EP1061800A1 - Compositions and methods for modulating vascularization - Google Patents

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.

Description

COMPOSITIONS AND METHODS FOR MODULATING

VASCULARIZATION

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of U S Provisional Application No 60/077,262, filed on March 9,1998, the disclosure of which is hereby incorporated by reference

STATEMENT OF GOVERNMENT INTEREST

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

FIELD OF THE INVENTION The present invention relates to methods for modulating vasculaπzation. particularly in a mammal In one aspect, methods are provided for modulating vasculaπzation that includes administrating to the mammal an effective amount of a vasculaπzation modulating agent, such as a granulocyte macrophage-colony stimulating factor (GM-CSF) Further provided are methods for treating or detecting damaged blood vessels m the mammal The invention has a wide spectrum of useful applications including inducing formation of new blood vessels m the mammal

BACKGROUND OF THE INVENTION Blood vessels help supply oxygen and nutπents 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 Bio! Chem 267 (16), 10931-10934 (1992) -2-

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

There have been reports that inappropriate angiogenesis can have severe consequences For example, it has been disclosed that solid tumor growth is facilitated by vasculaπzation 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 (le 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

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 m rapid proliferation occurs under tight regulation are found in the female reproduction system and duπng wound healing It has been reported that the rate of angiogenesis involves a change m the local equihbπum between positive and negative regulators of the growth of microvessels

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, psoπasis, rheumatoid arthπtis. as well as other -3-

medical conditions

The therapeutic implications of angiogenic growth factors were first descπbed by Folkman and colleagues over two decades ago (Folkman, -V Engl J Med , 85 1 182- 1 186 ( 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 Vase Sutg, 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 hindhmb ischemia (Takeshita, et al , Circulation, 90 228-234 (1994) and Takeshita. et al J Chn Invest. 93 662-70 (1994))

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 tπals 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 m the vessel wall subsequently augmented neovasculaπzation 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 ,

Cιrcιιlatιon,9 {\2) 3281-3290 (1996))

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 75

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There have been reports that at least some of these problems can be reduced by administeπng 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 πsk to both the patient and attending personnel in some circumstances

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 1 131 (1991) In particular, GM-CSF acts as a growth factor for granulocyte, monocyte and eosinophil progenitors

Administration of GM-CSF to human and non-human pπmates 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 m patients subjected to radiation or chemotherapy, or following bone marrow transplantation In addition, although GM-CSF is less potent than other cytokmes, e g , FGF, in promoting EC proliferation, GM-CSF activates a fully migrating phenotype (Bussohno, et al , J Chn Invent , 87 986 (1991)

ccordingly, it would be desirable to have methods for modulating vasculaπzation in a mammal and especially a human patient It would be particularly desirable to have methods that increase EPC mobilization and neovasculaπzation (formation of new blood vessels) in the patient that do not require isolation of EPC cells

SUMMARY OF THE INVENTION

The present invention generally relates to methods for modulating vasculaπzation in a mammal In one aspect, the invention provides methods for increasing vasculaπzation that includes administrating to the mammal an effective amount of a vasculaπzation modulating agent, such as granulocyte macrophage-colony stimulating factor (GM-CSF), VEGF. Steel factor (SLF, -5-

also known as Stem cell factor (SCF)), stromal cell-deπved 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, CH. 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: 11 1; 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.

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.

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.

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 75

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significant increase in the frequency and differentiation of EPCs as determined by assays disclosed herein In one embodiment, the method includes admimsteπng to the mammal an effective amount of a vasculaπzation modulating factor such as granulocyte macrophage-colony stimulating factor (GM-CSF), that is preferably sufficient to induce the neovasculaπzation in the mammal Preferably, that amount of GM-CSF is also capable of modulating and particularly increasing frequency of EPCs in the mammal A vaπety of methods for detecting and quantifying neovasculaπzation, EPC frequency, the effectiveness of vasculaπzation modulating agents, and other parameters of blood vessel growth are discussed below and in the examples

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 enπchment and is descπbed in detail below

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 descπbed 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 hindhmb ischemia assay as discussed below

In another particular embodiment of the method, the amount of vasculaπzation modulating agent administered to the mammal is sufficient to increase blood vessel size m 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 preterablv 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

In another particular embodiment of the method, the amount of administered vasculaπzation modulating agent is sufficient to increase neovascularization by at least about 5%, preferably from between about 50%o 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 descπbed below Additionally, prefeπed amounts of GM-CSF are sufficient to improve ischemic hindlimb blood pressure by at least about 5%, preferably between from about 10%o to 50% as determined by standard methods for measuπng the blood pressure of desired vessels More specific methods for measuπng blood pressure particularly with new or damaged vessels include techniques optimized to quantify vessel pressure m the mouse hindlimb assay discussed below

In another particular embodiment of the method, the amount of administered vasculaπzation modulating agent is sufficient to increase EPC bone marrow (BM) deπved EPC incorporation into foci by at least about 20% as determined by a standard muπne 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

The methods of this invention are suitable for modulating and especially inducing neovascularization in a vaπety 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 pπmates 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 aπse 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

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 expeπmental 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 vasculaπzation modulating agent and the angiogenic protein can positively impact neovasculaπzation 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-admimsteπng the vasculaπzation modulating agent and the angiogenic protein are descπbed below and will generally vary according to intended use

The present invention also provides methods for preventing or reducing the seventy of blood vessel damage in a mammal such as a human patient in need of such treatment In one embodiment, the method includes admimstenng 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 vasculaπzation 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 -9-

such as surgery, grafting, or angioplasty, infection or ischemia Additional conditions and methods for administering the vascularization modulating agent are discussed below

Preferred amounts of the vasculaπzation modulating agent to use m the methods are sufficient to prevent or reduce the seventy 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 neovasculaπzation 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

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

a) isolating endothelial progenitor cells (EPCs) from the mammal,

b) contacting the isolated EPCs with an effective amount of at least one factor sufficient to induce proliferation of the EPCs, and

c) admmisteπng the proliferated EPCs to the mammal in an amount sufficient to treat the injured blood vessel

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 denved from a pre- existing or on-go g medical condition

In another particular embodiment of the method, the vasculaπzation ____ „„_, PCT/US99/05130 99/45775

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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

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-labelmg cells are known m the field and include immunological or radioacti e tagging as well as specific recombinant methods disclosed below

In a particular embodiment of the method, the detectably-labeled EPCs can be used to "home-m" 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 vaπety of methods well- known in this field including those using tomography, magnetic resonance imaging, or related approaches

In another embodiment of the method, the tissue damage is facilitated by ischemia, particularly an ischemic vascular disease such as those specifically mentioned below

