BILIRUBIN SUPPRESSES NEOINTIMA FORMATION BY INTERACTING WITH THE RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN PATHWAY

Abstract

O386* Aims: Induction of heme oxygenase 1 (HO-1) ameliorates the development of chronic allograft arteriosclerosis, which normally involves oxidative stress and vascular smooth muscle cell (VSMC) proliferation. Bilirubin and biliverdin, generated during heme catabolism by HO-1, are potent antioxidants that play a role in maintenance of homeostasis. Based on the clinical observation that healthy individuals with higher levels of bilirubin develop less arteriosclerosis, we hypothesized that the bile pigments would inhibit VSMC proliferation in vitro and neointima formation in vivo. We used balloon injury (BI) as a model of the neointimal proliferation seen in transplant arteriosclerosis. Methods: BI was performed in the left common carotid artery of congenitally hyperbilirubinemic Gunn rats and congenic wild-type Wistar 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-thymidine 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. Immuncytochemistry was performed using antibody directed against the retinoblastoma tumor suppressor protein (pRb). 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 in a dose-dependent manner. Bilirubin (200 μM) arrested 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, and the resulting decreased phosphorylation of pRb. In nuclear extracts, lower total levels of pRb were found in bilirubin treated VSMCs, consistent with the findings that a significant portion of pRb was kept in the cytosol after bilirubin treatment as assessed by immunocytochemistry. Further, bilirubin treatment reduced expression levels of the transcription factor YY1, a zinc finger DNA-binding transcription factor known to regulate the expression of genes with important functions during cell growth and differentiation. The effects of bilirubin were independent of HO-1 expression, as demonstrated in HO-1−/− VSMCs. Conclusions: Biliverdin and bilirubin suppress both BI induced neointima formation in vivo and VSMC proliferation in vitro. Our data suggest that bilirubin treatment inhibits VSMC growth by impaired activation of p38 and JNK 1/2, resulting in two events. Firstly, the inhibition of pRb phosphorylation impedes the release of transcription factors such as YY1 that are important for VSMC growth. Secondly, there is reduced expression of the transcription factor YY1 itself, resulting in a suppressed capacity to induce DNA synthesis and transcriptional activity. Thus, administration of the bile pigments bilirubin and biliverdin may have a beneficial effect on VSMC proliferation in chronic allograft arteriopathy and may become a potent strategy for the treatment of chronic rejection.