Pericytes from Brain Microvessels Strengthen the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

(1) The blood–brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.     

Neurovascular Unit: a Focus on Pericytes

The blood–brain barrier (BBB) is a highly specialized system that controls the exchanges between the blood and the central nervous system (CNS). This barrier shields the CNS from toxic substances in the blood and provides nutrients to CNS, thus playing an essential role in the maintenance of homeostasis. The anatomical basis of the BBB is formed by the endothelial cells of brain microvasculature, with elaborated tight and adherens junctions, which together with pericytes, the basement membrane, and astrocytes, as well as neurons, microglia and oligodendrocytes form the neurovascular unit. The interaction between all these components guarantees a proper environment for neural function and a restricted permeability and transport. Pericytes were initially reported by Rouget in 1873 and since then they have been recognized as an important component of the BBB, despite the difficulty of their identification. Diverse functions have been assigned to pericytes, including a role in BBB properties, hemostasis, and angiogenesis, as well as a contractile, immune, and phagocytic function. These cells are also seen like multipotent cells and so with a great potential for therapy. Here, we review the neurovascular unit composition and the interplay between the diverse components, addressing pericytes with a particular detail.     

Pericyte-derived Glial Cell Line-derived Neurotrophic Factor Increase the Expression of Claudin-5 in the Blood–brain Barrier and the Blood-nerve Barrier

The destruction of blood–brain barrier (BBB) and blood-nerve barrier (BNB) has been considered to be a key step in the disease process of a number of neurological disorders including cerebral ischemia, Alzheimer’s disease, multiple sclerosis, and diabetic neuropathy. Although glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) facilitate neuronal or axonal regeneration in the brain or peripheral nerves, their action in the BBB and BNB remains unclear. The purpose of the present study was to elucidate whether these neurotrophic factors secreted from the brain or peripheral nerve pericytes increase the barrier function of the BBB or BNB, using our newly established human brain microvascular endothelial cell (BMEC) line or peripheral nerve microvascular endothelial cell (PnMEC) line. GDNF increased the expression of claudin-5 and the transendothelial electrical resistance (TEER) of BMECs and PnMECs, whereas BDNF did not have this effect. Furthermore, we herein demonstrate that the GDNF secreted from the brain and peripheral nerve pericytes was one of the key molecules responsible for the up-regulation of claudin-5 expression and the TEER value in the BBB and BNB. These results indicate that the regulation of GDNF secreted from pericytes may therefore be a novel therapeutic strategy to modify the BBB or BNB functions and promote brain or peripheral nerve regeneration.     

The effect of moderate hypothermia in acute ischemic stroke on pericyte migration: an ultrastructural study

Pericytes are essential components of the blood–brain barrier together with endothelial cells and astrocytes. Any disturbance of brain perfusion may result in blood–brain barrier dysfunction due to pericyte migration from the microvascular wall. The neuroprotective influence of hypothermia on ischemic brain injury has been clearly shown in models of both global and focal ischemia. Leakage of plasma proteins contributes to the extension of neuronal injury and hypothermia has a neuroprotective influence during the ischemic insult. This line of thinking impelled us to investigate the possible role of the pericytes in the occurrence of hypothermic protection during cerebral ischemia.In this study, we examined at the ultrastructural level the effect of moderate hypothermia on microvascular pericyte responses using a rat model of permanent middle cerebral artery occlusion. Twenty rats were divided into four groups. Middle cerebral artery occlusion was performed in all rats except the control group (first group), which was used to determine the pericyte morphology under normal conditions. In the second group, pericyte response to irreversible ischemia under normothermic conditions was examined at the end of the first hour. In the third group, pericyte response to hypoxia was examined under normothermic conditions three hours after ischemia. In the fourth group, temporalis muscle temperature was maintained at 27–29 °C for 1 h after middle cerebral artery occlusion and pericyte response was then examined at the ultrastructural level. In ischemic normothermic conditions at the end of the first hour (Group 2), a separation was observed between pericytes and the basement membrane and this was interpreted as pericyte migration from the microvascular wall. In ischemic normothermic conditions at the end of the third hour (Group 3), basement membrane disorganization and increased space between the basement membranes were seen in addition to the differentiation of second group. In ischemic hypothermic conditions at the end of the first hour (Group 4), pericyte separation or migration from basement membrane were not seen and the blood–brain barrier remained firm. These findings were interpreted by the authors as a possible relationship between pericyte behavior and neural protection during hypothermia. We suggest that hypothermia may delay the pericyte response but not necessarily attenuate it, and should be associated with hypothermic protection.     

