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.
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