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Introduction

The central nervothat maintain homeffects of internaentrance of plasma molecules and toxins able to induce

r constitutes thee alterations byis review.

in turn, establish the connection with the cytoskeleton. TJproteins form strains along the intercellular junctions,creating the connection with those of the opposing

dade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon,

Portugal; Phone: 351217946449; FAX: 351217946491; E-mail:abrito@ff.ulisboa.pt

Archives of Medical Researcactivation of glial cells and neural tissue damage. Simulta-neously, they also assure the uptake of nutrients, togetherproviding a stable environment for neural function. CNSbarriers exist at three key sites: the epithelial cells of thechoroid plexus, the arachnoid epithelium that lies underthe dura mater and completely encases the brain, andthe cerebral endothelium that constitutes the barrier

The endothelial cells of brain capillaries are considered theanatomic basis of the BBB. The brain microvascular endo-thelial cells (BMEC) are characterized by the presence ofelaborate junctional complexes that are crucial players inthe maintenance of a functional BBB. These structuresare formed by tight junctions (TJ) in the luminal area ofthe cell membrane and by adherens junctions (AJ) locatedin a basolateral position (4) (Figure 1). The TJ and AJ sharecommon characteristics because they are both formed bytransmembrane proteins linked to cytosolic proteins which,Address reprint requests to: Maria Alexandra Brito, PhD, Research

Institute for Medicines (iMed.ULisboa), Faculdade de Farmacia, Universi-0188-4409/$ - see frohttp://dx.doi.org/10components of the basement membrane, which together constitute the neurovascular unit.BBB disruption has been reported in a wide range of CNS pathologies, with an emergingrole in the onset and disease progression. Accordingly, recent studies revealed vasculardysfunction in neonatal jaundice, a common pathology in the early neonatal periodaffecting 1/10 children presenting values of total bilirubin O17 mg/dL (291 mM). Herewe summarize the clinical aspects of moderate to severe neonatal jaundice and providea comprehensive review of the literature regarding bilirubin-induced neurotoxicity froma vascular-centered approach. The collected evidence place endothelial dysfunction andpericyte demise as key players in the disruption of CNS homeostasis, mainly in cases oflasting hyperbilirubinemia, thus pointing to novel targets to prevent neurological dysfunc-tion due to severe neonatal jaundice. 2014 IMSS. Published by Elsevier Inc.Key Words: Bloodebrain barrier, Endothelial cells, Kernicterus, Neonatal jaundice, Neurovascularunit, Pericytes.

us system (CNS) contains cellular barrierseostasis by protecting the brain from thel and external changes, preventing the

between blood and brain (1e3). The lattebloodebrain barrier (BBB), of which thneonatal jaundice are the main focus of th

BBB Overviewactions with glial cells, neurons, and perivascular pericytes as well as with the acellularelaborate junctional complexes that nearly obliterate the intercellular space as well asthe presence of influx and efflux transporters. Endothelial cells establish important inter-BloodeBrain Barrier and Bilirubin: C

Maria Alexandra Brito,a,b Ine^s Palmela,a Filipa LoaResearch Institute for Medicines (iMed.ULisboa), bDepartment of Bioc

Lisbon

Received for publication November 10, 2014; ac

The bloodebrain barrier (BBB) is a complin central nervous system (CNS) homeostcules into the brain parenchyma and prevwhile promoting the efflux of several mocells are the anatomical basis of the BBBnt matter. Copyright 2014 IMSS. Published by Elsevier.1016/j.arcmed.2014.11.015TICLE

ical Aspects and Experimental Data

co Cardoso,a Ine^s Sa-Pereira,a and Dora Britesa,b

try and Human Biology, Faculty of Pharmacy, Universidade de Lisboa,

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d November 18, 2014 (ARCMED-D-14-00652).

nd dynamic structure that plays a key roleIt strictly regulates the entrance of mole-the access of neurotoxins and pathogensles. The brain microvascular endothelialich has unique characteristics such as the

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661BloodeBrain Barrier in Neonatal JaundiceFigure 1. Simplified representation of the bloodebrain barrier and of the inbarrier function of brain endothelial cells is achieved by the presence of tighmembrane and obliterating the intercellular space (5), anorganization that is more evident in the brain than in otherregions of the organism (6). Examples of TJ proteins are thecytosolic zonula occludens-1 (ZO-1) and the transmem-brane occludin and claudin-5, as well as the recentlydiscovered tricellulin (5). Among AJ proteins, the moststudied are the vascular endothelial-cadherin, an adhesivetransmembrane protein, and the cytosolic b-catenin (4,7).Alterations in TJ proteins are directly associated with bar-rier compromise. However, AJ also play an important rolein maintaining barrier function. In fact, they are essentialfor TJ formation as they directly activate signaling mole-cules and can regulate gene transcription (8e10). Accord-ingly, changes in the expression or distribution of AJproteins should also be considered to be involved in defectsof the endothelial barrier (11).

BMEC are additionally characterized by a number of re-ceptors and the presence of influx and efflux transportersthat assure the passage of substances into and out of thebrain parenchyma. Influx transporters are grouped accord-ingly with the type of the transported molecule, which in-cludes energy sources, amino acids, organic anions andneurotransmitters, among others (12). One of the most

ability. TJ are constituted by transmembrane proteins, such as claudins and occ

occludens (ZO) family. The adherens junctions are formed by the transmembrane

The cytoplasmic proteins from these two types of junctions establish the connect

brain endothelial cells to the basement membrane is mediated by integrins in the a

of endothelial cells responsible for transcytosis. Endothelial cells establish impo

microglia, neurons and, indirectly, with oligodendrocytes, giving rise to the concy between the different components of the neurovascular unit. The physical

ctions (TJ) and adherens junctions (AJ) that restrict the paracellular perme-crucial transporters, glucose transporter-1 (GLUT-1), isresponsible for the energy supply to the brain (4). BMECare also equipped with efflux transporters that export un-wanted compounds from the brain parenchyma (4). Themost studied group of efflux transporters is the ATP-binding cassette (ABC) family that mediates the export ofsubstrates from cells coupled with the hydrolysis of ATP(12,13), among which is the widely studied P-glycoprotein(P-gp) (14e16).

Transport across the endothelium may additionally occurvia vesicular mechanisms, also known as transcytosis (17).This type of transport describes the movement of moleculeswithin endocytic vesicles across endothelial cells, from theluminal to the abluminal side. BMEC contain two kinds ofvesicles, clathrin-coated vesicles and caveolae, the latterbeing the principal vesicular structure for transcytosis inthese cells (18,19), which are schematically depicted inFigure 1. These vesicles, also involved in various aspectsof signal transduction (20), are plasma membrane invagina-tions rich in caveolins, sphingolipids and cholesterol.Caveolin-1, the major structural protein of caveolae, is ex-pressed in several tissues with the highest levels of expres-sion in endothelial cells, adipocytes, fibroblasts and

ludin, as well as by cytoplasmic accessory proteins including the zonula

vascular endothelial cadherin (VE-cadherin) and the cytoplasmic catenins.

ion to the cytoskeleton by interacting with actin filaments. The adhesion of

bluminal surface of endothelial cells. Caveolae are membrane invaginations

rtant communication with the basement membrane, pericytes, astrocytes,

ept of neurovascular unit.

smooth-muscle cells (20). Soluble plasma molecules can be remain to be explored. Therefore, it is fundamental to un-

662 Brito et al./ Archives of Medical Research 45 (2014) 660e676b (TGF-b) (38,39). Importantly, addition of pericytes tocultured endothelial cells has shown to promote junctionformation and to improve endothelial barrier function(40e44). In accordance, several in vitro and in vivo studiesreport loss of pericytes as a key step in brain vascular dam-age, with changes in the structure of capillaries, in thelevels and cellular distribution of certain junctional pro-teins, and in morphological signs of increased endothelialpermeability (45e47). On the other hand, a link betweenpericyte coverage and susceptibility of distinct brain re-gions has been observed in the developing brain (48), sug-gesting that pericyte deficiency contributes to the fragilityof specific brain areas during development. Despite theclear role of pericytes in BBB modulation, the signalingmechanisms involved in the interaction of these cells withthe other components of the barrier interface are currentlyrandomly taken up by caveolae in a process known as bulk-phase or fluid-phase transcytosis (18,21), which is indepen-dent of any interaction between the transported moleculesand the vesicle membrane. In addition to ensuring themovement of substances via transcytosis, caveolae alsohave an active role in the regulation of BBB vesicularpermeability (20,22). Caveolin-1 has been associated withincreased BBB permeability and brain edema during injury(23). The effects induced by caveolin-1 seem to involve theinteraction with proteins such as vascular endothelialgrowth factor (VEGF) receptor 2 (VEGFR-2), P-gp andendothelial nitric oxide synthase (eNOS) (24e26). More-over, caveolin-1 affects the expression of junctional pro-teins because their decreased levels downregulate theexpression of occludin and ZO-1 with important implica-tions in the regulation of permeability (27,28).

Interactions Within the Neurovascular Unit

Endothelial cells are not alone in the formation of a properBBB. Indeed, BMEC are surrounded by a basement mem-brane and pericytes, which nearly embrace the capillaries intheir totality and are further enwrapped by astrocyte feetprocesses (4,7). The interaction of the endothelium withcellular and acellular components is crucial for the mainte-nance of barrier properties, giving rise to the concept ofneurovascular unit (NVU) (Figure 1).

Pericytes are cells rich in a-smooth muscle actin thatphysically interact with the endothelium (7,29) and playan important role in the control of blood flow (30,31). Inthe CNS, these cells present one of the highest pericyte toBMEC ratio (1:5), when compared to the lung (1:10) andthe skeletal muscle (1:100) (32e34), suggesting a criticalrole in the maintenance of BBB properties. The interactionof pericytes with BMEC is believed to control endothelialproliferation (35), as well as the differentiation and matura-tion of vessels (36,37), effects that are partially regulatedthrough pericyte secretion of transforming growth factor-derstand the multifaceted influence of pericytes not onlyunder physiological conditions but also in the multiple dis-orders that affect the CNS.

Another important component of the BBB is the base-ment membrane, which surrounds both the endothelial cellsand the pericytes (7). Because these cells are separated by asingle basement membrane, it allows direct signalingthrough cellular junctions (49). The basement membraneof the vasculature is a complex assembly of four majorglycoprotein families: laminins, collagen type IV, proteo-glycans, and nidogens (50). Extracellular matrix (ECM)molecules are involved in cell adhesion and signaling path-ways (51) and exist in different isoforms that can assumeseveral combinations and differ between tissues (52). Inaddition, some ECM molecules are associated with immunecell recruitment to the brain parenchyma during injury(53,54). CNS endothelial cells present several ECM recep-tors including dystroglycan and integrins, which act to an-chor these cells to the basement membrane. These receptorsare able to regulate BBB integrity through the formation ofa transmembrane link between the ECM and the endothelialcytoskeleton and by transducing extracellular stimuli intointracellular signals (55e57). In addition, increased integ-rin expression in endothelial cells has been associated withcerebral blood vessel maturation during brain development(58). Interestingly, both endothelial cells and pericytescontribute to the synthesis of the vascular basement mem-brane (59e61). Several in vitro studies have highlightedthe importance of basement membrane constituents suchas fibronectin, laminin and collagen IV in the maintenanceof the barrier integrity and the microvessel structure(52,62). Matrix metalloproteinases (MMPs) that can besecreted by brain cells, including endothelial cells, maydigest the basement membrane which, in turn, leads toreduced anchoring of the endothelium and impaired junc-tional integrity (63,64). The complex role of the basementmembrane in barrier integrity and cell recruitment rein-forces the importance of considering the effects of toxicstimuli on such structure. In addition, continuous studieson this BBB component may allow the identification ofimportant targets for modulation in neuropathology.