Phosphodiesterase-III inhibitor prevents hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA

The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA.     

Infection of human pericytes by HIV‐1 disrupts the integrity of the blood–brain barrier

Human immunodeficiency virus type 1 (HIV‐1) infection of the central nervous system (CNS) affects cross‐talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. Although the toxicity of HIV‐1 on neurons, astrocytes and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV‐1 on pericytes. Therefore, the purpose of this study was to evaluate whether or not pericytes can be infected with HIV‐1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV‐1 receptor CD4 and co‐receptors CXCR4 and CCR5. We also determined that HIV‐1 can replicate, although at a low level, in human brain pericytes as detected by HIV‐1 p24 ELISA. Pericytes were susceptible to infection with both the X4‐tropic NL4‐3 and R5‐tropic JR‐CSF HIV‐1 strains. Moreover, HIV‐1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV‐1 and suggest that infected pericytes are involved in the progression of HIV‐1‐induced CNS damage.     

The impact of glia-derived extracellular matrices on the barrier function of cerebral endothelial cells: an in vitro study

The blood-brain barrier (BBB) is composed of the cerebral microvascular endothelium, which, together with astrocytes, pericytes, and the extracellular matrix (ECM), contributes to a "neurovascular unit". It was our objective to clarify the impact of endogenous extracellular matrices on the barrier function of BBB microvascular endothelial cells cultured in vitro. The study was performed in two consecutive steps: (i) The ECM-donating cells (astrocytes, pericytes, endothelial cells) were grown to confluence and then removed from the growth substrate by a protocol that leaves the ECM behind. (ii) Suspensions of cerebral endothelial cells were seeded on the endogenous matrices and barrier formation was followed with time. In order to quantify the tightness of the cell junctions, all experiments were performed on planar gold-film electrodes that can be used to read the electrical resistance of the cell layers as a direct measure for endothelial barrier function (electric cell-substrate impedance sensing, ECIS). We observed that endogenously isolated ECM from both, astrocytes and pericytes, improved the tightness of cerebral endothelial cells significantly compared to ECM that was derived from the endothelial cells themselves as a control. Moreover, when cerebral endothelial cells were grown on extracellular matrices produced by non-brain endothelial cells (aorta), the electrical resistances were markedly reduced. Our observations indicate that glia-derived ECM - as an essential part of the BBB - is required to ensure proper barrier formation of cerebral endothelial cells.     

Blood-neural barrier: its diversity and coordinated cell-to-cell communication

The cerebral microvessels possess barrier characteristics which are tightly sealed excluding many toxic substances and protecting neural tissues. The specialized blood-neural barriers as well as the cerebral microvascular barrier are recognized in the retina, inner ear, spinal cord, and cerebrospinal fluid. Microvascular endothelial cells in the brain closely interact with other components such as astrocytes, pericytes, perivascular microglia and neurons to form functional 'neurovascular unit'. Communication between endothelial cells and other surrounding cells enhances the barrier functions, consequently resulting in maintenance and elaboration of proper brain homeostasis. Furthermore, the disruption of the neurovascular unit is closely involved in cerebrovascular disorders. In this review, we focus on the location and function of these various blood-neural barriers, and the importance of the cell-to-cell communication for development and maintenance of the barrier integrity at the neurovascular unit. We also demonstrate the close relation between the alteration of the blood-neural barriers and cerebrovascular disorders.     

