Effects of HIV PIs on CNS endothelial cells in NeuroAIDS

职位描述(申请人提供)：长期的职业目标是：1)开发一个研究项目，研究宿主-病原体相互作用引发的细胞信号变化，这些变化与HIV相关痴呆(HAD)等神经精神疾病有关；2)成为一家学术机构的独立生物医学科学家。针对高效抗逆转录病毒疗法(HAART)对神经艾滋病的潜在贡献，提出了详细的职业发展和科学计划。在血清转换后不久，HIV患者可能会被开出基于蛋白酶抑制剂(PI)的HAART方案。HAART已被证明在抑制全身性病毒负荷方面非常成功；然而，依赖于ATP的外排转运泵P-糖蛋白(P-gp)严重制约了PI治疗神经艾滋病。由于病毒反弹、耐药性突变以及PI与HIV蛋白和脑内皮细胞(CEC)的潜在相互作用，本研究的主要目的是确定慢性PI暴露对CEC反应宿主来源的生长因子(如成纤维细胞生长因子2)的能力的影响，这些生长因子是病毒反弹时产生的防御HIV蛋白的机制。我们假设长期暴露于PIs如沙奎那韦(SQV)、吲哚那韦(INV)、奈非那韦(NFV)和/或利托那韦(RTV)会改变P-gp外流依赖的表达和活性，从而改变FGF2介导的小窝蛋白/ERK/NO系统中P-gp外流非依赖的信号通路。为此，AIM I将通过与P-gp和小窝蛋白的相互作用来确定慢性PI治疗对CEC适合性和FGF2介导的信号转导的影响。CEC适合性的四个方面将被测试：1)活性，2)P-gp的表达和活性，3)P-gp介导的小窝蛋白和内皮型一氧化氮合酶(ENOS)信号和一氧化氮(NO)的产生，4)FGF2介导的细胞外调节激酶(ERK)信号和血管生成能力。AIM II将在体外研究CEC对PI的慢性暴露如何破坏P-gp/小窝蛋白/FGF2依赖的HIV蛋白gp120的保护。利用MSR-gp120和GFAPFGF2转基因小鼠，AIM III将在体内研究慢性PI治疗在与HIV蛋白相互作用过程中扰乱血脑屏障(BBB)信号的长期影响。了解慢性PI暴露后通过P-gp/Caveolae导致FGF2信号改变的机制，对于确定在病毒反弹或病毒学失败期间可能导致与NeuroAIDS相关的神经和神经行为变化的因素具有重要意义。

DESCRIPTION (provided by applicant): Long-term career goals are to: 1) develop a research program to investigate the cellular signaling alterations triggered by host-pathogen interactions relevant to neuro-psychiatric disorders such as HIV-associated Dementia (HAD) and 2) become an independent bio-medical scientist at an academic institution. Detailed career development and scientific plans addressing the potential contribution of highly active anti-retroviral therapy (HAART) to NeuroAIDS is proposed. Shortly after sero-conversion, HIV patients may be prescribed protease inhibitor (PI)-based HAART regimens. HAART has proven highly successful in suppressing systemic viral burden; however, the ATP-dependent efflux transport pump, P-glycoprotein (P-gp), imposes a serious constraint on PI treatment of NeuroAIDS. Because of viral rebound, resistance mutations and the potential interactions of PIs with HIV proteins and cerebral endothelial cells (CEC), the main objective of this proposal is to determine the effects of chronic PI exposure on the CEC's ability to respond to host-derived growth factors, such as fibroblast growth factor 2 (FGF2), that are generated as defense mechanisms against HIV proteins _resent in the blood stream at viral rebound. We hypothesize that long-term exposure to PIs such as saquinavir (SQV), indinavir (INV), nelfinavir (NFV) and/or ritonavir (RTV) will modify P-gp efflux-dependent expression and activity, thereby altering P-gp efflux-independent signaling pathways in the FGF2-mediated caveolin/ERK/NO systems. For this purpose, AIM I will determine the effects of chronic PI treatment on CEC fitness and signaling mediated by FGF2 via interactions with P-gp and caveolin. Four aspects of CEC fitness will be tested: 1) viability, 2) P-gp expression and activity, 3) P-gp-mediated caveolin and endothelial nitric oxide synthase (eNOS) signaling and nitric oxide (NO) production, 4) FGF2-mediated extracellular regulated kinase (ERK) signaling and angiogenic capacity. AIM II will address in vitro, how chronic exposure of CEC to PI disrupts P-gp/caveolin/FGF2-dependent protection from the HIV protein, gp120. Using MSR-gp120 and GFAPFGF2 transgenic mice, AIM III will investigate, in vivo, the long-term effects of chronic PI treatment in disrupting signaling at the blood-brain barrier (BBB) during interaction with HIV proteins. Understanding the mechanisms responsible for alterations in FGF2 signaling via P-gp/caveolae after chronic PI exposure is important for identifying factors that may contribute to the progression of neurological and neurobehavioral alterations associated with NeuroAIDS during viral rebound or at virologic failure.