Abstract 4139195: Tipping Point Analysis Identifies C-terminal src kinase-Collagen 22 Regulation by Inflammation in the Pathogenesis of Early-Stage Pulmonary Arterial Hypertension

Inflammatory injury to pulmonary artery endothelial cells (PAECs) is a key pathogenetic event that drives fibrotic vascular remodeling in pulmonary arterial hypertension (PAH), a progressive disease that lacks early stage-specific therapeutic targets. In an inflammatory rodent model of PAH, we established early-stage disease based on abnormalities in right ventricular (RV) afterload, pulmonary vascular resistance, and RV cardiomyocyte phenotype that were evident without substantial elevation in pulmonary artery pressure. We performed RNA-Seq on PAEC mRNA isolated ex vivo from control, early-stage, and advanced-stage PAH rats and deployed a ‘tipping point’ methodology to identify key pulmonary endothelial protein-protein interactions (PPIs) that regulate the transition from control to early-stage PAH in silico . Building on prior data on the intersection between glucose and proline signaling in fibrosis, we interrogated our early-stage PAH network for convergent PPIs involving metabolism and fibrosis endotypes. C-terminal src kinase (Csk), a conserved inhibitor of Src, was predicted to regulate PAEC fibrosis and was increased in early-stage PAH. In human PAECs, inflammatory stimuli affected markers of Src activation as well as collagen 22 (Col22A1). We observed that pulmonary arterial Col22A1 expression increased progressively across all PAH stages in vivo and that arterial Col22A1 expression is upregulated in human PAH. Further, although inflammation upregulated total Csk mRNA and protein expression in HPAECs, it decreased phosphorylation of Src at tyrosine 527 (pTyr527Src), a Csk-dependent brake on Src kinase activity. Targeting Src through small molecule inhibition or adenovirus overexpression of Csk to increase pTyr527Src, in turn, attenuated inflammation-induced Col22A1 in vitro . Taken together, these data suggest that acquired inhibition of Csk is a novel molecular mechanism by which inflammation induces pulmonary endothelial dysfunction, Src activation, and fibrosis. In early-stage PAH, disrupted Csk inhibition of Src may induce transcriptional upregulation of Csk as a negative feedback response to Src activation. These data suggest that the Csk-Src-Col22A1 axis is a novel target for advancing early-stage therapeutics in patients with PAH subtypes characterized by inflammatory vascular injury.