Neural Induction of the Blood–Brain Barrier: Still an Enigma

1. The study of the blood–brain barrier and its various realms offers a myriad of opportunities for scientific exploration. This review focuses on two of these areas in particular: the induction of the blood–brain barrier and the molecular mechanisms underlying this developmental process.2. The creation of the blood–brain barrier is considered a specific step in the differentiation of cerebral capillary endothelial cells, resulting in a number of biochemical and functional alterations. Although the specific endothelial properties which maintain the homeostasis in the central nervous system necessary for neuronal function have been well described, the inductive mechanisms which trigger blood–brain barrier establishment in capillary endothelial cells are unknown.3. The timetable of blood–brain barrier formation is still a matter of debate, caused largely by the use of varying experimental systems and by the general difficulty of quantitatively measuring the degree of blood–brain barrier tightness. However, there is a general consensus that a gradual formation of the blood–brain barrier starts shortly after intraneural neovascularization and that the neural microenvironment (neurons and/or astrocytes) plays a key role in inducing blood–brain barrier function in capillary endothelial cells. This view stems from numerous in vitro experiments using mostly cocultures of capillary endothelial cells and astrocytes and assays for easily measurable blood–brain barrier markers. In vivo, there are great difficulties in proving the inductive influence of the neuronal environment. Also dealt with in this article are brain tumors, the least understood in vivo systems, and the induction or noninduction of barrier function in the newly established tumor vascularization.4. Finally, this review tries to elucidate the question concerning the nature of the inductive signal eliciting blood–brain barrier formation in the cerebral microvasculature.     

Brain-Targeted Delivery of Docetaxel by Glutathione-Coated Nanoparticles for Brain Cancer

Gliomas are some of the most aggressive types of cancers but the blood–brain barrier acts as an obstacle to therapeutic intervention in brain-related diseases. The blood–brain barrier blocks the permeation of potentially toxic compounds into neural tissue through the interactions of brain endothelial cells with glial cells (astrocytes and pericytes) which induce the formation of tight junctions in endothelial cells lining the blood capillaries. In the present study, we characterize a glutathione-coated docetaxel-loaded PEG-PLGA nanoparticle, show its in vitro drug release data along with cytotoxicity data in C6 and RG2 cells, and investigate its trans-blood–brain barrier permeation through the establishment of a Transwell cellular co-culture. We show that the docetaxel-loaded nanoparticle’s size enables its trans-blood–brain barrier permeation; the nanoparticle exhibits a steady, sustained release of docetaxel; the drug is able to induce cell death in glioma models; and the glutathione-coated nanoparticle is able to permeate through the Transwell in vitro blood–brain barrier model.KEY WORDS: blood–brain barrier, brain cancer, docetaxel, glutathione, nanoparticle     

Effect of Baicalin on Matrix Metalloproteinase-9 Expression and Blood–Brain Barrier Permeability Following Focal Cerebral Ischemia in Rats

Focal cerebral ischemia results in an increased expression of matrix metalloproteinase-9 (MMP-9), which induces vasogenic brain edema via disrupting the blood–brain barrier (BBB) integrity. Recent studies from our laboratory showed that baicalin reduces ischemic brain damage by inhibiting inflammatory reaction and neuronal apoptosis in a rat model of focal cerebral ischemia. In the present study, we first explored the effect of baicalin on the neuronal damage, brain edema and BBB permeability, then further investigated its potential mechanisms. Sprague–Dawley rats underwent permanent middle cerebral artery occlusion (MCAO). Baicalin was administrated by intraperitoneally injected twice at 2 and 12 h after the onset of MCAO. Neuronal damage, brain edema and BBB permeability were measured 24 h following MCAO. Expression of MMP-9 protein and mRNA were determined by western blot and RT–PCR, respectively. Expression of tight junction protein (TJP) occludin was detected by western blot. Neuronal damage, brain edema and BBB permeability were significantly reduced by baicalin administration following focal cerebral ischemia. Elevated expression of MMP-9 protein and mRNA were significantly down-regulated by baicalin administration. In addition, MCAO caused the decreased expression of occludin, which was significantly up-regulated by baicalin administration. Our study suggested that baicalin reduces MCAO-induced neuronal damage, brain edema and BBB permeability, which might be associated with the inhibition of MMP-9 expression and MMP-9-mediated occludin degradation.     

