Cardiovascular and metabolic comorbidities shape endothelial dysfunction in pulmonary arterial hypertension and may influence treatment: insights from a multicenter translational network

Background Pulmonary arterial hypertension (PAH) is often associated with metabolic and cardiovascular comorbidities (CM), that exacerbate endothelial dysfunction and may affect treatment response. Methods We analyzed plasma from 120 patients with idiopathic or scleroderma-associated PAH enrolled at three Italian centers.Patients were stratified according to the (n=91) or absence (n=29) of comorbidities. Human pulmonary arterial endothelial cells (HPAECs) were exposed to patient plasma to evaluate autophagy (LC3-II/I, p62, Beclin-1), apoptosis, senescence (β-galactosidase, p16^INK4), mitophagy (PINK1, PARK2), endothelin signaling (ET-A/B receptors), eNOS activity, and BMPR2 expression. Circulating miRNAs and plasma proteomic profiles were also assessed. Results Plasma from PAH patients with comorbidities induced adaptive hyperautophagy, as demonstrated by increased autophagic flux (1.5 fold reduction in p62 levels and increased proportion of autophagic cells, p<0.015) without LC3 accumulation or Beclin-1 involvement (Figure 1 A). This response preserved cell viability and did not increase apoptosis. Senescence assays and p16^INK4 showed a significant increase in senescent cells (1.5 fold increase, p < 0.0001 and p<0.05, respectively). PINK1 expression was reduced by 1.5 fold (p<0.05), suggesting impaired mitophagy, whereas PARK2 levels were unchanged. Endothelin signaling was shifted toward vasoconstrictive profile, with a 1.5 fold increase in ET-A receptor expression (p<0.05) and no significant changes in ET-B receptor levels. p-eNOS levels were 1.5 fold lower compared with plasma from patients without comorbidities (p=0.012), indicating impaired nitric oxide signaling. BMPR2 expression was similarly reduced in both groups, with no significant influence of cardiovascular comorbidities. Circulating miRNAs analysis revealed a 3-fold increase in miR-21-5p (p<0.05), a 1.5-fold increase in miR-223-3p (p<0.05), and a 4-fold decrease in miR-126-3p (p<0.005, AUC 0.63–0.69), consistent with inflammation and endothelial dysfunction. Proteomic analysis demonstrated elevated Galectin-3 and activation of Ras/Raf/ERK pathway (2.24 z-score, p0.01), supporting a pro-inflammatory and dysregulated endothelial phenotype in comorbid PAH. Conclusions Cardiovascular and metabolic comorbidities exacerbate endothelial stress, promote senescence, and disrupt vasoregulatory balance in PAH, thereby influencing disease phenotype and potentially treatment response. These findingd may contribute to refine patient stratification within a precision medicine framework. Since BMPR2 expression was not affected by comorbidities, therapies targeting the activin/BMPR2 pathway are likely to remain effective and may be safely employed in PAH patients with coexisting cardiometabolic conditions.