Sex-Specific Role of Damage-Mediated Signaling in Endothelial Homeostasis

Abstract

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 a more severe disease progression and poorer outcomes. PAH initiates in response to PAEC damage, followed by inadequate repair and inflammation. The response to this damage includes the activation of the receptor for advanced glycation endproducts (RAGE), a multiligand receptor involved in inflammation, oxidative stress, and vascular injury, that mediates pro-survival and tissue-regeneration signaling. However, the precise role of RAGE in endothelial cell plasticity and remodeling remains poorly defined. This dissertation investigates how RAGE deficiency shapes endothelial fate and contributes to male-specific PAH pathogenesis. Using male and female whole-body RAGE knockout (RAGE KO) rats, we assessed PAH phenotype compared to age-matched wild-type (WT) rats as a control and examined the molecular, cellular, and physiological consequences of RAGE loss on the cardiopulmonary vasculature. Investigation of isolated endothelial cells from these rats revealed on a molecular level that RAGE deficiency activated both canonical (SMAD2/SMAD3 - SNAIL) and non-canonical (p38 MAPK - RhoA) TGF-β pathways, leading to Endothelial-to-Mesenchymal Transition (EndoMT), characterized by loss of CD31, upregulation of fibronectin, PDGFR-α, and enhanced cytoskeletal remodeling. These changes were associated with increased endothelial permeability and fibrotic deposition. Inhibition of TGF-β receptor I (TGFβ-RI) with SB431542 reversed these effects, restoring endothelial quiescence. PARSE single-cell transcriptomics analysis revealed broad sex-specific remodeling across endothelial, fibroblast, myofibroblast, and vascular smooth muscle cell populations, with male RAGE KO lungs showing expansion of proliferative, migratory, and ECM-remodeling cell states, while females maintained barrier-stabilizing and antioxidant programs. Functionally, male RAGE KO rats developed spontaneous pulmonary hypertension, evidenced by increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (measured by Fulton index), along with pulmonary vascular wall thickening and interstitial fibrosis. Females were mainly protected, with minimal vascular remodeling and preserved endothelial integrity. Notably, hemodynamic differences persisted after gonadectomy, suggesting that male susceptibility is only partly hormone dependent and may involve an underlying endothelial vulnerability. At the organ level, insufficient RAGE signaling produced a mixed cardiopulmonary phenotype of pulmonary hypertension (PH), including features of Group 1 PAH (occlusive vascular remodeling), Group 3 pulmonary hypertension (fibrotic lung parenchymal remodeling), and secondary Group 2 pathology (left ventricular dysfunction). Histological and morphometric analysis revealed right ventricular fibrosis, diffuse lung collagen deposition, and left ventricular hypertrophy. These structural changes were accompanied by immune activation and metabolic reprogramming, as T cells from RAGE KO rats exhibited a glycolytic shift and expansion of proliferative and naive subsets, linking endothelial dysfunction to inflammatory and metabolic remodeling. Therapeutically, inhibition with SB431542 reversed key features of the disease, including decreased right ventricular systolic pressure (RVSP) and significantly reduced left ventricular hypertrophy (LV/TL). These findings confirm that TGF-β activation downstream of RAGE loss drives both pulmonary vascular and cardiac remodeling, and that targeting this pathway restores cardiopulmonary structure and function. Taken together, these findings identify deficient RAGE signaling as a key driver of male susceptibility to vascular endothelial injury and PAH. Loss of RAGE triggers a sex-dependent cascade involving TGF-β hyperactivation, EndoMT, and immunometabolic activation, all contributing to progressive pulmonary hypertension and biventricular remodeling. The RAGE KO rat thus represents a novel, spontaneous, and mechanistically integrative model of pulmonary hypertension across Groups 1, 2, and 3, offering critical insights into the molecular basis of male-predominant disease severity.