Abstract Body (Do not enter title and authors here): Rationale: Endothelial dysfunction is a major hallmark of PAH whose underlying mechanism remains unexplored. We recently showed the vital role of the Wnt7a ligand, promotes pulmonary angiogenesis via ROR2, a tyrosine kinase receptor. We sought to elucidate how ROR2 activation orchestrates angiogenic responses in endothelial barrier establishment in pulmonary microvascular endothelial cells (PMVEC), and its potential contribution to endothelial dysfunction in PAH.
Hypothesis: ROR2 regulates lung angiogenesis, and its loss is associated with PAH.
Methods: Healthy and PAH PMVECs and lung tissues were obtained from transplants and commercial sources. PMVECs were transfected with ROR2 siRNA/constructs for functional and molecular studies. Bulk RNA-seq of control and siROR2 PMVECs was followed by bioinformatic analysis. Focal adhesion (FA) activation and force generation were measured using FRET-based total internal reflection microscopy (TIRF). Endothelial-specific conditional ROR2 KO (ROR2 ECKO) mice exposed to normoxia and hypoxia were phenotyped using echocardiography, hemodynamics, and lung morphometry.
Results: ROR2 expression was significantly reduced in PAH lesions versus healthy. ROR2 knockdown (siROR2) reduced vascular sprouting and VEGF response comparable to control PMVECs. siROR2 and ROR2-deficient PAH PMVECs shows increased adhesion to fibronectin and barrier permeability. Confocal and TIRF microscopy of siROR2 and ROR2-deficient PAH PMVECs showed increased FA number and force generated by individual FAs that inversely correlated with reduced VE-cadherin at cell-cell junctions. ROR2 overexpression in PAH PMVECs normalized ROR2 protein expression, FAs activity, and junctional integrity and promoted recovery. Proteomic studies showed, ROR2 interacts with Integrin b1 in focal adhesions, and its loss results in Integrin b1 activation, FA clustering, and high mechanical forces. RNA-seq of siROR2 PMVECs showed dysregulation of vesicular trafficking and adhesion genes and enriched vesicular trafficking and angiogenic pathways. In animal studies, ROR2 ECKO developed severe PH, right ventricular remodeling, microvascular reduction, and muscularization in hypoxia, correlating with increased integrin-b1 activation and decreased VE-cadherin expression.
Conclusions: ROR2 promotes vessel sprouting and barrier formation, and its loss is associated with dysfunctional angiogenesis in PAH. Restoring ROR2 signaling activity could be a novel therapeutic strategy in PAH.