- Email: [email protected]
VEGF-C adenovirus gene transfer reduces intima formation in rabbits
Tuesday June 27, 2000: Workshop Abstracts W:8 Gene Therapy and Other New Treatments of the wound healing process to minimize reocclusion of the vessel. I will discuss treatment strategies that have been shown to be efficacious in limiting post-angioplasty restenosis through evaluation with in vivo model systems. Candidate strategies that we have investigated utilize genes encoding factors that are either cytotoxic (Fas ligand and hammerhead ribozyme to Bcl-2) or cytostatic (Rb and p21). We have also investigated the therapeutic utility of a transcriptional regulator of integrin expression that was isolated from smooth muscle cells (Gax). I will also briefly discuss critical issues of delivery with devices and gene control that must be considered for successful application of this therapy.
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VEGF-C adenovirus gene transfer reduces intima formation in rabbits
Mikko O. Hiltunen I , Marja Laitinen I , Mikko P. Tunmen I , Michael Jeltsch 2, Juha Harfikainen 1, Tuomas T. Rissanen 1, Johanna Laukkanen I , Mad Niemi I , Maija KossilaI , Tomi E H~ikkinen t , Antti Kivel ~il, Berndt Enholm 2, Hannu Mansukoski I , Anna-Mad Turunen I , Karl Alitalo2, Seppo Y1 [i-Herttuala I . I A.I. Virtanen Institute, University of Kuopio, Kuopio;
2Molecular Cancer Biology Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland Background: Gene transfer may provide new possibilities for the treatment of postangioplasty restenosis. In this study we analyzed the effects of adenovirus-mediated VEGF-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Methods and Results: Aortas of cholesterol-fed New Zealand White rabbits were balloon denuded and gene transfer was performed three days later. Animals were sacrificed 2 and 4 weeks after the gene transfer and intima/media ratio (I/M), histology and cell proliferation were analyzed. Two weeks after the gene transfer I/M in the lacZ-transfected control group was 0.57 4- 0.04. VEGF-C gene transfer reduced I/M to 0.38 4- 0.02 (P < 0.05 vs. /acZ group). I/M in VEGF-A treated animals was 0.49 =t=0.17 (ns). Expression of VEGF receptors 1, 2 and 3 were detected in the vessel wall by using immunocytochemistry and in situ hybridization. Conclusions: VEGF-C adenovirus gene transfer is effective in reducing intimal thickening. VEGF-C may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.
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Feusibiltty of gene transfer through bone marrow ceils using lentiviral vector
S. Jovinge t,3, A. Harpf2 , L. Bdmtn 3 , P.K. Shah I , T. Rajavashisth1 .
t AtheroscL Res. Center, Cedars-Sinai MC, UCLA, Los Angeles, CA; 2 UCLA CVRL, Los Angeles, CA, USA; 3Dep of Med, Univ. Hospital MAS, University of Lund, Malmt, Sweden Objective: To establish a feasible model for gene-transfer for vascular disease using bone-marrow cell transduction. Methods: We investigated the hypothesis that lentiviral vectors transduce bone-marrow cells (BMC) which, when injected to totally body irradiated (TBI) C57BI/6 mice, result in successful engraftment. A shuttle expression plasmid encoding the enhanced green fluorescent protein (EGFP) under the cytomegalovirus major immediate early promoter/enhancer (CMV) was packaged into lentiviral particles. For, comparison a CD1 lb controlled expression system was used. Results: Transduced bone-marrow cells showed EGFP expression on flowcytometry. EGFP expression was also detected in peripheral blood cells (PBC). The EGFP expression in PBC was detected up to six months after bone marrow transplantation. To increase the specificity of gene-transfer to the vessel wall, the same vector-system was used substituting the CMV-promoter with the monocyte/macrophage specific CD 1 l b-promoter. The relative strength of this promoter was similar to the CMV-promoter in flow-cytometry based assay systems. This later system was used in bone marrow transplantation of apt E deficient mice and EGFP-expression in the atherosclerotic plaques was established demonstrated. Conclusion: We have demonstrated the successful transduction of murine bone marrow cells using lentiviral vectors encoding EGFP. Furthermore, transplantation of bone-marrow transduced with lentiviral vectors driven by CD1 lb promoter, we have also demonstrated successful EGFP expression in atherosclerotic plaques in apt E deficient mice. These findings thus demonstrate the feasibility of bone marrow selective gene transfer with lentiviral vectors targeting the vasculature.
