U.S. patent application number 10/714574 was filed with the patent office on 2004-11-18 for compositions and methods for modulating vascularization.
This patent application is currently assigned to St. Elizabeth's Medical Center. Invention is credited to Asahara, Takayuki, Isner, Jeffrey M..
Application Number | 20040228835 10/714574 |
Document ID | / |
Family ID | 29782166 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040228835 |
Kind Code |
A1 |
Isner, Jeffrey M. ; et
al. |
November 18, 2004 |
Compositions and methods for modulating vascularization
Abstract
The present invention generally provides methods for modulating
formation of new blood vessels. In one embodiment, the methods
include administering to a mammal an effective amount of
granulocyte macrophage-colony stimulating factor (GM-CSF)
sufficient to form the new blood vessels. Additionally provided are
methods for preventing or reducing the severity of blood vessel
damage in a mammal which methods preferably include administering
to the mammal an effective amount of GM-CSF. Provided also as part
of this invention are pharmaceutical products and kits for inducing
formation of new blood vessels in the mammal.
Inventors: |
Isner, Jeffrey M.; (Weston,
MA) ; Asahara, Takayuki; (Arlington, MA) |
Correspondence
Address: |
Edwards & Angell, LLP
Intellectual Property Practice Group
P.O. Box 55874
Boston
MA
02205
US
|
Assignee: |
St. Elizabeth's Medical
Center
|
Family ID: |
29782166 |
Appl. No.: |
10/714574 |
Filed: |
November 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10714574 |
Nov 14, 2003 |
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09698323 |
Oct 27, 2000 |
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09698323 |
Oct 27, 2000 |
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09265041 |
Mar 9, 1999 |
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6676937 |
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60077262 |
Mar 9, 1998 |
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Current U.S.
Class: |
424/85.1 ;
514/13.3; 514/8.1; 514/8.2; 514/8.5; 514/8.9; 514/9.1; 514/9.5;
514/9.6 |
Current CPC
Class: |
G01N 33/56972 20130101;
A61K 35/44 20130101; A61K 35/44 20130101; A61K 38/193 20130101;
A61K 38/1866 20130101; A61K 38/193 20130101; A61K 38/1866 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/085.1 ;
514/012 |
International
Class: |
A61K 038/19; A61K
038/18 |
Goverment Interests
[0002] Funding for the present invention was provided in part by
the Government of the United States by virtue of grants HL 40518,
HL02824 and HL57516 by the National Institutes of Health.
Accordingly, the Government of the United States has certain rights
in and to the invention claimed herein.
Claims
1-48. (canceled).
49. A method for inducing new blood vessel growth in myocardial
tissue of a mammal in need of such treatment comprising: a)
injecting an effective amount of a solution comprising a nucleic
acid encoding at least one angiogenic protein or an effective
fragment thereof into the myocardial tissue; and b) administering
to the mammal an effective amount of at least one of: stem cell
factor (SCF), colony stimulating factor (CSF) or an effective
fragment thereof, thereby inducing the new blood vessel growth in
the myocardial tissue of the mammal.
50. The method of claim 49, wherein the angiogenic factor is a
vascular endothelial growth factor (VEGF) or an effective fragment
thereof.
51. The method of claim 50, wherein the VEGF is VEGF-1 or
VEGF165.
52. The method of claim 49, further comprising expressing the
angiogenic protein or fragment in the myocardium.
53. The method of claim 52, wherein the method further comprises
increasing frequency of endothelial progenitor cells (EPC) in the
mammal.
54. The method of claim 52, wherein the increase in frequency of
the EPC is at least about 20% as determined by a standard EPC
isolation assay.
55. The method of claim 52, wherein the method further comprises
increasing EPC differentiation in the mammal.
56. The method of claim 55, wherein the increase in EPC
differentiation is at least about 20% as determined by a standard
EPC culture assay or a standard hindlimb ischemia assay.
57. The method of claim 50, wherein the level of VEGF or VEGF
fragment expression is sufficient to increase neovascularization by
at least about 5% as determined by a standard cornea micropocket
assay.
58. The method of claim 49, wherein the amount of administered SCF,
CSF or fragment is sufficient to increase EPC bone marrow derived
EPC incorporation into foci.
59. The method of claim 58, wherein the increase in EPC bone marrow
derived EPC incorporation into foci is at least about 20% as
determined by a standard rodent bone marrow (BM) transplantation
model.
60. The method of claim 49, wherein the method further comprises
administering at least one angiogenic protein or effective fragment
thereof before or after administration of the nucleic acid to the
mammal.
61. The method of claim 49, wherein the method further comprises
administering to the mammal an anti-coagulant before, during, or
after administration of the nucleic acid to the mammal.
62. The method of claim 61, wherein the anti-coagulant is one or
more of urokinase, plasminogen activator, and heparin.
63. The method of claim 49, wherein the nucleic acid is directly
injected with a catheter or stent.
64. The method of claim 49, wherein the nucleic acid is inserted
into a cassette operably linked to a promoter.
65. The method of claim 49, wherein the myocardial tissue is
ischemic or is associated with infarction or dysfunction.
66. The method of claim 49, wherein the angiogenic protein or
factor is one of acidic fibroblast growth factor (aFGF), basic
fibroblast growth factor (bFGF), epidermal growth factor (EGF),
transforming growth factor .alpha. and .beta. (TGF-.alpha. and
TFG-.beta.), platelet-derived endothelial growth factor (PD-ECGF),
platelet-derived growth factor (PDGF), tumor necrosis factor
.alpha. (TNF-.alpha.), hepatocyte growth factor (HGF), insulin like
growth factor (IGF), erythropoietin, colony stimulating factor
(CSF), macrophage-CSF (M-CSF), granulocyte/macrophage CSF (GM-CSF),
stem cell factor (SCF), angiopoetin-1 (Ang1), nitric oxidesynthase
(NOS); or a mutein or fragment thereof.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S.
Provisional Application No. 60/077,262, filed on Mar. 9, 1998; the
disclosure of which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to methods for modulating
vascularization particularly in a mammal. In one aspect, methods
are provided for modulating vascularization that includes
administrating to the mammal an effective amount of granulocyte
macrophage-colony stimulating factor (GM-CSF). Further provided are
methods for treating or detecting damaged blood vessels in the
mammal. The invention has a wide spectrum of useful applications
including inducing formation of new blood vessels in the
mammal.
BACKGROUND OF THE INVENTION
[0004] There is nearly universal recognition that blood vessels
help supply oxygen and nutrients to living tissues. Blood vessels
also facilitate removal of waste products. Blood vessels are
renewed by a process termed "angiogenesis". See generally Folkman
and Shing, J. Biol. Chem. 267 (16), 10931-10934 (1992).
[0005] Angiogenesis is understood to be important for the
well-being of most mammals. As an illustration, angiogenesis has
been disclosed as being an essential process for reproduction,
development and wound repair.
[0006] There have been reports that inappropriate angiogenesis can
have severe consequences. For example, it has been disclosed that
solid tumor growth is facilitated by vascularization. There is
broad support for the concept that mammals must regulate
angiogenesis extensively. There has been much attention directed to
understanding how angiogeneis is controlled. In particular,
angiogenesis is believed to begin with the degradation of the
basement membrane by proteases secreted from endothelial cells (EC)
activated by mitogens, e.g., vascular endothelial growth factor
(ie. VEGF-1), basic fibroblast growth factor (bFGF) and/or others.
The cells migrate and proliferate, leading to the formation of
solid endothelial cell sprouts into the stromal space, then,
vascular loops are formed and capillary tubes develop with
formation of tight junctions and deposition of new basement
membrane.
[0007] In adults, it has been disclosed that the proliferation rate
of endothelial cells is typically low, compared to other cell types
in the body. The turnover time of these cells can exceed one
thousand days. Physiological exceptions in which angiogenesis
results in rapid proliferation occurs under tight regulation are
found in the female reproduction system and during wound healing.
It has been reported that the rate of angiogenesis involves a
change in the local equilibrium between positive and negative
regulators of the growth of microvessels.
[0008] Abnormal angiogenesis is thought to occur when the body
loses its control of angiogenesis, resulting in either excessive or
insufficient blood vessel growth. For instance, conditions such as
ulcers, strokes, and heart attacks may result from the absence of
angiogenesis normally required for natural healing. In contrast,
excessive blood vessel proliferation can facilitate tumor growth,
blindness, psoriasis, rheumatoid arthritis, as well as other
medical conditions.
[0009] The therapeutic implications of angiogenic growth factors
were first described by Folkman and colleagues over two decades ago
(Folkman, N. Engl. J. Med., 85:1182-1186 (1971)). Recent work has
established the feasibility of using recombinant angiogenic growth
factors, such as fibroblast growth factor (FGF) family
(Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) and
Baffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial cell
growth factor (ECGF)(Pu, et al., J Surg Res, 54:575-83 (1993)), and
vascular endothelial growth factor (VEGF-1) to expedite and/or
augment collateral artery development in animal models of
myocardial and hindlimb ischemia (Takeshita, et al., Circulation,
90:228-234 (1994) and Takeshita, et al., J Clin Invest, 93:662-70
(1-994)).
[0010] The feasibility of using gene therapy to enhance
angiogenesis has received recognition. For example, there have been
reports that angiogenesis can facilitate treatment of ischemia in a
rabbit model and in human clinical trials. Particular success has
been achieved using VEGF-1 administered as a balloon gene delivery
system. Successful transfer and sustained expression of the VEGF-1
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 reported that direct intramuscular injection
of DNA encoding VEGF-1 into ischemic tissue induces angiogenesis,
providing the ischemic tissue with increased blood vessels (Tsurumi
et al., Circulation, 94(12):3281-3290 (1996)).
[0011] Alternative methods for promoting angiogenesis are desirable
for a number of reasons. For example, it is believed that native
endothelial progenitor cell (EPC) number and/or viability decreases
over time. Thus, in certain patient populations, e.g., the elderly,
EPCs capable of responding to angiogenic proteins may be limited.
Also, such patients may not respond well to conventional
therapeutic approaches.
[0012] There have been reports that at least some of these problems
can be reduced by administering isolated EPCs to patients and
especially those undergoing treatment for ischemic disease.
However, this suggestion is believed to be prohibitively expensive
as it can require isolation and maintenance of patient cells.
Moreover, handling of patient cells can pose a significant health
risk to both the patient and attending personnel in some
circumstances.
[0013] Granulocyte macrophage colony stimulating factor (GM-CSF)
has been shown to exert a regulatory effect on
granulocyte-committed progenitor cells to increase circulating
granulocyte levels (Gasson, J. C., Blood 77:1131 (1991). In
particular, GM-CSF acts as a growth factor for granulocyte,
monocyte ad eosinophil progenitors.
[0014] Administration of GM-CSF to human and non-human primates
results in increased numbers of circulating neutrophils, as well as
eosinophils, monocytes and lymphocytes. Accordingly, GM-CSF is
believed to be particularly useful in accelerating recovery from
neutropenia in patients subjected to radiation or chemotherapy, or
following bone marrow transplantation. In addition, although GM-CSF
is less potent than other cytokines, e.g., FGF, in promoting EC
proliferation, GM-CSF activates a fully migrating phenotype.
