p75/TNFR2 polypeptide in a
mammalian cell. In one embodiment, the vector is a viral vector. In
another embodiment, the promoter is sufficient to drive expression
in an endothelial progenitor cell or a bone marrow-derived
cell.
[0014] In yet another aspect, the invention generally features kit
for the treatment or prevention of ischemia, the kit comprising an
effective amount of an expression vector encoding a human p75/TNFR2
polypeptide or a fragment thereof in a pharmaceutically acceptable
excipient, where the p75/TNFR2 polypeptide is operably linked to a
promoter sufficient to drive expression of the p75/TNFR2
polypeptide in a mammalian cell.
[0015] In yet another aspect, the invention generally features a
method of monitoring a subject being treated for ischemia. The
method involves administering a treatment that enhances the
expression of a p75/TNFR2 polypeptide in a cell of the subject; and
measuring angiogenesis in a tissue of the subject relative to a
reference, where an increase in angiogenesis indicates a reduced
severity of ischemia in the subject.
[0016] In a related aspect, the invention generally features a
method of monitoring a subject being treated for ischemia. The
method involves administering a treatment that enhances the
expression of a p75/TNFR2 polypeptide in a cell of the subject; and
measuring apoptosis in a tissue of the subject relative to a
reference, where a decrease in apoptosis indicates a reduced
severity of ischemia in the subject. In one embodiment, the
reference is the level of angiogenesis or apoptosis previously
present in the subject or in a biological sample derived from the
subject at an earlier time point. In another embodiment, the
reference is a baseline level of apoptosis or angiogenesis present
prior to therapy. In yet another embodiment, the reference is the
level of angiogenesis or apoptosis present in a normal subject
(e.g., a human subject) sample.
[0017] In another aspect, the invention generally features a method
for identifying a candidate compound useful for the treatment of
ischemia. The method involves contacting a cell expressing
p75/TNFR2 nucleic acid molecule with a candidate compound; and
detecting an increase in p75/TNFR2 expression in the cell relative
to a reference, where an increase in p75/TNFR2 expression
identifies the candidate compound as a compound useful for the
treatment of ischemia. In one embodiment, the method identifies a
compound that increases transcription or translation of a p75/TNFR2
nucleic acid molecule.
[0018] In a related aspect, the invention generally features an
method for identifying a candidate compound a candidate compound
useful for the treatment of ischemia. The method involves
contacting a cell expressing p75/TNFR2 polypeptide with a candidate
compound; and detecting an increase in the level of p75/TNFR2
polypeptide in the cell relative to a reference level, where an
increase in the level of p75/TNFR2 polypeptide identifies a
candidate compound useful for the treatment of ischemia.
[0019] In another related aspect, the invention generally features
a method for identifying a candidate compound useful for the
treatment of ischemia. The method involves the steps of: contacting
a cell expressing a p75/TNFR2 polypeptide with a candidate
compound; and detecting an increase in the biological activity of
the p75/TNFR2 polypeptide in the cell contacted with the candidate
compound with a reference level of biological activity where the
candidate compound as a candidate compound that useful for the
treatment of ischemia.
[0020] In various embodiments of any of the above aspects, the
method further includes the step of delivering the cell to a
subject (e.g., a human or veterinary patient) having or at risk of
developing ischemia. In still other embodiments of the above
aspects, the cell is a mammalian cell (e.g., a human or murine
cell), such as an endothelial progenitor cell or bone marrow
derived cell. In yet other embodiments of the above aspects, the
method includes administering an angiogenic factor or an
endothelial cell mitogen. Factors that can be administered in
combination with a method of the invention include, for example,
TNF, TGF-.alpha., TGF-.beta., hemoglobin, interleukin-1,
interleukin-2, interleukin-3, interleukin-4, interleukin-5,
interleukin-6, interleukin-7, interleukin-8, interleukin-9,
interleukin-10, interleukin-11, interleukin-12 etc., GM-CSF, G-CSF,
M-CSF, human growth factor, co-stimulatory factor B7, insulin,
factor VIII, factor IX, PDGF, EGF, NGF, IL-ira, EPO, .beta.-globin.
Other factors that may be administered in combination with any of
the above methods include endothelial cell mitogens, acidic and
basic fibroblast growth factors, vascular endothelial growth factor
(VEGF), epidermal growth factor (EGF), platelet-derived endothelial
cell growth factor (PD-ECGF), hepatocyte growth factor (HGF),
insulin like growth factor (IGF), erythropoietin, colony
stimulating factor (CSF), macrophage-CSF (M-CSF),
granulocyte/macrophage CSF (GM-CSF), monocyte chemotactic
protein-1, and nitric oxide synthase (NOS).
Definitions
[0021] By "p75/TNFR2 polypeptide" is meant a protein or fragment
thereof having substantial identity to the amino acid sequence of
p75/TNFR2 provided at GenBank Accession No. NP.sub.--001057 that
promotes angiogenesis or has TNF binding activity.
[0022] By "p75/TNFR2 nucleic acid molecule" is meant a
polynucleotide that encodes a p75/TNFR2 polypeptide.
[0023] By "p75/TNFR2 biological activity" is meant TNF binding
activity or angiogenesis enhancing activity.
[0024] By "angiogenesis" is meant any alteration that benefits
tissue perfusion. Angiogenesis includes the growth by sprouting of
endothelial cells from existing blood vessels or the remodeling of
existing vessels to alter size, maturity, direction or flow
properties to improve blood perfusion of tissues. In one
embodiment, angiogenesis increases the density of an existing
vascular network. Angiogenesis is measured by any method known in
the art, including by determining the number of capillaries per
muscle fiber.
[0025] By "apoptosis" is meant the process of cell death wherein a
dying cell displays a set of well-characterized biochemical
hallmarks that include cell membrane blebbing, cell soma shrinkage,
chromatin condensation, and DNA laddering. Cells that die by
apoptosis include neurons (e.g., during the course of a stroke or
ischemic injury) and myocytes, such as cardiomyocytes (e.g., after
myocardial infarction or over the course of congestive heart
failure).
[0026] By "effective amount" is meant the amount of a compound
required to prevent, treat, or ameliorate the symptoms of a
disease.
[0027] By "enhance" is meant increase. For example, an increase of
at least 5%, 10%, 25%, 50%, 75% or 100% relative to a
reference.
[0028] By "ischemia" is meant reduced blood flow to a tissue or
organ relative to the level required for the maintenance of normal
cell metabolism. Exemplary ischemic events include primary
myocardial infarction, secondary myocardial infarction, angina
pectoris (including both stable and unstable angina), congestive
heart failure, sudden cardiac death, cerebral infarction,
restenosis, syncope, ischemia, reperfusion injury, vascular
occlusion, carotid obstructive disease, transient ischemic attack,
and the like.
[0029] By "angiogenic factor" is meant any polypeptide or fragment
thereof that enhances angiogenesis.
[0030] By "endothelial cell mitogen" is meant any polypeptide or
fragment thereof that supports the proliferation of an endothelial
cell.
[0031] By "ameliorate" is meant decrease, suppress, attenuate,
diminish, arrest, or stabilize the development or progression of a
disease.
[0032] By "disease" is meant any condition or disorder that damages
or interferes with the normal function of a cell, tissue, or organ.
Examples of diseases include bacterial invasion or colonization of
a host cell.
[0033] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, bearing a series of
specified nucleic acid elements that enable transcription of a
particular gene in a host cell. Typically, gene expression is
placed under the control of certain regulatory elements, including
constitutive or inducible promoters, tissue-preferred regulatory
elements, and enhancers.
[0034] By "fragment" is meant a portion of a protein or nucleic
acid that is substantially identical to a reference protein or
nucleic acid. In some embodiments the portion retains at least 50%,
75%, or 80%, or more preferably 90%, 95%, or even 99% of the
biological activity of the reference protein or nucleic acid
described herein.
[0035] By "immunological assay" is meant an assay that relies on an
immunological reaction, for example, antibody binding to an
antigen. Examples of immunological assays include ELISAs, Western
blots, immunoprecipitations, and other assays known to the skilled
artisan.
[0036] By "isolated nucleic acid molecule" is meant a nucleic acid
(e.g., a DNA) that is free of the genes which, in the
naturally-occurring genome of the organism from which the nucleic
acid molecule of the invention is derived, flank the gene.
[0037] By "operably linked" is meant that the polynucleotide of the
invention (e.g., a DNA molecule) is positioned adjacent to a DNA
sequence that directs transcription and translation of the sequence
(i.e., facilitates the production of, for example, a recombinant
polypeptide of the invention, or an RNA molecule).
[0038] By "promoter" is meant a polynucleotide sufficient to direct
transcription.
[0039] By "reduces" or "increases" is meant a negative or positive
alteration, respectively, of at least 10%, 25%, 50%, 75%, or
100%.
[0040] By "reference" is meant a standard or control condition.
[0041] By "subject" is meant a mammal, including, but not limited
to, a human or non-human mammal, such as a bovine, equine, canine,
ovine, or feline.
[0042] "Therapeutic compound" means a substance that has the
potential of affecting the function of an organism. A therapeutic
compound may decrease, suppress, attenuate, diminish, arrest, or
stabilize the development or progression of disease or disorder an
organism.
[0043] By "vector" is meant a DNA molecule, usually derived from a
plasmid or bacteriophage, into which fragments of DNA may be
inserted or cloned. A recombinant vector will contain one or more
unique restriction sites, and may be capable of autonomous
replication in a defined host or vehicle organism such that the
cloned sequence is reproducible. A vector contains a promoter
operably linked to a gene or coding region such that, upon
transfection into a recipient cell, an RNA is expressed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIGS. 1A-1G show that ischemia-induced angiogenesis is
impaired in old TNFR2 KO mice. FIG. 1A is a graph showing blood
flow recovery presented as the laser Doppler perfusion ratio up to
28 days after hind limb surgery in wild-type (WT) young mice (FIG.
1A) (black bars, n=15); wild-type old mice (gray bars, n=12), and
young mice lacking (i.e., having a p75 knock-out (p75KO)) (clear
bars, n=15). FIG. 1B shows blood flow recovery in old mice lacking
p75 (p75KO) (black bars, n=12) and (inset) a representative image
of 5 old p75KO mice showing autoamputation of operated limb. FIG.
1C (upper panel) shows representative images of hind limb muscles
immunostained with isolectin B4 to identify capillaries. FIG. 1C
(lower panel) is a graph showing relative capillary density at
post-operative day twenty-eight in young wild-type (WT), in young
mice lacking p75 (p75KO) and old wild-type mice. Capillary density
is expressed as a number of capillaries per muscle fiber. Results
represent the mean.+-.SEM of 12 randomly chosen areas (0.06
mm.sup.2) of muscle fiber from 3 mice per genotype. FIG. 1D is a
graph showing the kinetics of mobilization of bone marrow-derived
endothelial progenitor cells (EPCs) into peripheral blood (PB)
after hind limb surgery in wild-type (WT) and p75KO mice, measured
by staining the mononuclear (MN) fraction of PB with antibodies
against VEGF receptors Flk1 and Sca1. FIG. 1E shows five (41.6%)
p55KO mice that underwent hindlimb surgery and has intact limbs at
the time post-ischemic recovery was complete. Limbs were evaluated
28 days post-surgery. FIG. 1F shows two animals (16.8%) that
manifested distal toe necrosis (shown with white arrows). FIG. 1G
shows that only five (41.6%) old p55KO mice lost their limbs 2-3
weeks after hind limb surgery.
[0045] FIGS. 2A and B show that ischemia-induced VEGF expression is
lower in the limbs of p75KO mice. FIG. 2A is a series of eight
representative confocal images of ischemic muscles from young
wild-type (FIG. 2A, upper panel) and p75KO mice (FIG. 2A, lower
panel) after hind limb surgery stained with anti-VEGF antibodies.
Fluorescent cells are TUNEL-positive cells that are presumably
apoptotic. Blue fluorescent cells represent TopRo3-positive cells
used to visualize nuclei. FIG. 2B shows densitometric analysis of
VEGF, bFGF, and angiopoeitin-1 mRNA expression, using multiprobe
RPA, in post-ischemic HIND LIMB muscle tissue homogenates of
wild-type (clear bars) and p75KO (black bars) mice three and ten
days post-surgery.
[0046] FIGS. 3A and 3B show VEGF mRNA expression, determined by
RT-PCR, in post-ischemic hind limb muscle tissue homogenates. FIG.
3A is a graph showing mRNA expression in wild-type (WT) (clear
bars) and p75KO (black bars) mice 3 and 10 days post-surgery. FIG.
3B shows representative confocal images of ischemic muscles from
young (8-10 weeks old) WT and p75KO mice after hindlimb surgery
stained with anti-VEGF antibodies (fluorescence). Post-ischemic
muscles of at least 3 animals per genotype/time point were examined
and similar VEGF expression pattern, as shown in these
representative images, was observed in the tissues of WT vs. p75KO
mice.
[0047] FIGS. 4A-4C shows that ischemia-induced apoptosis is greater
in the limbs of p75KO mice. FIG. 4A is a series of twelve
representative merged images of triple stained hind limb muscle of
wild-type (upper panel) and p75KO (lower panel) mice up to 10 days
after hind limb ischemia (HU) surgery showing a significant
increase in the number of TUNEL/EC (+) cells in p75KO mice 1 and 10
days post-hind limb surgery. FIG. 4B is a graph that provides a
quantitative analysis of immunofluorescence (number of double
TUNEL/EC positive cells) in at least 8-10 randomly selected areas
of approximately 10,600 .mu.m2 (measured by computer assisted
software of the confocal microscope) of hind limb muscle from at
least 3 animals/treatment group. All samples were coded, and then
evaluated by a single blinded observer to eliminate the bias and
inter-observer variability. Compared to wild-type, by days 1 and 10
there was a statistically significant increase in the number of
TUNEL/EC (+) cells in p75KO mice after HL surgery (day 1--26.+-.5
vs. 7.+-.5; and day 10--37.+-.8 vs. 3.+-.0.5, p<0.0001, p75KO
vs. WT mice), representing a 67% and 93% increase in EC apoptosis
in p75KO mice on days 1 and 10, respectively. FIG. 4C is a series
of four representative confocal images of triple stained hindlimb
muscles of p75KO mice. TUNEL positive/endothelial cells (EC) are
visualized 1 day after hind limb surgery. Arrows show a few
examples of immunostaining with Isolectin B4, which identified
endothelial lineage cells, TUNEL-identified apoptotic cells,
TopRo3-visualized nuclei, and merged image to identify single,
double, and triple-stained cells.
[0048] FIGS. 5A-5D show that loss of p75 impairs the function of
cultured endothelial progenitor cells (EPCs). FIG. 5A is a graph
showing that no difference was found in the migration of wild-type
(WT) and P75KO bone marrow-derived EPCs toward specific chemotactic
agents. Graphs represent data pooled from independent experiments
completed in triplicate. FIG. 5B is a series of eight
photomicrographs showing that baseline and TNF-induced
tubulogenesis was inhibited in bone marrow-derived p75KO or
wild-type WT) EPCs, as assessed by the ability to form tube-like
structures on VEGF-enriched matrigel. FIG. 5C shows a
representative radiogram of multiprobe RPA after TNF stimulation
using 5 .mu.g total RNA/lane. FIG. 5D is a series of three graphs,
which provide a graphic representation of bFGF, VEGF and
angiopoetin-1 mRNA expression after densitometric analysis. All
values are adjusted relative to actin mRNA expression.