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81
Therapeutic angiogenesis induced by HGF: Potential gene therapy for ischemic diseases
Motokuni Aoki I , Ryuichi Morishita2, Yoshiaki Taniyarna I , Keita Yamasaki t , Yasufumi Kaneda2 , Toshio Ogihara 1. 1Department of Geriatric Medicine;
2Division of Gene Therapy Science, Osaka University Medical School, Suitu, Japan Objectives: The feasibility of a novel therapeutic strategy using angiogenic growth factors by expediting collateral artery development has recently entered the realm of treatment of ischemic diseases. In USA, human gene therapy for angina and ASO has already begun and it gives a surprising effect. We already reported that HGF has a powerful effect on the proliferation of endothelial cells in vitro. In this study, we hypothesized the transfection of HGF gene into ischemic hindlimbs and infarcted hearts could induce angiogenesis, potentially resulting in a beneficial response to ischemia. Methods & Results: Human HGF gene or control vector were transfected into ischemic limbs and myocardium by HVJ-liposome method. Although the concentration of endogenous HGF in ischemic hindiimbs and infarcted hearts were siginificantly decreased, this transfection showed a marked increase in rat immunoreactive HGF, accompanied by the over-expression of human HGF. In the myocardium transfected with HGF gene, a significant increase in PCNA-positive endothelial cells and the number of vessels could be observed at 14 days after transfection. Angiogenic activity was also confirmed by the activation of a transcription factor, ets, which is essential for angiogenesis, assessed by immunohistochemistry and electrophoretic mobility shift assay. Also, the complimented HGF by the transfection into the ischemic hindlimbs showed a significant increase in the number of vessels, resulting in a significant increase in blood flow assessed by Laser Doppler Image. Conclusion: The constant production of the local HGF will be considered as an innovative therapeutic angiogenesis strategy for ischemic diseases.
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Optimisation of in vivo arterial transfection
E. O'Brien, X. Ma, C: Glover, H. Miller. Ottawa Heart Institute, Canada
Objective: Gene therapy for the treatment of vascular disease is limited by low transfection efficiency and/or undesired biological responses (e.g., with viral vectors). The purpose of this study was to determine an efficient method of delivering liposome/DNA complexes into balloon-injured rabbit iliac arteries using a delivery catheter. Methods: Cationic liposomes were made from a 1:1 (wt/wt) mixture of DOTAP and DOPE. The plasmid pCMV-AP containing the human placenta alkaline phosphatase (AP) reporter gene was used as a marker gene for these experiments. Prior to initiating the in vivo experiments, the optimal ratio of liposome to DNA complex, as well as the persistence of transgene expression were determined in vitro using cultured vascular SMCs. The liposome/DNA complex was then delivered under pressure using a Dispatch catheter to rabbit iliac arteries that were balloon injured 5 days prior gene delivery. Transfection efficiency was defined as the percentage of transfected cells/total cells per high power field. Results: The optimal ratio of liposome to DNA was 8:1 (wt/wt). AP expression in transfected SMCs persisted for 28 days, although the percentage of transfected cells declined with time (e.g., at 24 hours: 27.3% =k 2.9%; at 28 days: 0.4% =t= 0.1%). The peak transfection efficiency in cultured smooth muscle cells was seen at 24 hours post-transfection. As well, smooth muscle cell proliferation in vitro enhanced the transfection efficiency (e.g., 12.6 fold higher than quiescent cells). In vivo experiments were performed on 9 balloon injured rabbit iliac arteries, with half of the arteries receiving the pCMV-AP plasmid and the other half receiving the liposome only (no plasmid). Low levels of transfection were observed in arteries harvested 1 day post delivery. However, 6/7 arteries harvested 3 days post-delivery had multiple regions of focal transgene expression involving all 3 arterial layers. No gene expression was found in the uninjured aorta. Conclusion: Liposome mediated gene transfectiun to all vessel layers can successfully be performed in vivo using local delivery, and may provide an ideal means of targeting vascular disease processes.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
