JP2002506008A - Compositions and methods for modulating angiogenesis - Google Patents

Abstract

  (57) [Summary] The present invention generally provides a method for regulating the formation of new blood vessels. In one embodiment, the method comprises administering to the mammal an effective amount of granulocyte macrophage colony stimulating factor (GM-CSF) sufficient to form new blood vessels. Further provided is a method of preventing or reducing severe vascular damage in a mammal, preferably comprising administering to the mammal an effective amount of GM-CSF. As part of the present invention, pharmaceutical products and kits for inducing the formation of new blood vessels in mammals are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is a continuation of US Provisional Application No. 60 / 077,262, filed March 9, 1998, the disclosure of which is incorporated herein by reference.

STATEMENT OF GOVERNMENT RIGHTS The funding for the present invention was provided in part by the United States Government through grants HL40518, HL02824, and HL57516 from the National Institutes of Health. Accordingly, the United States Government has certain rights in or to the invention claimed herein.

[0003] The present invention relates to methods for modulating angiogenesis, particularly in mammals. In one aspect, there is provided a method of modulating angiogenesis comprising administering to a mammal an effective amount of an angiogenesis regulating agent, such as granulocyte macrophage colony stimulating factor (GM-CSF). Further, there is provided a method of treating or detecting damaged blood vessels in a mammal. The invention has a wide range of useful applications, including inducing the formation of new blood vessels in mammals.

BACKGROUND OF THE INVENTION Blood vessels serve to supply oxygen and nutrients to living tissue. Blood vessels also facilitate the removal of waste products. Blood vessels are reborn by a process called "angiogenesis". Generally Folk
See man and Shing, J. Biol. Chem., 267 (16), 10931-10934 (1992).

[0005] Angiogenesis is believed to be important in the well-being of many mammals. By way of example, angiogenesis is disclosed as being an essential process of reproduction, development and wound repair.

[0006] Improper angiogenesis has been reported to have serious consequences. For example, angiogenesis promotes solid tumor growth. The notion that mammals must regulate angiogenesis extensively is widely supported.

[0007] There has been much interest in understanding how to regulate angiogenesis. In particular, angiogenesis is secreted, for example, from endothelial cells (EC) that are activated by vascular endothelial growth factor (ie, VEGF-1), basic fibroblast growth factor (bFGF) and / or other mitogens It is thought to begin with the degradation of the basement membrane by proteases. The cells migrate and proliferate, leading to the formation of solid endothelial cell buds in the matrix space, after which vascular loops are formed and the capillary develops due to the formation of tight junctions and the deposition of new basement membranes.

[0008] It is disclosed that in adult humans, endothelial cells typically have a lower proliferation rate than other cell types in the body. The period of turnover of these cells can be over a thousand days. Physiological exceptions where angiogenesis leads to rapid growth occurring under tight control are found within the female reproductive system and during wound healing. It has been reported that the rate of angiogenesis is related to changes in the local balance between positive and negative regulation of microvessel growth.

[0009] Aberrant angiogenesis occurs when the body loses control of angiogenesis and is thought to lead to excessive or insufficient vascular growth. For example, conditions such as ulcers, strokes, and heart attacks may be due to lack of angiogenesis that is usually required for spontaneous healing. In contrast, excessive vascular growth can promote tumor growth, blindness, psoriasis, rheumatoid arthritis, and other medical conditions.

The therapeutic effects of vascular growth factors were first described by Folkman and colleagues more than 20 years ago (Folkman, N. Engl. J. Med., 85: 1182-1186, 1971). Recent studies have shown that the fibroblast growth factor (FGF) family (Yanagisawa-Miwa et al., Science 257: 1401-
1403, 1992 and Baffour et al., J Vasc Surg, 16: 181-91, 1992), endothelial cell growth factor (ECGF) (Pu et al., J Surg Res, 54: 575-83, 1993), and vascular endothelial growth factor It has been established that recombinant angiogenic growth factors such as (VEGF-1) can promote and / or increase coronary artery development in animal models of myocardium and hind limb ischemia (Takeshita et al., Circulation 90: 228-234, 1994 and Takeshita et al., J Clin In
vest, 93: 662-70, 1994).

[0011] The potential use of gene therapy to promote angiogenesis has been recognized. For example, it has been reported that angiogenesis can enhance the treatment of ischemia in rabbit models and in human clinical trials. VEGF administered as a balloon gene delivery system
Using -1 has been particularly successful. Successful delivery and maintenance of the expression of the VEGF-1 gene in the vascular wall then increased neovascularization in the ischemic limb (Takeshita et al., L.
aboratory Investigation, 75: 487-502, 1996; Isner et al., Lancet, 348: 370, 1
996). It has further been reported that direct injection of DNA encoding VEGF-1 intramuscularly into ischemic tissue induces angiogenesis and provides increased blood vessels to the ischemic tissue (Tsur
umi et al., Circulation, 94 12: 3281-3290, 1996).

[0012] Alternative methods of promoting angiogenesis are desirable for a number of reasons. For example, it is believed that the original number of endothelial progenitor cells (EPCs) and / or viability decreases over time. Thus, within certain patient populations, such as the elderly, the EPCs that can respond to angiogenic proteins may be limited. In addition, such patients may not respond well to conventional treatment.

[0013] There are reports that administration of isolated EPCs to patients, especially patients undergoing treatment for ischemic disease, can reduce at least some of these problems. However, this proposal would be too costly because the patient's cells had to be isolated and maintained. In addition, handling the patient's cells may pose significant health risks to both the patient and the caregiver.

[0014] Granulocyte macrophage colony stimulating factor (GM-CSF) is a granulocyte committed progenitor cell (
It has been shown to exert a regulatory effect on granulocyte-committed progenitor cells and to increase the level of circulating granulocytes (Gasson, JC, Blood 77: 1)
131, 1991). In particular, GM-CSF acts as a growth factor for granulocytes, monocytes, and eosinophilic precursors.

[0015] Administration of GM-CSF to humans and non-human primates increases the number of circulating neutrophils, as well as eosinophils, monocytes, and lymphocytes. Therefore, GM-CSF is considered to be particularly effective in promoting recovery from neutropenia in patients receiving radiation or chemotherapy or following bone marrow transplantation. Furthermore, while GM-CSF is less likely to promote EC growth than other cytokines, such as FGF, GM-CSF activates a sufficiently mobile phenotype (Bussolino et al., J. Clin. Invest., 87: 986, 1991).

[0016] Accordingly, it is desirable to have a method of modulating angiogenesis in a mammal, especially a human patient. It would be particularly desirable to have a method of enhancing EPC mobilization and neovascularization (new blood vessel formation) in a patient that does not require isolation of EPC cells.

SUMMARY OF THE INVENTION The present invention generally relates to methods of modulating angiogenesis in a mammal. In one aspect, the invention relates to a granulocyte macrophage colony stimulating factor (GM-CSF)
, VEGF, Sl factor (SLF), also known as stem cell factor (SCF), stromal cell-derived factor (SDF-1), granulocyte colony stimulating factor (G-CSF), HGF, angiopoietin-1 (Ang
iopoietin-1), angiopoietin-2 (Angiopoietin-2), M-CSF, b-FGF, and
A method for increasing angiogenesis, which comprises administering to a mammal an effective amount of an angiogenesis regulator such as FLT-3 ligand, and an effective fragment thereof, or DNA encoding the angiogenesis regulator. I will provide a. Such materials were sometimes once described as "hematopoietic factors" and / or "hematopoietic proteins". Disclosures relating to these and other hematopoietic factors are described in Kim, CH and Broxmeyer, HE (1998) Blood, 91:10.
00; Turner, ML and Sweetenham, JW, Br. L. Haematol. (1996) 94: 592;
Aiuuti, A. et al. (1997) J. Exp. Med. 185: 111; Bleul, C. et al. (1996) J. Exp. Med.
184: 1101; Sudo, Y. et al. (1997) Blood, 89: 3166, as well as the references described herein. Prior to the present invention, it was not known that GM-CSF or other hematopoietic factors, as described herein, enhanced endothelial progenitor cells or modulated neovascularization.

Alternatively, instead of the protein or an effective fragment thereof, DNA encoding an angiogenesis regulator can be administered to a site where neovascularization is desired, as described further below. The invention also relates to a method of treating or detecting a damaged blood vessel in a mammal. The present invention has many uses, including to prevent or reduce severe vascular damage with ischemia or related conditions.

The present inventors have found that hematopoietic factors such as granulocyte macrophage colony stimulating factor (GM-CSF) modulate endothelial progenitor cell (EPC) mobilization and neovascularization (vascular formation). Was. In particular, the present inventors have found that GM-CSF and other hematopoietic factors enhance EPC mobilization and promote neovascularization. In light of previous reports showing the activity of GM-CSF in vitro and in vivo, this observation was surprising and unexpected. Thus, the present invention provides methods of using GM-CSF to promote EPC mobilization and promote neovascularization, particularly in tissues in need of EPC mobilization and / or neovascularization.

In one aspect, the invention provides a method for inducing neovascularization in a mammal. The term "induction" also means at least promoting the mobilization of EPCs, preferably promoting the formation of new blood vessels in mammals. EPC mobilization is understood to mean a significant increase in EPC frequency and differentiation as determined by the assays disclosed herein. In one embodiment, the method comprises administering to a mammal an effective amount of an angiogenesis regulator, such as granulocyte macrophage colony stimulating factor (GM-CSF), ie, an amount sufficient to induce neovascularization in the mammal. Including doing. Preferably, the amount of GM-CSF can modulate, in particular increase, the frequency of EPC in a mammal. Various methods for detecting and quantifying neovascularization, the frequency of EPCs, the efficacy of angiogenesis modulators, and other vascular growth parameters are discussed below and in the examples.

In one particular embodiment of the method, the enhancement of EPC mobilization, and especially the increase in the frequency of EPC, is determined by a standard EPC isolation assay and is at least about 20%, preferably 50% to 500%. %. This assay generally detects and quantifies the amount of EPC and is discussed in detail below.

In another particular embodiment of the method, the dosage of the modulator is sufficient to promote EPC mobilization and, in particular, increase EPC differentiation in the mammal. Methods for detecting and quantifying EPC differentiation include the following specific methods. Preferably, the increase in EPC differentiation is at least about 20%, preferably from about 100% to 10%, as measured by the standard EPC culture assay described below.
It is between 00%, more preferably between about 200% and 800%. More preferably, the dosage of the modulating agent is sufficient to increase the differentiation of EPCs after tissue ischemia, as determined by the standard hind limb ischemia assay described below, at approximately the above percentages.

