Abstract 4366423: Breaking the Barrier: RAGE Deficiency Promotes EndoMT and Male-Predominant Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a progressive and fatal disease initiated by injured pulmonary artery endothelial cells (PAECs). Although PAH disproportionately affects females, males consistently exhibit worse outcomes. Our prior work identified a greater susceptibility of male PAECs to necrosis and inflammatory damage. At the same time, the expression of the Receptor for Advanced Glycation Endproducts (RAGE) that controls cellular repair is inherently low in male’s pulmonary endothelial cells (ECs) and further reduced in male PAH PAECs. The resulting persistent endothelial injury is a known trigger of Endothelial-to-Mesenchymal Transition (EndoMT), a critical contributor to vascular remodeling. Hypothesis: We hypothesize that an insufficient RAGE signaling in male ECs promotes EndoMT, compromises endothelial barrier integrity, accelerates fibrosis, and drives PAH development. Methods: Twelve-week-old male and female RAGE knockout (KO) and wild-type (WT) rats were evaluated for pulmonary hemodynamics, endothelial barrier integrity, and fibrosis. Pulmonary ECs were isolated to assess EndoMT markers, angiogenic function, migration, and invasiveness. To investigate the potential contribution of sex hormones, RAGE KO and WT rats were gonadectomized 4 weeks before the hemodynamic assessment. Results: Only male RAGE KO rats developed spontaneous PAH, evidenced by elevated right ventricular systolic pressure, increased Fulton index, and small pulmonary artery remodeling. These effects persisted in orchiectomized KO males, indicating a sex hormone-independent mechanism. Lung histology revealed extensive perivascular fibrosis. Elevated extravasation of FITC-dextran in males confirmed a disrupted pulmonary endothelial barrier. Isolated male KO-derived PAECs showed a significant increase in TGF-β (1.63 ± 0.18 vs. 1.0 ± 0.14; p < 0.05), TGF-βR1 (3.0 ± 0.3 vs. 1.0 ± 0.1; p = 0.0017), and TGF-βR2 (5.4 ± 0.6 vs. 1.0 ± 0.1; p < 0.001), alongside a ~7-fold reduction in CD31. Functional assays confirmed an impaired angiogenesis and increased invasiveness of male KO PAECs. No significant changes were observed in females. Conclusion: Loss of endothelial RAGE in males initiates a cascade of pathological events, including EndoMT, endothelial barrier failure, and fibrosis, ultimately leading to spontaneous PAH. These effects highlight the increased sensitivity of males to inadequate endothelial repair, occurring independently of sex hormones.