Abstract 14438: Loss of the ROR2 Tyrosine Kinase Receptor is Associated With Dysfunctional Angiogenesis in Pulmonary Hypertension via Inappropriate Focal Adhesion Activation and Disruption of the Endothelial Barrier

Background: Dysfunctional angiogenesis is a hallmark of pulmonary arterial hypertension (PAH), but its mechanisms are unclear. We recently demonstrated a key role for Wnt7a in lung angiogenesis. Wnt7a acts through ROR2, a Wnt pathway tyrosine kinase receptor. We elucidate how ROR2 controls angiogenic functions in pulmonary microvascular endothelial cells (PMVECs).

Hypothesis: Loss of ROR2 activity is associated with PAH.

Methods: Healthy and PAH PMVECs were obtained from explanted lungs. PMVECs were transfected with ROR2 siRNA/constructs, followed by functional and molecular studies. Focal adhesion (FA) activation and force generation was measured using FRET-based total internal reflection microscopy (TIRF). Mice with endothelial-specific conditional ROR2 KO (ROR2 ECKO) were phenotyped in normoxia and hypoxia.

Results: ROR2 expression was found predominantly in the endothelium of healthy lung vessels but significantly reduced within PAH lesions. ROR2 knockdown (siROR2) reduced vascular sprouting comparable to PAH PMVECs. Interestingly, siROR2 and ROR2-deficient PAH PMVECs displayed increased fibronectin adhesion and hyperpermeability, which led us to look at focal adhesions (FA) and cell-cell junctions. Confocal and TIRF microscopy of siROR2 and ROR2-deficient PAH PMVECs revealed an increase in the FA number and force that inversely correlated with reduced VE-cadherin at cell junctions. However, transfection of a ROR2 construct in PAH PMVECs resulted in FAs activity normalization and junctional integrity accompanied by recovery of adhesion and permeability. Proteomic studies demonstrated that ROR2 interacts with Integrin b1 in focal adhesions, and its loss results in Integrin b1 activation, FA clustering, and high mechanical forces. In animal studies, we found that, compared to controls, ROR2 ECKO developed more severe pulmonary hypertension, right ventricular remodeling, microvascular reduction, and muscularization in hypoxia, which correlated with higher levels of integrin b1 activation and reduced VE-cadherin expression.

Conclusions: ROR2 promotes PMVEC adhesion and barrier formation, and its loss is associated with dysfunctional angiogenesis. Restoring ROR2 could be a novel therapeutic strategy in PAH.