Mazzone, Graciela Lujan
Role of unconjugated bilirubin in the endothelial dysfunction. [Tesi di dottorato]
Università degli studi di Trieste, 2008-05-07T15:48:22Z

Atherosclerosis, a progressive cardiovascular disease, is characterized by the accumulation of cholesterol in macrophage deposits (foam cells) and the formation of atherosclerotic plaques in the walls large- and medium- sized arteries. The earliest events in the development of atherosclerosis involve progressive modifications in the endothelial micro-environment. This endothelial dysfunction is a complex of multi-step mechanisms, for which reduced NO levels have been reported as a marker, is characterized by increasing expression of adhesion molecules (AMs), which mediate the diapedesis (migration) of inflammatory and immunocompetent cells through the endothelial layer into the arterial wall. NO is synthesized intracellularly by nitric oxide enzymes (eNOS and iNOS) and is regulated by a variety of stimuli. NO acts as an autocrine or paracrine hormone, as well as intracellular messenger, with a critical role in vascular endothelial growth factor-induced angiogenesis and vascular hyperpermeability in vitro. The over-expression of AMs is orchestrated by pro-inflammatory cytokines, particularly TNF-alpha. The two major subsets of AMs participating in these processes are the selectins, in particular E-selectin, and the immunoglobulin gene superfamily, in particular vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Transcriptional regulation of these inflammatory genes requires the participation of several proteins, inducible activators, as: NF-kappa B and (CRE)-binding protein (CREB). The most abundant form of NF-kappa B is an heterodimer of p50 and p65; which is sequestered in the cytoplasm in an inactive form through interaction with the I kappa B inhibitor proteins. Signals that induce NF-kappa B release dimmers to enter to the nucleus and induce gene expression. Pyrridoline dithiocarbamate (PDTC) a metal-chelating compound inhibits NF-kappa B by blocking ubiquitine ligase activity towards phosphorylated I kappa B, in turn downregulating the expression of E-selectin, VCAM-1 and ICAM-1. CREB is a widely expressed DNA-binding protein, downstream target of cAMP, activated by phosphorylation on serine 133. A regulatory site, on the gene promoters of both E-selectin and VCAM-1, binds both NF-kappa B and CREB transcription factors. Unconjugated bilirubin (UCB), long considered to be simply a waste end product of heme metabolism and a marker for hepatobiliary disorders, is now thought to function as an endogenous tissue protector by attenuating free radical-mediated damage to both lipids and proteins. There is increasing epidemiological evidence supporting an inverse association between cardiovascular disease and plasma levels of bilirubin. Recent studies indicated that bilirubin may be protective in the development of atherosclerotic diseases by inhibiting the proliferation of vascular smooth muscle cells by mechanisms yet to be established. It has been proposed that UCB can interfere with the atherosclerotic disease development by inhibiting the trans-endothelial vascular cell adhesion molecule (VCAM-1)-dependent migration of monocytes into the intima. The aim of this study is to investigate the effect of the UCB in the endothelial dysfunction. Specifically UCB effects on NO production, AMs expression and the regulatory transcription factors involve in the inflammatory response. Variable doses of free bilirubin (Bf) (the active form of UCB in plasma), simulating upper normal (15 nM) and modestly elevated levels (30 nM) for plasma, were evaluated in two models of endothelial cells. A) H5V, murine microvascular endothelial cell line, and B) HUVEC (Human Umbilical Vein Endothelial Cells), isolated from the vein of human umbilical cord. TNF-alpha (20 ng/mL) was added in order to reproduce, in vitro, the endothelial dysfunction and describe UCB contribution on its effects. UCB alone reduced the viability in H5V cells by MTT assay in a dose dependent manner after 24 hours while no effect was observed in the LDH released. In the first set of experiments NO production in H5V cells was evaluated, a time-depended increase on NO basal and a dose-dependent decrease on NO concentration after TNF-alpha (20 ng/mL) were observed. NO reduction related TNF-alpha was seen at all times studied. The effect of UCB was studied in co-treatments with TNF-alpha for 24 and 48 hours. UCB (Bf 15 and 30 nM) significantly reversed the reduction of nitrite content induced by TNF-alpha at 48 hours. The gene expression analysis was performed by Real Time PCR technology with specific primers for eNOS, iNOS, E-selectin, VCAM-1 and ICAM-1. In H5V cells, TNF-alpha increased the expression of all the genes studied (except eNOS) at 2, 6 and 24 hours. The co-treatment with UCB, at a Bf that did not themselves affect the expression of the three adhesion molecules, blunts the over-expression of E-selectin, Vcam-1 and iNOS induced by a pro-inflammatory cytokine such as TNF-alpha. The inhibitory effect of UCB was usually modest (20-30%) and detected at 2 and/or 6 hours, but had disappeared 24 hours. Furthermore, a synergistic effect between TNF-alpha and UCB was seen on the expression of iNOS at 24 hours, indicating a biphasic regulation. Moreover, no effects were seen on eNOS. Similar results were observed in the regulation of the gene expression of the AMs and viability in HUVEC cells, indicating the lack of species specific effect. However, no effect of TNF-alpha or UCB was seen in the expression of iNOS, eNOS or NO content. Western blot analysis in H5V cells confirmed that TNF-alpha induced the expression of E-selectin, VCAM-1 and ICAM-1 in a time-dependent manner. This effect was blunted after 24 hours by the presence of UCB (Bf 15 and 30 nM). The contribution of NF-kappa B pathway in UCB effects was investigated by addition of a specific inhibitor, PDTC. The co-treatment with PDTC and UCB for 2 hours produced an additive reduction of TNF-alpha effect on Eselectin, VCAM-1, and iNOS in H5V cells. In addition, UCB prevented the nuclear translocation of NF-kappa B induced by TNF-alpha. Failure of UCB to alter TNF-alpha -induced phosphorylation of CREB (at Ser 133) suggested that the CREB pathway was not involved in the UCB inhibition. The results obtained in the present study shows that unconjugated bilirubin, even at upper-normal physiological (15 nM) and mildly elevated (30 nM) Bf can modulate gene expression and endothelial cell function. Furthermore, UCB may regulate NO levels by a biphasic regulation of iNOS, and in addition influences the expression of the endothelial adhesion molecules. In summary, these data indicates that bilirubin limits the over-expression of the adhesion molecules and regulates the NO metabolism in the pro-inflammatory state induced by the cytokine TNF-alpha. Even though UCB alone does not alter these markers. UCB effects are mediated in part by a modulation of the NF-kappa B transcription factor. These results support the concept that modestly elevated concentrations of bilirubin may help prevent atherosclerotic disease as suggested by epidemiological studies.

Rigato, Igino
Tiribelli, Claudio

Tesi di dottorato. | Lingua: en. | Paese: | BID: TD13089495