Choroidal Neovascularization Is Provided by Bone Marrow Cells
http://www.100md.com
《干细胞学杂志》
a First Department of Pathology;
b Department of Ophthalmology;
c Regeneration Research Center for Intractable Disease;
d Department of Anatomical Pathology;
e Transplantation Center, Kansai Medical University, Moriguchi City, Osaka, Japan
Key Words. Choroidal neovascularization ? Bone marrow cells ? Age-related macular degeneration ? Bone marrow transplantation
Susumu Ikehara, M.D., First Department of Pathology, Kansai Medical University, Fumizono-cho, Moriguchi City, Osaka, Japan, 570-8506. Telephone: 81-6-6992-1001 (ext. 2474 or 2475); Fax: 81-6-6992-1219; e-mail: ikehara@takii.kmu.ac.jp
ABSTRACT
Recently, bone marrow cells (BMCs) have been used as a source of several kinds of mature cells in research for regenerative medicine. It has been reported that BMCs differentiate into various types of cells, including hepatocytes , epithelial cells (in the stomach, esophagus, small intestine, large intestine, and bronchus), cardiac muscle , and skeletal muscle . It has also been reported that BMCs differentiate into neural cells and astrocytes in vitro , and also into astrocytes in vivo when BMCs are transplanted into the normal or ischemic brain. Moreover, the intravenous injection of BMCs into mice has been shown to induce neural differentiation in the brain . BMCs have the capacity to differentiate into myelin-forming cells in vivo and to repair demyelinated spinal cord axons . We also have demonstrated that BMCs differentiate into retinal neural cells in the injured rat retina . Adult BMCs, which contain hematopoietic stem cells (HSCs), have the ability of self-renewal and provide individuals with mature hematopoietic cells throughout their life. Therefore, BMCs are utilized in the treatment of bone marrow failure states, including hematological malignancies (leukemia and lymphoma). Recently, it has been reported that endothelial progenitor cells (EPCs) are derived from the bone marrow , and that EPCs can differentiate into endothelial cells of blood vessels in the case of myocardial ischemia and hindlimb ischemic models . Thus, bone marrow-derived EPCs significantly contribute to blood vessel formation . A recent report has shown that retinal neovascularization is provided by HSCs . Choroidal neovascularization (CNV) is a known cause of age-related macular degeneration (ARMD); ARMD is the most common cause of new blindness in the elderly in advanced countries. It has been reported that the mechanisms underlying CNV are completely different from mechanisms underlying retinal neovascularization ; newly formed blood vessels in CNV have been shown to be provided by the existing choroidal vessels through a broken Bruch’s membrane . In this report, we use an experimental mouse model of CNV induced by krypton laser photocoagulation and clarify whether BMCs contribute to newly generated blood vessels in CNV.
MATERIALS AND METHODS
Analysis of Chimerism
To determine if BMCs contribute to CNV, we prepared complete bone marrow chimeric mice . C57BL/J recipient mice were lethally irradiated (6.0 Gy x 2 with a 4-hour interval the day before BMT), and whole BMCs from EGFP mice were transplanted intravenously via the tail vein. Three months after BMT, we examined the chimerism of recipients using a FACS scan (Fig. 1). More than 95% of the hematopoietic cells in the peripheral blood and hematolymphoid organs such as the spleen (data not shown) were positive for green fluorescence (donor derived). Thus, we confirmed the complete chimerism of the hematopoietic cells in the recipient mice.
Figure 1. Reconstitution of multilineage hematopoietic cells after BMT. Three months after BMT, the expression of green fluorescence and surface markers of hematopoietic cells in the peripheral blood were analyzed by flow cytometry. More than 95% of the hematopoietic cells in the peripheral blood were donor-derived cells, indicating that these BMCs had differentiated into multilineage hematopoietic cells.
Generation of Blood Vessels in CNV from Donor BMCs after BMT
After establishing complete chimerism, the mice were treated with laser photocoagulation to induce CNV of the ARMD model in one eye per mouse (three spots per eye). One eye from each of 10 mice was prepared in this experiment, and CNV was induced in all of the laser spots (n = 30). Two weeks after laser photocoagulation, one eye from each of 5 mice was enucleated under anesthesia and frozen sections were prepared. These frozen sections were stained with a PE-conjugated anti-CD31 antibody and vascular cell-selective lectin , which is known as a specific marker for endothelial cells of blood vessels in CNV. A minimum of 30 sections per choroid, with sampling on both sides of the optic nerve, were examined. In the sections stained with vascular cell-selective lectin, Bruch’s membrane was broken by krypton laser photocoagulation, and new blood vessels were generated in the CNVs (Fig. 2).