In another particular embodiment of the method, the dose of the angiogenesis regulating agent to the mammal is sufficient to increase the size of blood vessels in the mammal. For example, methods for measuring blood vessel size parameters such as length and circumference are known in the art and are described below. Preferably, the dosage of the modulator increases the length of the blood vessels by at least about 5%, preferably between about 10% and 50%, more preferably about 20%, as measured by the standard vessel length assay described below. Is enough. Preferably, the dosage of the modulator into the mammal is also measured by a standard vessel diameter assay, and said percentage amount is sufficient to increase the circumference or diameter of the vessel. Although other suitable assays can be used if desired, as described below, it will often be preferable to use standard corneal micropocket assays to detect and quantify changes in vessel size. .

In another particular embodiment of the method, the dosage of the angiogenesis modulating agent is at least about 5%, preferably between about 50% and 300%, as measured by a standard corneal micropocket assay. Preferably between about 100% and 200% is sufficient to increase neovascularization. Methods for performing this assay are known in the art and include the following specific methods. Further, a preferred amount of GM-CSF is measured by standard methods for measuring blood pressure in a desired blood vessel and improves ischemic hind limb blood pressure by at least about 5%, preferably between about 10% to 50%. Is enough. More specific methods for measuring blood pressure, especially for new or damaged blood vessels, include techniques optimized for measuring blood pressure in the mouse hind limb assay described below.

In another particular embodiment of the method, the dose of the angiogenesis modulating agent is at least about 20% EPC bone marrow (BM) -derived EP into the lesion as measured by a standard murine BM transplant model.
Enough to increase C uptake. The increase is preferably between about 50% and 400%, more preferably between about 100% and 300%, as measured by the standard model. E to the lesion
More specific methods of measuring increased PC uptake can be found in the discussion and examples below.

The method of the present invention is suitable for modulating, especially inducing, neovascularization in a variety of animals, including mammals. The term "mammal" is used herein to mean a warm-blooded animal, such as a rodent, rabbit, or primate, especially a human patient. Specific rodents and primates of interest include animals that represent adaptive models of human disease, including mice, rats, rabbits, and monkeys. Human patients of particular interest include patients having, suspected of having, or having, ischemic tissue. The ischemic tissue can be produced by almost any method, including surgical procedures or medical conditions. Ischemic tissue is often associated with ischemic vascular disease, such as the following specific conditions and diseases.

As will become more apparent from the following discussion and examples, the methods of the present invention are very adaptable and can be used in combination with established or experimental methods for modulating neovascularization. . In one embodiment, the invention includes a method of modulating, particularly inducing, neovascularization in a mammal that administers an effective amount of an angiogenesis modulating agent together with at least one vascular derived protein. In many settings, co-administration of an angiogenesis modulating agent and a vascular-derived protein will positively affect neovascularization in a mammal, for example, by providing an additive or conjugating effect. Preferred vascular derived proteins are recognized endothelial cell mitogens, such as the specific proteins described below. The method of co-administration of an angiogenesis regulator and a vascular-derived protein is described below, and generally varies depending on the intended use.

The present invention also provides a method of preventing or reducing severe vascular damage in a mammal, such as a human patient in need of such treatment. In one embodiment, the method comprises administering to a mammal an effective amount of an angiogenesis regulator, such as GS-CSF.
The mammal is exposed to conditions that promote vascular injury at about the same time as, or subsequent to, the administration. Alternatively, the angiogenesis regulator can be administered after exposure to conditions to prevent or reduce damage to the blood vessels. As described, various conditions that induce ischemic tissue in a mammal can lead to damage to blood vessels, such as invasive manipulation, such as surgery, transplantation, or angioplasty, infection or ischemia. Are known. The state and method of administering another angiogenesis regulator will be described later.

[0029] The preferred amount of the angiogenesis regulator used in the present method is sufficient to prevent or reduce severe vascular damage in the mammal. Specific amounts of GM-CSF are already described above, and include administering an effective amount of GM-CSF sufficient to induce neovascularization in a mammal. Exemplary methods for quantifying an effective amount of an angiogenesis modulating agent are discussed in this disclosure, including the discussion and examples below.

The present invention also provides a method of treating ischemic tissue, particularly damaged blood vessels in the tissue. Preferably, the method is practiced on a mammal, especially a human patient in need of such treatment. In one embodiment, the method comprises at least one, and preferably all, of the following steps: a) isolating endothelial progenitor tissue cells (EPC) from the mammal; b) isolating the EPC Contacting with an effective amount of at least one factor sufficient to induce proliferation; c) administering to the mammal an amount of proliferating EPC sufficient to treat damaged blood vessels.

[0031] In one particular embodiment of the method, the factor is a vascular derived protein, including cytokines known to induce the growth of EPCs, particularly in vitro. Exemplary factors and markers for the detection of EPC are described below. In one embodiment of the method, the vessel (or vessels) is damaged by almost any known method, including trauma, or invasive manipulation such as performing balloon angioplasty or deploying a stent or catheter. Can be. Particular stents are intravascular stents. Alternatively, the vascular injury may be organic and may derive from a once existing or presently occurring medical condition.

In another particular embodiment of the method, the angiogenesis modulating agent is administered to a mammal, especially a human patient, alone or at least one vascular derived protein (or an effective fragment thereof) as described below. (Co-administration).

Further, provided by the present invention is a method for detecting the presence of tissue damage in a mammal, especially a human patient. In one embodiment, the method comprises contacting the mammal with a population of detectably labeled EPCs and detecting detectable labeled cells at or near the damaged tissue site of the mammal. . In this example, the EP
C can be collected and, optionally, monitored or expanded in vitro by almost any acceptable route, including the specific methods described herein. By intravenous injection, the preferred route for most applications, one method or a combination of different methods,
The EPC can be administered to a mammal. Methods for applying detectable labels to cells are known in the art and include immunological or radioactive labels and specific recombinant methods described below.

In one particular embodiment of the method, the detectable labeled EPC can be used to “home-in” to a site of vascular injury, thereby allowing the site to be reduced even when the site is very small. A minimally invasive way of visualizing the site is provided. The detectable label EPC can be visualized by various methods well known in the art, including methods using tomography, magnetic resonance imaging or related means.

In another embodiment of the method, the tissue damage is promoted by ischemia, particularly an ischemic vascular disease as specified below.

Also comprising administering to the mammal an effective amount of at least one hematopoietic factor.
A method for adjusting the mobilization of an EPC is also provided by the present invention. Preferred are methods that promote EPC mobilization, as measured by any suitable assay disclosed herein. For example, in one particular embodiment of the method, the enhancement of EPC mobilization, particularly the increase in EPC frequency, is at least about 20%, preferably between 50% and 500%, as measured in a standard EPC isolation assay. It is.

In another particular embodiment of the method, the dose of hematopoietic factor is sufficient to promote EPC mobilization in a mammal, particularly to increase EPC differentiation. Methods for detecting and quantifying EPC differentiation include the following specific methods. Preferably, the increase in EPC differentiation is at least about 20%, preferably from about 100% to 100%, as measured by the standard EPC culture assay described below.
It is between 1000%, more preferably between about 200% and 800%. More preferably, the dose of hematopoietic factor is still sufficient to increase the differentiation of EPCs at about the above percentage after tissue ischemia, as measured by the standard hind limb ischemia assay described below.

As noted, EPC mobilization was found to facilitate significant induction of neovascularization in mammals. Thus, methods of modulating, particularly promoting, EPC mobilization can be used to induce neovascularization in mammals, especially human patients in need of such treatment. A method according to the present invention for promoting EPC mobilization comprising using at least one hematopoietic factor, which can be used alone or in combination with other methods, is disclosed herein. Include methods in which an effective amount of an angiogenesis regulator can be administered to a mammal alone or in combination with at least one vascular-derived protein (co-administration).

In particular, the invention is directed to mammals, particularly humans in need of such treatment, wherein the treatment comprises administering to the mammal an effective amount of at least one angiogenesis modulator, the one angiogenesis modulator. Provides a method of inducing neovascularization in a subject, the amount of which is sufficient to induce neovascularization in a mammal. This neovascularization can be detected and quantified, if desired, by standard assays disclosed herein, including the mouse corneal micropocket assay and the vessel size assay. Preferred methods promote neovascularization in mammals in the above percentage ranges.

In one embodiment of the method, an effective amount of angiogenic regulators (factors and the like) can be co-administered in combination with at least one vascular protein, preferably one vascular protein. The angiogenesis modulating agent can be administered to a mammal, particularly to a human patient in need of such treatment, with, after, or before the administration of vascular or other proteins.

The present invention also provides a pharmaceutical product preferably formulated for modulating, in particular, inducing neovascularization in a mammal. In a preferred embodiment, the product is provided sterile and optionally comprises an effective amount of GM-CSF, and optionally at least one vascular protein. In particular embodiments, the product is isolated in a formulation that is physiologically acceptable for mammals, particularly human patients in need of endothelial progenitor cells (EPCs).
Including EPC. Alternatively, the products can contain nucleic acids encoding GM-CSF and / or vascular proteins.

The present invention also provides kits preferably formulated for in vivo, and especially for systemic introduction of isolated EPCs. In one embodiment, the kit comprises the isolated EPC and, optionally, at least one vascular protein or nucleic acid encoding a vascular protein. Preferred is a kit that optionally includes a pharmacologically acceptable carrier solution, nucleic acid and mitogen, a means of transporting the EPC, and instructions for using the kit. Acceptable means of delivery of the EPC are well known in the art and include effective delivery by a stent, catheter, syringe, or related means.

[0043] Other aspects of the invention are disclosed below.

DETAILED DESCRIPTION As noted above, the present invention, in one embodiment, induces neovascularization in a human patient, comprising administering to the patient an effective amount of GM-CSF, or an effective fragment thereof. Provide a way to Also, as described above, the GM-CSF may be used alone or at least one angiogenesis regulator, preferably one of such factors, at least one vascular protein, preferably one vascular protein. It can be administered to a human patient in combination with one or more of the proteins (co-administration). Also provided is a method of promoting EPC mobilization comprising administering an effective amount of at least one angiogenesis modulating agent, preferably one such factor. Further provided is a method of treating or detecting damaged blood vessels in a human patient. The present invention has a wide range of uses, including the prevention or reduction of severe vascular damage in a patient.

The invention particularly provides a method of inducing angiogenesis in ischemic tissue of a patient in need of such treatment. In this aspect, the method generally comprises the GM disclosed herein.
-Including administration of an effective amount of CSF or other angiogenic agent to a patient. Administration of GM-CSF (or co-administration with other proteins) can be made as needed and can be performed before, during, or after formation of ischemic tissue. Further, GM-CSF can be administered as the sole active substance, or can be co-administered with at least one, preferably one, vascular derived protein, or other suitable protein or fragment provided herein.

