Figure 5.

The lectin–like oxidized low–density lipoprotein receptor-1 (LOX1) mediates endothelial recognition of danger signals on unesterified cholesterol–induced microvescicles (UCMVs). A, Cultured monolayers of human heart microvascular endothelial cells were incubated for 5 hours at 37°C with 0 (Control) or 3.0×10⁴ UCMVs per μL, followed by rinsing and then detergent extraction. Displayed are immunoblots for LOX1 and, as a loading control, GAPDH. B, Human endothelial monolayers were preincubated for 1 hour in unsupplemented medium (2 leftmost columns) or in a medium supplemented with a monoclonal blocking antibody against LOX1 (anti-LOX1 mAb) or a non-immune isotype control antibody (IgG2b Isotype). Additional medium without (Control) or with UCMVs was then added, followed by 5 more hours of incubation and then rinsing. Endothelial activation was assessed by the subsequent adherence of fluorescent-labeled monocytes. C, Thoracic aortic explants from wild-type or Lox1 knockout mice (Lox1 KO) were incubated ex vivo for 5 hours at 37°C without or with UCMVs, as indicated, followed by rinsing and then the adherence of fluorescently labeled monocytes was assessed. B and C, Counts of adherent, fluorescent monocytes per high-power field ([HPF]; mean±SEM, n=3–5). In these panels, P<0.001 by ANOVA, displayed are significance levels for individual pairwise comparisons by the Student-Newman-Keuls test.