Involvement of Endothelial Nitric Oxide in Sphingosine-1-Phosphate–Induced Angiogenesis

Abstract

Abstract-Nitric oxide (NO) has been implicated as a critical signaling molecule of angiogenesis. Recently, sphingosine-1-phosphate (S1P) has emerged as a mediator of angiogenesis, and S1P-induced NO synthesis in endothelial cells (ECs) has been reported. To analyze the signaling pathways involved in S1P-induced angiogenesis and clarify the role of NO in this process, we performed in vivo and in vitro angiogenesis assays. S1P activated the phosphatidylinositol-3-kinase (PI3K)/Akt/endothelial NO synthase (eNOS) pathway in ECs, since S1P-stimulated eNOS phosphorylation and NO production were blocked by inhibition of activities of PI3K and Akt. S1P increased capillary ingrowth into subcutaneously implanted Matrigel plugs in mice, and the effect of S1P was significantly reduced in mice that received N G -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS. S1P stimulated EC migration and tube formation on Matrigel, which processes were significantly decreased by inhibition of activities of PI3K, Akt, or eNOS, whereas treatment with LY294002, a PI3K inhibitor, but not L-NAME, inhibited EC viability and proliferation. Thus, our results demonstrate the crucial role of NO in S1P-induced angiogenesis in vivo and in vitro and suggest the divergent roles of NO in the S1P-induced angiogenic response.

Role of Afadin in Vascular Endothelial Growth Factor– and Sphingosine 1-Phosphate–Induced Angiogenesis

“…[23][24][25] In sparsely plated nonsilencing control siRNA-, Rap1 siRNA-and afadin siRNA-transfected HUVECs, VEGF and S1P increased the phosphorylation of Akt and eNOS. This increase was inhibited in Rap1-or afadin-knockdown HUVECs when compared with control cells ( Figure 5).…”

Angiopoietin1/TIE2 and VEGF/FLK1 Induced by MSC Treatment Amplifies Angiogenesis and Vascular Stabilization after Stroke

“…Briefly, 0.1 ml growth factor reduced Matrigel (Becton Dickinson, San Jose, CA, USA) was added per well of a 96-well plate, and then MBECs (2  10 4 cells) were added and incubated in (1) regular cell-culture medium (Dulbecco's modified Eagle's medium) for control, (2) MSC condition media alone, (3) astrocyte condition media alone, (4) MSCs coculture with astrocytes conditioned media, (5) MSCs coculture with MBECs conditioned media, (6) MSC condition media with neutralized Ang1 antibody (1 mg/mL, rabbit anti-Ang1 affinity-purified polycolonal antibody, Chemicon, Temecula, CA, USA), and (7) MSC conditioned media with neutralized Flk1 antibody (DC101, 1 mg/mL) for 24 h. All assays were performed in n = 6/group. For quantitative measurements of capillary tube formation, Matrigel wells were digitized under a  4 objective (Olympus BX40) for measurement of total tube length of capillary tube formation using a video camera (Sony DXC-970MD) interfaced with the MCID image analysis system (Imaging Research, St Catharines, Canada) at 5 and 24 h. Tracks of endothelial cells organized into networks of cellular cords (tubes) were counted and averaged in randomly selected three microscopic fields (Rikitake et al, 2002).…”

AMP-activated Protein Kinase (AMPK) Signaling in Endothelial Cells Is Essential for Angiogenesis in Response to Hypoxic Stress

“…Regulation of eNOS Ser-1177 Phosphorylation and NO Production by AMPK-The production of endothelial NO is important for vascular homeostasis and angiogenesis (42)(43)(44)(45)(46). VEGF stimulation promotes Akt signaling which, in turn, produces NO through the activating phosphorylation of eNOS at residue 1177 (27).…”

“…Endothelium-derived NO is a positive modulator of angiogenesis (42)(43)(44)(45)(46), although the mechanism by which NO exerts this effect is unknown. In the study reported here, dominant-negative AMPK diminished eNOS phosphorylation on Ser-1177 and NO output under conditions of low oxygen tension, whereas AMPK-inhibition had little if any effect on these parameters in normoxic cultures.…”