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Aims Erythropoietin ( EPO), the key hormone involved in erythropoiesis, beneficially affects endothelial cells ( ECs), but the detailed mechanisms are yet to be completely understood. In this study, we investigated the role of transient receptor potential vanilloid type 1 ( TRPV1), a ligand-gated non-selective calcium (Ca2+) channel, in EPO-mediated endothelial nitric oxide synthase ( eNOS) activation and angiogenesis. Methods and results In ECs, EPO time dependently increased intracellular levels of calcium; this increase was abrogated by the Ca2+ chelators and pharmacological inhibitors of TRPV1 in bovine aortic ECs (BAECs) and TRPV1-transfected HEK293 cells. In addition, EPO-induced nitrite oxide ( NO) production, phosphorylation of eNOS, Akt and AMP-activated protein kinase ( AMPK) and the formation of TRPV1-Akt- AMPK- eNOS complex as well as tube formation were diminished by the pharmacological inhibition of TRPV1 in BAECs. Moreover, EPO time dependently induced the phosphorylation of phospholipase C- γ1 ( PLC- γ1). Inhibition of PLC- γ1 activity blunted the EPO-induced Ca2+ influx, eNOS phosphorylation, TRPV1- eNOS complex formation and NO production. The phosphorylated level of eNOS increased in the aortas of EPO-treated wild-type ( WT) mice or EPO-transgenic (Tg) mice but not in those of EPO-treated TRPV1-deficient ( TRPV1−/−) mice or EPO-Tg/ TRPV1−/− mice. Matrigel plug assay showed that EPO-induced angiogenesis was abrogated in TRPV1 antagonist capsazepine-treated WT mice and TRPV1−/− mice. Conclusion These findings indicate the EPO-induced Ca2+ influx via the activation of the PLC- γ1 signalling pathway, which leads to TRPV1 activation and consequently increases the association of the TRPV1-Akt- AMPK- eNOS complex, eNOS activation, NO production and angiogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]