(Bussolino, et al., J. Clin. Invent., 87:986 (1991).
[0015] Accordingly, it would be desirable to have methods for
modulating vascularization in a mammal and especially a human
patient. It would be particularly desirable to have methods that
increase EPC mobilization and neovascularization-(formation of new
blood vessels) in the patient that do not require isolation of EPC
cells.
SUMMARY OF THE INVENTION
[0016] The present invention generally relates to methods for
modulating vascularization in a mammal. In one aspect, the
invention provides methods for increasing vascularization that
includes administrating to the mammal an effective amount of a
vascularization modulating agent, such as granulocyte
macrophage-colony stimulating factor (GM-CSF), VEGF, Steel factor
(SLF, also known as Stem cell factor (SCF)), stromal cell-derived
factor (SDF-1), granulocyte-colony stimulating factor (G-CSF), HGF,
Angiopoietin-1, Angiopoietin-2, M-CSF, b-FGF, and FLT-3 ligand, and
effective fragment thereof, or DNA coding for such vascularization
modulating agents. Such materials have sometimes previously been
described as "hematopoietic factors." and/or "hematopoietic
proteins." Disclosure relating to these and other hematopoietic
factors can be found in Kim, C. H. and Broxmeyer, H. E. (1998)
Blood, 91:100; Turner, M. L. and Sweetenham, J. W., Br. J.
Haematol. (1996) 94:592; Aiuuti, A. et al. (1997) J. Exp. Med.
185:111; Bleul, C. et al. (1996) J. Exp. Med. 184:1101; Sudo, Y. et
al. (1997) Blood, 89: 3166; as well as references disclosed
therein. Prior to the present invention, it was not kown that
GM-CSF or other hematopoietic factors could potentiate endothelial
progenitor cells, or modulate neovascularization as described
herein.
[0017] Alternatively, instead of the proteins themselves or
effective fragments thereof, the DNA coding for the vascularization
modulating agents can be administered to the site where
neovascularization is desired, as further discussed below. The
invention also relates to methods for treating or detecting damaged
blood vessels in the mammal. The invention has many uses including
preventing or reducing the severity of blood vessel damage
associated with ischemia or related conditions.
[0018] We have now discovered that hematopoietic factors such as
granulocyte-macrophage colony-stimulating factor (GM-CSF), modulate
endothelial progenitor cell (EPC) mobilization and
neovascularization (blood vessel formation). In particular, we have
found that GM-CSF and other hematopoietic factors increase EPC
mobilization and enhances neovascularization. This observation was
surprising and unexpected in light of prior reports addressing
GM-CSF activity in vitro and in vivo. Accordingly, this invention
provides methods for using GM-CSF to promote EPC mobilization and
to enhance neovascularization, especially in tissues in need of EPC
mobilization and/or neovascularization.
[0019] In one aspect, the present invention provides a method for
inducing neovascularization in a mammal. By the term "induction" is
meant at least enhancing EPC mobilization and also preferably
facilitating formation of new blood vessels in the mammal. EPC
mobilization is understood to mean a significant increase in the
frequency and differentiation of EPCs as determined by assays
disclosed herein. In one embodiment, the method includes
administering to the mammal an effective amount of a
vascularization modulating factor such as granulocyte
macrophage-colony stimulating factor (GM-CSF), that is preferably
sufficient to induce the neovascularization in the mammal.
Preferably, that amount of GM-CSF is also capable of modulating and
particularly increasing frequency of EPCs in the mammal. A variety
of methods for detecting and quantifying neovascularization, EPC
frequency, the effectiveness of vascularization modulating agents,
and other parameters of blood vessel growth are discussed below and
in the examples.
[0020] In a particular embodiment of the method, the enhancement in
EPC mobilization and particularly the increase in frequency of the
EPCs is at least about 20% and preferably from between 50% to 500%
as determined by a standard EPC isolation assay. That assay
generally detects and quantifies EPC enrichment and is described in
detail below.
[0021] In another particular embodiment of the method, the amount
of administered modulating agent is sufficient to enhance EPC
mobilization and especially to increase EPC differentiation in the
mammal. Methods for detecting and quantifying EPC differentiation
include those specific methods described below. Preferably, the
increase in EPC differentiation is at least about 20%, preferably
between from about 100% to 1000%, more preferably between from
about 200% to 800% as determined by a standard EPC culture assay
discussed below. More preferably, that amount of administered
modulating agent is additionally sufficient to increase EPC
differentiation by about the stated percent amounts following
tissue ischemia as determined in a standard hindlimb ischemia assay
as discussed below.
[0022] In another particular embodiment of the method, the amount
of vascularization modulating agent administered to the mammal is
sufficient to increase blood vessel size in the mammal. Methods for
determining parameters of blood vessel size, e.g., length and
circumference, are known in the field and are discussed below.
Preferably, the amount of administered modulating agent is
sufficient to increase blood vessel length by at least about 5%,
more preferably between from about 10% to 50%, even more preferably
about 20%, as determined by a standard blood vessel length assay
discussed below. Preferably, the amount of modulating agent
administered to the mammal is also sufficient to increase blood
vessel circumference or diameter by the stated percent amounts as
determined by a standard blood vessel diameter assay. As will be
discussed below, it will often be preferred to detect and quantify
changes in blood vessel size using a standard cornea micropocket
assay, although other suitable assays can be used as needed.
[0023] In another particular embodiment of the method, the amount
of administered vascularization modulating agent is sufficient to
increase neovascularization by at least about 5%, preferably from
between about 50% to 300%, and more preferably from between about
100% to 200% as determined by the standard cornea micropocket
assay. Methods for performing that assay are known in the field and
include those specific methods described below. Additionally,
preferred amounts of GM-CSF are sufficient to improve ischemic
hindlimb blood pressure by at least about 5%, preferably between
from about 10% to 50% as determined by standard methods for
measuring the blood pressure of desired vessels. More specific
methods for measuring blood pressure particularly with new or
damaged vessels include techniques optimized to quantify vessel
pressure in the mouse hindlimb assay discussed below.
[0024] In another particular embodiment of the method, the amount
of administered vascularization modulating agent is sufficient to
increase EPC bone marrow (BM) derived EPC incorporation into foci
by at least about 20% as determined by a standard murine BM
transplantation model. Preferably, the increase is between from
about 50% to 400%, more preferably between from about 100% to 300%
as determined by that standard model. More specific methods for
determining the increase in EPC incorporation into foci are found
in the discussion and Examples which follow.
[0025] The methods of this invention are suitable for modulating
and especially inducing neovascularization in a variety of animals
including mammals. The term "mammal" is used herein to refer to a
warm blooded animal such as a rodent, rabbit, or a primate and
especially a human patient. Specific rodents and primates of
interest include those animals representing accepted models of
human disease including the mouse, rat, rabbit, and monkey.
Particular human patients of interest include those which have, are
suspected of having, or will include ischemic tissue. That ischemic
tissue can arise by nearly any means including a surgical
manipulation or a medical condition. Ischemic tissue is often
associated with an ischemic vascular disease such as those specific
conditions and diseases discussed below.
[0026] As will become more apparent from the discussion and
Examples which follow, methods of this invention are highly
compatible and can be used in combination with established or
experimental methods for modulating neovascularization. In one
embodiment, the invention includes methods for modulating and
particularly inducing neovascularization in a mammal in which an
effective amount of vascularization modulating agent is
co-administered with an amount of at least one angiogenic protein.
In many settings, it is believed that co-administration of the
vascularization modulating agent and the angiogenic protein can
positively impact neovascularization in the mammal, e.g., by
providing additive or synergistic effects. A preferred angiogenic
protein is a recognized endothelial cell mitogen such as those
specific proteins discussed below. Methods for co-administering the
vascularization modulating agent and the angiogenic protein are
described below and will generally vary according to intended
use.
[0027] The present invention also provides methods for preventing
or reducing the severity of blood vessel damage in a mammal such as
a human patient in need of such treatment. In one embodiment, the
method includes administering to the mammal an effective amount of
vascularization modulating agent such as GM-CSF. At about the same
time or subsequent to that administration, the mammal is exposed to
conditions conducive to damaging the blood vessels. Alternatively,
administration of the vascularization modulating agent can occur
after exposure to the conditions to reduce or block damage to the
blood vessels. As discussed, many conditions are known to induce
ischemic tissue in mammals which conditions can be particularly
conducive to damaging blood vessels, e.g, invasive manipulations
such as surgery, grafting, or angioplasty, infection or ischemia.
Additional conditions and methods for administering the
vascularization modulating agent are discussed below.
[0028] Preferred amounts of the vascularization modulating agent to
use in the methods are sufficient to prevent or reduce the severity
of the blood vessel damage in the mammal. Particular amounts of
GM-CSF have already been mentioned above and include administration
of an effective amount of GM-CSF sufficient to induce
neovascularization in the mammal. Illustrative methods for
quantifying an effective amount of vascularization modulating
agents are discussed throughout this disclosure including the
discussion and Examples which follow.
[0029] The present invention also provides methods for treating
ischemic tissue and especially injured blood vessels in that
tissue. Preferably, the method is conducted with a mammal and
especially a human patient in need of such treatment. In one
embodiment, the method includes as least one and preferably all of
the following steps:
[0030] a) isolating endothelial progenitor cells (EPCs) from the
mammal,
[0031] b) contacting the isolated EPCs with an effective amount of
at least one factor sufficient to induce proliferation of the EPCs;
and
[0032] c) administering the proliferated EPCs to the mammal in an
amount sufficient to treat the injured blood vessel.
[0033] In a particular embodiment of the method, the factor is an
angiogenic protein including those cytokines known to induce EPC
proliferation especially in vitro. Illustrative factors and markers
for detecting EPCs are discussed below. In one embodiment of the
method, the blood vessel (or more than one blood vessel) can be
injured by nearly any known means including trauma or an invasive
manipulation such as implementation of balloon angioplasty or
deployment of a stent or catheter. A particular stent is an
endovascular stent. Alternatively, the vascular injury can be
organic and derived from a pre-existing or on-going medical
condition.
[0034] In another particular embodiment of the method, the
vascularization modulating agent is administered to the mammal and
especially the human patient alone or in combination
(co-administered) with at least one angiogenic protein (or
effective fragment thereof) such as those discussed below.
[0035] Additionally provided by this invention are methods for
detecting presence of tissue damage in a mammal and especially a
human patient. In one embodiment, the method includes contacting
the mammal with a detectably-labeled population of EPCs; and
detecting the detectably-labeled cells at or near the site of the
tissue damage in the mammal. In this example, the EPCs can be
harvested and optionally monitored or expanded in vitro by nearly
any acceptable route including those specific methods discussed
herein. The EPCs can be administered to the mammal by one or a
combination of different approaches with intravenous injection
being a preferred route for most applications. Methods for
detectably-labeling cells are known in the field and include
immunological or radioactive tagging as well as specific
recombinant methods disclosed below.
