Abstract P061: Identification Of Piezo1 Channels In Perivascular Adipose Tissue (pvat) And Their Potential Role In Vascular Function

The vasculature constantly experiences distension/pressure exerted by the blood and responds accordingly to maintain homeostasis. Perivascular adipose tissue (PVAT) is gaining support as a formal blood vessel layer and also experiences these changes. We hypothesized that activation of the mechanotransducer Piezo1 directly increases vascular contraction in a way that might be modified by PVAT. The presence of Piezo1 was investigated at the mRNA level via PCR; protein via immunohistochemistry; and contractility via isolated tissue bath. Rat superior and mesenteric arteries, thoracic aortae, human mesenteric vessels and their PVATs were studied. Piezo1 mRNA (beta2 microglobulin calibrator) was expressed in the aortic vessel (2 -ΔC T =0.011); aortic PVAT (2 -ΔC T =0.0172); mesenteric vessel (2 -ΔC T =.00302), and mesenteric PVAT (2 -ΔC T =0.0219). Both adipocytes (2 -ΔC T =0.0249) and stromal vascular fraction (2 -ΔC T =0.0159) of mesenteric PVAT expressed Piezo1 mRNA. Piezo1 mRNA expression was greater in magnitude (one-way ANOVA) than that of the mechanotransducers Piezo2, TRPV4, TMEM16, and Panx1. Piezo1 protein was present in rat aortic PVAT, rat mesenteric (mes) artery, vein, and PVAT, as well as in human artery, vein, and PVAT. The Piezo1 agonists Yoda and Jedi (1 nM - 10 μM) did not stimulate rat aortic contraction [max <10% phenylephrine (PE) 10 μM contraction] or relaxation independent from vehicle in tissues + or - PVAT (relaxation as % of half maximal PE contraction was: Veh-PVAT=45.3±7.0; Yoda-PVAT=46.7±25.6; Jedi-PVAT= 40.4±10.3; Veh+PVAT= 71.8±19.7; Jedi+PVAT=39.1±13.2; Yoda +PVAT=21.6±10.9). Slightly K+ depolarizing the aorta did not unmask contraction to Yoda. Finally, the Piezo1 antagonist Dooku [10 μM] did not shift the PE curve (-log EC50 values [M]: Veh-PVAT= 7.96±0.12; Dooku-PVAT=7.26±0.22, Veh+PVAT=7.29±0.08; Dooku+PVAT=6.96±0.07). Surprisingly, Dooku [10 μM] directly caused aortic contraction in the absence of PVAT (Dooku 27.2±11.7 vs vehicle 13.5±11.2 %PE contraction), but not in the presence of PVAT vs vehicle (Dooku 2.9±1.9 vs Vehicle 7.3±5.2% PE contraction). Thus, Piezo1 is present and functional in the isolated aorta, important knowledge given that this molecule may serve as a translator of vascular pressure.

Abstract MP06: The Protein Tyrosine Phosphatase Inhibitors Induce The Vasoconstriction Via Src/epidermal growth factor/rho-kinase Cascade

Orthovanadate (OVA), a protein tyrosine phosphatase (PTPase) inhibitor, exerts contractile effects on smooth muscle in a Rho-kinase-dependent manner, but the precise mechanisms are not elucidated. The aim of this study was to determine the potential roles of Src and epidermal growth factor receptor (EGFR) in the OVA-induced contraction of rat aortas and the phosphorylation of myosin phosphatase target subunit 1 (MYPT1; an index of Rho-kinase activity) in vascular smooth muscle cells (VSMCs). The concentration-response curves for OVA (8–1000μM) were constructed in endothelium-denuded rings using stepwise increases in the concentration of OVA, and the maximal force was observed at concentrations of >500 μM (16 ± 2 in % of 60 mmol/L KCl). OVA (500 μM) evoked rapid contraction of endothelium-denuded aortas, presenting as a hyperbolic rise in tension development within 10 min followed by a slow linear rise. Aortic contraction by OVA was significantly blocked not only by Rho kinase inhibitors Y-27632 and hydroxyfasudil, but also by Src inhibitors PP2 and Src inhibitor No. 5 and the EGFR inhibitors AG1478 and EGFR inhibitor 1. OVA induced rapid increases in the phosphorylation of MYPT1 (Thr-853; 180 ± 24 % in OVA vs 98 ± 22 in vehicle), Src (Tyr-416; 225 ± 34 % in OVA vs 102 ± 30 in vehicle), and EGFR (Tyr-1173; 173 ± 31 % in OVA vs 101 ± 18 in vehicle) in VSMCs, and Src inhibitors abolished these effects. OVA-induced Src phosphorylation was abrogated by Src inhibitors, but not affected by inhibitors of EGFR and Rho-kinase. Inhibitors of Src and EGFR, but not Rho-kinase, also blocked OVA-induced EGFR phosphorylation. Furthermore, a metalloproteinase inhibitor TAPI-0 and an inhibitor of heparin-binding EGF not only abrogated the OVA-induced aortic contraction, but also OVA-induced EGFR and MYPT1 phosphorylation, suggesting the involvement of EGFR transactivation. OVA also induced EGFR phosphorylation at Tyr-845, one of residues phosphorylated by Src. These results suggest that OVA-induced vasocontraction is mediated by the Rho-kinase-dependent inactivation of myosin light chain phosphatase via signaling downstream of Src-induced transactivation of EGFR.