The role of complement in the adherence of microbubbles to dysfunctional arterial endothelium and atherosclerotic plaque

Objective: To determine whether serum complement C3 mediates adherence of albumin-encapsulated microbubbles to vascular endothelium in the development of atherosclerotic plaques. Methods: Adherence of microbubbles to aortic endothelium was examined with scanning electron microscopy following intravenous injection of 20% intralipid in wild-type mice, genetic complement-deficient mice (C3^-/-), and in pharmacologic C3-depleted wild-type mice. In a second experimental model, atherosclerostic plaque was induced in apolipoprotein E-deficient mice (apoE^-/-), and adherence of microbubbles to atherosclerotic plaques was evaluated using fluorescent microscopy of fluorescein isothiocynate-conjugated microbubbles. Finally, imaging of aortas was performed in eight rats (four JCR:LA-cp atherosclerosis-prone rats on high cholesterol diets; four controls) following intravenous albumin microbubble injections (PESDA) to determine whether microbubble adherence to the endothelium could be detected with low mechanical index pulse sequence schemes. Results: Scanning electron microscopy confirmed the adherence of microbubbles to the endothelial cells of the aorta in wild-type mice following induction of hypertriglyceridemia but not in C3-depleted mice. Microbubble adherence to the endothelial surface of atherosclerotic plaque was confirmed in all apoE^-/- mice (median 172 microbubbles/field; compared to a median of 3 microbubbles/field in cobra venom factor-treated apoE^-/- mice; p < 0.001). Low mechanical index ultrasound imaging detected microbubble adherence in all JCR atherosclerosis prone rats even in the absence of vasomotor or phenotypical evidence of endothelial dysfunction. The numbers of adherent microbubbles correlated with serum triglyceride levels, and were seen in conjunction with increased endothelial nitric oxide synthase activity. Conclusions: Complement C3 binds to albumin-encapsulated microbubbles and mediates microbubble adherence to vascular endothelium both early and late in the atherosclerotic process.