Longitudinal studies of HIV-1 Nef in Pulmonary Hypertension

描述（由申请人提供）： 肺动脉高压（PAH）的发病机制和导致其在HIV感染患者中更高患病率的因素知之甚少。慢性暴露于肺中的病毒产物以及HIV诱导的免疫失调可能导致肺血管疾病。本资助申请中提出的实验是对标题为“HIV相关肺部感染和并发症的纵向研究”的RFA-HL-07-008的回应，并将扩展我们发现HIV-1 Nef（阴性因子）蛋白与HIV相关肺动脉高压（HRPAH）之间相关性的初步研究。与法国的Marc Humbert博士合作，我们从法国HRPAH患者中获得外周血单核细胞DNA（PBMC），并对这些样本中的nef等位基因进行测序。系统发育分析表明，大部分序列与B亚型序列聚在一起。因此，我们将法国Nef序列与已发表数据库中的所有HIV-1亚型B序列进行了比较。我们发现，特定的nef分子签名更频繁地发生在法国HRPAH患者比数据库。因此，我们假设nef的等位基因变体（可通过独特的分子特征识别）与已诊断患者的肺动脉高压（PAH）发生相关，并预测超声心动图评估的高危患者人群的疾病进展。这些变异体可能在肺中触发级联反应，从而选择快速生长的抗肺纤维化内皮细胞群。我们将依赖于两个不同的，完善的HIV-1感染受试者的队列-一组通过右心导管插入术（RHC）诊断为HRPAH的法国患者，诊断金标准，以及一组通过超声心动图诊断为肺动脉收缩压（PASP）升高的弗朗西斯科HIV-1感染者，他们可能处于高死亡风险中。来自两个临床研究中心的回顾性和前瞻性血浆、PBMC和支气管肺泡灌洗液细胞（BALF）将被送往丹佛的研究中心，在那里将获得、分析和表征nef序列。我们的目的是评估来自有和没有 HRPAH以确定常见序列中断或相似性是否与疾病相关 发病机制;评价来自San弗朗西斯科具有升高的PASP和处于发展PAH风险的HIV-1感染患者的nef等位基因是否具有与在法国患者中鉴定的序列相似的特征序列，并且其可以预测进展为HRPAH;以及鉴定和比较从BALF分离的nef序列与在San弗朗西斯科具有和不具有升高的PASP的患者的PBMC和血浆中发现的序列，以获得区室化的证据。最后，我们将评估在这些患者中鉴定的nef等位基因是否诱导培养的人肺微血管内皮细胞增殖。 对于所有队列，DNA、RNA、血浆、重组载体、BALF细胞样本、免疫表型和数据以及这些研究产生的分子工具将保存在数据协调中心（DCC），并可用于未来的机制研究。

DESCRIPTION (provided by applicant): Little is known about the pathogenesis of pulmonary arterial hypertension (PAH) and the factors that contribute to its higher prevalence in HlV-infected patients. Chronic exposure to viral products in the lung as well as HlV-induced immune dysregulation may contribute to pulmonary vascular disease. The experiments proposed in this grant application are in response to the RFA-HL-07-008 titled "Longitudinal Studies of HIV- Associated Lung Infections and Complications'' and will expand our initial studies that found an association between the HIV-1 Nef (negative factor) protein and HIV-related pulmonary arterial hypertension (HRPAH). In collaboration with Dr. Marc Humbert from France, we obtained peripheral blood mononuclear cell DNA (PBMC) from French HRPAH patients and sequenced nef alleles in these samples. Phylogenetic analyses revealed that the majority of sequences clustered with subtype B sequences. We therefore compared the French Nef sequences with all HIV-1 subtype B sequences in published databases. We found that particular nef molecular signatures occurred more frequently in French HRPAH patients than in the databases. We therefore hypothesize that allelic variants of nef, which can be identified by unique molecular signatures, are associated with the development of pulmonary arterial hypertension (PAH) in patients already diagnosed and predict disease progression in a population of patients at risk as assessed by echocardiography. These variants may trigger a cascade of events in the lungs that allows for the selection of a rapidly growing, apoptosis-resistant endothelial cell population. We will rely upon two distinct, well-established cohorts of HIV-1-infected subjects - a group of French patients with HRPAH diagnosed by right heart catheterization (RHC), the diagnostic gold standard, and a cohort of HIV-1- infected individuals in San Francisco who have been diagnosed with elevated pulmonary artery systolic pressures (PASP) by echocardiography and who may be at a high mortality risk. Retrospective and prospective plasma, PBMC and bronchoalveolar lavage fluid cells (BALF) from the two clinical sites will be sent to the research site in Denver where nef sequences will be obtained, analyzed and characterized. We aim to evaluate the amino acid sequence and known domain structures from patients with and without HRPAH to determine if common sequence disruptions or similarities are associated with disease pathogenesis; to evaluate whether nef alleles from HIV-1 infected patients with elevated PASP and at risk of developing PAH in San Francisco have signature sequences similar to sequences identified in French patients and that may predict progression to HRPAH; and to identify and compare nef sequences isolated from BALF with sequences found in PBMC and plasma in patients with and without elevated PASP in San Francisco for evidence of compartmentalization. Finally, we will evaluate whether nef alleles identified in these patients induce proliferation in cultured human lung microvascular endothelial cells. For all cohorts, DNA, RNA, plasma, recombinant vectors, BALF cell samples, immunophenotypes and data as well as molecular tools generated from these studies will be deposited with the Data Coordinating Center (DCC) and made available for future mechanistic studies.