G-protein coupled receptor 35 activation mediates human vascular smooth muscle migration

Abstract

Introduction: GPR35 is a poorly characterised G protein-coupled receptor endogenously activated by kynurenic acid and selectively coupled to Gα13, a G protein involved in regulation of cellular motility and proliferation via the Rho kinase pathway. The role of GPR35 in cardiovascular disease (CVD) is poorly explored, though it has been proposed to regulate blood pressure in rodents. Ligands which activate GPR35 are orthologue selective however, we have identified potent novel human GPR35 specific ligands; agonist pamoic acid and antagonists, CID-2745687 and ML145 for further dissection of GPR35 pharmacology. Based on GPR35's coupling to Gα13, here we investigated the role of GPR35 in primary human saphenous vein vascular smooth muscle cell (HSVSMC) migration and proliferation; a pathological process integral to the development of vein graft failure and restenosis. Methods: Human selective GPR35 ligands were pharmacologically characterised in β-arrestin translocation assays via bioluminenscence resonence energy transfer (BRET) in cells stably expressing FLAG-hGPR35-eYFP (Flp-In TREx 293). Endogenous expression of GPR35 was quantified in HSVSMC via QRT-PCR and migration was measured in HSVSMCs stimulated with 10-500nM pamoic acid, +/- CID-2745687 and ML145 (100nM) or ROCK1/2 inhibitor Y-27632 (10μM) in scratch wound assays using ImageJ. HSVSMC proliferation was assessed via MTS assay. Changes in HSVSMC morphology and actin-cytoskeletal structure were assessed via tetramethyl rhodamine isothiocyanate (TRITC) actin phalloidin staining. Results: BRET assays in FLAG-hGPR35-eYFP cells demonstrated that pamoic acid was a potent, human selective GPR35 agonist (pEC50 7.28±0.007) while CID-2745687 (pIC50 7.16±0.12) and ML145 (pIC50 8.08±0.12) were potent antagonists. QRT-PCR demonstrated HSVSMC endogenously express GPR35 (ΔCT=19.97±0.23, normalised to 18s). Analysis of the actin cytoskeleton revealed GPR35 activation induced a rearrangement of actin filaments consistent with HSVSMC migration. Quantification of HSVSMC migration revealed the GPR35 agonist pamoic acid induced a 30% increase in migration (p 0.05 vs -serum control). GPR35 activation did not affect HSVSMC proliferation. Conclusion: Here, we have described a novel role for GPR35 activation in the migratory capacity of HSVSMC via its downstream effector ROCK1/2. This suggests GPR35 might be a therapeutic target in the setting of vein graft failure and restenosis.