Elevated Levels of Bilirubin and Long-Term Exposure Impair Human Brain Microvascular Endothelial Cell Integrity

Ines      Palmela; Filipa      L. Cardoso; Michael      Bernas; Leonor      Correia; Ana      R. Vaz; Rui      F. M. Silva; Adelaide      Fernandes; Kwang      S. Kim; Dora      Brites; Maria      A. Brito

Abstract

The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of high levels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, it remains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brain microvascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primary cultures of HBMEC were exposed to 50 or 100 μM UCB, in the presence of 100 μM human serum albumin, to mimic moderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCB inhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor at early time points, but enhanced their secretion later on. Upregulation of mRNA expression, particularly by 100 μM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis and the increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCB upregulated the expression of β-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrity of HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression of caveolin-1, which has been associated with BBB breakdown, and β-catenin, probably as an attempt to circumvent that impairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.

Keywords: Blood-brain barrier, β-catenin, caveolin-1, cytokines, endothelial cells, oxidative stress, vascular endothelial growth factor, unconjugated bilirubin, bilirubin, microvascular, endothelial, neuronal cells, monolayers, HBMEC, chemoattractant, protein-1, jaundice, interleukin-6