Mechanisms of HAART-Induced Endothelial Dysfunction

描述(由申请人提供)： 拟议研究的主要目标是探讨高效抗逆转录病毒疗法(HAART)诱导的内皮细胞功能障碍的分子机制/S。我们发现HIV-1蛋白抑制物(PI)抑制成脂分化，炎性细胞因子TNFpha和HIV-1 Tat蛋白影响内皮祖细胞间充质祖细胞(MPC)的克隆形成能力和分化潜能。我们还发现，在治疗浓度下，HAART(齐多夫定、伊法韦仑、吲哚那韦或奈非那韦)可抑制胰岛素诱导的人主动脉内皮细胞(HAECs)NO的产生和eNOS基因的表达，这一作用可被TNFpha进一步降低。HAART增加白细胞/单核细胞黏附并诱导细胞黏附分子(CAM)基因表达，而在TNFpha存在下，这一作用进一步增强。因此，我们建议测试这一假说，即长期暴露于HAART药物改变了胰岛素调节血管内皮细胞产生NO的作用，而血管内皮细胞产生的NO进一步被TNFpha和HIV-1Tat蛋白抑制。为了验证这一假说，将达到以下特定目的：(1)确定HAART和TNFpha对内皮细胞-白细胞黏附和迁移的影响。HUVECs和HAECs在存在或不存在TNFpha和/或Tat蛋白的情况下，将随时间暴露于不同浓度的抗逆转录病毒药物。将测定白细胞/单核细胞与内皮细胞的黏附和跨内皮细胞迁移的变化。细胞黏附分子(V-CAM、I-CAM和E-选择素)的表达和金属蛋白酶(MMPs)的激活将分别用RT-PCR和ELISA法以及酶谱法检测。CAM特异性中和抗体和/或基质金属蛋白酶抑制剂的作用将被确定。(2)观察HAART对骨髓基质细胞增殖和分化的影响。细胞将暴露于单一药物和/或HAART、TNFpha和HIV-1 Tat蛋白的组合中，并将监测向内皮细胞系的分化。将确定在分化MPC过程中对白细胞黏附和NO产生的影响。(3)阐明HAART致内皮细胞功能障碍的分子机制。我们将检验这样一种假设，即HAART药物单独或联合使用可阻断胰岛素信号，从而减少内皮细胞中NO的产生并增加氧化应激。HAART药物对胰岛素信号转导的影响将通过胰岛素受体底物(IRS-1)磷酸化的免疫标记以及转录因子、核因子-kappaB和AP-1的瞬时转染和凝胶迁移率改变分析来确定，并将通过逆转HAART诱导的白细胞与胰岛素增敏剂的黏附来确定。HAART药物对内皮细胞NO和ROS产生的影响将在NO供体存在或不存在的情况下进行监测，NO合成酶抑制剂、自由基猝灭剂和胰岛素增敏剂将被确定在克服HAART和TNFpha诱导的EC功能障碍方面的作用。(4)研究慢性HAART暴露对小鼠内皮细胞功能障碍的影响。为了在体内验证所提出的假设，将使用三组不同的小鼠(A)对照(B)TNFpha受体敲除和(C)Tat转基因。在给予或不给予HAART和/或NO供体或NOS抑制剂的情况下，将确定每组小鼠的EC功能障碍、高胰岛素血症和高血糖。这些研究应该为我们的假设提供证据，即HAART干扰NO的产生会导致内皮功能障碍，而在存在TNFpha和/或TAT蛋白时，内皮功能障碍会进一步加剧。

DESCRIPTION (provided by applicant): The primary objective of the proposed research is to investigate the molecular mechanism/s involved in highly active anti-retroviral therapy (HAART)-induced dysfunctions in endothelial cells (ECs). We have shown that HIV-1 protease inhibitors (PIs) suppress adipogenic differentiation and that the inflammatory cytokine TNFalpha and HIV-1 Tat protein affect the clonogenic ability and differentiation potential of the endothelial precursors, mesenchymal progenitor cells (MPCs). We have also shown that at therapeutic concentrations, HAART (zidovudine, efavirenz, and indinavir or nelfinavir) exposure of human aortic ECs (HAECs) suppressed insulin induced NO production and eNOS gene expression, which was further decreased by TNFalpha HAART increased leukocyte/monocyte adhesion and induced cell adhesion molecule (CAM) gene expression, which was further enhanced in the presence of TNFalpha. We are therefore proposing to test the hypothesis that chronic exposure to HAART drugs alters the action of insulin in regulating NO production by the vascular endothelial cells which is further inhibited by TNFalpha and HIV-1 Tat protein. To test this hypothesis the following specific aims will be achieved: (1) To determine the effects of HAART and TNFalpha on endothelial-leukocyte adhesion and migration. HUVECs and HAECs will be exposed to varying concentrations of anti-retroviral drugs as a function of time in the presence or absence of TNFalpha and/or Tat protein. The changes in leukocyte/monocyte adhesion to ECs and trans-endothelial migration (TEM) will be determined. Expression of cell adhesion molecules (V-CAM, I-CAM and E-selectin), and activation of metalloproteases (MMPs) in ECs will be determined by RTPCR and ELISA, and zymography, respectively. The role of CAM specific neutralizing antibodies and/or MMP inhibitors will be determined. (2) To determine the effects of HAART on proliferation and differentiation of marrow derived MPCs. Cells will be exposed to single drugs and/or combinations of HAART, TNFalpha and HIV-1 Tat protein and differentiation towards endothelial lineage will be monitored. Effects on leukocyte adhesion and NO production in differentiating MPCs will be determined. (3) To delineate the molecular mechanisms of HAART induced endothelial dysfunction. We will test the hypothesis that HAART drugs, individually or in combination abrogate insulin signaling thus decreasing NO production and increasing oxidative stress in the ECs. Effect of HAART drugs on insulin signaling will be determined by immunolabeling of insulin receptor substrate (IRS-1) phosphorylation and by transient transfection and gel mobility shift assays for transcription factors, NF-kappaB and AP-1 and reversal of HAART induced leukocyte adhesion with insulin sensitizers will be determined. Effect of HAART drugs on NO and ROS production in ECs will be monitored in the presence or absence of NO-donors, NO synthase inhibitor, free radical quencher and insulin sensitizers in overcoming the HAART and TNFalpha induced EC dysfunctions will be determined. (4) To determine the in vivo effects of chronic HAART exposure on EC dysfunction in mice. To test the proposed hypotheses in vivo, three different groups of mice will be used (a) control (b) TNFalpha receptor knockout and (c) Tat transgenic. EC dysfunction, hyperinsulinemia and hyperglycemia in each group of mice will be determined with or without the administration of HAART and/or NO donor or NOS inhibitor. These studies should provide evidence in support of our hypotheses that disruption of NO production by HAART results in endothelial dysfunction, which is further exacerbated in the presence of TNFalpha and/or Tat protein.