Project Summary Pulmonary arterial hypertension (PAH) is an enigmatic vascular disease characterized by pulmonary vascular remodeling, an increased pulmonary vascular resistance, and subsequent right ventricular hypertrophy and failure. There is an increased predisposition to PAH in HIV-infected populations, and as a historically neglected vascular disease, the pathogenesis of HIV-induced PAH (HIV-PAH) remains largely unknown. My mentor's laboratory has demonstrated that stiffening of the extracellular matrix induces the mechanosensitive transcriptional co-activators YAP/TAZ, resulting in the upregulation of the microRNA (miR) cluster miR-130/301 and glutaminase (GLS1) in human pulmonary arterial endothelial cells (HPAECs). Furthermore, his lab has demonstrated HPAEC metabolic reprogramming via a shift from oxidative phosphorylation to glycolysis in support of neoplastic-like growth. The implications of this discovery are two-fold: increased miR-130/301 further promotes matrix remodeling, while upregulated GLS1 increases glutaminolysis?an anaplerotic reaction that sustains proliferation. In addition, my mentor's laboratory has demonstrated that miR-21 is upregulated in the plasma of HIV-PAH patients, and that miR-21 is linked to the miR-130/301 cluster to exert broad influence in PAH. Of note, however, is that the YAP/TAZ-miR-130/301-GLS1 axis in GLS1 upregulation may not be the entire mechanism by which increased metabolic demands are met, for HIV-infected cells of myeloid origin actively secrete GLS1 into the extracellular environment, thereby increasing extracellular glutamate concentration. Taken together, I hypothesize that HIV-infected macrophages actively secrete both miR-21 and GLS1 to promote vessel stiffening, glutaminolysis, and the pathogenesis of HIV-PAH. This model will be tested by co-culturing HPAECs and human monocyte-derived macrophages (MDMs) infected or uninfected with HIV. HPAEC dysfunction will be assessed via functional assays, and metabolic reprogramming?that is a shift from oxidative phosphorylation to glycolysis with accompanying anaplerosis? will be measured in both cell types for aberrations. GLS1 transmission will be assessed using lentiviral transduction of a GLS1-tagged vector into MDMs, and upregulation of extracellular metabolites will be analyzed using mass spectrometry. Lastly, the effects of viral characteristics will be evaluated using various artificial strains and using MDMs isolated from HIV-positive patients. This proposal leverages unique experimental techniques that will expand my technical repertoire, while simultaneously elucidating a novel paradigm of HIV-PAH. The execution of the proposal in question will enhance my pre-doctoral training and establish a niche for me as a physician-scientist in addressing the cardiopulmonary manifestations of chronic HIV infection.