[0036] In a particular embodiment of the method, the
detectably-labeled EPCs can be used to "home-in" to a site of
vascular damage, thereby providing a minimally invasive means of
visualizing that site even when it is quite small. The
detectably-labeled EPCs can be visualized by a variety of methods
well-known in this field including those using tomography, magnetic
resonance imaging, or related approaches.
[0037] In another embodiment of the method, the tissue damage is
facilitated by ischemia, particularly an ischemic vascular disease
such as those specifically mentioned below. Also provided by this
invention are methods for modulating the mobilization of EPCs which
methods include administering to the mammal an effective amount of
at least one hematopoietic factor. Preferred are methods that
enhance EPC mobilization as determined by any suitable assay
disclosed herein. For example, in a particular embodiment of the
method, the enhancement in EPC mobilization and particulary the
increase in frequency of the EPCs is at least about 20% and
preferably from between 50% to 500% as determined by a standard EPC
isolation assay.
[0038] In another particular embodiment of the method, the amount
of administered hematopoietic factor is sufficient to enhance EPC
mobilization and especially to increase EPC differentiation in the
mammal. Methods for detecting and quantifying EPC differentiation
include those specific methods described below. Preferably, the
increase in EPC differentiation is at least about 20%, preferably
between from about 100% to 1000%, more preferably between from
about 200% to. 800% as determined by a standard EPC culture assay
discussed below. More preferably, that amount of administered
hematopoietic factor is additionally sufficient to increase EPC
differentiation by about the stated percent amounts following
tissue ischemia as determined in a standard hindlimb ischemia assay
as discussed below.
[0039] As discussed, it has been found that EPC mobilization
facilitates significant induction of neovascularization in mammals.
Thus, methods that modulate EPC mobilization and particularly
enhance same can be used to induce neovascularization in the mammal
and especially a human patient in need of such treatment. Methods
of this invention which facilitate EPC mobilization including those
employing at least one hematopoietic factor which use can be alone
or in combination with other methods disclosed herein including
those in which an effective amount of vascularization modulating
agent is administered to the mammal alone or in combination
(co-administered) with at least one angiogenic protein.
[0040] In particular, the invention provides methods for inducing
neovascularization in a mammal and especially a human patient in
need of such treatment which methods include administering to the
mammal an effective amount of at least one vascularization
modulating agent, preferably one vascularization modulating agent,
which amount is sufficient to induce neovascularization in the
mammal. That neovascularization can be detected and quantified if
desired by the standard assays disclosed herein including the mouse
cornea micropocket assay and blood vessel size assays. Preferred
methods will enhance neovascularization in the mammal by the stated
percent ranges discussed previously.
[0041] In one embodiment of the method, the effective amount of the
vascularization modulating agent (s) is co-administered in
combination with at least one angiogenic protein, preferably one
angiogenic protein. The vascularization modulating agent can be
administered to the mammal and especially a human patient in need
of such treatment in conjunction with, subsequent to, or following
administration of the angiogenic or other protein.
[0042] The invention also provides a pharmaceutical product that is
preferably formulated to modulate and especially to induce
neovascularization in a mammal. In a preferred embodiment, the
product is provided sterile and optionally includes an effective
amount of GM-CSF and optionally at least one angiogenic protein. In
a particular embodiment, the product includes isolated endothelial
progenitor cells (EPCs) in a formulation that is preferably
physiologically acceptable to a mammal and particularly a human
patient in need of the EPCs. Alternatively, the product can include
a nucleic acid that encodes the GM-CSF and/or the angiogenic
protein.
[0043] Also provided by this invention are kits preferably
formulated for in vivo and particularly systemic introduction of
isolated EPCs. In one embodiment, the kit includes isolated EPCs
and optionally at least one angiogenic protein or nucleic acid
encoding same. Preferred is a kit that optionally includes a
pharmacologically acceptable carrier solution, nucleic acid or
mitogen, means for delivering the EPCs and directions for using the
kit. Acceptable means for delivering the EPCs are known in the
field and include effective delivery by stent, catheter, syringe or
related means.
[0044] Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIGS. 1A-D are representations of photomicrographs showing
neovascularization following GM-CSF and VEGF-1 treatment in control
(FIGS. 1A, 1C) and treated (FIGS. 1B and 1D) mice in a cornea
micropocket assay.
[0046] FIGS. 2A-B are graphs showing quantitation of increases in
vessel length (2A) and vessel angle (2B) observed in the cornea
micropocket assay.
[0047] FIGS. 3A-C are graphs showing EPC frequency (3A), EPC
differentiation (3B), blood pressure and capillary density (3C)
following GM-CSF treatment in the rabbit hindlimb ischemia
assay.
[0048] FIGS. 4A-4J are representations of photomicrographs showing
that EPCs can home and incorporate into foci of neovascularization.
(4A) cultured murine cells, (4B-D) homing of Sca-1.sup.+ cells
administered to the mouse, (4E-G) immunostaining of rabbit hindlimb
muscle showing accumulation and colonization of EPCs, (4H-J)
colonized TBM.sup.- cells establishing new vessels.
[0049] FIGS. 5A-B are graphs showing EPC kinetics in relation to
development of hindlimb ischemia.
[0050] FIGS. 5C-F are representations of photomicrographs showing
results of the mouse cornea micropocket assay with hindlimb
ischemia. (5C-D) slit-lamp biomicroscopy, (5E-F) demonstration of
neovascularization.
[0051] FIGS. 5G-H are graphs illustrating quantitation of vessel
length and circumferential distribution of neovascularization.
[0052] FIGS. 6A-C are graphs showing effect of GM-CSF-induced EPC
mobilization on neovascularization in the rabbit ischemic hindlimb
model.
[0053] FIGS. 6D-G are representations of photomicrographs showing
the GM-CSF induced effects described in FIGS. 6A-C. (6D, E)
slit-lamp biomicroscopy, (6F, G) fluorescent photomicrographs.
[0054] FIGS. 6H and 6I are graphs showing measurements of vessel
length (6H) and vessel circumference (6I) taken from the experiment
shown in FIGS. 6D-G.
[0055] FIGS. 7A-C are graphs showing that detectably-labeled
bone-marrow derived EPCs contribute to corneal neovascularization.
(7A) corneal neovascularization in mice with hindlimb ischemia,
(7B) rabbits pre-treated with GM-CSF, (7C) beta-galactosidase
activity in GM-CSF control group.
DETAILED DESCRIPTION OF THE INVENTION
[0056] As discussed, the present invention provides, in one aspect,
methods for inducing neovascularization in a human patient that
include administrating to the patient an effective amount of GM-CSF
or an effective fragment thereof. As also discussed, that GM-CSF
can be administered to the human patient alone or in combination
(c-administered) with one or more of: at least one vascularization
modulating agent, preferably one of such factors; at least one
angiogenic protein, preferably one angiogenic protein; or an
effective fragment thereof. Also provided are methods for enhancing
EPC mobilization which methods include administration of an
effective amount of at least one vascularization modulating agent,
preferably one of such factors. Further provided are methods for
treating or detecting damaged blood vessels in the human patient.
The invention has a wide spectrum of uses including preventing or
reducing the severity of blood vessel damage in the patient.
[0057] The invention particularly provides methods for inducing
angiogenesis in ischemic tissue of a patient in need such
treatment. In this embodiment, the methods generally include
administering to the patient an effective amount of GM-CSF or other
vascularization modulating agent disclosed herein. Administration
of the GM-CSF (or co-adminstration with other another protein or
proteins) can be as needed and may be implemented prior to, during
or after formation of the ischemic tissue. Additionally, the GM-CSF
can be administered as the sole active compound or it can be
co-administered with at least one and preferably one angiogenic
protein or other suitable protein or fragment as provided
herein.
[0058] Administration of an effective amount GM-CSF or other
vascularization modulating agent disclosed herein in accord with
any of the methods disclosed herein can be implemented by one or a
combination of different strategies including administering a DNA
encoding same.
[0059] As discussed, methods of this invention have a wide spectrum
of uses especially in a human patient, e.g., use in the prevention
or treatment of at least one of trauma, graft rejection,
cerebrovascular ischemia, renal ischemia, pulmonary ischemia,
ischemia related to infection, limb ischemia, ischemic
cardiomyopathy, cerebrovascular ischemia, and myocardial ischemia.
Impacted tissue can be associated with nearly any physiological
system in the patient including the circulatory system or the
central nervous system, e.g., a limb, graft (e.g., muscle or nerve
graft), or organ (e.g., heart, brain, kidney and lung). The
ischemia may especially adversely impact heart or brain tissue as
often occurs in cardiovascular disease or stroke, respectively.
[0060] In embodiments in which an effective amount of the
vascularization modulating agent is administered to a mammal and
especially a human patient to prevent or reduce the severity of a
vascular condition and particularly ischemia, the vascularization
modulating agent will preferably be administered at least about 12
hours, preferably between from about 24 hours to 1 week up to about
10 days prior to exposure to conditions conducive to damaging blood
vessels. If desired, the method can further include administering
the vascularization modulating agent to the mammal following
exposure to the conditions conducive to damaging the blood vessels.
As discussed, the vascularization modulating agent can be
administered alone or in combination with at least one angiogenic
protein preferably one of such proteins.
[0061] Related methods for preventing or reducing the severity of
the vascular condition can be employed which methods include
administering alone or in combination (co-administration) with the
GM-CSF one or more of: at least one hematopoietic factor,
preferably one of such factors; or at least one angiogenic protein,
preferably one of such proteins. Preferred methods of
administration are disclosed herein.
[0062] Vessel injury is known to be facilitated by one or a
combination of different tissue insults. For example, vessel injury
often results from tissue trauma, surgery, e.g., balloon
angioplasty and use of related devices (e.g., directional
atherectomy, rotational atherectomy, laser angioplasty,
transluminal extraction, pulse spray thrombolysis); and deployment
of an endovascular stent or a vascular graft.
[0063] Specific EPCs in accord with this invention will be
preferably associated with cell markers that can be detected by
conventional immunological or related strategies. Preferred are
EPCs having at least one of the following markers: CD34.sup.+,
flk-1.sup.+ or tie-2.sup.+. Methods for detecting EPCs with these
markers are discussed in the Examples below.
[0064] As discussed above and in the Examples following, we have
discovered means to promote angiogenesis and reendothelialize
denuded blood vessels in mammals. These methods involve the use of
vascularization modulating agent to mobilize endothelial cell (EC)
progenitors. In accordance with the present invention, GM-CSF and
other vascularization modulating agents can be used in a method for
enhancing angiogenesis in a selected patient having an ischemic
tissue i.e., a tissue having a deficiency in blood as the result of
an ischemic disease such as cerebrovascular ischemia, renal
ischemia, pulmonary ischemia, limb ischemia, ischemic
cardiomyopathy and myocardial ischemia.
[0065] Additionally, in another embodiment, the vascularization
modulating agent, alone or in combination with at least one other
factor disclosed herein can be used to induce reendothelialization
of an injured blood vessel, and thus reduce restenosis by
indirectly inhibiting smooth muscle cell proliferation.