Figure 2. The structure of CNV as created by laser photocoagulation. The CNVs were stained by the vascular cell-selective lectin, which is usually used to observe the structure of the CNV. New blood vessels in the CNVs were completely generated by laser photocoagulation. In the center of each CNV, Bruch’s membrane was broken. The arrow heads show the CNV (x50). The tubular structure of EGFP-positive cells in the same part was observed using a confocal microscope in the CNV.
To examine the pattern of vascular development in CNV, one eye from each of the other five mice was perfused with red fluorescent-labeled dextran (n = 5) and was observed in the flat-mount preparation of choroids. The CNV in the laser-photocoagulated lesions of the mounted choroids was observed using confocal microscopy. EGFP-positive cells existed along the blood vessels, and the shapes of those cells were compatible with endothelial cells; EGFP-positive cells existed along the red-fluorescence-positive lumen of the CNV (n = 15 spots; Fig. 3). As shown in Figure 4, the CNVs were stained with PE-conjugated anti-CD31, and over 70% of EGFP-positive cells, which were in the CNVs, were also positive for CD31 (n = 15 spots). These results suggest that EGFP-positive donor BMCs differentiate into CD31-positive endothelial cells, and that these endothelial cells constitute functional blood vessels.
Figure 3. Confocal microscopic examination of whole mounted choroids after BMT. Three months after BMT, CNV was induced by laser photocoagulation. Two weeks later, the eyes of the mice that had received BMT were perfused with 1 ml PBS containing TRITC-conjugated dextran. A and D) EGFP-positive cells derived from transplanted BMCs. B and E) Blood flow in CNVs, which are TRITC positive. C) Superimposed figure of (A) on (B). F) Superimposed figure of (D) on (E). (D), (E), and (F) show high magnification of (A), (B), and (C), respectively. EGFP-positive donor-derived cells show the structure and shape of vessel walls and endothelial cells (A and D). The vessels with red fluorescence (TRITC-conjugated dextran) are surrounded with donor-derived EGFP-positive cells (arrows), which show the structure of vessel walls and endothelial cells in the CNV (C and F).
Figure 4. Confocal microscopic examination of CNV section after BMT. Three months after BMT, CNV was induced by laser photocoagulation. Two weeks later, newly generated blood vessels in the CNV, which were stained with PE-conjugated anti-CD31 antibody, were observed with a confocal microscope. (A) and (B) show the expression of EGFP and CD31, respectively. Donor-derived EGFP-positive cells exist in CNVs (A) and CD31-positive cells exist in the CNV (B). (C) shows a superimposed figure of (A) on (B). EGFP-positive cells express CD31.
DISCUSSION
We thank professor M. Okabe (Osaka University) for the donation of EGFP-transgenic mice. We also thank Hilary Eastwick-Field and K. Ando for the preparation of this manuscript.
REFERENCES
Lagasse E, Conors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Petersen BE, Bowen WC, Patrene KC et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168–1170.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow derived myogenic progenitors. Science 1998;279:1528–1530.
Woodbury D, Schwarz EJ, Prockop DJ et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364–370.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.
Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999;96:10711–10716.
Azizi SA, Stokes D, Augelli BJ et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts. Proc Natl Acad Sci USA 1998;95:3908–3913.
Eglitis MA, Dawson D, Park KW et al. Targeting of marrow-derived astrocytes to the ischemic brain. Neuroreport 1999;10:1289–1292.
Brazelton TR, Rossi FMV, Keshet GI et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:1775–1779.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
Eglitis MA, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci USA 1997;94:4080–4085.
Sakaki M, Honmou O, Akiyama Y et al. Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia 2001;35:26–34.
Tomita M, Adachi Y, Yamada H et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. STEM CELLS 2002;20:279–283.
Lin Y, Weisdorf DJ, Solovey A et al. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000;105:71–77.
Murayama T, Tepper OM, Silver M et al. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo. Exp Hematol 2002;30:967–972.
Bhattacharya V, McSweeney PA, Shi Qun et al. Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34 bone marrow cells. Blood 2000;95:581–585.
Kocher AA, Schuster MD, Szabolcs MJ et al. Neovascularization of ischemic myocardium by human bone marrow derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7:430–436.