In accordance with any of the methods disclosed herein, administration of an effective amount of a GM-CSF or an angiogenesis modulating agent disclosed herein, alone or by administration of a DNA encoding the same. It can be implemented in combination with different means including:

As described above, the method of the present invention may be used, for example, for trauma, transplant rejection, cerebrovascular ischemia, renal ischemia, glomerular ischemia, ischemia associated with infection, limb ischemia, ischemic cardiomyopathy, Cerebrovascular ischemia,
And has a wide range of uses in human patients, such as use in at least one prevention or treatment of myocardial ischemia. The affected tissue may be substantially any physiological system of the patient, including the circulatory or central nervous system, eg, limbs, grafts (eg, muscle or nerve grafts), organs (eg, heart, brain, kidney, and lung). Can be related to Because ischemia often occurs in cardiovascular disease or stroke, ischemia can have a particularly detrimental effect on heart or brain tissue, respectively.

In embodiments where an effective amount of an angiogenesis regulator is administered to a mammal and especially a human patient to prevent or reduce severe vascular conditions, especially ischemia, the subject is exposed to conditions that promote vascular damage. The angiogenesis modulating agent is preferably administered at least about 12 hours before, preferably about 24 hours to 1 week, up to about 10 days before. If desired, the method can further comprise administering to the mammal an anti-angiogenic agent after exposure to conditions that promote vascular damage. As mentioned above, the angiogenesis regulating agent can be administered alone or in combination with at least one vascular derived protein, preferably one of such proteins.

GM-CSF alone or at least one hematopoietic factor, preferably one of such factors, or one of at least one vascular-derived protein, preferably one of such proteins Alternatively, methods associated with preventing or reducing severe vascular conditions, including administration in combination with more than one, can be performed. Preferred methods of administration are disclosed herein.

It is known that vascular damage is promoted by one or a combination of damages to different tissues. For example, vascular injuries are often trauma, eg, balloon angioplasty, or surgery, such as the use of related devices (eg, directional atherectomy, rotational atheroma resection, laser angioplasty, transluminal extraction, pulse spray thrombolysis),
And deployment of intravascular stents or vascular grafts.

The specific EPC according to the invention is preferably associated with a cell marker which can be detected by conventional immunological or related methods. Next CD34 ⁺ , flk-1 ⁺ or tie
-2 ⁺ EPC having at least one of is preferred. The method for detecting EPC with these markers is described in the Examples below.

As described above and in the examples, the present inventors have discovered a method for promoting angiogenesis and re-endothelializing exposed blood vessels in mammals. These methods involve the use of angiogenesis regulators for mobilization of endothelial cell (EC) precursors. In accordance with the present invention, GM-CSF and other angiogenesis modulating agents are used in ischemic tissues, for example, cerebrovascular ischemia, renal ischemia, glomerular ischemia, limb ischemia, ischemic cardiomyopathy, and myocardial ischemic As a result of an ischemic disease such as blood, it can be used in a method to promote angiogenesis in selected patients with tissues having a blood deficiency.

In another aspect, the angiogenesis modulator alone or in combination with at least one of the other factors disclosed herein, induces reendothelialization of damaged blood vessels and thus smooth muscle cell proliferation Can be used to reduce restenosis by indirect suppression of restenosis.

[0054] In one preferred embodiment, the angiogenesis modulating agent alone or in combination with at least one of the other factors disclosed herein can be used to modulate angiogenesis in a patient. Some patient populations, especially the elderly population, may have either a limited number of ECs or a limited number of functional ECs. Thus, for example, VEGF
Stimulating angiogenesis with a potential pro-angiogenic agent such as -1,
Where enhanced angiogenesis is desired, such angiogenesis is more limited by the lack of EPC. However, prior to administration of an angiogenesis-promoting agent sufficient to allow EC mobilization, for example, GM-CSF can be administered to synergize these with angiogenesis in the patient. Preferably, GM-CSF is administered about one week prior to angiogenesis treatment.

The term “GM-CSF” is used herein, for example, in published International Application WO86 / 00639.
It is understood to mean a native or recombinantly prepared protein substantially identical to the amino acid sequence of the disclosed human GM-CSF, herein incorporated by reference.

Human GM-CSF (hGM-CSF) has been isolated and cloned, and is filed on July 4, 1985, International Application No. PCT / EP85 / 00326 (published as WO86 / 00639). Please refer to.

Escherichia coli-derived non-glycosylated rhGM-CSF is obtained by the method described in the publication of International Application No. PCT / EP85 / 00326, which describes two natural GM-CSFs differing by a single amino acid. Have been.

The natural GM-CSF used in the present invention can be modified by changing their amino acid sequence. For example, it is possible to change from one to five amino acids in their sequence without changing their basic characteristics, or to lengthen these sequences, which are sufficient for the original protein Functionally equivalent modified proteins can be supplied. Such functional equivalents can also be used in the practice of the present invention. A common GM-CSF that differs from the original sequence by only one amino acid is described in U.S. Pat.
No. 6, produced in glycosylated form in yeast and clinically shown to be the physiological equivalent of GM-CSF, such a modified form is GM-CSF (L
eu-23).

[0059] in the name of GM-CSF is a trade mark LEUKINE ^R (Immunex Corporation), as an analog peptides are commercially also clinically available. The generic name for recombinant human Leu ²³ GM-CSF analog peptide expressed in yeast is salgramostin Sargramos
tim. The cloning and expression of native sequence human GM-CSF is described in Cantrell et al., Proc.
atl. Acad. Sci. USA 82: 6250 (1985).

The native and recombinantly prepared proteins and functional equivalents used in the present invention are preferably purified, substantially cell-free and can be obtained by well-known methods. .

Another protein and sequence related to the factors disclosed herein, including GM-CSF, is
National Center for Biotechnology Information (NCBI) -Genetic Sequence Da
It can be obtained through ta Bank (Genbank). In particular, the Sequence Listing is the National Li
Genry Bank of brary of Medicine, 38A, 8N05, Rockville Pike, Bethesda, MD20894
Can be obtained from Genbank also has a website http: //www.ncbi.n
Also available at lm.nih.gov. In general, see Benson, DA for a description of Genbank.
(1997) Nucl. Acids. Res. 25: 1. Proteins and nucleic acid sequences not specifically referred to can be found in Genbank or other sources disclosed herein.

According to the method of the present invention, GM-CSF can be administered to a mammal, especially a patient in need of such treatment. As exemplified, GM-CSF and therapeutic ingredients comprising GM-CSF are preferably administered parenterally. More specific examples of parenteral administration include subcutaneous, intravenous, intraarterial, intramuscular, and intraperitoneal administration, with subcutaneous administration being preferred.

In embodiments of the invention where parenteral administration is chosen, the GM-CSF will generally be in a unit dose injectable form (solution, suspension, emulsion), preferably genetically non-toxic. Formulated in a non-therapeutic pharmaceutically acceptable carrier medium. Examples of such vehicles include, but are not limited to, saline, Ringer's solution, dextrose solution, mannitol, and normal serum albumin. Neutral buffered saline or serum albumin-mixed saline is a typical suitable vehicle. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. Other additives include substances to improve isotonicity and chemical stability, such as, for example, buffers, preservatives, and surfactants, such as polysorbate 80. Adjustment of peritoneally acceptable protein solutions consisting of appropriate pH, isotonicity, stability, etc. is within the skill of the art.

Preferably, the product can be formulated in a known manner as a lyophilizate using suitable excipients (eg sucrose) as diluents.

Preferred in vivo angiogenesis modulator doses are from about 1 μg / kg / day to about 100 μg / kg / day.
Until the day. The use of a more specific dose will be guided by parameters well known to those skilled in the art, such as the specific state of the treatment and the general health of the subject. See U.S. Patent No. 5,578,301 for another method of administering GM-CSF. Preferred in vivo hematopoietic and vascular derived protein doses disclosed herein are GM-C
Within the same or similar range as SF.

As noted, for some uses, co-administration with at least one hematopoietic protein, at least one vascular derived protein, or one or more of their effective fragments, results in the modulation of angiogenesis. It is effective to add to the administration of the agent.
This method is desirable, especially when increased (boost) angiogenesis is required. For example, in one embodiment, at least one vascular-derived protein, preferably one of the vascular-derived proteins, is administered to the patient at the same time, after, or before the administration of GM-CSF. The vascular-derived protein can be administered directly, for example, intra-arterially, intramuscularly, or intravenously, or amino acids encoding mitogens can be used. Baffour et al. (BFGF), Pu et al., Circulation, 88:20.
8-215 (1993) (aFGF); Yanagisama-Miwa et al., Supra, (bFGF); Ferrara et al., Bioch.
em. Biophys. Res. Commun., 161: 851-855 (1989) (VEGF-1) (Takeshita et al., Ci.
rculation, 90: 228-234, (1994); Takeshita et al., Laboratory, 75: 487-502,
(1996); Tsusumi et al., Circulation, 94 (12): 3281-3290 (1996).

As another example, at least one hematopoietic protein, preferably one of such proteins, is administered to a human patient in need of such treatment, simultaneously with, subsequent to, administration of GM-CSF, or It can be administered before that. As noted, at least one vascular derived protein can also be co-administered with GM-CSF or hematopoietic proteins. Although other modes of administration may be appropriate for some purposes, the method of administering hematopoietic proteins generally follows that described for the administration of GM-CSF.

The term “co-administration” refers to the preferred administration of at least two proteins disclosed herein to a mammal, ie, one protein is administered simultaneously with, after, or before the administration of another protein. It is understood to mean to administer.

In embodiments where co-administration with DNA encoding a pulse-derived protein or hematopoietic protein is desired, the amino acids encoding them can be found in US Pat. No. 5,652,225, the disclosure of which is incorporated herein by reference. No. 1 can be administered to ischemic tissue perfused vessels through catheters such as, for example, hydrogel catheters. Amino acids can also be injected into ischemic tissue and delivered using the methods described in PCT WO 97/14307.

As used herein, related terms such as “vascular-derived protein” or “angiogenic protein” include any protein, polypeptide that can directly or indirectly induce vascular growth , Their muteins, or parts.
Such proteins include, for example, acidic or basic fibroblast growth factors (aFGF and bFGF), vascular endothelial growth factor (VEGF-1), VEGF165, epidermal growth factor (EGF), transforming growth factors α and β (TGF -α and TGF-β), platelet-derived endothelial growth factor (PD-ECGF), platelet-derived 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), angiopoietin-1 (Ang1) and NO synthase (NOS). Klagsburn et al., Annu. Rev. Physiol., 53: 217-239 (1991); Folkman et al., J. B.
iol. Chem. 267: 10931-10934 (1992) and Symes et al., Current Opinion in Lipid.
ology 5: 305-312 (1994). As long as they induce or promote vascular growth, muteins or fragments of the mitogen can be used.