[0066] In one preferred embodiment, the vascularization modulating
agent, alone or in combination with at least one other factor
disclosed herein can be used to prepare a patient for angiogenesis.
Some patient populations, typically elderly patients, may have
either a limited number of ECs or a limited number of functional
ECs. Thus, if one desires to promote angiogenesis, for example, to
stimulate vascularization by using a potent angiogenesis promotor
such as VEGF-1, such vascularization can be limited by the lack of
EPCs. However, by administering e.g., GM-CSF at a time before
administration of the angiogenesis promoter sufficient to allow
mobilization of the ECs, one can potentiate the vascularization in
those patients. Preferably, GM-CSF is administered about one week
prior to treatment with the angiogenesis promoter.
[0067] The term "GM-CSF" as used herein shall be understood to
refer to a natural or recombinantly prepared protein having
substantial identity to an amino acid sequence of human GM-CSF as
disclosed, for example, in published international application WO
86/00639, which is incorporated herein by reference. Recombinant
human GM-CSF is hereinafter also referred to as "hGM-CSF."
[0068] Human GM-CSF (hGM-CSF) has been isolated and cloned, see
published International Application No. PCT/EP 85/00326, filed Jul.
4, 1985 (published as WO 86/00639). E. coli derived,
non-glycosylated rhGM-CSF can be obtained by the methods described
in publication of the International Application No. PCT/EP
85/00326, wherein two native GM-CSFs differing in a single amino
acid are described.
[0069] The natural GM-CSF proteins used in the invention may be
modified by changing the amino acid sequence thereof. For example,
from 1 to 5 amino acids in their sequences may be changed, or their
sequences may be lengthened, without changing the fundamental
character thereof and provide modified proteins which are the full
functional equivalents of the native proteins. Such functional
equivalents may also be used in practicing the present invention. A
GM-CSF differing by a single amino acid from the common native
sequence is disclosed in U.S. Pat. No. 5,229,496 and has been
produced in glycosylated form in yeast, and has been clinically
demonstrated to be a biological equivalent of native GM-CSF, such
modified form known as GM-CSF (Leu-23).
[0070] GM-CSF is commercially and clinically available as an analog
polypeptide (Leu.sup.23) under the trademark LEUKINE.RTM. (Immunex
Corporation). The generic name for recombinant human Leu.sup.23
GM-CSF analog protein expressed in yeast is Sargramostim. Cloning
and expression of native sequence human GM-CSF was described in
Cantrell et al., Proc Natl. Acad. Sci. U.S.A. 82:6250(1985).
[0071] The natural or recombinantly prepared proteins, and their
functional equivalents used in the method of the invention are
preferably purified and substantially cell-free, which may be
accomplished by known procedures.
[0072] Additional protein and nucleic sequences relating to the
factors disclosed herein including GM-CSF can be obtained through
the National Center for Biotechnology Information (NCBI)--Genetic
Sequence Data Bank (Genbank). In particular, sequence listings can
be obtained from Genbank at the National Library of Medicine, 38A,
8N05, Rockville Pike, Bethesda, Md. 20894. Genbank is also
available on the internet at http://www.ncbi.nlm.nih.gov. See
generally Benson, D. A. et al. (1997) Nucl. Acids. Res. 25: 1 for a
description of Genbank. Protein and nucleic sequences not
specifically referenced can be found in Genbank or other sources
disclosed herein.
[0073] In accord with the methods of this invention, GM-CSF can be
administered to a mammal and particularly a human patient in need
of such treatment. As an illustration, GM-CSF as well as
therapeutic compositions including same are preferably administered
parenterally. More specific examples of parenteral administration
include subcutaneous, intravenous, intra-arterial, intramuscular,
and intraperitoneal, with subcutaneous being preferred.
[0074] In embodiments of this invention in which parenteral
administration is selected, the GM-CSF will generally be formulated
in a unit dosage injectable form (solution, suspension, emulsion),
preferably in a pharmaceutically acceptable carrier medium that is
inherently non-toxic and non-therapeutic. Examples of such vehicles
include without limitation saline, Ringer's solution, dextrose
solution, mannitol and normal serum albumin. Neutral buffered
saline or saline mixed with serum albumin are exemplary appropriate
vehicles. Non-aqueous vehicles such as fixed oils and ethyl oleate
may also be used. Additional additives include substances to
enhance isotonicity and chemical stability, e.g., buffers,
preservatives and surfactants, such as Polysorbate 80. The
preparation of parenterally acceptable protein solutions of proper
pH, isotonicity, stability, etc., is within the skill of the
art.
[0075] Preferably, the product is formulated by known procedures as
a lyophilizate using appropriate excipient solutions (e.g.,
sucrose) as a diluent.
[0076] Preferred in vivo dosages the vascularization modulating
agents are from about 1 .mu.g/kg/day to about 100 .mu.g/kg/day. Use
of more specific dosages will be guided by parameters well-known to
those in this field such as the specific condition to be treated
and the general health of the subject. See also U.S. Pat. No.
5,578,301 for additional methods of administering GM-CSF.
[0077] Preferred in vivo dosages for the hematopoietic proteins and
angiogenic proteins disclosed herein will be within the same or
similar range as for GM-CSF. As discussed, for some applications it
will be useful to augment the vascularization modulating agent
administration by co-administering one or more of: at least one
hematopoietic protein, at least one angiogenic protein; or an
effective fragment thereof. This approach may be especially
desirable where an increase (boost) in angiogenesis is needed. For
example, in one embodiment, at least one angiogenic protein and
preferably one of same will be administered to the patient in
conjunction with, subsequent to, or prior to the administration of
the GM-CSF. The angiogenic protein can be administered directly,
e.g., intra-arterially, intramuscularly, or intravenously, or
nucleic acid encoding the mitogen may be used. See, Baffour, et
al., supra (bFGF); Pu, et al, Circulation, 88:208-215 (1993)
(aFGF); Yanagisawa-Miwa, et al., supra (bFGF); Ferrara, et al.,
Biochem. Biophys. Res. Commun., 161:851-855 (1989) (VEGF-1);
(Takeshita, et al., Circulation, 90:228-234 (1994); Takeshita, et
al., Laboratory, 75:487-502 (1996); Tsusumi, et al., Circulation,
94 (12):3281-3290 (1996)).
[0078] As another illustration, at least one hematopoietic protein
and preferably one of such proteins can be administered to the
human patient in need of such treatment in conjunction with,
subsequent to, or prior to the administration of the GM-CSF. As
discussed, at least one angiogenic protein can also be
co-administered with the GM-CSF and hematopoietic protein. Methods
for administering the hematopoietic protein will generally follow
those discussed for adminstering the GM-CSF although other modes of
administration may be suitable for some purposes.
[0079] It will be understood that the term "co-administration" is
meant to describe preferred administration of at least two proteins
disclosed herein to the mammal, ie., administration of one protein
in conjunction with, subsequent to, or prior to administration of
the other protein.
[0080] In embodiments in which co-administration of a DNA encoding
and angiogenic or hematopoietic protein is desired, the nucleic
acid encoding same can be administered to a blood vessel perfusing
the ischemic tissue via a catheter, for example, a hydrogel
catheter, as described by U.S. Pat. No. 5,652,225, 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 PCT WO 97/14307.
[0081] As used herein the term "angiogenic protein" or related term
such as "angiogenesis protein" means any protein, polypeptide,
mutein or portion that is capable of, directly or indirectly,
inducing blood vessel growth. Such proteins include, for example,
acidic and basic fibroblast growth factors (aFGF and bFGF),
vascular endothelial growth factor (VEGF-1), VEGF165, epidermal
growth factor (EGF), transforming growth factor .alpha. and .beta.
(TGF-.alpha. and TFG-.beta.), platelet-derived endothelial growth
factor (PD-ECGF), platelet-derived growth factor (PDGF), tumor
necrosis factor .alpha.(TNF-.alpha.), hepatocyte growth factor
(HGF), insulin like growth factor (IGF), erythropoietin, colony
stimulating factor (CSF), macrophage-CSF (M-CSF),
granulocyte/macrophage CSF (GM-CSF), angiopoetin-1 (Ang1) and
nitric oxidesynthase (NOS). 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). Muteins or fragments of a mitogen may
be used as long as they induce or promote blood vessel growth.
[0082] Preferred angiogenic proteins include vascular endothelial
growth factors. One of the first of these was termed VEGF, now
called VEGF-1, exists in several different isoforms that are
produced by alternative splicing from a single gene containing
eight exons (Tischer, et al., J. Biol. Chem., 806, 11947-11954
(1991), Ferrara, Trends Cardio. Med., 3, 244-250 (1993), Polterak,
et al., J. Biol. Chem., 272, 7151-7158 (1997)). Human VEGF isoforms
consists of monomers of 121 (U.S. Pat. No. 5,219,739), 145, 165
(U.S. Pat. No. 5,332,671), 189 (U.S. Pat. No. 5,240,848) and 206
amino acids, each capable of making an active homodimer (Houck, et
al., Mol. Endocrinol., 8, 1806-1814 (1991)).
[0083] Other vascular endothelial growth factors include VEGF-B and
VEGF-C (Joukou, et al., J. of Cell. Phys. 173:211-215 (1997),
VEGF-2 (WO 96/39515), and VEGF-3 (WO 96/39421).
[0084] Preferably, the angiogenic protein contains a secretory
signal sequence that facilitates secretion of the protein. Proteins
having native signal sequences, e.g., VEGF-1, 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).
[0085] Reference herein to a "vascularization modulating agent",
"hematopoietic factor" or related term, e.g., "hematopoietic
protein" is used herein to denote recognized factors that increase
mobilization of hematopoietic progenitor cells (HPC). Preferred
hematopoietic factors include granulocyte-macrophage
colony-stimulating factor (GM-CSF), VEGF, Steel factor (SLF, also
known as Stem cell factor (SCF)), stromal cell-derived factor
(SDF-1), granulocyte-colony stimulating factor (G-CSF), HGF,
Angiopoietin-1, Angiopoietin-2, M-CSF, b-FGF, and FLT-3 ligand.
Disclosure relating to these and other hematopoietic factors can be
found in Kim, C. H. and Broxmeyer, H. E. (1998) Blood, 91: 100;
Turner, M. L. and Sweetenham, J. W., Br. J. Haematol. (1996) 94:
592; Aiuuti, A. et al. (1997) J. Exp. Med. 185: 111; Bleul, C. et
al. (1996) J. Exp. Med. 184: 1101; Sudo, Y. et al. (1997) Blood,
89: 3166; as well as references disclosed therein.
[0086] The nucleotide sequence of numerous angiogenic proteins, are
readily available through a number of computer databases, 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.
[0087] In certain situations, it may be desirable to use nucleic
acids encoding two or more different proteins in order optimize
therapeutic outcome. For example, DNA encoding two proteins, e.g.,
VEGF-1 and bFGF, can be used, and provides an improvement over the
use of bFGF alone. Or an angiogenic factor 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.
