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629. Recombinant AAV Vector-Mediated Human VEGF165 Gene Transfer Stimulates Agiogenesis and Wound Healing in the Genetically Diabetic Mice
CARDIOVASCULAR of vascular endothelial growth factor 121 (AdVEGF121) would increase perfusion and function of the non-ischemic myocardium. Normal rabbits were administered AdVEGF121 or an empty expression (AdNull) vector (2x109 pu in 25 μl) or saline (25 μl) via direct myocardial injection. Histologic and myocardial perfusion studies [2x106 microspheres (15 μl) via left atrial injection] were performed 4 wk later. Microsphere data were used to generate a relative perfusion index calculated as the ratio of microspheres/gm of tissue from the injected region versus the number of microspheres/ gm of tissue from an untreated region (upper septum). Exercise treadmill testing was performed 2 wk after a similar injection regimen in rats (n=8/group) to assess cardiac performance. Four wk after treatment, the myocardial perfusion index was significantly greater (p < 0.05) in AdVEGF121 treated animals (n=14, 1.02 ± 0.04) compared to AdNull (n=13, 0.88 ± 0.05) or saline-treated animals (n=9, 0.81 ± 0.03). Mean endothelial cell number per high power field in the treated myocardium was also significantly greater (p <0.001) in AdVEGF121-treated animals (143 ± 4) compared to AdNull (121 ± 4) or saline-injected animals (120 ± 4). AdVEGF121treated rats also demonstrated significantly prolonged exercise tolerance (p<0.01) 2 wk post-injection (average time to exhaustion 26 ± 2 min) compared to AdNull (19 ± 1 min) or saline-injected control rats (20 ± 1 min). We conclude that adenovirus-mediated transfer of VEGF 121 enhances myocardial perfusion in the nonischemic heart, which appears to allow for greater exercise tolerance. Enhancement of myocardial perfusion with AdVEGF may be useful for inducing angiogenesis in the non-ischemic state, and may consequently represent a prophylactic therapy for patients at risk for coronary artery disease. Dr. Crystal has equity in, is a consultant to, and receives sponsored research funds from, GenVec, Inc., Gaithersburg, Maryland, a publicly-traded biotechnology company.
629. Recombinant AAV Vector-Mediated Human VEGF165 Gene Transfer Stimulates Agiogenesis and Wound Healing in the Genetically Diabetic Mice Barbara Deodato,1 Mariarosaria Galeano,2 Domenica Altavilla,1 Domenico Cucinotta,3 Nikola Arsic,4 Herbert Marini,1 Valerio Torre,5 Mauro Giacca,4 Francesco Squadrito.1 1 Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, University of Messina, Messina, Italy; 2Department of Surgical Sciences, Section of Plastic Surgery, University of Messina, Messina, Italy; 3 Department of Internal Medicine, Section of Metabolic Diseases, University of Messina, Messina, Italy; 4Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 5Unit of Pathology, F. Veneziale Hospital, Isernia, Italy. It has been widely demonstrated that inefficient angiogenesis is one of the main mechanisms underlying the wound healing disorders associated with diabetes. Experimental evidences indicate that in such conditions an altered expression pattern of Vascular Endothelial Growth Factor (VEGF) plays a pivotal role in the impairment of neovascularization observed in chronic diabetic ulcers. Here we explore the efficacy of gene therapy of diabetes-related skin repair disorders with a recombinant Adeno-Associated Virus (AAV) vector expressing the 165 amino acid isoform of VEGF-A (rAAVVEGF165). Diabetic C57BL/KsJ db+/+ mice and their normal littermates (db+/ + m) were randomized to receive intradermally into the edges of fullthickness incisional skin wounds, either recombinant AAV-Lac-Z (rAAV-LacZ) or rAAV-VEGF165. Wounded skin specimens, collected at different time points after the delivery of the vectors, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
were used for gene marker studies, histological and immunohistochemical evaluation and wound breaking strength analysis. By β-galactosidase activity assay, we found that AAV vectors are highly efficient for gene transfer to the mouse skin. Moreover, injection of rAAV-VEGF165 into the wounds resulted in a remarkable increase of the tissue content of the mature protein. This phenomenon was associated with a significant induction of new vessel formation, with consequent reduction of the healing time. Histological examination of rAAV-VEGF165 treated wounds revealed an improvement in reepithelization, granulation tissue formation, synthesis and organization of extracellular matrix and wound breaking strength. Our study suggests that VEGF165 gene transfer might represent a novel therapeutic approach to treat wound healing disorders associated with diabetes.
