Endothelial SHMT2 Drives Pulmonary Vascular Remodeling Through Noncanonical Pathway in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive, life-threatening disease characterized primarily by pulmonary vascular remodeling in which endothelial dysfunction plays a vital role. However, the molecular factors contributing to this pathological process remain incompletely understood. Through proteomic analysis of hypoxia-treated human pulmonary artery endothelial cells, we identified serine hydroxymethyltransferase 2 (SHMT2) as a potential target in PH, but its role in disease pathogenesis and the underlying mechanisms remain unclear. The expression and function of SHMT2 were assessed in lung samples from patients with PH and in rodent PH models, including hypoxia-exposed mice and monocrotaline- or Sugen 5416/hypoxia-induced rats. Endothelial cell-specific SHMT2 loss and gain of function were achieved by conditional knockout and adeno-associated virus 9-mediated gene modulation. In vitro studies were performed in hypoxia-treated human pulmonary artery endothelial cells and HEK-293T cells. Virtual screening was used to identify a small-molecule inhibitor targeting the nonmetabolic function of SHMT2, and its therapeutic potential was further evaluated in rodent PH models. SHMT2 was upregulated predominantly in pulmonary vascular endothelium of patients with PH and multiple rodent PH models. In vivo, endothelial cell-specific deletion of Shmt2 markedly attenuated pulmonary vascular remodeling and right ventricular dysfunction in PH mice, whereas endothelial cell-specific Shmt2 overexpression aggravated PH development. Consistently, adeno-associated virus 9-mediated endothelial Shmt2 knockdown alleviated PH phenotypes in rat models. Mechanistically, SHMT2 promoted hypoxia-induced endothelial barrier dysfunction mainly through a noncanonical function by blocking the K63-ubiquitin-mediated lysosomal degradation of ras homolog family member B (RhoB). Additional in vivo studies supported an important role of the endothelial SHMT2-RhoB axis in pulmonary vascular remodeling of PH. Through virtual screening, Namodenoson was identified as a small-molecule inhibitor targeting the SHMT2-RhoB pathway. In vivo, Namodenoson showed both preventive and therapeutic effects against PH. This study highlights endothelial SHMT2 as an important contributor to PH pathogenesis and reveals a noncanonical SHMT2-RhoB pathway that promotes endothelial dysfunction. Targeting this pathway may represent a potential therapeutic strategy for PH.