| Project/Area Number |
08457215
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Jichi Medical School |
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Principal Investigator |
SHIMADA Kazuyuki Jichi Medical School, Professer, 医学部, 教授 (90145128)
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| Co-Investigator(Kenkyū-buntansha) |
IROKAWA Masahiko Jichi Medical School, Lecturer, 医学部, 助手 (80296087)
TAKAHASHI Masafumi Jichi Medical School, Lecturer, 医学部, 助手 (40296108)
FUJIKAWA Hideyuki Jichi Medical School, Assistant, 医学部, 講師 (00238544)
IKEDA Uichi Jichi Medical School, Associated Professor, 医学部, 助教授 (30221063)
小口 朝彦 自治医科大学, 医学部, 助手 (10233488)
西永 正典 自治医科大学, 医学部, 助手 (50265245)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥7,900,000 (Direct Cost: ¥7,900,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Keywords | vasular endothelial growth factor / endothelial cell / adeno-associated virus vectors / cardiac cell / nitric oxide synthase / gene transfer / 血管 |
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| Research Abstract |
Backgrounds and Aims. We have previously reported that Adeno-associated virus (AAV) vectors can efficiently transduce vasular and cardiac cells. The purposes of this study are to investigate whether AAV-mediated endothelial constitutive nitric oxide synthase (ecNOS) or vasular endothelial growth factor (VEGF) genes transfer could modulate the vasoconstrictive response and promote regeneration of endothelial cells, respectively.
Methods. We produced ecNOS- and VEGE-expressing AAV vectors (AAV-ecNOS and AAV-VEGF). (1) Excised rat aortas were incubated with medium containing AAV-ecNOS.Expression of ecNOS in aortic segments were evaluated by immunohistochemical staining. Isometric tension of aortic segments transduced with AAV-ecNOS was also measured.
(2) Cultured neonatal rat cardiac myocytes were incubated with AAV-VEGF.VECK expression was analysed by immunoblotting and immunohistochemical staining. Concentration of VEGF' in the cultured medium was measured by ELISA.Human umbilical vein en
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dothelial cells (HUVEC) proliferation was measured by thyinidine incorporation.
Results. (1) The vasoconstrictive response induced by 30 mM KCl was enhanced in endothelium(EC)-denuded aortic segments compared with intact aortic segments. However, in BC-denuded aortic segments transduced with AAV-ecNOS, there was no enhancement of vasoconstrictive response, and the isometric tension of the vessel in response to 30mM KCl returned to the level of intact aortic segments. This effect of ecNOS gene transfer was abolished in the presence of 1mM L-NMMA.(2) Immunoblotting revealed VECF protein expression in transduced cardiac myocytes. Immunohistochemical staining using a VEGF antibody demonstrated that about 60% of cardiac myocytes were stained positively. Concentration of VEGF in the cultured medium increased in a multipecities of infection-dependent manner and reached upto 10 ng/ml. Thymidine incorporation into HUVEC was significantly increased (601.5% of control) by the conditioned medium from transduced cardiac myocytes. This increased thymidine uptake was inhibited in the presense of a VEGF-neutralizing antibody.
Conclusions. ecNOS gene transfer using AAV vectors abolished the pathological enhancement of vasoconstrictive response of EC-denuded aortic segments. This finding suggest that ecNOS gene transfer into vascular structures using AAV vectors may be a feasible approach for gene therapy of vasospastic and atherosclerotic vascular diseases. AAV-mediated VEGF gene transfer into cardiac myocytes induced functional VEGF secretion resulting HUVEC proliferation. Thus VBGF gene transfer into the myocardium might be useful for endothelial regeneration therapy. These gene transfer approaches might be useful to stabilize the unstable plaque. Less
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