PROJECT SUMMARY/ABSTRACT Pulmonary arterial hypertension (PAH) is characterized by inflammation, endothelial dysfunction, and metabolic dysregulation, which promote fibroproliferative pulmonary arterial remodeling, right ventricular failure (RV), and early death despite current PAH therapies. Mild pulmonary hypertension is associated with increased mortality and RV dysfunction, emphasizing a need to understand metabolic drivers of vessel remodeling in PAH. Using network medicine, we identify proline metabolism as functionally relevant to PAH. In a model of advanced-stage PAH in vivo, multi-isotope imaging mass spectrometry (MIMS) reveals increased endothelial incorporation of stable isotope proline tracer as well as the presence of “hotspots” of proline signal in collagen fibrils, proliferating cells, and extracellular matrix—which we define collectively as, “biomass.” As histological analysis of early-stage inflammatory PAH shows significant deposition of collagen prior to the development of severe pulmonary hypertension, it follows that endothelial proline dysregulation may be present in early-stage PAH. The central goal of the proposed R03 is to understand proline dysregulation in control and PAH pulmonary artery endothelial cells (PAECs) in vitro, early-stage PAH (esPAH) in vivo, and in human PAH. Using systems biology, we identify C-terminal src kinase (Csk), as a regulator of fibrosis and metabolism in early- stage PAH. Csk is known inhibit the metabolic regular Src through phosphorylation of Src tyrosine 527 (pTyr527Src). We demonstrate that inflammation reduces Csk expression on the plasma membrane, which may have implications for Src-dependent regulation of proline. We hypothesize that inflammation impairs Csk recruitment to the plasma membrane, leading to decreased pTyr527Src, and enhanced Src-dependent proline incorporation by HPAECs to support cell proliferation, synthesis of endothelial biomass, and vasculopathy in esPAH. We propose the following specific aims: 1) Establish Csk-Src regulation of HPAEC proline availability and fibroproliferative biomass in vitro, 2) Establish Csk-Src dependent proline dysregulation in experimental esPAH and human PAH in vivo. Understanding Csk-Src dependent proline dysregulation will complement K08 career development activities, establish a novel metabolic driver of PAH inception, and support a future R01 application to study proline dysregulation in early-stage PAH.