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Figure 3 Plaque MP-Induced Endothelial Cell Proliferation Is Mainly Dependent on CD40L Signaling
(A) Exposure of MPs to an anti-CD40L or exposure of HUVECs to an anti-CD40 or the combination of these 2 neutralizing antibodies decreases the endothelial cell 3H-thymidine (thym.) incorporation (n = 12). Control experiments (dotted line) were performed with the respective immunoglobulin G isotypes. (B) Endothelial proliferation induced by either plaque MPs or recombinant CD40L are abolished in CD40-silenced HUVECs (n = 3). (C) The inhibitor of cyclooxygenase 2, NS-398 (10–6 mol/l), has no effect on plaque MP-induced endothelial proliferation (n = 6). (D) Plaque MP-induced proliferation is impaired by either the PI3-kinase/Akt inhibitors (LY294002, 10–6 mol/l and wortmanin, 10–6 mol/l) or an antivascular endothelial growth factor (VEGF)-R2 blocking antibody (5 µg/ml, n = 3); the corresponding immunoglobulin G isotype did not significantly affect MP-induced proliferation. (E) Exposure of HUVECs to increasing concentrations of MPs (red bars) or FCS (black bar) for 24 h stimulated VEGF release in medium; VEGF levels were also determined in the medium containing the highest concentration of MPs, but not exposed to HUVEC (n = 6). %/cont = percent of control experiments performed with the respective immunoglobulin G isotypes; LY = LY-294002; recCD40L = recombinant CD40 ligand; si = small interfering; w/o = without cells; wort = wortmanin; other abbreviations as in Figure 1.
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