WO1999045775A1 - Compositions et methodes modulant la vascularisation - Google Patents

Compositions et methodes modulant la vascularisation Download PDF

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Publication number
WO1999045775A1
WO1999045775A1 PCT/US1999/005130 US9905130W WO9945775A1 WO 1999045775 A1 WO1999045775 A1 WO 1999045775A1 US 9905130 W US9905130 W US 9905130W WO 9945775 A1 WO9945775 A1 WO 9945775A1
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Prior art keywords
mammal
csf
epc
growth factor
epcs
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PCT/US1999/005130
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English (en)
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Jeffrey M. Isner
Takayuki Asahara
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St. Elizabeth's Medical Center
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Priority to JP2000535201A priority Critical patent/JP2002506008A/ja
Priority to EP99912344A priority patent/EP1061800A4/fr
Priority to CA2322559A priority patent/CA2322559C/fr
Priority to AU30737/99A priority patent/AU766238B2/en
Publication of WO1999045775A1 publication Critical patent/WO1999045775A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to methods for modulating vascula ⁇ zation. particularly in a mammal
  • methods for modulating vascula ⁇ zation that includes administrating to the mammal an effective amount of a vascula ⁇ zation modulating agent, such as a granulocyte macrophage-colony stimulating factor (GM-CSF)
  • a vascula ⁇ zation modulating agent such as a granulocyte macrophage-colony stimulating factor (GM-CSF)
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • the invention has a wide spectrum of useful applications including inducing formation of new blood vessels m the mammal
  • Blood vessels help supply oxygen and nut ⁇ ents to living tissues Blood vessels also facilitate removal of waste products Blood vessels are renewed by a process termed "angiogenesis" See generally Folkman and Shing, J Bio! Chem 267 (16), 10931-10934 (1992) -2-
  • Angiogenesis is understood to be important for the well-being of most mammals As an illustration, angiogenesis has been disclosed as being an essential process for reproduction, development and wound repair
  • angiogenesis is believed to begin with the degradation of the basement membrane by proteases secreted from endothelial cells (EC) activated by mitogens, e g , vascular endothelial growth factor (le VEGF-1), basic fibroblast growth factor (bFGF) and/or others
  • EC endothelial cells
  • mitogens e g
  • VEGF-1 vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • Abnormal angiogenesis is thought to occur when the body loses its control of angiogenesis, resulting in either excessive or insufficient blood vessel growth For instance, conditions such as ulcers, strokes, and heart attacks may result from the absence of angiogenesis normally required for natural healing In contrast, excessive blood vessel proliferation can facilitate tumor growth, blindness, pso ⁇ asis, rheumatoid arth ⁇ tis. as well as other -3-
  • angiogenic growth factors such as fibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al , Science, 257 1401-1403 (1992) and Baffour, et al , J Vase Sutg, 16 181-91 (1992)), endothelial cell growth factor (ECGF)(Pu, et al , J Surg Res, 54 575-83 (1993)), and vascular endothelial growth factor (VEGF-1) to expedite and or augment collateral artery development in animal models of myocardial and hindhmb ischemia (Takeshita, et al , Circulation, 90 228-234 (1994) and Takeshita. et al J
  • FGF fibroblast growth factor
  • angiogenesis can facilitate treatment of ischemia in a rabbit model and in human clinical t ⁇ als
  • VEGF-1 administered as a balloon gene delivery system
  • Successful transfer and sustained expression of the VEGF-1 gene m the vessel wall subsequently augmented neovascula ⁇ zation in the ischemic limb (Takeshita, et al , Laboratory Investigation, 75 487-502 (1996).
  • EPC endothelial progenitor cell
  • Granulocyte macrophage colony stimulating factor has been shown to exert a regulatory effect on granulocyte-committed progenitor cells to increase circulating granulocyte levels (Gasson, J C , Blood 77 1 131 (1991)
  • GM-CSF acts as a growth factor for granulocyte, monocyte and eosinophil progenitors
  • GM-CSF is believed to be particularly useful in accelerating recovery from neutropenia m patients subjected to radiation or chemotherapy, or following bone marrow transplantation
  • GM-CSF is less potent than other cytokmes, e g , FGF, in promoting EC proliferation
  • GM-CSF activates a fully migrating phenotype (Bussohno, et al , J Chn Invent , 87 986 (1991)
  • the present invention generally relates to methods for modulating vascula ⁇ zation in a mammal
  • the invention provides methods for increasing vascula ⁇ zation that includes administrating to the mammal an effective amount of a vascula ⁇ zation modulating agent, such as granulocyte macrophage-colony stimulating factor (GM-CSF), VEGF. Steel factor (SLF, -5-
  • SCF Stem cell factor
  • SDF-1 stromal cell-de ⁇ ved factor
  • G-CSF granulocyte-colony stimulating factor
  • HGF Angiopoietin-1
  • Angiopoietin-2 Angiopoietin-2
  • M-CSF, b-FGF, and FLT-3 ligand and effective fragment thereof, or DNA coding for such vascularization modulating agents.
  • Such materials have sometimes previously been described as “hematopoietic factors.” and/or "hematopoietic proteins.” Disclosure relating to these and other hematopoietic factors can be found in Kim, CH. and Broxmeyer, H.E. (1998) Blood, 91: 100; Turner, M.L. and Sweetenham, J.W., Br.
  • the DNA coding for the vascularization modulating agents can be administered to the site where neovascularization is desired, as further discussed below.
  • the invention also relates to methods for treating or detecting damaged blood vessels in the mammal.
  • the invention has many uses including preventing or reducing the severity of blood vessel damage associated with ischemia or related conditions.
  • hematopoietic factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), modulate endothelial progenitor cell (EPC) mobilization and neovascularization (blood vessel formation).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • EPC endothelial progenitor cell
  • the present invention provides a method for inducing neovascularization in a mammal.
  • induction is meant at least enhancing EPC mobilization and also preferably facilitating formation of new blood vessels in the mammal.