[0049] FIGS. 6A-6E show that signaling through TNFR2 p75 is
required for NF.kappa.B VEGF gene expression. FIG. 6A is a pair of
representative confocal images of NF.kappa.B nuclear translocation
in wild-type and p75KO EPCs 30 minutes after treatment with TNF.
NF.kappa.B nuclear translocation was evaluated by immunostaining
with NF.kappa.B (p65) antibodies. FIG. 6B shows NF.kappa.B DNA
binding activity in TNF-treated wild-type and p75KO EPCs up to two
hours after TNF treatment. Specificity of NF.kappa.B bands was
confirmed by (25.times. mutant NF_B/p65 competition--lane 5 and
25.times. cold probe competition--lane 6). FIG. 6C shows VEGF
promoter activity in TNF-treated wild-type (WT) and p75KO EPCs
transfected with VEGF/luciferase reporter plasmid deletion
constructs. Results are expressed relative to control pGL2 plasmid
activity and represent data pooled from independent experiments
completed in triplicate. FIG. 6D is a schematic diagram showing
VEGF promoter deletion constructs (kind gift of Dr. Debabrata
Mukhopadhyay, Beth Israel Deaconess Medical Center, Boston, Mass.).
A 2.6 kb promoter fragment represents complete promoter region of
VEGF gene and contains several consensus transcriptional response
elements: AP-1, AP-2, GATA, IL-6RE, hypoxia-inducible enhancer
sequences, several SP1 sites and clusters and several estrogen
response elements (EREs) and NFkB putative but not classical
response elements. A 0.35 kb promoter contains only two putative
NFkB sites (bp -220 and -125, relative to the transcription
origination site) and one Sp1 cluster and a 0.07 kb promoter
contains an incomplete Sp1 cluster and has no remaining promoter
activity. FIG. 6E is a graph that shows a concentration-dependent
increase in VEGF promoter activity in wild-type cells treated with
1 and 20 ng/ml TNF, whereas 40 ng/ml TNF treatment inhibited the
activity of VEGF promoter, consistent with the previous reports of
low TNF doses being angiogenic and high doses being antiangiogenic.
To determine an angiogenic TNF concentration in EPCs from WT and
p75KO mice these cells were transfected with 2.6 kb VEGF/Luciferase
promoter constructs and treated with different concentrations of
TNF (1 ng, 20 ng and 40 ng/ml) for 16 hours. No increase in VEFG
promoter activity was observed in p75KO EPCs after treatment with
any of the TNF concentrations. Hence, in subsequent studies 1 ng/ml
TNF concentration was used. Moreover, 0.35 kb VEGF promoter
construct was used to elucidate the TNF-mediated NFkB-dependent
regulation of VEGF gene expression in EPCs from WT and p75KO
mice.
[0050] FIG. 7 provides a diagram of murine bone marrow
transplantation models.
[0051] FIGS. 8A and 8B are graphs showing the evaluation of bone
barrow engraftment and peripheral blood mononuclear cells in
wild-type and p75KO mice. FIG. 8A is a graph showing that by day 28
recipients bone marrow (BM) was almost completely (88-93%)
reconstituted with donor marrow and no difference between wild-type
or p75KO engraftment was observed. FIG. 8B is a graph showing that
although, percent of GFP (+) mononuclear cells in wild-type and
p75KO bone marrow-transplanted mice was 26-34% lower than in
wild-type GFP control mice the number of bone marrow -derived
peripheral blood mononuclear cells (PB MNCs) were similar in
recipient-mice of both genotypes, as evaluated by FACS
analysis.
[0052] FIGS. 9A-9D show that p75/TNFR2 is required for efficient
contribution of bone marrow-derived EPCs in post-ischemic recovery.
FIG. 9A is a graph showing limb autoamputation after hind limb
surgery in old p75KO mice transplanted 28 days prior to hind limb
surgery with bone marrow-derived mononuclear cells from young
wild-type (WT) and p75KO mice (n=4 per transplanted group). FIG. 9B
is a graph showing loss of muscle tissue after hind limb surgery in
old wild-type (WT) mice transplanted twenty-eight days prior to
hind limb surgery with bone marrow-derived mononuclear cells from
young wild-type and p75KO mice (n=7-8 per transplanted group). FIG.
9C shows representative images of old (10-12 months) p75KO mice
transplanted with bone marrow from young (3-4 weeks) p75KO and WT
mice (n=4/BMT group). 50% of old p75KO mice transplanted with bone
marrow from p75KO mice (upper panel) have lost limbs (indicated by
arrows) by day 14 post-HL surgery, whereas 100% of mice
transplanted with WT BM (lower panel) preserved limbs. FIG. 9D
shows representative images of old (8-10 months) WT mice
transplanted with bone marrow from young (3-4 weeks) WT and p75KO
mice (n=8/ bone marrow transplant (BMT) group). Although, no limb
loss was observed in mice of either bone marrow transplant group,
compared to the WT bone marrow transplant group there was more than
50% statistically significant muscle loss in p75KO bone marrow
transplant group (indicated by arrow).
[0053] FIG. 10A shows representative confocal images of
non-ischemic (right) mouse limb 56 days after transplantation with
bone marrow-derived mononuclear cells from WT/GFP mice and 28 days
after hind limb surgery on contralateral (left) limb.
Immunofluorescence in the far left panels is TopRo3 that was used
to visualize nuclei. No GFP-positive cells were detected in
non-ischemic limbs of operated mice. Intact muscle fibers emit
negligible autofluorescence. FIG. 10B shows representative confocal
images of operated ischemic limb in the border zone (indicated by
dotted line). Intact and ischemic areas of operated limb muscle
fibers were first delineated after hematoxylin and eosin (H&E)
staining in adjacent sections. GFP-positive cells (green
fluorescence) horned only to the ischemic areas of operated limbs.
FIG. 10C shows a representative confocal images of operated limbs
showing abundance of GFP-positive bone marrow-derived cells in
ischemic areas.
[0054] FIGS. 11A and 11B show representative confocal images of
ischemic and ischemic border zone muscles of operated limbs of mice
transplanted with WT/GFP bone marrow-derived mononuclear cells 28
days prior to hind limb surgery. Immunostaining with isolectin B4
(middle panels) identifies endothelial lineage cells and
GFP-positive cells (left panels) identifies bone marrow-derived
cells. In merged images, double-positive positive (right panels)
cells represent bone marrow-derived EPCs.
DETAILED DESCRIPTION OF THE INVENTION
[0055] In general, the invention provides compositions and methods
for modulating p75 receptor/TNFR2 expression or activity for the
prevention or treatment of ischemia.
[0056] As reported in more detail below, the invention is based in
part on the discovery that signaling through the p75 receptor/TNFR2
was required for collateral vessel development in ischemia-induced
neovascularization. Accordingly, the invention provides methods and
compositions for treating or preventing ischemia by enhancing p75
receptor/TNFR2 signaling to increase the growth of collateral blood
vessels.
p75/TNFR2
[0057] The p75 receptor/TNFR2 is a 415-amino acid polypeptide with
a single membrane-spanning domain and has an extracellular domain
with sequence similarity to nerve growth factor receptor (Schall et
al., "Molecular cloning and expression of a receptor for human
tumor necrosis factor," Cell 61: 361-370, 1990). Human p75/TNFR2
gene spans nearly 43 kb and consists of 10 exons and 9 introns
(Beltinger et al., Physical mapping and genomic structure of the
human TNFR2 gene. Genomics 35: 94-100, 1996). The amino acid
sequence of p75/TNFR2 is provided at GenBank Accession No.
NP.sub.--001057. A nucleic acid sequence encoding the p75/TNFR2
polypeptide is provided at NM.sub.--001066.
P75/TNFR2 Polypeptides and Analogs
[0058] Overexpression of a P75/TNFR2 polypeptide or fragment
thereof promotes angiogenesis and is useful for the treatment of
ischemia. Included in the invention are P75/TNFR2 polypeptides,
analogs, or fragments thereof, that are modified in ways that
enhance their ability to promote angiogenesis. In one embodiment,
the invention provides methods for optimizing a P75/TNFR2 amino
acid sequence or nucleic acid sequence by producing an alteration
in the sequence. Such alterations may include certain mutations,
deletions, insertions, or post-translational modifications. The
invention further includes analogs of any naturally-occurring
polypeptide of the invention. Analogs can differ from a
naturally-occurring polypeptide of the invention by amino acid
sequence differences, by post-translational modifications, or by
both. Analogs of the invention will generally exhibit at least 85%,
more preferably 90%, and most preferably 95% or even 99% identity
with all or part of a naturally-occurring amino, acid sequence of
the invention. The length of sequence comparison is at least 5, 10,
15 or 20 amino acid residues, preferably at least 25, 50, or 75
amino acid residues, and more preferably more than 100 amino acid
residues. Again, in an exemplary approach to determining the degree
of identity, a BLAST program may be used, with a probability score
between e.sup.-3 and e.sup.-100 indicating a closely related
sequence. Modifications include in vivo and in vitro chemical
derivatization of polypeptides, e.g., acetylation, carboxylation,
phosphorylation, or glycosylation; such modifications may occur
during polypeptide synthesis or processing or following treatment
with isolated modifying enzymes. Analogs can also differ from the
naturally-occurring polypeptides of the invention by alterations in
primary sequence. These include genetic variants, both natural and
induced (for example, resulting from random mutagenesis by
irradiation or exposure to ethanemethylsulfate or by site-specific
mutagenesis as described in Sambrook, Fritsch and Maniatis,
Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989,
or Ausubel et al., supra). Also included are cyclized peptides,
molecules, and analogs which contain residues other than L-amino
acids, e.g., D-amino acids or non-naturally occurring or synthetic
amino acids, e.g., .beta. or .gamma. amino acids.
[0059] In addition to full-length polypeptides, the invention also
includes fragments of any one of the polypeptides of the invention.
As used herein, the term "a fragment" means at least 10, 25, 50,
75, 100, 150, or 200 amino acids. In other embodiments a fragment
is at least 20 contiguous amino acids, at least 30 contiguous amino
acids, or at least 50 contiguous amino acids, and in other
embodiments at least 60 to 80 or more contiguous amino acids.
Fragments of the invention can be generated by methods known to
those skilled in the art or may result from normal protein
processing (e.g., removal of amino acids from the nascent
polypeptide that are not required for biological activity or
removal of amino acids by alternative mRNA splicing or alternative
protein processing events).
[0060] Non-protein p75/TNFR2 analogs having a chemical structure
designed to mimic p75/TNFR2 functional activity can be administered
according to methods of the invention. p75/TNFR2 analogs may exceed
the physiological activity of the original polypeptide. Methods of
analog design are well known in the art, and synthesis of analogs
can be carried out according to such methods by modifying the
chemical structures such that the resultant analogs exhibit the
angiogenesis promoting activity of a reference p75/TNFR2
polypeptide. These chemical modifications include, but are not
limited to, substituting alternative R groups and varying the
degree of saturation at specific carbon atoms of a reference
p75/TNFR2 polypeptide. Preferably, the p75/TNFR2 analogs are
relatively resistant to in vivo degradation, resulting in a more
prolonged therapeutic effect upon administration. Assays for
measuring functional activity include, but are not limited to,
those described in the Examples below.
Treatment of an Ischemic Disease
[0061] The increased expression of p75/TNFR2 in a cell prevents or
treats ischemia. Ischemia results when blood flow to a cell,
tissue, or organ is interrupted. Tissue damage related to apoptotic
cell death often results. Ischemic diseases are characterized by
cell or tissue damage related to hypoxia. Exemplary ischemic
diseases include, but are not limited to, ischemic injuries caused
by a myocardial infarction, a stroke, a transient ischemic episode,
a reperfusion injury, physical injury, renal failure, a secondary
exsanguination, or blood flow interruption resulting from any other
primary diseases. The effects of ischemia are particularly
devastating in the brain, when stroke, traumatic brain injury,
myocardial infarction, or a transient ischemic attack limits blood
flow to the tissues of the CNS. Accordingly, the invention provides
therapeutic and prophylactic compositions useful for the treatment
of ischemia that affects a variety of cells, tissues, or organs.
Such therapies include polynucleotide therapies, polypeptide
therapies, as well as the delivery of endothelial progenitor cells
(EPCs) expressing a heterologous p75/TNFR2. If desired, such cells
may be removed from the patient, transfected, and then returned to
the patient for the treatment of ischemia.
[0062] Delivery of therapeutic agents to ischemic tissues (e.g.,
muscle tissue, including cardiac tissue, and neural tissue,
including the CNS) can be achieved by several methods. One method
relies on neurosurgical techniques. In the case of gravely ill
patients, surgical intervention is warranted despite its attendant
risks. For instance, therapeutic agents can be delivered by direct
physical introduction into the CNS, such as intraventricular,
intralesional, or intrathecal injection. Intraventricular injection
can be facilitated by an intraventricular catheter, for example,
attached to a reservoir, such as an Ommaya reservoir. Methods of
introduction are also provided by rechargeable or biodegradable
devices.
[0063] In addition, the invention provides methods of screening for
compounds that increase the biological activity or expression of
p75/TNFR2 or that inhibit the biological activity or expression of
p75/TNFR2. Such compounds are useful for enhancing angiogenesis,
limiting the tissue damage associated with ischemia (e.g., by
enhancing the survival of cells at risk of cell death associated
with ischemia). Compounds that enhance p75/TNFR2 biological
activity (e.g., angiogenesis enhancing activity) or expression may
be used to treat or prevent ischemia in cells, tissues, or organs.
Individuals at increased risk of an ischemic disease due to a
hereditary condition are also candidates for such treatment.
p75/TNFR2 Polynucleotide Therapy
[0064] Therapy featuring a nucleic acid molecule encoding a p75
receptor/TNFR2 polypeptide, variant, or fragment thereof is one
therapeutic approach for treating ischemia. Such nucleic acid
molecules can be delivered to cells (e.g., endothelial progenitor
cells, or bone marrow derived cells) of a subject before, during,
or after an ischemic episode. Such delivery may take place in vivo
or ex vivo. In one embodiment, a human EPC is removed from a donor,
tranfected with a polynucleotide ex vivo, and then injected into a
recipient patient in need thereof. Polynucleotide therapy has been
successfully used to enhance angiogenesis. For example, the
promotion of angiogenesis in the treatment of ischemia was
demonstrated in a rabbit model and in human clinical trials with
VEGF using a Hydrogel-coated angioplasty balloon as the gene
delivery system (Takeshita, et al., Laboratory Investigation,
75:487-502 (1996); Isner, et al., Lancet, 348:370 (1996)).
