Stabiligation of vulnerable plaque by gene transter.

背景和目标。我们以前报道过腺相关病毒（AAV）载体可以有效地转导血管和心脏细胞。本研究旨在探讨aav介导的内皮型一氧化氮合酶（ecNOS）和血管内皮生长因子（VEGF）基因的转移是否能分别调节血管收缩反应和促进内皮细胞再生。我们制备了表达ecNOS和vegf的AAV载体（AAV-ecNOS和AAV- vegf）。(1)将切除的大鼠主动脉用含AAV-ecNOS的培养基孵育。免疫组化染色检测主动脉段ecNOS的表达。AAV-ecNOS诱导的主动脉段等长张力也被测量。(2)用AAV-VEGF培养新生大鼠心肌细胞。免疫印迹和免疫组织化学染色分析VECK的表达。采用酶联免疫吸附法测定培养液中VEGF'的浓度。用胸腺苷掺入法测定人脐静脉内皮细胞（HUVEC）增殖。(1)与完整主动脉段相比，30 mM KCl诱导的内皮剥脱主动脉段血管收缩反应增强。然而，在用AAV-ecNOS转导的bc -剥脱主动脉段中，血管收缩反应没有增强，血管的等长张力在30mM KCl的作用下恢复到完整主动脉段的水平。在1mM L-NMMA的存在下，这种效应被消除。(2)免疫印迹法显示了VECF蛋白在转导心肌细胞中的表达。VEGF抗体免疫组化染色显示约60%的心肌细胞呈阳性。VEGF在培养液中的浓度呈多种感染依赖性增加，最高可达10 ng/ml。通过转导心肌细胞的条件培养基，胸苷在HUVEC中的掺入量显著增加（为对照组的601.5%）。这种增加的胸苷摄取在vegf中和抗体的存在下被抑制。AAV载体转染ecNOS基因可消除ec剥脱主动脉段血管收缩反应的病理增强。这一发现提示，利用AAV载体将ecNOS基因转移到血管结构中可能是一种可行的血管痉挛和动脉粥样硬化性血管疾病的基因治疗方法。aav介导的血管内皮生长因子基因转入心肌细胞，诱导血管内皮生长因子的功能分泌，导致HUVEC增殖。因此，将VBGF基因转移到心肌中可能有助于内皮细胞再生治疗。这些基因转移方法可能有助于稳定不稳定的斑块。少

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 … More 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