5 Also provided by this invention are methods for modulating the mobilization of EPCs which methods include admimsteπng to the mammal an effective amount of at least one hematopoietic factor Prefeπed are methods that enhance EPC mobilization as determined by any suitable assay disclosed herein For example, m a particular embodiment of the method, the 0 enhancement m 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 5775 __u_

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 descπbed 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

As discussed, it has been found that EPC mobilization facilitates significant induction of neovasculaπzation in mammals Thus, methods that modulate EPC mobilization and particularly enhance same can be used to induce neovasculaπzation 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 vasculaπzation modulating agent is administered to the mammal alone or m combination (co- administered) with at least one angiogenic protein

In particular, the invention provides methods for inducing neovasculaπzation in a mammal and especially a human patient in need of such treatment which methods include administeπng to the mammal an effective amount of at least one vasculaπzation modulating agent, preferably one vasculanzation modulating agent, which amount is sufficient to induce neovasculaπzation in the mammal That neovasculaπzation 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 neovasculaπzation in the mammal by the stated percent ranges discussed previously

In one embodiment of the method, the effective amount of the - 12 -

vasculaπzation modulating agent (s) is co-administered m combination with at least one angiogenic protein, preferably one angiogenic protein The vasculaπzation 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

The invention also provides a pharmaceutical product that is preferably formulated to modulate and especially to induce neovasculaπzation in a mammal In a preferred embodiment, the product is provided steπle 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

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 Prefeπed is a kit that optionally includes a pharmacologically acceptable earner solution, nucleic acid or mitogen, means for dehveπng the EPCs and directions for using the kit Acceptable means for dehveπng the EPCs are known in the field and include effective delivery by stent, catheter, syπnge or related means

Other aspects of the invention are disclosed infia

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-D are representations of photomicrographs showing neovasculaπzation following GM-CSF and VEGF-1 treatment m control (Figs 1A, 1C) and treated (Figs IB and ID) mice in a cornea micropocket assay

Figures 2A-B are graphs showing quantitation of increases in vessel length (2A) and vessel angle (2B) observed in the cornea micropocket assay 5775

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Figures 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

Figures 4A-4J are representations of photomicrographs showing that EPCs can home and incorporate into foci of neovasculaπzation (4A) cultured muπne cells, (4B-D) homing of Sea- 1 ⁺ cells administered to the mouse, (4E-G) lmmunostaining of rabbit hindlimb muscle showing accumulation and colonization of EPCs, (4H-J) colonized TBM cells establishing new vessels

Figures 5A-B are graphs showing EPC kinetics in relation to development ot hindlimb ischemia

Figures 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 neovasculaπzation

Figures 5G-H are graphs illustrating quantitation of vessel length and circumferential distribution of neovasculanzation

Figures 6A-C are graphs showing effect of GM-CSF-induced EPC mobilization on neovascularization in the rabbit ischemic hindlimb model

Figures 6D-G are representations of photomicrographs showing the

GM-CSF induced effects descπbed in Figures 6A-C (6D, E) slit-lamp biomicroscopy, (6F, G) fluorescent photomicrographs

Figures 6H and 61 are graphs showing measurements of vessel length (6H) and vessel circumference (61) taken from the expeπment shown in

Figures 6D-G

Figures 7A-C are graphs showing that detectably-labeled bone-marrow deπved EPCs contribute to corneal neovasculanzation (7 A) corneal -14-

neovasculaπzation 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 As discussed, the present invention provides, in one aspect, methods for inducing neovasculanzation m 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 vasculaπzation 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 seventy of blood vessel damage in the patient.

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 admmisteπng to the patient an effective amount of GM-CSF or other vasculaπzation 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 pπor to. dunng or after formation of the ischemic tissue. Additionally, the GM-CSF can be administered as the sole active compound or it can be co-admmistered with at least one and preferably one angiogenic protein or other suitable protein or fragment as provided herein.

Administration of an effective amount GM-CSF or other vasculaπzation 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 admimstenng a DNA encoding same As discussed, methods of this invention ha e 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 m 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

In embodiments in which an effective amount of the vasculaπzation modulating agent is administered to a mammal and especially a human patient to prevent or reduce the seventy 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 pπor to exposure to conditions conducive to damaging blood vessels If desired, the method can further include admmisteπng the asculaπzation modulating agent to the mammal following exposure to the conditions conducive to damaging the blood vessels As discussed, the v asculanzation modulating agent can be administered alone or in combination with at least one angiogenic protein preferably one of such proteins

Related methods for preventing or reducing the seventy of the vascular condition can be employed which methods include administenng alone or m 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

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, translummal extraction, pulse spray thrombolysis), and deployment of an endovascular stent or a vascular graft

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⁺, flk-l⁺ or tιe-2 Methods for detecting EPCs with these markers are discussed in the Examples below

As discussed above and m 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 m a method for enhancing angiogenesis in a selected patient having an ischemic tissue i.e , a tissue having a deficiency blood as the result of an ischemic disease such as cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia Additionally, in another embodiment, the vasculanzation 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

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 vasculanzation by using a potent angiogenesis promotor such as VEGF-1, such vasculaπzation can be limited by the lack of EPCs However, by administenng e g . GM-CSF at a time before administration of the angiogenesis promoter sufficient to allow mobilization of the ECs. one can potentiate the vasculanzation in those patients Preferably, GM-CSF is administered about one week pπor to treatment with the angiogenesis promoter 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 "

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 coh deπved, non-glycosylated rhGM-CSF can be obtained by the methods descπbed in publication of the International Application No PCT/EP 85/00326, wherein two native GM-CSFs differing in a single ammo acid are descπbed

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 diffeπng by a single ammo 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)

GM-CSF is commercially and clinically available as an analog polypeptide (Leu^"J) under the trademark LEUKINE® (Immunex Corporation) The genenc name for recombinant human Leu^" GM-CSF analog protein expressed in yeast is Sargramostim Cloning and expression of native sequence human GM-CSF was descπbed m Cantrell et al , Proc Natl Acad Sci USA 82 6250(1985)

The natural or recombinantly prepared proteins, and their functional equivalents used in the method of the invention are preferably punfied and substantially cell-free, which may be accomplished by known procedures

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 descπption of Genbank Protein and nucleic sequences not specifically referenced can be found in Genbank or other sources disclosed herein

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, mtra- arteπal, intramuscular, and mtrapeπtoneal, with subcutaneous being prefeπed

In embodiments of this invention m which parenteral administration is selected, the GM-CSF will generally be formulated in a unit dosage mjectable form (solution, suspension, emulsion), preferably in a pharmaceutically acceptable earner medium that is inherently non-toxic and non-therapeutic Examples of such vehicles include without limitation saline, Ringer's solution, dextrose solution, manmtol and normal serum albumin Neutral buffered saline or salme mixed with serum albumin are exemplary appropπate 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

Preferably, the product is formulated by known procedures as a lyophilizate using appropπate excipient solutions (e g , sucrose) as a diluent

Preferred in vzvo dosages the vasculanzation modulating agents are from about 1 μg/ka'day to about lOOμg/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 Patent No 5,578,301 for additional methods of administering GM-CSF Prefeπed 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 vasculanzation modulating agent administration by co-administeπng 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 pπor to the administration of the GM-CSF The angiogenic protein can be administered directly, e g , intra-arteπally, 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), Feπara. et al , Bwchem Biophvs 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))

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 pnor to the administration of the GM-CSF As discussed, at least one angiogenic protein can also be co-admimstered with the GM-CSF and hematopoietic protein Methods for administenng the hematopoietic protein will generally follow those discussed for admmstenng the GM-CSF although other modes of administration may be suitable for some purposes -20-

It will be understood that the term "co-administration" is meant to descπbe prefeπed administration of at least two proteins disclosed herein to the mammal, le , administration of one protein in conjunction with, subsequent to, or prior to administration of the other protein

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 descπbed by U S. Patent 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 descπbed in PCT WO 97/14307

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 α and β (TGF-α and TFG-β), platelet-deπved endothelial growth factor (PD-ECGF), platelet- deπved growth factor (PDGF), tumor necrosis factor α (TNF-α), 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 (Angl) 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

Prefeπed 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,

1 1947-1 1954 (1991), Feπara, Trends Cardw Med , 3, 244-250 (1993), Polterak. et aL. y 5zo/ Chem , 272, 7151-7158 (1997)) Human VEGF -21-

lsoforms consists of monomers of 121 (U S Patent No 5,219,739), 145, 165 (U S Patent No 5,332,671), 189 (U S Patent No 5,240,848) and 206 amino acids, each capable of making an active homodimer (Houck, et al , Mol Endocnnol , 8, 1806-1814 (1991))

Other vascular endothelial growth factors include VEGF-B and VEGF-

C (Joukou, et al , J of Cell Phys 173 21 1-215 (1997), VEGF-2 (WO 96/39515), and VEGF-3 (WO 96/39421)

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 prefeπed 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)

Reference herein to a "vasculanzation 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-deπved factor (SDF-1), granulocyte-colony stimulating factor (G-CSF), HGF, Angιopoιetιn-1, Angιopoιetιn-2, M-CSF, b-FGF, and FLT-3 hgand 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) / Exp Med 185 111, Bleul, C et al (1996) 7 Exp Med 184 1101 , Sudo, Y et al (1997) Blood, 89 3166, as well as references disclosed therein

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

In certain situations, it may be desirable to use nucleic acids encoding 5775 - 22 -

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, mtπc oxide synthase, L-argmme, fibronectm, urokinase, plasminogen activator and hepann

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, vasculanzation modulating agent, hematopoietic protein, angiogenic protein) l e , levels capable of inducing endothelial cell growth and/or inducing angiogenesis as determined bv standard assays disclosed throughout this application Thus, the important aspect is the level of protein expressed Accordingly, one can use multiple transcπpts 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 coπesponding tar element

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 dπving expression of the protein cells of the desired target tissue The selection of appropπate 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 pUCl 18, pBR322, or other known plasmid vectors, that includes, for example, an E coh oπgin of replication See, Sambrook, et al , Molecular Cloning A Laboratorv Manual, Cold Spnng Harbor Laboratory press, (1989) The plasmid vector may also include a selectable marker such as the β- lactamase gene tor ampicilhn 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

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, pπmary 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 pπmary 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 artenes 3 3 mm or larger If patients undergo stent deployment in arteπes smaller than this the incidence of sub-acute thrombosis is even higher Sub- acute thrombosis is cuπently prevented only by the aggressive use of anticoagulation The combination of vascular intervention and intense anticoagulation creates significant nsks with regard to peπpheral 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 pnor 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 pπor to the denuding of the vessel wall

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

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 -24-

factor (CSF) VEGF- 1 is prefeπed

In addition, the methods of the present invention may be used to accelerate the healing of graft tissue, e g , vascular grafts, by potentiating vasculanzation

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

a) obtaining a penpheral blood sample from a subject mammal, preferably a rodent and especially a mouse, b) separating from the blood sample light-density mononuclear cells, c) contacting the separated mononuclear cells with beads that include a sequence capable of specifically binding Sea- 1^~ cells and separating same from the mononuclear cells, and d) quantitating the Sca-1^* cells, eg , by counting those cells manually

See the following discussion and Examples for more specific disclosure relating to the standard EPC isolation assay

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 a) isolating Sca-1+ and Sca-1- cells from the penpheral blood of mouse, or TBM+ and TBM- cells from the penpheral blood of a rabbit, and detectably-labelhng the cells (Sca-1+ and TBM- ), e g , with Di-I as provided herein, b) cultunng the cells in a suitable dish or plate in medium for several days and usually for about 4 days, 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+, d) and quantitating specific positive cells as being indicative of EPCs

More specific disclosure relating to the standard EPC culture assay can be found in the discussion and Examples that follow

Reference herein to a "standard hmd 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 -25-

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.

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.

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.

a) creating a corneal micropocket in at least one eye of a mouse, b) adding to the pocket a pellet including an acceptable polymer and at least one angiogenic protein, preferably VEGF-1, 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), d) marking EC cells in the eye, e.g., with BS-1 lectin; and e) quantitating vascularization and optionally EC cell counts in the eye.

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.

Reference herein to a "standard murine bone maπow (BM) transplantation model" or similar phrase is meant at least one and preferably all of the following steps. -26-

a) obtaining detectably-labeled BM cells from a donor mammal and typically a mouse, b) isolating low-density BM mononuclear cells from the mouse, c) removing BM cells from a suitable recipient mouse, e.g, by irradiation, d) administering the isolated and detectably- labeled BM cells to the recipient mouse, e) exposing the recipient mouse to conditions conditions conducive to damaging blood vessels in the mouse, e.g., hindlimb ischemia, f) administering an effective amount of GM-CSF to the recipient mouse, g) harvesting at least one cornea from the recipient mouse; and h) detecting and quantitating any labeled BM cells in the cornea.

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.

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%o 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 prefeπed 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.

All documents mentioned herein are incorporated by reference herein in their entirety.

The present invention is further illustrated by the following examples. These examples are provided to aid in the understanding of the invention and -27-

are not construed as a limitation thereof.

Example 1- Modulation of EPC kinetics by Cytokme Adminstration

Circulating EPCs may constitute a reparative response to injury. The hypothesis that cytokine-administration may mobilize EPCs and thereby augment therapeutic neovasculanzation was investigated as follows.

GM-CSF, which induces proliferation and differentiation of hematopoietic progmtor cells (Socmski, 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 (Bussohm, et al., J Clin. Invest., 1991;87:986-995), was used to promote cytokine-mduced EPC mobilization. To avoid a direct mitogemc effect on ECs, GM-CSF was administered for 7 days pnor to creating the stimulus for neovasculanzation. De novo vascular formation was initially examined m the mouse cornea pocket assay descπbed above. GM-GSF-pretreatment (mtrapentoneal [ι.p.] rmGM- CSF [R&D Systems] 500 ng/day) increased circulating EPCs (221% of untreated controls) at day 0, i.e., pπor to creation of the cornea micropocket and insertion of VEGF pellet; coπespondmgly, neovasculanzation at day 6

(Figures 1A-C) was augmented in compaπson to control mice (length = 0 67 + 0.04 vs 0.53 + 0.04, p<0.05; angle (circumferential degrees occupied by neovasculanty) = 155 ± 13 vs 117 + 12, p.<0.05) (Figures IB-ID). See also Figures 2A and 2B

Example 2- Cytokine-induced EPC mobilization Enhances Neovasculanzation of Ischemic Tissues

To determine if cytokine-induced EPC mobilization could enhance neovasculanzation 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; 50μg/day s.c), EPC- enπched cell population was increased ( 189%₀ compared to control animals), and EPC differentiation was enhanced (421% compared to control) at day 0 of (i.e., prior to) surgery (Figure 3). Morphometnc analysis of capillary density disclosed extensive neovascularization induced by GM-CSF pre-treatment compared to control (ischemia, no GM-CSF) group (249 vs 146/mm², 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) (Figures 3A-3C).

Example 3- EPC Kinetics During Tissue Ischemia.

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-T ) cells, and from rabbits as the cell population depleted of T-lymphocytes, B-lymphocytes and monocytes (TBM^"), denoted by the antigen repertoire CD5-/Igμ-/CD1 lb-.

The frequency of EPC-enriched population marked by Sca-1 in the circulation was 10J±1.0% in C57/6JBL normal mice. A subset of Sca-1⁺ cells plated on rat vitronectin attached and became spindle-shaped within 5 days. Co-cultures of Sca-1 and Sca-1 negative (Sca-1^") cells were examined after marking Sca-1^" cells with Dil fluorescence. Sca-1⁺ cells developed a spindle- shaped morphology. Mouse adherent cells in co-culture were found to be principally derived from Dil-marked Sca-1⁺ cells (65-84%) and showed evidence of EC lineage by reaction with BS-1 lectin and uptake of acLDL (Figure 4A). To determine if Sca-1 ⁺ cells can differentiate into ECs in vivo, a homogeneous population of Dil-marked Sca-1⁺ 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. Dil-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 (Figures 4A-4D).

For the rabbit model, mature HCs were depleted using antibodies to T and B lymphocytes and monocytes, yielding an EPC-enriched (TBM^") fraction. The frequency of TBM^" 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 denved pnncipally from Dil-marked TBM^" cells (71~92%o) and showed evidence of EC lineage by positive reaction with BS-1 lect and uptake of acLDL

TBM^" cells were shown to differentiate into ECs in vivo by administration of autologous Dil-marked TBM^" cells, isolated from 40 ml penpheral blood, to rabbits with unilateral hmdhmb ischemia (Takeshita. S., et dλ J Clin Invest (1994) at 0, 3 and 7 days post-operatively Dil-labeled EPC- deπved cells differentiated in situ into ECs, shown by co-stainmg for CD31 and incorporation into colonies, sprouts, and capillaries (Figures 4E-4J)

Figures 4A-4D are more particularly explained as follows The figures provide fluorescent microscopic evidence that EPCs derived from isolated populations of Sca-1 cells in mice, and TBM^" cells in rabbit, can home and incorporate into foci of neovasculaπzation. In particular, in Figure 4A cultured muπne cells are shown, double-stained for acLDL-DiI (red) and BS-1 lectin (green) 4 days after EPC culture assay (Figures 4B-D) Sca-1⁺ cells administered to mouse with hindlimb ischemia have homed, differentiated and incorporated into foci of neovasculanzation in mouse ischemic hmdhmb muscles 2 wks after surgery Figures 4B and 4C document that Dil-labelled Sca-1⁺ denved cells (red) co-localize with CD31 (green) indicdatmg that these EPCs have incorporated into CD31 -positive vascularture Aπows indicate cells positive for Dil and CD31 (denved from delivered EPCs), while aπowheads indicate CD31 -positive, Dil-negative (autologous ECs) Non- fluorescent, phase contrast photograph in Figure Id documents vascular foci of EPCs (aπows) are within interstitial sites adjacent to skeletal myocytes Figures 4E-G show lmmunostaining of rabbit ischemic hindlimb muscle 2 wks after ischemia surgery shows accumulation and colonization of EPCs, in this case isolated as TBM^" cells (red) (Figure 4E), these cells were marked with Dil and leinjected at day 0, 3 and 7 Figure 4F shows that these cells co-label with CDS 1 , within neov ascular toci DAPI stains cell nuclei (blue) (Figure 1G) (Figures 4H-J) Colonized TBM cells are incorporated into developing sprouts, establishing new capillaπes among skeletal myocytes

Example 4- Confirmation of EPC Kinetics Duπng Tissue Ischemia

EPC kinetics during severe tissue ischemia were assayed for frequency and differentiation The EPC-ennched 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 xlO'/ml in mouse [p<0 05], 1 1 4=0 6 vs 6 7=0 3 xl0³/ml in rabbit [p<0 05]) (Figures 5 A, 6 A) 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 in mouse [p<0 05], 539=73 vs 100±19 in rabbit [p<0 05]) (Figures 5B, 6B) Neither the frequency of the EPC- ennched population nor the EPC culture assay showed a significant increase in EPC kinetics in either sham-operated animal model at 7 days following surgery

Figures 5A and 5B are more specifically explained as follows The figures show EPC kinetics in relation to development of hindlimb ischemia (Figure 5A) Following surgery to create ischemic hindlimb, frequency of mouse EPC-ennched population (Sca-T) in circulating blood increases, becoming maximum by day 7 (n=5 mice at each time point) (Figure 5B) Adherent cells in EPC culture are denved pπncipally from Dil-marked Sca-1 ⁺ cells Assay culture demonstrates enhanced EPC differentiation after surgically induced ischemia with a peak at day 7 (n=5 each time point)