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VEGF-C adenovirus gene transfer reduces intima formation in rabbits
Mikko O. Hiltunen I , Marja Laitinen I , Mikko P. Tunmen I , Michael Jeltsch 2, Juha Harfikainen 1, Tuomas T. Rissanen 1, Johanna Laukkanen I , Mad Niemi I , Maija KossilaI , Tomi E H~ikkinen t , Antti Kivel ~il, Berndt Enholm 2, Hannu Mansukoski I , Anna-Mad Turunen I , Karl Alitalo2, Seppo Y1 [i-Herttuala I . I A.I. Virtanen Institute, University of Kuopio, Kuopio;
2Molecular Cancer Biology Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland Background: Gene transfer may provide new possibilities for the treatment of postangioplasty restenosis. In this study we analyzed the effects of adenovirus-mediated VEGF-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Methods and Results: Aortas of cholesterol-fed New Zealand White rabbits were balloon denuded and gene transfer was performed three days later. Animals were sacrificed 2 and 4 weeks after the gene transfer and intima/media ratio (I/M), histology and cell proliferation were analyzed. Two weeks after the gene transfer I/M in the lacZ-transfected control group was 0.57 4- 0.04. VEGF-C gene transfer reduced I/M to 0.38 4- 0.02 (P < 0.05 vs. /acZ group). I/M in VEGF-A treated animals was 0.49 =t=0.17 (ns). Expression of VEGF receptors 1, 2 and 3 were detected in the vessel wall by using immunocytochemistry and in situ hybridization. Conclusions: VEGF-C adenovirus gene transfer is effective in reducing intimal thickening. VEGF-C may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.
•
Feusibiltty of gene transfer through bone marrow ceils using lentiviral vector
S. Jovinge t,3, A. Harpf2 , L. Bdmtn 3 , P.K. Shah I , T. Rajavashisth1 .
t AtheroscL Res. Center, Cedars-Sinai MC, UCLA, Los Angeles, CA; 2 UCLA CVRL, Los Angeles, CA, USA; 3Dep of Med, Univ. Hospital MAS, University of Lund, Malmt, Sweden Objective: To establish a feasible model for gene-transfer for vascular disease using bone-marrow cell transduction. Methods: We investigated the hypothesis that lentiviral vectors transduce bone-marrow cells (BMC) which, when injected to totally body irradiated (TBI) C57BI/6 mice, result in successful engraftment. A shuttle expression plasmid encoding the enhanced green fluorescent protein (EGFP) under the cytomegalovirus major immediate early promoter/enhancer (CMV) was packaged into lentiviral particles. For, comparison a CD1 lb controlled expression system was used. Results: Transduced bone-marrow cells showed EGFP expression on flowcytometry. EGFP expression was also detected in peripheral blood cells (PBC). The EGFP expression in PBC was detected up to six months after bone marrow transplantation. To increase the specificity of gene-transfer to the vessel wall, the same vector-system was used substituting the CMV-promoter with the monocyte/macrophage specific CD 1 l b-promoter. The relative strength of this promoter was similar to the CMV-promoter in flow-cytometry based assay systems. This later system was used in bone marrow transplantation of apt E deficient mice and EGFP-expression in the atherosclerotic plaques was established demonstrated. Conclusion: We have demonstrated the successful transduction of murine bone marrow cells using lentiviral vectors encoding EGFP. Furthermore, transplantation of bone-marrow transduced with lentiviral vectors driven by CD1 lb promoter, we have also demonstrated successful EGFP expression in atherosclerotic plaques in apt E deficient mice. These findings thus demonstrate the feasibility of bone marrow selective gene transfer with lentiviral vectors targeting the vasculature.