[0088] The term "effective amount" means a sufficient amount of a
compound, e.g. protein or nucleic acid delivered to produce an
adequate level of the subject protein (e.g., GM-CSF,
vascularization modulating agent, hematopoietic protein, angiogenic
protein) i.e., levels capable of inducing endothelial cell growth
and/or inducing angiogenesis as determined by standard assays
disclosed throughout this application. Thus, the important aspect
is the level of protein expressed. Accordingly, one can use
multiple transcripts or one can have the gene under the control of
a promoter that will result in high levels of expression. In an
alternative embodiment, the gene would be under the control of a
factor that results in extremely high levels of expression, e.g.,
tat and the corresponding tar element.
[0089] To simplify the manipulation and handling of the nucleic
acid encoding the protein, the nucleic acid is preferably inserted
into a cassette where it is operably linked to a promoter. The
promoter must be capable of driving expression of the protein in
cells of the desired target tissue. The selection of appropriate
promoters can readily be accomplished. Preferably, one would use a
high expression promoter. An example of a suitable promoter is the
763-base-pair cytomegalovirus (CMV) promoter. The Rous sarcoma
virus (RSV) (Davis, et al., Hum Gene Ther 4:151 (1993)) and MMT
promoters may also be used. Certain proteins can be expressed using
their native promoter. Other elements that can enhance expression
can also be included such as an enhancer or a system that results
in high levels of expression such as a tat gene and tar element.
This cassette can then be inserted into a vector, e.g., a plasmid
vector such as pUC118, pBR322, or other known plasmid vectors, that
includes, for example, an E. coli origin of replication. See,
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory press, (1989). The plasmid vector may also
include a selectable marker such as the .beta.-lactamase gene for
ampicillin resistance, provided that the marker polypeptide does
not adversely effect the metabolism of the organism being treated.
The cassette can also be bound to a nucleic acid binding moiety in
a synthetic delivery system, such as the system disclosed in WO
95/22618.
[0090] Particular methods of the present invention may be used to
treat blood vessel injuries that result in denuding of the
endothelial lining of the vessel wall. For example, primary
angioplasty is becoming widely used for the treatment of acute
myocardial infarction. In addition, endovascular stents are
becoming widely used as an adjunct to balloon angioplasty. Stents
are useful for rescuing a sub-optimal primary result as well as for
diminishing restenosis. To date, however; the liability of the
endovascular prosthesis has been its susceptibility to thrombotic
occlusion in approximately 3% of patients with arteries 3.3 mm or
larger. If patients undergo stent deployment in arteries smaller
than this the incidence of sub-acute thrombosis is even higher.
Sub-acute thrombosis is currently prevented only by the aggressive
use of anticoagulation. The combination of vascular intervention
and intense anticoagulation creates significant risks with regard
to peripheral vascular trauma at the time of the stent/angioplasty
procedure. Acceleration of reendothelialization by administration
of GM-CSF alone or in combination with other factors disclosed
herein to a patient prior to undergoing angioplasty and/or stent
deployment can stabilize an unstable plaque and prevent
re-occlusion. In this example, GM-CSF is preferably administered
about 1 week prior to the denuding of the vessel wall.
[0091] The methods of the present invention may be used in
conjunction a DNA encoding an endothelial cell mitogen in
accordance with the method for the treatment of vascular injury
disclosed in PCT/US96/15813.
[0092] As used herein the term "endothelial cell mitogen" means any
protein, polypeptide, mutein or portion that is capable of inducing
endothelial cell growth. Such proteins include, for example,
vascular endothelial growth factor (VEGF-1), acidic fibroblast
growth factor (aFGF), basic fibroblast growth factor (bFGF),
hepatocyte growth factor (scatter factor), and colony stimulating
factor (CSF). VEGF-1 is preferred.
[0093] In addition, the methods of the present invention may be
used to accelerate the healing of graft tissue, e.g., vascular
grafts, by potentiating vascularization.
[0094] Reference herein to a "standard EPC isolation assay" or
other similar phrase means an assay that includes at least one of
and preferably all of the following steps:
[0095] a) obtaining a peripheral blood sample from a subject
mammal, preferably a rodent and especially a mouse,
[0096] b) separating from the blood sample light-density
mononuclear cells,
[0097] c) contacting the separated mononuclear cells with beads
that include a sequence capable of specifically binding Sca-1 cells
and separating same from the mononuclear cells; and
[0098] d) quantitating the Sca-1.sup.+ cells, eg., by counting
those cells manually.
[0099] See the following discussion and Examples for more specific
disclosure relating to the standard EPC isolation assay.
[0100] By the term "standard EPC culture assay" or related term is
meant an assay that includes at least one of and preferably all of
the following steps.
[0101] a) isolating Sca-1+ and Sca-1- cells from the peripheral
blood of mouse, or TBM+ and TBM- cells from the peripheral blood of
a rabbit, and detectably-labelling the cells (Sca-1+ and TBM-),
e.g., with Di-I as provided herein,
[0102] b) culturing the cells in a suitable dish or plate in medium
for several days and usually for about 4 days,
[0103] c) counting any attached spreading cells in the dish or
plate as being Di-I labeled Sca-1+ or TBM- or non-labeled Sca-1- or
TBM+,
[0104] d) and quantitating specific positive cells as being
indicative of EPCs.
[0105] More specific disclosure relating to the standard EPC
culture assay can be found in the discussion and Examples that
follow.
[0106] Reference herein to a "standard hind limb ischemia assay" or
related term is meant to denote a conventional assay for inducing
hindlimb ishemica in accepted animal models and particularly the
mouse or rabbit. Disclosure relating to conducting the assay can be
found in the Examples and Materials and Methods section that
follows. See also Couffinhal, T. et al. (1998) Am. J. Pathol.,
infra; and Takeshita, S. et al. (1994) J. Clinical. Invest. 93: 662
for more disclosure relating to performing the assay.
[0107] Reference herein to a "standard blood vessel length assay"
or "standard blood vessel diameter assay" generally means exposing
a blood vessel of interest in the subject mammal (e.g., mouse or
rabbit) and measuring the length or diameter of that vessel by
conventional means following inspection of that vessel.
Illustrative blood vessels such as certain arteries or veins which
can be measured are provided below.
[0108] The phrase "standard cornea micropocket assay" or related
term is used herein in particular reference to a mouse corneal
neovascularization assay. The assay generally involves one and
preferably all of the following steps.
[0109] a) creating a corneal micropocket in at least one eye of a
mouse,
[0110] b) adding to the pocket a pellet including an acceptable
polymer and at least one angiogenic protein, preferably VEGF-1,
[0111] c) examining the mouse eye, e.g, by slit-lamp biomicroscopy
for vascularization, typically a few days, e.g., 5 to 6 days
following step b),
[0112] d) marking EC cells in the eye, e.g., with BS-1 lectin;
and
[0113] e) quantitating vascularization and optionally EC cell
counts in the eye.
[0114] For more specific disclosure relating to the standard cornea
micropocket assay, see the discussion and Examples which follow. If
desired, the assay can include a control as a reference which
control will include performing steps a)-e) above, except that step
b) will include adding a pellet without the angiogenic protein.
[0115] Reference herein to a "standard murine bone marrow (BM)
transplantation model" or similar phrase is meant at least one and
preferably all of the following steps.
[0116] a) obtaining detectably-labeled BM cells from a donor mammal
and typically a mouse,
[0117] b) isolating low-density BM mononuclear cells from the
mouse,
[0118] c) removing BM cells from a suitable recipient mouse, e.g,
by irradiation,
[0119] d) administering the isolated and detectably-labeled BM
cells to the recipient mouse,
[0120] e) exposing the recipient mouse to conditions conditions
conducive to damaging blood vessels in the mouse, e.g., hindlimb
ischemia,
[0121] f) administering an effective amount of GM-CSF to the
recipient mouse,
[0122] g) harvesting at least one cornea from the recipient mouse;
and
[0123] h) detecting and quantitating any labeled BM cells in the
cornea.
[0124] An illustrative detectable-label is beta-galactosidase
enzyme activity. More specific information relating to the assay
can be found in the discussion and Examples which follow.
[0125] Reference herein to an "effective fragment" of
vascularization modulating agents such as GM-CSF, a hemopoietic
protein, or angiogenic protein means an amino acid sequence that
exhibits at least 70%, preferably between from about 75% to 95% of
the vessel promoting activity of the corresponding full-length
protein as determined by at least one standard assay as disclosed
herein. Preferred are those assays which detect and preferably
quantify EPC mobilization although other standard assays can be
used. As an illustration, a preferred effective fragment of GM-CSF
will have at least 70% and preferably from about 75% to 95% of the
vessel promoting activity of full-length human GM-CSF (see the
published International Application No. PCT/EP/85/00376
(WO86/00639)) as determined in the standard corneal micropocket
assay and especially the standard blood vessel length or diameter
assays.
[0126] All documents mentioned herein are incorporated by reference
herein in their entirety.
[0127] The present invention is further illustrated by the
following examples. These examples are provided to aid in the
understanding of the invention and are not construed as a
limitation thereof.
EXAMPLE 1
Modulation of EPC Kinetics by Cytokine Adminstration
[0128] Circulating EPCs may constitute a reparative response to
injury. The hypothesis that cytokine-administration may mobilize
EPCs and thereby augment therapeutic neovascularization was
investigated as follows.
[0129] GM-CSF, which induces proliferation and differentiation of
hematopoietic prognitor cells (Socinski, et al., Lancet, 1988;
1:1194-1198, Gianni, et al., Lancet, 1989;2:580-584) and cells of
myeloid lineage (Clark, et al., Science 1987;236:1229-1237, Sieff,
C., J. Clin. Invest. 1987;79:1549-1557), as well as
non-hematopoietic cells including BM stroma cells (Dedhar, et al.,
Proc. Natl. Acad. Sci USA 1988;85:9253-9257) and ECs (Bussolini, et
al., J. Clin. Invest., 1991;87:986-995), was used to promote
cytokine-induced EPC mobilization. To avoid a direct mitogenic
effect on ECs, GM-CSF was administered for 7 days prior to creating
the stimulus for neovascularization. De novo vascular formation was
initially examined in the mouse cornea pocket assay described
above. GM-GSF-pretreatment (intraperitoneal [i.p.] rmGM-CSF
[R&D Systems] 500 ng/day) increased circulating EPCs (221% of
untreated controls) at day 0, i.e., prior to creation of the cornea
micropocket and insertion of VEGF pellet; correspondingly,
neovascularization at day 6 (FIGS. 1A-C) was augmented in
comparison to control mice (length=0.67.+-.0.04 vs 0.53.+-.0.04,
p<0.05; angle (circumferential degrees occupied by
neovascularity)=155.+-.13 vs 117.+-.12, p.<0.05) (FIGS. 1B-1D).
See also FIGS. 2A and 2B.
EXAMPLE 2
Cytokine-Induced EPC Mobilization Enhances Neovascularization of
Ischemic Tissues
[0130] To determine if cytokine-induced EPC mobilization could
enhance neovascularization of ischemic tissues, we employed the
rabbit hindlimb ischemia model (Takeshita, et al., J. Clin. Invest.