Kawamoto A, Gwon HC, Iwaguro H et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001;103:634–637.
Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.
Crosby JR, Kaminski WE, Schatteman G et al. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res 2000;87:728–730.
Takakura N, Watanabe T, Suenobu S et al. A role for hematopoietic stem cells in promoting angiogenesis. Cell 2000;102:199–209.
Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607–612.
Campochiaro PA. Retinal and choroidal neovascularization. J Cell Phys 2000;184:301–310.
Ishibashi T, Miller M, Orr G et al. Morphologic observation on experimental subretinal neovascularization in the monkey. Invest Ophthalmol Vis Sci 1987;28:1116–1130.
Dobi ET, Puliafito CA, Destro M. A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol 1989;107:264–269.
Tobe T, Ortega S, Luna JD et al. Target distribution of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am J Pathol 1998;153:1641–1646.
Okabe M, Ikawa M, Kominami K et al. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.
Cui YZ, Hisha H, Yang GX et al. Optimal protocol for total body irradiation for allogenic bone marrow transplantation in mice. Bone Marrow Transplant 2002;30:843–849.
Mori K, Ando A, Gehlbach P et al. Inhibition of choroidal neovascularization by intravenous injection of adenoviral vectors expressing secretable endostain. Am J Pathol 2001;159:313–320.
Mori K, Gehlbach P, Yamamoto S et al. AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization. Invest Opthalmol Vis Sci 2001;43:1994–2000.
Kvanta A, Algvere PV, Berglin L et al. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci 1996;37:1929–1934.
Kwak N, Okamoto N, Wood JM et al. VEGF is major stimulator in model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2000;41:3158–3164.
Frank RN, Amin RH, Eliott D et al. Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthalmol 1996;122:393–403.
Lopez PF, Sippy BD, Lambert HM et al. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1996;37:855–868.
Asahara T, Takahashi T, Matsuda H et al. VEGF contribute to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 1999;18:3964–3972.
Iwaguro H, Yamaguchi J, Kalka C et al. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 2002;105:732–738.
Kalka C, Masuda H, Takahashi T et al. Vascular endothelial growth factor165 gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res 2000;86:1198–1202.(Minoru Tomitaa,b, Haruhik)
b Department of Ophthalmology;
c Regeneration Research Center for Intractable Disease;
d Department of Anatomical Pathology;
e Transplantation Center, Kansai Medical University, Moriguchi City, Osaka, Japan
Key Words. Choroidal neovascularization ? Bone marrow cells ? Age-related macular degeneration ? Bone marrow transplantation
Susumu Ikehara, M.D., First Department of Pathology, Kansai Medical University, Fumizono-cho, Moriguchi City, Osaka, Japan, 570-8506. Telephone: 81-6-6992-1001 (ext. 2474 or 2475); Fax: 81-6-6992-1219; e-mail: ikehara@takii.kmu.ac.jp
ABSTRACT
Recently, bone marrow cells (BMCs) have been used as a source of several kinds of mature cells in research for regenerative medicine. It has been reported that BMCs differentiate into various types of cells, including hepatocytes , epithelial cells (in the stomach, esophagus, small intestine, large intestine, and bronchus), cardiac muscle , and skeletal muscle . It has also been reported that BMCs differentiate into neural cells and astrocytes in vitro , and also into astrocytes in vivo when BMCs are transplanted into the normal or ischemic brain. Moreover, the intravenous injection of BMCs into mice has been shown to induce neural differentiation in the brain . BMCs have the capacity to differentiate into myelin-forming cells in vivo and to repair demyelinated spinal cord axons . We also have demonstrated that BMCs differentiate into retinal neural cells in the injured rat retina . Adult BMCs, which contain hematopoietic stem cells (HSCs), have the ability of self-renewal and provide individuals with mature hematopoietic cells throughout their life. Therefore, BMCs are utilized in the treatment of bone marrow failure states, including hematological malignancies (leukemia and lymphoma). Recently, it has been reported that endothelial progenitor cells (EPCs) are derived from the bone marrow , and that EPCs can differentiate into endothelial cells of blood vessels in the case of myocardial ischemia and hindlimb ischemic models . Thus, bone marrow-derived EPCs significantly contribute to blood vessel formation . A recent report has shown that retinal neovascularization is provided by HSCs . Choroidal neovascularization (CNV) is a known cause of age-related macular degeneration (ARMD); ARMD is the most common cause of new blindness in the elderly in advanced countries. It has been reported that the mechanisms underlying CNV are completely different from mechanisms underlying retinal neovascularization ; newly formed blood vessels in CNV have been shown to be provided by the existing choroidal vessels through a broken Bruch’s membrane . In this report, we use an experimental mouse model of CNV induced by krypton laser photocoagulation and clarify whether BMCs contribute to newly generated blood vessels in CNV.
MATERIALS AND METHODS
Analysis of Chimerism
To determine if BMCs contribute to CNV, we prepared complete bone marrow chimeric mice . C57BL/J recipient mice were lethally irradiated (6.0 Gy x 2 with a 4-hour interval the day before BMT), and whole BMCs from EGFP mice were transplanted intravenously via the tail vein. Three months after BMT, we examined the chimerism of recipients using a FACS scan (Fig. 1). More than 95% of the hematopoietic cells in the peripheral blood and hematolymphoid organs such as the spleen (data not shown) were positive for green fluorescence (donor derived). Thus, we confirmed the complete chimerism of the hematopoietic cells in the recipient mice.
Figure 1. Reconstitution of multilineage hematopoietic cells after BMT. Three months after BMT, the expression of green fluorescence and surface markers of hematopoietic cells in the peripheral blood were analyzed by flow cytometry. More than 95% of the hematopoietic cells in the peripheral blood were donor-derived cells, indicating that these BMCs had differentiated into multilineage hematopoietic cells.
Generation of Blood Vessels in CNV from Donor BMCs after BMT
After establishing complete chimerism, the mice were treated with laser photocoagulation to induce CNV of the ARMD model in one eye per mouse (three spots per eye). One eye from each of 10 mice was prepared in this experiment, and CNV was induced in all of the laser spots (n = 30). Two weeks after laser photocoagulation, one eye from each of 5 mice was enucleated under anesthesia and frozen sections were prepared. These frozen sections were stained with a PE-conjugated anti-CD31 antibody and vascular cell-selective lectin , which is known as a specific marker for endothelial cells of blood vessels in CNV. A minimum of 30 sections per choroid, with sampling on both sides of the optic nerve, were examined. In the sections stained with vascular cell-selective lectin, Bruch’s membrane was broken by krypton laser photocoagulation, and new blood vessels were generated in the CNVs (Fig. 2).
Figure 2. The structure of CNV as created by laser photocoagulation. The CNVs were stained by the vascular cell-selective lectin, which is usually used to observe the structure of the CNV. New blood vessels in the CNVs were completely generated by laser photocoagulation. In the center of each CNV, Bruch’s membrane was broken. The arrow heads show the CNV (x50). The tubular structure of EGFP-positive cells in the same part was observed using a confocal microscope in the CNV.
To examine the pattern of vascular development in CNV, one eye from each of the other five mice was perfused with red fluorescent-labeled dextran (n = 5) and was observed in the flat-mount preparation of choroids. The CNV in the laser-photocoagulated lesions of the mounted choroids was observed using confocal microscopy. EGFP-positive cells existed along the blood vessels, and the shapes of those cells were compatible with endothelial cells; EGFP-positive cells existed along the red-fluorescence-positive lumen of the CNV (n = 15 spots; Fig. 3). As shown in Figure 4, the CNVs were stained with PE-conjugated anti-CD31, and over 70% of EGFP-positive cells, which were in the CNVs, were also positive for CD31 (n = 15 spots). These results suggest that EGFP-positive donor BMCs differentiate into CD31-positive endothelial cells, and that these endothelial cells constitute functional blood vessels.
Figure 3. Confocal microscopic examination of whole mounted choroids after BMT. Three months after BMT, CNV was induced by laser photocoagulation. Two weeks later, the eyes of the mice that had received BMT were perfused with 1 ml PBS containing TRITC-conjugated dextran. A and D) EGFP-positive cells derived from transplanted BMCs. B and E) Blood flow in CNVs, which are TRITC positive. C) Superimposed figure of (A) on (B). F) Superimposed figure of (D) on (E). (D), (E), and (F) show high magnification of (A), (B), and (C), respectively. EGFP-positive donor-derived cells show the structure and shape of vessel walls and endothelial cells (A and D). The vessels with red fluorescence (TRITC-conjugated dextran) are surrounded with donor-derived EGFP-positive cells (arrows), which show the structure of vessel walls and endothelial cells in the CNV (C and F).
