Loss of ROR2 Tyrosine Kinase Receptor Is Associated With Endothelial Dysfunction in PAH via Inappropriate Integrin β1 Activation

BACKGROUND: Endothelial dysfunction is a key feature of pulmonary arterial hypertension (PAH). Previously, we demonstrated decreased Wnt7a transcript levels, causing reduced angiogenesis in PAH. Wnt7a expression correlates with tip formation via ROR2 (receptor tyrosine kinase-like orphan receptor 2), a tyrosine kinase receptor. We hypothesized that ROR2 activation in pulmonary microvascular endothelial cells (PMVECs) promotes angiogenesis, particularly endothelial barrier establishment, and its loss causes PAH. METHODS: Endothelial-specific ROR2 knockout (ROR2 ECKO) and wild-type mice were studied under normoxia and chronic hypoxia using echocardiography, hemodynamics, and lung morphometry. PMVECs from healthy and PAH lungs were transfected with ROR2 siRNA/constructs for functional and molecular studies. Focal adhesion activation and force generation were assessed via Förster resonance energy transfer-based methods. Bulk and single-cell transcriptomic analyses were performed on siROR2 (ROR2 siRNA) PMVECs and ROR2 ECKO lungs. RESULTS: ROR2 ECKO mice exacerbated pulmonary hypertension and vascular remodeling in hypoxia. Single-cell RNA-sequencing of lung endothelial cells revealed dysregulated barrier formation and angiogenesis. Evans blue dye extravasation confirmed reduced endothelial barrier integrity in ROR2 ECKO mice. ROR2-deficient PAH PMVECs displayed increased adhesion, permeability, and focal adhesion numbers, with reduced VE-cadherin at cell junctions. Confocal imaging and foster resonance energy transfer revealed ROR2 localization in focal adhesions, interacting with ITGB1 (integrin β1) which remained in an active, adhesion-promoting state in ROR2-deficient cells. Restoring ROR2 in PAH PMVECs normalized adhesion, barrier function, and focal adhesion abundance. Transcriptomic analysis revealed Rab12 mediated ROR2-ITGB1 crosstalk, whose knockdown mimicked ROR2 deficiency in PMVECs. CONCLUSIONS: ROR2 regulates pulmonary angiogenesis by maintaining endothelial barrier integrity and facilitating integrin recycling. ROR2 restoration could be a potential therapeutic approach for PAH.