  • EPC mobilization is understood to mean a 75
  • the method includes admimste ⁇ ng to the mammal an effective amount of a vascula ⁇ zation modulating factor such as granulocyte macrophage-colony stimulating factor (GM-CSF), that is preferably sufficient to induce the neovascula ⁇ zation in the mammal
  • a vascula ⁇ zation modulating factor such as granulocyte macrophage-colony stimulating factor (GM-CSF)
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • that amount of GM-CSF is also capable of modulating and particularly increasing frequency of EPCs in the mammal
  • the enhancement in EPC mobilization and particularly the increase in frequency of the EPCs is at least about 20% and preferably from between 50% to 500%) as determined by a standard EPC isolation assay That assay generally detects and quantifies EPC en ⁇ chment and is desc ⁇ bed in detail below
  • the amount of administered modulating agent is sufficient to enhance EPC mobilization and especially to increase EPC differentiation in the mammal
  • Methods for detecting and quantifying EPC differentiation include those specific methods desc ⁇ bed below
  • the increase in EPC differentiation is at least about 20%), preferably between from about 100% to 1000%, more preferably between from about 200%) to 800%> as determined by a standard EPC culture assay discussed below
  • that amount of administered modulating agent is additionally sufficient to increase EPC differentiation by about the stated percent amounts following tissue ischemia as determined in a standard hindhmb ischemia assay as discussed below
  • the amount of vascula ⁇ zation modulating agent administered to the mammal is sufficient to increase blood vessel size m the mammal
  • the amount of administered modulating agent is sufficient to increase blood vessel length by at least about 5%, more preterablv between from about 10% to 50%, even more preferably about 20%, as determined by a standard blood vessel length assay discussed below
  • the amount of modulating agent administered to the mammal is also sufficient to increase blood vessel circumference or diameter by the stated percent amounts as determined by a standard blood vessel diameter assay.
  • the amount of administered vascula ⁇ zation modulating agent is sufficient to increase neovascularization by at least about 5%, preferably from between about 50%o to 300%), and more preferably from between about 100% to 200%> as determined by the standard cornea micropocket assay Methods for performing that assay are known in the field and include those specific methods desc ⁇ bed below Additionally, prefe ⁇ ed amounts of GM-CSF are sufficient to improve ischemic hindlimb blood pressure by at least about 5%, preferably between from about 10%o to 50% as determined by standard methods for measu ⁇ ng the blood pressure of desired vessels More specific methods for measu ⁇ ng blood pressure particularly with new or damaged vessels include techniques optimized to quantify vessel pressure m the mouse hindlimb assay discussed below
  • the amount of administered vascula ⁇ zation modulating agent is sufficient to increase EPC bone marrow (BM) de ⁇ ved EPC incorporation into foci by at least about 20% as determined by a standard mu ⁇ ne BM transplantation model
  • the increase is between from about 50% to 400%, more preferably between from about 100%) to 300% as determined by that standard model More specific methods for determining the increase in EPC incorporation into foci are found in the discussion and Examples which follow
  • the methods of this invention are suitable for modulating and especially inducing neovascularization in a va ⁇ ety of animals including mammals
  • mammals including mammals
  • the term "mammal” is used herein to refer to a warm blooded animal such as a rodent, rabbit, or a primate and especially a human patient
  • Specific rodents and p ⁇ mates of interest include those animals representing accepted models of human disease including the mouse, rat, rabbit, and monkey
  • Particular human patients of interest include those which have, are suspected of having, or will include ischemic tissue That ischemic tissue can a ⁇ se by nearly any means including a surgical manipulation or a medical condition Ischemic tissue is often associated with an ischemic vascular disease such as those specific conditions and diseases discussed below
  • the invention includes methods for modulating and particularly inducing neovascularization in a mammal in which an effective amount of vascularization modulating agent is co-administered with an amount of at least one angiogenic protein
  • an effective amount of vascularization modulating agent is co-administered with an amount of at least one angiogenic protein
  • co-administration of the vascula ⁇ zation modulating agent and the angiogenic protein can positively impact neovascula ⁇ zation in the mammal, e g , by providing additive or synergistic effects
  • a preferred angiogenic protein is a recognized endothelial cell mitogen such as those specific proteins discussed below Methods for co-admimste ⁇ ng the vascula ⁇ zation modulating agent and the angiogenic protein are desc ⁇ bed below and will generally vary according to intended use
  • the present invention also provides methods for preventing or reducing the seventy of blood vessel damage in a mammal such as a human patient in need of such treatment
  • the method includes admimstenng to the mammal an effective amount of vascularization modulating agent such as GM-CSF
  • vascularization modulating agent such as GM-CSF
  • administration of the vascula ⁇ zation modulating agent can occur after exposure to the conditions to reduce or block damage to the blood vessels
  • many conditions are known to induce ischemic tissue in mammals which conditions can be particularly conducive to damaging blood vessels, e g, invasive manipulations -9-
  • vascularization modulating agent such as surgery, grafting, or angioplasty, infection or ischemia Additional conditions and methods for administering the vascularization modulating agent are discussed below
  • vascula ⁇ zation modulating agent to use m the methods are sufficient to prevent or reduce the seventy of the blood vessel damage in the mammal
  • Particular amounts of GM-CSF have already been mentioned above and include administration of an effective amount of GM- CSF sufficient to induce neovascula ⁇ zation in the mammal
  • Illustrative methods for quantifying an effective amount of vascularization modulating agents are discussed throughout this disclosure including the discussion and Examples which follow
  • the present invention also provides methods for treating ischemic tissue and especially injured blood vessels in that tissue
  • the method is conducted with a mammal and especially a human patient in need of such treatment
  • the method includes as least one and preferably all of the following steps
  • EPCs endothelial progenitor cells
  • the factor is an angiogenic protein including those cytokines known to induce EPC proliferation especially in vitro Illustrative factors and markers for detecting EPCs are discussed below
  • the blood vessel (or more than one blood vessel) can be injured by nearly any known means including trauma or an invasive manipulation such as implementation of balloon angioplasty or deployment of a stent or catheter
  • a particular stent is an endovascular stent
  • the vascular injury can be organic and denved from a pre- existing or on-go g medical condition
  • vascula ⁇ zation _____ prepared for injection into a patient.
  • PCT/US99/05130 99/45775 prepared for injection into a patient.
  • modulating agent is administered to the mammal and especially the human patient alone or in combination (co-administered) with at least one angiogenic protein (or effective fragment thereof) such as those discussed below
  • the method includes contacting the mammal with a detectably- labeled population of EPCs, and detecting the detectably-labeled cells at or near the site of the tissue damage in the mammal
  • the EPCs can be harvested and optionally monitored or expanded in vitro by nearly any acceptable route including those specific methods discussed herein
  • the EPCs can be administered to the mammal by one or a combination of different approaches with intravenous injection being a preferred route for most applications.
