Identification and use og human bone marrow-derived endothelial progenitor cells to improve myocardial function after ischemic injury

Abstract

The present invention provides a method of stimulating vasculogenesis of myocardial infarct damaged tissue in a subject comprising: (a) removing stem cells from a location in the subject; (b) recovering endothelial progenitor cells in the stem cells; (c) introducing the endothelial progenitor cells from step (b) into a different location in the subject such that the precursors migrate to and stimulate revascularization of the tissue. The stem cells may be removed directly or by mobilization. The endothelial progenitor cells may be expanded before introduction into the subject. The present invention further provides a method of inducing angiogenesis in peri-infarct tissue. The present invention further provides a method of selectively increasing the trafficking of human bone marrow-derived endothelial cell precursors to the site of tissue damaged by ischemic injury which comprises: (a) administering endothelial progenitor cells to a subject; (b) administering chemokines to the subject so as to thereby attract endothelial cell precursors to the ischemic tissue. The present invention provides a method of stimulating vasculogenesis or angiogenesis of myocardial infarct damaged tissue in a subject comprising injecting allogeneic stem cells into a subject. The present invention further provides a method of improving myocardial function in a subject that has suffered a myocardial infarct comprising any of the instant methods. The present invention further provides a method of improving myocardial function in a subject that has suffered a myocardial infarct comprising injecting G-CSF or anti-CXCR4 antibody into the subject in order to mobilize endothelial progenitor cells.

1 . A method of stimulating vasculogenesis in ischemia-damaged tissue of a subject comprising: 
 (a) removing stem cells from a location within the subject;    (b) recovering endothelial progenitor cells from the stem cells removed in step (a); and    (c) introducing the endothelial progenitor cells from step (b) into a different location within the subject such that the endothelial progenitor cells stimulate vasculogenesis in the subject's ischemia-damaged tissue.    
     
     
         2 . The method of  claim 1 , wherein the endothelial progenitor cells are autologous.  
     
     
         3 . The method of  claim 1 , wherein subsequent to step (b), but before step (c), the endothelial progenitor cells are expanded by contacting them with a growth factor.  
     
     
         4 . The method of  claim 3 , wherein the growth factor is specific for, or primarily has effects upon endothelial cells.  
     
     
         5 . The method of  claim 3 , wherein the growth factor is a cytokine.  
     
     
         6 . The method of  claim 5 , wherein the cytokine is VEGF, PGF, G-CSF, IGF, M-CSF, or GM-CSF.  
     
     
         7 . The method of  claim 3 , wherein the growth factor is a chemokine.  
     
     
         8 . The method of  claim 7 , wherein the chemokine is Interleukin-8.  
     
     
         9 . The method of  claim 3 , wherein the endothelial progenitor cells are separated from other stem cells before expansion.  
     
     
         10 . The method of  claim 1 , wherein the ischemia-damaged tissue is myocardium.  
     
     
         11 . The method of  claim 1 , wherein the subject has suffered a myocardial infarct.  
     
     
         12 . The method of  claim 1 , wherein the ischemia-damaged tissue is nervous system tissue.  
     
     
         13 . The method of  claim 1 , wherein the subject has suffered a cerebral ischemic event.  
     
     
         14 . The method of  claim 1 , wherein the subject is deemed at risk from a cerebral ischemic event.  
     
     
         15 . The method of  claim 1 , wherein the stem cells are removed from the subject's bone marrow.  
     
     
         16 . The method of  claim 15 , wherein the removal of the stem cells from the bone marrow is effected by aspiration from the subject's bone marrow.  
     
     
         17 . The method of  claim 1 , wherein the removal of the stem cells from the subject is effected by a method comprising: 
 (a) introducing a growth factor into the subject to mobilize the stem cells into the subject's blood; and    (b) removing a sample of blood containing the stem cells from the subject.    
     
     
         18 . The method of  claim 17 , wherein the growth factor is introduced into the subject subcutaneously, orally, intravenously or intramuscularly.  
     
     
         19 . The method of  claim 17 , wherein the growth factor is a chemokine that induces mobilization.  
     
     
         20 . The method of  claim 19 , wherein the chemokine is Interleukin-8.  
     
     
         21 . The method of  claim 17 , wherein the growth factor is a cycokine.  
     
     
         22 . The method of  claim 21 , wherein the cytokine is G-CSF, M-CSF, or GM-CSF.  
     
     
         23 . The method of  claim 1 , wherein the endothelial progenitor cells are recovered based upon their expression of CD117.  
     
     
         24 . The method of  claim 1 , wherein the endothelial progenitor cells are recovered based upon their expression of a GATA-2 activated gene product.  
     