Successful transfer and sustained expression of the VEGF gene in
the vessel wall subsequently augmented neovascularization in the
ischemic limb (Takeshita, et al., Laboratory Investigation,
75:487-502 (1996); Isner, et al., Lancet, 348:370 (1996)). In
addition, it has been demonstrated that direct intramuscular
injection of DNA encoding VEGF into ischemic tissue induces
angiogenesis, providing the ischemic tissue with increased blood
vessels (U.S. Ser. No. 08/545,998; Tsurumi et al., Circulation
94(12):3281-90, 1996). The nucleic acid molecules must be delivered
to the cells of a subject in a form in which they can be taken up
so that therapeutically effective levels of an p75/TNFR2
polypeptide or fragment thereof can be produced.
[0065] Transducing viral (e.g., retroviral, adenoviral, and
adeno-associated viral) vectors can be used for somatic cell gene
therapy, especially because of their high efficiency of infection
and stable integration and expression (see, e.g., Cayouette et al.,
Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye
Research 15:833-844, 1996; Bloomer et al., Journal of Virology
71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and
Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For
example, a polynucleotide encoding a p75/TNFR2 receptor polypeptide
variant, or a fragment thereof, can be cloned into a retroviral
vector and expression can be driven from its endogenous promoter,
from the retroviral long terminal repeat, or from a promoter
specific for a target cell type of interest. Other viral vectors
that can be used include, for example, a vaccinia virus, a bovine
papilloma virus, or a herpes virus, such as Epstein-Barr Virus
(also see, for example, the vectors of Miller, Human Gene Therapy
15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al.,
BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion
in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278,
1991; Cornetta et al., Nucleic Acid Research and Molecular Biology
36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood
Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990,
1989; Le Gal La Salle et al., Science 259:988-990, 1993; and
Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are
particularly well developed and have been used in clinical settings
(Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al.,
U.S. Pat. No. 5,399,346). In one embodiment, a viral vector is used
to administer a p75/TNFR2 receptor nucleic acid molecule
systemically.
[0066] Non-viral approaches can also be employed for the
introduction of a therapeutic nucleic acid molecule to a cell of a
subject requiring modulation of angiogenesis or vascularization.
For example, a nucleic acid molecule can be introduced into a cell
by administering the nucleic acid in the presence of lipofection
(Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono
et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J.
Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology
101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et
al., Journal of Biological Chemistry 263:14621, 1988; Wu et al.,
Journal of Biological Chemistry 264:16985, 1989), or by
micro-injection under surgical conditions (Wolff et al., Science
247:1465, 1990). Preferably the nucleic acids are administered in
combination with a liposome and protamine.
[0067] Gene transfer can also be achieved using non-viral means
involving transfection in vitro. Such methods include the use of
calcium phosphate, DEAE dextran, electroporation, and protoplast
fusion. Liposomes can also be potentially beneficial for delivery
of DNA into a cell. Transplantation of genes into the affected
tissues of a subject (e.g., tissues subject to ischemia or
requiring enhanced vascularization) can also be accomplished by
transferring a normal nucleic acid into a cultivatable cell type ex
vivo (e.g., an autologous or heterologous primary cell or progeny
thereof), after which the cell (e.g., a bone marrow-derived cell or
EPC) (or its descendants) are injected into a targeted tissue
(e.g., an ischemic tissue).
[0068] cDNA expression for use in polynucleotide therapy methods
can be directed from any suitable promoter (e.g., the human
cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein
promoters), and regulated by any appropriate mammalian regulatory
element. For example, if desired, enhancers known to preferentially
direct gene expression in specific cell types can be used to direct
the expression of a nucleic acid. The enhancers used can include,
without limitation, those that are characterized as tissue- or
cell-specific enhancers. Alternatively, if a genomic clone is used
as a therapeutic construct, regulation can be mediated by the
cognate regulatory sequences or, if desired, by regulatory
sequences derived from a heterologous source, including any of the
promoters or regulatory elements described above.
[0069] Another therapeutic approach included in the invention
involves administration of a recombinant therapeutic, such as a
recombinant p75 receptor/TNFR2 polypeptide, variant, or fragment
thereof, either directly to the site of a potential or actual
disease-affected tissue or systemically (for example, by any
conventional recombinant protein administration technique). The
dosage of the administered polypeptide depends on a number of
factors, including the size and health of the individual subject.
For any particular subject, the specific dosage regimes should be
adjusted over time according to the individual need and the
professional judgment of the person administering or supervising
the administration of the compositions.
Endothelial Progenitor Cell Therapy
[0070] Transplantation of hematopoietic stem cells derived from
peripheral blood can provide sustained hematopoietic recovery.
(See, for example, Messinger et al., Blood 77, 211 (1991); Sheridan
et al., Lancet 339, 640 (1992); Shpall et al., J. Clin. Oncol. 12,
28 (1994). This observation is now being exploited clinically as an
alternative to bone marrow transplantation. By using techniques
similar to those employed for hematopoietic stem cells, endothelial
progenitor cells can be isolated from circulating blood. Such
cells, once isolated, can be expanded in vitro and engineered to
express one or more heterologous nucleic acid molecules. The cells
are then delivered back to the donor, or to another subject, to
achieve a therapeutic result.
[0071] To obtain the endothelial progenitor cell from peripheral
blood about 5 ml to about 500 ml of blood is taken from a donor.
Preferably, about 50 ml to about 200 ml of blood is taken.
Endothelial progenitor cells are expanded in vivo by administration
of recruitment growth factors, e.g., GM-CSF and IL-3, to the donor
prior to removing the progenitor cells. Methods for obtaining and
using hematopoietic progenitor cells in autologous transplantation
are disclosed in U.S. Pat. No. 5,199,942, the disclosure of which
is incorporated by reference. Alternatively, the cells are expanded
ex vivo using, for example, the method disclosed by U.S. Pat. No.
5,541,103. Endothelial progenitor cells may be obtained from human
mononuclear cells obtained from peripheral blood or bone marrow of
the subject before treatment. Such cells may also be obtained from
heterologous or autologous umbilical cord blood. In particular,
endothelial progenitor cells may be obtained from the leukocyte
fraction of peripheral blood. Endothelial progenitor cells may be
isolated using antibodies that recognize endothelial progenitor
cell specific antigens on immature human hematopoietic progenitor
cells. For example, CD34 is commonly shared by endothelial
progenitor cells and hematopoietic stem cells. CD34 is expressed by
all hematopoietic stem cells but is lost by hematopoietic cells as
they differentiate. Flk-1, a receptor for vascular endothelial
growth factor (VEGF) is also expressed by both early hematopoietic
stem cells and endothelial cells, but ceases to be expressed in the
course of hematopoietic differentiation.
[0072] In vitro, endothelial progenitor cells differentiate into
endothelial cells. Indeed, one can use a multipotentiate
undifferentiated cell as long as it is still capable of becoming an
endothelial cell in the presence of agents that promote its
differentiation. In vivo, heterologous, homologous, and autologous
endothelial cell progenitor grafts incorporate into sites of active
angiogenesis or blood vessel injury by selectively migrating to
such locations. Angiogenesis can be promoted in a subject by
administering a potent angiogenesis factor, such as VEGF, alone or
in combination with endothelial progenitor cells. Once the
progenitor cells are obtained by a particular separation technique,
they may be administered to a selected subject to treat a number of
conditions including, for example, unregulated angiogenesis or
blood vessel injury. The cells may also be stored in cryogenic
conditions.
[0073] The progenitor cells are administered to a subject by any
suitable means, including, for example, intravenous infusion, bolus
injection, and site directed delivery via a catheter. Preferably,
the progenitor cells obtained from the subject are readministered.
Generally, from about 10.sup.6 to about 10.sup.18 progenitor cells
are administered to a subject for transplantation. Depending on the
use of the progenitor cells, various genetic material may be
delivered to the cell (e.g., a polynucleotide encoding p75/TNFR2).
Such genetic material includes nucleic acid sequences both
exogenous and endogenous to cells into which a virus vector, for
example, a pox virus such as swine pox containing the human TNF
gene may be introduced. Additionally, it is of interest to use
genes encoding polypeptides for secretion from the endothelial
progenitor cells so as to provide for a systemic effect by the
protein encoded by the gene. Specific genes of interest include
those encoding TNF, TGF-.alpha., TGF-.beta., hemoglobin,
interleukin-1, interleukin-2, interleukin-3, interleukin-4,
interleukin-5, interleukin-6, interleukin-7, interleukin-8,
interleukin-9, interleukin-10, interleukin-11, interleukin-12 etc.,
GM-CSF, G-CSF, M-CSF, human growth factor, co-stimulatory factor
B7, insulin, factor VIII, factor IX, PDGF, EGF, NGF, IL-ira, EPO,
.beta.-globin, endothelial cell mitogens, as well as biologically
active variants of these proteins. The gene may further encode a
product that regulates expression of another gene product or blocks
one or more steps in a biological pathway. Similarly, the gene may
encode a therapeutic protein fused to a targeting polypeptide, to
deliver a therapeutic effect to a diseased tissue or organ. To
further enhance angiogenesis, endothelial cell mitogens may also be
administered to the subject in conjunction with, or subsequent to,
the administration of the endothelial progenitor cells. Endothelial
cell mitogens can be administered directly, e.g., intra-arterially,
intramuscularly, or intravenously, or a nucleic acid molecule
encoding the mitogen may be used. See, Baffour, et al., J Vase
Surg. 16(2):181-91 (1992). (bFGF); Pu, et al, Circulation,
88:208-215 (1993) (aFGF); Yanagisawa-Miwa, et al., Science.
257(5075):1401-3 (1992). (bFGF); Ferrara, et al., Biochem. Biophys.
Res. Commun., 161:851-855 (1989) (VEGF); (Takeshita, et al.,
Circulation, 90:228-234 (1994)).
[0074] The nucleic acid encoding the endothelial cell mitogen can
be administered to a blood vessel by perfusing the ischemic tissue
or to a site of vascular injury via a catheter, for example, a
hydrogel catheter, as described by U.S. Ser. No. 08/675,523, the
disclosure of which is herein incorporated by reference. The
nucleic acid also can be delivered by injection directly into the
ischemic tissue using the method described in U.S. Ser. No.
08/545,998.
[0075] As used herein the term "endothelial cell mitogen" means any
protein, polypeptide, variant or portion thereof that is capable
of, directly or indirectly, inducing endothelial cell growth. Such
proteins include, for example, acidic and basic fibroblast growth
factors (aFGF) (GenBank Accession No. NP.sub.--149127) and bFGF
(GenBank Accession No. AAA52448), vascular endothelial growth
factor (VEGF) (GenBank Accession No. AAA35789 or
NP.sub.--001020539), epidermal growth factor (EGF) (GenBank
Accession No. NP.sub.--001954), transforming growth factor .alpha.
(TGF-.alpha.) (GenBank Accession No. NP.sub.--003227) and
transforming growth factor .beta. (TFG-.beta.) (GenBank Accession
No. 1109243A), platelet-derived endothelial cell growth factor
(PD-ECGF) (GenBank Accession No. NP.sub.--001944), platelet-derived
growth factor (PDGF) (GenBank Accession No. 1109245A), tumor
necrosis factor .alpha. (TNF-.alpha.) (GenBank Accession No.
CAA26669), hepatocyte growth factor (HGF) (GenBank Accession No.
BAA14348), insulin like growth factor (IGF) (GenBank Accession No.
P08833), erythropoietin (GenBank Accession No. P01588), colony
stimulating factor (CSF), macrophage-CSF (M-CSF) (GenBank Accession
No. AAB59527), granulocyte/macrophage CSF (GM-CSF) (GenBank
Accession No. NP.sub.--000749), monocyte chemotactic protein-1
(GenBank Accession No. P13500) and nitric oxide synthase (NOS)
(GenBank Accession No. AAA36365). See, Klagsbrun, et al., Annu.
Rev. Physiol., 53:217-239 (1991); Folkman, et al., J. Biol. Chem.,
267:10931-10934 (1992) and Symes, et al., Current Opinion in
Lipidology, 5:305-312 (1994). Variants or fragments of a mitogen
may be used as long as they induce or promote endothelial cell or
endothelial progenitor cell growth. Preferably, the endothelial
cell mitogen contains a secretory signal sequence that facilitates
secretion of the protein. Proteins having native signal sequences,
e.g., VEGF, are preferred. Proteins that do not have native signal
sequences, e.g., bFGF, can be modified to contain such sequences
using routine genetic manipulation techniques. See, Nabel et al.,
Nature, 362:844 (1993).
[0076] The nucleotide sequence of numerous endothelial cell
mitogens, are readily available through a number of computer data
bases, for example, GenBank, EMBL and Swiss-Prot. Using this
information, a DNA segment encoding the desired may be chemically
synthesized or, alternatively, such a DNA segment may be obtained
using routine procedures in the art, e.g, PCR amplification. A DNA
encoding VEGF is disclosed in U.S. Pat. No. 5,332,671, the
disclosure of which is herein incorporated by reference.
[0077] In certain situations, it may be desirable to use nucleic
acids encoding two or more different proteins in order optimize the
therapeutic outcome. For example, DNA encoding two proteins, e.g.,
VEGF and bFGF, can be used, and provides an improvement over the
use of bFGF alone, or an angiogenic factor (e.g., fibroblast growth
factor (bFGF), acidic FGF (aFGF), FGF-5, vascular endothelial
growth factor isoforms (VEGF), angiopoietin-1 (Ang-1),
angiopoietin-2 (Ang-2), platelet-derived endothelial cell growth
factor (PD-ECGF), hepatocyte growth factor (HGF) (GenBank Accession
No. BAA14348), interleukin-8 (IL-8) (GenBank Accession No.
NP.sub.--000575), granulocyte-colony stimulating factor (G-CSF),
placental growth factor (GenBank Accession No. NP.sub.--002623.),
proliferin (GenBank Accession No. S48671), angiogenin
(NP.sub.--001136), TNF.alpha., Transforming growth factor-.beta.
(GenBank Accession No. 1109243A)) can be combined with other genes
or their encoded gene products to enhance the activity of targeted
cells, while simultaneously inducing angiogenesis, including, for
example, nitric oxide synthase, L-arginine, fibronectin, urokinase,
plasminogen activator and heparin.
[0078] The effective dose of the nucleic acid will be a function of
the particular expressed protein, the target tissue, the subject
and his or her clinical condition. Effective amount of DNA are
between about 1 and 4000 .mu.g, more preferably about 1000 and
2000, most preferably between about 2000 and 4000.
Kits
[0079] The invention provides kits for the treatment or prevention
of ischemia or related tissue damage. In one embodiment, the kit
includes a therapeutic or prophylactic composition containing an
effective amount of a p75 receptor/TNFR2 polypeptide or an
expression vector encoding the p75 receptor/TNFR2 polypeptide in
unit dosage form. In some embodiments, the kit comprises a sterile
container which contains a therapeutic or prophylactic composition;
such containers can be boxes, ampules, bottles, vials, tubes, bags,
pouches, blister-packs, or other suitable container forms known in
the art. Such containers can be made of plastic, glass, laminated
paper, metal foil, or other materials suitable for holding
medicaments.
