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Published on: September 18, 2013
Loss of ROR2 Tyrosine Kinase Receptor Is Associated With Endothelial Dysfunction in PAH via Inappropriate Integrin β1
Ankita Mitra1, Stuti Agarwal1, Ananya Chakraborty1
1Division of Pulmonary and Critical Care (A.M., S.A., A.C., A.R., A.B., A.P., N.A., P.C., A.J., J.A.M.M., E.S.G., V.A.d.J.P.), Stanford University, Palo Alto, CA.
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.
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