Filipa Lourenço Cardoso

Looking at the blood–brain barrier: Molecular anatomy and possible investigation approaches

The blood-brain barrier (BBB) is a dynamic and complex interface between blood and the central nervous system that strictly controls the exchanges between the blood and brain compartments, therefore playing a key role in brain homeostasis and providing protection against many toxic compounds and pathogens. In this review, the unique properties of brain microvascular endothelial cells and intercellular junctions are examined. The specific interactions between endothelial cells and basement membrane as well as neighboring perivascular pericytes, glial cells and neurons, which altogether constitute the neurovascular unit and play an essential role in both health and function of the central nervous system, are also explored. Some relevant pathways across the endothelium, as well as mechanisms involved in the regulation of BBB permeability, and the emerging role of the BBB as a signaling interface are addressed as well. Furthermore, we summarize some of the experimental approaches that can be used to monitor BBB properties and function in a variety of conditions and have allowed recent advances in BBB knowledge. Elucidation of the molecular anatomy and dynamics of the BBB is an essential step for the development of new strategies directed to maintain or restore BBB integrity and barrier function and ultimately preserve the delicate interstitial brain environment.

Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVECs). HBMVECs are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size filtered using polyester meshes. The resulting microvessel fragments are placed onto type I collagen-coated flasks to allow HBMVECs to migrate and proliferate. The overall process takes less than 3 h and does not require specialized equipment or enzymatic processes. HBMVECs are typically cultured for approximately 1 month until confluent. Cultures are highly pure (∼97% endothelial cells; ∼3% pericytes), are reproducible, and show characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1) and robust expression of tight and adherens junction proteins as well as caveolin-1 and efflux protein P-glycoprotein. Monolayers of HBMVECs show characteristically high transendothelial electric resistance and have proven useful in multiple functional studies for in vitro modeling of the human blood-brain barrier.

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.

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.

Impact of Neuroinflammation on Hippocampal Neurogenesis: Relevance to Aging and Alzheimer’s Disease

The cognitive reserve is associated with the capacity of the brain to maintain cognitive performance in spite of being challenged by stressful degenerative insults related to aging. Hippocampal neurogenesis is a life-long process of continuous addition of functional new neurons in the memory processing circuits. Accordingly, adult hippocampal neurogenesis is increasingly seen as a key determinant of cognitive reserve robustness. On the other side, neuroinflammation, by releasing a plethora of proinflammatory cytokines and other inflammatory molecules, is increasingly shown to be one of the key determinant pathophysiological factors that negatively impact on neurogenesis and on the cognitive reserve, playing a detrimental role in hippocampal neurogenic niche dynamics and in the progression of neurodegenerative diseases, such as Alzheimers disease. In the present manuscript, we highlight the functional interplay between neuroinflammation, dynamics of the neurogenic niche, and spatial memory performance in healthy and age-related pathological processes, including progression of Alzheimers disease.

Impact of developmental exposure to methylphenidate on rat brain’s immune privilege and behavior: Control versus ADHD model

Attention deficit hyperactivity disorder (ADHD) is the most prevalent childhood mental disorders that often persists into adulthood. Moreover, methylphenidate (MPH) is the mainstay of medical treatment for this disorder. Yet, not much is known about the neurobiological impact of MPH on control versus ADHD conditions, which is crucial to simultaneously clarify the misuse/abuse versus therapeutic use of this psychostimulant. In the present study, we applied biochemical and behavioral approaches to broadly explore the early-life chronic exposure of two different doses of MPH (1.5 and 5 mg/kg/day) on control and ADHD rats (Wistar Kyoto and Spontaneously Hypertensive rats, respectively). We concluded that the higher dose of MPH promoted blood-brain barrier (BBB) permeability and elicited anxiety-like behavior in both control and ADHD animals. BBB dysfunction triggered by MPH was particularly prominent in control rats, which was characterized by a marked disruption of intercellular junctions, an increase of endothelial vesicles, and an upregulation of adhesion molecules concomitantly with the infiltration of peripheral immune cells into the prefrontal cortex. Moreover, both doses of MPH induced a robust neuroinflammatory and oxidative response in control rats. Curiously, in the ADHD model, the lower dose of MPH (1.5 mg/kg/day) had a beneficial effect since it balanced both immunity and behavior relative to vehicle animals. Overall, the contrasting effects of MPH observed between control and ADHD models support the importance of an appropriate MPH dose regimen for ADHD, and also suggest that MPH misuse negatively affects brain and behavior.