(A) Plaque microparticles (MPs) induce a dose-dependent increase in ³H-thymidine incorporation in human umbilical vein endothelial cells (HUVECs), whereas both MPs' vehicle (Dulbecco's modified eagle medium [DMEM]) and the 20,500 g supernatant (Sn) obtained after pelleting MPs from plaque homogenate did not. (B) Plaque MPs induce a dose-dependent increase in endothelial cell number (n = 6). (C) Plaque MPs (6,000 Annexin V+ MPs/µl) from symptomatic (Sympto) patients induce more proliferation than those from asymptomatic (AS) patients (n = 6). FCS = fetal calf serum.

(A) Representative flow cytometry fluorescence histogram showing the absence of CD40 on plaque MPs (left) and its presence on endothelial cells under basal conditions and after exposure to plaque MPs (right). The black line corresponds to the negative control with an isotypic antibody, and the blue line reflects CD40-phycoerythrin-cyanin 5 (PC5) labeling. Endothelial CD40 expression is augmented when HUVECs are exposed to plaque MPs, as shown by the red line (right). CD40 silencing of HUVECS with small interfering ribonucleic acid (siRNA) abolished PC5 labeling (gray line, right). (B) Endothelial CD40 expression is augmented when HUVECs are exposed to increasing numbers of plaque MPs (n = 3). (C) Representative flow cytometry fluorescence histogram showing the presence of CD40L on plaque MPs (left) and its absence on endothelial cells under basal conditions or after exposure to plaque MPs (right). The black line corresponds to negative control and the blue line to CD40L-phycoerythrin (PE)–positive MPs. Tumor necrosis factor (TNF)-stimulated HUVECS express CD40L (green line), whereas HUVECs exposed to MPs do not (blue line, right). (D) Plaque MPs from Sympto patients express more CD40L than those from AS patients (n = 13 each). Abbreviations as in Figure 1.

(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.

(A) Matrigel sections stained with Masson Trichrome (green = collagen, red = cells) show infiltration of endothelial cells and red blood cells in presence of plaque MPs. (B) CD31 staining of Matrigel sections indicates the presence of endothelial cells; similar results were obtained with isolectin B4 staining (not shown). Antimouse Ter-119 staining was used to identify red blood cells in Matrigel. (C) Plaque MPs induce neovessel formation in Matrigel plugs from BalbC/Nude and wild-type (WT) mice, whereas MPs supernatant (Sn) has no effect. Microparticles effect on neovessel formation was abolished either in the presence of neutralizing antibody CD40L or when experiments were performed in CD40^–/– mice. FITC = fluoroisothiocyanate; other abbreviations as in Figure 1.