Abstract Rationale Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease characterized by elevated mean pulmonary arterial pressure and increased pulmonary vascular resistance as a function of vascular remodeling and luminal obliteration of the precapillary circulation. Altogether, these features lead to right heart failure and a 40% 5-year mortality rate. PAH vessels are marked by aberrant proliferation and extracellular matrix (ECM) remodeling by pulmonary artery smooth muscle cells (PASMCs). The transforming growth factor-β (TGF-β) superfamily is essential to these processes as aberrant TGF-β/bone morphogenetic protein (BMP) signaling promotes vascular pathology. While loss of BMP signaling drives heritable PAH, a void remains in our understanding of TGF-β-induced vascular remodeling as merely one FDA-approved therapy targets this axis in PAH. Here, we demonstrate that Endosialin (CD248), a transmembrane glycoprotein elevated in PAH, promotes cell proliferation and ECM remodeling through activation of TGF-β signaling. Methods We evaluated CD248 expression in human and murine lung tissue from PAH and control subjects via western blot (WB) and immunofluorescence (IF) imaging. CD248 expression was validated via WB and RNAseq of primary human vascular cells. Consequence of CD248 knock down (siRNA) on PASMC proliferation/migration was evaluated via WB, MTT, EdU-488 incorporation (flow cytometry), and wound healing scratch assays. CD248-TGF-β signaling effects were evaluated via WB or RT-qPCR for receptor (TβRI/RII), intermediate (SMAD3), and transcriptional (Fn1, Col1a1, Acta2) responses to CD248 loss. CD248-TβRI interaction was assessed via co-immunoprecipitation (co-IP). Results CD248 levels were elevated in human PAH lung tissue relative to controls. CD248 expression was localized to PASMCs as mRNA and protein levels were significantly elevated in PAH. CD248 was also increased in the hypoxia-sugen mouse lung, a preclinical PAH model, where it was expressed by α-SMA+ PASMCs. Cell Proliferation: Following CD248 siRNA KD in PAH PASMCs, we identified a reduction in PCNA levels, MTT proliferative metabolic potential, and EdU-AF488+ proliferative cells. TGF-β Signaling: TβRI but not TβRII was reduced in CD248-deficient PASMCs. TGF-β1-induced phosphorylation of TβRI and SMAD3 was attenuated by CD248 loss, which reduced the expression of downstream TGF-β-responsive genes (Fn1, Col1a1, Acta2). Interestingly, co-IP of CD248 and TβRI confirmed a direct physical interaction between these proteins. Conclusion CD248 is critical for proliferation and migration of PAH PASMCs. CD248 enhances TGF-β-induced vascular remodeling in PAH, likely via physical interaction and activation of TβRI. Future studies are needed to determine the therapeutic relevance of CD248 blockade on pulmonary vascular remodeling in PAH. This abstract is funded by: NIH NHLBI R01
Jones et al. (Fri,) studied this question.