A TRPC3 Blocker, Ethyl-1-(4-(2,3,3-Trichloroacrylamide)Phenyl)-5-(Trifluoromethyl)-1H-Pyrazole-4-Carboxylate (Pyr3), Prevents Stent-Induced Arterial Remodeling

Abstract

TRPC-mediated Ca 21 entry has been implicated in the control of smooth muscle proliferation and might represent a pivotal mechanism underlying in-stent restenosis. As we have observed significant expression of TRPC3 in human smooth muscle from the coronary artery as well as the aorta, we tested the efficiency of a recently discovered TRPC3 selective Ca 21 entry blocker Pyr3 to prevent vascular smooth muscle proliferation and stent implantation-induced hyperplasia of human aorta. The effect of Pyr3 on proliferation was measured by detection of BrdU incorporation and PCNA expression in human coronary smooth muscle and microvascular endothelium, which displays significantly smaller expression levels of TRPC3 as compared with smooth muscle. Pyr3 inhibited smooth muscle proliferation but lacked detectable effects on endothelial proliferation. Measurements of ATP-induced Ca 21 signals revealed that Pyr3 suppressed agonist-induced Ca 21 entry more effectively in vascular smooth muscle than in endothelial cells. Inhibitory effects of Pyr3 on stent implantation-induced arterial injury was tested using a novel in vitro model of in-stent hyperplasia in human arteries based on organ typical culture of human aortic constructs. Pyr3 effectively prevented increases in tissue levels of PCNA and Ki-67 at 2 weeks after stent implantation into human aortae. Similarly, proliferation markers were significantly suppressed when implanting a Pyr3-releasing stent prototype as compared with a bare metal stent (BMS) control. Our results suggest TRPC3 as a potential target for pharmacological control of smooth muscle proliferation. Selectively inhibition of TRPC Ca 21 entry channels in vascular smooth muscle is suggested as a promising strategy for instent restenosis prevention.

Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis

“…42 The action site of pyr3 is located on the external side of the TRPC3 protein, and similar acute actions of pyr3 were noted in human coronary artery smooth muscle cells and microvascular endothelial cells. 42,43 DAG analog OAG has been routinely used as a direct activator of receptor operated channels TRPC3 and -6 independently of protein kinases C and without the generation of inositol trisphosphate. 59 Nonselective blockers SKF96365, Gd 3+ , and 2-APB were also used to further validate the implication of TRP channels.…”

“…This might be because of a positive feed-forward regulation of TRPC3; in fact, this positive feed-forward regulation of TRPC signaling was reported for excessive TRPC activation in other cell types. 43,99 During tissue injuries, such as those occurring in kidney diseases, mesenchymal and inflammatory cells secrete a wide array of profibrotic and inflammatory cytokines, such as TGF-b1, Ang II, and endothelin I, contributing to the differentiation of fibroblasts into myofibroblasts. 71,86 Furthermore, the ERK signaling pathway is activated in UUO and may provide approaches in preventing progression of renal fibrosis.…”

“…[40][41][42][43] When ERK inhibitors were used, cells were treated after 3 days of culture for 24 hours with 50 mM PD98059 or 10 mM U0126 (Tocris Bioscience). Sixteen cultures from eight rats were performed for each condition.…”

“…40,41 The TRPC3-selective inhibitor, ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1 H-pyrazole-4-carboxylate (pyr3), is reported to block cardiac hypertrophy in mice subjected to pressure overload, with a marked selectivity for TRPC3 over other TRPs from the same family, 42 and prevent stentinduced arterial remodeling. 43 More recently, TRPC3 has been found in human cardiac fibroblasts and regulated cell proliferation and differentiation in atrial fibrillation. 44,45 Few other studies reported the presence of TRPC channels in normal rat kidney cell lines 46,47 and identified their localization in the adult rat and human kidney.…”

Characteristics of Transient Receptor Potential Canonical Calcium-Permeable Channels and Their Relevance to Vascular Physiology and Disease

“…28, 32 The idea that TRPCs contribute particularly in inflammatory and remodeling contexts is supported by studies of Trpc gene-disrupted mice and effects of TRPC chemical inhibitor. [33][34][35] One such study has suggested that TRPC3 is a driver of the nuclear translocation of zyxin, 36 an important signal for stretch-induced gene expression. from cells or animals carrying long-term disruption of a gene or several genes encoding TRPCs.…”