Inês Palmela

Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells.

Background Sepsis 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 Findings Monolayers 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. Conclusions LPS 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.

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

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.

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

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 at later time points. 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.

The TNF-α/NF-κB signaling pathway has a key role in methamphetamine–induced blood–brain barrier dysfunction

Methamphetamine (METH) is a psychostimulant that causes neurologic and psychiatric abnormalities. Recent studies have suggested that its neurotoxicity may also result from its ability to compromise the blood–brain barrier (BBB). Herein, we show that METH rapidly increased the vesicular transport across endothelial cells (ECs), followed by an increase of paracellular transport. Moreover, METH triggered the release of tumor necrosis factor-alpha (TNF-α), and the blockade of this cytokine or the inhibition of nuclear factor-kappa B (NF-κB) pathway prevented endothelial dysfunction. Since astrocytes have a crucial role in modulating BBB function, we further showed that conditioned medium obtained from astrocytes previously exposed to METH had a negative impact on barrier properties also via TNF-α/NF-κB pathway. Animal studies corroborated the in vitro results. Overall, we show that METH directly interferes with EC properties or indirectly via astrocytes through the release of TNF-α and subsequent activation of NF-κB pathway culminating in barrier dysfunction.

Tricellulin expression in brain endothelial and neural cells.

Tricellulin is a tight junction (TJ) protein, which is not only concentrated at tricellular contacts but also present at bicellular contacts between epithelial tissues. We scrutinized the brain for tricellulin expression in endothelial and neural cells by using real-time polymerase chain reaction, Western blot and immunohistochemical and immunocytochemical analysis of cultured brain cells and paraffin sections of brain. Tricellulin mRNA was detected in primary cultures and in a cell line of human brain microvascular endothelial cells. Protein expression was confirmed by Western blot and immunofluorescence analysis, which further highlighted the localization of tricellulin in the cell membrane at tricellular and along bicellular contacts, and in the nucleus and perinuclear region. Compared with the well-studied TJ protein, zonula occludens-1, tricellulin expression was less marked at the cell membrane but more evident in the nuclear and perinuclear regions. The presence of tricellulin in cultured endothelial cells was corroborated by immunohistochemical and immunofluorescence staining in brain blood vessels, where it was colocalized with another TJ protein, claudin-5. Tricellulin mRNA was detected in neurons and astrocytes, whereas protein expression was observed in astrocytes but not in neurons, as shown by immunofluorescence analysis. This study reveals the presence and subcellular distribution of tricellulin in brain endothelial cells, both in vitro and in situ and its colocalization with other relevant TJ proteins. Moreover, it demonstrates the expression of the protein in astrocytes opening new avenues for future research to establish the biological significance of tricellulin expression in glial cells.

Polyphenols journey through blood-brain barrier towards neuronal protection.

Age-related complications such as neurodegenerative disorders are increasing and remain cureless. The possibility of altering the progression or the development of these multifactorial diseases through diet is an emerging and attractive approach with increasing experimental support. We examined the potential of known bioavailable phenolic sulfates, arising from colonic metabolism of berries, to influence hallmarks of neurodegenerative processes. In silico predictions and in vitro transport studies across blood-brain barrier (BBB) endothelial cells, at circulating concentrations, provided evidence for differential transport, likely related to chemical structure. Moreover, endothelial metabolism of these phenolic sulfates produced a plethora of novel chemical entities with further potential bioactivies. Pre-conditioning with phenolic sulfates improved cellular responses to oxidative, excitotoxicity and inflammatory injuries and this attenuation of neuroinflammation was achieved via modulation of NF-κB pathway. Our results support the hypothesis that these small molecules, derived from dietary (poly)phenols may cross the BBB, reach brain cells, modulate microglia-mediated inflammation and exert neuroprotective effects, with potential for alleviation of neurodegenerative diseases.

Potential for brain accessibility and analysis of stability of selected flavonoids in relation to neuroprotection in vitro.

Natural food sources constitute a promising source of new compounds with neuroprotective properties, once they have the ability to reach the brain. Our aim was to evaluate the brain accessibility of quercetin, epigallocatechin gallate (EGCG) and cyanidin-3-glucoside (C3G) in relation to their neuroprotective capability. Primary cortical neuron cultures were exposed to oxidative insult in the absence and presence of the selected compounds, and neuroprotection was assessed through evaluation of apoptotic-like and necrotic-like cell death. The brain accessibility of selected compounds was assessed using an optimised human blood-brain barrier model. The blood-brain barrier model was crossed rapidly by EGCG and more slowly by C3G, but not by quercetin. EGCG protected against oxidation-induced neuronal necrotic-like cell death by ~40%, and apoptosis by ~30%. Both quercetin and C3G were less effective, since only the lowest quercetin concentration was protective, and C3G only prevented necrosis by ~37%. Quercetin, EGCG and C3G effectively inhibited α-synuclein fibrillation over the relevant timescale applied here. Overall, EGCG seems to be the most promising neuroprotective compound. Thus, inclusion of this polyphenol in the diet might provide an affordable means to reduce the impact of neurodegenerative diseases.

Blood-brain barrier and bilirubin: clinical aspects and experimental data.

The blood-brain barrier (BBB) is a complex and dynamic structure that plays a key role in central nervous system (CNS) homeostasis. It strictly regulates the entrance of molecules into the brain parenchyma and prevents the access of neurotoxins and pathogens while promoting the efflux of several molecules. The brain microvascular endothelial cells are the anatomical basis of the BBB, which has unique characteristics such as the elaborate junctional complexes that nearly obliterate the intercellular space as well as the presence of influx and efflux transporters. Endothelial cells establish important interactions with glial cells, neurons, and perivascular pericytes as well as with the acellular components of the basement membrane, which together constitute the neurovascular unit. BBB disruption has been reported in a wide range of CNS pathologies, with an emerging role in the onset and disease progression. Accordingly, recent studies revealed vascular dysfunction in neonatal jaundice, a common pathology in the early neonatal period affecting 1/10 children presenting values of total bilirubin>17 mg/dL (291 μM). Here we summarize the clinical aspects of moderate to severe neonatal jaundice and provide a comprehensive review of the literature regarding bilirubin-induced neurotoxicity from a vascular-centered approach. The collected evidence place endothelial dysfunction and pericyte demise as key players in the disruption of CNS homeostasis, mainly in cases of lasting hyperbilirubinemia, thus pointing to novel targets to prevent neurological dysfunction due to severe neonatal jaundice.

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: An in vitro study

Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells.

LDL-Cholesterol Increases the Transcytosis of Molecules through Endothelial Monolayers.

Cholesterol has been identified as a causative factor in numerous pathologies including atherosclerosis and cancer. One of the frequent effects of elevated cholesterol levels in humans is the compromise of endothelial function due to activation of pro-inflammatory signalling pathways. While the mechanisms involved in endothelial activation by cholesterol during an inflammatory response are well established, less is known about the mechanisms by which cholesterol may affect endothelial barrier function, which were the subject of the present study. Here we show that low density lipoprotein (LDL) increases the permeability of endothelial monolayers to high molecular weight dextrans in an LDL receptor and cholesterol-dependent manner. The increased permeability seen upon LDL treatment was not caused by disruption of cell-to-cell junctions as determined by a normal localization of VE-Cadherin and ZO-1 proteins, and no major alterations in transendothelial electrical resistance or permeability to fluorescein. We show instead that LDL increases the level of high molecular weight transcytosis and that this occurs in an LDL receptor, cholesterol and caveolae-dependent way. Our findings contribute to our understanding of the systemic pathological effects of elevated cholesterol and the transport of cargo through endothelial monolayers.