The influence of glial cells in the maintenance of aproper barrier is fundamental, particularly when it comesto astrocytes. The specialized foot-processes of perivascularastrocytes have a particular role in inducing and regulatingthe BBB (65). The positive role of astrocytes on BBB prop-erties is believed to be achieved through the contact of theirend-feet with BMEC and through secretion of mediators.Indeed, the presence of astrocytes or of astrocyte-conditioned media in endothelial cultures has been shownto promote barrier integrity and the correct assembly ofthe intercellular junctions (42,66,67). Furthermore, astro-cytes are able to induce the expression and polarized local-ization of transporters including GLUT-1 (68) and

specialized enzyme systems (69,70). In addition, astrocytes barrier function. These cells are responsible for the forma-

663BloodeBrain Barrier in Neonatal Jaundiceare suggested to be necessary for the correct association ofendothelial cells and pericytes in tube-like structures (71),indicating that interaction among the three cell types isimportant for proper cerebral capillary differentiation. As-trocytes are, thus, crucial players in the formation andmaintenance of this barrier interface and are major contrib-utors to brain homeostasis.

CNS endogenous microglia share many properties withmacrophages, including expression of innate immune recep-tors and the ability to phagocyte pathogens, cells or cellulardebris (72). Microglial cells have been considered to takepart in the many interactions of BMEC because they arefound in the perivascular space (73). However, and despitethis proximity, their role in the regulation of BBB propertiesis still controversial. Indeed, in vitro studies by Prat and col-laborators (74) showed that endothelial permeabilitydecreased in the presence of conditioned medium collectedfrom astrocytes and microglia, probably through the induc-tion of junction formation. Studies by Willis (75) alsoshowed that the activation of glial cells, including micro-glia, guides junctional proteins to paracellular domainsand restores barrier integrity. On the contrary, activated mi-croglia were shown to damage the endothelium as well as toinduce vascular permeability (76e78) and transporter pro-tein dysfunction (79). Thus, microglial cells seem to befundamental players in the modulation of BBB properties.However, more studies on this interaction are needed tofully clarify their influence on the NVU dynamics.

The proximity of neurons to microvessels, |15 mm (80),points to the influence of neurons on BBB properties, butmore precise information is needed. The neuronal influenceon BBB properties may involve a direct interaction with theendothelium or derive from an indirect action mediated byother brain cells such as astrocytes. One of the striking ef-fects of neuronal activity and metabolism on the vascula-ture is the regulation of cerebral blood flow by amechanism known as neurovascular coupling (81,82).Some of the acknowledged effects on the BBB attributedto neurons include the ability to transform neuronal signalsinto vascular responses (83), together with the induction ofbarrier integrity (84) and occludin synthesis (85). In fact,in vitro studies with endothelial cells in the presence of neu-rons and astrocytes highlight the importance of both braincells in the induction of endothelial occludin expressionand its correct localization at the membrane level (86).The proximity of neurons to microvessels allows the endo-thelial cells to actively participate in the regulation of anoptimal environment for neuronal activity. In pathologieswhere the BBB is affected, especially the barrier properties,brain homeostasis and neuronal function often becomealtered, making the study of this cellular interaction anessential topic to be further explored.

Other glial cells, the oligodendrocytes, have beenignored regarding the interactions that they may exert ontion of myelin sheets in the CNS that are necessary for theproper conduction of the neuronal stimuli. Interestingly,BBB influence on oligodendrocytes has been reported,especially in demyelinating disorders like multiple sclerosis(MS) where BBB dysfunction is often perceived as animportant initiating step for oligodendrocyte damage(87e89). In contrast, oligodendrocyte effect on BBB prop-erties is still an unexplored issue. A recent study has shownthat these glial cells are able to secrete MMP-9, which playsan important role in the events that lead to BBB disruptionafter white matter injury (90). In addition, in vitro evalua-tion of the effect of oligodendrocyte-conditioned mediumshowed that the tightness of the endothelial barrier wasnoticeably weakened. These important findings indicatethat the impact of oligodendrocytes on BBB properties isfar more complex than previously thought, and this interac-tion clearly needs to be further explored. As so, it appearsrelevant to consider oligodendrocytes as another constituentof the NVU as we recently proposed (7).

BBB in Neuropathology

Because BMEC are the first cells of the CNS to contactwith toxic molecules in the systemic circulation, it is plau-sible that they may be the first players in the signalingmechanisms that trigger neuropathology. During injury,BMEC may produce elevated levels of inflammatory mole-cules such as nitric oxide (NO) and cytokines (91e95),which can influence both brain and blood compartmentsand contribute to injury aggravation. In addition, cytokinesand other inflammatory mediators are known to influencecaveolae, deregulating the passage of molecules betweencompartments. They may also induce gaps between endo-thelial cells by disassembly of intercellular junctions, eitherthrough alterations in the cellular cytoskeleton structure orby means of indirect damage on cell monolayer (96,97).Accordingly, BBB disruption has been reported in a widerange of pathologies that affect the CNS, such as MS(98,99), Alzheimers disease (100,101), Parkinsons disease(102,103), amyotrophic lateral sclerosis (104,105) andbrain tumors (106,107), implicating that at some point ofthe disease progression the release of inflammatory media-tors influence the balance between a beneficial role in main-taining or recovering brain homeostasis and the initiation ofneurological injury.

Neonatal Jaundice

Several diseases are particularly relevant in neonatal lifebecause this is a period of great vulnerability for the devel-oping CNS. Among them is neonatal hyperbilirubinemia,commonly known as neonatal jaundice, which is consideredto be a significant and risky condition for 1/10 children dueto elevated concentrations of unconjugated bilirubin (UCB)(108). Although jaundice is usually benign and normalizes

at the end of the first week of life, levels of total serum bili-

0

extending beyond the first week of life and deserving acareful management in order to prevent BIND and kernic-terus. In the same Figure 2 (black line), TSB levels of 17infants selected in Lisbon Maternity Wards are within therange considered to be susceptible of causing bilirubin en-cephalopathy (119).

Risk Factors for Severe Jaundice

The yellowish skin discoloration characteristic of neonataljaundice progresses in a cephalocaudal direction, beingeasily detected in the face and sclera when TSB reaches6 mg/dL and in the rest of the body at values O10 mg/dL (120). Although TSB is the most widely used parameterto assess the severity of jaundice, the range of TSB levelsresponsible for the occurrence of pathological jaundicefrequently varies among studies and clinical observations.The lack of consistency is justified by the fact that the bili-rubin species responsible for brain toxicity is in fact Bf(121,122), mostly due to its ability to easily cross theBBB (123). For this reason, several studies argue that Bfmeasurement, or even bilirubin/albumin ratio, are betterpredictors of BIND (124e127). Thus, assessing the riskof developing kernicterus is a complex subject and should

ond day of life and close to 20 or even 30 mg/dL at the 5 e6 days of life,

and still elevated at the end of the first week of life. Data are mean values

664 Brito et al./ Archives of Medical Research 45 (2014) 660e676cally reduced rate of UCB conjugation by uridine 5 -di-phosphoglucuronosyltransferase 1A1 (116). The excretionof bilirubin is also noticeably decreased in the neonatedue to the poor intestinal flora that usually metabolizesUCB in excretable products. Also, elevated b-glucuroni-dase levels, an enzyme that deconjugates the conjugatedbilirubin species, favor UCB reabsorption into the entero-hepatic circulation (117). Moreover, such reabsorption ofUCB can be promoted by breastfeeding, which is believedto increase the concentration of b-glucuronidase (118).Consequently, TSB levels increase in the first days of life,mainly due to UCB species, a condition that progressivelynormalizes towards the end of the first week of life with notreatment required and usually referred to as physiologicaljaundice of the neonate. In a previous study performed atour laboratory at the Faculdade de Farmacia, Universidadede Lisboa (119), we evaluated TSB levels in 134 healthyinfants selected by neonatologists at Hospital D. Estefa^nea(Lisbon) during the first 3 days and in the seventh day ofrubin (TSB) may increase dramatically and triggerbilirubin-induced neurologic dysfunction (BIND) with mi-nor brain deficits or more severe encephalopathy with ker-nicterus and even death (109,110) that is more frequent ininfants presenting a TSB of at least 20 mg/dL (342 mM)(108). Kernicterus continues to occur nowadays, even indeveloped countries, with variable prevalence rates rangingfrom 0.6e2.7/100 000 (111). Interestingly, in low- andmiddle-income countries, jaundice is one of the top fivecauses of neonatal death and a significant proportion of sur-vivors manifest signs of kernicterus (112). In 249 infantsadmitted to Cairo University Childrens Hospital with aTSB of $25 mg/dL during a 12-month period, 44 children(14%) presented bilirubin encephalopathy (113). In thesame study it was shown that sepsis should be considereda risk factor. Thus, understanding the steps involved inbrain injury progression during severe hyperbilirubinemiathat lead to the occurrence of kernicterus is critical in orderto prevent this devastating condition.

Bilirubin Metabolism in Perinatal Life

UCB is a tetrapyrrolic molecule formed by the catabolismof heme, which is mostly present in erythrocyte hemoglo-bin. Due to its low aqueous solubility, UCB has to be trans-ported in the blood bound to a carrier protein, albumin,before conjugation in the liver. The most neurotoxic speciesis believed to be the small fraction that circulates as un-bound species, termed free bilirubin (Bf) (114,115).

A series of events concerning bilirubin metabolism makejaundice a very common occurrence in the neonatal period.Particularly, neonates have an increased number of erythro-cytes that due to their shorter lifespan contribute to bili-rubin production. In addition, UCB clearance in thenewborn is less effective than in the adult, with a dramati-life. As depicted in Figure 2 (grey line), the average values(SD) are !10 mg/dL (171 mM) and tend to normalize atthe seventh day of life. However, in some newborn infantsUCB levels may increase more markedly, starting earlier or

(SD) from 134 healthy infants selected by neonatologists at HospitalD. Estefa^nea (Lisbon) and 17 cases of infants evaluated in Dora Brites lab-

oratory at the Faculdade de Farmacia, Universidade de Lisboa, selected in

Lisbon Maternity Wards by showing values of TSB considered to be sus-

ceptible of causing bilirubin encephalopathy. Data derived from Brites D

(119).Figure 2. Total serum bilirubin (TSB) concentrations in the first week of

life in physiological and pathological jaundice. TSB concentrations in

the first week of life greatly differ between healthy infants with levels usu-

ally!10 mg/dL, and normalizing at the seventh day of life (physiologicaljaundice), and severely ill infants with TSB values of 12 mg/dL at the sec-

th th

be based on the estimation of Bf, TSB and bilirubin/albu- in both neurons and glial cells (143) as reviewed by Brites

665BloodeBrain Barrier in Neonatal Jaundicemin ratio.Neonatal jaundice has many aggravating factors that in-

crease the risk of developing kernicterus with low TSBvalues, including prematurity, low birth weight, and thesimultaneous presence of other injurious conditions, suchas sepsis (128e130). Moreover, the development of highlevels of TSB in the first hours of life increases this riskdramatically. Nowadays, formulas based on nomogramsmay be found in the current literature, facilitating the deter-mination of the clinical risk of an icteric infant (131).Recently, the plotting of transcutaneous bilirubin measure-ment on a TSB nomogram also has been proposed as a toolfor predictive characteristics (132), but further studies areneeded.