Figure 4. Confocal microscopic examination of CNV section after BMT. Three months after BMT, CNV was induced by laser photocoagulation. Two weeks later, newly generated blood vessels in the CNV, which were stained with PE-conjugated anti-CD31 antibody, were observed with a confocal microscope. (A) and (B) show the expression of EGFP and CD31, respectively. Donor-derived EGFP-positive cells exist in CNVs (A) and CD31-positive cells exist in the CNV (B). (C) shows a superimposed figure of (A) on (B). EGFP-positive cells express CD31.
DISCUSSION
We thank professor M. Okabe (Osaka University) for the donation of EGFP-transgenic mice. We also thank Hilary Eastwick-Field and K. Ando for the preparation of this manuscript.
REFERENCES
Lagasse E, Conors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Petersen BE, Bowen WC, Patrene KC et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168–1170.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow derived myogenic progenitors. Science 1998;279:1528–1530.
Woodbury D, Schwarz EJ, Prockop DJ et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364–370.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.
Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999;96:10711–10716.
Azizi SA, Stokes D, Augelli BJ et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts. Proc Natl Acad Sci USA 1998;95:3908–3913.
Eglitis MA, Dawson D, Park KW et al. Targeting of marrow-derived astrocytes to the ischemic brain. Neuroreport 1999;10:1289–1292.
Brazelton TR, Rossi FMV, Keshet GI et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:1775–1779.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
Eglitis MA, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci USA 1997;94:4080–4085.
Sakaki M, Honmou O, Akiyama Y et al. Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia 2001;35:26–34.
Tomita M, Adachi Y, Yamada H et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. STEM CELLS 2002;20:279–283.
Lin Y, Weisdorf DJ, Solovey A et al. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000;105:71–77.
Murayama T, Tepper OM, Silver M et al. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo. Exp Hematol 2002;30:967–972.
Bhattacharya V, McSweeney PA, Shi Qun et al. Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34 bone marrow cells. Blood 2000;95:581–585.
Kocher AA, Schuster MD, Szabolcs MJ et al. Neovascularization of ischemic myocardium by human bone marrow derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7:430–436.
Kawamoto A, Gwon HC, Iwaguro H et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001;103:634–637.
Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.
Crosby JR, Kaminski WE, Schatteman G et al. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res 2000;87:728–730.
Takakura N, Watanabe T, Suenobu S et al. A role for hematopoietic stem cells in promoting angiogenesis. Cell 2000;102:199–209.
Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8:607–612.
Campochiaro PA. Retinal and choroidal neovascularization. J Cell Phys 2000;184:301–310.
Ishibashi T, Miller M, Orr G et al. Morphologic observation on experimental subretinal neovascularization in the monkey. Invest Ophthalmol Vis Sci 1987;28:1116–1130.
Dobi ET, Puliafito CA, Destro M. A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol 1989;107:264–269.
Tobe T, Ortega S, Luna JD et al. Target distribution of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am J Pathol 1998;153:1641–1646.
Okabe M, Ikawa M, Kominami K et al. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.
Cui YZ, Hisha H, Yang GX et al. Optimal protocol for total body irradiation for allogenic bone marrow transplantation in mice. Bone Marrow Transplant 2002;30:843–849.
Mori K, Ando A, Gehlbach P et al. Inhibition of choroidal neovascularization by intravenous injection of adenoviral vectors expressing secretable endostain. Am J Pathol 2001;159:313–320.
Mori K, Gehlbach P, Yamamoto S et al. AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization. Invest Opthalmol Vis Sci 2001;43:1994–2000.
Kvanta A, Algvere PV, Berglin L et al. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci 1996;37:1929–1934.
Kwak N, Okamoto N, Wood JM et al. VEGF is major stimulator in model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2000;41:3158–3164.
Frank RN, Amin RH, Eliott D et al. Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthalmol 1996;122:393–403.
Lopez PF, Sippy BD, Lambert HM et al. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1996;37:855–868.
Asahara T, Takahashi T, Matsuda H et al. VEGF contribute to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 1999;18:3964–3972.
Iwaguro H, Yamaguchi J, Kalka C et al. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 2002;105:732–738.
Kalka C, Masuda H, Takahashi T et al. Vascular endothelial growth factor165 gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res 2000;86:1198–1202.(Minoru Tomitaa,b, Haruhik)