title: The essential role of transient receptor potential vanilloid 1 in simvastatin-induced activation of endothelial nitric oxide synthase and angiogenesis.
abstract: OBJECTIVE
  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 Ca(2+) 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 Ca(2+). Pharmacological inhibition or genetic disruption of TRPV1 abrogated simvastatin-mediated elevation of intracellular Ca(2+) 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-Ca(2+) signalling and in the consequent NO production and angiogenesis as evidence by that re-expression of TRPA1 further augmented simvastatin-elicited Ca(2+) 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.
  
  
  CONCLUSIONS
  Simvastatin-induced Ca(2+) 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.
authors.affiliation: Institute of Physiology, National Yang-Ming University, Taipei, Taiwan.
authors.forename: K-H
authors.initials: KH
authors.lastname: Su
authors.forename: S-J
authors.initials: SJ
authors.lastname: Lin
authors.forename: J
authors.initials: J
authors.lastname: Wei
authors.forename: K-I
authors.initials: KI
authors.lastname: Lee
authors.forename: J-F
authors.initials: JF
authors.lastname: Zhao
authors.forename: S-K
authors.initials: SK
authors.lastname: Shyue
authors.forename: T-S
authors.initials: TS
authors.lastname: Lee
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D019821:Simvastatin / Q000494:pharmacology*; D050916:TRPV Cation Channels / Q000378:metabolism*
chemical_list: D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; D019821:Simvastatin; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
keywords: Ca2+; TRPV1 channels; angiogenesis; eNOS; simvastatin