Das Gallepigment Bilirubin: ein natürlicher Hemmer der Arteriosklerose?

Abstract

Introduction: Induction of heme oxygenase 1 (HO-1) ameliorates the development of neointimal hyperplasia, which normally involves oxidative stress and vascular smooth muscle cell (VSMC) proliferation [1]. Bilirubin and biliverdin, generated during heme catabolism by HO-1, are potent anti-oxidants that play a role in maintenance of homeostasis [2]. Based on the clinical observation that healthy individuals with higher levels of bilirubin develop less arteriosclerosis³, we hypothesized that the bile pigments would inhibit VSMC proliferation in vitro and neointima formation in vivo. Methods: Balloon injury (BI) was performed in the left common carotid artery of congenitally hyperbilirubinemic Gunn rats. The artery was harvested two weeks after BI, and neointima formation was assessed by quantifying intima/media ratio and luminal cross-sectional area narrowing (LCAN). In vitro, proliferation of primary vascular smooth muscle cells (VSMC) was measured by [3H]-thymi-dine incorporation in the presence or absence of bilirubin/biliverdin. Cell cycle progression was monitored by DNA content analysis; apoptosis by the Annexin V binding assay. Protein levels were detected by western blot. Immunocytochemistry was performed using primary antibodies directed against cdk2 and phospho-p38 in growth stimulated primary VSMCs, in the presence or absence of bilirubin/biliverdin. To further delineate the pathways of bilirubin/biliverdin action on VSMC proliferation, we used VSMCs derived from p53 KO, p21 KO and HO-1 KO mice. Results: BI mediated neointima formation was significantly reduced in hyperbilirubinemic Gunn rats (serum bilirubin levels: 12.0 ± 2.5 mg/dl) as compared to control Wistar rats (LCAN 0.18 vs. 0.44; p<0.05). Systemic administration of biliverdin, the precursor of bilirubin, to normal rats also suppressed neointima proliferation (LCAN 0.29 vs. 0.43; p<0.05). In vitro, bilirubin inhibited VSMC proliferation by arresting VSMC cell cycle progression at the G₀/G₁ phase, while it did not increase apoptosis (p=0.2816). Inhibition of cell cycle progression was mediated by reduced phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase 1/2 (JNK 1/2), down-regulation of cdk2, cyclins D1, E and A resulting in hypophosphorylation of pRb as demonstrated in nuclear extracts of cultured cells. Further the effect of bilirubin on VSMC proliferation was dependent on p53, but independent of p21 and HO-1. Conclusions: Our studies implicate bilirubin as the molecule to explain the observed associations between higher levels of plasma bilirubin in humans and a lesser incidence of atherosclerosis-related diseases and suggest the future use of bilirubin/biliverdin as a therapeutic to prevent restenosis, chronic rejection and atherosclerosis.