Figures 5C-H show results of the mouse cornea micropocket assay as applied to mice with hindlimb ischemia 7 days after surgery Slit-lamp biomicroscopy (Figures 5C and 5D) and fluorescent photomicrographs

(Figures 5E and 5F) demonstrate that neovasculanzation in avascular area of -31-

mouse cornea is enhanced by EPC mobilization induced by ischemia, shown with the same magnification (Figures 5G and 5H) Quantitative analysis of two parameters, vessel length and circumferential distπbution of neovasculanzation, indicates that corneal neovasculanzation was more profound in animals with hindlimb ischemia (n=7 mice) than m non-ischemic, sham control mice (n=9) (*=p<0 05)

Example 5- Analysis of Impact of Enhanced EPC Mobilization on Neovasculanzation

To investigate the impact on neovasculanzation of enhanced EPC mobilization induced by ischemia, the mouse cornea micropocket assay was applied to animals which hmdhmb ischemia had been surgically created 3 days earlier Slit-lamp (Figures 5C and 6D) and fluorescent (Figures 5E, 6F) photomicrographs documented that neovasculanzation of avascular mouse cornea was enhanced in animals with hmdhmb ischemia compared to non- lschemic sham-operated controls Measurements of vessel length and circumference showed a significant effect of EPC mobilization on neovasculanzation 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) (Figures 5G, 5H)

Example 6- Confirmation of Enhanced Neovasculanzation with Cytokine-induced EPC Mobilization

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 neovasculaπzation of ischemic tissues To effect GM-CSF-mduced 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 (50μg/day s c ) increased the EPC-ennched population (12 5±0 8 vs 6 7±0 3 xlO ml, -32-

p<0 01 ) and enhanced EPC differentiation (423=90 vs 100±19 /mm², p<0 01) at day 0 (day 7 of pre-treatment pπor 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 1 1 3=2 5 xlθ^"7ml [p<0 05], 813±54 vs 539=73 /mm² [p<0 01 ]) respectively (Figures 6A, 6B) Capillary density analysis documented extensive neovasculanzation induced by GM-CSF pre-treatment (249±18 vs 146=18 /mm^" in untreated controls, p<0 01), as well as improved ischemic/normal hmdhmb blood pressure ratio (0 71-tO 03 vs 0 49±0 03, P<0 01) (Figure 6C)

Figures 6 A-I are explained in more detail as follows The figures show the effect of GM-CSF-mduced EPC mobilization on neovasculanzation in rabbit ischemic hmdhmb model (Figures 6A,B) Following pre-treatment with GM-CSF, circulating EPC-ennched population (TBM^") is increased in number compared to control (ischemic, untreated) animals beginning at day 0 (pnor to surgery) through day 7 (Figure 6A), as is EPC differentiation in culture

(Figure 5B) (n=5 mice at each time point) (Figure 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 01, **=p<0 05)

Slit-lamp biomicroscopy (Figures 6D and 6E) and fluorescent photomicrographs (Figures 6F and 6G, same magnification) show that neovasculanzation in avascular area of mouse cornea is also enhanced by EPC mobilization induced by GM-CSF pretreatment (Figures 6H and 61) Measurements of vessel length and circumference indicate significant effect of EPC mobilization on neovasculanzation in GM-CSF pretreated (n=6) versus control mice (n= 10) (*=p<0 05) -33-

Example 7-Confirmatιon of Enhanced Neovasculanzation Using The Mouse Comea Micropocket Assay

These results descπbed above were coπoborated by assessment of de novo vasculanzation in the mouse comea micropocket assay. GM-CSF-pretreated mice (rmGM-CSF, 500ng/day l.p.) developed more extensive corneal neovasculanzation 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) (Figures 6D-6I).

Example 8- Enhanced BM-deπved EPC incorporation in the BM Transplantation Model

A munne BM transplantation (BMT) model was employed to establish direct evidence of enhanced BM-denved EPC incorporation into foci of corneal neovasculanzation 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 m 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-fclJ, p<0 01) (Figures 7 A. 7B). Corneas from control mice (post-BMT) disclosed no cells expressing β-galactosidase. Quantitative chemical detection confirmed a statistically significant increase in β-galactosidase activity among mice receiving GM-CSF vs controls (2.90±0.30 vs 2.1 1±0.09 X10³, p<0 05) (Figure 7C).

Figures 7A-C are explained in more detail as follows. The figures illustrate that Bone maπow-deπved EPCs contnbute to comeal neovasculanzation. Photomicrographs shown as inserts document incorporation of BM-deπved EPCs expressing endothehal-specific Tιe-2/lacZ (blue cells) into foci of corneal neovasculaπzation, both in mice with hmdhmb ischemia (Figure 7A), as well as in rabbits pretreated with GM-CSF (Figure 7B). The frequency of incorporated EPCs stained by X-gal was manually counted under light microscopy (Figure 7A) Incorporated EPCs were significantly more frequent in mice with hindlimb ischemia vs the sham- operated mice, (Figure 7B) the same was true for rabbits receiving GM-CSF group vs control rabbits (*=p<0 01 for each condition) (Figure 7C) β- galalactosidase activity was significantly higher in GM-CSF group than control group **=p<0 05)

The development of limb ischemia was observed to induce EPC mobilization, and these EPCs consequently contribute to "vasculogemc" neovasculanzation Ledney et al (Ledney, G D , et al J S rg Res (1985) reported that wound trauma causes mobilization of HCs including plunpotent stem or progenitor cells m spleen, BM. and peripheral blood Because EPCs are denved from BM and EPC mobilization is enhanced duπng 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

The results indicate that GM-CSF exerts a potent stimulatory effect on EPC kinetics and that such cytokine-induced EPC mobilization can enhance neovasculanzation of severely ischemic tissues as well as de novo vasculanzation of previously avascular sites In particular, the Examples show mobilization of EPCs in response to endogenous and exogenous stimuli

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 neovasculaπzation 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 neovasculanzation 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 coπesponding improvement in hmdhmb neovasculanzation. Direct evidence that EPCs which contributed to enhanced corneal neovasculanzation 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 _-galacotsιdase transcnptionally regulated by the endothelial cell (EC) specific Tιe-2 promoter. These findings indicate that circulating EPCs are mobilized endogenously in response to tissue ischemia or exogenously by cytokine therapy and thereby augment neovasculanzation of ischemic tissues.

In particular, the concept of EPC mobilization and subsequent neovasculanzation 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 vanety of ischemic vascular diseases including those specifically mentioned herein.

General Comments- The following Matenals and Methods were used as needed in the Examples above.