  • Methods for detectably-labelmg cells are known m the field and include immunological or radioacti e tagging as well as specific recombinant methods disclosed below
  • the detectably-labeled EPCs can be used to "home-m" to a site of vascular damage, thereby providing a minimally invasive means of visualizing that site even when it is quite small
  • the detectably-labeled EPCs can be visualized by a va ⁇ ety of methods well- known in this field including those using tomography, magnetic resonance imaging, or related approaches
  • the tissue damage is facilitated by ischemia, particularly an ischemic vascular disease such as those specifically mentioned below
  • Also provided by this invention are methods for modulating the mobilization of EPCs which methods include admimste ⁇ ng to the mammal an effective amount of at least one hematopoietic factor Prefe ⁇ ed are methods that enhance EPC mobilization as determined by any suitable assay disclosed herein
  • the 0 enhancement m EPC mobilization and particulary the increase in frequency of the EPCs is at least about 20% and preferably from between 50% to 500% as determined by a standard EPC isolation assay 5775 _ u _
  • the amount of administered hematopoietic factor is sufficient to enhance EPC mobilization and especially to increase EPC differentiation in the mammal
  • Methods for detecting and quantifying EPC differentiation include those specific methods desc ⁇ bed below
  • the increase in EPC differentiation is at least about 20%, preferably between from about 100% to 1000%, more preferably between from about 200%> to 800% as determined by a standard EPC culture assay discussed below
  • that amount of administered hematopoietic factor is additionally sufficient to increase EPC differentiation by about the stated percent amounts following tissue ischemia as determined in a standard hindlimb ischemia assay as discussed below
  • EPC mobilization facilitates significant induction of neovascula ⁇ zation in mammals
  • methods that modulate EPC mobilization and particularly enhance same can be used to induce neovascula ⁇ zation in the mammal and especially a human patient in need of such treatment
  • Methods of this invention which facilitate EPC mobilization including those employing at least one hematopoietic factor which use can be alone or in combination with other methods disclosed herein including those in which an effective amount of vascula ⁇ zation modulating agent is administered to the mammal alone or m combination (co- administered) with at least one angiogenic protein
  • the invention provides methods for inducing neovascula ⁇ zation in a mammal and especially a human patient in need of such treatment which methods include administe ⁇ ng to the mammal an effective amount of at least one vascula ⁇ zation modulating agent, preferably one vasculanzation modulating agent, which amount is sufficient to induce neovascula ⁇ zation in the mammal That neovascula ⁇ zation can be detected and quantified if desired by the standard assays disclosed herein including the mouse cornea micropocket assay and blood vessel size assays Preferred methods will enhance neovascula ⁇ zation in the mammal by the stated percent ranges discussed previously
  • vascula ⁇ zation modulating agent (s) is co-administered m combination with at least one angiogenic protein, preferably one angiogenic protein
  • the vascula ⁇ zation modulating agent can be administered to the mammal and especially a human patient in need of such treatment in conjunction with, subsequent to, or following administration of the angiogenic or other protein
  • the invention also provides a pharmaceutical product that is preferably formulated to modulate and especially to induce neovascula ⁇ zation in a mammal
  • the product is provided ste ⁇ le and optionally includes an effective amount of GM-CSF and optionally at least one angiogenic protein
  • the product includes isolated endothelial progenitor cells (EPCs) in a formulation that is preferably physiologically acceptable to a mammal and particularly a human patient in need of the EPCs
  • the product can include a nucleic acid that encodes the GM-CSF and or the angiogenic protein
  • kits preferably formulated for in vivo and particularly systemic introduction of isolated EPCs
  • the kit includes isolated EPCs and optionally at least one angiogenic protein or nucleic acid encoding same
  • Prefe ⁇ ed is a kit that optionally includes a pharmacologically acceptable earner solution, nucleic acid or mitogen, means for dehve ⁇ ng the EPCs and directions for using the kit Acceptable means for dehve ⁇ ng the EPCs are known in the field and include effective delivery by stent, catheter, sy ⁇ nge or related means
  • Figures 1A-D are representations of photomicrographs showing neovascula ⁇ zation following GM-CSF and VEGF-1 treatment m control (Figs 1A, 1C) and treated (Figs IB and ID) mice in a cornea micropocket assay
  • Figures 2A-B are graphs showing quantitation of increases in vessel length (2A) and vessel angle (2B) observed in the cornea micropocket assay 5775
  • Figures 3A-C are graphs showing EPC frequency (3A), EPC differentiation (3B), blood pressure and capillary density (3C) following GM- CSF treatment in the rabbit hindlimb ischemia assay
  • Figures 4A-4J are representations of photomicrographs showing that EPCs can home and incorporate into foci of neovascula ⁇ zation (4A) cultured mu ⁇ ne cells, (4B-D) homing of Sea- 1 + cells administered to the mouse, (4E-G) lmmunostaining of rabbit hindlimb muscle showing accumulation and colonization of EPCs, (4H-J) colonized TBM cells establishing new vessels
  • Figures 5A-B are graphs showing EPC kinetics in relation to development ot hindlimb ischemia
  • Figures 5C-F are representations of photomicrographs showing results of the mouse cornea micropocket assay with hindlimb ischemia (5C-D) slit- lamp biomicroscopy, (5E-F) demonstration of neovascula ⁇ zation
  • Figures 5G-H are graphs illustrating quantitation of vessel length and circumferential distribution of neovasculanzation
  • Figures 6A-C are graphs showing effect of GM-CSF-induced EPC mobilization on neovascularization in the rabbit ischemic hindlimb model
  • Figures 6D-G are representations of photomicrographs showing the
  • Figures 6H and 61 are graphs showing measurements of vessel length (6H) and vessel circumference (61) taken from the expe ⁇ ment shown in
  • Figures 7A-C are graphs showing that detectably-labeled bone-marrow de ⁇ ved EPCs contribute to corneal neovasculanzation (7 A) corneal -14-
  • the present invention provides, in one aspect, methods for inducing neovasculanzation m a human patient that include administrating to the patient an effective amount of GM-CSF or an effective fragment thereof.
  • GM-CSF can be administered to the human patient alone or in combination (c-administered) with one or more of at least one vascularization modulating agent, preferably one of such factors; at least one angiogenic protein, preferably one angiogenic protein, or an effective fragment thereof
  • at least one vascularization modulating agent preferably one of such factors
  • at least one angiogenic protein preferably one angiogenic protein, or an effective fragment thereof
  • methods for enhancing EPC mobilization which methods include administration of an effective amount of at least one vascula ⁇ zation modulating agent, preferably one of such factors
  • methods for treating or detecting damaged blood vessels in the human patient The invention has a wide spectrum of uses including preventing or reducing the seventy of blood vessel damage in the patient.
  • the invention particularly provides methods for inducing angiogenesis in ischemic tissue of a patient in need such treatment.
  • the methods generally include admmiste ⁇ ng to the patient an effective amount of GM-CSF or other vascula ⁇ zation modulating agent disclosed herein
  • Administration of the GM-CSF (or co-adminstration with other another protein or proteins) can be as needed and may be implemented p ⁇ or to. dunng or after formation of the ischemic tissue.