     
         25 . The method of  claim 1 , wherein the endothelial progenitor cells are recovered based upon their expression of one or more of CD34, VEGF-R, Tie-2, GATA-3 or AC133.  
     
     
         26 . The method of  claim 1 , wherein the subject has suffered or is suffering from one or more of the following: myocardial infarction, chronic heart failure, ischemic heart disease, coronary artery disease, diabetic heart disease, hemorrhagic stroke, thrombotic stroke, embolic stroke, limb ischemia, or another disease in which tissue is rendered ischemic.  
     
     
         27 . A method of treating acute myocardial infarct comprising the method of  claim 1 , wherein the subject is suffering acute myocardial infarct.  
     
     
         28 . The method of  claim 1 , wherein step (a) occurs prior to the subject suffering ischemia-damaged tissue and wherein step (c) occurs after the subject has suffered ischemia-damaged tissue.  
     
     
         29 . The method of  claim 1 , wherein the endothelial progenitor cells are frozen for a period of time between steps (b) and (c).  
     
     
         30 . The method of  claim 3 , wherein the endothelial progenitor cells are frozen for a period of time after being expanded but before step (c) is performed.  
     
     
         31 . The method of  claim 1 , wherein the endothelial progenitor cells are introduced into the subject by injection directly into the peripheral circulation, heart muscle, left ventricle, right ventricle, coronary artery, cerebro-spinal fluid, neural tissue, ischemic tissue, or post-ischemic tissue.  
     
     
         32 . The method of  claim 1 , further comprising administering to the subject one or more of the following: an inhibitor of Plasminogen Activator Inhibitor, Angiotensin Converting Enzyme Inhibitor or a beta blocker, wherein such administration occurs prior to, concomitant with, or following step (c).  
     
     
         33 . A method of stimulating angiogenesis in peri-infarct tissue in a subject comprising: 
 (a) removing stem cells from a location within the subject;    (b) recovering endothelial progenitor cells from the stem cells removed in step (a);    (c) expanding the endothelia progenitor cells recovered in step (b) by contacting the progenitor cells with a growth factor; and    (d) introducing the expanded endothelial progenitor cells from step (c) into a different location in the subject such that the endothelial progenitor cells stimulate angiogenesis in peri-infarct tissue in the subject.    
     
     
         34 . A method of selectively increasing the trafficking of endothelial progenitor cells to ischemia-damaged tissue in a subject comprising administering to the subject endothelial progenitor cells and a chemokine so as to thereby attract the endothelial progenitor cells to the ischemia-damaged tissue.  
     
     
         35 . The method of  claim 34 , wherein the endothelial progenitor cells have been derived from the subject's bone marrow.  
     
     
         36 . The method of  claim 34 , wherein the chemokine is a CXC chemokine.  
     
     
         37 . The method of  claim 36 , wherein the CXC chemokine is Interleukin-8, Gro-Alpha, or Stromal-Derived Factor-1.  
     
     
         38 . The method of  claim 34 , wherein the chemokine is a CC chemokine.  
     
     
         39 . The method of  claim 38 , wherein the CC chemokine is RANTES, EOTAXIN, MCP-1, MCP-2, MCP-3, or MCP-4.  
     
     
         40 . The method of  claim 34 , wherein the chemokine is administered to the subject by injection into the subject's peripheral circulation, heart muscle, left ventricle, right ventricle, a coronary artery, spinal fluid, neural tissue, ischemic tissue, or post-ischemic tissue.  
     
     
         41 . The method of  claim 34 , wherein the endothelial progenitor cells are human cells.  
     
     
         42 . The method of  claim 34 , wherein the ischemia-damaged tissue is myocardium.  
     
     
         43 . The method of  claim 34 , wherein the ischemia-damaged tissue is neural tissue.  
     
     
         44 . The method of  claim 34 , wherein the endothelial progenitor cells express CD117.  
     
     
         45 . The method of  claim 34 , wherein the endothelial progenitor cells express at least one of CD34, GATA-2, GATA-3 or AC133.  
     
     
         46 . The method of  claim 34 , wherein the chemokine is administered to the subject by injection into the subject's peripheral circulation, heart muscle, left ventricle, right ventricle, coronary arteries, cerebro-spinal fluid, neural tissue, ischemic tissue, or post-ischemic tissue.  
     
     
         47 . The method of  claim 1 ,  33  or  34 , wherein the subject is mammalian.  
     
     
         48 . The method of  claim 47 , wherein the subject is human.  
     
