Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis

doi:10.1152/ajpendo.90780.2008

Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis

Fina Lovren,¹^,5 Yi Pan,¹ Praphulla C. Shukla,¹ Adrian Quan,¹^,5 Hwee Teoh,¹^,3^,4^,5 Paul E. Szmitko,²^,5 Mark D. Peterson,¹ Milan Gupta,¹^,3^,4^,6 Mohammed Al-Omran,⁷ and Subodh Verma¹^,3^,4^,7

¹Division of Cardiac Surgery, Keenan Research Centre, Li Ka Shing Knowledge Institute, ²Division of General Internal Medicine, and ³Cardiometabolic Risk Initiative, St. Michael's Hospital, Toronto; ⁴Canadian Cardiovascular Research Network, Toronto; ⁵Translational Traineeship Program in Atherosclerosis, Toronto; ⁶Division of Cardiology, William Osler Health Centre, Brampton, Ontario, Canada; and ⁷Division of Vascular Surgery, College of Medicine and King Khalid University Hospital, King Saud University-Li Ka Shing Collaborative Research Program, Riyadh, Kingdom of Saudi Arabia

Submitted 20 September 2008 ; accepted in final form 6 April 2009

Improving endothelial nitric oxide synthase (eNOS) bioactivityand endothelial function is important to limit native, veingraft, and transplant atherosclerosis. Visfatin, a NAD biosyntheticenzyme, regulates the activity of the cellular survival factor,Sirt1. We hypothesized that visfatin may improve eNOS expression,endothelial function, and postnatal angiogenesis. In human umbilicalvein (HUVEC) and coronary artery endothelial cells, we evaluatedthe effects of recombinant human visfatin on eNOS protein andtranscript expression and mRNA stability, in the presence andabsence of visfatin RNA silencing. We also assessed visfatin-inducedprotein kinase B (Akt) activation and its association with src-tyrosinekinases, phosphorylation of Ser¹¹⁷⁷ within eNOS in the presenceand absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibitionwith LY-294002, and evaluated the contributory role of extracellularsignal-regulated kinase 1/2. Finally, we determined the impactof visfatin on HUVEC migration, proliferation, inflammation-inducedpermeability, and in vivo angiogenesis. Visfatin (100 ng/ml)upregulated and stabilized eNOS mRNA and increased the productionof nitric oxide and cGMP. Visfatin-treated HUVEC demonstratedgreater proliferation, migration, and capillary-like tube formationbut less tumor necrosis factor- ${alpha}$ -induced permeability; theseeffects were decreased in visfatin gene-silenced cells. Visfatinincreased total Akt and Ser⁴⁷³-phospho-Akt expression with concomitantrises in eNOS phosphorylation at Ser¹¹⁷⁷; these effects wereblocked by LY-2940002. Studies with PP2 showed that the nonreceptortyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Aktpathway. Visfatin also activated mitogen-activated protein (MAP)kinase through PI 3-kinase, and mitogen/extracellular signal-regulatedkinase inhibition attenuated visfatin-elicited Akt and eNOSphosphorylation. Visfatin-filled Matrigel implants showed anelevated number of infiltrating vessels, and visfatin treatmentproduced significant recovery of limb perfusion following hindlimbischemia. These results indicate a novel effect of visfatinto stimulate eNOS expression and function in endothelial cells,via a common upstream, src-mediated signaling cascade, whichleads to activation of Akt and MAP kinases. Visfatin representsa translational target to limit endothelial dysfunction, native,vein graft and transplant atherosclerosis, and improve postnatalangiogenesis.

nitric oxide; mice; endothelium; atherosclerosis; visfatin; protein kinase B; phosphatidylinositol 3-kinase; mitogen-activated protein kinase; endothelial nitric oxide synthase

Address for reprint requests and other correspondence: S. Verma, Canada Research Chair in Atherosclerosis, Director, Traineeship in Atherosclerosis, Translational Atherosclerosis Research Program, Cardiac Surgeon & Associate Professor, Division of Cardiac Surgery, St. Michael's Hospital, 8th Floor Bond Wing, 30 Bond St., Toronto, ON, Canada M5B 1W8 (e-mail: subodh.verma{at}sympatico.ca)