Pulmonary arterial hypertension (PAH) is characterized by vascular fibrosis and right ventricular (RV) failure. The role of oxidant stress in advanced PAH is well-established; however, the pathogenetic implications of pro-fibrotic redox pathways in early-stage PAH are unknown. We hypothesized that unique redox-sensitive molecular targets in pulmonary artery endothelial cells (PAECs) distinguish early- from advanced-stage PAH. Sprague-Dawley rats were administered monocrotaline (MCT) (60 mg/kg) (day 0) and invasive hemodynamics, histopathology, and PAEC transcriptomics were analyzed on days 15 and 23 for early-stage and advanced PAH, respectively.
Compared to untreated control, early-stage PAH demonstrated impaired RV-PA coupling (1.13 ± 0.05 vs. 0.90 ± 0.06 Ees/Ea, P=0.02), increased indexed pulmonary vascular resistance (50 ± 8 vs. 213 ± 29 mmHg*min*g-1*mL-1, P<0.001), and a 2.7-fold increase in arteriolar collagen by picrosirius stain (P<0.0001) without substantial pulmonary hypertension (25 ± 1.2 vs. 33.2 ± 2.7 mmHg PA systolic pressure, P=0.02). The PAEC transcriptome in early-stage MCT-PAH included N=1,058 unique, differentially expressed genes (FDR<0.05), which was enriched for fibrosis genes (P=0.019). These data were used to develop a novel protein-protein interaction (PPI) network for predicting mediators of endothelial fibrosis in early-stage PAH. Dynamic network biomarker and gene ontology analyses suggested PPIs involving c-terminal src kinase (Csk) are important in early-stage PAH fibrosis in silico. Compared to control, Csk mRNA (log fold-change=+0.72; FDR<0.0001) and protein expression (123 ± 12 vs. 260 ± 25, a.u., P=0.01) were increased in PAECs in early-stage PAH by RNASEQ ex vivo and anti-Csk immunofluorescence in situ, respectively. Compared to control, hydrogen peroxide (250 μM) treatment increased PAEC Csk by 2.1-fold (P=0.02). Further, Csk correlated positively with arteriolar ollagen quantity in MCT-PAH (r=+0.87, P=0.006).
Collectively, early-stage PAH is defined by a unique endothelial pathobiology relative to advanced PAH. Redox-regulation of endothelial fibrosis by Csk may have important therapeutic implications for early-stage PAH.