Project Summary/Abstract HIV1 infected individuals in both pre- and post-antiretroviral eras develop a variety of vasculopathies. Consistently, both morphologic and functional derangements in endothelial cells are found, revealing dysregulated endothelial-mesenchymal transition, activation, and regeneration failure. The endothelium in AIDS is thought to be a bystander target of a variety of viral products, including the exported transcriptional activator Tat, a known prooxidative protein. This latter point is relevant to HIV1-associated vasculopathies, since biochemically, a common denominator seen in these and other vasculopathies is an increase in oxidative activity. Oxidant stress in general is a poorly understood concept, and the mechanisms by which oxidants mediate their numerous cellular effects remain unclear. With respect to HIV1 Tat, we have established in the prior funding period that Tat activates an NADPH oxidase (Nox) in the endothelium, leading to activation of the stress- activated protein kinase JNK, and to distinct cytoskeletal rearrangements. Our preliminary data now reveal that Tat simultaneously activates both Nox2 and Nox4 pathways downstream of Rac1, leading to independent PAK/JNK and Ras/ERK activation. We further show that these two pathways lead to distinct cellular responses with alterations in cellular function. These data strongly suggest a novel paradigm in which differential activation of Nox paralogs by Tat specifies distinct downstream cellular events by activating different oxidative signaling cassettes. The identification of such dual Nox pathways allows a unique opportunity to dissect Tat-stimulated vascular cell signaling at a subcellular level. We hypothesize that Tat signaling diverges into separate Nox2 and Nox4-containing cellular microdomains built on endosomal and endoplasmic reticulum membrane platforms, to mediate transitions toward a mesenchymal or endothelial phenotype, respectively. Inappropriate activation of this Nox2/4 oxidative molecular switch may explain abnormal EMT transitions seen in the vasculature of AIDS patients. In this proposal, we will use a combination of advanced microscopy and biochemical techniques to identify the Nox2 and Nox4 specific subcellular platforms and signaling pathways activated by HIV1 Tat. The broad aim of this grant is to understand the biochemical and cellular basis for Tat-induced oxidative signaling, which may reveal protein targets for intervention in various AIDS vasculopathic syndromes.