[0080] If desired an expression vector of the invention is provided
together with instructions for administering it to a subject having
or at risk of developing ischemia. The instructions will generally
include information about the use of the composition for the
treatment or prevention of ischemia or for enhancing angiogenesis
to a tissue in need thereof. In other embodiments, the instructions
include at least one of the following: description of the
expression vector; dosage schedule and administration for treatment
or prevention of ischemia or symptoms thereof; precautions;
warnings; indications; counter-indications; overdosage information;
adverse reactions; animal pharmacology; clinical studies; and/or
references. The instructions may be printed directly on the
container (when present), or as a label applied to the container,
or as a separate sheet, pamphlet, card, or folder supplied in or
with the container.
Screening Assays
[0081] Signaling through the p75/TNFR2 polypeptide was required for
collateral vessel development in ischemia-induced
neovascularization. Based in part on this discovery, compositions
of the invention are useful for the high-throughput low-cost
screening of candidate compounds that enhance p75/TNFR2 expression
or activity. Such compounds are useful for the treatment or
prevention of ischemia. Tissues or cells treated with a candidate
compound are compared to untreated control samples to identify
therapeutic agents that enhance the p75 receptor/TNFR2 expression
or activity. If desired, such compounds are further tested in vitro
or in vivo for their effects on angiogenesis using any method known
in the art. Any number of methods are available for carrying out
screening assays to identify new candidate compounds that bind a
p75 receptor/TNFR2 polypeptide and enhance the angiogenesis
promoting activity of the receptor.
[0082] In one working example, candidate compounds are added at
varying concentrations to the culture medium of cultured cells. p75
receptor/TNFR2 expression (e.g., polypeptide or mRNA expression) is
then measured using standard methods. The expression of a p75
receptor/TNFR2 in the presence of the candidate compound is
compared to the level measured in a control culture medium lacking
the candidate molecule. A compound that increases the expression of
a p75 receptor/TNFR2 is useful for promoting an increase in
angiogenesis. Such compounds are considered useful in the
invention; such a compound may be used, for example, as a
therapeutic to prevent, delay, ameliorate, stabilize, reduce the
severity of, or treat ischemia. In other embodiments, the candidate
compound prevents, delays, ameliorates, stabilizes, or treats a
disease or disorder related to ischemia or reduces tissue damage or
apoptosis associated with ischemia. Such therapeutic compounds are
useful in vivo as well as ex vivo.
[0083] In some embodiments, a compound that promotes an increase in
the biological activity of a p75 receptor/TNFR2 of the invention is
considered useful. Such compounds are added to a culture containing
p75 receptor/TNFR2 expressing cells. The effect of the compound on
p75 receptor/TNFR2 biological activity is measured and compared to
p75 receptor/TNFR2 biological activity in the absence of the
candidate compound. Again, a candidate compound that enhances the
biological activity of a p75 receptor/TNFR2 may be used, for
example, as a therapeutic to treat or prevent ischemia.
[0084] One skilled in the art appreciates that the effects of a
candidate compound on the p75 receptor/TNFR2 expression or
biological activity are typically compared to the expression or
activity of the p75 receptor/TNFR2 in the absence of the candidate
compound. Thus, the screening methods include comparing the value
of a cell modulated by a candidate compound to a reference value of
an untreated control cell.
[0085] Expression levels can be compared by procedures well known
in the art such as RT-PCR, Northern blotting, Western blotting,
flow cytometry, immunocytochemistry, binding to magnetic and/or
antibody-coated beads, in situ hybridization, fluorescence in situ
hybridization (FISH), flow chamber adhesion assay, and ELISA,
microarray analysis, or colorimetric assays, such as the Bradford
Assay and Lowry Assay. Changes in angiogenesis can be assayed by
methods described herein or by any method known in the art,
including Angiogram, Computed Tomography Angiography (CTA), Duplex
Ultrasound, magenetic resonance angiography, vascular ultrasound,
or angiogram.
[0086] Molecules that increase the p75 receptor/TNFR2 expression or
activity include organic molecules, peptides, peptide mimetics,
polypeptides, nucleic acids, and antibodies that bind to a the p75
receptor/TNFR2 nucleic acid sequence or polypeptide and increase
its expression or biological activity are preferred.
[0087] In yet another example, candidate compounds are screened for
those that specifically bind to a p75 receptor/TNFR2. The efficacy
of such a candidate compound is dependent upon its ability to
interact with the p75 receptor/TNFR2, or with functional
equivalents thereof. Such an interaction can be readily assayed
using any number of standard binding techniques and functional
assays (e.g., those described in Ausubel et al., supra). In one
embodiment, the compound is assayed in vitro for receptor
binding.
[0088] In one particular working example, a candidate compound that
binds to a p75 receptor/TNFR2 is identified using a
chromatography-based technique. For example, a recombinant
polypeptide of the invention may be purified by standard techniques
from cells engineered to express the polypeptide (e.g., those
described above) and may be immobilized on a column. A solution of
candidate compounds is then passed through the column, and a
compound specific for the p75 receptor/TNFR2 is identified on the
basis of its ability to bind to the polypeptide and be immobilized
on the column. To isolate the compound, the column is washed to
remove non-specifically bound molecules, and the compound of
interest is then released from the column and collected. Similar
methods may be used to isolate a compound bound to a polypeptide
microarray. Compounds identified using such methods are then
assayed for their effect on angiogenesis as described herein.
[0089] In another example, the compound, e.g., the substrate, is
coupled to a radioisotope or enzymatic label such that binding of
the compound to the p75 receptor/TNFR2 can be determined by
detecting the labeled compound, e.g., substrate, in a complex. For
example, compounds can be labeled with .sup.125I, .sup.35S,
.sup.14C, or .sup.3H, either directly or indirectly, and the
radioisotope detected by direct counting of radioemmission or by
scintillation counting. Alternatively, compounds can be
enzymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[0090] In yet another embodiment, a cell-free assay is provided in
which the p75 receptor/TNFR2 or a biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the polypeptide thereof is evaluated.
[0091] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al.,
U.S. Pat. No. 4,868,103). A fluorophore label on the first, `donor`
molecule is selected such that its emitted fluorescent energy will
be absorbed by a fluorescent label on a second, `acceptor`
molecule, which in turn is able to fluoresce due to the absorbed
energy. Alternately, the `donor` protein molecule may simply
utilize the natural fluorescent energy of tryptophan residues.
Labels are chosen that emit different wavelengths of light, such
that the `acceptor` molecule label may be differentiated from that
of the `donor`. Since the efficiency of energy transfer between the
labels is related to the distance separating the molecules, the
spatial relationship between the molecules can be assessed. In a
situation in which binding occurs between the molecules, the
fluorescent emission of the `acceptor` molecule label in the assay
should be maximal. An FET binding event can be conveniently
measured through standard fluorometric detection means well known
in the art (e.g., using a fluorimeter).
[0092] In another embodiment, determining the ability of a test
compound to bind to the p75 receptor/TNFR2 can be accomplished
using real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S. and Urbaniczky, C., Anal. Chem. 63:2338-2345, 1991;
and Szabo et al., Curr. Opin. Struct. Biol. 5:699-705, 1995).
"Surface plasmon resonance" or "BIA" detects biospecific
interactions in real time, without labeling any of the interactants
(e.g., BIAcore). Changes in the mass at the binding surface
(indicative of a binding event) result in alterations of the
refractive index of light near the surface (the optical phenomenon
of surface plasmon resonance (SPR)), resulting in a detectable
signal that can be used as an indication of real-time reactions
between biological molecules.
[0093] It may be desirable to immobilize either the candidate
compound or its p75 receptor/TNFR2 target to facilitate separation
of complexed from uncomplexed forms of one or both of the proteins,
as well as to accommodate automation of the assay. Binding of a
candidate compound to the p75 receptor/TNFR2, or interaction of a
test compound with a target molecule in the presence and absence of
a candidate compound, can be accomplished in any vessel suitable
for containing the reactants. Examples of such vessels include
microtiter plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a domain
that allows one or both of the proteins to be bound to a matrix.
For example, glutathione-S-transferase/p75 receptor/TNFR2 fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtiter
plates, which are then combined with the test compound or the test
compound and a sample comprising the GST-tagged p75 receptor/TNFR2
polypeptide, and the mixture incubated under conditions conducive
to complex formation (e.g., at physiological conditions for salt
and pH). Following incubation, the beads or microtiter plate wells
are washed to remove any unbound components, the matrix immobilized
in the case of beads, complex determined either directly or
indirectly, for example, as described above.
[0094] Other techniques for immobilizing a complex of a compound
and the p75 receptor/TNFR2 polypeptide on matrices include using
conjugation of biotin and streptavidin. For example, biotinylated
proteins can be prepared from biotin-NHS (N-hydroxy-succinimide)
using techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
[0095] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0096] In one embodiment, a p75 receptor/TNFR2 antibody is
identified that reacts with an epitope on the p75 receptor/TNFR2.
Methods for detecting binding of expression antibody to the
receptor are known in the art and include immunodetection of
complexes, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the channel. Antibodies that
bind the p75 receptor/TNFR2 are then tested for the ability to
activate the receptor. Such antibodies may tested for angiogenesis
promoting activity as, described herein.
[0097] Alternatively, cell free assays can be conducted that assay
the interaction of a compound with a p75 receptor/TNFR2 or that
assay the activity of a p75 receptor/TNFR2. In such an assay, the
reaction products are separated from unreacted components, by any
of a number of standard techniques, including but not limited to:
differential centrifugation (see, for example, Rivas, G., and
Minton, A. P., Trends Biochem Sci 18:284-7, 1993); chromatography
(gel filtration chromatography, ion-exchange chromatography);
electrophoresis and immunoprecipitation (see, for example, Ausubel,
F. et al., eds. (1999) Current Protocols in Molecular Biology, J.
Wiley: New York). Such resins and chromatographic techniques are
known to one skilled in the art (see, e.g., Heegaard, N. H., J Mol
Recognit 11:141-8, 1998; Hage, D. S., and Tweed, S. A., J
Chromatogr B Biomed Sci Appl. 699:499-525, 1997). Further,
fluorescence energy transfer may also be conveniently utilized, as
described herein, to detect binding without further purification of
the complex from solution. Preferably, cell free assays preserve
the structure of the the p75 receptor/TNFR2, e.g., by including a
membrane component or synthetic membrane components.
[0098] In a specific embodiment, the assay includes contacting the
p75 receptor/TNFR2 polypeptide or biologically active portion
thereof with a known compound which binds the p75 receptor/TNFR2
polypeptide to form an assay mixture, contacting the assay mixture
with a test compound, and determining the ability of the test
compound to interact with a the p75 receptor/TNFR2, wherein
determining the ability of the test compound to interact with a the
p75 receptor/TNFR2 includes determining the ability of the test
compound to preferentially bind to the p75 receptor/TNFR2, or to
modulate the activity of the p75 receptor/TNFR2, as compared to the
known compound.
[0099] Compounds isolated by this method (or any other appropriate
method) may, if desired, be further purified (e.g., by high
performance liquid chromatography). In addition, these candidate
compounds may be tested for their ability to increase the activity
of a p75 receptor/TNFR2 (e.g., as described herein). Compounds
isolated by this approach may also be used, for example, as
therapeutics to treat or prevent ischemia in a subject. Compounds
that are identified as binding to the p75 receptor/TNFR2 with an
affinity constant less than or equal to 10 mM are considered
particularly useful in the invention. Alternatively, any in vivo
protein interaction detection system, for example, any two-hybrid
assay may be utilized.
[0100] In another embodiment, a the p75 receptor/TNFR2 nucleic acid
described herein is expressed as a transcriptional or translational
fusion with a detectable reporter, and expressed in an isolated
cell (e.g., mammalian or insect cell) under the control of an
endogenous or a heterologous promoter. The cell expressing the
fusion protein is then contacted with a candidate compound, and the
expression of the detectable reporter in that cell is compared to
the expression of the detectable reporter in an untreated control
cell. A candidate compound that increases the expression of the
detectable reporter is a compound that is useful for the treatment
of ischemia. In preferred embodiments, the candidate compound
increases the expression of a reporter gene fused to a the p75
receptor/TNFR2 nucleic acid molecule.
[0101] Each of the DNA sequences listed herein may also be used in
the discovery and development of a therapeutic compound for the
treatment of intestinal inflammation or an inflammatory bowel
disease. The encoded protein, upon expression, can be used as a
target for the screening of drugs. Additionally, the DNA sequences
encoding the amino terminal regions of the encoded protein or
Shine-Delgarno or other translation facilitating sequences of the
respective mRNA can be used to construct sequences that promote the
expression of the coding sequence of interest. Such sequences may
be isolated by standard techniques (Ausubel et al., supra).
[0102] Small molecules of the invention preferably have a molecular
weight below 2,000 daltons, more preferably between 300 and 1,000
daltons, and most preferably between 400 and 700 daltons. It is
preferred that these small molecules are organic molecules.
Test Compounds and Extracts
[0103] In general, compounds capable of increasing the activity of
the p75 receptor/TNFR2 are identified from large libraries of both
natural product or synthetic (or semi-synthetic) extracts or
chemical libraries or from polypeptide or nucleic acid libraries,
according to methods known in the art. Those skilled in the field
of drug discovery and development will understand that the precise
source of test extracts or compounds is not critical to the
screening procedure(s) of the invention. Compounds used in screens
may include known compounds (for example, known therapeutics used
for other diseases or disorders). Alternatively, virtually any
number of unknown chemical extracts or compounds can be screened
using the methods described herein. Examples of such extracts or
compounds include, but are not limited to, plant-, fungal-,
prokaryotic- or animal-based extracts, fermentation broths, and
synthetic compounds, as well as modification of existing
compounds.
[0104] Numerous methods are also available for generating random or
directed synthesis (e.g., semi-synthesis or total synthesis) of any
number of chemical compounds, including, but not limited to,
saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
Synthetic compound libraries are commercially available from
Brandon Associates (Merrimack, N.H.) and Aldrich Chemical
(Milwaukee, Wis.). Alternatively, chemical compounds to be used as
candidate compounds can be synthesized from readily available
starting materials using standard synthetic techniques and
methodologies known to those of ordinary skill in the art.
[0105] Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds identified by the methods described herein are known
in the art and include, for example, those such as described in R.
Larock, Comprehensive Organic Transformations, VCH Publishers
(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser
and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis,
John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of
Reagents for Organic Synthesis, John Wiley and Sons (1995), and
subsequent editions thereof.
[0106] Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant, and animal extracts are commercially
available from a number of sources, including Biotics (Sussex, UK),
Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.
Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In
addition, natural and synthetically produced libraries are
produced, if desired, according to methods known in the art, e.g.,
by standard extraction and fractionation methods. Examples of
methods for the synthesis of molecular libraries can be found in
the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.
U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA
91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho
et al., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int.
Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl.
33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233, 1994.
Furthermore, if desired, any library or compound is readily
modified using standard chemical, physical, or biochemical
methods.
[0107] Libraries of compounds may be presented in solution (e.g.,
Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature
354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria
(Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No.
5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA
89:1865-1869, 1992) or on phage (Scott and Smith, Science
249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al.
Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol.
222:301-310, 1991; Ladner supra.).
[0108] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their activity should be employed whenever possible.
[0109] When a crude extract is found to increase the activity of a
p75 receptor/TNFR2, or to bind the p75 receptor/TNFR2, further
fractionation of the positive lead extract is necessary to isolate
chemical constituents responsible for the observed effect. Thus,
the goal of the extraction, fractionation, and purification process
is the careful characterization and identification of a chemical
entity within the crude extract that increases the activity of a
the p75 receptor/TNFR2. Methods of fractionation and purification
of such heterogenous extracts are known in the art. If desired,
compounds shown to be useful as therapeutics for the treatment of
ischemia are chemically modified according to methods known in the
art.
Pharmaceutical Therapeutics
[0110] The invention provides a simple means for identifying
compositions (including nucleic acids, peptides, small molecule
inhibitors, and mimetics) capable of acting as therapeutics for the
treatment of ischemia. Accordingly, a chemical entity discovered to
have medicinal value using the methods described herein is useful
as a drug or as information for structural modification of existing
compounds, e.g., by rational drug design. Such methods are useful
for screening compounds having that treat or prevent ischemia.
[0111] For therapeutic uses, the compositions or agents of the
invention described herein may be administered systemically, for
example, formulated in a pharmaceutically-acceptable buffer such as
physiological saline. Alternatively, compositions or agents of the
invention described herein are delivered directly to a tissue in
need of such treatment, such a tissue suffering from or at risk of
ischemia. Preferable routes of administration include, for example,
subcutaneous, intravenous, interperitoneally, intramuscular, or
intradermal injections that provide continuous, sustained levels of
the drug in the subject. Treatment of human subjects or other
animals will be carried out using a therapeutically effective
amount of an ischemia therapeutic in a physiologically-acceptable
carrier. Suitable carriers and their formulation are described, for
example, in Remington's Pharmaceutical Sciences by E. W. Martin.
The amount of the therapeutic agent to be administered varies
depending upon the manner of administration, the age and body
weight of the subject, and with the clinical symptoms of ischemia.
Generally, amounts will be in the range of those used for other
agents used in the treatment of other diseases associated with
ischemia, although in certain instances lower amounts will be
needed because of the increased specificity of the compound. A
compound is administered at a dosage that controls the clinical or
physiological symptoms of ischemia or that reduces tissue damage
associated with ischemia as determined by a diagnostic method known
to one skilled in the art, or using any that assay that measures
the expression or the biological activity of a the p75
receptor/TNFR2 polypeptide.
Formulation of Pharmaceutical Compositions
[0112] The administration of a compound for the treatment of
ischemia may be by any suitable means that results in a
concentration of the therapeutic that, combined with other
components, is effective in preventing, ameliorating, reducing, or
stabilizing an ischemic disease. The compound may be contained in
any appropriate amount in any suitable carrier substance, and is
generally present in an amount of 1-95% by weight of the total
weight of the composition. The composition may be provided in a
dosage form that is suitable for parenteral (e.g., subcutaneously,
intravenously, intramuscularly, or intraperitoneally)
administration route. The pharmaceutical compositions may be
formulated according to conventional pharmaceutical practice (see,
e.g., Remington: The Science and Practice of Pharmacy (20th ed.),
ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and
Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.
C. Boylan, 1988-1999, Marcel Dekker, New York).
[0113] Pharmaceutical compositions according to the invention may
be formulated to release the active compound substantially
immediately upon administration or at any predetermined time or
time period after administration. The latter types of compositions
are generally known as controlled release formulations, which
include (i) formulations that create a substantially constant
concentration of the drug within the body over an extended period
of time; (ii) formulations that after a predetermined lag time
create a substantially constant concentration of the drug within
the body over an extended period of time; (iii) formulations that
sustain action during a predetermined time period by maintaining a
relatively, constant, effective level in the body with concomitant
minimization of undesirable side effects associated with
fluctuations in the plasma level of the active substance (sawtooth
kinetic pattern); (iv) formulations that localize action by, e.g.,
spatial placement of a controlled release composition adjacent to
or in the central nervous system or cerebrospinal fluid; (v)
formulations that allow for convenient dosing, such that doses are
administered, for example, once every one or two weeks; and (vi)
formulations that target an ischemic disease by using carriers or
chemical derivatives to deliver the therapeutic agent to a
particular cell type (e.g., cell subject to reduced blood flow
related to ischemia) whose function is perturbed in an ischemia
disease. For some applications, controlled release formulations
obviate the need for frequent dosing during the day to sustain the
plasma level at a therapeutic level.
[0114] Any of a number of strategies can be pursued in order to
obtain controlled release in which the rate of release outweighs
the rate of metabolism of the compound in question. In one example,
controlled release is obtained by appropriate selection of various
formulation parameters and ingredients, including, e.g., various
types of controlled release compositions and coatings. Thus, the
therapeutic is formulated with appropriate excipients into a
pharmaceutical composition that, upon administration, releases the
therapeutic in a controlled manner. Examples include single or
multiple unit tablet or capsule compositions, oil solutions,
suspensions, emulsions, microcapsules, microspheres, molecular
complexes, nanoparticles, patches, and liposomes.
[0115] Human dosage amounts for any therapy described herein can
initially be determined by extrapolating from the amount of
compound used in mice, as a skilled artisan recognizes it is
routine in the art to modify the dosage for humans compared to
animal models. In certain embodiments it is envisioned that the
dosage may vary from between about 1 mg compound/Kg body weight to
about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body
weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body
weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body
weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg
body weight to about 1000 mg/Kg body weight; or from about 150
mg/Kg body weight to about 500 mg/Kg body weight. In other
embodiments this dose may be about 1, 5, 10, 25, 50, 75, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,
1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500,
4000, 4500, 5000 mg/Kg body weight. In other embodiments, it is
envisaged that higher does may be used, such doses may be in the
range of about 5 mg compound/Kg body to about 20 mg compound/Kg
body. In other embodiments the doses may be about 8, 10, 12, 14, 16
or 18 mg/Kg body weight. Of course, a dosage amount may be adjusted
upward or downward, as is routinely done in such treatment
protocols, depending on the results of the initial clinical trials
and the needs of a particular patient.
Patient Treatment and Monitoring
[0116] The present invention provides methods of treating an
ischemic disease and/or disorders or symptoms thereof (e.g., tissue
damage related to ischemia) by enhancing angiogenesis or reducing
apoptosis. The methods comprise administering a therapeutically
effective amount of a pharmaceutical composition comprising a
polypeptide, nucleic acid molecule or compound that enhances the
expression or activity of p75/TNFR2 as described herein to a
subject (e.g., a mammal such as a human). Thus, one embodiment is a
method of treating a subject suffering from or susceptible to an
ischemic disease or disorder or symptom thereof. The method
includes the step of administering to the mammal a therapeutic
amount of an amount of a polypeptide, nucleic acid molecule or
compound herein sufficient to treat the disease or disorder or
symptom thereof, under conditions such that the disease or disorder
is treated.
[0117] The methods herein include administering to the subject
(including a subject identified as in need of such treatment) an
effective amount of a compound described herein, or a composition
described herein to produce such effect. Identifying a subject in
need of such treatment can be in the judgment of a subject or a
health care professional and can be subjective (e.g. opinion) or
objective (e.g. measurable by a test or diagnostic method).
[0118] The therapeutic methods of the invention, which include
prophylactic treatment, in general comprise administration of a
therapeutically effective amount of the compounds herein, such as a
compound of the formulae herein to a subject (e.g., animal, human)
in need thereof, including a mammal, particularly a human. Such
treatment will be suitably administered to subjects, particularly
humans, suffering from, having, susceptible to, or at risk for a
disease, disorder, or symptom thereof. Determination of those
subjects "at risk" can be made by any objective or subjective
determination by a diagnostic test or opinion of a subject or
health care provider (e.g., genetic test, enzyme or protein marker,
Marker (as defined herein), family history, and the like). The
compounds herein may be also used in the treatment of any other
disorders in which ischemia may be implicated.
[0119] In one embodiment, the invention provides a method of
monitoring treatment progress. The method includes the step of
determining a level of diagnostic marker (Marker) (e.g., any target
delineated herein modulated by a compound herein, a protein or
indicator thereof, etc.) or diagnostic measurement
(e.g.,angiogenesis or tissue perfusion) in a subject suffering from
or susceptible to a disorder or symptoms thereof associated with
ischemia, in which the subject has been administered a therapeutic
amount of a compound herein sufficient to treat the disease or
symptoms thereof. The level of Marker or diagnostic measurement
determined in the method can be compared to known levels of Marker
or diagnostic measurement in either healthy normal controls or in
other afflicted patients to establish the subject's disease status.
In preferred embodiments, a second level of Marker or diagnostic
measurement in the subject is determined at a time point later than
the determination of the first level, and the two levels are
compared to monitor the course of disease or the efficacy of the
therapy. In certain preferred embodiments, a pre-treatment level of
Marker in the subject is determined prior to beginning treatment
according to this invention; this pre-treatment level of Marker can
then be compared to the level of Marker in the subject after the
treatment commences, to determine the efficacy of the
treatment.
EXAMPLES
[0120] The TNF receptor p55 is largely believed to mediate
cytotoxic effects of TNF-.alpha., whereas p75 (TNFR2) is thought to
mediate the protective effects of the cytokine..sup.1,2 Since aging
is associated with the increased expression of p55 and the
decreased expression of p75 receptor/TNFR2 in lymphocytes,.sup.3
p75 receptor/TNFR2 may be needed in angiogenic signaling in adults.
To test this hypothesis neovascularization in the hind limb
ischemia model was studied in young and old mice lacking the p75
receptor (p75KO) mice and age-matched wild-type controls. With
advanced age, signaling through p75 receptor/TNFR2 was required for
collateral vessel development in ischemia-induced
neovascularization.
[0121] In animal models of ischemia.sup.4-7 and in limited human
clinical trials,.sup.23 transplantation of bone marrow cells or
bone marrow-derived endothelial progenitor cells (EPCs) leads to
the migration and homing of these cells to areas of ischemia. In
the ischemic tissue, these cells contribute to the process of
neovascularization by locally releasing angiogenic growth factors
and cytokines.sup.6 and further differentiation of EPCs into mature
endothelial cells. These activities lead to the development of
collateral vessels that then contribute to the faster recovery of
blood flow in the ischemic areas.sup.4-8.
Example 1
Neovascularization and EPC Mobilization is Mediated by p75 TNFR2
Signaling
[0122] To evaluate the effect of bone marrow transplantation on
neovascularization in old mice and to examine the role of
functional p75 receptor/TNFR2 in post-ischemic recovery, two bone
marrow transplantation murine models were established. The first
model was used to evaluate whether the replacement of old p75KO
bone marrow with young wild-type marrow would prevent limb
autoamputation in old p75KO mice. This model provides for the
evaluation of the contribution of wild-type bone marrow-derived
cells to the processes of post-ischemic recovery in p75KO tissue.
To evaluate the contribution of bone marrow-derived p75KO cells to
post-ischemic recovery in otherwise wild-type tissue, a second
murine model was established. In this model, old wild type mice
were treated with lethal irradiation to destroy their endogenous
bone marrow and the old wild-type mice were subsequently
transplanted with bone marrow from young p75KO mice.
Transplantation of young wild-type bone marrow into old p75KO mice
rescued the limbs of these old animals from autoamputation. This
result was not observed when bone marrow mononuclear cells from
young p75KO mice was used. Furthermore, in the old wild-type bone
marrow transplantation model functional p75 receptor was necessary
for the bone marrow-derived EPCs to properly contribute to the
post-ischemic recovery. These results indicated that
ischemia-induced neovascularization and mobilization of EPCs from
bone marrow is mediated, at least in part, by p75 TNFR2
signaling.
Example 2
Ischemia-Induced Angiogenesis is Impaired in Old TNFR2 KO Mice
[0123] When assessing the relationship between age and TNFR1 and 2
levels in EPCs, a 25-30% statistically non-significant decrease in
p55 levels was found in EPCs from elderly mice compared with
younger animals. In contrast, there was a more than 55% (P<0.01)
decrease in p75 mRNA levels in EPCs from elderly mice compared with
younger animals.
[0124] Mean blood flow in young wild-type mice twenty-eight days
after hind limb surgery reached 80% of the pre-ischemic flow (FIG.
1A, black bars). In contrast, recovery of blood flow was delayed up
to fourteen days in old wild-type (gray bars) and young p75KO
(clear bars) mice (40% of pre-ischemic value vs. 80% in young
wild-type mice, P<0.03), but was similar to the recovery in
young wild-type mice thereafter (FIG. 1A, days 21 and 28). These
results suggested that old wild-type and young p75KO mice exhibit a
partial and temporal insufficiency of post-ischemic recovery
compared with young wild-type mice.
[0125] All of the old p75KO mice (n=12, FIG. 1B, black bars)
experienced autoamputation of the operated limb between days 7-10
after hind limb surgery, suggesting an absolute requirement of
TNFR2 p75 for post-ischemic blood flow recovery in adult mice.
Surprisingly, at day 14 after surgery post-ischemic blood flow in
young p55KO mice was 75% of the pre-surgery level (FIG. 1C, black
bars), approaching the rate of recovery of age-matched young
wild-type controls (FIG. 1A, black bars). In contrast, old p55KO
mice demonstrated significantly slower recovery of post-ischemic
blood flow between days 7-14 compared with young p55KO mice (FIG.
1C, black bars=young p55KO mice, gray bars=old p55KO mice). The old
p55KO mice also demonstrated no improvement in blood flow recovery
from immediately post-surgery blood flow for the first 7 to 21 days
after surgery.
[0126] On day 21 after surgery, only 60% (n=7) of old p55KO mice
preserved the ischemic limb (FIG. 1F) (with two animals exhibiting
distal toe necrosis) (FIG. 1G). At this time, old p55KO mice showed
a 60% lower rate of blood flow recovery than the young p55KO mice
(P<0.04), whereas about 40% (n=5) of the old TNFR1 p55KO mice
lost an ischemic limb between days 14-21 after hind limb surgery
(approximately 1-2 weeks later than age-matched old p75KOs) (FIG.
1H), suggesting that the p55 receptor may make a smaller
contribution to the processes of ischemia-induced
neovascularization than the p75 receptor in old mice.
[0127] There was a 50% decrease in capillary density at 28 days
after hind limb ischemia in young p55KO mice compared to young
wild-type mice. Similarly, capillary density in young p75KO mice
was also 50% lower than in young wild-type mice (1.83.+-.0.3 vs.
0.92.+-.0.1 and 0.99.+-.0.14, respectively, P<0.05; FIG. 1D).
There was a statistically non-significant 27% lower capillary
density in young wild-type mice compared with old wild-type mice
(1.83.+-.0.3 vs. 1.34.+-.0.42, P=NS). The difference in capillary
density between old p55KO mice and old wild-type mice was also not
statistically significant (1.34.+-.0.42 vs. 0.75.+-.0.25, P=NS).
Due to autoamputation, there was insufficient tissue remaining to
assess capillary density in old p75KO mice (FIG. 1B). Because of
the severity of the post-ischemic outcome in old p75KO mice and
reported age-associated decrease in p75 receptor expression,
subsequent studies focused on elucidating the role of TNFR2 p75 in
the processes of post-ischemic recovery.