  • the GM-CSF can be administered as the sole active compound or it can be co-admmistered with at least one and preferably one angiogenic protein or other suitable protein or fragment as provided herein.
  • GM-CSF or other vascula ⁇ zation modulating agent disclosed herein in accord with any of the methods disclosed herein can be implemented by one or a combination of different strategies including admimstenng a DNA encoding same
  • methods of this invention ha e a wide spectrum of uses especially in a human patient, e g , use in the prevention or treatment of at least one of trauma, graft rejection, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, ischemia related to infection, limb ischemia, ischemic cardiomyopathy. cerebrovascular ischemia, and myocardial ischemia
  • Impacted tissue can be associated with nearly any physiological system m the patient including the circulatory system or the central nervous system, e g , a limb, graft (e g , muscle or nerve graft), or organ (e g , heart, brain, kidney and lung)
  • the ischemia may especially adversely impact heart or brain tissue as often occurs in cardiovascular disease or stroke, respectively
  • the vascularization modulating agent will preferably be administered at least about 12 hours, preferably between from about 24 hours to 1 week up to about 10 days p ⁇ or to exposure to conditions conducive to damaging blood vessels
  • the method can further include admmiste ⁇ ng the ascula ⁇ zation modulating agent to the mammal following exposure to the conditions conducive to damaging the blood vessels
  • the v asculanzation modulating agent can be administered alone or in combination with at least one angiogenic protein preferably one of such proteins
  • vascular condition preventing or reducing the seventy of the vascular condition
  • methods include administenng alone or m combination (co-administration) with the GM-CSF one or more of at least one hematopoietic factor, preferably one of such factors, or at least one angiogenic protein, preferably one of such proteins
  • methods of administration include administenng alone or m combination (co-administration) with the GM-CSF one or more of at least one hematopoietic factor, preferably one of such factors, or at least one angiogenic protein, preferably one of such proteins
  • Vessel injury is known to be facilitated by one or a combination of different tissue insults
  • vessel injury often results from tissue trauma, surgery, e g , balloon angioplasty and use of related devices (e g , directional atherectomy, rotational atherectomy, laser angioplasty, translummal extraction, pulse spray thrombolysis), and deployment of an endovascular stent or a vascular graft
  • EPCs in accord with this invention will be preferably associated with cell markers that can be detected by conventional immunological or related strategies
  • EPCs having at least one of the following markers CD34 + , flk-l + or t ⁇ e-2 Methods for detecting EPCs with these markers are discussed in the Examples below
  • vascularization modulating agent to mobilize endothelial cell (EC) progenitors
  • EC endothelial cell
  • GM-CSF and other vascularization modulating agents can be used m a method for enhancing angiogenesis in a selected patient having an ischemic tissue i.e , a tissue having a deficiency blood as the result of an ischemic disease such as cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia
  • the vasculanzation modulating agent alone or in combination with at least one other factor disclosed herein can be used to induce reendothelialization of an injured blood vessel, and thus reduce restenosis by indirectly inhibiting smooth muscle cell proliferation
  • the vascularization modulating agent alone or in combination with at least one other factor disclosed herein can be used to prepare a patient for angiogenesis
  • Some patient populations typically elderly patients, may have either a limited number of ECs or a limited number of functional ECs
  • a potent angiogenesis promotor such as VEGF-1
  • vascula ⁇ zation can be limited by the lack of EPCs
  • administenng e g . GM-CSF at a time before administration of the angiogenesis promoter sufficient to allow mobilization of the ECs.
  • GM-CSF is administered about one week p ⁇ or to treatment with the angiogenesis promoter
  • GM-CSF as used herein shall be understood to refer to a natural or recombinantly prepared protein having substantial identity to an amino acid sequence of human GM-CSF as disclosed, for example, in published international application WO 86/00639, which is incorporated herein by reference Recombinant human GM-CSF is hereinafter also referred to as "hGM-CSF"
  • hGM-CSF Human GM-CSF
  • E coh de ⁇ ved, non-glycosylated rhGM-CSF can be obtained by the methods desc ⁇ bed in publication of the International Application No PCT/EP 85/00326, wherein two native GM-CSFs differing in a single ammo acid are desc ⁇ bed
  • the natural GM-CSF proteins used in the invention may be modified by changing the amino acid sequence thereof For example, from 1 to 5 amino acids in their sequences may be changed, or their sequences may be lengthened, without changing the fundamental character thereof and provide modified proteins which are the full functional equivalents of the native proteins Such functional equivalents may also be used in practicing the present invention
  • a GM-CSF diffe ⁇ ng by a single ammo acid from the common native sequence is disclosed in U S Pat No 5,229,496 and has been produced in glycosylated form in yeast, and has been clinically demonstrated to be a biological equivalent of native GM-CSF, such modified form known as GM-CSF (Leu-23)
  • GM-CSF is commercially and clinically available as an analog polypeptide (Leu "J ) under the trademark LEUKINE® (Immunex Corporation)
  • LEUKINE® International trademark of LuUKINE®
  • the genenc name for recombinant human Leu " GM-CSF analog protein expressed in yeast is Sargramostim Cloning and expression of native sequence human GM-CSF was desc ⁇ bed m Cantrell et al , Proc Natl Acad Sci USA 82 6250(1985)
  • the natural or recombinantly prepared proteins, and their functional equivalents used in the method of the invention are preferably punfied and substantially cell-free, which may be accomplished by known procedures
  • Genbank National Center for Biotechnology Information
  • sequence listings can be obtained from Genbank at the National Library of Medicine, 38A, 8N05, Rockville Pike. Bethesda.
  • MD 20894 Genbank is also available on the internet at http //www ncbi nlm nih gov See generally Benson, D A et al (1997) Nucl Acids Res 25 1 for a desc ⁇ ption of Genbank Protein and nucleic sequences not specifically referenced can be found in Genbank or other sources disclosed herein
  • GM-CSF can be administered to a mammal and particularly a human patient in need of such treatment
  • GM-CSF as well as therapeutic compositions including same are preferably administered parenterally More specific examples of parenteral administration include subcutaneous, intravenous, mtra- arte ⁇ al, intramuscular, and mtrape ⁇ toneal, with subcutaneous being prefe ⁇ ed
  • the GM-CSF will generally be formulated in a unit dosage mjectable form (solution, suspension, emulsion), preferably in a pharmaceutically acceptable earner medium that is inherently non-toxic and non-therapeutic
  • a pharmaceutically acceptable earner medium that is inherently non-toxic and non-therapeutic
  • examples of such vehicles include without limitation saline, Ringer's solution, dextrose solution, manmtol and normal serum albumin
  • Neutral buffered saline or salme mixed with serum albumin are exemplary approp ⁇ ate vehicles
  • Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used
  • Additional additives include substances to enhance isotonicity and chemical stability, e g , buffers, preservatives and surfactants, such as Polysorbate 80
  • the preparation of parenterally acceptable protein solutions of proper pH. isotonicity, stability, etc is within the skill of the art
  • the product is formulated by known procedures as a lyophilizate using approp ⁇ ate excipient solutions (e g , sucrose) as a diluent
  • vasculanzation modulating agents are from about 1 ⁇ g/ka'day to about lOO ⁇ g/kg/day
  • Use of more specific dosages will be guided by parameters well-known to those in this field such as the specific condition to be treated and the general health of the subject See also U S Patent No 5,578,301 for additional methods of administering GM-CSF Prefe ⁇ ed in vivo dosages for the hematopoietic proteins and angiogenic proteins disclosed herein will be within the same or similar range as for GM- CSF
  • At least one angiogenic protein and preferably one of same will be administered to the patient in conjunction with, subsequent to, or p ⁇ or to the administration of the GM-CSF
  • the angiogenic protein can be administered directly, e g , intra-arte ⁇ ally, intramuscularly, or intravenously, or nucleic acid encoding the mitogen may be used See, Baffour, et al , supra (bFGF), Pu, et al.
  • Circulation, 88 208-215 (1993) (aFGF), Yanagisawa-Miwa, et al . supra (bFGF), Fe ⁇ ara. et al , Bwchem Biophvs Res Commun , 161 851-855 (1989) (VEGF-1), (Takeshita. et al , Circulation, 90 228-234 (1994), Takeshita, et al , Laboratory, 75 487-502 (1996), Tsusumi, et al , Circulation, 94 (12) 3281- 3290 (1996))
  • At least one hematopoietic protein and preferably one of such proteins can be administered to the human patient in need of such treatment in conjunction with, subsequent to, or pnor to the administration of the GM-CSF
  • at least one angiogenic protein can also be co-admimstered with the GM-CSF and hematopoietic protein
  • Methods for administenng the hematopoietic protein will generally follow those discussed for admmstenng the GM-CSF although other modes of administration may be suitable for some purposes -20-
  • co-administration is meant to desc ⁇ be prefe ⁇ ed administration of at least two proteins disclosed herein to the mammal, le , administration of one protein in conjunction with, subsequent to, or prior to administration of the other protein
  • the nucleic acid encoding same can be administered to a blood vessel perfusing the ischemic tissue via a catheter, for example, a hydrogel catheter, as desc ⁇ bed by U S. Patent No 5,652,225, the disclosure of which is herein incorporated by reference
  • a catheter for example, a hydrogel catheter
  • the nucleic acid also can be delivered by injection directly into the ischemic tissue using the method desc ⁇ bed in PCT WO 97/14307
  • angiogenic protein or related term such as “angiogenesis protein” means any protein, polypeptide, mutein or portion that is capable of, directly or indirectly, inducing blood vessel growth.
  • proteins include, for example, acidic and basic fibroblast growth factors (aFGF and bFGF), vascular endothelial growth factor (VEGF-1), VEGF165, epidermal growth factor (EGF), transforming growth factor ⁇ and ⁇ (TGF- ⁇ and TFG- ⁇ ), platelet-de ⁇ ved endothelial growth factor (PD-ECGF), platelet- de ⁇ ved growth factor (PDGF), tumor necrosis factor ⁇ (TNF- ⁇ ), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF).
  • aFGF and bFGF acidic and basic fibroblast growth factors
  • VEGF-1 vascular endothelial growth factor
  • GM-CSF granulocyte/ macrophage CSF
  • Angl angiopoetin- 1
  • NOS nitric oxidesynthase
  • vascular endothelial growth factors include vascular endothelial growth factors
  • VEGF-1 vascular endothelial growth factors
  • lsoforms consists of monomers of 121 (U S Patent No 5,219,739), 145, 165 (U S Patent No 5,332,671), 189 (U S Patent No 5,240,848) and 206 amino acids, each capable of making an active homodimer (Houck, et al , Mol Endocnnol , 8, 1806-1814 (1991))
  • vascular endothelial growth factors include VEGF-B and VEGF-
  • the angiogenic protein contains a secretory signal sequence that facilitates secretion of the protein Proteins having native signal sequences, e g , VEGF-1, are prefe ⁇ ed Proteins that do not have native signal sequences, e g , bFGF, can be modified to contain such sequences using routine genetic manipulation techniques See, Nabel et al , Nature, 362 844 (1993)
  • hematopoietic factor or related term, e g , "hematopoietic protein” is used herein to denote recognized factors that increase mobilization of hematopoietic progenitor cells (HPC)
  • Preferred hematopoietic factors include granulocyte- macrophage colony-stimulating factor (GM-CSF), VEGF, Steel factor (SLF, also known as Stem cell factor (SCF) ), stromal cell-de ⁇ ved factor (SDF-1), granulocyte-colony stimulating factor (G-CSF), HGF, Ang ⁇ opo ⁇ et ⁇ n-1, Ang ⁇ opo ⁇ et ⁇ n-2, M-CSF, b-FGF, and FLT-3 hgand Disclosure relating to these and other hematopoietic factors can be found in Kim, C H and Broxmeyer, H E (1998) Blood, 91 100, Turner, M L and Sweetenham
  • nucleotide sequence of numerous angiogenic proteins are readily available through a number of computer databases, for example, GenBank. EMBL and Swiss-Prot Using this information, a DNA segment encoding the desired may be chemically synthesized or, alternatively, such a DNA segment may be obtained using routine procedures in the art, e g, PCR amplification
  • DNA encoding two proteins e g , VEGF-1 and bFGF
  • VEGF-1 and bFGF can be used, and provides an improvement over the use of bFGF alone
  • an angiogenic factor can be combined with other genes or their encoded gene products to enhance the activity of targeted cells, while simultaneously inducing angiogenesis, including, for example, mt ⁇ c oxide synthase, L-argmme, fibronectm, urokinase, plasminogen activator and hepann
  • the term "effective amount” means a sufficient amount of a compound, e g protein or nucleic acid delivered to produce an adequate level of the subject protein (e g . GM-CSF, vasculanzation modulating agent, hematopoietic protein, angiogenic protein) l e , levels capable of inducing endothelial cell growth and/or inducing angiogenesis as determined bv standard assays disclosed throughout this application