[0071] Preferred angiogenic proteins include vascular endothelial growth factor. The first one of these is termed VEGF and is now called VEGF-1 and exists in several different isoforms produced by alternate splicing from a single gene containing eight exons (
Tischer et al., J. Biol. Chem. 806, 11947-11954 (1991) Ferrara, Trends Cardio
Med., 3, 244-250 (1993); Polterak et al., J. Biol. Chem., 272, 7151-7158 (
1997). Human VGEF isoforms consist of monomers of 121 (US Pat. No. 5,219,739), 145, 165 (US Pat. No. 5,332,671), 189 (US Pat. No. 5,240,848) and 206 amino acids, each forming an active homodimer. (Houck et al., Mol. Endo
crinol., 8, 1806-1814, 1991).

Other vascular endothelial growth factors include VEGF-B and VEGF-C (Joukou et al., J. of Cell. Phys.
., 173: 211-215, 1997), VEGF-2 (WO96 / 39515) and VEGF-3 (WO96 / 39421).

Preferably, the vascular protein has a secretory signal sequence that promotes secretion of the protein. For example, a protein having a natural signal sequence such as VEGF-1 is preferred. For example, a protein having no natural signal sequence, such as bFGF, can be modified by common genetic engineering techniques to have such a sequence. Nabel et al., Natur
e, 362: 844 (1993).

Reference herein to “angiogenesis modulators,” “hematopoietic factors,” or related terms such as, for example, “hematopoietic proteins” refers to a recognized factor that increases hematopoietic progenitor cell (HPC) mobilization. Used to mean. Preferred hematopoietic factors are granulocyte macrophage colony stimulating factor (GM-CSF), VEGF, Sl factor (SLF), also known as stem cell factor (SCF), stromal cell-derived factor (SDF-1), granulocyte colony-stimulating factor ( GC
SF), HGF, Angiopoietin-1, Angiopoietin-2, M-CSF, b-FGF, and FLT
Includes -3 ligand. Disclosures relating to these and other hematopoietic factors are provided by Kim, CH and B
roxmeyer, HE (1998), Blood 91: 100; Turner, ML, and Sweetenham, J
W., Br. J. Haematol. (1996) 94: 592; Aiuuti, A. et al. (1997) J. Exp. Med.,
185: 111: Bleul, C. et al. (1996) J. Exp. Med., 184: 1101; Sudo, Y. et al., Blood, 8
9: 3166; and individually disclosed references.

The nucleotide sequences of many vascular proteins can be conveniently determined, for example, by GenBank, EMBL and S
It is available from computer databases such as wiss-Prot. Using this information, the DNA segment encoding the desired can be chemically synthesized, or such DNA segments can be obtained by routine methods in the art, such as, for example, PCR amplification.

In certain situations, it may be desirable to use nucleic acids encoding two or more different proteins to optimize therapeutic outcome. For example, DNA encoding two proteins, such as VEGF-1 and bFGF, can be used, and the use of bFGF alone can be improved. Vascular factors can be combined with other genes or their encoded gene products,
It can promote the activity of target cells while inducing angiogenesis, such as NO synthase, L-
Includes arginine, fibronectin, urokinase, plasminogen activator and heparin.

The term “effective amount” refers to a protein of interest (eg, GM-CSF, an angiogenesis regulator,
Hematopoietic protein, vascular-derived protein), that is, sufficient to produce levels capable of inducing endothelial cell proliferation and / or angiogenesis, as measured by standard assays disclosed in the present application. The amount of a compound such as a protein or nucleic acid to be delivered. Thus, an important aspect is the level of the expressed protein. Thus, multiple transcripts can be used, or the gene can be under the control of a promoter that causes high expression levels. In other embodiments, for example, ta
The gene can also be under the control of factors that cause very high expression levels, such as the t and the corresponding tar element.

To facilitate manipulation and handling of the nucleic acid encoding the protein, the nucleic acid is preferably inserted into a cassette that is engineered and linked to a promoter. The promoter can drive expression of the protein in cells of the desired target tissue. Selection of an appropriate promoter can be conveniently achieved. Preferably, a high expression promoter can be used. An example of a suitable promoter is 763-base pair cytomegalovirus (CMV). Rous sarcoma virus (RSV) (Davis et al., Hum Gene Ther 4: 151, 1993)
) And the MMT promoter can also be used. Certain proteins can be expressed with their natural promoter. Other elements that enhance expression may also include enhancers or systems that cause high levels of expression, such as the tat gene or tar element. This cassette can then be inserted into a vector such as pUC118, pBR322 or other well-known plasmid vectors containing, for example, the E. coli origin of replication. Sambrook
Et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laborator
See y press (1989). The plasmid vector can also include a selectable marker, such as ampicillin-resistant beta-lactamase, provided that the marker polypeptide does not adversely affect the metabolism of the organism being treated. The cassette can also bind to a nucleic acid binding portion of a synthetic delivery system such as the system disclosed in WO95 / 22618.

The particular method of the present invention can be used to treat vascular damage that causes the endothelial contents of the vascular wall to be exposed. For example, primary angiogenesis has become widely used in the treatment of acute myocardial infarction. In addition, intravascular stents have also become widely used as an adjunct to balloon angioplasty. Stents are effective in rescuing near-optimal initial results and in reducing restenosis. However, at present, the downside of endovascular prostheses is that about 3% of patients with arteries greater than 3.3 mm are more susceptible to thrombotic occlusion. Subacute thrombosis is more likely if the patient is deploying a stent in a smaller artery. Subacute thrombosis can currently only be prevented by overuse of anticoagulation. The combination of vascular intervention and overuse of anticoagulation creates a significant risk for peripheral vascular trauma during stent / angioplasty procedures. Promoting re-endothelialization by administering GM-CSF alone or in combination with the factors disclosed herein to patients prior to performing angioplasty and / or stent deployment stabilizes unstable plaques And prevent re-thrombosis. In this example, it is desirable to administer GM-CSF about one week before the exposure of the vessel wall.

The method of the present invention can be used with DNA encoding an endothelial mitogen, according to the method for treating vascular injury disclosed in PTC / US96 / 15813.

As used herein, the term “endothelial cell mitogen” means any protein, polypeptide, mutein, or moiety that can induce endothelial cell proliferation. Such proteins include, for example, vascular endothelial growth factor (VEGF-1), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (dispersion factor), and colony stimulating Factor (CSF). VEGF-1 is preferred.

In addition, the methods of the present invention can be used to allow angiogenesis and promote the healing of transplanted cells, for example, vascular grafts.

Reference herein to “a standard EPC isolation assay” or other similar phrase means an assay that includes at least one, and preferably all, of the following steps. a) obtaining a peripheral blood sample from the mammal of interest, preferably a rodent, especially a mouse; b) separating low-density mononuclear cells from a blood sample; c) separating the isolated mononuclear cells, specifically Sca-1. ⁺ to be binding, also quantification of Sca-1 ⁺ cells with Sca-1 ⁺ cell number of measurements at the contact d) for example the hand of the bead can be separated Sca-1 ⁺ mononuclear cells

See the following discussion and examples for more specific disclosure relating to standard EPC isolation assays.

The term “standard EPC isolation assay” or related terms means an assay that includes at least one, and preferably all, of the following steps. Sca-1 ⁺ and Sca-1 from a) Mouse peripheral blood ^- from isolation, or rabbit peripheral blood
Isolation of TBM ⁺ and TBM ⁻ and detectable labeling of the cells (Sca-1 ⁺ and TBM ⁻ ), for example with Di-I as provided herein b) for several days, usually about Culturing the cells in a suitable dish or medium in a plate for 4 days c) Di-I labeled Sca-1 ⁺ or TBM ⁻ , or Di-I unlabeled Sca-1 ⁻ or TBM ⁺ , a dish or Count of all attached dispersed cells in the plate d) Quantification of specific positive cells indicating EPC

More specific disclosures relating to standard EPC culture assays can be found in the discussion and examples below.

Reference herein to “standard murine hind limb ischemia assay” or related terms indicates a conventional assay for inducing hind limb ischemia in an acceptable animal model, particularly a mouse or rabbit. Means to do. Disclosures relevant to performing the assay can be found in the Examples and Materials and Methods section below. Also,
For further disclosure regarding the assay, see Couffinhal, T. et al (199).
8) Am. J. Pathol.,; And Takeshita, S. et al. (1994) J. Clinical. Invest. 93: 6
See 62.

A reference herein to a “standard vascular length assay” or “standard vascular diameter assay” generally exposes the vessel of interest in the mammal (eg, mouse or rabbit) of interest, and It refers to measuring the vessel length or diameter in a conventional manner after inspecting the vessel. Examples of blood vessels such as certain arteries or veins that can be measured are given below.

The phrase “standard corneal micropocket assay”, or related terms, is used herein with particular reference to the mouse corneal micropocket assay. The assay generally includes at least one, and preferably all, of the following steps. a) creating a corneal micropocket in at least one eye of the mouse b) adding a pellet containing an acceptable polymer and at least one intervening protein to the pocket c) characteristically stepwise, e.g. by slit-lamp biomicroscopy b) After several days, e.g. 5-6 days, examine mouse angiogenesis d) Mark eye EC cells, e.g. with BS-1, e) Eye angiogenesis, and optionally EC cells Is quantified.

For a more specific disclosure regarding the standard corneal micropocket assay, see
See discussion and examples below. If desired, the assay may be performed as described above, except that the control comprises a pellet containing no pulse-derived protein in step b).
A control can be included as a reference, including the performance of steps a) to e).

Reference herein to a “standard murine bone marrow (BM) transplant model” or similar term means at least one, and preferably all, of the following steps. a) Obtaining detectable labeled BM cells from a donor mammal and, characteristically, a mouse. b) Isolation of low density BM mononuclear cells from the mouse. c) Removal of BM cells from appropriate recipient mice, for example by irradiation. d) Administration of isolated and detectably labeled BM cells to the recipient mouse. e) Exposure of the recipient mouse to a condition that promotes vascular injury in the mouse, eg, hind limb ischemia. f) Administration of an effective amount of GM-CSF to the recipient mouse. g) obtaining at least one cornea from the recipient mouse; and h) detecting and quantifying labeled BM cells in the cornea.

[0092] An exemplary detectable label is beta-galactosidase enzyme activity. More specific information on the assay can be found in the discussion and examples below.

[0093] Reference herein to an "effective amount" of an angiogenic agent, such as GM-CSF, a hematopoietic protein, or a pulse-derived protein, refers to at least one standard assay disclosed herein. As determined, the corresponding full-length protein has at least 70
%, Preferably about 75% to 95%. Although other standard assays can be used, those that detect and preferably measure EPC mobilization are preferred. As an example, a preferred effective fragment of GM-CSF is determined by a standard corneal mini-pocket assay, in particular a standard vascular length or diameter assay, and is expressed in full length human GM.
-Has at least 70%, preferably about 75% to 95% of the vascular stimulatory activity of CSF (see published International Application No. PCT / EP / 85/00376 (WO86 / 00639)).