1994;93:662-670). In GM-CSF pretreated rabbits (subcutaneous [s.c.]
rhGM-CSF; 50 .mu.g/day s.c.), EPC-enriched cell population was
increased (189% compared to control animals), and EPC
differentiation was enhanced (421% compared to control) at day 0 of
(i.e., prior to) surgery (FIG. 3) Morphometric analysis of
capillary density disclosed extensive neovascularization induced by
GM-CSF pre-treatment compared to control (ischemia, no GM-CSF)
group (249 vs 146/mm.sup.2, p<0.01). GM-CSF pre-treatment also
markedly improved ischemic limb/normal limb blood pressure ratio
(0.71 vs 0.49, p<0.01) FIGS. 3A-3C).
EXAMPLE 3
EPC Kinetics During Tissue Ischemia
[0131] To investigate EPC kinetics during tissue ischemia, the
frequency and differentiation of EPCs were assessed by EPC
isolation from peripheral blood and EPC culture assay. EPC-enriched
fractions were isolated from mice as Sca-1 antigen-positive
(Sca-1.sup.+) cells, and from rabbits as the cell population
depleted of T-lymphocytes, B-lymphocytes and monocytes (TBM.sup.-),
denoted by the antigen repertoire CD5-/Ig.mu.-/CD11b-.
[0132] The frequency of EPC-enriched population marked by Sca-1 in
the circulation was 10.7.+-.1.0% in C57/6JBL normal mice. A subset
of Sca-1.sup.+ cells plated on rat vitronectin attached and became
spindle-shaped within 5 days. Co-cultures of Sca-1.sup.+ and Sca-1
negative (Sca-1.sup.-) cells were examined after marking
Sca-1.sup.+ cells with DiI fluorescence. Sca-1.sup.+ cells
developed a spindle-shaped morphology. Mouse adherent cells in
co-culture were found to be principally derived from DiI-marked
Sca-1.sup.+ cells (65.about.84%) and showed evidence of EC lineage
by reaction with BS-1 lectin and uptake of acLDL.sup.1 (FIG. 4A).
To determine if Sca-1.sup.+ cells can differentiate into ECs in
vivo, a homogeneous population of DiI-marked Sca-1.sup.+ cells,
isolated from peripheral blood of the same genetic background, was
administered intravenously to mice with hindlimb ischemia
(Couffinhal, T., et al. Am. J. Pathol. (1998) day after ischemic
surgery. DiI-labeled EPC-derived cells were shown to be
differentiated in situ into ECs by co-staining for CD31 (PECAM) and
were found incorporated into colonies, sprouts, and capillaries
(FIGS. 4A-4D).
[0133] For the rabbit model, mature HCs were depleted using
antibodies to T and B lymphocytes and monocytes, yielding an
EPC-enriched (TBM.sup.-) fraction. The frequency of TBM.sup.-
EPC-enriched population in rabbit peripheral blood was
22.0.+-.1.4%. Differentiation of EPCs was assayed by counting
adherent cultured mononuclear blood cells. Adherent cells in EPC
culture were found again to be derived principally from DiI-marked
TBM.sup.- cells (71.about.92%) and showed evidence of EC lineage by
positive reaction with BS-1 lectin and uptake of acLDL.
[0134] TBM.sup.- cells were shown to differentiate into ECs in vivo
by administration of autologous DiI-marked TBM.sup.- cells,
isolated from 40 ml peripheral blood, to rabbits with unilateral
hindlimb ischemia (Takeshita, S., et al. J. Clin. Invest (1994) at
0, 3 and 7 days post-operatively. DiI-labeled EPC-derived cells
differentiated in situ into ECs, shown by co-staining for CD31 and
incorporation into colonies, sprouts, and capillaries (FIGS.
4E-4J).
[0135] FIGS. 4A-4D are more particularly explained as follows. The
figures provide fluorescent microscopic evidence that EPCs derived
from isolated populations of Sca-1.sup.+ cells in mice, and
TBM.sup.- cells in rabbit, can home and incorporate into foci of
neovascularization. In particular, in FIG. 4A cultured murine cells
are shown, double-stained for acLDL-DiI (red) and BS-1 lectin
(green) 4 days after EPC culture assay. (FIGS. 4B-D) Sca-1.sup.+
cells administered to mouse with hindlimb ischemia have homed,
differentiated and incorporated into foci of neovascularization in
mouse ischemic hindlimb muscles 2 wks after surgery. FIGS. 4B and
4C document that DiI-labelled Sca-1.sup.+ derived cells (red)
co-localize with CD31 (green) indicdating that these EPCs have
incorporated into CD31-positive vascularture. Arrows indicate cells
positive for DiI and CD31 (derived from delivered EPCs), while
arrowheads indicate CD31-positive, DiI-negative (autologous ECs).
Non-fluorescent, phase contrast photograph in FIG. 1d documents
vascular foci of EPCs (arrows) are within interstitial sites
adjacent to skeletal myocytes.
[0136] FIGS. 4E-G show immunostaining of rabbit ischemic hindlimb
muscle 2 wks after ischemia surgery shows accumulation and
colonization of EPCs, in this case isolated as TBM.sup.- cells
(red) (FIG. 4E); these cells were marked with DiI and reinjected at
day 0, 3 and 7. FIG. 4F shows that these cells co-label with CD31,
within neovascular foci. DAPI stains cell nuclei (blue) (FIG. 1G).
(FIGS. 4H-J). Colonized TBM.sup.- cells are incorporated into
developing sprouts, establishing new capillaries among skeletal
myocytes.
EXAMPLE 4
Confirmation of EPC Kinetics During Tissue Ischemia
[0137] EPC kinetics during severe tissue ischemia were assayed for
frequency and differentiation. The EPC-enriched population in
circulating blood increased following the onset of ischemia,
peaking at day 7 post-operatively (day 7 vs day 0: 17.5.+-.2.4 vs
3.8.+-.0.6.times.10.sup.- 5/ml in mouse [p<0.05], 11.4.+-.0.6 vs
6.7.+-.0.3.times.10.sup.5/ml in rabbit [p<0.05]) (FIGS. 5A, 6A).
EPC assay culture demonstrated dramatic enhancement of EPC
differentiation after ischemia, peaking at day 7 (day 7 vs day 0:
263.+-.39 vs 67.+-.14/mm.sup.2 in mouse [p<0.05], 539.+-.73 vs
100.+-.19 in rabbit [p<0.05]) (FIGS. 5B, 6B). Neither the
frequency of the EPC-enriched population nor the EPC culture assay
showed a significant increase in EPC kinetics in either
sham-operated animal model at 7 days following surgery.
[0138] FIGS. 5A and 5B are more specifically explained as follows.
The figures show EPC kinetics in relation to development of
hindlimb ischemia. (FIG. 5A) Following surgery to create ischemic
hindlimb, frequency of mouse EPC-enriched population (Sca-1) in
circulating blood increases, becoming maximum by day 7 (n=5 mice at
each time point). (FIG. 5B) Adherent cells in EPC-culture are
derived principally from DiI-marked Sca-1.sup.+ cells. Assay
culture demonstrates enhanced EPC differentiation after surgically
induced ischemia with a peak at day 7 (n=5 each time point).
[0139] FIGS. 5C-H show results of the mouse cornea micropocket
assay as applied to mice with hindlimb ischemia 7 days after
surgery. Slit-lamp biomicroscopy (FIGS. 5C and 5D) and fluorescent
photomicrographs (FIGS. 5E and 5F) demonstrate that
neovascularization in avascular area of mouse cornea is enhanced by
EPC mobilization induced by ischemia, shown with the same
magnification. (FIGS. 5G and 5H) Quantitative analysis of two
parameters, vessel length and circumferential distribution of
neovascularization, indicates that corneal neovascularization was
more profound in animals with hindlimb ischemia (n=7 mice) than in
non-ischemic, sham control mice (n=9) (*=p<0.05).
EXAMPLE 5
Analysis of Impact of Enhanced EPC Mobilization on
Neovascularization
[0140] To investigate the impact on neovascularization of enhanced
EPC mobilization induced by ischemia, the mouse cornea micropocket
assay was applied to animals in which hindlimb ischemia had been
surgically created 3 days earlier. Slit-lamp (FIGS. 5C and 6D) and
fluorescent (FIGS. 5E, 6F) photomicrographs documented that
neovascularization of avascular mouse cornea was enhanced in
animals with hindlimb ischemia compared to non-ischemic
sham-operated controls. Measurements of vessel length and
circumference showed a significant effect of EPC mobilization on
neovascularization in ischemic animals versus sham control mice
(length 0.67.+-.0.04 vs 0.53.+-.0.04 mm, p<0.05;
circumference=43.3.+-.3.5 vs 32.4.+-.3.4%, p<0.05) (FIGS. 5G,
5H).
EXAMPLE 6
Confirmation of Enhanced Neovascularization with Cytokine-Induced
EPC Mobilization
[0141] The rabbit model of hindlimb ischemia (Takeshita, S., et al.
J. Clin. Invest. (1994)) was employed to determine if
cytokine-induced EPC mobilization could enhance neovascularization
of ischemic tissues. To effect GM-CSF-induced EPC mobilization
while avoiding a direct effect on ECs, recombinant human GM-CSF was
administered daily for 7 days prior to to development of hindlimb
ischemia. Such GM-CSF pre-treatment (50 .mu.g/day s.c.) increased
the EPC-enriched population (12.5.+-.0.8 vs
6.7.+-.0.3.times.10.sup.5/ml, p<0.01) and enhanced EPC
differentiation (423.+-.90 vs 100.+-.19/mm.sup.2, p<0.01) at day
0 (day 7 of pre-treatment prior to surgery). By post-operative day
7, the frequency of circulating EPCs and EPC differentiation in
GM-CSF-pretreated group exceeded control values (20.9.+-.1.0 vs
11.3.+-.2.5.times.10.sup.5/ml [p<0.05], 813.+-.54 vs
539.+-.73/mm.sup.2 [p<0.01]) respectively (FIGS. 6A, 6B).
Capillary density analysis documented extensive neovascularization
induced by GM-CSF pre-treatment (249.+-.118 vs 146.+-.18/mm.sup.2
in untreated controls, p<0.01), as well as improved
ischemic/normal hindlimb blood pressure ratio (0.71.+-.0.03 vs
0.49.+-.0.03, P<0.01) (FIG. 6C).
[0142] FIGS. 6A-I are explained in more detail as follows. The
figures show the effect of GM-CSF-induced EPC mobilization on
neovascularization in rabbit ischemic hindlimb model. (FIGS. 6A,B)
Following pre-treatment with GM-CSF, circulating EPC-enriched
population (TBM.sup.-) is increased in number compared to control
(ischemic, untreated) animals beginning at day 0 (prior to surgery)
through day 7 (FIG. 6A), as is EPC differentiation in culture (FIG.
