Effects of HIV Protease Inhibitors of Endothelial Barrie

描述(由申请人提供)： 作为高效抗逆转录病毒疗法(HAART)的一部分，HIV蛋白酶抑制剂已经改善了HIV的病程。然而，治疗的结果是出现了新的临床并发症，包括外周和冠状动脉疾病，以及代谢障碍。我们的初步研究表明，HIV蛋白酶抑制剂可能损害内皮屏障功能，下调occludin的表达，增加活性氧(ROS)的产生，并激活某些类型的Motigen激活的蛋白激酶(MAPKs)。因此，进一步的研究有三个特定的目的：1.确定HIV蛋白酶抑制剂对内皮细胞通透性的影响。根据我们的初步数据，我们假设HIV蛋白酶抑制剂会增加内皮细胞的通透性。因此，我们将在体外进一步研究培养的人冠状动脉内皮细胞(HCAECs)细胞内紧密连接的形成和维持。利用该系统，我们将评估HIV蛋白水解酶抑制剂对跨内皮细胞电阻(TER)和细胞旁流量的影响。接下来，使用完整的猪动脉灌流模型，我们将通过测量金颗粒向血管壁的运输并对这些结果进行模拟，来定量评估内皮层通透性的变化。2.评价HIV蛋白水解酶抑制剂对内皮细胞紧密连接结构和基因表达的影响。具体地说，我们认为HIV蛋白酶抑制剂改变了紧密连接分子occludin、ZO-1和肌动蛋白的基因表达。这些假说得到了我们初步发现的支持。在这项拟议的研究中，我们将进一步表征HIV蛋白酶抑制剂对这些紧密连接分子定位的影响。基因表达水平将通过蛋白质和信使核糖核酸测量来评估。还将在HCAEC单层和猪颈动脉灌注培养模型中研究基因转录速率、mRNA稳定性和启动子活性。3.明确HIV蛋白水解酶抑制剂诱导内皮细胞通透性改变的信号转导途径。我们假设HIV蛋白酶抑制剂可能：1)改变紧密连接蛋白的磷酸化水平；2)通过ROS、MAPKs、蛋白激酶C(PKC)、蛋白酪氨酸激酶(PTK)和蛋白酪氨酸磷酸酶(PTPh)等信号转导途径诱导内皮细胞通透性增加。我们的初步结果再次为这一假设提供了支持。在这项拟议的研究中，我们将使用HCAEC单层和猪颈动脉灌注模型来研究occludin、ZO-1和肌动蛋白的磷酸化。我们将测量ROS的产生，并评估蛋白激酶和PTPh在观察到的通透性变化中的作用。综上所述，这些研究将有助于识别和建立HIV蛋白酶抑制剂诱导的内皮通透性改变的新机制，从而提高我们对HAART相关心血管疾病机制的理解。我们的发现还可能提出新的治疗策略，最终可能被证明对预防和治疗这些并发症有效。

DESCRIPTION (provided by applicant): The HIV protease inhibitors as a part of highly active antiretroviral therapy (HAART) have improved the course of HIV disease. However, new clinical complications have emerged as a result of the therapy, including peripheral and coronary arterial diseases, and metabolic disturbances. Our preliminary studies suggest that the HIV protease inhibitors may impair endothelial barrier function, down regulate occludin expression, increase reactive oxygen species (ROS) production, and activate certain type of motigen activated protein kinases (MAPKs). Thus, further investigations are proposed with three specific aims: 1. Determine the effect of HIV protease inhibitors on endothelial permeability. Based upon our preliminary data, we hypothesize that HIV protease inhibitors will increase endothelial cell permeability. Accordingly, we will further characterize in vitro the formation and maintenance of tight intracellular junctions by cultured human coronary artery endothelial cells (HCAECs). Using this system we will evaluate the effects of HIV protease inhibitors on transendothelial electrical resistance (TER) and paracellular flux. Next, using an intact porcine artery perfusion model, we will quantitatively assess changes in endothelial layer permeability by measuring gold particle transport into the vessel wall and modeling of these results. 2. Assess the effects of HIV protease inhibitors on endothelial tight junction structures and gene expression. Specifically, we propose that HIV protease inhibitors alter gene expression of the tight junction molecules occludin, ZO-1, and actin. These hypotheses are supported by our preliminary findings. In the proposed study, we will further characterize the effects of HIV protease inhibitors on the localization of these tight junction molecules. Gene expression levels will be assessed by protein and mRNA measurements. Gene transcription rate, mRNA stability, and promoter activity will be also investigated in both the HCAEC monolayer and pig carotid artery perfusion culture models. 3. Identify the signal transduction pathways involved in HIV protease inhibitor-induced endothelial permeability changes. We hypothesize that HIV protease inhibitors may: 1) alter the phosphorylation levels of tight junction proteins, and 2) induce increased endothelial permeability through signal transduction pathways involving ROS, MAPKs, protein kinase C (PKC), protein tyrosine kinase (PTK), and protein tyrosine phosphatase (PTPh). Again, our preliminary results provide support for this hypothesis. In the proposed study, we will use both the HCAEC monolayer and porcine carotid artery perfusion models to investigate the phosphorylation of occludin, ZO-1 and actin. We will measure the production of ROS, and assess the roles of protein kinases and PTPh in the observed permeability changes. In summary, these studies will help identify and establish new mechanisms of HIV protease inhibitor-induced endothelial permeability changes, thereby improving our understanding of the mechanisms of HAART-associated cardiovascular disorders. Our findings may also suggest new therapeutic strategies, which may eventually prove effective for the prevention and treatment of these complications.