Neurological Manifestations of Severe Jaundice

In the course of a severe jaundice condition there is a pref-erential UCB brain deposition in specific regions such asthe basal ganglia, hippocampus and cerebellum(133e136). The selective vulnerability of these brain re-gions has been further supported by the observation ofextensive neuronal loss and myelination defects in autopsycases of kernicterus (133,135,137). Perlman et al. (133)also reported the presence of brain edema in autopsy casesof kernicteric infants, suggesting an enhanced permeabilityof the vascular walls. Thus, it is well established that thereis a region-specific vulnerability to UCB harmful effects,but the underlying reasons for the preferential depositionof UCB in selective brain regions are still unclear andshould be clarified in order to better understand the under-lying mechanisms and to prevent BIND.

The consequences of BIND may be either reversible orirreversible. Some of the reversible effects include lethargyand decreased feeding, high-pitched cry, fever, and sei-zures. On the other hand, the irreversible sequelae are char-acterized by the classical manifestations of choreoathetoidcerebral palsy, isolated auditory loss and dental enamel hy-poplasia that may culminate in death (110,138). In addition,several studies revealed that neonatal jaundice could havean impact on learning and memory and in long-term cogni-tive disabilities (139) and may even increase the risk of psy-chological development outcome, especially autisticdisorders (140).

Mechanisms of UCB Neurotoxicity

The mechanisms of UCB toxicity have been extensivelystudied and include a general impairment of the cell mem-brane structure, properties and function (141,142). Bili-rubin toxicity to brain cells has been widely studied andinvolves some common features such as the induction ofcell death (by both apoptosis and necrosis), production ofpro-inflammatory cytokines (mainly by astrocytes and mi-croglia) and oxidative stress, as well as specialized features(144) and Brites and Brito (143). In fact, several studieshave shown that elevated levels of UCB are responsiblefor neuronal oxidative stress (145,146). UCB is able tointeract with the mitochondrial membrane, triggering theswelling of the organelle and the release of cytochrome c,with consequent caspase-3 activation and apoptotic celldeath (147). Interestingly, these neuronal effects appear tobe mediated by the increased expression of neuronal NOsynthase (nNOS) and consequent NO production(148,149). In addition, UCB induction of oxidative stressin the brain involves the inhibition of cytochrome c oxidaseactivity (146,150) together with superoxide anion radicalproduction, ATP release and disruption of glutathione redoxstatus (146). Other neuronal effects of UCB include neuriticatrophy and reduced neuronal arborization (151,152).Moreover, studies on rodent neurons exposed to UCB haveshown that the most affected neurons were those from thehippocampus (153), and that this increased susceptibilitywas reflected in compromised neuronal differentiation,development and plasticity (152). Together these resultsshow the extreme vulnerability of neurons to UCB, whichmight ultimately lead to the impairment of the formationof neuronal circuits in the brain, thus compromising brainfunction and eventually facilitating the development ofneurological disorders.

UCB neurotoxicity also involves the induction of celldeath in astrocytes, microglia and oligodendrocytes(154e156). To note that immature astrocytes and neuronsare more susceptible to UCB-induced cell death than matureor old cells (151,157) what may explain the aggravatingrole of prematurity in UCB toxicity and the susceptibilityof the neonatal period to UCB brain injury. Astrocytes andoligodendrocytes exposed to UCB show signs of mitochon-drial dysfunction and oxidative stress, which influence theapoptotic process (156,158). UCB interaction with astrocytesand microglia triggers the release of cytokines such as TNF-a, IL-1b and IL-6 (154,155), which may initiate an inflam-matory condition with potentiating effects on UCB damageto nerve cells. In addition to the common effects of UCBon several glial cell types, these studies reported specific ef-fects, especially on microglia and oligodendrocytes. Particu-larly, microglia seem to have a first neuroprotective effectwhen exposed to UCB by showing an increased phagocyticability (155). This property was enhanced when microgliawere treated with conditioned media from neurons exposedto UCB (159). However, if exposed for long periods toUCB, microglial cells revealed a phenotype considered tobe senescent and dystrophic, lose their functional defensivedynamic properties and ultimately die (155,160). Regardingoligodendrocytes, their differentiation and myelinating ca-pacity are also compromised in the presence of UCB (161)and may relate with the attention-deficit disorders found ininfants who suffered from severe neonatal hyperbilirubine-mia (162). Collectively, these studies highlight the diverse

range of action of UCB regarding glial cells, which might ul- glucose transport (168). The increased influx of glucose

666 Brito et al./ Archives of Medical Research 45 (2014) 660e676timately negatively influence the optimal environment forneuronal function and BBB properties, thus criticallycontributing to bilirubin encephalopathy.

BBB in Neonatal Jaundice

Initial studies by Cashore and coworkers in newborn piglets(163,164) provided evidence for a clear BBB permeabilityto unbound bilirubin in the first days of life. In fact, theseauthors showed that the BBB is permeable to Bf and thatthe ratio of bilirubin to albumin was higher in the brain thanin plasma after infusion of bilirubin to 2-day-old piglets.They further showed that the permeability was higher insubcortical regions than in the cortex and that such perme-ability was no longer evident in 2-week-old animals. Theseobservations provided a basis for the bilirubin entrance inbrain parenchyma, underlying the neurological injury bybilirubin and for the kernicterus characteristic regionalpattern, as well as for the increased susceptibility of prema-ture infants. Later on, Zucker et al. (165) corroborated thatUCB, mainly the uncharged diacid unbound species, wasable to diffuse spontaneously through the hydrophobicmembrane core. In addition, it was considered that a fastdeposition of Bf in the brain may be followed by a slowerUCB passage across the intact BBB and that the BBBdisruption may even facilitate transport of bilirubin/albu-min into the brain (166). Despite these indications ofBBB permeation by Bf and UCB and the widely establishedimpairment of several brain cell types, involvement of theBBB itself in UCB-induced neurotoxicity has been under-estimated. Here, we will present and discuss the literatureshowing that relevant microvascular-associated alterationsalso occur, thus adding the BBB as an additional potentialplayer in UCB neuropathology.