     
         49 . A method of increasing trafficking of endothelial progenitor cells or angioblasts to ischemia-damaged tissue in a subject comprising inhibiting any interaction between Stromal-Derived Factor-1 and CXCR4.  
     
     
         50 . The method of  claim 49 , wherein interaction between Stromal-Derived Factor-1 (SDF-1)and CXCR4 is inhibited by administration of an anti-SDF-1 or an anti-CXCR4 monoclonal antibody to the subject.  
     
     
         51 . The method of  claim 50 , further comprising administering to the subject an Angiotensin Converting Enzyme Inhibitor, an AT 1 -receptor blocker, or a beta blocker.  
     
     
         52 . A method of reducing trafficking of endothelial progenitor cells to bone marrow in a subject comprising inhibiting production of Stromal-Derived Factor-1 in the subject's bone marrow.  
     
     
         53 . The method of  claim 52 , wherein SDF-1 production is inhibited by administration of an anti-SDF-1 or anti-CXCR4 monoclonal antibody to the subject.  
     
     
         54 . A method for treating a cancer in a subject comprising administering to the subject a monoclonal antibody directed against an epitope of a specific chemokine produced by proliferating cells associated with the cancer so as to reduce trafficking of endothelial progenitor cells to such proliferating cells and thereby treat the cancer in the subject.  
     
     
         55 . A method for treating a cancer in a subject comprising administering to the subject a monoclonal antibody directed against an epitope of a specific receptor located on an endothelial progenitor cell, for a chemokine produced by proliferating cells associated with the cancer, so as to reduce trafficking of the endothelial progenitor cell to such proliferating cells and thereby treat the cancer in the subject.  
     
     
         56 . The method of  claim 55 , wherein the receptor is CXCR1, CXCR2 or VEGF-R.  
     
     
         57 . A method for treating a tumor in a subject comprising administering to the subject an antagonist to a specific receptor on an endothelial progenitor cell so as to reduce the progenitor cell's ability to induce vasculogenesis in the subject's tumor and thereby treat the tumor.  
     
     
         58 . A method for treating a tumor in a subject comprising administering to the subject an antagonist to a specific receptor on an endothelial progenitor cell so as to reduce the progenitor cell's ability to induce angiogenesis in the subject's tumor and thereby treat the tumor.  
     
     
         59 . The method of  claim 57  or  58 , wherein the receptor is a CD117 receptor.  
     
     
         60 . The method of  claim 54 ,  55 ,  57 , or  58 , wherein the subject is mammalian.  
     
     
         61 . The method of  claim 60 , wherein the subject is human.  
     
     
         62 . A method for expressing a gene of interest in an endothelial progenitor cell or a mast progenitor cell which comprises inserting into the cell a vector comprising a promoter containing a GATA-2 motif and the gene of interest.  
     
     
         63 . The method of  claim 62 , wherein the vector is inserted into the cell by transfection.  
     
     
         64 . The method of  claim 62  wherein the promoter is a preproendothelin-1 promoter.  
     
     
         65 . The method of  claim 64 , wherein the promoter is of mammalian origin.  
     
     
         66 . The method of  claim 65 , wherein the promoter is of human origin.  
     
     
         67 . A composition comprising an amount of a monoclonal antibody directed against an epitope of a specific chemokine produced by a cancer effective to reduce trafficking of endothelial progenitor cells to the cancer, and a pharmaceutically acceptable carrier.  
     
     
         68 . A method of treating an abnormality in a subject wherein the abnormality is treated by the expression of a GATA-2 activated gene product in the subject comprising: 
 (a) removing stem cells from a location within the subject;    (b) recovering endothelial progenitor cells from the stem cells removed in step (a);    (c) recovering those endothelial progenitor cells recovered in step (b) that express GATA-2;    (d) inducing the cells recovered in step (c) as expressing GATA-2 to express a GATA-2 activated gene product; and    (e) introducing the cells expressing a GATA-2 activated gene product from step (d) into a different location in the subject such as to treat the abnormality.    
     
     
         69 . A method of treating an abnormality in a subject wherein the abnormality is treated by the expression of a GATA-2 activated gene product in the subject comprising: 
 (a) removing stem cells from a location within the subject;    (b) recovering mast progenitor cells from the stem cells removed in step (a);    (c) recovering those mast progenitor cells recovered in step (b) that express GATA-2;    (d) inducing the cells recovered in step (c) as expressing GATA-2 to express a GATA-2 activated gene product; and    (e) introducing the cells expressing a GATA-2 activated gene product from step (d) into a different location in the subject such as to treat the abnormality    
     
     
         70 . The method of claims  68  or  69 , wherein the abnormality is ischemia-damaged tissue.  
     
     
         71 . The method of claims  68  or  69 , wherein the gene product is proendothelin.  
     
     
         72 . The method of claims  68  or  69 , wherein the gene product is endothelin.  
     
     
         73 . A method of improving myocardial function in a subject that has suffered a myocardial infarct comprising: 
 (a) removing stem cells from a location in the subject;    (b) recovering cells that express CD117 from the stem cells; and    (c) introducing the recovered cells into a different location in the subject such that the cells improve myocardial function in the subject.    
     
     
         74 . The method of any of claims  68 ,  69 , or  73 , wherein the subject is of mammalian origin.  
     
     
         75 . The method of  claim 74 , wherein the mammal is of human origin.  
     
     
         76 . A method of stimulating vasculogenesis in ischemia-damaged tissue in a subject comprising: 
 (a) obtaining allogeneic stem cells;    (b) recovering endothelial progenitor cells from the stem cells removed in step (a); and    (c) introducing the endothelial progenitor cells recovered in step (b) into the subject such that the endothelial progenitor cells stimulate vasculogenesis in the subject's ischemia-damaged tissue.    
     
     
         77 . The method of  claim 76 , wherein the allogeneic stem cells are obtained from embryonic, fetal or cord blood sources.  
     
     
         78 . A method of stimulating angiogenesis in ischemia-damaged tissue in a subject comprising: 
 (a) obtaining allogeneic stem cells;    (b) recovering endothelial progenitor cells in the stem cells removed in step (a); and    (c) introducing the endothelial progenitor cells recovered in step (b) into the subject such that the endothelial progenitor cells stimulate angiogenesis in the subject's ischemia-damaged tissue.    
     
     
         79 . The method of  claim 78 , wherein the allogeneic stem cells are obtained from embryonic, fetal or cord blood sources.  
     
     
         80 . A method of improving myocardial function in a subject that has suffered a myocardial infarct comprising injecting G-CSF into the subject in order to mobilize endothelial progenitor cells.  
     
     
         81 . A method of improving myocardial function in a subject that has suffered a myocardial infarct comprising injecting anti-CXCR4 antibody into the subject.  
     
     
         82 . The method of  claim 81  further comprising introducing endothelial progenitor cells into the subject.  
     
     
         83 . The method of  claim 82  further comprising introducing G-CSF into the subject in order to mobilize endothelial progenitor cells.  
     
     
         84 . An endothelial progenitor cell that expresses CD117.  
     
     
         85 . An endothelial progenitor cell that expresses one or more of the group consisting of GATA-2, GATA-3, CD34, AC133, CD34 and CD117.  
     
     
         86 . The endothelial progenitor cell of  claim 85 , wherein the progenitor cell is derived from bone marrow.  
     
     
         87 . A method of expanding an endothelial progenitor cell population comprising contacting an endothelial progenitor cell with a cytokine.  
     
     
         88 . The method of  claim 87 , wherein the cytokine is selected from the group consisting of G-CSF, GM-CSF, M-CSF, VEGF, and FGF.  
     
     
         89 . A method of expanding an endothelial progenitor cell population comprising contacting an endothelial progenitor cell with a chemokine.  
     
     
         90 . The method of  claim 89 , wherein the chemokine is a CC chemokine.  
     
     
         91 . The method of  claim 90 , wherein the CC chemokine is selected from the group consisting of RANTES, EOTAXIN, MCP-1, MCP-2, MCP-3, and MCP-4.  
     
     
         92 . A method of identifying bone marrow-derived endothelial progenitor cells comprising recovering progenitor cells based on their expression of CD117.  
     
     
         93 . A method of identifying bone marrow-derived endothelial progenitor cells comprising recovering progenitor cells based on their expression of any or all of the group consisting of GATA-2, GATA-3, CD34, AC133, CD34 and CD117.  
     
     
         94 . The method of  claim 1 , wherein the removal of the stem cells from the subject is effected by a method comprising; 
 (a) eliciting production of growth factor in the subject to mobilize the stem cells into the subject's blood; and    (b) removing a sample of blood containing the stem cells from the subject.    
     
     
         95 . The method of  claim 94 , wherein the growth factor production is elicited by a gene therapy technique.  
     
     
         96 . A method of selectively increasing the trafficking of endothelial progenitor cells to ischemia-damaged tissue in a subject comprising eliciting chemokine production in the subject so as to thereby attract the endothelial progenitor cells to the ischemia-damaged tissue.  
     
     
         97 . The method of  claim 96 , wherein the chemokine production is elicited by a gene therapy technique.  
     
     
         98 . The method of  claim 97 , further comprising administering endothelial progenitor cells to the subject.