[0094] All documents mentioned herein are hereby incorporated by reference in their entirety.

The invention is further described by the following examples. These examples are provided to aid the understanding of the present invention and are not to be construed as limiting.

Example 1 Modulation of EPC Dynamics by Administration of Cytokines Circulating EPCs may build a recovery response to injury. The hypothesis that administration of cytokines mobilizes EPCs and thereby enhances therapeutic neovascularization was studied as follows.

GM-CSF is a hematopoietic progenitor cell (Socinski et al., Lancet, 1988; 1: 1194-1198, Gianni
Et al., Lancet, 1989; 2: 580-584), and myeloid lineage cells (Clark et al., Science 198).
7; 236: 1229-1237, Sieff, C., J. Clin. Invest. 1987; 79: 1549-1557), and BM substrate cells (Dedhar et al., Proc. Natl. Acad. Sci. USA, 1988; 85: 9253-9257) and EC (Bussolini et al., J. Clin. Invest., 1991; 87: 986-995) to induce the proliferation and differentiation of non-hematopoietic cells and promote cytokine-induced EPC mobilization. Used. To avoid a direct mitogenic effect on EC, GM-CSF was administered 7 days prior to the onset of stimulating neovascularization. New angiogenesis was first tested in the mouse corneal pocket assay described above. Pretreatment of GM-CSF (ip, (ip), rmGM-CSF [
R & D system] 500 ng / day) at day 0, ie, creation of corneal micropockets and VEG
Increase the circulating EPC before insertion of the F pellet (221% of untreated controls),
At day, neovascularization was increased (FIGS. 1A-C) compared to control mice (length = 0.67 ± 0).
.04 vs. 0.53 ± 0.04, p <0.05; angle (peripheral extent occupied by neovascular distribution)
= 155 ± 13 vs. 117 ± 12, p <0.05) (FIGS. 1B-1D). See FIGS. 2A and 2B.

Example 2 Cytokine-induced EPC Mobilization Promotes Neovascularization of Ischemic Tissue [0098] To determine whether cytokine-induced EPC mobilization promotes neovascularization of ischemic tissue, a rabbit hindlimb ischemia model (Takeshita et al., J. Clin. Inve
st. 1994; 93: 662-670). Rabbits pretreated with GM-CSF (subcutaneous, (sc), rhGM-C
SF; 50 μg / day sc) increased the EPC-enriched cell population (189% compared to control animals) and promoted EPC differentiation at day 0 (ie, prior to surgery) (421 compared to control animals). %) (Figure 3). Capillary density morphometric analysis showed greater neovascularization induced by pretreatment with GM-CSF compared to controls (ischemia, non-GM-CSF) (249 vs. 146 / mm ² ,
p <0.01). Pretreatment with GM-CSF also greatly improved the ischemic limb / normal limb blood pressure ratio (0.71 to 0.49, p <0.01) (FIGS. 3A-3C).

Example 3 Dynamics of EPC in Ischemic Tissue In order to examine the kinetics of EPC in ischemic tissue, the frequency and differentiation of EPC were examined by EPC isolation from peripheral blood and EPC culture assay. . Eca-rich fractions of EPC can be obtained from mice using Sca-
1 As antigen-positive (Sca-1 ⁺ ) cells, from rabbits, the antigen repertoire CD5- / Igμ-
/ CD11b- called T- lymphocytes, B- lymphocytes and monocytes ^- was isolated as a depletion cell population (TBM).

The frequency of the EPC-rich population in the circulation marked with Sca-1 was 10.7 ± 1.0% in C57 / 6JBL normal mice. A subset of Sca-1 ⁺ cells plated on rat vitronectin adhered and became spindles within 5 days. Co-culture of cells, Sca-1 with DiI fluorescent ^- Sca-1 ⁺ and Sca-1 negative (Sca-1) ^- was tested after marking the cells. In Sca-1 ⁺ , spindle morphology was developed. Mouse adherent cells in co-culture are mainly S marked with DiI
The cells were found to be ca-1 ⁺ cells (65-84%) and proved to be EC lines by reaction with BS-1 lectin and uptake of acLDL (FIG. 4A). To determine whether Sca-1 ⁺ cells can differentiate into ECs in vivo, Di isolated from peripheral blood with similar genetic background
A homogenous population of Sca-1 ⁺ cells, marked I, was administered intravenously in hindlimb ischemic mice following ischemic surgery (Couffinhal, T. et al., Am. J. Pathol., 1998). DiI-labeled EPC-derived cells were shown to differentiate into EC in situ by CD31 (PECAM) co-staining and found to integrate into colonies, buds, and capillaries (FIGS. 4A-4D).

In a rabbit model, antigens on T and B lymphocytes and monocytes were used to deplete mature HC, yielding an EPC-rich (TBM ⁻ ) fraction. The frequency of the TBM ^- EPC-rich population in rabbit peripheral blood was 22.0 ± 1.4%. EPC differentiation was determined by counting adherently cultured mononuclear blood cells. Adherent cells in EPC cultures have again been found to be mainly derived from DiI-marked TBM ^- cells (71-92%) and are EC lines due to reaction with BS-1 lectin and uptake of acLDL Prove that.

TBM ^- cells were isolated from 40 ml of peripheral blood 0, 3, 7 days after surgery in vivo.
Autologous TBM ^- cells marked with DiI were administered to rabbits with unilateral hind limb ischemia and shown to differentiate into EC (Takeshita, S. et al., J. Clin. Invest. 1994). DiI-labeled EPC-derived cells were shown to differentiate into EC in situ by co-staining with CD31 and incorporation into colonies, buds, and capillaries (FIGS. 4E-4J).

FIGS. 4A to 4D are specifically described below. The figure provided fluorescence microscopy evidence that EPCs from the mouse Sca-1 ⁺ cell population and the rabbit TBM ^- cell population could be sent to and integrated into foci of neovascularization. In particular, FIG. 4A shows double-stained cultured murine cells of acLDL-DiI (red) and BS-1 lectin (green) 4 days after the EPC culture assay. (FIGS. 4B-D) Two weeks after surgery, Sca-1 ⁺ cells administered to mice with hind limb ischemia were sent to neovascularized foci within the mouse ischemic hind limb, differentiated and integrated. FIGS. 4B and 4C demonstrate that DiI-labeled Sca-1 ⁺ cells (red) co-localize with CD31 (green), indicating that these EPCs have been incorporated into CD31-positive vascular structures. Arrows indicate cells positive for DiI and CD31, wedges indicate CD31 positive, DiI
Indicates negative (self EC) cells. The non-fluorescence phase contrast photograph in FIG. 1d shows that the vascular lesion of EPC (arrow) is in the intestinal site adjacent to skeletal muscle cells.

FIGS. 4E-G show that two weeks after ischemic surgery, immunostaining of rabbit ischemic hind limb muscles was performed in this case.
Isolated as TBM ^- cells (red) (FIG. 4E), showing EPC accumulation and colonization. These cells were marked with DiI and re-injected on days 0, 3, and 7. FIG. 4F shows that these cells are co-labeled with CD31 in neovascular lesions. DAPI stains cell nuclei (blue) (Fig. 1G). (FIGS. 4H-J). The colonized TBM ^- cells integrate into the developing buds and form new capillaries between skeletal muscle cells.

Example 4 Confirmation of EPC Dynamics in Ischemic Tissues EPC kinetics between tissues with severe ischemia was examined for frequency and differentiation. The EPC-rich population in the circulating blood increased after the onset of ischemia and peaked on day 7 of surgery (day 7 vs. day 0: 17.5 ± 2.4 vs. 3.8 ± 0.6 × 10 ⁵ / ml [mice]. p <0.05], 11.1 for rabbits
4 ± 0.6 vs. 6.7 ± 0.3 × 10 ⁵ / ml [p <0.05],) (FIGS. 5A, 6A). The EPC culture assay showed a dramatic improvement in EPC differentiation after ischemia and peaked at day 7 (Day 7 vs. Day 0: 263 ± 39 vs. 67 ± 14 / mm ^{2 in} mice [263]. p <0.05], 539 ± 73 vs 100 ± 19 [p <
0.05])) (FIGS. 5B, 6B). Both the frequency of the EPC-rich population and the EPC culture assay
There was no significant increase in EPC kinetics in any of the sham animal models at day.

Next, FIGS. 5A and 5B will be specifically described. The figure shows the kinetics of EPCs related to the development of hind limb ischemia. (FIG. 5A) After surgery for ischemic hindlimb creation, the EPC-enriched population of mice in the circulating blood (Sca-1 ⁺ ) increased and peaked at day 7 (n = 5 at each time point). mouse of). (FIG. 5B) The adherent cells in the EPC culture are mainly derived from Sca-1 ⁺ cells marked with DiI. Culture assays show that EPC differentiation is enhanced after surgery-induced ischemia, peaking at day 7 (n = 5 at each time point).

FIGS. 5C-H show the results of a mouse corneal micropocket assay applied to hind limb ischemic mice 7 days after surgery. Slit-lamp biomicroscopy (Figures 5C and 5D) and fluorescence micrographs (Figures 5E and 5F) show that neovascularization of non-vascular areas of the mouse cornea was enhanced by ischemia-induced EPC mobilization This is shown at the same magnification. (FIGS. 5G and 5H) Quantitative analysis of the two parameters of vascular length and perivascular distribution of neovascularization showed that corneal neovascularization in hindlimb ischemic animals was greater than in non-ischemic sham-operated control mice (n = 9) Indicates significant formation (* = p <0.05)

Example 5 Analysis of the Effect of Enhanced EPC Mobilization on Neovascularization In order to examine the effect of ischemia-induced enhanced EPC mobilization on neovascularization, surgery was performed 3 days earlier to achieve hindlimb ischemia. Mouse corneal micropockets were applied to animals that produced blood. Slit-lamp (FIGS. 5C and 6D) and Fluorescence (FIGS. 5E and 6F) micrographs show improved nonvascular mouse cornea neovascularization in hindlimb ischemic animals compared to non-ischemic sham-operated controls . Compared to non-ischemic sham surgery controls,
A significant effect of EPC mobilization on neovascularization in ischemic animals was shown (length = 0.67 ±
0.04 vs 0.53 ± 0.04mm, p <0.05, circumference = 43.3 ± 3.5 vs 32.4 ± 3.4%, p <0.05) (Fig. 5G
And 5H).