Maintaining blood–brain barrier integrity: Pericytes perform better than astrocytes during prolonged oxygen deprivation

The blood–brain barrier (BBB), consisting of specialized endothelial cells surrounded by astrocytes and pericytes, plays a crucial role in brain homeostasis. Many cerebrovascular diseases are associated with BBB breakdown and oxygen (O₂) deprivation constitutes a critical factor that onsets its disruption. We investigated the impact of astrocytes and pericytes on brain endothelial cell permeability and survival during different degrees of O₂ deprivation. Prolonged exposure to 1% O₂ caused barrier breakdown and exposure to 0.1% O₂ dramatically accelerated disruption and induced cell death, mediated at least in part via caspase‐3 activation. Reoxygenation allowed only cells exposed to 1% O₂ to re‐establish barrier function. Notably co‐culture with astrocytes and pericytes substantially enhanced barrier function under normoxic conditions, and produced differential responses during O₂ deprivation. At 1% O₂ astrocytes partially maintained barrier integrity whereas pericytes accelerated its disruption in the short‐term, having positive effects only after prolonged exposure. Unexpectedly, at 0.1% O₂ pericytes were more effective than astrocytes in preserving barrier function although the protection afforded by both cells involved inhibition of caspase‐3 pathways. Furthermore, cell‐specific regulation of auto‐ and paracrine VEGF signaling pathways were also in part responsible for the differential modulation of barrier function. Our data suggests that cellular cross‐talk within the neurovascular unit is crucial for preservation of barrier integrity and that pericytes, not astrocytes, play a significant role during severe and prolonged O₂ deprivation. J. Cell. Physiol. 218: 612–622, 2009. © 2008 Wiley‐Liss, Inc.     

Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats

Matrix metalloproteinases (MMPs) play an important role in reperfusion-induced brain injury following ischemia. To define the effects of peroxynitrite decomposition catalyst on MMP activation and neurovascular reperfusion injury, 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin iron (III) (FeTMPyP) was administered intravenously 30?min prior to reperfusion following a middle cerebral artery occlusion. Activation of MMP was assessed by in situ and gel zymography. Neurovascular injury was assessed using endothelial barrier antigen, collagen IV immunohistochemistry and Cresyl violet staining. Results were compared with sham and ischemia alone groups. We found that administration of FeTMPyP just before reperfusion after ischemia inhibited MMP-9 activation and total MMP-2 increases in the cortex and decreased active MMP-9 along with the total amounts of active MMP-9 and active MMP-2 in the striatum. Reperfusion-induced injury to the basal lamina of collagen IV-immunopositive microvasculature and neural cells in cortex and striatum was ameliorated by FeTMPyP. Losses of blood vessel endothelium produced by ischemia or reperfusion were also decreased in the cortex. These results suggest that administration of FeTMPy prior to reperfusion decreases MMP activation and neurovascular injury after prolonged cerebral ischemia. This strategy may be useful for future therapies targeted at preventing breakdown of the blood-brain barrier and hemorrhagic transformation.     

Lipopolysaccharide-Activated Microglia Induce Dysfunction of the Blood–Brain Barrier in Rat Microvascular Endothelial Cells Co-Cultured with Microglia

The blood–brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase.     

