Abstract 16436: Targeting Store Operated Calcium Entry in Vascular Smooth Muscle Cells by a Novel Small-molecule Inhibitor

Abstract

Vascular smooth muscle cells (VSMC) are physiologically quiescent cells optimised for vascular contractility and the modulation of vascular tone. Platelet-derived growth factor (PDGF) signalling is a critical driver of pathological remodelling of native VSMC to a proliferative synthetic state, which is a process associated with vascular pathologies. Here, we studied the role of store operated calcium entry (SOCE) through Orai1 channels in VSMC plasticity utilizing novel small molecule inhibitor of Orai1 channels. We developed small-molecule Orai1 inhibitor, 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline, called JPIII, evaluated its effects on SOCE, and its functional impact in human primary VSMC (hVSMC) from saphenous vein and rat thoracic aorta VSMC cell line. Our Orai1 inhibitor potently suppressed SOCE in hVSMC (IC 50 of 297 nM, n=3) and rat VSMC (IC 50 of 819.5 nM, n=3), leading to significant impairment of PDGF-induced cell migration in hVSMC by 31% (P=0.0071, n=6) and rat VSMC by 24% (P=0.04, n=3). Orai1 inhibition also reduced PDGF-induced proliferation of hVSMC by 20% (P=0.02, n=5) and of rat VSMC by 26% (P=0.003, n=4) at concentration of 30 μM JPIII relative to controls. RNA-Seq analysis of PDGF-stimulated primary human aortic VSMC (n=4/group) was performed to investigate the effects of Orai1 inhibition on the transcriptional response of cells and showed significant downregulation of genes involved in inflammation (C-X-C motif chemokine ligand (CXCL) 1, CXCL3, CXCL5, CXCL6), downregulation of matrix degrading enzymes (metalloproteinase (MMP) 12 and MMP16), and upregulation of cholesterol biosynthesis pathways by Orai1 inhibition. Consistent with the observed functional phenotype, our results revealed an inhibitory effect on transcriptional networks involved in cell proliferation and migration. The effects of Orai1 inhibition on the transcription of genes involved in inflammation, MMPs expression, cholesterol metabolism, cell proliferation, and migration powerfully associate Orai1 signalling with VSMC remodelling. Our results provide insight into the role of Orai1 in VSMC remodelling and the possible therapeutic potential of a small molecule inhibition approach in targeting pathologic remodelling.