Time-dependent dual effects of high levels of unconjugated bilirubin on the human blood-brain barrier lining

Abstract

In neonatal jaundice, high levels of unconjugated bilirubin (UCB) may induce neurological dysfunction (BIND). Recently, it was observed that UCB induces alterations on brain microvasculature, which may facilitate its entrance into the brain, but little is known about the steps involved. To evaluate if UCB damages the integrity of human brain microvascular endothelial cells (HBMECs), we used 50 or 100 μM UCB plus human serum albumin, to mimic the neuropathological conditions where levels of UCB free species correspond to moderate and severe neonatal jaundice, respectively. Our results point to a biphasic response of HBMEC to UCB depending on time of exposure. The early response includes increased number of caveolae and caveolin-1 expression, as well as upregulation of vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR-2) with no alterations of the paracellular permeability. In contrast, effects by sustained hyperbilirubinemia are the reduction in zonula occludens (ZO)-1 and β-catenin levels and thus of tight junctions (TJ) strands and cell-to-cell contacts. In addition, reduction of the transendothelial electrical resistance (TEER) and increased paracellular permeability are observed, revealing loss of the barrier properties. The 72 h of HBMEC exposure to UCB triggers a cell response to the stressful stimulus evidenced by increased autophagy. In this later condition, the UCB intracellular content and the detachment of both viable and non-viable cells are increased. These findings contribute to understand why the duration of hyperbilirubinemia is considered one of the risk factors of BIND. Indeed, facilitated brain entrance of the free UCB species will favor its parenchymal accumulation and neurological dysfunction.

Inflammatory signature of cerebellar neurodegeneration during neonatal hyperbilirubinemia in Ugt1 -/- mouse model

“…8). These observations are in line with experiments performed in neuroblastoma and endothelial cell lines exposed to high bilirubin levels [35, 65]. In both cases, the autophagy pathway was activated at very high levels of bilirubin (Bf = 100–140 nM) and at later stages (24–72 h) when cell death was evident.…”

Heme Oxygenase Dependent Bilirubin Generation in Vascular Cells: A Role in Preventing Endothelial Dysfunction in Local Tissue Microenvironment?

“…Moreover, immortalized endothelial cell lines, such as bEnd.3 cells used as a model of BBB, undergo oxidative stress and apoptosis when treated with 20-40 µM UCB, while no significant damage occurs in endothelial cells derived from pancreatic islets of Langerhans (Kapitulnik et al, 2012). The peculiar sensitivity of BBB-like endothelial cells has been confirmed by other authors (Palmela et al, 2012), underlining the marked sensitivity of brain endothelial cells and neurons to UCB, as previously reviewed (Ostrow et al, 2003;Brito et al, 2014).…”

Endotoxemia-mediated inflammation potentiates aminoglycoside-induced ototoxicity

“…Immune cells are capable of secreting cytotoxic molecules that disrupt tight junctions between adjacent endothelial cells, opening paracellular trafficking routes (68). Alternatively, the tight junction-coupling between adjacent endothelial cells could break down independently of immune cell activity allowing paracellular flux through disrupted endothelial tight junctions (69). Although we did not use markers for immune cell-mediated injury or electron-dense tracers, the lack of hTR flux into the intra-strial tissues of adult mice contraindicated a major contribution by paracellular trafficking during acute endotoxemia.…”