Novel role of occludin and pericytes in ischemic stroke in HIV infection

 描述（由申请人提供）：HIV感染的心脏、肺和/或血液（HBL）并发症是HIV相关病理学中的新问题。在目前的提案中，我们将研究HIV在脑血管周细胞中复制的机制，解决基本的机制问题，重点是HIV感染对血管组织功能障碍的影响，从而导致缺血性卒中的发生。血脑屏障（BBB）的完整性和功能性由紧密连接（TJ）和神经血管单元的细胞之间的紧密界面维持，包括与微血管内皮细胞相互作用的周细胞。虽然脑血管周细胞可以说是血管组织中研究最少的细胞，但最近的文献报道提供了令人信服的证据，证明它们参与了卒中的发病机制。此外，我们还报道了他们易受HIV感染，对血管内皮的完整性产生负面影响。我们最近获得的重要的初步数据表明，HIV在脑血管周细胞中的复制与 与TJ蛋白闭合蛋白水平相关。本申请建立在这些新发现的基础上，我们提出闭合蛋白的细胞水平调节脑血管周细胞中的HIV复制，从而影响HIV感染的脑中缺血性中风的发展。 从机制上讲，我们确定闭合蛋白具有NADH氧化酶活性，导致NAD+的细胞池增加。在目的1中，我们假设这种活性导致沉默调节蛋白1的激活 （SIRT 1），一种NAD（+）依赖性脱乙酰酶，然后抑制NF-κB的活化并抑制脑血管周细胞中的HIV复制。在目标2中，我们将重点关注occludin作为一个因素，限制了HIV复制的后期阶段，通过干扰ESCRT（运输所需的内体复合物）机制，这是负责HIV出芽和释放的作用。这些研究得到了另一组令人兴奋的初步发现的支持，表明occludin可以干扰阿利克斯，一种早期作用的ESCRT因子。虽然增加的细胞闭合蛋白防止HIV复制，但通过降低闭合蛋白水平观察到相反的效果。这一点很重要，因为HIV感染中occludin的上调对脑血管周细胞来说是非常独特的，其他细胞类型通过降低这种蛋白质的水平来应对感染。因此，我们的建议的目的3是集中在缺血性中风的动物模型中，我们将利用新的周细胞和闭合蛋白缺陷小鼠感染最近开发的小鼠嗜性EcoHIV株。拟议的研究是高度创新的，其重点是新的机制，以了解和控制的机制和发病机制的发展HBL条件。这项提案的完成有可能改变我们对occludin和血管周细胞在HIV感染及其HBL并发症（包括中风）中的作用的理解。

 DESCRIPTION (provided by applicant): Heart, lung, and/or blood (HBL) complications of HIV infection are the emerging problem in HIV-related pathology. In the current proposal, we will study the mechanisms of HIV replication in cerebrovascular pericytes, addressing fundamental mechanistic questions focused on the impact of HIV infection on vascular tissue dysfunction contributing to the development of ischemic stroke. The integrity and functionality of the blood-brain barrier (BBB) are maintained by tight junctions (TJs) and the close interface between the cells of the neurovascular units, including pericytes interacting with microvascular endothelial cells. While cerebrovascular pericytes are arguably the most understudied cells of vascular tissue, recent literature reports provide compelling evidence on their involvement in the pathogenesis of stroke. In addition, we reported that they are susceptible to HIV infection, negatively affecting the integrity of the vascular endothelium. Our recently obtained critical preliminary data indicate that HIV replication in cerebrovascular pericytes is inversely correlated with the levels TJ protein occludin. The present application is built on these novel findings and we propose that cellular levels of occludin regulate HIV replication in cerebrovascular pericytes and thus influence the development of ischemic stroke in HIV-infected brain. Mechanistically, we identified that occludin has an NADH oxidase activity, resulting in increased cellular pool of NAD+. In Aim 1, we hypothesize that this activity leads to activation of sirtuin 1 (SIRT1), an NAD(+)-dependent deacetylase, which then suppresses activation of NF-κB and inhibits HIV replication in cerebrovascular pericytes. In Aim 2, we will focus on the role of occludin as a factor that limits a late stage of HIV replication by interfering with the ESCRT (endosomal complex required for transport) machinery, which is responsible for HIV budding and release. These studies are supported by another set of our exciting preliminary findings, indicating that occludin can interfere with ALIX, an early-acting ESCRT factor. While increased cellular occludin protects against HIV replication, an opposite effect is observed by decreased occludin levels. This is important because occludin upregulation in HIV infection is highly unique to cerebrovascular pericytes, with other cell types responding to infection by decreasing levels of this protein. Therefore, Aim 3 of our proposal is focused on an animal model of ischemic stroke in which we will utilize novel pericyte and occludin deficient mice infected with recently developed mouse-tropic EcoHIV strain. The proposed research is highly innovative by its focus on novel mechanisms to understand and control the mechanisms and pathogenesis underlying the development of HBL conditions. The completion of this proposal has the potential to change our understanding of the role of occludin and vascular pericytes in HIV infection and its HBL complications, including stroke.