[0128] Fluorescence activated cell sorter (FACS) analysis of
circulating bone marrow-derived EPCs in peripheral blood after hind
limb surgery in wild-type and P75 KO mice revealed a greater
increase in circulating bone marrow-derived EPCs in the wild-type
mice than in the p75KO mice between days 1 and 3, with a maximal
4-fold increase by day 3 (11.1.+-.3.1 vs 2.6.+-.1.8, P<0.05;
FIG. 1E). Circulating EPCs remained about two-fold higher
(7.6.+-.0.5 vs 3.21.+-.0.7, P<0.01) in wild-type than in p75KO
mice up to seven days after hind limb surgery, suggesting that
ischemia-induced mobilization of EPC from bone marrow depends, at
least in part, on TNFR2 p75 signaling. By day ten the EPC levels
were similar in wild-type and P75KO mice (4.75.+-.3.3 vs
4.5.+-.0.9, P=NS; FIG. 1E).
Example 3
Ischemia-Induced VEGF Expression is Lower in the Limbs of p75KO
Mice
[0129] When VEGF expression in the muscles of operated limbs was
assessed by immunofluorescence, it was highly expressed in the
muscle tissue of wild-type mice between days 3 and 7 post-surgery
(FIG. 2A, upper panel). In contrast, VEGF expression was minimal in
the tissues of p75KO mice between days 3 and 7 (FIG. 2A, lower
panel), suggesting that ischemia-induced VEGF expression is
impaired in p75KO mice.
[0130] Densitometric analysis of total RNA from hind limb muscle
revealed that VEGF expression was not detectable in p75KO mice 3
days after hind limb surgery (FIG. 2B, upper panel), whereas VEGF
was highly expressed in wild-type tissue (FIG. 2B, upper panel,
clear bars), confirming the immunofluorescent staining results
(FIG. 2A). By day 10 VEGF was also detectable in p75KO mice (FIG.
2B, upper panel, black bars), but was about half the level of
wild-type controls.
[0131] These results differed from those obtained when mRNA
expression of two other known angiogenic factors, bFGF and
angiopoietin-1 was examined. In p75KO mice by day 3 expression of
bFGF mRNA was 50% of bFGF levels compared to wild-type controls
(FIG. 2B, middle panel, clear bars). By day 10 the level of bFGF
was similar in both genotypes (FIG. 2B, middle panel).
Interestingly, mRNA levels of angiopoeitin-1 were consistently and
substantially higher in p75KO mice (FIG. 2B, bottom panel, black
bars) than in wild-type mice up to day 10 after surgery, suggesting
that ischemia-induced angiopoeitin-1 gene expression does not
require signaling through p75 TNFR2.
[0132] Real-time PCR analysis of homogenized hind limb muscle on
days 3 and 10 after hind limb surgery revealed that, compared to
wild-type tissue, VEGF expression was decreased about 40 to 50%
(p<0.05) in p75KO mice between days 3 and 10 after HL surgery
(FIG. 3A). The mRNA expression of bFGF was also decreased by 15 and
36% (day 3 and 10, respectively) in p75KO compared to wild-type
tissue, but differences were not significant statistically.
Interestingly, mRNA levels of angiopoeitin-1 were comparable in
p75KO than in wild-type mice up to day 10 after surgery, suggesting
that ischemia-induced angiopoeitin-1 gene expression does not
require signaling through p75 TNFR2.
Example 4
Ischemia-Induced Apoptosis is Greater in the Limbs of p75KO
Mice
[0133] To evaluate the viability of ECs, sections of operated hind
limb muscle were triple stained for TUNEL (TdT-dUTP terminal
nick-end labeling) a marker of apoptosis, for Isolectin B4, a
marker of endothelial cells, and TopRo3 to visualize nuclei. When
sections of operated hind limb muscle were stained for TUNEL, no
apoptosis was detectable in wild-type mice before hind limb surgery
and up to day 3 post-surgery (FIG. 4A, upper panel). A few
TUNEL-positive cells were detected at day 7 post-surgery. In
contrast, p75KO mice demonstrated extensive apoptosis by day 1
after hind limb surgery (FIG. 4A, lower panel). The extent of
apoptosis remained greater in the p75KO mice compared with
wild-type controls up to day 7 post-surgery, suggesting that
ischemia-induced apoptosis was attenuated by p75 expression. There
was a statistically significant increase in the number of TUNEL
positive endothelial cells in p75KO mice following hind limb
surgery, representing a 67% and 93% increase in endothelial cell
apoptosis in p75KO mice on days 1 and 10 respectively (FIG. 4B).
Representative images of TUNEL positive endothelial cells that are
also stained for isolectin B4 and TopRo3 (to visualize nuclei) are
shown in FIG. 4C.
Example 5
Loss of p75 Impairs the Function of Cultured EPCs
[0134] Our in vivo studies showed that loss of p75 TNFR2 impairs
post-ischemic recovery, by affecting angiogenesis (FIGS. 1A and B)
and ischemia-induced mobilization of EPCs from bone marrow is
decreased in p75 KO mice. To examine whether endothelial cell
functions may be altered in p75KO cells ex-vivo expanded cultures
of BM-derived EPCs from wild-type and p75KO mice were used. The
identity of ex-vivo expanded cells was confirmed before functional
experiments. In addition, RT-PCR expression of TNF receptors p55R1
and p75R2 was confirmed in ex-vivo expanded endothelial progenitor
cells.
[0135] Up to 14 days after initial plating of ex vivo expanded
cultures of bone marrow-derived EPCs from wild-type and p75KO mice,
no difference was detected between the two genotypes in morphologic
phenotype or rate of proliferation of ex vivo expanded EPCs. Seven
days after initial plating and 2 days after re-plating almost 100%
of cells were identified as EPCs by Isolectin B4-positive staining
and by incorporation of DiI-labeled acetylated low density
lipoprotein (LDL). These characteristics were indicative of the
endothelial origin of these cells. No differences were detected in
cells from mice of different genotypes.
[0136] One of the functional features of endothelial cells is their
ability to migrate towards chemotactic stimuli.sup.9. No difference
in chemotactic activity was found between wild-type and p75KO EPCs
in in vitro migration toward TNF, VEGF, and GM-CSF (FIG. 5A). The
ability to form tube-like structures on VEGF-enriched matrigel is
another functional characteristic of ECs. EPCs from wild-type mice
formed tube-like structures in control chambers and those treated
with TNF, whereas EPCs from p75KO mice failed to form tube-like
structures in either type of chamber, indicating a functional loss
in the bone marrow-derived EPCs of p75KO mice (FIG. 5B).
[0137] Because induction of endothelial growth factors and
cytokines (i.e., VEGF, bFGF, platelet-derived growth factor (PDGF),
IL-8, TNF-.alpha. etc) is required for initiation of angiogenesis
and neovascularization,.sup.10-13 the effect of TNF treatment on
the expression mRNA transcripts using custom-made multiprobe
ribonuclease protection assay (RPA) for bFGF, VEGF, angiopoeitin-1
and actin (FIG. 5C) was evaluated. Densitometric analysis revealed
that TNF-induced expression of bFGF mRNA was counteracted in p75KO
mice up to 16 hours after treatment and at 24 hours after treatment
was only a third the level of the bFGF mRNA in wild-type cells
(FIG. 5D, black bar=P75KO; clear bar=WT). The difference between
wild-type and P75KO mice in VEGF expression was even more dramatic.
VEGF expression was nearly undetectable in p75KO cells up to 8
hours after TNF treatment, and remained 8-fold lower at 24 hours
after surgery in p75KO compared with wild-type EPCs (FIG. 5D).
Interestingly, TNF-induced mRNA levels of angiopoeitin-1 were
comparable at 8 and 16 hours in EPCs from p75KO and wild-type
cells, but by twenty-four hours the mRNA levels in the cells from
the p75KO mice were reduced to half the levels observed in
wild-type cells (FIG. 5D). This paralleled the in vivo observation
of angiogenic factor expression in tissue homogenates of wild-type
and p75KO mice after hind limb surgery (FIG. 2B) and confirming the
loss of several functions characteristic of endothelial cells in
p75KO cells.
Example 6
Signaling Through TNFR2 p75 is Required for NF.kappa.B-Mediated
VEGF Gene Expression
[0138] Because treatment with TNF activates transcription factor
NFB.kappa.and NF.kappa.B is known to regulate VEGF
expression.sup.10,14, NF.kappa.B nuclear translocation and DNA
binding activity was compared in wild-type and p75KO cells. Thirty
minutes after TNF treatment, no NF.kappa.B nuclear translocation
was observed in EPCs from p75KO mice, whereas NF.kappa.B was
translocated in the nucleus in 100% of EPCs from wild-type mice
(FIG. 6A). Electrophoretic Mobility Shift Assay (EMSA) using
NF.kappa.B consensus sequence probe showed that constitutive
NF.kappa.B DNA binding was higher in p75KO EPCs (FIG. 6B). TNF
treatment failed to activate (and in fact decreased) NF.kappa.B DNA
binding activity in p75KO EPCs up to 120 minutes post-stimulation.
In contrast, TNF treatment increased (3- to -4-fold) the NF.kappa.B
DNA binding activity in wild-type cells from 30 to 120 minutes
post-stimulation. These findings suggested that in the absence of
TNF stimulation, signaling via the p55 receptor maintained a
slightly higher NF.kappa.B DNA binding activity in p75KO cells than
in wild-type cells. Following TNF stimulation, NF.kappa.B signaling
in p75KO EPCs is counteracted.
[0139] To further investigate the molecular mechanisms of TNF
signaling in the regulation of VEGF expression, serial VEGF
promoter-luciferase reporter constructs were transfected into EPCs
from wild-type and p75KO mice. When the effects of 1 ng/ml TNF (a
known angiogenic concentration of TNF) were examined, a 2- to
-3-fold increase in the activity of full-length (2.6 kb) VEGF
promoter was observed in p75KO EPCs compared with wild-type EPCs
(FIG. 6C). A schematic diagram of the VEGF luciferase constructs is
provided at FIG. 6D. Interestingly, in wild-type cells transfected
with an NF.kappa.B construct (0.35 kb) there was a 3-fold increase
in VEGF promoter activity compared with wild-type cells transfected
with a full-length construct. A schematic diagram of the
VEGF/luciferase reporter deletion constructs is provided at FIG.
6D. This suggested that in wild-type cells under similar TNF
treatment conditions, NF.kappa.B alone could activate VEGF promoter
to the same degree as the full length promoter (FIG. 6C). In
contrast, VEGF promoter activity was completely counteracted in
p75KO cells transfected with an NF.kappa.B construct, indicating
that signaling through p75 receptor was required for TNF-induced
activation of VEGF promoter (FIG. 6E). In addition, NF.kappa.B may
mediate the induction of VEGF expression through the TNF.alpha./p75
pathway. VEGF/Luciferase activity is quantitated in FIG. 6C. No
difference was observed between wild-type and P75KO EPCs
transfected with an inactive (0.07 kb) VEGF promoter construct.
Example 7
Transplantation of Bone Marrow Cells from Young Wild-Type Mice
Preserved Limbs of Old Mice from Ischemia-Induced
Autoamputation
[0140] A schematic diagram of the bone marrow transplantation
models described herein is provided at FIG. 7. Lethally irradiated
old p75KO mice with BM MNCs isolated from WT GFP (+) and control
p75KO DiI-labeled cells from young (4 weeks old) mice (for detailed
BMT protocol please refer to the diagram in FIG. 7). Four weeks
after BMT, to allow for complete engraftment of transplanted BM (by
day 28 recipients BM was completely reconstituted with donor marrow
and no difference between wild-type or p75KO engraftment was
observed and the number BM-derived PB EPCs were also similar in
recipient-mice of both genotypes, as evaluated by FACS analysis
(FIGS. 8A and 8B). Evaluation of bone marrow engraftment and
peripheral blood mononuclear cells in wild-type and p75KO mice is
provided at FIGS. 8A and 8B.
[0141] Because bone marrow-derived mononuclear cells contributed to
post-natal neovascularization, and because post-ischemic recovery
was substantially impaired in p75KO mice, the question of whether
restoration of p75 receptor expression in bone marrow of old p75KO
mice could augment ischemic recovery was examined. Twenty-eight
days after hind limb surgery, 100% of old p75KO mice transplanted
with wild-type bone marrow had a preserved ischemic limb, whereas
only half the old P75KO mice transplanted with bone marrow from
young p75KO mice had a preserved ischemic limb (FIGS. 9A and 9C).
These results suggested that bone marrow-derived EPCs play an
important role in to post-ischemic recovery.
Example 7
p75/TNFR2 is Required for Efficient Contribution of Bone
Marrow-Derived EPCs in Post-Ischemic Recovery
[0142] To explore further the contribution of bone marrow-derived
EPCs old wild-type mice were transplanted with bone marrow
mononuclear cells from young wild-type or p75KO mice. Although, old
wild-type mice that received either wild-type or P75KO bone marrow
avoided post-ischemic limb loss at 28 days after hind limb surgery,
the mice that received P75KO bone marrow experienced a greater loss
of total muscle tissue in the ischemic limb (50% vs. 23%; FIGS. 9B
and 9D). These data indicated that even in wild-type ischemic
tissue, bone marrow-derived EPCs with functional p75 TNFR2 are
required for efficient post-ischemic recovery.
[0143] Homing of bone marrow-derived EPCs to ischemic tissue was
also examined. Confocal microscopy of hind limb tissue from the
operated limbs of GFP-labeled bone marrow-transplanted mice showed
that bone marrow-derived cells homed only into ischemic areas of
operated limbs (FIGS. 10A-10C). In addition, homing of endothelial
lineage cells into the areas of ischemia was examined. Twenty-eight
days after hind limb surgery, more that 60-70% of GFP-positive
cells in the ischemic limbs were identified as bone marrow-derived
endothelial lineage cells (FIGS. 11A and 11B, merged image:
double-positive cells), strongly suggesting a substantial
contribution of bone marrow-derived EPCs to post-ischemic
recovery.
[0144] Age-related impairment of angiogenesis has been documented
in previous research.sup.11,15-18. Specifically, investigators have
delineated deficiencies in several components of ischemia-induced
neovascularization, including inhibition of endothelial cell
proliferation and function.sup.11,19-21; impaired expression of
angiogenic growth factors, such as VEGF, bFGF, transforming growth
factor-beta (TGF-.beta.), and PDGF .sup.11,22-24. In addition, some
studies have also demonstrated a significant contribution of
ischemia-induced inflammatory responses to the delayed cutaneous
wound healing associated with age.sup.25,26.
[0145] Interestingly, impaired signaling by TNF-.alpha. and other
cytokines in endothelial cells has been correlated with enhanced
apoptotic responses in cutaneous microvasculature in adult
tissue.sup.27. It is well known that TNF-.alpha. can induce the
expression of many important immune- and angiogenesis-related
genes.sup.10,28, through two different TNF-.alpha. receptors: p55
and p75.sup.29-31. In various vascular endothelial cells,
TNF-.alpha. increased the expression of the well-known angiogenic
factors VEGF, bFGF, IL-6.sup.10, and PDGF..sup.32 The role of two
distinct TNF-.alpha. receptors in mediating these responses remains
obscure. Although the distribution of p55 is more widespread than
that of p75, p75 is present in greater amounts on cells of
endothelial and hematopoietic lineage than is p55. Also, expression
of p55 is constitutive in most of the cells, whereas expression of
p75 appears to be inducible.sup.33. The p55 receptor is largely
believed to mediate the cytotoxic effects of TNF-.alpha., whereas
signaling through p75 is thought to mediate the protective effects
of TNF-.alpha...sup.1,2 Aging is associated with increased
expression of p55 and decreased expression of p75 in human
cells,.sup.3 and a decrease in the expression of p75 receptor in
peripheral blood EPCs from adult donors, as reported herein. The
p75 may be needed in angiogenic signaling and post-ischemic
recovery in adults.
