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)仍然是病毒的主要目标和储存库，导致 HIV-1相关神经认知障碍的发展(手)。重要的是，自年初以来， 艾滋病毒流行以来，吸毒仍然是感染、传播和恶化艾滋病毒的主要危险因素 艾滋病毒的后果。事实上，在发病机制方面，精神刺激剂可以诱导更高的病毒载量，降低CD4 并增加抗逆转录病毒的抵抗率。此外，个体的神经心理衰退更大。 可卡因吸毒者和艾滋病毒血清阳性者。细胞外微泡(EVS)，包括微泡 (MVS)和外体，已经成为一种新的生物现象，几乎由所有类型的细胞释放 身体。我们已经证明脑微血管内皮细胞(BMVEC)来源的EV含有TRAIL 连接蛋白(TJP)和转运蛋白，这是血脑屏障的主要成分。此外，a HAND的特点是血脑屏障的破坏和TJ波群的丢失。在这份提案中，我们表明 BMVEC对HIV病毒毒素和精神刺激剂的反应是排出EV。因此，我们假设艾滋病毒 感染和/或滥用药物触发EV释放，导致血脑屏障不稳定和促进神经侵袭 被感染的免疫细胞感染。这项提议的创新性体现在三个独立的目标上。在 第一个目标是，我们将EV的产生程度(MVS和Exosome)表征为以下函数 使用具有原代人类细胞的神经血管单位的新型微流体模型的精神刺激剂类型。我们 还将调查接触艾滋病毒病毒毒素和抗逆转录病毒药物对BMVEC-EV的影响 制作。此外，我们将在体内自我给药模型中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的释放与成瘾阶段相关联 行政管理。在第二个目标中，我们引入了BMVEC衍生的电动汽车与激活的电动汽车绑定的新概念 或受感染的单核细胞，从而触发单核细胞跨内皮细胞迁移增加。因此，我们假设 单核细胞利用EVS上的TJP结合内皮紧密连接复合体，促进免疫 中枢神经系统的渗透。在第三个目标中，我们将探索一种可以将电动汽车的产量降至最低的治疗策略 从而挽救BBB的完整性。上述作用将通过靶向抑制ARF6来实现，这是 参与MV和外切体的生物发生。使用新的药理工具抑制ARF6，我们的目标是稳定 血脑屏障在神经炎症期间预防血脑屏障功能障碍。这里提出的研究将提供 对脑内皮细胞EV的产生，免疫细胞渗透到中枢神经系统的机制，以及 揭示促进艾滋病毒和药物使用中血脑屏障保护的药物干预目标 无序。

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.