5B) (n=5 mice at each time point). (FIG. 6C) Two weeks after onset
of rabbit ischemia, physiological assessment using blood pressure
ratio of ischemic to healthy limb indicates significant improvement
in rabbits receiving GM-CSF versus control group. Moreover,
histologic examination with alkaline phosphatase staining
documented increased capillary density in GM-CSF treated rabbits
compared to control group (n=9 mice in each group). (*=p<0.0,
**=p<0.05).
[0143] Slit-lamp biomicroscopy (FIGS. 6D and 6E) and fluorescent
photomicrographs (FIGS. 6F and 6G, same magnification) show that
neovascularization in avascular area of mouse cornea is also
enhanced by EPC mobilization induced by GM-CSF pretreatment. (FIGS.
6H and 61) Measurements of vessel length and circumference indicate
significant effect of EPC mobilization on neovascularization in
GM-CSF pretreated (n=6) versus control mice (n=10)
(*=p<0.05).
EXAMPLE 7
Confirmation of Enhanced Neovascularization Using the Mouse Cornea
Micropocket Assay
[0144] These results described above were corroborated by
assessment of de novo vascularization in the mouse cornea
micropocket assay. GM-CSF-pretreated mice (rmGM-CSF, 500 ng/day
i.p.) developed more extensive corneal neovascularization than
control mice (length=0.65.+-.0.05 vs 0.53.+-.0.04, p<0.05 mm;
circumference=38.0.+-.3.5 vs 28.3.+-.2.7%, p<0.05) (FIGS.
6D-6I).
EXAMPLE 8
Enhanced BM-Derived EPC Incorporation in the BM Transplantation
Model
[0145] A murine BM transplantation (BMT) model was employed to
establish direct evidence of enhanced BM-derived EPC incorporation
into foci of corneal neovascularization in response to ischemia and
GM-CSF. Corneas excised 6 days after micropocket implantation and
examined by light microscopy demonstrated a statistically
significant increase in cells expressing beta-galactosidase in the
ischemic limb versus sham group (3.5.+-.0.6 vs 10.5.+-.1.7,
p<0.01); the same was true for BMT recipients treated with
GM-CSF vs control (3.2.+-.0.3 vs 12.4.+-.1.7, p<0.01) (FIGS. 7A,
7B). Corneas from control mice (post-BMT) disclosed no cells
expressing .beta.-galactosidase. Quantitative chemical detection
confirmed a statistically significant increase in p-galactosidase
activity among mice receiving GM-CSF vs controls (2.90.+-.0.30 vs
2.11.+-.0.09.times.10.sup.3, p<0.05) (FIG. 7C).
[0146] FIGS. 7A-C are explained in more detail as follows. The
figures illustrate that Bone marrow-derived EPCs contribute to
corneal neovascularization. Photomicrographs shown as inserts
document incorporation of BM-derived EPCs expressing
endothelial-specific Tie-2/lacZ (blue cells) into foci of corneal
neovascularization, both in mice with hindlimb ischemia (FIG. 7A),
as well as in rabbits pretreated with GM-CSF (FIG. 7B). The
frequency of incorporated EPCs stained by X-gal was manually
counted under light microscopy. (FIG. 7A) Incorporated EPCs were
significantly more frequent in mice with hindlimb ischemia vs the
sham-operated mice; (FIG. 7B) the same was true for rabbits
receiving GM-CSF group vs control rabbits (*=p<0.01 for each
condition). (FIG. 7C) .beta.-galalactosidase activity was
significantly higher in GM-CSF group than control group.
**=p<0.05).
[0147] The development of limb ischemia was observed to induce EPC
mobilization, and these EPCs consequently contribute to
"vasculogenic" neovascularization. Ledney et al (Ledney, G. D., et
al J. Surg. Res. (1985) reported that wound trauma causes
mobilization of HCs including pluripotent stem or progenitor cells
in spleen, BM, and peripheral blood. Because EPCs are derived from
BM and EPC mobilization is enhanced during tissue ischemia,
circulating EPCs may constitute a reparative response to ischemic
injury, controlled by BM via circulating cytokines and soluble
receptors and/or adhesive molecules.
[0148] The results indicate that GM-CSF exerts a potent stimulatory
effect on EPC kinetics and that such cytokine-induced EPC
mobilization can enhance neovascularization of severely ischemic
tissues as well as de novo vascularization of previously avascular
sites. In particular, the Examples show mobilization of EPCs in
response to endogenous and exogenous stimuli.
[0149] The discussion and Examples above addressed the significance
of We investigated the endogenous stimuli, namely tissue ischemia,
and exogenous cytokine therapy, specifically granulocyte
macrophage-colony stimulating factor (GM-CSF), in the mobilization
of EPCs and induction of neovascularization of ischemic tissues.
Development of regional ischemia in both mice and rabbits was found
to increase the frequency of circulating EPCs. In mice, the impact
of ischemia-induced EPC mobilization was shown by enhanced ocular
neovascularization following cornea micropocket surgery in animals
with hindlimb ischemia compared to non-ischemic controls. In
rabbits with hindlimb ischemia, circulating EPCs were further
augmented following GM-CSF pre-treatment, with a corresponding
improvement in hindlimb neovascularization. Direct evidence that
EPCs which contributed to enhanced corneal neovascularization were
specifically mobilized from the bone marrow (BM) in response to
ischemia and GM-CSF was documented in mice transplanted with BM
from transgenic donors expressing .quadrature.-galacotsidase
transcriptionally regulated by the endothelial cell (EC) specific
Tie-2 promoter. These findings indicate that circulating EPCs are
mobilized endogenously in response to tissue ischemia or
exogenously by cytokine therapy and thereby augment
neovascularization of ischemic tissues.
[0150] In particular, the concept of EPC mobilization and
subsequent neovascularization as disclosed herein and in the
co-pending U.S. Provisional Application No. 60/077,262 is believed
to represent a potent strategy for the prevention and treatment of
a variety of ischemic vascular diseases including those
specifically mentioned herein.
[0151] General Comments--The following Materials and Methods were
used as needed in the Examples above.
1. Isolation of Mouse EPC-Enriched Fraction from Peripheral
Blood
[0152] Peripheral blood samples of mice were obtained from the
heart immediately before sacrifice, and separated by
Histopaque-1083 (Sigma, St. Louis, Mo.) density gradient
centrifugation at 400 g for 20 min. The light-density mononuclear
cells were harvested, washed twice with Dulbecco's phosphate
buffered saline supplemented with 2 mM EDTA (DPBS-E) and counted
manually. Blood mononuclear cells in each animal were suspended in
500 .mu.l of DPBS-E buffer supplemented with 0.5% bovine serum
albumin (Sigma) with 50 .mu.l of Sca-1 microbeads (Miltenyi Biotec,
Auburn, Calif.) for 15 min at 4.degree. C. After washing cells with
buffer, Sca-1 antigen positive (Sca-1.sup.+) cells were separated
with a magnetic stainless steel wool column (Miltenyi Biotec) and
counted. Cells which did not bind to antibodies for Sca-1 passed
through the column, while Sca-1.sup.+ cells were retained. The
Sca-1.sup.+ cells were eluted from the column and both cell
fractions were counted manually.
2. Isolation of Rabbit EPC-Enriched Fraction from Peripheral
Blood
[0153] Rabbit peripheral blood samples were obtained from either
ear vein through a 20 G infusion catheter and separated by
Histopaque-1077 (Sigma) density gradient centrifugation at 400 g
for 20 min. The light-density mononuclear cells were harvested,
washed twice by DPBS-E and counted manually. As an appropriate
antibody for rabbit hematopoietic stem/precursor cells is not
available, immatureHCs were isolated by depletion of matureHCs. The
cells were incubated with mixed primary antibodies (Serotec) of
mouse anti-rabbit CD5, anti-rabbit IgM (.mu. chain) and CD11b to
recognize mature T and B lymphocytes and monocytes respectively.
After washing antibodies, the cells were incubated with secondary
rat anti-mouse IgG microbeads (Miltenyi Biotec) and placed in a
magnetic separation column (Miltenyi Biotec). Cells which did not
bind to antibodies for mature T and B lymphocytes and monocytes
(TBM.sup.-), identical to hematopoietic stem/precursor cells,
passed through the column, while cells positive for cocktail
antibodies were retained. The positive cells (TBM.sup.+),
matureHCs, were eluted from the column and both cell fractions were
counted manually.
[0154] 3. EPC Differentiation Assay
[0155] To evaluate EPC differentiation from circulating blood
cells, Sca-1.sup.+ and Sca-1.sup.- cells isolated from 700 .mu.l
peripheral blood of each mouse, as well as TBM.sup.- and TBM.sup.+
cells isolated from 2 ml peripheral blood of each rabbit, were
co-cultured in one well of a 24-well plate coated with rat plasma
vitronectin (Sigma) after DiI-labeling of Sca-1.sup.+ or TBM.sup.-
cells in EBM-II media supplemented with 5% FBS (Clonetics, San
Diego, Calif.). After four days in culture, cells were washed twice
with media, and attached spreading cells were counted according to
the frequency of DiI-labeled Sca-1.sup.+ or TBM.sup.- cell-derived
cells and non-labeled Sca-1.sup.- or TBM.sup.+ cell-derived
cells.
[0156] To determine the cell type of attached spindle shaped cells
in the above assay, identical cells were assayed by acLDL-DiI
uptake and BS-1 lectin reactivity. Double-positive cells were
judged as EPCs and counted (96.2.+-.1.8% in mouse and 95.5.+-.2.4%
in rabbit).
[0157] 4. Study Design for Evaluation of Circulating EPC Kinetics
Following Ischemia
[0158] C57BL/6J mice (n=40) with hindlimb ischemia were sacrificed
at days 0 (before surgery), 3, 7 and 14 post-operatively (10 mice
at each timepoint). Sham-operated mice were sacrificed at day 7
post-operatively as well (n=4). Peripheral blood mononuclear cells
were prepared for counting of Sca-1.sup.+ cells, as an EPC-enriched
fraction, by magnetic bead selection (n=5) and EPC culture assay
(n.+-.5).
[0159] In New Zealand White rabbits (n=24) with hindlimb ischemia,
peripheral blood mononuclear cells were isolated at post-operative
days 0, 3, 7 and 14 in order to prepare for counting of TBM.sup.-
cells by magnetic bead selection and EPC culture assay.
Sham-operated rabbits were examined at day 7 post-operatively as
well (n=4).
[0160] To evaluate the effect of ischemia-induced circulating EPCs
on neovascularization, a corneal neovascularization assay (Kenyon,
B. M., et al. Invest Ophthalmol Vis Sci (1996) and Asahara, T. et
al. Circ. Res. (1998) was performed in mice with hindlimb ischemia.
Three days after ischemia or sham surgery, C57BL/6J mice (n=5 each)
underwent corneal assay microsurgery, including measurement of
neovasculature length and circumference 6 days after corneal
surgery (9 days after ischemia). In situ BS-1 lectin staining was
performed prior to sacrifice.
[0161] 5. Study Design for GM-CSF Effect on Circulating EPC
Kinetics and Neovascularization
[0162] These experiments were intended to demonstrate the effect of
GM-CSF on EPC kinetics and consequent vasculogenic contribution to
neovascularization.
[0163] a. Rabbit model. Animals with hindlimb ischemia were divided
into 2 groups. GM-CSF treatment, administered to 8 rabbits,
consisted of recombinant human GM-CSF (70 .mu.g/day) injected
subcutaneously daily for one week, beginning 7 days before surgery
(GM-CSF group). The ischemic control group consisted of 8 rabbits
receiving subcutaneous injections of saline daily for one week
before surgery (control group).
[0164] Rabbits were investigated on the day immediately before
initial injection (day [-]7), the day of ischemic surgery (day 0),
and 3, 7, 14 days post-operatively (days 3, 7, 14), at which time
peripheral blood was isolated from the central ear artery. At each
timepoint, 5 ml of blood was isolated for cell counting and culture
assay. In all animals from each group, the blood pressure ratio
between the ischemic and healthy limb was measured and on day 14
(at sacrifice), capillary density of ischemic muscles was
determined as well (vide b. Mouse model Following recombinant
murine GM-CSF (0.5 .mu.g/day) or control saline by i.p. injection
daily for one week, beginning at day [-]7 through day [-]1,
C57BL/6J mice (n=5 each) underwent corneal micropocket surgery at
day 0 and the length and circumference of the consequent
neovasculature was measured at day 6. In situ BS-1 lectin staining
was performed before sacrifice.
[0165] 6. Murine Bone Marrow Transplantation Model
[0166] FVB/N mice underwent BMT from transgenic mice constitutively
expressing .quadrature.-galactosidase encoded by lacZ under the
transcriptional regulation of an EC-specific promoter, Tie-2
(Schlaeger, T.m. et al. Development (1995). Reconstitution of the
transplanted BM yielded Tie-2/LZ/BMT mice in which expression of
lacZ is restricted to BM-derived cells expressing Tie-2; lacZ
expression is not observed in other somatic cells. The Tie-2/LZ/BMT
mice then underwent corneal assay microsurgery (Kenyon, B. M. et
al. Invest Ophthalmol Vis Sci (1996) and (Asahara, T. et al. Circ.
Res. (1998), 3 days following ischemia or sham operation, or 1 day
following completion of a 7-day course of GM-CSF or control
vehicle.
[0167] BM cells were obtained by flushing the tibias and femurs of
age-matched (4 wk), donor Tie-2 transgenic mice
(FVB/N-TgN[TIE2LacZ]182Sa- to, Jackson Lab). Low-density BM
mononuclear cells were isolated by density centrifugation over
Histopaque-1083 (Sigma). BM transplantation (BMT) was performed in
FVB/N mice (Jackson Lab) lethally irradiated with 12.0 Gy and
intravenously infused with approximately 2.times.10.sup.6 donor BM
mononuclear cells each. At 4 wks post-BMT, by which time the BM of
the recipient mice was reconstituted, the mice underwent surgery to
create hindlimb ischemia (vide infra) or a sham operation; 3 days
later, microsurgery for assay of corneal neovascularization was
performed. Likewise, at 4 wks post-BMT, GM-CSF or control vehicle
was administered for a period of 7 days; 1 day after completion of
GM-CSF or control pre-treatment, surgery for cornea
neovascularization assay was performed Corneas of BMT animals were
harvested at 6 days after corneal microsurgery for light
microscopic evidence of .beta.-galactosidase expression or chemical
detection of .beta.-galactosidase activity.
[0168] 7. Detection of .beta.-Galactosidase Expression in Corneal
Tissue
[0169] For histological detction of .beta.-galactosidase-expressing
cells, the whole eye of the mouse was enucleated, fixed in 4%
paraformaldehyde for 2 hours at 4.degree. C., and incubated in
X-gal solution overnight at 37.degree. C. The sample was then
placed in PBS and the hemisphered cornea was excised under the
dissecting microscope and embedded for histologic processing.
Histologic samples were counterstained with light hematoxylin- and
-eosin and examined by light microscopy to manually count the
number of X-gal positive cells per cross-section. Three sections
were examined from each tissue sample and averaged for evaluation
of X-gal stained cell frequency.
[0170] For chemical detection of .beta.-galactosidase activity, the
enucleated eye was placed into liquid nitrogen and stored at
-80.degree. C. The assay was performed using Chemiluminescence
Reporter Gene Assay System, Galacto-Light Plus.TM. (Tropix Inc.,
Bedford Mass.) according to the modified protocol. Briefly, the eye
was placed in 1 ml of supplemented lysis buffer, and after adding
0.5 mM DTT was homogenized with a Tissuemizer Mark II (Tekmar Co.,
Cincinatti, Ohio). Homogenized lysis solution was centrifuged to
remove debris. An aliquot of the supernatant from homogenized lysis
buffer was used for protein measurement using a BCA Protein Assay
kit (PERCE, Rockford, Ind.). The supernatant was assayed after
treatment with ion exchange resin, Chelex100, and
beta-galactosidase activity was measured using a chemiluminometer
(Lumat LB9501, Berthold, Nashua, N.H.). beta-galactosidase activity
was standardized according to protein concentration.
[0171] 8. Mouse Model of Hindlimb Ischemia
[0172] We used age-mached (8 wks) C57BL/6J male mice (Jackson Lab,
Bar Harbor, Me.) to create a mouse model of hindlimb ischemia
(Couffinhal, T. et al. Am. J. Pathol (1998). All animals were
anesthetized by intraperitoneal (i.p.) pentobarbital injection (160
mg/kg) for subsequent surgical procedures. A skin incision was
performed at the middle portion of the left hindlimb overlying the
femoral artery. The femoral artery then was gently isolated and the
proximal portion of the femoral artery was ligated with a 3-0 silk
ligature. The distal portion of the saphenous artery was ligated,
and other arterial branches as well as veins were all dissected
free, then excised. The overlying skin was closed using two
surgical staples. After surgery, mice were kept on a heating plate
at 37.degree. C., and special care was taken to monitor the animals
until they had completely recovered from anesthesia.
[0173] 9. Rabbit Model of Hindlimb Ischemia
[0174] We used a rabbit ischemic hindlimb model described
previously (Takeshita, S. et al. J. Clin. Invest. (1994). A total
of 20 New Zealand White rabbits (3.8-4.2 kg) (Pine Acre Rabbitry,
Norton, Mass.) were anesthetized with a mixture of ketamine (50
mg/kg) and acepromazine (0.8 mg/kg) following premedication with
xylazine (2 mg/kg). A longitudinal incision was then performed,
extending inferiorly from the inguinal ligament to a point just
proximal to the patella. The limb in which the incision was
performed was determined randomly at the time of surgery by the
operator. Through this incision, using surgical loupes, the femoral
artery was dissected free along its entire length; all branches of
the femoral artery, including the inferior epigastric, deep
femoral, lateral circumflex, and superficial epigastric, were also
dissected free. After dissecting the popliteal and saphenous
arteries distally, the external iliac artery and all of the above
arteries were ligated with 4.0 silk (Ethicon, Sommerville, N.J.).
Finally, the femoral artery was completely excised from its
proximal origin as a branch of the external iliac artery, to the
point distally where it bifurcates to form the saphenous and
popliteal arteries. Following excision of the femoral artery,
retrograde propagation of thrombus leads to occlusion of the
external iliac artery. Blood flow to the ischemic limb consequently
becomes dependent upon collateral vessels issuing from the internal
iliac artery.
[0175] 10. Mouse Corneal Neovascularization Assay
[0176] Age-mached (8 wk) C57 BL/6J male mice (Jackson Lab) were
used to evaluate mouse corneal neovascularization. All animals were
anesthetized by i.p. pentobarbital injection (160 mg/kg) for
subsequent surgical procedures. Corneal micropockets were created
with a modified von Graefe cataract knife in the eyes of each
mouse. Into each pocket, a 0.34.times.0.34 mm sucrose aluminum
sulfate (Bukh Meditec, Denmark) pellet coated with hydron polymer
type NCC (LFN Science, New Brunswick, N.J.) containing 150 ng of
vascular endothelial growth factor (VEGF) was implanted. The
pellets were positioned 1.0 mm from the corneal limbus and
erythromycin ophthalmic ointment (E. Foufera, Melville, N.Y.) was
applied to each operated eye. The corneas of all mice were
routinely examined by slit-lamp biomicroscopy on postoperative days
5 through 6 after pellet implantation. Vessel length and
circumference of neovascularization were measured on the sixth
postoperative day when all corneas were photographed. After these
measurements, mice received 500 .mu.p of Bandeiraea Simplicifolia
lectin-1 (BS-1) conjugated with FITC (Vector Lab, Burlingame,
Calif.), an EC-specific marker, intravenously, and were then
sacrificed 30 minutes later. The eyes were enucleated and fixed in
1% paraformaldehyde solution. After fixation, the corneas were
placed on glass slides and studied by fluorescent microscopy.
[0177] 11. Lower Limb Blood Pressure Ratio
[0178] These in vivo physiologic studies were performed on
anesthetized rabbits. Blood pressure was measured in both
hindlimbs. On each occasion, the hindlimbs were shaved and cleaned,
the pulse of the posterior tibial artery was identified with a
Doppler probe, and the systolic blood pressure in each limb was
measured using standard techniques. The blood pressure ratio was
defined for each rabbit as the ratio of systolic pressure of the
ischemic limb to the systolic pressure of the normal limb.
[0179] 12. Capillary Density
[0180] The extent of neovascularization was assessed by measuring
the frequency of capillaries in light microscopic sections taken
from the normal and ischemic hindlimbs. Tissue specimens were
obtained as transverse sections from muscles of both limbs of each
animal at the time of sacrifice. Muscle samples were embedded in
O.C.T. compound (Miles, Elkhart, Ind.) and snap-frozen in liquid
nitrogen. Multiple frozen sections 5 .mu.m in thickness were then
cut from each specimen so that the muscle fibers were oriented in a
transverse fashion. The tissue sections were stained for alkaline
phosphatase with an indoxyl-tetrazolium method to detect capillary
ECs as previously described and counterstained with eosin.
Capillaries were counted under a 20.times. objective to determine
the capillary density (mean number of capillaries/mm.sup.2). Ten
different fields were randomly selected for the capillary counts.
The counting scheme used to compute the capillary/muscle fiber
ratio was otherwise identical to that used to compute capillary
density. See Prokop, D. J. (1997) Science, 276: 71; Perkins, S. and
Fleischman, R. A. (1988) J. Clinical Invest. 81: 1072; Perkins, S.
and Fleischman, R. A. (1990) Blood 75: 620.
[0181] 13. Statistical Analysis
[0182] All results are expressed as mean.+-.standard error
(m.+-.SE). Statistical significance was evaluated using unpaired
Student's t test for comparisons between two means. The
multiple-comparison between more than 3 groups was performed with
the use of ANOVA. A value of p<0.05 was interpreted to denote
statistical significance.
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* * * * *
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