Alterations of BBB Endothelium

BMEC are the first brain cells to contact with UCB in theblood circulation, either bound to albumin or in the freeform. Thus, it is reasonable to believe that this first contact,in addition to providing UCB passage into the brain, mayalso be directly involved in the pathogenesis of UCB en-cephalopathy if we consider that endothelial response anddysfunction can be produced in such process. Indeed, theBBB may be envisaged as having an active participationin the pathological process resulting from severe hyperbilir-ubinemia. Three decades ago it was described for the firsttime that during conditions of severe hyperbilirubinemia,binding of UCB to isolated brain capillaries may occur(167). More recently, Cohen and collaborators (168) furtherindicated that UCB toxicity to bovine aortic endothelialcells involves an altered expression of GLUT-1, the trans-porter assuring the brain uptake of glucose (4). Moreover,expression of this transporter was enhanced, especially inthe plasma membrane, with matching increased rate ofmay constitute a compensatory ATP synthesis by glycolysiswhen mitochondrial energy production is impaired, asobserved in neurons exposed to UCB (146). Even thoughthis outcome alone disturbs the homeostasis of the CNS,other findings suggest that high glucose levels enhanceUCB toxicity to BMEC by generating increased productionof reactive oxygen species (ROS) (169).

ROS modulate cerebrovascular permeability through adiverse extent of inflammatory mediators such as NO andcytokines (170,171). In our own studies using primary cul-tures and a cell line of human BMEC (HBMEC) as a simplemodel of the BBB (172,173) exposed to UCB/human serumalbumin ratios (UCB/HSA) mimicking moderate (UCB/HSA 5 0.5) and severe (UCB/HSA 5 1.0), we have shownthat UCB disturbs endothelial homeostasis (174). In fact,the interaction of UCB with HBMEC induced a fast andsustained elevation of eNOS expression, followed by amarked accumulation of nitrites, a widely used biomarkerof NO production (Figure 3A). These results indicate thatHBMEC are exposed to nitrosative stress to which mayaccount the sustained disruption of glutathione metabolismas revealed by the elevated ratio of oxidized glutathione(GSSG) relative to total glutathione (GSx), also suggestingoxidative stress (Figure 3A). UCB additionally affected thesecretion of cytokines as depicted in Figure 3B. In fact, ashort exposure to UCB inhibited the release of interleukin(IL)-6, IL-8 and VEGF, which is followed by an increasedrelease, first of IL-6 already at 4 h, followed by IL-8 andlater on by VEGF (174). Interestingly, a similar effectwas observed for IL-6 secretion by astrocytes, with an earlyinhibition followed by the cytokine upregulation by moreprolonged exposure to UCB (154). Overall, these eventsshow that UCB is able to trigger an endothelial injuriousresponse with the production of important mediators thatmay aggravate the inflammatory condition in the brain pa-renchyma or even impact the endothelial integrity itself byacting in an autocrine manner.

eNOS, VEGF and VEGFR-2 may interact with caveolin-1 (24e26). Caveolin-1, the major structural protein of cav-eolae as reported above, not only ensures the movement ofsubstances via transcytosis but also has an active role in theregulation of BBB vesicular permeability (20,22).Caveolin-1 is also involved in various aspects of signaltransduction pathways and has been associated withincreased BBB permeability and brain edema during injury(23). Interestingly, UCB caused an increase in the levels ofcaveolin-1 (Figure 4A), as well as in the number of caveo-lae (Figure 4B), reflecting an augmented intracellular trans-port and permeability (175).

Caveolin-1 is known to interact with the ABC trans-porter P-gp (24e26). This efflux transporter has shown animportant role in the transport of UCB across the endothe-lial monolayer through a significant active efflux of thismolecule in the basolateral to apical direction (176). These

observations are corroborated by the disrupted junctions and

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667BloodeBrain Barrier in Neonatal Jaundicedata support the notion of UCB as a substrate of effluxtransporters such as P-gp (177,178). Interestingly, recentdata from our group showed that BMEC exposed to UCBpresent an inhibition of P-gp activity (179). This observa-tion may justify the increased expression of the proteinobserved in human brain tissue samples obtained from akernicterus case (136), probably as a compensatory mecha-nism created by the brain to overcome the decreased activ-ity and to maintain the efflux of UCB.

ROS can regulate the transcription of MMPs. These areproteolytic enzymes with the ability to degrade all types of

Figure 3. Unconjugated bilirubin (UCB) interferes with the secretion patter

thelial cells (HBMEC). Data ensued from treatment of a cell line of HBME

mimics a severe neonatal jaundice, for the indicated time points. Results wer

expression of endothelial nitric oxide synthase (eNOS), as indicated by the e

oxide (NO). In parallel, the cell defense system provided by glutathione is im

total glutathione (GSx). There is also an early inhibition of cytokines secreti

to be released, followed by IL-8, whereas vascular endothelial growth factoECM proteins, thus participating in tissue regeneration, re-modeling or even angiogenesis (180). However, in patholog-ical conditions MMPs may lead to ECM degradation andweakened anchoring of the endothelium to the basementmembrane (181). This may occur in cases of severe jaundiceonce we demonstrated that UCB leads to MMP-2 and -9 acti-vation in the extracellular media of treated BMEC (179).

Increased levels of UCB may then lead to HBMEC reac-tivity that ultimately can result in BBB impairment, facili-tating UCB passage into the brain. Together it appears thatthe UCB-induced expression and release of NO and cytokinesaffects the endothelial cells as a stable barrier. This contributesto alterations in AJ and TJ proteins (175,179) as observed inFigure 4C for the TJ protein ZO-1, leading to weakness ofthe TJ strands and to the retraction of cell-to-cell contacts(Figure 4D and E). In accordance, UCB caused a decreasein transendothelial electrical resistance (TEER) and an in-crease in paracellular permeability to the low molecularweight compound sodium fluorescein (376 Da) (175). Theseare widely used indicators of barrier integrity (4), reinforcingthat the compromise of the BBB integrity may be one of thecompromised barrier integrity obtained in rat BMEC (179)and intestinal epithelial cells (182).

Ultimately, the damage of UCB to HBMEC lead to the in-duction of cell death by apoptosis and necrosis (174,179,183),towhichmay account the increased cellmembrane fragility byUCB, as suggested by the release of cellular fragments andeven entire cells from the monolayer (175).

Impairment of Pericytesevents additionally contributing to UCB neurotoxicity. These

ytokines and disrupts the redox status of human brain microvascular endo-

th 100 mM UCB in the presence of 100 mM human serum albumin, which

ressed as fold change from the respective controls. UCB stimulates an early

ed expression at 1 h, which is followed by an increased production of nitric

d, as shown by the elevation of the ratio of oxidized glutathione (GSSG) to

llowed by their later increased production. Interleukin (IL)-6 is the first one

GF) presents a delayed response. Data derived from Palmela et al. (174).Brain microvascular pericytes extensively enwrap capil-laries, sharing basement membrane with BMEC and influ-encing their properties (7). So, pericyte deficiency in theCNS contributes to BBB breakdown and brain hypoperfu-sion resulting in neurodegenerative changes (184). A paucityof pericytes in germinal matrix vasculature of premature in-fants has been suggested to contribute to hemorrhage pro-pensity (48). Despite the relevance that pericytes may havein the mechanisms of BBB disruption by UCB, no informa-tion in addition to what is presented here is available.

Our recent and unpublished studies showed that UCB inconditions mimicking a moderate and severe hyperbilirubi-nemia induces a rapid increase in eNOS expression byhuman brain vascular pericytes (HBVP), already noticedat 1-h incubation (Figure 5A). Such effect may be behindthe increase in NO production by UCB, which was noto-rious (|4-fold increase) at 72 h after UCB addition(Figure 5B). As already mentioned, NO is among the stim-uli that influence endothelial properties (171). Therefore,the release of NO by HBVP adds to that produced by

668 Brito et al./ Archives of Medical Research 45 (2014) 660e676BMEC, reinforcing the homeostasis disruption. Moreover,it may impact on neurons where NO contributes to celldeath as demonstrated in experiments where the NOS in-hibitor L-NAME prevented neuronal death (148).

Figure 4. Unconjugated bilirubin (UCB) induces caveolae formation and disrupt

Data ensued from treatment of a cell line of HBMEC with 50 or 100 mMUCB in t

a severe neonatal jaundice, respectively. UCB induces the expression of caveolin-1

(arrows) as shown by transmission electron microscopy (B). It also decreases th

shown by fluorescence microscopy (C), decreases the tight junction strands (arr

the intercellular spaces (arrows), as shown by scanning electron microscopy (E)UCB also induced the secretion of cytokines by HBVP.Indeed, the expression of IL-6 mRNAwas upregulated at 45min and that of VEGF mRNA at 4 h, with the subsequentcytokine secretion, already marked at 1 h for IL-6 and

s tight junctions in human brain microvascular endothelial cells (HBMEC).

he presence of 100 mM human serum albumin, which mimic a moderate and

as observed by fluorescence microscopy (A) and the formation of caveolae

e expression of the tight junction protein zonula-occludens-1 (arrows) as

ows) visualized by freeze-fracture electron microscopy (D), and increases

. Adapted from Palmela et al. (175) with permission.

669BloodeBrain Barrier in Neonatal Jaundicepeaking at 24 h for VEGF (Figure 5C and D). It is inter-esting to point out that the HBVP response to UCB occursfaster than that observed for BMEC (Figure 3B) where IL-6secretion was only detected at 4 h and that of VEGF at 72 h

Figure 5. Unconjugated bilirubin (UCB) induces nitrosative stress, a pro-inflamm

cytes (HBVP). Data ensued from treatment of primary cultures of HBVP with 50

mimics a moderate and a severe neonatal jaundice, respectively, for the indicated

synthase, as indicated by the elevated expression at 1 h (A), which is followed b

There is also upregulation of interleukin (IL)-6 and vascular endothelial growth fa

(D). A loss of HBVP was observed by phase contrast microscopy (E). Nuclear fea

Hoechst 33258 dye (F) and the percentage of apoptotic cells was quantified (G)(174). Also interesting is the fact that the HBVP release ofIL-6 even precedes the one by astrocytes and microgliatreated with UCB (154,155). Therefore, secretion of thesemediators by HBVP may further contribute to the injurious

atory response and compromises the viability of human brain vascular peri-

or 100 mM UCB in the presence of 100 mM human serum albumin, which

time points. UCB stimulates an early expression of endothelial nitric oxide

y an increased production of nitrites (B), the end product of nitric oxide.

ctor (VEGF) mRNA (C), followed by the corresponding cytokines secretion

tures of apoptosis (arrow) were observed by fluorescence microscopy with

. *p !0.05 and **p !0.01 vs. respective control.

670 Brito et al./ Archives of Medical Research 45 (2014) 660e676effects by directly promoting BBB disruption, to whichshall also account the proper death of pericytes. Indeed, atime- and concentration-dependent loss of cell viabilitywas visualized by phase contrast microscopy (Figure 5E),and apoptotic features of apoptosis were significantlyenhanced (Figure 5F and G).

