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582. VEGF Gene Transfer Using AAV Vectors Induces Postnatal Neovascularization through Bone Marrow Stem Cell Recruitment
CARDIOVASCULAR CARDIOVASCULAR 581. Cytokine-Mobilized Bone Marrow Cells Re-Endothelialize Injured Blood Vessel and Inhibit Neointimal Formation Deling Kong,1 Lunan Zhang,1 Massimiliano Gnecchi,1 Luis G. Melo,1 Chong C. Liew,1 Richard E. Pratt,1 Victor J. Dzau.1 1 Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States. Bone marrow stem cells can be mobilized by cytokines such as stem cell factor (SCF), granulocyte-colony-stimulating factor (GCSF), or vascular endothelial growth factor (VEGF). These enriched bone marrow cells in the circulation can home to the injured tissues and differentiate, thereby contribute to neovascularization and cardiac repair. In this study, we hypothesize systemic cytokine administration at the time of vascular injury mobilizes these cells to the injured blood vessel. These progenitor cells differentiate into endothelial cells, provide enhanced endothelialization, and inhibit neointimal formation. Sprague-Dawley male rats (body weight 200g) were splenectomized and 2 weeks later were injected s.c. with recombinant human G-CSF, 50 mg/kg/day, once a day for 8 days. At the fifth day, animals were subjected to balloon angioplasty of the carotid artery, and followed by three more days of G-CSF injection. The control animals received injection of normal saline. Animals were killed at varied time points, and carotid arteries were harvested and processed for morphometric analysis, immunochemistry, scan electronic microscopy (SEM) and vascular reactivity measurement. The neointimal formation at 2 weeks after injury was significantly inhibited by 70% in the G-CSF treated animals compared to control. Staining with endothelium specific marker, PECAM-1 shows enhanced endothelialization (>90%) in the G-CSF treated arteries compared to less than 20% in the control. SEM data further confirmed this difference of endothelium coverage between the two groups of animals. The expression of vascular cell adhesion molecule-1 (VCAM-1) was much lower in the G-CSF treated than the control. The effect of G-CSF on restoration of vascular reactivity and proliferation rate of vascular smooth muscle cell were also studied. These findings provide evidence that mobilized bone marrow cells by G-CSF effectively inhibit neointimal formation by homing to the injured site and by facilitating endothelialization, and suggest the possibility of administration of G-CSF as a simple but effective method to prevent neointimal hyperplasia.
582. VEGF Gene Transfer Using AAV Vectors Induces Postnatal Neovascularization through Bone Marrow Stem Cell Recruitment Lorena Zentilin,1 Nikola Arsic,1 Sabrina Tafuro,1 Genaro RamirezCorrea,1 Serena Zacchigna,1 Sara Tomasi,1 Lucia Pattarini,1 Gianfranco Sinagra,2 Alessandro Salvi,2 Mauro Giacca.1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 2Cardiology Unit, Ospedale Maggiore, Trieste, Italy. Gene therapy has emerged as a promising new strategy to promote angiogenesis in the treatment of myocardial and peripheral ischemia. In this setting, recombinant AAV-based vectors stand as one of the most efficient systems to achieve stable and safe gene delivery to the myocardium and the skeletal muscle. Using an AAV vector expressing human vascular endothelial growth factor (VEGF), we previously demonstrated a significant increase in capillary density and formation of α-SMA-positive arteriolae in vector-injected normoperfused skeletal muscle (Arsic et al 2003). Moreover, as soon as 7 days after intramuscular injection of the recombinant vector, we observed a remarkable increase in cellularity, S226
