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.