630. Transfection of Decoy for Transcription Factor CCAAT/Enhancer Binding Protein Attenuates Vascular Inflammation and Neointima Formation in Mice Weihua Ni,1 Deling Kong,1 Lunan Zhang,1 Choong-Chin 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. Neointimal thickening and restenosis after angioplasty remain a major problem limiting the efficacy of PTCA and bypass surgery. Recently we and others have reported that vascular injury induces inflammatory gene expression which plays an important role in the development of vascular disease. It is noteworthy that many of these inflammatory genes bear the binding sites for CCAAT/enhancer binding protein (C/EBP) in their promoter regions. We have developed synthetic double stranded cis-element oligonucleotides decoy to C/EBP and demonstrated its effectiveness in blocking C/ EBP interaction with its binding sequence of target genes in vitro. In this study, we hypothesized that vascular injury-induced inflammatory response may be partially mediated by the activation of C/EBP signaling pathway, and that C/EBP decoy could inhibit the transactivation of target genes and attenuate vascular inflammation and proliferation in vivo. Non-constricting cuff placed around the femoral arteries of C57BL/6J mice resulted in rapid and extended induction of several inflammatory gene expressions such as IL-6, MCP-1, TNFalpha, IL-1beta as shown by a semiquantitative RT-PCR. Cuff-placement also induced marked neointimal hyperplasia after 2 or 4 weeks after cuff-placement. Perivascular transfection of FITC-labeled C/EBP decoy oligos (5 or 10 uM) using a Polyxamer Pluronic F127 gel resulted in a widespread distribution of fluorescent nuclear signals in the medial layer and adventitia. C/EBP decoy treatment also markedly decreased cuff injury-induced IL-6 and MCP-1 gene expression at day 1, 3, 5, 7, 14 and 28 after injury. Morphometric analysis after 28 days revealed that C/EBP decoy treatment significantly reduced neointimal area compared with untreated or scrambled decoy treated vessels. In summary, our data provide new evidence that C/EBP family of transcription factors play an important role in vascular injuryinduced inflammation and subsequent neointimal hyperplasia. The data also suggest that C/EBP may be a useful molecular target for the therapy of inflammatory vascular diseases.
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629. Recombinant AAV Vector-Mediated Human VEGF165 Gene Transfer Stimulates Agiogenesis and Wound Healing in the Genetically Diabetic Mice Barbara Deodato,1 Mariarosaria Galeano,2 Domenica Altavilla,1 Domenico Cucinotta,3 Nikola Arsic,4 Herbert Marini,1 Valerio Torre,5 Mauro Giacca,4 Francesco Squadrito.1 1 Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, University of Messina, Messina, Italy; 2Department of Surgical Sciences, Section of Plastic Surgery, University of Messina, Messina, Italy; 3 Department of Internal Medicine, Section of Metabolic Diseases, University of Messina, Messina, Italy; 4Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 5Unit of Pathology, F. Veneziale Hospital, Isernia, Italy. It has been widely demonstrated that inefficient angiogenesis is one of the main mechanisms underlying the wound healing disorders associated with diabetes. Experimental evidences indicate that in such conditions an altered expression pattern of Vascular Endothelial Growth Factor (VEGF) plays a pivotal role in the impairment of neovascularization observed in chronic diabetic ulcers. Here we explore the efficacy of gene therapy of diabetes-related skin repair disorders with a recombinant Adeno-Associated Virus (AAV) vector expressing the 165 amino acid isoform of VEGF-A (rAAVVEGF165). Diabetic C57BL/KsJ db+/+ mice and their normal littermates (db+/ + m) were randomized to receive intradermally into the edges of fullthickness incisional skin wounds, either recombinant AAV-Lac-Z (rAAV-LacZ) or rAAV-VEGF165. Wounded skin specimens, collected at different time points after the delivery of the vectors, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
were used for gene marker studies, histological and immunohistochemical evaluation and wound breaking strength analysis. By β-galactosidase activity assay, we found that AAV vectors are highly efficient for gene transfer to the mouse skin. Moreover, injection of rAAV-VEGF165 into the wounds resulted in a remarkable increase of the tissue content of the mature protein. This phenomenon was associated with a significant induction of new vessel formation, with consequent reduction of the healing time. Histological examination of rAAV-VEGF165 treated wounds revealed an improvement in reepithelization, granulation tissue formation, synthesis and organization of extracellular matrix and wound breaking strength. Our study suggests that VEGF165 gene transfer might represent a novel therapeutic approach to treat wound healing disorders associated with diabetes.
630. Transfection of Decoy for Transcription Factor CCAAT/Enhancer Binding Protein Attenuates Vascular Inflammation and Neointima Formation in Mice Weihua Ni,1 Deling Kong,1 Lunan Zhang,1 Choong-Chin 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. Neointimal thickening and restenosis after angioplasty remain a major problem limiting the efficacy of PTCA and bypass surgery. Recently we and others have reported that vascular injury induces inflammatory gene expression which plays an important role in the development of vascular disease. It is noteworthy that many of these inflammatory genes bear the binding sites for CCAAT/enhancer binding protein (C/EBP) in their promoter regions. We have developed synthetic double stranded cis-element oligonucleotides decoy to C/EBP and demonstrated its effectiveness in blocking C/ EBP interaction with its binding sequence of target genes in vitro. In this study, we hypothesized that vascular injury-induced inflammatory response may be partially mediated by the activation of C/EBP signaling pathway, and that C/EBP decoy could inhibit the transactivation of target genes and attenuate vascular inflammation and proliferation in vivo. Non-constricting cuff placed around the femoral arteries of C57BL/6J mice resulted in rapid and extended induction of several inflammatory gene expressions such as IL-6, MCP-1, TNFalpha, IL-1beta as shown by a semiquantitative RT-PCR. Cuff-placement also induced marked neointimal hyperplasia after 2 or 4 weeks after cuff-placement. Perivascular transfection of FITC-labeled C/EBP decoy oligos (5 or 10 uM) using a Polyxamer Pluronic F127 gel resulted in a widespread distribution of fluorescent nuclear signals in the medial layer and adventitia. C/EBP decoy treatment also markedly decreased cuff injury-induced IL-6 and MCP-1 gene expression at day 1, 3, 5, 7, 14 and 28 after injury. Morphometric analysis after 28 days revealed that C/EBP decoy treatment significantly reduced neointimal area compared with untreated or scrambled decoy treated vessels. In summary, our data provide new evidence that C/EBP family of transcription factors play an important role in vascular injuryinduced inflammation and subsequent neointimal hyperplasia. The data also suggest that C/EBP may be a useful molecular target for the therapy of inflammatory vascular diseases.
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