Exposure to Lipopolysaccharide and/or Unconjugated Bilirubin Impair the Integrity and Function of Brain Microvascular Endothelial Cells

Abstract

BackgroundSepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC).Methodology/Principal FindingsMonolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB.ConclusionsLPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

β2 Integrin–Mediated Crawling on Endothelial ICAM-1 and ICAM-2 Is a Prerequisite for Transcellular Neutrophil Diapedesis across the Inflamed Blood–Brain Barrier

“…Although LPS stimulation induced a proinflammatory phenotype of the BBB in vitro and in vivo, it did not alter barrier characteristics of the pMBMEC monolayers. LPSinduced impairment of barrier characteristics of pMBMECs could, however, be observed after stimulation with 10-fold higher concentration of LPS (1 mg/ml) (data not shown) in accordance with a previous study in which stimulation of rat brain microvascular endothelial cells with LPS (1 mg/ml) impaired barrier characteristics of the cell monolayer (45). In addition, prolonged exposure of brain endothelial cells to LPS over 12 h might lead to LPSinduced cell death (46).…”

The TNF-α/Nf-κB Signaling Pathway has a Key Role in Methamphetamine–Induced Blood–Brain Barrier Dysfunction

“…3 Though its impact on BBB properties was highlighted more recently, 9 the intracellular mechanisms involved in METH-induced cerebral endothelium dysfunction remain unknown, as well as its effect on the crosstalk between different neurovascular unit cells. Thus, to reach our goal we first took advantage of a wellestablished BBB in vitro model, 19 and we observed that METH increased ECs permeability at concentrations relevant to human abuse. Considering the differences between rodents and humans, we further used human ECs 20 to reproduce the results obtained with rat primary cultures.…”

“…Primary cultures of rat brain microvascular ECs (RBMVECs) were prepared from 2-week-old Wistar rats, as previously described 19 with minor modifications. Specifically, forebrains were freed of meninges, dissociated into small pieces, and digested in Dulbecco's modified Eagle's medium/ Ham's F12 (DMEM/F12; Biochrom AG, Berlin, Germany), containing 1 mg/ mL collagenase CLS2 (Sigma-Aldrich, St. Louis, MO, USA) and 14 μg/mL DNase (Sigma-Aldrich) during 90 minutes at 37°C.…”

“…Hydrocortisone was used to improve barrier proprieties of ECs, namely by the induction of TJs expression and enhancement of TEER. 19 …”

Doxycycline Hyclate Protects Lipopolysaccharide-Induced Endothelial Barrier Dysfunction by Inhibiting the Activation of p38 Mitogen-Activated Protein Kinase

“…5,6) The disruption of endothelial barrier in those tissues will result in acute lung injury (ALI) and acute respiratory distressed syndrome (ARDS), as well as the dysfunction of central nerve system. 6,7) The blockade of hyperpermeability response could improve the survival of LPS-challenged mice 8) and allogeneic human mesenchymal stem cells (MSCs) treatment could reduced the degree of edema and cellularity in the endotoxin-injured human lung lobe. 9) Despite progress in diagnosis and supportive care, sepsis is still challenging to the field of medicine, and its occurrence appears to be increasing.…”