  • the important aspect is the level of protein expressed Accordingly, one can use multiple transc ⁇ pts or one can have the gene under the control of a promoter that will result in high levels of expression In an alternative embodiment, the gene would be under the control of a factor that results in extremely high levels of expression, e g , tat and the co ⁇ esponding tar element
  • the nucleic acid is preferably inserted into a cassette where it is operably linked to a promoter
  • the promoter must be capable of d ⁇ ving expression of the protein cells of the desired target tissue
  • the selection of approp ⁇ ate promoters can readily be accomplished Preferably, one would use a high expression promoter
  • An example of a suitable promoter is the 763- base-pair cytomegalovirus (CMV) promoter
  • a plasmid vector such as pUCl 18, pBR322, or other known plasmid vectors, that includes, for example, an E coh o ⁇ gin of replication See, Sambrook, et al , Molecular Cloning A Laboratorv Manual, Cold Spnng Harbor Laboratory press, (1989)
  • the plasmid vector may also include a selectable marker such as the ⁇ - lactamase gene tor ampicilhn resistance, provided that the marker polypeptide does not adversely effect the metabolism of the organism being treated
  • the cassette can also be bound to a nucleic acid binding moiety in a synthetic delivery system, such as
  • Particular methods of the present invention may be used to treat blood vessel injuries that result in denuding of the endothelial lining of the vessel wall
  • p ⁇ mary angioplasty is becoming widely used for the treatment of acute myocardial infarction
  • endovascular stents are becoming widely used as an adjunct to balloon angioplasty Stents are useful for rescuing a sub-optimal p ⁇ mary result as well as for diminishing restenosis
  • the liability of the endovascular prosthesis has been its susceptibility to thrombotic occlusion in approximately 3% of patients with artenes 3 3 mm or larger If patients undergo stent deployment in arte ⁇ es smaller than this the incidence of sub-acute thrombosis is even higher Sub- acute thrombosis is cu ⁇ ently prevented only by the aggressive use of anticoagulation
  • the combination of vascular intervention and intense anticoagulation creates significant nsks with regard to pe ⁇ pheral vascular trauma at the time of the stent/angi
  • the methods of the present invention may be used in conjunction a
  • endothelial cell mitogen means any protein, polypeptide, mutein or portion that is capable of inducing endothelial cell growth
  • proteins include, for example, vascular endothelial growth factor
  • VEGF- 1 acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (scatter factor), and colony stimulating -24-
  • CSF CSF factor
  • the methods of the present invention may be used to accelerate the healing of graft tissue, e g , vascular grafts, by potentiating vasculanzation
  • standard EPC culture assay or related term is meant an assay that includes at least one of and preferably all of the following steps a) isolating Sca-1+ and Sca-1- cells from the penpheral blood of mouse, or TBM+ and TBM- cells from the penpheral blood of a rabbit, and detectably-labelhng the cells (Sca-1+ and TBM- ), e g , with Di-I as provided herein, b) cultunng the cells in a suitable dish or plate in medium for several days and usually for about 4 days, c) counting any attached spreading cells in the dish or plate as being Di-I labeled Sca-1+ or TBM- or non-labeled Sca-1- or TBM+, d) and quantitating specific positive cells as being indicative of EPCs
  • standard blood vessel length assay or “standard blood vessel diameter assay” generally means exposing a blood vessel of interest in the subject mammal (e.g., mouse or rabbit) and measuring the length or diameter of that vessel by conventional means following inspection of that vessel.
  • Illustrative blood vessels such as certain arteries or veins which can be measured are provided below.
  • standard cornea micropocket assay or related term is used herein in particular reference to a mouse corneal neovascularization assay.
  • the assay generally involves one and preferably all of the following steps.
  • a pellet including an acceptable polymer and at least one angiogenic protein preferably VEGF-1
  • the assay can include a control as a reference which control will include performing steps a)-e) above, except that step b) will include adding a pellet without the angiogenic protein.
  • BM bone ma ⁇ ow
  • An illustrative detectable-label is beta-galactosidase enzyme activity. More specific information relating to the assay can be found in the discussion and Examples which follow.
  • an "effective fragment" of vascularization modulating agents such as GM-CSF, a hemopoietic protein, or angiogenic protein means an amino acid sequence that exhibits at least 70%>, preferably between from about 75% to 95%o of the vessel promoting activity of the corresponding full-length protein as determined by at least one standard assay as disclosed herein. Preferred are those assays which detect and preferably quantify EPC mobilization although other standard assays can be used.
  • a prefe ⁇ ed effective fragment of GM-CSF will have at least 70% and preferably from about 75% to 95%> of the vessel promoting activity of full- length human GM-CSF (see the published International Application No. PCT/EP/85/00376 (WO86/00639)) as determined in the standard corneal micropocket assay and especially the standard blood vessel length or diameter assays.
  • Circulating EPCs may constitute a reparative response to injury.
  • the hypothesis that cytokine-administration may mobilize EPCs and thereby augment therapeutic neovasculanzation was investigated as follows.
  • GM-CSF which induces proliferation and differentiation of hematopoietic progmtor cells (Socmski, et al., Lancet, 1988; 1.1194-1198, Gianni, et al., Lancet, 1989;2:580-584) and cells of myeloid lineage (Clark, et al., Science 1987;236:1229-1237, Sieff, C., J. Clin. Invest. 1987;79: 1549-
  • BM stroma cells As well as non-hematopoietic cells including BM stroma cells (Dedhar, et al., Proc. Natl. Acad. Sci USA 1988;85:9253-9257) and ECs (Bussohm, et al., J Clin. Invest., 1991;87:986-995), was used to promote cytokine-mduced EPC mobilization.
  • GM-CSF was administered for 7 days pnor to creating the stimulus for neovasculanzation. De novo vascular formation was initially examined m the mouse cornea pocket assay desc ⁇ bed above.
  • GM-GSF-pretreatment (mtrapentoneal [ ⁇ .p.] rmGM- CSF [R&D Systems] 500 ng/day) increased circulating EPCs (221% of untreated controls) at day 0, i.e., p ⁇ or to creation of the cornea micropocket and insertion of VEGF pellet; co ⁇ espondmgly, neovasculanzation at day 6
  • Morphometnc analysis of capillary density disclosed extensive neovascularization induced by GM-CSF pre-treatment compared to control (ischemia, no GM-CSF) group (249 vs 146/mm 2 , p ⁇ 0.01). GM-CSF pre-treatment also markedly improved ischemic limb/normal limb blood pressure ratio (0.71 vs 0.49, p ⁇ 0.01) ( Figures 3A-3C).
  • EPC isolation was assessed by EPC isolation from peripheral blood and EPC culture assay.
  • EPC-enriched fractions were isolated from mice as Sca-1 antigen-positive (Sca-T ) cells, and from rabbits as the cell population depleted of T-lymphocytes, B-lymphocytes and monocytes (TBM " ), denoted by the antigen repertoire CD5-/Ig ⁇ -/CD1 lb-.
  • the frequency of EPC-enriched population marked by Sca-1 in the circulation was 10J ⁇ 1.0% in C57/6JBL normal mice.
  • Co-cultures of Sca-1 and Sca-1 negative (Sca-1 " ) cells were examined after marking Sca-1 " cells with Dil fluorescence.
  • Sca-1 + cells developed a spindle- shaped morphology.
  • Mouse adherent cells in co-culture were found to be principally derived from Dil-marked Sca-1 + cells (65-84%) and showed evidence of EC lineage by reaction with BS-1 lectin and uptake of acLDL ( Figure 4A).
  • TBM EPC-enriched fraction
  • the frequency of TBM " EPC-enriched population in rabbit peripheral blood was 22 0-1 4 % Differentiation of EPCs was assayed by counting adherent cultured mononuclear blood cells Adherent cells in EPC culture were found again to be denved pnncipally from Dil-marked TBM " cells (71 ⁇ 92%o) and showed evidence of EC lineage by positive reaction with BS-1 lect and uptake of acLDL
  • TBM " cells were shown to differentiate into ECs in vivo by administration of autologous Dil-marked TBM " cells, isolated from 40 ml penpheral blood, to rabbits with unilateral hmdhmb ischemia (Takeshita. S., et d ⁇ J Clin Invest (1994) at 0, 3 and 7 days post-operatively Dil-labeled EPC- de ⁇ ved cells differentiated in situ into ECs, shown by co-stainmg for CD31 and incorporation into colonies, sprouts, and capillaries ( Figures 4E-4J)
  • FIGS. 4A-4D are more particularly explained as follows
  • the figures provide fluorescent microscopic evidence that EPCs derived from isolated populations of Sca-1 cells in mice, and TBM " cells in rabbit, can home and incorporate into foci of neovascula ⁇ zation.
  • FIG. 4A cultured mu ⁇ ne cells are shown, double-stained for acLDL-DiI (red) and BS-1 lectin (green) 4 days after EPC culture assay (Figures 4B-D) Sca-1 + cells administered to mouse with hindlimb ischemia have homed, differentiated and incorporated into foci of neovasculanzation in mouse ischemic hmdhmb muscles 2 wks after surgery
  • Figures 4B and 4C document that Dil-labelled Sca-1 + denved cells (red) co-localize with CD31 (green) indicdatmg that these EPCs have incorporated into CD31 -positive vascularture
  • a ⁇ ows indicate cells positive for Dil and CD31 (denved from delivered EPCs), while a ⁇ owheads indicate CD31 -positive, Dil-negative (autologous ECs)
  • Figure Id documents vascular foci of EPCs (a ⁇ ows) are within interstitial sites adjacent
  • EPC kinetics during severe tissue ischemia were assayed for frequency and differentiation
  • Figures 5B, 6B Neither the frequency of the EPC- ennched population nor the EPC culture assay showed a significant increase in EPC kinetics in either sham
  • Figures 5A and 5B are more specifically explained as follows
  • Adherent cells in EPC culture are denved p ⁇ ncipally from Dil-marked Sca-1 + cells
  • Figures 5C-H show results of the mouse cornea micropocket assay as applied to mice with hindlimb ischemia 7 days after surgery Slit-lamp biomicroscopy ( Figures 5C and 5D) and fluorescent photomicrographs
  • the rabbit model of hindlimb ischemia (Takeshita, S , et al J Clin Invest (1994)) was employed to determine if cytokine-induced EPC mobilization could enhance neovascula ⁇ zation of ischemic tissues
  • recombinant human GM-CSF was administered daily for 7 days prior to to development of hindlimb ischemia
  • Such GM-CSF pre-treatment 50 ⁇ g/day s c
  • FIGS. 6 A-I show the effect of GM-CSF-mduced EPC mobilization on neovasculanzation in rabbit ischemic hmdhmb model ( Figures 6A,B) Following pre-treatment with GM-CSF, circulating EPC-ennched population (TBM " ) is increased in number compared to control (ischemic, untreated) animals beginning at day 0 (pnor to surgery) through day 7 ( Figure 6A), as is EPC differentiation in culture
  • BMT munne BM transplantation
  • Corneas from control mice disclosed no cells expressing ⁇ -galactosidase. Quantitative chemical detection confirmed a statistically significant increase in ⁇ -galactosidase activity among mice receiving GM-CSF vs controls (2.90 ⁇ 0.30 vs 2.1 1 ⁇ 0.09 X10 3 , p ⁇ 0 05) (Figure 7C).
  • FIGS. 7A-C are explained in more detail as follows.
  • the figures illustrate that Bone ma ⁇ ow-de ⁇ ved EPCs contnbute to comeal neovasculanzation.
  • mice were obtained from the heart immediately before sac ⁇ fice, and separated by H ⁇ stopaque-1083 (Sigma, St. Louis, MO) density gradient cent ⁇ fugation at 400g for 20 mm The light- density mononuclear cells were harvested, washed twice with Dulbecco's phosphate buffered saline supplemented with 2mM EDTA (DPBS-E) and counted manually. Blood mononuclear cells in each animal were suspended in
  • Rabbit peripheral blood samples were obtained from either ear vein through a 20G infusion catheter and separated by H ⁇ stopaque-1077 (Sigma) density gradient centnfugation at 400g for 20 min The light -density mononuclear cells were harvested, washed twice by DPBS-E and counted manually As an approp ⁇ ate antibody for rabbit hematopoietic stem/precursor cells is not available, lmmatureHCs were isolated by depletion of matureHCs The cells were incubated with mixed p ⁇ mary antibodies (Serotec) of mouse anti-rabbit CD5.
  • T and B lymphocytes and monocytes were incubated with secondary rat anti-mouse IgG microbeads (Miltenyi Biotec) and placed in a magnetic separation column (Miltenyi Biotec) Cells which did not bind to antibodies for mature T and B lymphocytes and monocytes (TBM " ), identical to hematopoietic stem/precursor cells, passed through the column, while cells positive for cocktail antibodies were retained. The positive cells (TBM + ), matureHCs. were eluted from the column and both cell fractions were counted manually
  • EPC differentiation assay To evaluate EPC differentiation from circulating blood cells, Sca-1 and Sca-1 " cells isolated from JOO ⁇ l peripheral blood of each mouse, as well as TBM and TBM " cells isolated from 2 ml penpheral blood of each rabbit, were co-cultured in one well of a 24-well plate coated with rat plasma vitronectin (Sigma) after Dil-labehng of Sea- 1 * or TBM " cells in EBM-II media supplemented with 5% FBS (Clonetics, San Diego, CA) After four days in culture, cells were washed twice with media, and attached spreading cells were counted according to the frequency of Dil-labeled Sca-l " or TBM " cell-denved cells and non-labeled Sca-1 ' or TBM cell-denved cells
  • a comeal neovascula ⁇ zation assay (Kenyon, B.M., et al. Invest Ophthalmol Vis Sci (1996) and Asahara, T. et al. Ore. Res. (1998) was performed m mice with hmdhmb ischemia.