[0146] To test this hypothesis neovascularization and the
underlying molecular, cellular and tissue repair processes in the
hind limb ischemia models of young and old p55 and p75KO mice and
age-matched wild-type controls was studied. TNF-.alpha.-mediated
cell signaling pathways were examined in wild-type and p75KO bone
marrow-derived mouse EPCs in vitro physiologic post-ischemia
recovery after hind limb surgery in vivo, and wild-type and p75KO
EPC function in vitro.
[0147] These results demonstrated that a deficiency in p75 TNFR2
expression lead to the failure of post-ischemic recovery in adult
mice, manifested by 100% limb loss in old p75KO, compared with only
40% limb loss in old p55 mice, which occurred one to two weeks
later than that observed in p75KO mice. This suggested that the p55
receptor contributed less than the p75 receptor to the processes of
ischemia-induced neovascularization in old p55KO mice. The negative
effect of p75 receptor deficiency on post-ischemic recovery was
also evident in young animals. Recovery in young p55 mice after
hind limb surgery was similar to recovery in age-matched young
wild-type controls, whereas recovery in young p75KO mice was
delayed up to 14 days and was similar to the recovery in old
wild-type mice. These data strongly suggested that decreased
angiogenesis in aging.sup.11,17,18 is due to impaired signaling
through TNFR2 p75. Likewise, the number of capillaries per muscle
fiber in young p75KO, old p55KO, and old wild-type mice were
significantly lower than in young wild-type mice.
[0148] Numerous reports suggest that VEGF is a useful growth
factors in therapeutic angiogenesis..sup.34-37 In the present
study, VEGF expression in muscle tissue from ischemic limbs of
p75KO mice was shown to be lower in both mRNA and protein levels
than in wild-type mice. In addition, expression of bFGF, another
potent angiogenic growth factor,.sup.13,38 was lower in p75KO than
in wild-type mice. VEGF, which is present early in the response to
ischemia,.sup.39 has been shown to mobilize bone marrow-derived
EPCs in murine models and in humans.sup.9,40. It is conceivable
that significantly lower mobilization of bone marrow-derived EPCs
into peripheral blood that was observed in p75KO mice was a direct
result of decreased VEGF and bFGF expression.
[0149] Aging is associated with alterations in cytokine signaling
pathways that result in enhanced apoptosis.sup.27,41,42. The
reduced expression of TNFR2 p75 associated with increasing age,
coupled with post-ischemic increases in the systemic levels of
TNF-.alpha., favor apoptosis in adult endothelial cells,.sup.42
which could subsequently lead to inhibition of angiogenesis. The
present studies observed an exaggerated apoptotic response in the
hind limbs of p75KO mice. It is possible that unopposed signaling
through the p55 receptor in p75KO mice, and conceivably in human
adult tissue with decreased p75 receptor expression,.sup.41
increased a predominantly proapoptotic cascade via increases in
Fas-associating protein with death domain (FADD), TNFR1-associated
death domain protein (TRADD), and Fas death domain
(FASDD),.sup.3,41 as impaired p75-mediated anti-apoptotic signaling
via NF.kappa.B potentiated further apoptotic responses in the same
tissue.
[0150] Aged endothelials cells show impaired proliferation and
migration in response to various cytokines.sup.12,43. As reported
herein, while bone marrow-derived EPCs from wild-type and p75KO
mice, did not show impaired in vitro proliferation or migration in
response to several cytokine and growth factor stimuli, suggesting
that these endothelial cell functions may not depend on TNFR2 p75
signaling, a substantial decrease in VEGF and fibroblast growth
factor (FGF) mRNA expression was observed following TNF-.alpha.
stimulation in vitro of bone marrow-derived EPCs from wild-type and
p75KO mice. These results strongly suggest that ischemia-induced
expression of the angiogenic growth factors VEGF and FGF, depends,
at least in part, on signaling through p75 TNFR2. Similar to
previous findings in adult tissue,.sup.11 a bifactorial reduction
in VEGF expression was observed in the p75KO mouse model. First,
lower VEGF protein expression by immunostaining in the ischemic
hind limb was observed in p75KO mice as compared with wild-type
mice; and second, VEGF promoter activity was lower in bone
marrow-derived EPCs from p75KO than in wild-type EPCs, strongly
suggesting that previously reported decreases in VEGF reporter
activity in adult cells may be a direct consequence of
age-associated decreases in the expression/signaling via p75
TNFR2.
[0151] To support these findings and to identify the molecular
mechanisms by which TNFR2 p75 mediates decreased VEGF promoter
activity, NF.kappa.B nuclear translocation and DNA binding activity
was studied in wild-type and p75KO EPCs. TNF-induced NF.kappa.B
nuclear translocation and DNA binding activity were inhibited in
p75KO EPCs, whereas NF.kappa.B was readily translocated to the
nucleus in wild-type cells, where it showed enhanced DNA binding
activity. To further elucidate the role of TNF signaling in
NF.kappa.B-mediated VEGF gene expression, serial transfection
assays were performed using VEGF promoter deletion constructs. In
wild-type EPCs transfected with full-length promoter, a 3-fold
increase in VEGF promoter activity was observed, and a 2-fold
increase in p75KO EPCs transfected with full length-promoter.
[0152] Interestingly, in cells transfected with a VEGF promoter
deletion construct that contained only NF.kappa.B response
elements, the VEGF promoter was completely counteracted in p75KO
EPCs. In contrast, there was a 3-fold increase in VEGF promoter
activity in wild-type EPCs that had been transfected with the same
deletion construct. These findings suggest that intact signaling
through both p55 and p75 receptors assures proper NF.kappa.B
activation and subsequent transcriptional activation of VEGF in
cells from young donors, whereas signaling through p55 receptor
alone is not sufficient for proper NF.kappa.B activation and
regulation of VEGF transcription in cells from adult donors having
decreased p75 receptor expression.sup.3,41. These findings may
explain, at least in part, the impaired angiogenic response
observed in adult tissue.sup.11,15,16,44,18.
[0153] Research in animal models of ischemia.sup.4-7 and in limited
human clinical trials.sup.28 has shown that transplantation of bone
marrow cells or bone marrow-derived EPCs significantly augmented
ischemia-induced neovascularization by recruitment and retention of
these cells to the areas of ischemia. Further, researchers have
shown that transplanted bone marrow cells or bone marrow-derived
EPCs contribute to the processes of neovascularization through
local release of angiogenic growth factors and cytokines.sup.6 and
further differentiation of EPCs into mature endothelial cells,
which leads to the development of collateral vessels that
contribute to more rapid recovery of blood flow in the ischemic
areas.sup.4-8.
[0154] In an attempt to prevent total loss of the ischemic limb of
old p75KO mice and to further confirm the role of EPCs in
neovascularization, a series of bone marrow-derived mononuclear
cell transplantation experiments was performed in which bone marrow
from young wild-type and young p75KO was transplanted into old
p75KO mice. Bone marrow transplant was followed by hind limb
surgery, hind limb blood flow recovery was evaluated one month
after hind limb surgery. Transplantation of young wild-type but not
p75KO bone marrow-derived mononuclear cells into old p75KO mice
rescued ischemic limbs from autoamputation, strongly suggesting
that ischemia-induced neovascularization and mobilization of EPCs
from bone marrow is mediated, at least in part, by p75 TNFR2
signaling.
[0155] Since numerous component of the neovascular response may be
affected in the tissue of old p75KO mice (i.e. the proliferation,
survival, migration, and altered angiogenic growth factor supply of
endothelial cells and vascular smooth muscle cell (VSMC) in the
local hind limb tissue), a second bone marrow transplant model was
established that allowed the evaluation of the contribution of bone
marrow-derived tissue from young wild-type and young p75KO mice in
post-ischemic recovery in old wild-type tissue. While neither
wild-type nor p75KO bone marrow transplanted mice experienced
post-ischemic limb loss after hind limb surgery, there was a
statistically significant (50%; P<0.0001) loss of total muscle
tissue in old wild-type mice that had been transplanted with young
p75KO cells, compared to insignificant (P=ns) 23% muscle loss in
old wild-type mice transplanted with cells from young wild-type
mice. These data suggest that bone marrow-derived EPCs with
functional p75 TNFR2 are required even in wild-type ischemic tissue
for efficient post-ischemic recovery. In addition, this finding
clearly substantiates the importance of bone marrow- and peripheral
blood-derived EPCs in ischemia-induced neovascularization.
[0156] These results indicate, first, that the loss of p75/TNFR2
expression impairs post-ischemic recovery; second, p75/TNFR2 is
important for ischemic recovery in part via up-regulation of VEGF
gene expression; third, post-ischemic apoptotic responses are
exaggerated in the absence of p75/TNFR2; fourth, ischemia-induced
neovascularization and mobilization of EPCs from bone marrow is
mediated, at least in part, via p75/TNFR2; fifth, p75/TNFR2
expression by bone marrow-derived mononuclear cells is important
for ischemia-induced neovascularization; sixth, expression of
p75/TNFR2 is decreased in EPC from elderly donors; and seventh,
signaling through p75/TNFR2 is required for collateral vessel
development in models of advanced age. Moreover, based on these
results it is likely that augmentation of p75/TNFR2 in older
subjects will improve recovery after ischemia and prevent the
development of severe ischemia-induced damage in adult coronary and
peripheral vascular disease.
[0157] These experiments were carried out using the following
materials and methods.
Mouse Strains
[0158] Wild-type and mutant mice strains that lack TNFR1 (p55) and
p75 (TNFR2), as described herein, are commercially available from
The Jackson Laboratory, Bar Harbor, Me.
EPC Culture and In Vitro Functional Assays
[0159] Bone marrow-derived EPCs from young wild-type p75KO mice
were isolated and expanded ex vivo as previously described.sup.5.
Cells were grown on 1.5% gelatin-coated plates (Sigma, St. Louis,
Mo.) and cultured with commerciall available cell culture media,
endothelial cell basal medium 2 (EBM-2) with 10% fetal bovine serum
(Clonetics, San Diego, Calif.). EBM-2 was supplemented with growth
factors provided by the manufacturer of the gelatin-coated plates.
For in vitro studies bone marrow-derived ex vivo expanded EPCs were
used 6 to 8 days after initial plating as described.sup.5,9.
[0160] Chemotaxis and chemokinesis of wild-type and TNFR2 EPCs in
response to TNF-.alpha. (1 and 10 ng/ml), rmVEGF (20 ng/ml) and
GM-CSF (50 ng/ml) were evaluated using a modified checkerboard
assay with commercially available microplates, Costar Transwell
chambers (6.5 mm diameter, 5 .mu.m pore) as described
previously.sup.9. Cells migrating into the lower chamber were
collected in 50 .mu.l of buffer and counted manually using
hemocytometer and Coulter Counter.
[0161] Circulating peripheral blood EPCs in wild-type and TNFR2 KO
mice were evaluated using EPC culture assay as described
previously.sup.9. Briefly, mononuclear cells isolated from 500
.mu.l of peripheral blood were cultured in EBM2 medium (Clonetics,
San Diego, Calif.) conditioned with growth factor on 4-well glass
slides coated with 0.5% gelatin solution (Sigma, St. Louis, Mo.).
After 4 days in culture, EPCs were identified morphologically and
were stained with antibodies against acetylated low density
lipoprotein-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate-labeled (DiI-AcLDL) (Molecular Probes, Eugene, Oreg.)
and fluorescein isothiocyanate (FITC)-labeled Isolectin B4 (Vector
Laboratories, Inc, Burlingame, Calif.). Double-positive staining is
characteristic for cells of the endothelial lineage. On fluorescent
microscopy, double-positive cells were identified as EPCs. In
addition, the kinetics of EPC mobilization into peripheral blood
was evaluated by Fluorescence Activated Cell Sorting (FACS)
analysis as described.sup.45.
[0162] To examine the formation of tube-like structures, ex vivo
expanded wild-type and p75KO EPCs were seeded on 4-well chamber
slides (at 5.times.10.sup.4 cells/well), which were coated with a
commercially available solubilized basement membrane preparation,
MATRIGEL (Collaborative Biomedical Products, Bedford, Mass.). Cells
were incubated for 12 hours in medium containing 5% fetal bovine
serum and supplemented with medium alone, 1 ng/ml, or 10 ng/ml of
recombinant (rm)TNF-.alpha. (BD PharMingen, Los Angeles, Calif.).
Cells in the chambers were examined and photographs were taken 12
hours post-stimulation.
[0163] To examine mRNA expression of angiogenic factors in vivo in
the homogenized tissue of wild-type and p75KO mice, ribonuclease
protection assay (RPA) was performed using commercially available
multiprobe custom-made angiogenic factor (VEGF, bFGF and
angiopoeitin DNA templates (PharMingen, Los Angeles, Calif.). The
operated limbs were examined on days 3 and 10 after hind limb
surgery in ex vivo expanded wild-type and p75KO EPCs treated with
10 ng/ml of mrTNF-.alpha.. Total cellular RNA was isolated using
Trizol reagent (Life Technologies, Inc., St. Paul, Minn.). [.alpha.
.sup.32P]UTP (NEN) was used to synthesize in vitro transcribed
antisense riboprobes. RNAse Protection Assays were performed using
commercially available hybridization and digestion buffer reagents
provided in the RPA III TM kit (Ambion, Austin, Tex.) following
manufacturer instructions.
[0164] To examine the effect of TNF on NF.kappa.B nuclear
translocation and NF.kappa.B DNA binding activity, EPCs (grown in
4-well chamber slides) from wild-type and p75KO mice were treated
with TNF (10 ng/ml) for 30, 60, and 120 min. EPCs were then
processed for immunostaining with NF.kappa.B p65 (Santa Cruz
Biotechnology, Santa Cruz, Calif.) to evaluate nuclear
translocation and electromobility shift assay with NF.kappa.B
consensus sequence to evaluate NF.kappa.B DNA binding activity
(Santa Cruz Biotechnology, Santa Cruz, Calif.) as
described.sup.26.
[0165] To examine the role of TNF on NF.kappa.B-mediated VEGF
promoter activity, EPCs from wild-type and p75KO mice were
transfected with full length (2.6 kb) VEGF promoter-reporter
construct (in basic pGL2 plasmid backbone), a deletion construct
(0.35 kb) containing two putative NF.kappa.B sites and one Sp1
cluster, and an inactive deletion construct. The inactive deletion
construct spanned up to -70 from transcription origination site and
containing an incomplete Sp1 cluster. (All constructs were kind
gift from Dr. Debabrata Mukhopadhyay, Beth Israel Medical Center,
Boston, Mass.), which are described in Mukhopadhyay et al., Mol
Cell Biol. 1997 September; 17(9):5629-39). Twenty-four hours after
transfection cells were treated with TNF (1 ng/ml) and evaluated
the VEGF promoter activity 18 hours later by measuring luciferase
activity as described.sup.46.
