Brain endothelial EVs role in the neuropathology of drugs of abuse and HIV

用抗逆转录病毒疗法（ART）治疗艾滋病毒感染者，有效地抑制了病毒复制; 然而，中枢神经系统（CNS）仍然是病毒的主要靶点和储存库，导致 HIV-1相关的神经认知障碍（HAND）。重要的是，自2001年以来， 艾滋病毒流行，吸毒仍然是感染，传播和恶化的主要风险因素， 艾滋病毒的结果。事实上，关于发病机制，精神兴奋剂可以诱导更高的病毒载量，降低CD 4 计数，并增加ART耐药率。此外，神经心理学的衰退在个体中更大， 可卡因使用者和艾滋病毒血清阳性者。细胞外微泡（EV），其包括微泡 (MVs)和外泌体，已经成为一种新的生物学现象，几乎每种细胞类型都可以释放， 身体我们已经表明，脑微血管内皮细胞（BMVEC）衍生的EV含有紧密的 连接蛋白（TJPs）和转运蛋白，它们是血脑屏障的主要成分。而且有 HAND的标志性特征是BBB的破坏和TJ复合物的损失。在这个建议中，我们表明， BMVEC对HIV病毒毒素和精神兴奋剂作出反应而脱落EV。因此，我们假设艾滋病毒 感染和/或药物滥用触发EV释放，导致BBB不稳定和促进神经侵入 被感染的免疫细胞感染该提案的创新性体现在三个独立的目标中。在 第一个目标，我们将表征EV产生（MV和外来体）的程度作为以下因素的函数： 精神兴奋剂类型使用具有原代人类细胞的神经血管单元的新型微流体模型。我们 还将研究暴露于HIV病毒毒素和ART药物对BMVEC-EV的影响 生产此外，我们将在体内自我给药模型以及在有或没有药物的HIV感染的人源化小鼠模型中将EC-EV释放与成瘾阶段相关联 局在第二个目标中，我们引入了新的概念，即BMVEC衍生的EV与激活的 或感染的单核细胞，其触发增加的单核细胞跨内皮迁移。因此，我们假设 单核细胞利用EV上的TJPs与内皮紧密连接复合物结合， 中枢神经系统浸润。在第三个目标中，我们将探索一种治疗策略， 从而挽救血脑屏障的完整性。上述将通过靶向抑制ARF 6来实现， 参与MV和外泌体生物发生。使用新的药理学工具来抑制ARF 6，我们的目标是稳定 BBB以防止神经炎症期间的BBB屏障功能障碍。本文提出的研究将提供 对脑内皮EV产生、免疫细胞浸润到CNS的机制以及 揭示药物干预的目标，促进BBB保护艾滋病毒和物质使用 disorder.

Treatment of HIV-infected patients with antiretroviral therapy (ART) has effectively suppressed viral replication; however, the central nervous system (CNS) is still a major target and reservoir of the virus leading to the development of HIV-1-associated neurocognitive disorders (HAND). Importantly, since the beginning of the HIV epidemic, drug use has remained a primary risk factor for contracting, transmitting, and worsening the outcomes of HIV. In fact, regarding pathogenesis, psychostimulants can induce higher viral loads, reduce CD4 counts, and increase rates of ART resistance. Furthermore, neuropsychological decline is greater in individuals that are cocaine users and HIV-seropositive. Extracellular microvesicles (EVs), which includes microvesicles (MVs) and exosomes, have emerged as a novel biological phenomenon, released by virtually every cell type in the body. We have shown that brain microvasculature endothelial cell (BMVEC)-derived EVs contain tight junction proteins (TJPs) and transporter proteins, which are main constituents of the BBB. Furthermore, a hallmark feature of HAND is the disruption of the BBB and loss of TJ complexes. In this proposal, we show that BMVECs shed EVs in response to HIV virotoxins and psychostimulants. Thus, we hypothesize that HIV infection and/or drugs of abuse triggers EV release leading to BBB instability and facilitation of neuroinvasion by infected immune cells. The innovative nature of this proposal is featured in three independent aims. In the first aim, we will characterize the degree of EV production (MVs and exosomes) as a function of psychostimulant type using a novel microfluidic model of the neurovascular unit with primary human cells. We will also investigate the effects of exposure to HIV virotoxins and ART pharmacologic agents on BMVEC-EV production. Furthermore, we will correlate EC-EV release to the phases of addiction in an in-vivo self-administration model as well as in a humanized mouse model of HIV infection with or without drug administration. In the second aim, we introduce the novel concept that BMVEC-derived EVs bind to activated or infected monocytes, which triggers increased monocytic transendothelial migration. Thus, we hypothesize that monocytes utilize TJPs on EVs to engage endothelial tight junction complexes and facilitate immune infiltration of the CNS. In the third aim, we will explore a therapeutic strategy that could minimize EV production and thus rescue BBB integrity. The above will be accomplished by targeted inhibition of ARF6, which is involved in MV and exosome biogenesis. Using new pharmacological tools to inhibit ARF6, we aim to stabilize the BBB to prevent BBB barrier dysfunction during neuroinflammation. The studies proposed herein will offer crucial insight to brain endothelial EV production, mechanisms of immune cell infiltration into the CNS, and also reveal targets for pharmacological interventions that promote BBB protection in HIV and substance use disorder.