The essential role of transient receptor potential vanilloid 1 in simvastatin-induced activation of endothelial nitric oxide synthase and angiogenesis

Aims We investigated the role of transient receptor potential vanilloid receptor type 1 (TRPV1) in simvastatin‐mediated activation of endothelial nitric oxide synthase (eNOS) and angiogenesis. Methods Fluo‐8 NW assay was for Ca2+ detection; Griess's assay was for NO bioavailability; Western blotting and immunoprecipitation were for protein phosphorylation and interaction; tube formation and Matrigel plug assay were for angiogenesis. Results In endothelial cells (ECs), treatment with simvastatin time‐dependently increased intracellular level of Ca2+. Pharmacological inhibition or genetic disruption of TRPV1 abrogated simvastatin‐mediated elevation of intracellular Ca2+ in ECs or TRPV1‐transfected HEK293 cells. Loss of TRPV1 function abolished simvastatin‐induced NO production and phosphorylation of eNOS and calmodulin protein kinase II (CaMKII) in ECs and in aortas of mice. Inhibition of TRPV1 activation prevented the simvastatin‐elicited increase in the formation of TRPV1–Akt–CaMKII–AMPK–eNOS complex. In mice, Matrigel plug assay showed that simvastatin‐evoked angiogenesis was abolished by TRPV1 antagonist and genetic ablation of TRPV1. Additionally, our results demonstrated that TRP ankyrin 1 (TRPA1) is the downstream effector in the simvastatin‐activated TRPV1‐Ca2+ signalling and in the consequent NO production and angiogenesis as evidence by that re‐expression of TRPA1 further augmented simvastatin‐elicited Ca2+ influx in TRPV1‐expressed HEK293 cells and ablation of TRPA1 function profoundly inhibited the simvastatin‐induced increase in the phosphorylation of eNOS and CaMKII, formation of TRPV1–Akt–CaMKII–AMPK–eNOS complex, NO bioavailability, tube formation and angiogenesis in ECs or mice. Conclusion Simvastatin‐induced Ca2+ influx may through the activation of TRPV1–TRPA1 signalling, which leads to phosphorylation of CaMKII, increases in the formation of TRPV1–CaMKII–AMPK–eNOS complex, eNOS activation, NO production and, ultimately, angiogenesis in ECs.