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）载体可以有效地转染血管和心肌细胞。本研究旨在探讨腺相关病毒（AAV）介导的内皮型一氧化氮合酶（ecNOS）和血管内皮生长因子（VEGF）基因转染是否能分别调节血管收缩反应和促进内皮细胞再生。我们产生了表达ecNOS和VEGF的AAV载体（AAV-ecNOS和AAV-VEGF）。(1)将离体大鼠腹主动脉与含AAV-ecNOS的培养液共同孵育，免疫组化染色观察ecNOS在主动脉段的表达。还测量了用AAV-ecNOS转导的主动脉段的等距张力。(2)培养的乳鼠心肌细胞与AAV-VEGF共同孵育，用免疫印迹和免疫组化染色法检测VECK的表达。ELISA法检测培养液中VEGF的含量。 关于我们 用胸腺嘧啶核苷比色法检测内皮细胞（HUVEC）增殖。(1)与完整的主动脉段相比，30 mM KCl诱导的血管收缩反应在内皮（EC）剥脱的主动脉段中增强。然而，在去BC的主动脉段中，AAV-ecNOS转导的血管收缩反应没有增强，并且血管对30 mM KCl的反应的等长张力恢复到完整主动脉段的水平。在1 mM L-NMMA存在下，ecNOS基因转移的这种作用被消除。(2)免疫印迹显示VECF蛋白在转导的心肌细胞中表达。用VEGF抗体进行免疫组化染色，结果显示约60%的心肌细胞呈阳性染色。培养液中VEGF浓度呈多特异性感染依赖性增加，最高可达10 ng/ml。胸苷掺入HUVEC显着增加（601.5%的控制）由转导心肌细胞的条件培养基。这种增加的胸苷摄取在VEGF中和抗体的存在下被抑制。用AAV载体转移ecNOS基因可消除去内皮主动脉段血管收缩反应的病理性增强。这一发现表明，ecNOS基因转移到血管结构，利用AAV载体可能是一个可行的方法，基因治疗血管痉挛和动脉粥样硬化性血管疾病。AAV介导的VEGF基因转移到心肌细胞中诱导功能性VEGF分泌，导致HUVEC增殖。因此，VBGF基因转染心肌可能是有用的内皮再生治疗。这些基因转移方法可能有助于稳定不稳定斑块。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