Example 6 Confirmation of Enhanced Neovascularization by Cytokine-Induced EPC Mobilization To determine whether cytokine-induced EPC mobilization promotes neovascularization of ischemic tissue, a hind limb ischemia rabbit model (Takeshita, S. et al., J. Clin. Invest. 1994) was used. Recombinant human GM-CSF was administered daily for 7 days prior to the development of hind limb ischemia to induce GM-CSF-induced EPC mobilization while avoiding direct effects on EC. Such GM-CSF pretreatment (50 μg / day sc) increases the EPC-rich population (12.5 ± 0.
8 vs. 6.7 ± 0.3 × 10 ^{5 /} ml, p <0.01), promoted EPC differentiation at day 0 (day 7 of pre-operative pretreatment) (423 ± 90 vs. 100 ± 19 / mm ² , p <0.01). By 7 days after surgery, GM-CS
The frequency of circulating EPC and the differentiation of EPC in the F pretreatment group exceeded the control value (20.
9 ± 1.0 vs 11.3 ± 2.5 × 10 ⁵ / ml [p <0.05], 813 ± 54 vs 539 ± 73 / mm ² [p <0.01]) (
Figures 6A and 6B). Analysis of capillary density shows large neovascularization induced by GM-CSF pretreatment (249 ± 18 vs. 146 ± 218 mm ² p <0.01 in untreated group) and increased ischemia / normal hind limb blood pressure ratio ( 0.71 ± 0.03 vs 0.49 ± 0.03, p <0.01) (FIG. 6C).

Next, FIGS. 6A to 6I will be described in more detail. The figure shows GM- in a rabbit ischemic hind limb model.
Figure 4 shows the effect of CSF-induced EPC mobilization on neovascularization. (Figure 6A, B) after GM-CSF pretreatment, from day 0 (before surgery) day 7 control animals (ischemic untreated) compared to, EPC rich number population of circulating (TBM ^- ) Increased (FIG. 6A), as did EPC differentiation in culture (FIG. 5B) (n = 5 mice at each time point). (FIG. 6C) Two weeks after the start of rabbit ischemia, physiological measurements using the ratio of blood pressure of the ischemic limb to blood pressure of the healthy limb showed a significant improvement in rabbits receiving GM-CSF compared to the control group. It has been shown. In addition, histological examination using basic phosphatase staining
Rabbits treated with -CSF showed increased capillary density (n = 9 mice in each group) (* = p <0.01, ** = p <0.05).

Slit-lamp biomicroscopy (FIGS. 6D and 6E) and fluorescence micrographs (FIGS. 6F and 6G, same magnification) show that EPC mobilization induced by pretreatment with GM-CSF showed non-vascularization of mouse corneas. It indicates that it promotes neovascularization in the area. (FIGS. 6H and 6I) In the measurement of blood vessel length and blood vessel circumference, GM-CSF pre-treated mice (n = 10) were compared with control mice (n = 10).
In 6), a significant effect of EPC mobilization on neovascularization was shown (* = p <0.05).

Example 7 Confirmation of Enhanced Neovascularization Using Mouse Corneal Micropocket Assay The above results were confirmed by measuring new angiogenesis in the mouse corneal micropocket assay. GM-CSF pre-treated mice (rmGM-CSF, 500 ng / day, ip) developed greater corneal neovascularization than control mice (length = 0.65 ± 0.05 vs. 0.53 ± 0.04, p <0.05, mm; Circumference = 38.0 ± 3.5 vs 28.3 ± 2.7%, p <0.05) (FIGS. 6D-6I).

Example 8 Enhancement of Uptake of BM-Derived EPC in BM Transplant Model Establishes direct evidence of enhanced uptake of BM-derived EPC into foci of corneal neovascularization in response to ischemia and GM-CSF Therefore, the rat BM transplant model (BMT) was applied. The cornea was excised 6 days after micropocket transplantation and examined by light microscopy. As a result, a significant increase in beta-galactosidase-expressing cells was shown in the ischemic limb compared to the sham-operated group (3.5 ± 0.6 vs. 10.5 ± 1.7, p <0.01). Treated with GM-CSF compared to control group
The same was true for BMT recipients (3.2 ± 0.3 vs. 12.4 ± 1.7, p <0.01) (FIGS. 7A, 7B). No beta-galactosidase-expressing cells were found in the cornea of control mice (after BMT). Quantitative chemical detection confirmed a statistically significant increase in beta-galactosidase activity in GM-CSF-treated mice compared to controls (2.90 ± 0.30 vs.
2.11 ± 0.09 × 10 ³ , p <0.05) (FIG. 7C).

Next, details of FIGS. 7A to 7C will be described. The figure shows that bone marrow-derived EPCs contribute to corneal neovascularization. The micrographs shown in the inset show the endothelium-specific Tie-2 / LacZ-expressing BM-derived EPC (blue cells) in both hindlimb ischemic mice (FIG. 7A) and GM-CSF pretreated rabbits (FIG. 7B). Figure 4 shows uptake into corneal neovascularization lesions. EPC stained with X-gal
The frequency of incorporation was counted visually under a light microscope. (FIG. 7A) Incorporated EPCs were significantly higher in hind limb ischemic mice than in sham-operated mice; (FIG. 7B) also in GM-CSF pretreated rabbits compared to control rabbits (each * = P <0.0 in the situation
1). (FIG. 7C) Beta-galactosidase activity was significantly higher in the GM-CSF group than in the control group (** p = <0.05).

The development of limb ischemia has been observed to promote EPC mobilization, and these EPCs thus contribute to “angiogenic” neovascularization. Ledney et al. (Ledney, GD et al., J. Surg.
Res., 1985) Wound trauma has been reported to cause mobilization of HCs, including pluripotent stem or progenitor cells in the spleen, BM, and peripheral blood. EPC is derived from BM and
Because PC mobilization is promoted between ischemic tissues, it is BM-controlled through the circulation of cytokines and soluble receptors and / or adhesion molecules, and circulating EPCs may build a repair response to ischemic damage. is there.

The results indicate a potential stimulatory effect of GM-CSF on EPC kinetics, and that such cytokine-induced EPC mobilization could result in severe ischemic tissue and the renewal of previously non-vascular sites. It is shown to promote live blood vessel formation. In particular, the examples show the mobilization of EPCs in response to exogenous and intrinsic stimuli.

The above discussion and examples demonstrate that the inventors studied endogenous stimuli in mobilizing EPCs and inducing neovascularization of ischemic tissue, ie, tissue ischemia, and exogenous cytokine treatment, especially granulogenesis. Shows the importance of sphere macrophage colony stimulating factor (GM-CSF). The development of focal ischemia in mice and rabbits was found to increase the frequency of circulating EPC. In mice, the effect of ischemia-induced EPC mobilization was demonstrated by enhanced ocular neovascularization following corneal micropocket surgery in hindlimb ischemic animals compared to non-ischemic controls. In hindlimb ischemic rabbits, circulating EPCs were further enhanced following GM-CSF pretreatment, consistent with promotion of hindlimb angiogenesis. EPCs that contributed to the promotion of corneal neovascularization by mice transplanted with BM from recombinant donors expressing beta-galactosidase that are transcriptionally regulated to endothelial cell (EC) -specific Tie-2 promoters were stimulated by ischemia and GM-CSF. In response, there was direct evidence of specific mobilization from bone marrow (BM). These findings indicate that circulating EPCs are mobilized endogenously in response to tissue ischemia or exogenously by cytokine therapy, thereby enhancing neovascularization of ischemic tissue.

In particular, the concepts of EPC mobilization and subsequent neovascularization disclosed herein and in US Provisional Application No. 60 / 077,262, both of which are pending, are specifically described herein. It is considered that there is a possibility as a method for preventing and treating various ischemic interpulsion diseases including ischemic interpulsion disease.

General Comments-The following materials and methods are used in the above examples as needed.

1. Isolation of Mouse EPC- enriched Fraction from Peripheral Blood Peripheral blood samples from mice were obtained from the heart immediately before sacrifice and Histopark-1083 (Sigma,
(St. Louis, MO) Density gradient centrifugation was performed at 400 g for 20 minutes to separate. Low density mononuclear cells were harvested, washed twice with Dulbecco's phosphate buffered saline supplemented with 2 mM EDTA (DPBS-E) and counted by eye. Blood mononuclear cells from individual animals were placed in 500 μl DPBS-E buffer supplemented with 50 μl Sca-1 microbeads (Miltenyi Biotec, Auburn, Calif.) And 0.5% bovine serum albumin (Sigma) for 15 minutes at 4 ° C. Suspended. The cells were washed with a buffer, and Sca-1 antigen-positive (Sca-1 ⁺ ) cells were separated on a magnetic stainless steel wool column (Miltenyi Biotec) and counted. Cells that did not bind to the Sca-1 antibody passed through the column, while Sca-1 ⁺ cells remained. The Sca-1 ⁺ cells were eluted from the column and both cell fractions were counted visually.

2. Isolation of Rabbit EPC- enriched Fractions from Peripheral Blood Rabbit peripheral blood samples were obtained from one ear vein with a 20 G perfusion catheter and Histo Park-1077 (Sigma) density gradient centrifugation at 400 g for 20 minutes. Minutes and separated. Low density mononuclear cells were harvested, washed twice with DPBS-E and counted by eye. Immature HCs were isolated by loss of mature HCs due to the lack of suitable antibodies for rabbit hematopoietic stem / progenitor cells. The cells were incubated with a mixed primary antibody of mouse-rabbit CD5, anti-rabbit IgM (μ chain) and CD11 to identify mature T and B lymphocytes and monocytes, respectively. After washing the antibodies, the cells were incubated with secondary rat-mouse IgG microbeads (Miltenyi Biotec) and placed on a magnetic separation column (Miltenyi Biotec). Cells identical to hematopoietic stem / progenitor cells that did not bind to mature T and B lymphocyte and monocyte antibodies passed through the column, while positive cells to the antibody mixture remained. The positive cells (TBM ⁺ ), mature HC
s was eluted from the column and both cell fractions were counted visually.

3. EPC Differentiation Assay To evaluate EPC differentiation from circulating blood cells, 5% FBS (Clontecs, San Diego)
Sca-1 ⁺ and Sca-1 ⁻ isolated from 700 μl of peripheral blood from individual mice after Di-I labeling of Sca-1 ⁺ or TBM ⁻ in EBM-II medium supplemented with TBM ^- and TBM ⁺ isolated from 2 ml of peripheral blood of a rabbit were co-cultured in one well of a 24-well plate coated with rat plasma vitronectin (Sigma). After four days in culture, the cells were washed two degree medium, Di-I-labeled Sca-1 ⁺ or TBM ^- cells derived cells, and unlabeled Sca-1 ^- or
Spreading adherent cells were counted according to the frequency of TBM ⁺ cell-derived cells.

