eNOS activity and NO-induced proliferation of lymphatic endothelial cells
(A) VEGF-C and VEGF-C (C156S) induce PI3K-dependent activation of eNOS in LECs. Signaling through VEGFR-2 and VEGFR-3 leads to activation of eNOS, Akt and p42/p44 in LECs as detected by Western blot analysis for phosphorylated eNOS (Ser1177), Akt (473) and p42/p44 (Thr202/Tyr204) proteins after LEC stimulation with VEGF-A, VEGF-C and VEGF-C (C156S). eNOS activation after VEGFR-2 or VEGFR-3 stimulation is dependent on PI3K activity as shown by decreased eNOS phosphorylation after pretreatment of LECs with PI3K inhibitor Wortmannin. (B) Effect of exogenous NO donors on lymphatic endothelial cell proliferation and/or survival. LEC proliferation and/or survival in response to increasing concentrations of NO donors DETA-NONOate and Glyco-SNAP-2 in growth factor reduced media was analyzed after 72h treatment using WST-1 cell proliferation reagent. Absorbance (450nm) obtained for cells incubated with full endothelial cell growth media was set to 100%. Bars represent mean ± SEM of quadruplicate samples. *P<0.05, Student t test.