with a large number of proliferating cells accumulating and surrounding muscle fibers; only a few of these cells were of inflammatory origin. Some of these infiltrating cells showed positive for the endothelial cell marker CD31, and, most notably, for markers of endothelial progenitor cells and bone-marrow derived stem cells, including Tie-2, Flk-1, c-Kit and Sca-1. Moreover, some cells in these proliferating bulks displayed a tendency to align in cord-like structures and to organize into small, CD31-positive vessels, suggestive of a vasculogenesis process. To understand whether the cells that are locally recruited by constitutive expression of VEGF might originate from the bone marrow, we transplanted unfractionated male bone marrow cells into lethally irradiated female mice. In situ hybridization analysis on AAV-VEGF injected muscle sections of transplanted females actually showed that more than 70% of the infiltrating cells were positive for Y chromosome sequences. This result clearly supports the notion that local expression of VEGF recruits vascular progenitors from the circulation. The capacity of VEGF to mobilize bone marrow precursors was further assessed by flow cytometry on bone marrow and peripheral blood mononuclear cells in mice at 15 and 30 days from AAV-VEGF injection. We obtained evidence that, in peripheral blood, cells positive for c-Kit, Flk-1, CD34 and CD31(high) were more abundant than controls at 30 days from vector injection. No significant variations were detected in the pattern of bone marrow cells and of peripheral blood cells at earlier time points. This work demonstrates that new blood vessel formation in the adult, normoperfused muscle can be achieved by constitutive expression of VEGF using AAV vectors. Neovascularization occurs not only by proliferation and migration of endothelial cells from the preexisting vasculature, but also involves a vasculogenesis process through recruitment and differentiation of bone marrow vascular progenitors.
583. Stimulation of Lymphatic Vessel Growth Reduces Edema caused by Adenoviral VEGF Gene Transfer Johanna E. Markkanen,1 Tuomas T. Rissanen,1 Marcin Gruchala,1 Antti Puranen,1 Tommi T. Heikura,1 Marc G. Achen,2 Steven A. Stacker,2 Seppo Ylä-Herttuala.1 1 Department of Molecular Medicine, A.I.V. Institute, Kuopio, Finland; 2Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Australia. Background Vascular permeability and subsequent edema is a considerable side effect of therapeutic angiogenesis. We have previously shown that the proteolytically processed form of VEGFD, VEGF-DΔNΔC, is a powerful angiogenesis factor via VEGFR-2 activation. On the other hand, full length VEGF-Dfull predominantly stimulates lymphangiogenesis through VEGFR-3 and could be used to reduce interstitial edema. Angiopoietin-1 (Ang-1) has been proposed to stabilize newly formed vessels and prevent vascular leakage. Here we compared naked plasmid-mediated (p) gene transfer of VEGF-DΔNΔC with adenoviral (Ad) VEGF-DΔNΔC for therapeutic angiogenesis and vascular permeability in rabbit skeletal muscle. Furthermore, we sought to prevent VEGF-DΔNΔC induced vascular leakage with combination gene therapy using AdAng-1 or to reduce edema with AdVEGF-Dfull. Methods and results AdVEGF-DΔNΔC (1011vp, 1ml) induced 3fold increase in tissue perfusion, 10-fold increase in capillary size and up to 30-fold increase in protein extravasation in rabbit hindlimb muscles compared to control (AdLacZ). Naked plasmid -mediated VEGF-DΔNΔC (500μg) gene transfer was ineffective by all these measures. Although some positive cells were found in needle track areas, human VEGF-DΔNΔC protein levels were under detection limit Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
582. VEGF Gene Transfer Using AAV Vectors Induces Postnatal Neovascularization through Bone Marrow Stem Cell Recruitment Lorena Zentilin,1 Nikola Arsic,1 Sabrina Tafuro,1 Genaro RamirezCorrea,1 Serena Zacchigna,1 Sara Tomasi,1 Lucia Pattarini,1 Gianfranco Sinagra,2 Alessandro Salvi,2 Mauro Giacca.1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 2Cardiology Unit, Ospedale Maggiore, Trieste, Italy. Gene therapy has emerged as a promising new strategy to promote angiogenesis in the treatment of myocardial and peripheral ischemia. In this setting, recombinant AAV-based vectors stand as one of the most efficient systems to achieve stable and safe gene delivery to the myocardium and the skeletal muscle. Using an AAV vector expressing human vascular endothelial growth factor (VEGF), we previously demonstrated a significant increase in capillary density and formation of α-SMA-positive arteriolae in vector-injected normoperfused skeletal muscle (Arsic et al 2003). Moreover, as soon as 7 days after intramuscular injection of the recombinant vector, we observed a remarkable increase in cellularity, S226