To determine the cell type of spindle cells attached in the above assay, identical cells were measured by asLDL-DiI uptake and BS-1 lectin reactivity. Double positive cells were counted as EPCs (96.2 ± 1.8% in mice, 95.5 ± 2.4% in rabbits)

4. Study setting for evaluation of post-ischemic circulating EPC kinetics Hind limb ischemia C57BL / 6J mice (n = 40) were sacrificed on days 0 (before surgery), 3, 7, and 14 after surgery (10 animals at each time point). Sham-operated mice were also sacrificed 7 days after surgery (n = 4). Peripheral blood mononuclear cells were prepared for Sca-1 ⁺ cell measurement, and the EPC enriched fraction was prepared by magnetic bead selection (n = 5) and EPC culture assay (n = 5).

[0125] TBM with magnetic beads selection and EPC culture assay ^- in preparation for the measurement of postoperative 0,
Peripheral blood mononuclear cells of hind limb ischemia New Zealand white rabbits were isolated on days 3, 7, and 14. Sham-operated rabbits were examined in the same manner 7 days after surgery (n = 4).

To evaluate the effect of ischemia-induced circulating EPCs on neovascularization, corneal neovascularization assays (Kenyon, BM et al., Invest Ophthalmol Vis Sci, 1996 and Asahara, T.
Et al., Circ Res, 1998) in hind limb ischemic mice. Three days after ischemic surgery or sham surgery, C57BL / 6J mice (n = 5 each) underwent corneal assay microsurgery, including neovascular length 6 days after corneal surgery (9 days after ischemia). And blood vessel circumference measurements. In situ BS-1 lectin staining was performed prior to sacrifice.

5. Study Settings for Effects of GM-CSF on Circulating EPC Kinetics and Neovascularization These experiments demonstrate the effects of GM-CSF on EPC kinetics and subsequent angiogenesis on neovascularization. Intended to show contribution.

A. Rabbit model hind limb ischemic animals were divided into two groups. GM consisting of recombinant human GM-CSF (70 μg / day) injected subcutaneously daily from 7 days before surgery to 1 week after administration to 8 rabbits
-CSF treatment (GM-CSF group). An ischemic control group (control group) consisting of eight rabbits given saline subcutaneously every day for one week before surgery.

The day immediately before the first injection ([−] day 7), the day of ischemic surgery (day 0), and 3,
Rabbits were examined 7, 14 days later (days 3, 7, 14) and at each time point, peripheral blood was isolated from the central ear artery. At each time point, 5 ml of blood was isolated for cell counting and cell culture assays. In all animals of each group, the blood pressure ratio between ischemia and normal limbs was measured, and similarly, on day 14 (at the time of sacrifice), the capillary density of the ischemic muscle was measured (
See below).

B. Mouse model [-] Recombinant murine GM-CSF (0.
5 μg / day) or control saline was injected intraperitoneally, and on day 0, C57BL / 6J mice (n = 5 each) were subjected to corneal micropocket surgery and the resulting length and circle of intervascular structures The circumference was measured on day 6. In situ BS-1 lectin staining was performed prior to sacrifice.

6. BMT was performed on FVB / N mice from a recombinant mouse expressing by construction of beta-galactosidase encoded by LacZ under the transcriptional control of Tie-2, a murine bone marrow transplantation model EC-specific promoter (Schlaeger, TM et al., Development, 1995). By reconstructing the transplanted BM, La
We generated Tie-2 / LZ / BMT mice in which cZ expression was restricted to Tie-2-expressing BM-derived cells and LacZ expression was not observed in other somatic cells. Then, three days before ischemia or sham surgery, or one day after completing the 7-day treatment with GM-CSF or control vehicle, Tie-2 / LZ
Corneal assay microsurgery (Kenyon, BM et al., Invest Ophthalmol Vi
s Sci, 1996 and Asahara, T. et al., Circ. Res. 1998).

[0132] The donor Tie-2 recombinant mouse (FVB / N-TgN [TIE2LacZ] 182Sa) matching the age (4 weeks)
To, Jackson Lab), the tibia and femur were washed away to obtain BM cells. Low density BM mononuclear cells were isolated by density centrifugation on Histopark-1083 (Sigma). FVB / N mice (12.0 Gy) irradiated with a lethal dose and perfused with approximately 2 × 10 ⁶ donor BM mononuclear cells each (
Jackson Lab). Four weeks after BMT, when the BM of the recipient mouse has been reconstructed, the mouse undergoes surgery or sham surgery to produce hind limb ischemia (see below) and 3 days later, assays for corneal neovascularization Microsurgery was performed for the procedure. Similarly, 4 weeks after BMT, GM-CSF or a control vehicle was administered for 7 days, and GM-C
Surgery for corneal neovascularization assay was performed one day after completion of pretreatment with SF or control. Six days after corneal microsurgery, corneas from BMT animals were harvested for light microscopic demonstration of beta-galactosidase expression and for chemical detection of beta-galactosidase activity.

7. Detection of beta- galactosidase expression in corneal tissue In order to histologically detect beta-galactosidase-expressing cells, the entire mouse eyes were excised and fixed in 4% paraformaldehyde at 4 ° C. for 2 hours. X-gal overnight at ℃
The solution was incubated. The specimen is then placed in PBS and the corneal hemisphere (kemispher
ed cornea) were excised by dissecting microscopy and embedded for histological processing. Tissue specimens were counterstained with light hematoxylin- and -eosin and examined by light microscopy to visually count the number of X-gal positive cells per section. Three sections of each tissue were examined and averaged for evaluation of X-gal stained cell frequency.

For chemical detection of beta-galactosidase activity, excised eyes were placed in liquid nitrogen and stored at -80 ° C. Chemiluminescence reporter gene assay system (Chemilumi
nescence Reporter Gene Asay System), Galactolite PlusTM (Galacto-Li)
ght Plus ™) (Tropix Inc., Bedford MA) according to a modified protocol.
The assay was performed. Briefly, the eyes are placed in 1 ml of supplemented lysis buffer and 0.5 mM DTT is added, followed by Tissuemizer Mark II (Tekmar Co., Cincinatti, O.D.).
H). The homogenized lysis solution was centrifuged to remove debris. Using an aliquot of the supernatant of the homogenized lysis buffer, use the BCA Protein Assay Kit (PIE
RCE, Rockford, IN). This supernatant is ion-exchange resin Che
After treatment with lex100, quantification was performed and beta-galactosidase activity was measured with a chemiluminometer (Lumat LB9501, Berthold, Nashua, NH). Beta-galactosidase was normalized by protein concentration.

8. Mouse hind limb ischemia model The same age (8 weeks) C57BL / 6J male mouse (Jackson Lab, Bar Harbor, ME) was used.
A mouse hind limb ischemia model was created (Couffinhal, T. et al., Am. J. Pathol, 1998).
All animals were injected intraperitoneally with pentobarbital (160 mg) for subsequent surgical procedures.
/ kg). The skin was excised in the middle of the left hind limb lying on the femoral artery. The femoral artery was then gradually isolated and the base of the femoral artery was ligated with a 3-0 silk ligature. The distal portion of the saphenous artery was ligated and the other arterial branch vein was dissected and resected. The overlying skin was closed with two surgical staples. After surgery, the mice were placed on a heating plate at 37 ° C and the animals were monitored with special care until fully awake from anesthesia.

9. Rabbit hind limb ischemia model The above rabbit hind limb ischemia model was used (Takeshita, S. et al., J. Clin. Invest. 1
994). After premedication with dirazine, ketamine (50 mg / kg) and acepromazine (0.8
mg / kg) mixture and a total of 20 New Zealand white rabbits (3.8-7.2 kg) (Pi
ne Acre Rabbitry, Norton, MA). A longitudinal incision was then made from below to the inguinal ligament to a point just adjacent to the patella. The limbs at which the surgeon made random incisions at the time of surgery were determined. At this incision using a surgical loupe, the femoral artery is dissected and separated over the entire length, and all femoral artery branches, including the inferior abdominal wall artery, the deep femoral artery, the lateral femoral circumflex artery, and the superficial abdominal wall artery, are also dissected. Released. After dissecting and releasing the popliteal and saphenous arteries, the external iliac artery and all of the above arteries were ligated with 4.0 silk ligatures (Ethicon, Sommervill).
e, NJ). Finally, the femoral artery was completely dissected from the origin of the base as a branch of the external iliac artery to the terminal point where it diverged to form the saphenous and external iliac arteries. After incision of the femoral artery, retrograde propagation of the thrombus caused occlusion of the external iliac artery. Blood flow was consequently dependent on collateral draining from the external iliac artery.

10. Mouse Corneal Neovascularization Assay Mouse corneal neovascularization was evaluated using the same age (8 weeks) of C57BL / 6J male mice (Jackson Lab). All animals were anesthetized with an intraperitoneal pentobarbital injection (160 mg / kg) for subsequent surgical procedures. Corneal micropockets were created in each mouse eye with a modified Banglafefe cataract knife. In each pocket,
Hydron polymer type NCC containing 150 ng of vascular endothelial growth factor (VEGF) (IFN Sci
ence, New Brunswick, NJ) were implanted with 0.34 x 0.34 mm aluminum sucrose sulfate (Buck Meditec, Denmark) pellets. Pellet is located 1.0 mm from the limbus of the cornea and is an erythromycin ophthalmic ointment (E. Foufera, Melville, NY
) Was added to each surgical eye. The corneas of all mice were examined daily by slit-lamp biomicroscopy 5 to 6 days after pellet implantation. Neovascularization vessel length and vessel circumference were measured 6 days after surgery and all corneas were photographed. After these measurements, 500 ng of Bandeiraea Simplicifolia lectin (BS-1) conjugated to the EC-specific marker FITC (Vector Lab, Burlingame, CA) was administered intravenously and sacrificed 30 minutes later. The eyes were removed and fixed in a 1% paraformaldehyde solution. After fixation, the cornea was mounted on a glass slide and examined by fluorescence microscopy.

11. Low limb blood pressure ratio These in vivo studies were performed on anesthetized rabbits. Blood pressure in both hind limbs was measured. In each case, the hind limbs were shaved and cleaned, the pulse of the posterior tibial artery was identified with a Doppler probe, and the systolic blood pressure of each limb was measured by standard methods.
The blood pressure ratio was determined in each rabbit as the ratio of the systolic blood pressure of the ischemic limb to the systolic blood pressure of the normal limb.

12. Capillary density The extent of neovascularization was examined by measuring the frequency of capillaries in light microscopic sections taken from normal and ischemic hind limbs. Tissue specimens were obtained at sacrifice as cross sections from muscles of both limbs. Muscle specimens were embedded in OCT compounds (Miles, Elkhart, Ind.) And snap frozen in liquid nitrogen. Thereafter, a plurality of frozen sections having a thickness of 5 μm were cut from each specimen so that the muscle fibers were in the lateral direction. Tissue sections were stained for basic phosphatase using the indoxyltetrazolium method, capillary ECs were detected as described above, and counterstained with eosin. Capillaries were counted at 20 × magnification to determine capillary density (average number of capillaries per mm ² ). Randomly, 10 different regions were selected for capillary counting. The calculation method used to calculate the capillary / muscle fiber ratio is otherwise identical to the method used for calculating the capillary. Prokop, D.
J. (1997) Science, 276: 71; Perkins, S. and Fleischman, RA (1998) J.
Clinical Invest. 81: 1072; Perkins, S. and Fleischman, RA (1990) Blo.
See od 75: 620.

13. Statistical Analysis All results are expressed as mean ± standard error. Statistical significance was assessed by unpaired Student's T-test for comparison of the two means. ANOV for multiple comparisons of 3 groups or more
I went in A. A value of p <0.05 was interpreted as indicating a statistically significant difference.

The following references are specifically incorporated herein.

[Brief description of the drawings]

1A-D show neovascularization after GM-CSF and VEGF-1 treatment in controls (FIGS. 1A, 1C) and treated mice (FIGS. 1B, 1D) in a corneal micropocket assay. A micrograph is shown.

FIGS. 2A-B are graphs showing quantification of the increase in vessel length (2A) and vessel angle (2B) observed in the corneal micropocket assay.

3A-C are graphs showing EPC frequency (3A), EPC differentiation (3B), blood pressure and capillary density (3C) after GM-CSF treatment in rabbit hind limb ischemia assay. .

4A-4J show photomicrographs showing that EPCs can be induced (returned) and taken up into neovascular lesions. (4A) cultured murine cells, (4B-D) induction (homing) of Sca-1 cells administered to the mice, (4E-G) immunostaining of rabbit hind limb muscles showing EPC accumulation and colonization, (4H- J) Colonized TBM ^- cells that establish new blood vessels.

FIGS. 5A-B are graphs showing EPC kinetics associated with the development of hind limb ischemia.

FIGS. 5C to 5F show micrographs showing the results of a mouse corneal micropocket assay with hind limb ischemia.

FIGS. 5G-H are graphs showing quantification of the distribution of vessel length and circumference for neovascularization.

6A-C show G to neovascularization in a rabbit ischemic hind limb model.
It is a graph which shows the influence of M-CSF induced EPC.

6D-G show photomicrographs showing the GM-CSF-induced effects described in FIGS. 6A-C. (6D, E) Slit-lamp biomicroscopy, (6F, G) Fluorescence microscopy.

6H-I are graphs showing measurements of vessel length (6H) and vessel circumference (6I) taken from the experiments shown in FIGS. 6D-G.

7A-C are graphs showing that detectable labeled bone marrow-derived EPCs contribute to corneal neovascularization. (7A) Corneal neovascularization in mice with hind limb ischemia, (7B) GM-CSF pretreated rabbits, (7C) GM-CSF control group beta gactosidase activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) C12Q 1/02 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, G , GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU , ZWF term (reference) 4B063 QA01 QA19 QQ02 QQ03 QQ08 QQ35 QR77 QS24 QX01 4C084 AA02 AA03 AA17 AA20 DA19 DA27 DB27 DB54 DB55 DB56 DB58 DB62 DC01 MA02 NA14 ZA362 ZA812 ZB212

Claims (48)

[Claims] 1. A method for inducing new blood vessel formation in a mammal, comprising administering to the mammal an effective amount of an angiogenesis regulator sufficient to form new blood vessels in the mammal. The vascularization regulator is GM-CSF, M-CSF, b-FGF, SCF, SDF-1, G-
2. The method according to claim 1, which is CSF, HGF, Angiopoietin-1, Angiopoietin-2, FTL-3 ligand, or an effective fragment thereof. 2. The method of claim 1, wherein the angiogenesis regulator is GM-CSF and the dose of GM-CSF to the mammal is sufficient to increase the frequency of endothelial progenitor cells (EPC) in the mammal. The described method. 3. The method of claim 2, wherein the increase in EPC frequency is at least about 20% as measured by a standard EPC isolation assay. 4. The amount of an angiogenesis regulator to be administered to a mammal is
2. The method of claim 1, wherein the method is sufficient to increase EPC differentiation. 5. The method of claim 4, wherein the increase in EPC differentiation as measured by a standard EPC culture assay is at least about 20%. 6. The method of claim 1, wherein the dose of the angiogenesis regulator to the mammal is sufficient to increase blood vessel length in the mammal. 7. The method of claim 6, wherein the increase in vessel length is at least about 5% as measured by a standard vessel length assay. 8. The method of claim 6, wherein the amount of the angiogenesis regulator 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 vessel diameter is at least about 5% as measured by a standard vessel diameter assay. 10. The method according to claim 10, wherein the amount of the angiogenesis regulator administered to the mammal is EP after tissue ischemia.
2. The method of claim 1, wherein the method is sufficient to increase C differentiation. 11. The method of claim 10, wherein the increase in EPC differentiation is at least about 20% as measured by a standard hind limb ischemia assay. 12. The method of claim 1, wherein the dosage of the angiogenesis modulating agent is sufficient to increase neovascularization by at least about 5% as measured by a standard corneal micropocket assay. 13. The method of claim 1, wherein the dosage of the angiogenesis regulator is sufficient to increase the uptake of EPC bone marrow-derived EPC into the lesion. 14. The method of claim 13, wherein the increase in EPC bone marrow-derived EPC in the lesion is at least about 20% as measured in a standard rodent bone marrow (BM) transplantation model. 15. The method of claim 1, wherein the mammal has, is suspected of having, or is believed to have, ischemic tissue. 16. The method of claim 15, wherein the ischemic tissue is associated with ischemic vascular disease. 17. The ischemic tissue comprises tissue from a limb, graft, or organ.
The method of claim 15. 18. The method of claim 15, wherein the tissue is associated with a circulatory system or a central nervous system.
The described method. 19. The method of claim 15, wherein said tissue is heart or brain tissue. 20. The method of claim 1, wherein the method is co-administered with at least one vascular-derived protein. 21. The method of claim 20, wherein the vascular-derived protein is an endothelial cell mitogen. 22. The blood vessel-derived protein is an acidic fibroblast growth factor (aFGF).
Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF-1), epidermal growth factor (EGF), transforming growth factors α and β (TGF-α and TGF-β), platelet-derived endothelial proliferation Factor (PD-ECGF), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), 21. The method according to claim 20, which is macrophage-CSF (M-CSF), angiopoietin-1 (Ang1) or NO synthase (NOS), or a fragment thereof. 23. The method of claim 22, wherein the protein is VEGF-B, VEGF-C, VEGF-2, VEGF-3, or an effective fragment thereof. 24. An amount of GM-CSF, comprising: administering an effective amount of granulocyte macrophage colony stimulating factor (GM-CSF) to the mammal; and exposing the mammal to a condition that promotes vascular damage. A method of preventing or reducing severe vascular damage in a mammal, wherein the is sufficient to prevent or reduce severe vascular damage in the mammal. 25. The condition that promotes vascular injury is invasive manipulation or ischemia.
25. The method according to claim 24. 26. The method of claim 25, wherein the invasive operation is a surgery. 27. The ischemia is associated with at least one of infection, trauma, graft rejection, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy or myocardial ischemia. 26. The method of claim 25, wherein 28. The method of claim 24, wherein the GM-CSF is administered to the mammal at least about 12 hours prior to exposing the mammal to a condition that promotes vascular injury. 29. Approximately one day to one hour of exposing the mammal to conditions that promote vascular injury
29. The method of claim 28, wherein GM-CSF is administered to the mammal during the day before. 30. The method of claim 28, further comprising administering GM-CSF to the mammal after exposure to a condition that promotes vascular injury. 31. A method of treating ischemic tissue in a mammal in need of treatment comprising the steps of: a) isolating endothelial progenitor cells (EPC) from the mammal, b) isolating the isolated EPC Contacting an amount of a vascular-derived protein sufficient to induce proliferation of the EPC, and c) administering to the mammal an amount of the expanded EPC sufficient to treat the ischemic tissue. 32. The method of claim 31, wherein the EPC has at least one of the following markers: CD34 ⁺ , flk-1 ⁺ , or tie-2 ⁺ . 33. The method of claim 31, wherein the ischemic tissue has damaged blood vessels. 34. The method of claim 33, wherein the blood vessel is damaged by invasive manipulation. 35. The method of claim 34, wherein the invasive manipulation is balloon angioplasty, or placement of a stent or catheter. 36. The method of claim 35, wherein the stent is an intravascular stent. 37. The method of claim 31, further comprising co-administering at least one vascular derived protein. 38. The method of claim 37, wherein the vascular-derived protein is an endothelial cell mitogen or a nucleic acid encoding an endothelial cell mitogen. 39. The blood vessel-derived protein is an acidic fibroblast growth factor (aFGF).
Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF-1), epidermal growth factor (EGF), transforming growth factors α and β (TGF-α and TGF-β), platelet-derived endothelial proliferation Factor (PD-ECGF), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), 39. The method of claim 38, wherein the method is macrophage-CSF (M-CSF), angiopoietin-1 (Ang1) or NO synthase (NOS), or a fragment thereof. 40. The method of claim 39, wherein the protein is VEGF-B, VEGF-C, VEGF-2, VEGF-3, or an effective fragment thereof. 41. contacting the mammal with a detectably labeled population of endothelial progenitor cells (EPC) and detecting the labeled cells at or near the site of tissue damage in the mammal. A method for detecting the presence of tissue damage in a mammal. 42. The method of claim 41, wherein the tissue damage is ischemia or ischemic vascular disease. 43. A pharmaceutical product for inducing neovascularization in a mammal, comprising isolated endothelial progenitor cells (EPC) and formulated to be physiologically acceptable to the mammal. 44. The pharmaceutical product of claim 43, wherein the pharmaceutical product is sterile and further comprises at least one vascular derived protein or a nucleic acid encoding said protein. 45. An isolated EPC and optionally at least one of a vascular derived protein or a nucleic acid encoding said protein, and optionally a pharmaceutically acceptable carrier solution, nucleic acid or A kit for systemic introduction of isolated endothelial progenitor cells (EPC), further comprising a mitogen, a means for delivering the EPC, and instructions for using the kit. 46. The kit of claim 45, wherein the means for delivering the EPC is a stent, catheter, or syringe. 47. A method of enhancing EPC mobilization in a mammal, comprising administering an effective amount of at least one hematopoietic factor sufficient to promote endothelial progenitor cell (EPC) mobilization in the mammal. . 48. Co-administering an effective amount of one or more of granulocyte macrophage colony stimulating factor (GM-CSF), at least one vascular derived protein, or an effective fragment thereof to a mammal. 50. The method of claim 47, further comprising.