TWEAK and the Central Nervous System

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily that acts on responsive cells via binding to a cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK can regulate numerous cellular responses in vitro and in vivo. Recent studies have indicated that TWEAK and Fn14 are expressed in the central nervous system (CNS), and that in response to a variety of stimuli, including cerebral ischemia, there is an increase in TWEAK and Fn14 expression in perivascular astrocytes, microglia, endothelial cells, and neurons with subsequent increase in the permeability of the blood–brain barrier (BBB) and cell death. Furthermore, there is a growing body of evidence indicating that TWEAK induces the activation of the NF-κB in the CNS with release of proinflammatory cytokines and matrix metalloproteinases. In addition, inhibition of TWEAK activity by either treatment with a Fn14-Fc fusion protein or neutralizing anti-TWEAK antibodies has shown therapeutic efficacy in animal models of ischemic stroke, cerebral edema, and multiple sclerosis.     

The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes and neurons. In this study we analyzed the differentiation stage dependent influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells. At first, we were able to induce two distinct differentiation stages of the primary pericytes in vitro. TGFβ treated pericytes expressed more α-SMA and actin while desmin, vimentin and nestin expression was decreased when compared to bFGF induced cells. Further analysis of α-SMA revealed that most of the pericytes differentiated with TGFβ expressed functional α-SMA while only few cells expressed functional α-SMA in the presence of bFGF. In addition the permeability factors VEGF, MMP-2 and MMP-9 were higher secreted by the α-SMA positive phenotype indicating a proangiogenic role of this TGFβ induced pericyte differentiation stage. Higher level of VEGF, MMP-2 and MMP-9 were as well detected in the TGFβ pretreated pericyte coculture with endothelial cells when compared to the influence of the bFGF pretreated pericytes. The TEER measurement of the barrier integrity of endothelial cells revealed that bFGF induced α-SMA negative pericytes stabilize the barrier integrity while α-SMA positive pericytes differentiated by TGFβ decrease the barrier integrity. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity in a differentiation stage dependant pathway.     

Oxygen-glucose deprivation and interleukin-1α trigger the release of perlecan LG3 by cells of neurovascular unit

Two of the main stresses faced by cells at the neurovascular unit (NVU) as an immediate result of cerebral ischemia are oxygen-glucose deprivation (OGD)/reperfusion and inflammatory stress caused by up regulation of IL-1. As a result of these stresses, perlecan, an important component of the NVU extracellular matrix, is highly proteolyzed. In this study, we describe that focal cerebral ischemia in rats results in increased generation of laminin globular domain 3 (LG3), the c-terminal bioactive fragment of perlecan. Further, in vitro study of the cells of the NVU was performed to locate the source of this increased perlecan-LG3. Neurons, astrocytes, brain endothelial cells and pericytes were exposed to OGD/reperfusion and IL-1α/β. It was observed that neurons and pericytes showed increased levels of LG3 during OGD in their culture media. During in vitro reperfusion, neurons, astrocytes and pericytes showed elevated levels of LG3, but only after exposure to brief durations of OGD. IL-1α and IL-1β treatment tended to have opposite effects on NVU cells. While IL-1α increased or had minimal to no effect on LG3 generation, high concentrations of IL-1β decreased it in most cells studied. Finally, LG3 was determined to be neuroprotective and anti-proliferative in brain endothelial cells, suggesting a possible role for the generation of LG3 in the ischemic brain.     

Transient Expression of Iron Transport Proteins in the Capillary of the Developing Rat Brain

Iron is essential for normal brain function and its uptake in the developing rat brain peaks during the first two weeks after birth, prior to the formation of the blood–brain barrier (BBB). The first step of iron transport from the blood to the brain is transferrin receptor (TfR)-mediated endocytosis in the capillary endothelial cells. However, the subsequent step from the endothelium into interstitium has not been fully described. The goal of this study was to examine the expression of iron transport proteins by immunodetection and RT–PCR in the developing rat brain. Tf and TfR are transiently expressed in perivascular NG2+ cells of the capillary wall during the early postnatal weeks in the rat brain. However, MTP-1 and hephaestin were expressed in endothelial cells, but not in the NG2+ perivascular cells. Immunoblot analysis for these iron transfer proteins in the developing brain generally confirmed the immunochemical findings. Furthermore, the expression of Tf and TfR in the blood vessels precedes its expression in oligodendrocytes, the main iron-storing cells in the vertebrate brain. RT–PCR analysis for the primary culture of endothelial cells and pericytes revealed that Tf and TfR were highly expressed in the pericytes while MTP-1 and hephaestin were expressed in the endothelial cells. The specific expression of Tf and TfR in brain perivascular cells and MTP-1 and hephaestin in endothelial cells suggest the possibility that trafficking of elemental iron through perivascular cells may be instrumental in the distribution of iron in the developing central nervous system.     

Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression

Blood–brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier; however, they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein, we employed oxygen–glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3‐h before co‐culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8‐D1A cells). Incubation of OGD‐treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin‐5 proteins as well as increased vascular endothelial growth factor (VEGF) secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.

     

Inflammatory Mediators and Modulation of Blood–Brain Barrier Permeability

1. Unlike some interfaces between the blood and the nervous system (e.g., nerve perineurium), the brain endothelium forming the blood–brain barrier can be modulated by a range of inflammatory mediators. The mechanisms underlying this modulation are reviewed, and the implications for therapy of the brain discussed.2. Methods for measuring blood–brain barrier permeability in situ include the use of radiolabeled tracers in parenchymal vessels and measurements of transendothelial resistance and rate of loss of fluorescent dye in single pial microvessels. In vitro studies on culture models provide details of the signal transduction mechanisms involved.3. Routes for penetration of polar solutes across the brain endothelium include the paracellular tight junctional pathway (usually very tight) and vesicular mechanisms. Inflammatory mediators have been reported to influence both pathways, but the clearest evidence is for modulation of tight junctions.4. In addition to the brain endothelium, cell types involved in inflammatory reactions include several closely associated cells including pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. In situ it is often difficult to identify the site of action of a vasoactive agent. In vitro models of brain endothelium are experimentally simpler but may also lack important features generated in situ by cell:cell interaction (e.g. induction, signaling).5. Many inflammatory agents increase both endothelial permeability and vessel diameter, together contributing to significant leak across the blood–brain barrier and cerebral edema. This review concentrates on changes in endothelial permeability by focusing on studies in which changes in vessel diameter are minimized.6. Bradykinin (Bk)² increases blood–brain barrier permeability by acting on B₂ receptors. The downstream events reported include elevation of [Ca²⁺]_i, activation of phospholipase A₂, release of arachidonic acid, and production of free radicals, with evidence that IL-1 potentiates the actions of Bk in ischemia.7. Serotonin (5HT) has been reported to increase blood–brain barrier permeability in some but not all studies. Where barrier opening was seen, there was evidence for activation of 5-HT₂ receptors and a calcium-dependent permeability increase.8. Histamine is one of the few central nervous system neurotransmitters found to cause consistent blood–brain barrier opening. The earlier literature was unclear, but studies of pial vessels and cultured endothelium reveal increased permeability mediated by H₂ receptors and elevation of [Ca²⁺]_i and an H₁ receptor-mediated reduction in permeability coupled to an elevation of cAMP.9. Brain endothelial cells express nucleotide receptors for ATP, UTP, and ADP, with activation causing increased blood–brain barrier permeability. The effects are mediated predominantly via a P_2U (P2Y₂) G-protein-coupled receptor causing an elevation of [Ca²⁺]_i; a P2Y₁ receptor acting via inhibition of adenyl cyclase has been reported in some in vitro preparations.10. Arachidonic acid is elevated in some neural pathologies and causes gross opening of the blood–brain barrier to large molecules including proteins. There is evidence that arachidonic acid acts via generation of free radicals in the course of its metabolism by cyclooxygenase and lipoxygenase pathways.11. The mechanisms described reveal a range of interrelated pathways by which influences from the brain side or the blood side can modulate blood–brain barrier permeability. Knowledge of the mechanisms is already being exploited for deliberate opening of the blood–brain barrier for drug delivery to the brain, and the pathways capable of reducing permeability hold promise for therapeutic treatment of inflammation and cerebral edema.