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81
Therapeutic angiogenesis induced by HGF: Potential gene therapy for ischemic diseases
Motokuni Aoki I , Ryuichi Morishita2, Yoshiaki Taniyarna I , Keita Yamasaki t , Yasufumi Kaneda2 , Toshio Ogihara 1. 1Department of Geriatric Medicine;
2Division of Gene Therapy Science, Osaka University Medical School, Suitu, Japan Objectives: The feasibility of a novel therapeutic strategy using angiogenic growth factors by expediting collateral artery development has recently entered the realm of treatment of ischemic diseases. In USA, human gene therapy for angina and ASO has already begun and it gives a surprising effect. We already reported that HGF has a powerful effect on the proliferation of endothelial cells in vitro. In this study, we hypothesized the transfection of HGF gene into ischemic hindlimbs and infarcted hearts could induce angiogenesis, potentially resulting in a beneficial response to ischemia. Methods & Results: Human HGF gene or control vector were transfected into ischemic limbs and myocardium by HVJ-liposome method. Although the concentration of endogenous HGF in ischemic hindiimbs and infarcted hearts were siginificantly decreased, this transfection showed a marked increase in rat immunoreactive HGF, accompanied by the over-expression of human HGF. In the myocardium transfected with HGF gene, a significant increase in PCNA-positive endothelial cells and the number of vessels could be observed at 14 days after transfection. Angiogenic activity was also confirmed by the activation of a transcription factor, ets, which is essential for angiogenesis, assessed by immunohistochemistry and electrophoretic mobility shift assay. Also, the complimented HGF by the transfection into the ischemic hindlimbs showed a significant increase in the number of vessels, resulting in a significant increase in blood flow assessed by Laser Doppler Image. Conclusion: The constant production of the local HGF will be considered as an innovative therapeutic angiogenesis strategy for ischemic diseases.
•
Optimisation of in vivo arterial transfection
E. O'Brien, X. Ma, C: Glover, H. Miller. Ottawa Heart Institute, Canada
Objective: Gene therapy for the treatment of vascular disease is limited by low transfection efficiency and/or undesired biological responses (e.g., with viral vectors). The purpose of this study was to determine an efficient method of delivering liposome/DNA complexes into balloon-injured rabbit iliac arteries using a delivery catheter. Methods: Cationic liposomes were made from a 1:1 (wt/wt) mixture of DOTAP and DOPE. The plasmid pCMV-AP containing the human placenta alkaline phosphatase (AP) reporter gene was used as a marker gene for these experiments. Prior to initiating the in vivo experiments, the optimal ratio of liposome to DNA complex, as well as the persistence of transgene expression were determined in vitro using cultured vascular SMCs. The liposome/DNA complex was then delivered under pressure using a Dispatch catheter to rabbit iliac arteries that were balloon injured 5 days prior gene delivery. Transfection efficiency was defined as the percentage of transfected cells/total cells per high power field. Results: The optimal ratio of liposome to DNA was 8:1 (wt/wt). AP expression in transfected SMCs persisted for 28 days, although the percentage of transfected cells declined with time (e.g., at 24 hours: 27.3% =k 2.9%; at 28 days: 0.4% =t= 0.1%). The peak transfection efficiency in cultured smooth muscle cells was seen at 24 hours post-transfection. As well, smooth muscle cell proliferation in vitro enhanced the transfection efficiency (e.g., 12.6 fold higher than quiescent cells). In vivo experiments were performed on 9 balloon injured rabbit iliac arteries, with half of the arteries receiving the pCMV-AP plasmid and the other half receiving the liposome only (no plasmid). Low levels of transfection were observed in arteries harvested 1 day post delivery. However, 6/7 arteries harvested 3 days post-delivery had multiple regions of focal transgene expression involving all 3 arterial layers. No gene expression was found in the uninjured aorta. Conclusion: Liposome mediated gene transfectiun to all vessel layers can successfully be performed in vivo using local delivery, and may provide an ideal means of targeting vascular disease processes.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000