1. Isolation of mouse EPC-ennched fraction from penpheral blood Penpheral blood samples of mice were obtained from the heart immediately before sacπfice, and separated by Hιstopaque-1083 (Sigma, St. Louis, MO) density gradient centπfugation at 400g for 20 mm The light- density mononuclear cells were harvested, washed twice with Dulbecco's phosphate buffered saline supplemented with 2mM EDTA (DPBS-E) and counted manually. Blood mononuclear cells in each animal were suspended in

500 μl of DPBS-E buffer supplemented with 0.5% bovine serum albumin (Sigma) with 50μl of Sca-1 microbeads (Miltenyi Biotec, Aubum, CA) for 15 min at 4°C. After washing cells with buffer, Sca-1 antigen positive (Sca-1 ) 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-l^"" cells were retained. The Sca-1⁺ cells were eluted from the column and both cell fractions were counted manually. 2 Isolation of rabbit EPC-ennched fraction from peripheral blood

Rabbit peripheral blood samples were obtained from either ear vein through a 20G infusion catheter and separated by Hιstopaque-1077 (Sigma) density gradient centnfugation at 400g for 20 min The light -density mononuclear cells were harvested, washed twice by DPBS-E and counted manually As an appropπate antibody for rabbit hematopoietic stem/precursor cells is not available, lmmatureHCs were isolated by depletion of matureHCs The cells were incubated with mixed pπmary antibodies (Serotec) of mouse anti-rabbit CD5. anti-rabbit IgM (μ chain) and CDl lb 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^"), identical to hematopoietic stem/precursor cells, passed through the column, while cells positive for cocktail antibodies were retained. The positive cells (TBM⁺), matureHCs. were eluted from the column and both cell fractions were counted manually

3. EPC differentiation assay To evaluate EPC differentiation from circulating blood cells, Sca-1 and Sca-1^" cells isolated from JOOμl peripheral blood of each mouse, as well as TBM and TBM^" cells isolated from 2 ml penpheral blood of each rabbit, were co-cultured in one well of a 24-well plate coated with rat plasma vitronectin (Sigma) after Dil-labehng of Sea- 1^* or TBM^" cells in EBM-II media supplemented with 5% FBS (Clonetics, San Diego, CA) After four days in culture, cells were washed twice with media, and attached spreading cells were counted according to the frequency of Dil-labeled Sca-l^" or TBM^" cell-denved cells and non-labeled Sca-1^' or TBM cell-denved cells

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 lectm reactivity Double-positive cells were judged as EPCs and counted (96 2±1 8% in mouse and 95 5±2 4% in rabbit) 4 Study design for evaluation of circulating EPC kinetics following ischemia

C57BL/6J mice (n=40) with hmdhmb ischemia were sacnficed 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) Penpheral blood mononuclear cells were prepared for counting of Sca-1 ^τ cells, as an EPC-ennched fraction, by magnetic bead selection (n=5) and EPC culture assay (n=5).

In New Zealand White rabbits (n=24) with hmdhmb ischemia, penpheral blood mononuclear cells were isolated at post-operative days 0, 3, 7 and 14 in order to prepare for counting of TBM^" cells by magnetic bead selection and EPC culture assay Sham-operated rabbits were examined at day 7 post- operatively as well (n=4)

To evaluate the effect of ischemia-induced circulating EPCs on neovasculanzation, a comeal neovasculaπzation assay (Kenyon, B.M., et al. Invest Ophthalmol Vis Sci (1996) and Asahara, T. et al. Ore. Res. (1998) was performed m mice with hmdhmb 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 comeal surgery (9 days after ischemia) In situ BS-1 lectm staining was performed pπor to saenfice.

5 Study design for GM-CSF effect on circulating EPC kinetics and neovasculanzation These experiments were intended to demonstrate the effect of GM-CSF on

EPC kinetics and consequent vasculogenic contπbution to neovasculanzation.

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 (70μg/ 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).

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 penpheral 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 sacπfice), capillary density of ischemic muscles was determined as well (vide infra) b Mouse model Following recombinant muπne GM-CSF (0 5 μg/day) or control saline by l p. injection daily for one week, beginning at day [-]7 through day [-] 1 , C57BL/6J mice (n=5 each) underwent comeal micropocket surgery at day 0 and the length and circumference of the consequent neovasculature was measured at day 6 In situ BS-1 lectm staining was performed before sacπfice

6. Muπne bone maπow transplantation model FVB/N mice underwent BMT from transgenic mice constitutively expressing __ -galactosidase encoded by lacZ under the transcπptional regulation of an EC-specific promoter, Tιe-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 restπcted to BM-deπved cells expressing Tιe-2; lacZ expression is not observed in other somatic cells The Tιe-2/LZ/BMT mice then underwent comeal assay microsurgery (Kenyon, B M. et al. Invest Ophthalmol Vis Sci (1996) and (Asahara, T et al Ore. 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. BM cells were obtained by flushing the tibias and femurs of age-matched

(4wk), donor Tιe-2 transgenic mice (FVB/N-TgN[TIE2LacZ] 182Sato, Jackson Lab) Low-density BM mononuclear cells were isolated by density centπfugation over Hιstopaque-1083 (Sigma). BM transplantation (BMT) was performed in FVB/N mice (Jackson Lab) lethally irradiated with 12.0 Gy and intravenously infused with approximately 2X10 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 comeal -39-

neovasculaπzation was performed Likewise, at 4 wks post-BMT, GM-CSF or control vehicle was administered for a penod of 7 days, 1 day after completion of GM-CSF or control pre-treatment, surgery for comea neovasculanzation assay was performed Corneas of BMT animals were harvested at 6 days after comeal microsurgery for light microscopic evidence of β-galactosidase expression or chemical detection of β-galactosidase activity

7 Detection of β-galactosidase expression in comeal tissue For histological detction of β-galactosidase-expressing cells, the whole eye of the mouse was enucleated, fixed m 4% paraformaldehyde for 2 hours at 4 °C, and incubated in X-gal solution overnight at 37 °C The sample was then placed in PBS and the hemisphered comea was excised under the dissecting microscope and embedded for histologic processing Histologic samples were counterstained with light hematoxyhn-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

For chemical detection of β-galactosidase activity, the enucleated eye was placed into liquid nitrogen, and stored at -80°C The assay was performed using Chemiluminescence Reporter Gene Assay System, Galacto- Light Plus TM (Tropix Ine , Bedford MA) according to the modified protocol Bπefly, the eye was placed in 1 ml of supplemented lysis buffer, and after adding 0 5mM DTT was homogenized with a Tissuemizer Mark II (Tekmar Co , Cincinatti, OH) Homogenized lysis solution was centnfuged to remove debπs An aliquot of the supernatant from homogenized lysis buffer was used for protein measurement using a BCA Protein Assay kit (PIERCE, Rockford, IN). The supernatant was assayed after treatment with ion exchange resin, ChelexlOO, and beta- galactosidase activity was measured using a chemiluminometer (Lumat LB9501 , Berthold, Nashua, NH) beta- galactosidase activity was standardized according to protein concentration

8 Mouse model of hindlimb ischemia

We used age-mached (8wks) 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 intrapentoneal (l p ) pentobarbital injection (160 mg/kg) for subsequent surgical procedures A skin incision was performed at the middle portion of the left hmdhmb 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 artenal branches as well as veins were all dissected free, then excised The overlying skm was closed using two surgical staples After surgery, mice were kept on a heating plate at 37°C, and special care was taken to monitor the animals until they had completely recovered from anesthesia

9 Rabbit model of h dhmb ischemia

We used a rabbit ischemic hindlimb model descnbed 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, MA) were anesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (0 8 mg/kg) following premedication with xylaz e (2mg/kg) A longitudinal incision was then performed, extending mfenorly 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 infeπor epigastπc, deep femoral, lateral circumflex, and superficial epigastπc, were also dissected free After dissecting the popliteal and saphenous artenes distally, the external iliac artery and all of the above artenes were ligated with

4 0 silk (Ethicon, Sommerville, NJ) Finally, the femoral artery was completely excised from its proximal ongin as a branch of the external iliac artery, to the point distally where it bifurcates to form the saphenous and popliteal artenes 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 775

-41-

10 Mouse co eal neovasculanzation assay

Age-mached (8wk) C57BL/6J male mice (Jackson Lab) were used to evaluate mouse comeal neovasculanzation. All animals were anesthetized by l.p pentobarbital injection (160 mg/kg) for subsequent surgical procedures. Comeal micropockets were created with a modified von Graefe cataract knife in the eyes of each mouse. Into each pocket, a 0 34X0 34 mm sucrose aluminum sulfate (Bukh Meditec, Denmark) pellet coated with hydron polymer type NCC (IFN Science, New Brunswick, NJ) containing 150 ng of vascular endothelial growth factor (VEGF) was implanted. The pellets were positioned 1.0mm from the comeal limbus and erythromycm ophthalmic ointment (E.Foufera, Melville, NY) 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 neovasculanzation were measured on the sixth postoperative day when all corneas were photographed. After these measurements, mice received 500μg of Bandeiraea Simphcifolia lectιn-1 (BS-1) conjugated with FITC (Vector Lab, Burlmgame, CA), an EC-specific marker, intravenously, and were then sacnficed 30 minutes later The eyes were enucleated and fixed m 1% paraformaldehyde solution. After fixation, the corneas were placed on glass slides and studied by fluorescent microscopy.

11. Lower limb blood pressure ratio

These in vivo physiologic studies were performed on anesthetized rabbits. Blood pressure was measured in both hindhmbs. On each occasion, the hmdlimbs were shaved and cleaned, the pulse of the postenor 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

12. Capillary density

The extent of neovasculaπzation was assessed by measuπng the frequency of capillanes in light microscopic sections taken from the normal and ischemic hindhmbs Tissue specimens were obtained as transverse sections from -42-

muscles of both limbs of each animal at the time of sacrifice. Muscle samples were embedded in O.C.N compound (Miles, Elkhart, Ind.) and snap-frozen in liquid nitrogen. Multiple frozen sections 5 μm 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 20X objective to determine the capillary density (mean number of capillaries/mm^). 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.

13. Statistical Analysis

All results are expressed as mean ± standard eπor (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.

The following references are specifically incorporated herein by reference:

(1) Asahara, T., Murohara, T., Sullivan, A., et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275,965-967

(1997).

(2) Folkman, J. & Klagsbrun, M. Angiogenic factors. Science 235,442-447 (1987).

(3) Soldi, R., Pπmo, L., Brizzi. M.F., et al. Activation of JAK2 in human vascular endothelial cells by granulocyte-macrophage colony- stimulating factor. Blood 89,863-872 (1997).

(4) Bussolino, F., Wang, J.M., Turrini, F., et al. Stimulation of the Na+/H+ exchanger in human endothelial cells activated by granulocyte- and -43-

granulocyte-macrophage-colony stimulating factor Evidence for a role in proliferation and migration. J Biol. Chem. 264,188284-18287 (1989).

(5) Aghetta, M., Piacibello, W., Sanavio, F., et al. Kinetics of human hematopoietic cells after in vivo admmstration of granulocyte- macrophage colony-stimulating factor. J.Clin. Invest. 83,551-557 (1989).

(6) Fleischman, R., Simpson, A.F., Gallardo, T., Jin. X.L. & Perkins, S. Isolation of endothelial-like stromal cells that express Kit hgand and support in vitro hematopoiesis. Exp Hematol 23,1407-1416 (1995).

(7) Flanagan, M.F., Fujii, A.M., Colan, S.D., Flanagan, R.G. & Lock, J.E. Myocardial angiogenesis and coronary perfusion in left ventncular pressure-overload hypertrophy in the young lamb: evidence for inhibition with chronic protamine administration. Ore. Res. 68,1458-1470 (1991)

(8) Takahashi, T. et al. (1998) Ischemia-and cytokine-induced mobilization of bone maπow-deπved endothelial progenitor cells for neovasulanzation. Nature Medicine 5: 1-7.

Claims

What is claimed is

1 A method for inducing formation of new blood vessels in a mammal, wherein the method compπses administenng to the mammal an effective amount of a vasculanzation modulating agent sufficient to form the new blood vessels in the mammal

1A The method of claim 1, wherein the vasculanzation modulating agent is GM-CSF, M-CSF, b-FGF, SCF, SDF-1, G-CSF, HGF, Angiopoietm- 1 , Ang╬╣opo╬╣etm-2, FLT-3 hgand, or an effective fragment thereof

2 The method of claim 1, wherein the vasculanzation modulating agent is GM-CSF, and amount of the GM-CSF administered to the mammal is sufficient to increase frequency of endothelial progenitor cells (EPC) in the mammal

3 The method of claim 2, wherein the increase in frequency of the

EPC is at least about 20% as determined by a standard EPC isolation assay

4 The method of claim 1 , wherein the amount of vasculanzation modulating agent administered to the mammal is sufficient to increase EPC differentiation in the mammal

5 The method of claim 4, wherein the increase in EPC differentiation is at least about 20%> as determined by a standard EPC culture assay

6 The method of claim 1 , wherein the amount of vasculanzation modulating agent administered to the mammal is sufficient to increase blood -45-

vessel length in the mammal

7. The method of claim 6, wherein the increase in blood vessel length is at least about 5% as determined by a standard blood vessel length assay

8. The method of claim 6, wherein the amount of vasculanzation modulating agent administered to the mammal is further sufficient to increase blood vessel diameter in the mammal.

9 The method of claim 9, wherein the increase in blood vessel diameter is at least about 5% as determined by a standard blood vessel diameter assay.

10. The method of claim 1, wherein the amount of vasculanzation mmoodduullaattiinngg aaggeenntt aaddmmiinniisstteerreedd ttoo tthhee mmaanmmal is sufficient to increase EPC differentiation following tissue ischemia.

11. The method of claim 10, wherein the increase in EPC differentiation is at least about 20%> as determined by a standard hindlimb ischemia assay

12. The method of claim 1, wherein the amount of administered vasculaπzation modulating agent is sufficient to increase neovasculaπzation by at least about 5% as determined by a standard comea micropocket assay

13 The method of claim 1, wherein the amount of administered vasculanzation modulating agent is sufficient to increase EPC bone marrow denved EPC incorporation into foci

14 The method of claim 13, wherein the increase in EPC bone marrow denved EPC incorporation into foci is at least about 20% as determined by a standard rodent bone maπow (BM) transplantation model

15 The method of claim 1 , wherein the mammal has, is suspected of having, or will have ischemic tissue

16 The method of claim 15, wherein the ischemic tissue is associated with an ischemic vascular disease

17 The method of claim 15, wherein the ischemic tissue compnses tissue from a limb, graft, or organ

18 The method of claim 15, wherein the tissue is associated with the circulatory system or the central nervous system

19 The method of claim 15, wherein the tissue is heart or brain tissue

20 The method of claim 1, wherein the is co-admmistered with at least one angiogenic protein

21 The method of claim 20, wherein the angiogenic protein is an endothelial cell mitogen -47-

22 The method of claim 20, wherein the angiogenic protein is acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF-1), epidermal growth factor (EGF), transforming growth factor α and β (TGF-α and TFG-β), platelet-denved endothelial growth factor (PD-ECGF), platelet-denved growth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF), angiopoetin- 1 (Angl) or nitπc oxidesynthase (NOS), or a fragment thereof

23 The method of claim 22, wherein the protein is one of VEGF-B, VEGF-C, VEGF-2, VEGF-3, or an effective fragment thereof

24 A method for preventing or reducing the seventy of blood vessel damage in a mammal, wherein the method compnses administenng to the mammal an effective amount of granulocyte macrophage-colony stimulating factor (GM-CSF), and exposing the mammal to conditions conducive to damaging the blood vessels, the amount of GM-CSF being sufficient to prevent or reduce the seventy of the blood vessel damage in the mammal

25 The method of claim 24, wherein the conditions conducive to the blood vessel damage are an invasive manipulation or ischemia

26 The method of claim 25, wherein the invasive manipulation is surgery

27 The method of claim 25, wherein the ischemic is associated with at least one ot infection, trauma, graft rejection, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, or myocardial ischemia

28 The method of claim 24, wherein the GM-CSF is administered to the mammal at least about 12 hours before exposing the mammal to the conditions conducive to damaging the blood vessels

29 The method of claim 28, wherein the GM-CSF is administered to the mammal between from about 1 to 10 days before exposing the mammal to the conditions conducive to damaging the blood vessels

30 The method of claim 28, wherein the method further compπses administenng the GM-CSF to the mammal following the exposure to the conditions conducive to damaging the blood vessels

31 A method for treating ischemic tissue in a mammal in need of such treatment, wherein the method compnses

a) isolating endothelial progenitor cells (EPCs) from the mammal,

b) contacting the isolated EPCs with an amount of an angiogenic protein sufficient to induce proliferation of the EPCs, and

c) administenng the proliferated EPCs to the mammal in an amount sufficient to treat the ischemic tissue

32 The method of claim 31 , wherein the EPCs have at least one of the following markers CD34 , flk-l⁺ or t╬╣e-2⁺

33 The method of claim 31, wherein the ischemic tissue compπses injured blood vessels

34 The method of claim 33, wherein the blood vessels are injured by an invasive manipulation

35 The method of claim 34, wherein the invasive manipulation is balloon angioplasty, or deployment of a stent or catheter

36 The method of claim 35, wherein the stent is an endovascular stent

37 The method of claim 31 further compnsmg co-administenng at least one angiogenic protein

38 The method of claim 37, wherein the angiogenic protein is an endothelial cell mitogen or a nucleic acid encoding the endothelial cell mitogen

39 The method of claim 38, wherein the angiogenic protein is acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF-1), epidermal growth factor (EGF), transforming growth factor ╬▒ and ╬▓ (TGF-╬▒ and TFG-╬▓), platelet-denved endothelial growth factor (PD-ECGF), platelet-denved growth factor (PDGF), tumor necrosis factor ╬▒ (TNF-╬▒), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietm, colony stimulating factor (CSF), macrophage-CSF (M-CSF), ang╬╣opoet╬╣n-1 (Angl) or nitnc oxidesynthase (NOS); or a fragment thereof

40 The method of claim 39, wherein the protein is one of VEGF-B, VEGF-C. VEGF-2, VEGF-3, or a fragment thereof

41 A method for detecting presence of tissue damage in a mammal, wherein the method compπses contacting the mammal with a detectably- labeled population of endothehcal progenitor cells (EPCs), and detecting the labeled cells at or near the site of the tissue damage in the mammal

42 The method of claim 41, wherein the tissue damage is ischemia or an ischemic vascular disease

43 A pharmaceutical product for inducing neovasculanzation in a mammal, wherein the product compnses isolated endothelial progenitor cells (EPCs) and is formulated to be physiologically acceptable to a mammal

44 The pharmaceutical product of claim 43, wherein the product is steπle and further compπses at least one angiogenic protein or nucleic acid encoding the protein

45 A kit for the systemic introduction of a isolated endothelial progenitor cells (EPCs), wherein the kit compnses the isolated EPCs and optionally at least one angiogenic protein or nucleic acid encoding same, the kit further optionally compnsmg a pharmacologically acceptable earner solution, nucleic acid or mitogen, means for dehvenng the EPCs and directions for using the kit

46. The kit of claim 45, wherein the means for delivering the EPCs is a stent, catheter or syringe.

47. A method for enhancing endothelial progenitor cell (EPC) mobilization in a mammal, wherein the method comprises administering an effective amount of at least one hematopoietic factor sufficient to enhance the EPC mobilization in the mammal.

48. The method of claim 47 further comprising co-administering to the mammal an effective amount of one or more of: granulocyte macrophage- colony stimulating factor (GM-CSF); at least one angiogenic protein; or an effective fragment thereof.