with a large number of proliferating cells accumulating and surrounding muscle fibers; only a few of these cells were of inflammatory origin. Some of these infiltrating cells showed positive for the endothelial cell marker CD31, and, most notably, for markers of endothelial progenitor cells and bone-marrow derived stem cells, including Tie-2, Flk-1, c-Kit and Sca-1. Moreover, some cells in these proliferating bulks displayed a tendency to align in cord-like structures and to organize into small, CD31-positive vessels, suggestive of a vasculogenesis process. To understand whether the cells that are locally recruited by constitutive expression of VEGF might originate from the bone marrow, we transplanted unfractionated male bone marrow cells into lethally irradiated female mice. In situ hybridization analysis on AAV-VEGF injected muscle sections of transplanted females actually showed that more than 70% of the infiltrating cells were positive for Y chromosome sequences. This result clearly supports the notion that local expression of VEGF recruits vascular progenitors from the circulation. The capacity of VEGF to mobilize bone marrow precursors was further assessed by flow cytometry on bone marrow and peripheral blood mononuclear cells in mice at 15 and 30 days from AAV-VEGF injection. We obtained evidence that, in peripheral blood, cells positive for c-Kit, Flk-1, CD34 and CD31(high) were more abundant than controls at 30 days from vector injection. No significant variations were detected in the pattern of bone marrow cells and of peripheral blood cells at earlier time points. This work demonstrates that new blood vessel formation in the adult, normoperfused muscle can be achieved by constitutive expression of VEGF using AAV vectors. Neovascularization occurs not only by proliferation and migration of endothelial cells from the preexisting vasculature, but also involves a vasculogenesis process through recruitment and differentiation of bone marrow vascular progenitors.
583. Stimulation of Lymphatic Vessel Growth Reduces Edema caused by Adenoviral VEGF Gene Transfer Johanna E. Markkanen,1 Tuomas T. Rissanen,1 Marcin Gruchala,1 Antti Puranen,1 Tommi T. Heikura,1 Marc G. Achen,2 Steven A. Stacker,2 Seppo Ylä-Herttuala.1 1 Department of Molecular Medicine, A.I.V. Institute, Kuopio, Finland; 2Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Australia. Background Vascular permeability and subsequent edema is a considerable side effect of therapeutic angiogenesis. We have previously shown that the proteolytically processed form of VEGFD, VEGF-DΔNΔC, is a powerful angiogenesis factor via VEGFR-2 activation. On the other hand, full length VEGF-Dfull predominantly stimulates lymphangiogenesis through VEGFR-3 and could be used to reduce interstitial edema. Angiopoietin-1 (Ang-1) has been proposed to stabilize newly formed vessels and prevent vascular leakage. Here we compared naked plasmid-mediated (p) gene transfer of VEGF-DΔNΔC with adenoviral (Ad) VEGF-DΔNΔC for therapeutic angiogenesis and vascular permeability in rabbit skeletal muscle. Furthermore, we sought to prevent VEGF-DΔNΔC induced vascular leakage with combination gene therapy using AdAng-1 or to reduce edema with AdVEGF-Dfull. Methods and results AdVEGF-DΔNΔC (1011vp, 1ml) induced 3fold increase in tissue perfusion, 10-fold increase in capillary size and up to 30-fold increase in protein extravasation in rabbit hindlimb muscles compared to control (AdLacZ). Naked plasmid -mediated VEGF-DΔNΔC (500μg) gene transfer was ineffective by all these measures. Although some positive cells were found in needle track areas, human VEGF-DΔNΔC protein levels were under detection limit Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy