Abstract 15706: Loss of Wnt/Ror2 Signaling is Associated With Reduced Pericyte Recruitment and Impaired Pulmonary Angiogenesis in Idiopathic Pulmonary Arterial Hypertension
Pericytes are vascular cells that interact with endothelial cells (ECs) to provide mural support and promote vessel maturation. While abnormalities in EC-pericyte interactions could contribute to small vessel loss in IPAH, little is known about the role of pericytes in the pulmonary vasculature. Using a novel magnetic bead-based assay, we isolated pericytes from lungs of control and IPAH patients followed by lineage validation via FACS. When seeded with pulmonary microvascular ECs (PMVECs) in matrigel coated plates, IPAH pericytes failed to associate with endothelial tubes resulting in smaller vascular networks compared to control. Live microscopy showed that IPAH pericytes were unable to migrate towards endothelial tubes suggesting a defect in polarization and motility. Quantitative PCR of healthy pericytes co-cultured with PMVECs demonstrated a 34-fold increase in the expression of ROR2, a receptor of the Wnt/planar cell polarity (PCP) pathway. Given Wnt/PCP’s involvement in coordinating cell polarity and motility, we asked whether ROR2 was necessary for pericyte recruitment to endothelial tubes. To test this, we co-cultured pericytes treated with either non-targeting or ROR2 specific siRNA with healthy PMVECs in both Boyden Chamber and wound healing assays. We found that not only ROR2 deficient pericytes demonstrated significantly less polarization and motility but also reduced activation of RhoA/Rac1, mediators of Wnt/PCP signaling. Assessment of ROR2 expression in IPAH pericytes demonstrated that, while ROR2 mRNA was >4-fold downregulated in IPAH pericytes vs. control, there was no significant difference in protein level between the two groups. However, analysis of ROR2 phosphorylation (i.e. activation) via nanoimmunoassay demonstrated significant less phosphorylation in IPAH pericytes and RhoA/Rac1 activation in response to Wnt5a, a known ROR2 ligand. Taken together, these studies suggest that recruitment and formation of EC-pericyte interactions during pulmonary angiogenesis is dependent on ROR2-mediated Wnt/PCP activation. Thus, future treatment strategies aimed at restoring ROR2 activation may be useful to prevent small vessel loss in IPAH.
- © 2013 by American Heart Association, Inc.