Collectively, data revealed impairment of HBVP byUCB in relevant physiopathological conditions. Due tothe importance of these cells in CNS homeostasis, futurestudies should explore the mechanisms involved in pericytedemise and their reflex in the NVU compromise in order todevise ways to modulate them.

Compromise of the NVU

One of the most interesting findings obtained in the in vitrostudies was that several players that mediate the formation

Figure 6. The brain parenchyma of a kernicterus case presents vascular and neu

topsied premature neonate with kernicterus and in an age-matched control with

marker cluster of differentiation (CD) 34 shows an increase in microvascular den

of immature vessels (inserts). (B) A poor Luxol-Periodic Acid Schiff (Luxol-PA

Decreased neurofilament immunoreactivity shows the impairment of neuritic arb

permission of Journal of Child Neurology.of new vessels were modified by UCB. Particularly, theinteraction of UCB with HBMEC revealed to induce anincreased production of IL-6, NO and VEGF, upregulatedlevels of VEGFR-2 and augmented endothelial perme-ability (174,175), parameters that have been implicated inthe angiogenic process (185e190). In accordance withthese evidences, our studies on human neonatal brain sam-ples from autopsy material of a kernicterus case revealed amarked increase in vessel density in the cerebellum (137)(Figure 6A). Such effect was also observed in the corpusstriatum, in the brainstem and in the hippocampus (136).Interestingly, the microvessels presented a poorly definedlumen, a characteristic of immature and hyperpermeablevessels. This observation is in line with the data obtainedin our in vitro studies with HBMEC, suggesting an angio-genic process that might function to compensate theUCB-induced dysfunction.

ronal alterations. Immunohistochemical and histological analysis in an au-

out brain pathology. (A) Immunohistochemical analysis of the endothelial

sity, and the presence of vessels with a poorly defined lumen, characteristic

S) staining in the kernicterus case indicates a loss of myelin fibers. (C)

orization in the kernicterus case. Reproduced from Brito et al. (137) with

Curiously, the kernicteric brain showed an increased as prematurity, hypoxia and sepsis, making difficult the

the detachment of endothelial cells from the monolayer, whichconstitute signs of barrier fragility. UCB damage is extended to

Together theBBBappears as a possibleCNSplayer tomodulate

7. Sa-Pereira I, Brites D, Brito MA. Neurovascular unit: a focus on peri-

671BloodeBrain Barrier in Neonatal Jaundiceexpression of VEGF in cerebellar neurons (137), remainingto clarify whether these cells are the producers or the maintargets of the vascular permeability factor. The accumulationof VEGF in Purkinje neurons may underline the preferentialpoisoning effect of bilirubin to such neurons already sug-gested (191) and contribute to the abnormal tone and motorcoordination in children with kernicterus (110). Upregulationof VEGF was paralleled by an increased expression ofVEGFR-2 (137), suggesting an autocrine/paracrine effectof UCB on neurons that can perpetuate homeostatic pertur-bations. The presence of the blood component albumin inthe brain parenchyma was also noticed (136), pointing to mi-crovessel leakage that was previously reported to be respon-sible for neurotoxic manifestations (192e194).

Vascular alterations in the kernicterus case were accom-panied by neuronal dysfunction, characterized by the lossof myelin fibers (Figure 6B). In addition, there was animpairment of the dendritic arborization in the cerebellum,and particularly in the Purkinje neurons (Figure 6C),corroborating both in vitro findings (151,161) and thosereported in a mouse model of hyperbilirubinemiawhere loss of Purkinje neurons was observed (195). Dam-age is not restricted to the cerebellum, and impairment ofthe basal ganglia was a common feature in otherhuman neonatal brain samples from infants with kernic-terus (196).

Increased expression of the efflux transporters multidrugresistance-associated proptein 1 (MRP1) and P-gpdescribed in the kernicterus case (136,137) is in line witha compensatory mechanism of protection against UCBneurotoxicity (197). Indeed, the inhibition of MRP-1 byMK571 enhanced UCB neurotoxic effects (198). A signif-icant upregulation of P-gp expression in microvessels ofthe jj (jaundiced) Gunn rats (a kernicterus model) ascompared with Jj (not jaundiced) littermates additionallyhighlights the relevance that such efflux transporter mayhave on bilirubin encephalopathy (199). Furthermore, ifwe consider that the expression of P-gp increases untiladulthood (15,199), we may speculate that the blood ves-sels of the mouse pups have a low capacity to accomplishthe efflux UCB from the brain parenchyma. CompromisedNVU in neonatal life may similarly result from thedecreased expression of MRP1 previously observed inimmature neurons in primary culture (198).

Collectively, upregulation of the efflux transporters mayrepresent an attempt to protect the brain from UCBentrance, but the leakiness of the microvasculature clearlyseems to counteract such process. Collected data are impor-tant to open new avenues for future research aiming tobetter understand whether vascular dysfunction is a deter-minant or a secondary event in bilirubin encephalopathy.Added to that, the evaluation of additional cases of kernic-terus is fundamental. These cases frequently occur ininfants who present concomitant risk factors such cytes. Mol Neurobiol 2012;45:327e347.whenever brain dysfunction by lasting hyperbilirubinemia issuspected in order to prevent neurological deficits by UCB.

AcknowledgmentsWe acknowledge our funding support from Fundac~ao para aCie^ncia e a Tecnologia (FCT), Lisbon, Portugal, through grantsPTDC/SAU-NEU/64385/2006 (to DB), PTDC/SAU-FCF/68819/2006 (to MAB), and the strategic project PEst-OE/SAU/UI4013/2011e2014, as well as FEDER.

Conflicts of interest: The authors declare no conflict of interest.

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