Bilirubin inhibits the TNFa-related induction of three endothelial adhesion molecules Graciela Luján Mazzone a,b , Igino Rigato a,c, * , J. Donald Ostrow d , Fleur Bossi e , Alessia Bortoluzzi a , Caecilia H.C. Sukowati a,b , Francesco Tedesco e , Claudio Tiribelli a,b a Centro Studi Fegato, AREA Science Park, Basovizza, Bldg Q, SS 14, Km 163.5, 34012 Trieste, Italy b University of Trieste, piazzale Europa 1, 34127 Trieste, Italy c Emergency Department, San Vito al Tagliamento Hospital, via Savorgnano 2, 33078 San Vito al Tagliamento (PN), Italy d Gastroenterology/Hepatology Division, University of Washington and Veterans Affairs Medical Center, 1660 South Columbian Way, 98108-1597 Seattle, WA, USA e Department of Life Sciences, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy article info Article history: Received 5 June 2009 Available online 10 June 2009 Keywords: Endothelial cell activation Bilirubin Adhesion molecules TNFa Atherosclerosis abstract Since an increased serum unconjugated bilirubin (UCB) level has been proposed as an independent pro- tective factor against atherosclerotic disease, we investigated the molecular events at the basis of this effect. HUVEC and H5V cells were treated with TNFa and UCB and the effects assessed on E-selectin, VCAM-1 and ICAM-1. In HUVEC cells, UCB blunted the TNFa-induced gene upregulation of E-selectin VCAM-1 and ICAM-1. The same pattern was observed in H5V cells except for ICAM-1. UCB also inhibited the PMN endothelial adhesion in HUVEC H5V cells. Western blot and FACS analysis confirmed that UCB prevented TNFa-induced over-expression of adhesion molecules proteins in H5V cells. These data con- tribute to further explain the protective effect of bilirubin against development of atherosclerosis. Ó 2009 Elsevier Inc. All rights reserved. Introduction Atherosclerosis, a progressive cardiovascular disease, is charac- terized by the accumulation of cholesterol in macrophage deposits (foam cells) and the formation of atherosclerotic plaques in the walls of large- and medium-sized arteries. The deposition of the oxidized lipids leads to cell proliferation within the arterial wall that gradually impinges on the vessel lumen and impedes blood flow. This process may insidiously last for decades until an athero- sclerotic plaque ruptures and components deep in the arterial wall are exposed to flowing blood, leading to thrombosis and compro- mised oxygen supply in organs such as the heart and brain [1]. The earliest events in the development of atherosclerosis in- volve progressive modifications in the endothelial micro-environ- ment. This endothelial cell activation, a complex of multi-step mechanisms, is characterized by increasing expression of adhesion molecules, which mediate the diapedesis (migration) of inflamma- tory and immunocompetent cells through the endothelial layer into the arterial wall. The over-expression of adhesion molecules is orchestrated by pro-inflammatory cytokines, particularly TNFa [2,3]. The two major subsets of adhesion molecules participating in these processes are the selectins (in particular E-selectin) and the immunoglobulin gene superfamily (in particular cell vascular adhesion molecule 1, VCAM-1, and intercellular adhesion molecule 1, ICAM-1). Unconjugated bilirubin (UCB), long considered to be simply a waste end product of heme metabolism and a marker for hepatob- iliary disorders, is now thought to function as an endogenous tis- sue protector by attenuating free radical-mediated damage to both lipids and proteins [4]. There is increasing epidemiological evidence supporting an inverse association between cardiovascu- lar disease and plasma levels of bilirubin [5]. Recent studies indi- cated that bilirubin may be protective against the development of atherosclerotic diseases by inhibiting the proliferation of vascu- lar smooth muscle cells by mechanisms yet to be established [6]. It has been proposed that UCB can interfere with the development of atherosclerotic disease by inhibiting the (VCAM-1)-dependent trans-endothelial migration of monocytes into the intima [7]. The present study investigates the in vitro effects of UCB on the expres- sion of three adhesion molecules and the consequent adhesion of polymorphonuclear leukocytes (PMN). Materials and methods Materials. Dulbecco’s Phosphate saline (DPS), Dulbecco’s modi- fied Eagle’s medium high glucose (DME/HIGH), penicillin and 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.06.029 * Corresponding author. Address: Centro Studi Fegato, AREA – Science Park, Basovizza Bldg. Q, SS 14 Km 163.5, 34012 Trieste, Italy. Fax: +39 040 375 7832. E-mail address: igino.rigato@csf.units.it (I. Rigato). Biochemical and Biophysical Research Communications 386 (2009) 338–344 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc