Abstract 661: Bilirubin Prevents Atherosclerotic Plaque Formation in LDLR -/- Mice by Inhibiting Monocyte Migration Through the Disruption of Endothelial Vascular Cell Adhesion Molecule 1 (VCAM-1) and Intercellular Adhesion Molecule 1 (ICAM-1) Signaling

Abstract

Background: Epidemiologic studies support an inverse association between serum bilirubin levels and the risk of cardiovascular disease. Monocyte infiltration into the vascular endothelium, a critical early step in atherosclerotic plaque formation, is mediated primarily by the adhesion molecules VCAM-1 and ICAM-1. We postulate that bilirubin, a potent antioxidant, inhibits plaque formation by preventing monocyte transendothelial migration through the scavenging of VCAM-1 and ICAM-1 reactive oxygen species (ROS) signaling intermediaries. Methods: The effect of physiological concentrations (≤ 1.2 mg/dL) of bilirubin on the migration of THP-1 monocytes across HUVEC monolayers activated with TNFα to express VCAM-1 and ICAM-1 was assessed by Boyden chamber assay. The influence of bilirubin on monocyte adhesion, cytokine production, and VCAM-1- and ICAM-1-mediated ROS production was quantified by fluorescence assay, RT-PCR, and time-lapse confocal microscopy, respectively. To evaluate the effect on plaque formation in vivo , LDLR -/- mice were fed a Western diet and administered i.p. bilirubin (30 mg/kg), vehicle, or sham for 8 weeks. Aortic roots were stained with Oil Red O (lipids), Sirius Red (collagen), α-smooth muscle actin (SMCs), chlorotyrosine and nitrotyrosine (oxidation), CD68 (macrophages), and for VCAM-1 and ICAM-1. Results: THP-1 migration across activated HUVEC monolayers is dose-dependently inhibited by bilirubin. Bilirubin did not alter monocyte binding or cytokine production by activated HUVEC, but effectively abolished cellular ROS production induced by crosslinking anti-VCAM-1 and anti-ICAM-1 antibodies. Bilirubin treatment markedly inhibited plaque formation in the aortic root of LDLR -/- mice, and was associated with significant reductions in plaque lipid and collagen deposition, SMC infiltration, oxidation, and macrophage infiltration, without altering VCAM-1 or ICAM-1 protein expression. Conclusion: Bilirubin prevents atherosclerotic plaque formation in LDLR -/- mice by disrupting endothelial VCAM-1- and ICAM-1-mediated monocyte migration through the scavenging of ROS signaling intermediaries. These findings suggest a potential mechanism for the purported cardioprotective effects of bilirubin.