  • In situ BS-1 lectm staining was performed p ⁇ or to saenfice.
  • GM-CSF treatment administered to 8 rabbits, consisted of recombinant human GM-CSF (70 ⁇ g/ day) injected subcutaneously daily for one week, beginning 7 days before surgery (GM-CSF group).
  • the ischemic control group consisted of 8 rabbits receiving subcutaneous injections of saline daily for one week before surgery (control group).
  • Mu ⁇ ne bone ma ⁇ ow transplantation model FVB/N mice underwent BMT from transgenic mice constitutively expressing __ -galactosidase encoded by lacZ under the transc ⁇ ptional regulation of an EC-specific promoter, T ⁇ e-2 (Schlaeger, T m et al. Development (1995) Reconstitution of the transplanted BM yielded Tie- 2/LZ/BMT mice in which expression of lacZ is rest ⁇ cted to BM-de ⁇ ved cells expressing T ⁇ e-2; lacZ expression is not observed in other somatic cells The T ⁇ e-2/LZ/BMT mice then underwent comeal assay microsurgery (Kenyon, B M. et al.
  • BM cells were obtained by flushing the tibias and femurs of age-matched
  • donor T ⁇ e-2 transgenic mice FVB/N-TgN[TIE2LacZ] 182Sato, Jackson Lab
  • Low-density BM mononuclear cells were isolated by density cent ⁇ fugation over H ⁇ stopaque-1083 (Sigma).
  • BM transplantation (BMT) was performed in FVB/N mice (Jackson Lab) lethally irradiated with 12.0 Gy and intravenously infused with approximately 2X10 donor BM mononuclear cells each.
  • mice At 4 wks post-BMT, by which time the BM of the recipient mice was reconstituted, the mice underwent surgery to create hindlimb ischemia (vide infra) or a sham operation, 3 days later, microsurgery for assay of comeal -39-
  • neovascula ⁇ zation was performed Likewise, at 4 wks post-BMT, GM-CSF or control vehicle was administered for a penod of 7 days, 1 day after completion of GM-CSF or control pre-treatment, surgery for comea neovasculanzation assay was performed Corneas of BMT animals were harvested at 6 days after comeal microsurgery for light microscopic evidence of ⁇ -galactosidase expression or chemical detection of ⁇ -galactosidase activity
  • the enucleated eye was placed into liquid nitrogen, and stored at -80°C The assay was performed using Chemiluminescence Reporter Gene Assay System, Galacto- Light Plus TM (Tropix Ine , Bedford MA) according to the modified protocol B ⁇ efly, the eye was placed in 1 ml of supplemented lysis buffer, and after adding 0 5mM DTT was homogenized with a Tissuemizer Mark II (Tekmar Co , Cincinatti, OH) Homogenized lysis solution was centnfuged to remove deb ⁇ s An aliquot of the supernatant from homogenized lysis buffer was used for protein measurement using a BCA Protein Assay kit (PIERCE, Rockford, IN).
  • the supernatant was assayed after treatment with ion exchange resin, ChelexlOO, and beta- galactosidase activity was measured using a chemiluminometer (Lumat LB9501 , Berthold, Nashua, NH) beta- galactosidase activity was standardized according to protein concentration
  • mice We used age-mached (8wks) C57BL/6J male mice (Jackson Lab, Bar Harbor, ME) to create a mouse model of hindlimb ischemia (Couffinhal. T et al Am J Pathol (1998) All animals were anesthetized by intrapentoneal (l p ) pentobarbital injection (160 mg/kg) for subsequent surgical procedures A skin incision was performed at the middle portion of the left hmdhmb overlying the femoral artery The femoral artery then was gently isolated and the proximal portion of the femoral artery was ligated with a 3-0 silk ligature The distal portion of the saphenous artery was ligated.
  • VEGF vascular endothelial growth factor
  • the pellets were positioned 1.0mm from the comeal limbus and erythromycm ophthalmic ointment (E.Foufera, Melville, NY) was applied to each operated eye
  • the corneas of all mice were routinely examined by slit-lamp biomicroscopy on postoperative days 5 through 6 after pellet implantation Vessel length and circumference of neovasculanzation were measured on the sixth postoperative day when all corneas were photographed.
  • mice received 500 ⁇ g of Bandeiraea Simphcifolia lect ⁇ n-1 (BS-1) conjugated with FITC (Vector Lab, Burlmgame, CA), an EC-specific marker, intravenously, and were then sacnficed 30 minutes later
  • FITC Vector Lab, Burlmgame, CA
  • the eyes were enucleated and fixed m 1% paraformaldehyde solution. After fixation, the corneas were placed on glass slides and studied by fluorescent microscopy.
  • neovascula ⁇ zation was assessed by measu ⁇ ng the frequency of capillanes in light microscopic sections taken from the normal and ischemic hindhmbs Tissue specimens were obtained as transverse sections from -42-
  • Muscle samples were embedded in O.C.N compound (Miles, Elkhart, Ind.) and snap-frozen in liquid nitrogen. Multiple frozen sections 5 ⁇ m in thickness were then cut from each specimen so that the muscle fibers were oriented in a transverse fashion. The tissue sections were stained for alkaline phosphatase with an indoxyl- tetrazolium method to detect capillary ECs as previously described and counterstained with eosin. Capillaries were counted under a 20X objective to determine the capillary density (mean number of capillaries/mm ⁇ ). Ten different fields were randomly selected for the capillary counts.

Abstract

L'invention porte sur des procédés modulant la formation de nouveaux vaisseaux sanguins. Dans l'une des exécutions, les procédés consistent à administrer à un mammifère une dose efficace de GM-CST (sargramostim) suffisante pour former de nouveaux vaisseaux sanguins. L'invention porte en outre sur des procédés prévenant ou réduisant la sévérité des lésions des vaisseaux sanguins d'un mammifère consistant de préférence à lui administrer une dose efficace de GM-CST, ainsi que sur des produits pharmaceutiques et des trousses induisant la formation de vaisseaux sanguins chez les mammifères.
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EP1061800A1 (fr) 2000-12-27
CA2322559A1 (fr) 1999-09-16
AU3073799A (en) 1999-09-27
CA2322559C (fr) 2012-07-17
JP2010265301A (ja) 2010-11-25
EP1061800A4 (fr) 2004-10-06
AU766238B2 (en) 2003-10-09
JP2002506008A (ja) 2002-02-26

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