[0166] To evaluate the constitutive mRNA expression of TNF
receptors p75 and p55 in peripheral blood EPCs from adults, human
peripheral blood EPCs were isolated from peripheral blood of donors
of various ages first by separating mononuclear cell fraction using
density centrifugation over Histopaque-1083 (Sigma, St. Louis, Mo.)
and then growing mononuclear cells on selective medium as described
previously.sup.9. After 7 days in culture (standard time for
selection of EPC), cells were harvested and processed for
evaluation of mRNA expression of TNF receptors p75 and p55 by RPA
using a custom-made (p75, p55 and actin) multiprobe DNA template
(BD PharMingen, Los Angeles, Calif.).
Ischemia Studies
[0167] Ligation and removal of femoral artery was used as
previously described to induce unilateral hind limb ischemia in
male young and old wild-type C57BL/6J and p75KO mice.sup.27. Serial
assessments of hind limb blood flow was performed with a PIM 2.0
Laser Doppler perfusion imager (LDPI) (Lisca) as previously
described.sup.27. Perfusion was expressed as the ratio of left
(ischemic) to right (control) limb. Results represent the
mean.+-.SEM of at least 10-15 mice per group.
[0168] To determine capillary density, on day 28 post-hind limb
surgery whole nonischemic and ischemic limbs of at least 5 mice
from each group were immediately fixed in methanol overnight and
processed for histology. Endothelial cells were identified by
histochemical staining with biotinylated isolectin B4 according to
the manufacturer's directions (Vector Labs, Burlingame,
Calif.).sup.47. Capillary networks were evaluated as described
previously.sup.47.
[0169] To evaluate the kinetics of VEGF expression in operated
limbs of wild-type and p75KO mice after hind limb surgery, tissue
from 5 mice per group was collected before, and 1, 3, and 7 days
after surgery, immediately fixed in methanol overnight and then
processed for immunofluorescent staining with VEGF (Santa Cruz
Biotechnology, Santa Cruz, Calif.). In addition, continuous
sections of hind limb tissue from the same animals were
immunostained with TUNEL to assess the viability of hind limb
tissue in the operated limbs in wild-type and p75KO mice. Both
VEGF- and TUNEL-stained slides were evaluated using laser scanning
confocal microscopy (Zeiss, Axiovert 100, Thornwood, N.Y.).
Murine Bone Marrow Transplantation Studies
[0170] To evaluate the effect of bone marrow-derived cells in
ischemic recovery in p75KO tissue, a bone marrow transplantation
model was established in which bone marrow mononuclear cells from
young (3 to 4 week old) wild-type and young p75KO mice were
injected into old (12 to 14 month old) p75KO animals. To evaluate
the effect of bone marrow-derived p75KO mononuclear cells in
ischemic recovery in wild-type tissue, bone marrow-derived
mononuclear cells from young wild-type and young p75KO mice were
transplanted into old wild-type mice. Bone marrow cells were
obtained by flushing the tibias and femurs of young (3-4 weeks)
donor wild-type (C57BL/6J)/GFP (green fluorescent protein) mice and
p75KO (on C57BL/6J background) mice (both from The Jackson
Laboratory, Bar Harbor, Me.). Low-density bone marrow mononuclear
cells were isolated by density centrifugation using a commercially
available medium containing polysucrose and sodium diatrizoate that
facilitates the recovery of large numbers of viable mononuclear
cells, Histopaque-1083 (Sigma, St. Louis, Mo.). A schematic diagram
showing murine bone marrow transplantation models is provided at
FIG. 6.
[0171] To evaluate the impact of bone marrow-derived mononuclear
cell transplantation on therapeutic neovascularization, old
TNFR2/p75KO mice were lethally irradiated with 9.about.11 Gy and
subsequently received intracardiac injections of 3.times.10.sup.6
of donor bone marrow mononuclear cells. These mononuclear cells
were either WT/GFP or TNFR2, and were labeled prior to intracardiac
injection with
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide (DiI)
(Molecular Probes, Eugene, Oreg.). At 4 weeks post-bone marrow
transplantation, by which time the bone marrow of the recipient
mice has usually regenerated with donor bone marrow cells, hind
limb surgery was performed. Animals were evaluated at different
times post-hind limb surgery for physiologic recovery using LDPI,
histologic assessment of capillary network (CD31 and/or isolectin
B4 staining).sup.28, evaluation of total muscle loss using the
ratio of operated vs. non-operated limbs on day 28, and homing of
WT/GFP and TNFR2/DiI-labeled bone marrow-derived mononuclear cells
in the areas of ischemia by confocal microscopy.sup.26.
[0172] To evaluate the recipient bone marrow engraftment before
hind limb surgery and at the end of the post-hind limb surgery
evaluation period (4 weeks after surgery), 5 animals from each
transplantation group were sacrificed. Bone marrow from tibias and
femurs were flushed and after density centrifugation and methanol
fixation, cells were processed for FACS analysis.sup.25. In
addition, 500 .mu.l of peripheral blood was drawn from the same
animals. Circulating peripheral blood mononuclear cells were
isolated by lysing red blood cells with an ammonium chloride
solution (Stem Cell Technologies, Seattle, Wash.) then using
density centrifugation with Histopaque-1083 (Sigma, St. Louis,
Mo.). After centrifugation, mononuclear cells were fixed with 1%
PFA and processed for FACS analysis.sup.45.
Statistical Analysis
[0173] Results are expressed as mean.+-.SEM. Differences among
groups were evaluated by ANOVA and Fisher's PLSD post hoc test
using StatView software (SAS Institute Inc., Gary, N.C.).
Differences were considered significant at P<0.05.
Other Embodiments
[0174] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0175] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0176] All patents and publications mentioned in this specification
are herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and
individually indicated to be incorporated by reference.
[0177] A review of the following specific references will help
advance appreciation of the present invention.
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Sequence CWU 1
1
21461PRTHomo sapiens 1Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala
Val Gly Leu Glu Leu1 5 10 15Trp Ala Ala Ala His Ala Leu Pro Ala Gln
Val Ala Phe Thr Pro Tyr 20 25 30Ala Pro Glu Pro Gly Ser Thr Cys Arg
Leu Arg Glu Tyr Tyr Asp Gln 35 40 45Thr Ala Gln Met Cys Cys Ser Lys
Cys Ser Pro Gly Gln His Ala Lys 50 55 60Val Phe Cys Thr Lys Thr Ser
Asp Thr Val Cys Asp Ser Cys Glu Asp65 70 75 80Ser Thr Tyr Thr Gln
Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95Gly Ser Arg Cys
Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100 105 110Glu Gln
Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120
125Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg
130 135 140Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp
Val Val145 150 155 160Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn
Thr Thr Ser Ser Thr 165 170 175Asp Ile Cys Arg Pro His Gln Ile Cys
Asn Val Val Ala Ile Pro Gly 180 185 190Asn Ala Ser Met Asp Ala Val
Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205Met Ala Pro Gly Ala
Val His Leu Pro Gln Pro Val Ser Thr Arg Ser 210 215 220Gln His Thr
Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser225 230 235
240Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly
245 250 255Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr Ala
Leu Gly 260 265 270Leu Leu Ile Ile Gly Val Val Asn Cys Val Ile Met
Thr Gln Val Lys 275 280 285Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala
Lys Val Pro His Leu Pro 290 295 300Ala Asp Lys Ala Arg Gly Thr Gln
Gly Pro Glu Gln Gln His Leu Leu305 310 315 320Ile Thr Ala Pro Ser
Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser 325 330 335Ala Leu Asp
Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly 340 345 350Val
Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser 355 360
365Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile
370 375 380Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser
Ser Gln385 390 395 400Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser
Pro Ser Glu Ser Pro 405 410 415Lys Asp Glu Gln Val Pro Phe Ser Lys
Glu Glu Cys Ala Phe Arg Ser 420 425 430Gln Leu Glu Thr Pro Glu Thr
Leu Leu Gly Ser Thr Glu Glu Lys Pro 435 440 445Leu Pro Leu Gly Val
Pro Asp Ala Gly Met Lys Pro Ser 450 455 46023682DNAHomo sapiens
2gcgagcgcag cggagcctgg agagaaggcg ctgggctgcg agggcgcgag ggcgcgaggg
60cagggggcaa ccggaccccg cccgcaccca tggcgcccgt cgccgtctgg gccgcgctgg
120ccgtcggact ggagctctgg gctgcggcgc acgccttgcc cgcccaggtg
gcatttacac 180cctacgcccc ggagcccggg agcacatgcc ggctcagaga
atactatgac cagacagctc 240agatgtgctg cagcaaatgc tcgccgggcc
aacatgcaaa agtcttctgt accaagacct 300cggacaccgt gtgtgactcc
tgtgaggaca gcacatacac ccagctctgg aactgggttc 360ccgagtgctt
gagctgtggc tcccgctgta gctctgacca ggtggaaact caagcctgca
420ctcgggaaca gaaccgcatc tgcacctgca ggcccggctg gtactgcgcg
ctgagcaagc 480aggaggggtg ccggctgtgc gcgccgctgc gcaagtgccg
cccgggcttc ggcgtggcca 540gaccaggaac tgaaacatca gacgtggtgt
gcaagccctg tgccccgggg acgttctcca 600acacgacttc atccacggat
atttgcaggc cccaccagat ctgtaacgtg gtggccatcc 660ctgggaatgc
aagcatggat gcagtctgca cgtccacgtc ccccacccgg agtatggccc
720caggggcagt acacttaccc cagccagtgt ccacacgatc ccaacacacg
cagccaactc 780cagaacccag cactgctcca agcacctcct tcctgctccc
aatgggcccc agccccccag 840ctgaagggag cactggcgac ttcgctcttc
cagttggact gattgtgggt gtgacagcct 900tgggtctact aataatagga
gtggtgaact gtgtcatcat gacccaggtg aaaaagaagc 960ccttgtgcct
gcagagagaa gccaaggtgc ctcacttgcc tgccgataag gcccggggta
1020cacagggccc cgagcagcag cacctgctga tcacagcgcc gagctccagc
agcagctccc 1080tggagagctc ggccagtgcg ttggacagaa gggcgcccac
tcggaaccag ccacaggcac 1140caggcgtgga ggccagtggg gccggggagg
cccgggccag caccgggagc tcagattctt 1200cccctggtgg ccatgggacc
caggtcaatg tcacctgcat cgtgaacgtc tgtagcagct 1260ctgaccacag
ctcacagtgc tcctcccaag ccagctccac aatgggagac acagattcca
1320gcccctcgga gtccccgaag gacgagcagg tccccttctc caaggaggaa
tgtgcctttc 1380ggtcacagct ggagacgcca gagaccctgc tggggagcac
cgaagagaag cccctgcccc 1440ttggagtgcc tgatgctggg atgaagccca
gttaaccagg ccggtgtggg ctgtgtcgta 1500gccaaggtgg gctgagccct
ggcaggatga ccctgcgaag gggccctggt ccttccaggc 1560ccccaccact
aggactctga ggctctttct gggccaagtt cctctagtgc cctccacagc
1620cgcagcctcc ctctgacctg caggccaaga gcagaggcag cgagttgtgg
aaagcctctg 1680ctgccatggc gtgtccctct cggaaggctg gctgggcatg
gacgttcggg gcatgctggg 1740gcaagtccct gactctctgt gacctgcccc
gcccagctgc acctgccagc ctggcttctg 1800gagcccttgg gttttttgtt
tgtttgtttg tttgtttgtt tgtttctccc cctgggctct 1860gccccagctc
tggcttccag aaaaccccag catccttttc tgcagagggg ctttctggag
1920aggagggatg ctgcctgagt cacccatgaa gacaggacag tgcttcagcc
tgaggctgag 1980actgcgggat ggtcctgggg ctctgtgcag ggaggaggtg
gcagccctgt agggaacggg 2040gtccttcaag ttagctcagg aggcttggaa
agcatcacct caggccaggt gcagtggctc 2100acgcctatga tcccagcact
ttgggaggct gaggcgggtg gatcacctga ggttaggagt 2160tcgagaccag
cctggccaac atggtaaaac cccatctcta ctaaaaatac agaaattagc
2220cgggcgtggt ggcgggcacc tatagtccca gctactcaga agcctgaggc
tgggaaatcg 2280tttgaacccg ggaagcggag gttgcaggga gccgagatca
cgccactgca ctccagcctg 2340ggcgacagag cgagagtctg tctcaaaaga
aaaaaaaaag caccgcctcc aaatgccaac 2400ttgtcctttt gtaccatggt
gtgaaagtca gatgcccaga gggcccaggc aggccaccat 2460attcagtgct
gtggcctggg caagataacg cacttctaac tagaaatctg ccaatttttt
2520aaaaaagtaa gtaccactca ggccaacaag ccaacgacaa agccaaactc
tgccagccac 2580atccaacccc ccacctgcca tttgcaccct ccgccttcac
tccggtgtgc ctgcagcccc 2640gcgcctcctt ccttgctgtc ctaggccaca
ccatctcctt tcagggaatt tcaggaacta 2700gagatgactg agtcctcgta
gccatctctc tactcctacc tcagcctaga ccctcctcct 2760cccccagagg
ggtgggttcc tcttccccac tccccacctt caattcctgg gccccaaacg
2820ggctgccctg ccactttggt acatggccag tgtgatccca agtgccagtc
ttgtgtctgc 2880gtctgtgttg cgtgtcgtgg gtgtgtgtag ccaaggtcgg
taagttgaat ggcctgcctt 2940gaagccactg aagctgggat tcctccccat
tagagtcagc cttccccctc ccagggccag 3000ggccctgcag aggggaaacc
agtgtagcct tgcccggatt ctgggaggaa gcaggttgag 3060gggctcctgg
aaaggctcag tctcaggagc atggggataa aggagaaggc atgaaattgt
3120ctagcagagc aggggcaggg tgataaattg ttgataaatt ccactggact
tgagcttggc 3180agctgaacta ttggagggtg ggagagccca gccattacca
tggagacaag aagggttttc 3240caccctggaa tcaagatgtc agactggctg
gctgcagtga cgtgcacctg tactcaggag 3300gctgagggga ggatcactgg
agcccaggag tttgaggctg cagcgagcta tgatcgcgcc 3360actacactcc
agcctgagca acagagtgag accctgtctc ttaaagaaaa aaaaagtcag
3420actgctggga ctggccaggt ttctgcccac attggaccca catgaggaca
tgatggagcg 3480cacctgcccc ctggtggaca gtcctgggag aacctcaggc
ttccttggca tcacagggca 3540gagccgggaa gcgatgaatt tggagactct
gtggggcctt ggttcccttg tgtgtgtgtg 3600ttgatcccaa gacaatgaaa
gtttgcactg tatgctggac ggcattcctg cttatcaata 3660aacctgtttg
ttttaaaaaa aa 3682
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