Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis.
Improving endothelial nitric oxide synthase (eNOS) bioactivity and endothelial function is important to limit native, vein graft, and transplant atherosclerosis. Visfatin, a NAD biosynthetic enzyme, regulates the activity of the cellular survival factor, Sirt1. We hypothesized that visfatin may improve eNOS expression, endothelial function, and postnatal angiogenesis. In human umbilical vein (HUVEC) and coronary artery endothelial cells, we evaluated the effects of recombinant human visfatin on eNOS protein and transcript expression and mRNA stability, in the presence and absence of visfatin RNA silencing. We also assessed visfatin-induced protein kinase B (Akt) activation and its association with src-tyrosine kinases, phosphorylation of Ser(1177) within eNOS in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibition with LY-294002, and evaluated the contributory role of extracellular signal-regulated kinase 1/2. Finally, we determined the impact of visfatin on HUVEC migration, proliferation, inflammation-induced permeability, and in vivo angiogenesis. Visfatin (100 ng/ml) upregulated and stabilized eNOS mRNA and increased the production of nitric oxide and cGMP. Visfatin-treated HUVEC demonstrated greater proliferation, migration, and capillary-like tube formation but less tumor necrosis factor-alpha-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser(473)-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser(1177); these effects were blocked by LY-2940002. Studies with PP2 showed that the nonreceptor tyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Akt pathway. Visfatin also activated mitogen-activated protein (MAP) kinase through PI 3-kinase, and mitogen/extracellular signal-regulated kinase inhibition attenuated visfatin-elicited Akt and eNOS phosphorylation. Visfatin-filled Matrigel implants showed an elevated number of infiltrating vessels, and visfatin treatment produced significant recovery of limb perfusion following hindlimb ischemia. These results indicate a novel effect of visfatin to stimulate eNOS expression and function in endothelial cells, via a common upstream, src-mediated signaling cascade, which leads to activation of Akt and MAP kinases. Visfatin represents a translational target to limit endothelial dysfunction, native, vein graft and transplant atherosclerosis, and improve postnatal angiogenesis.
Division of Cardiac Surgery, Keenan Research Centre, Li Ka Shing Knowledge Institute, Riyadh, Kingdom of Saudi Arabia.
Fina
F
Lovren
Yi
Y
Pan
Praphulla C
PC
Shukla
Adrian
A
Quan
Hwee
H
Teoh
Paul E
PE
Szmitko
Mark D
MD
Peterson
Milan
M
Gupta
Mohammed
M
Al-Omran
Subodh
S
Verma
D000818:Animals; D050197:Atherosclerosis / Q000188:drug therapy* / Q000503:physiopathology / Q000601:surgery; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D003094:Collagen; D003331:Coronary Vessels / Q000166:cytology; D004338:Drug Combinations; D004343:Drug Implants; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007511:Ischemia / Q000188:drug therapy* / Q000503:physiopathology / Q000601:surgery; D007797:Laminin; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D054409:Nicotinamide Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011509:Proteoglycans; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D014680:Veins / Q000502:physiology / Q000637:transplantation
D004338:Drug Combinations; D004343:Drug Implants; D007797:Laminin; D011509:Proteoglycans; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C059485:matrigel; D003094:Collagen; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D054409:Nicotinamide Phosphoribosyltransferase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases