Blood–brain Barrier: Structural Components and Function Under Physiologic and Pathologic Conditions
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The blood–brain barrier (BBB) is the specialized system of brain microvascular endothelial cells (BMVEC) that shields the brain from toxic substances in the blood, supplies brain tissues with nutrients, and filters harmful compounds from the brain back to the bloodstream. The close interaction between BMVEC and other components of the neurovascular unit (astrocytes, pericytes, neurons, and basement membrane) ensures proper function of the central nervous system (CNS). Transport across the BBB is strictly limited through both physical (tight junctions) and metabolic barriers (enzymes, diverse transport systems). A functional polarity exists between the luminal and abluminal membrane surfaces of the BMVEC. As a result of restricted permeability, the BBB is a limiting factor for the delivery of therapeutic agents into the CNS. BBB breakdown or alterations in transport systems play an important role in the pathogenesis of many CNS diseases (HIV-1 encephalitis, Alzheimer's disease, ischemia, tumors, multiple sclerosis, and Parkinson's disease). Proinflammatory substances and specific disease-associated proteins often mediate such BBB dysfunction. Despite seemingly diverse underlying causes of BBB dysfunction, common intracellular pathways emerge for the regulation of the BBB structural and functional integrity. Better understanding of tight junction regulation and factors affecting transport systems will allow the development of therapeutics to improve the BBB function in health and disease.
Key wordsblood–brain barrier leukocyte migration tight junctions multidrug resistance proteins neurovascular unit
The blood–brain barrier (BBB) is composed of a microvascular endothelium, astrocytes, basement membrane, and pericytes and neurons that are in physical proximity to the endothelium. All these elements are part of the functional neurovascular unit. Under physiologic conditions, the BBB ensures constant supply of nutrients (oxygen, glucose, and other substances) for brain cells and guides the inflammatory cells to respond to the changes of local environment. Glial cells and neurons can regulate the function of blood vessels in response to metabolic requirements. The impairment of the neurovascular unit is present in a variety of neurodegenerative (Alzheimer's disease, Parkinson's disease) or inflammation-related diseases in the brain (infections, stroke, vascular dementia, and multiple sclerosis). Emerging concepts of the neurovascular unit allows better understanding of neuropathogenesis of pathologic conditions. This review addresses structural and functional aspects of the BBB under physiologic conditions and its alterations during neuroinflammatory and neurodegenerative diseases.
Structural components of BBB: neurovascular unit
Brain microvascular endothelial cells (BMVEC), situated at the interface between the blood and the brain, perform essential biological functions, including barrier, transport of micronutrients and macronutrients, receptor-mediated signaling, leukocyte trafficking, and osmoregulation. The BMVEC structural components responsible for these unique properties include the following: (1) tight junctions (TJ) composed of TJ proteins [occludin, claudins, zonula occludens (ZO)-1, ZO-2, ZO-3, cingulin, AF6, 7H6]; (2) adherent junctions (AJ) composed of cadherins, catenins, vinculin, and actinin; and (3) junctional adhesion molecules (JAM; Doolittle et al. 2005). The endothelial cell cytoplasm has uniform thickness with very few pinocytotic vesicles (the hollowed-out portion of the cell membrane filled with fluid, forming a vacuole that allows for nutrient transport) and lacks fenestrations (Abbott 2005). BMVEC have greater number and volume of mitochondria as compared with endothelium of other organs. This increased content of mitochondria enhances the energy potential and is thought to be required for active transport of nutrients to the brain. It is estimated that cerebral capillaries have five to six times more mitochondria per capillary section than rat skeletal muscle capillaries (Oldendorf et al. 1977).
There is an enzymatic barrier at the cerebral endothelia, capable of metabolizing drug and nutrients. These enzymes include γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase (AP), and aromatic acid decarboxylase. They metabolize neuroactive bloodborne solutes; when compared with nonneuronal capillaries, their concentration is high in cerebral microvessels (Pardridge 2005). A polarity exists between the luminal and abluminal membrane surfaces of the BMVEC contributing to the barrier function. The concept of the functional polarity of the BBB emerged from quantitative biochemical studies. The enzymes γ-GTP and AP are present at the luminal endothelium, whereas Na+-K+ ATPase and the sodium-dependent neutral amino acid transporter are associated with the abluminal surface of the endothelium. Immunogold labeling and electron microscopy show that the glucose transporter, GLUT-1, have a 3:1 ratio of distribution from the abluminal to the luminal surface (Abbott 2005). Drug efflux transporters, such as P-glycoprotein (P-gp), are mainly present on the luminal membrane surface (Loscher and Potschka 2005b). Structural, pharmacological, and biochemical evidence of luminal and abluminal polarization of receptors, enzymes, and ion channels on the cerebral endothelium shows that the BBB is a working nonstagnant membrane that maintains the brain homeostasis.
Astrocytes are glial cells that envelop >99% of the BBB endothelium (Hawkins and Davis 2005). Astrocytes and endothelial cells influence each other's structure; their interactions induce and modulate the development of the BBB and unique BMVEC phenotype. Interaction of astrocytes with BMVEC greatly enhanced endothelial cell TJ and reduced gap junctional area (Tao-Cheng and Brightman 1988), and this interaction increases the number of astrocytic membrane particle assemblies and astrocyte density (Tao-Cheng et al. 1987; Tao-Cheng and Brightman 1988; Abbott 2002). Astrocytes are essential for proper neuronal function and the close proximity of neuronal cell bodies to brain capillaries suggests that astrocyte–BMVEC interactions are essential for a functional neurovascular unit (Abbott et al. 2006).
In vitro studies suggest that coculture of brain endothelial cells with astrocytes is important in the maintenance of BBB tightness and function (Tao-Cheng et al. 1987; Tao-Cheng and Brightman 1988; Holash et al. 1993). Other studies showed that cerebral microvessels could maintain barrier integrity in areas that underwent extensive astrocyte loss (Willis et al. 2004b). A subsequent study also showed loss and restoration of barrier integrity in vivo following a temporary focal loss of astrocytes (Willis et al. 2004a). It is plausible that astrocytes may modulate the BBB phenotype without being directly involved in the physical BBB properties.
The association of pericytes to blood vessels has been suggested to regulate endothelial cell proliferation, survival, migration, differentiation, and vascular branching (Lai and Kuo 2005). Pericytes are flat, undifferentiated, contractile connective tissue cells that develop around capillary walls. Microvascular pericytes lack the α-actin isoform (Lai and Kuo 2005), indicating that these cells may not be involved in capillary contraction. Part of pericytes of the BBB might be of macrophage lineage, possessing capacity to phagocytize exogenous proteins and present antigen (Williams et al. 2001).
Pericytes have a close physical association with the endothelium, and gap junction communication between pericyte and endothelial cells was shown in vitro (Lai and Kuo 2005). Pericytes send out cellular projections, which penetrate the basal lamina and cover approximately 20 –30% of the microvascular circumference. Furthermore, pericytes migrate away from brain microvessels in response to hypoxia (Gonul et al. 2002) or brain trauma (Dore-Duffy et al. 2000), conditions associated with increased BBB permeability. A lack of pericytes results in endothelial hyperplasia and abnormal vascular morphogenesis in the brain. There is evidence that pericytes are able to mimic astrocyte ability to induce BBB “tightness.” These evidences support the hypothesis that pericyte play an important role in maintaining the structural integrity of the BBB.
High level of neuronal activity and the dynamic nature of their metabolic needs require tight regulation of the microcirculation and the tissue it supplies. A close relationship between regional brain activity and blood flow was demonstrated by functional neuroimaging (Paemeleire 2002), but the exact mechanism remains unknown. BBB disruption may occur in pathological conditions decreasing cerebral blood flow or perfusion pressure (i.e., ischemia, hemorrhage, or traumatic injury) (Petty and Wettstein 2001). Lee et al. (1999) demonstrated that increased BBB permeability may be a specific compensatory event, suggesting that neuronal–microvascular communications can modulate BBB permeability. The BMVEC and/or associated astrocytic processes are innervated by noradrenergic (Cohen et al. 1997a), serotonergic (Cohen et al. 1997b), cholinergic (Tong and Hamel 1999), and GABA-ergic neurons (Vaucher et al. 2000). Chemically induced injury of the locus coeruleus, which sends the noradrenergic projections to the microvasculature, enhances vulnerability of the BBB to acute hypertension (Ben-Menachem et al. 1982). Significant loss of cholinergic innervation of cortical microvessels seen in Alzheimer's disease is associated with impaired cerebrovascular function (Tong and Hamel 1999). There are evidences that neurons induce expression of enzymes unique for BMVEC (Tontsch and Bauer 1991). In summary, significant evidence exists that neurons can regulate BBB function.
The extracellular matrix
Extracellular matrix of the basement membrane also interacts with the cerebral microvascular endothelium. Disruption of the extracellular matrix is strongly associated with increased BBB permeability in pathological states (Rascher et al. 2002; Jian Liu and Rosenberg 2005). The matrix provides an anchor for BMVEC via interaction of laminin, collagen type IV, and other matrix proteins with integrin receptors on BMVEC (del Zoppo and Hallenbeck 2000). Cell–matrix interactions mediate several intracellular signaling pathways (Tilling et al. 2002) and matrix proteins promote the expression of endothelial TJ proteins (Savettieri et al. 2000). Thus, the basement membrane is likely involved in TJ maintenance.
Structural integrity of BBB: junctional complexes
The integrity of the TJ assembly determines the paracellular permeability of water-soluble molecules across the BBB. A TJ is composed of the integral transmembranous proteins, occludin, the claudins, and the JAMs. These TJ proteins are connected to the actin cytoskeleton by TJ accessory/anchoring proteins, ZO-1, ZO-2, and ZO-3.
Occludin is a 65-kDa protein highly expressed in BMVEC, and it is detected consistently along the cell margins (Wolburg and Lippoldt 2002; Hawkins and Davis 2005). Occludin content is much lower in endothelial cells of non–central nervous system (CNS) origin (Hirase et al. 1997; Vorbrodt and Dobrogowska 2003), suggesting that occludin is actively involved in BBB function. It has been shown that high levels of occludin ensure high electrical resistance (tightness) of the epithelial cell monolayers (McCarthy et al. 1996), and this increase in electrical resistance was regulated by the second extracellular loop domain (Wong and Gumbiner 1997). Occludin is composed of four transmembranous domains with the carboxyl and amino terminals oriented to the cytoplasm and two extracellular loops (44 and 45 amino acids) spanning the intercellular cleft (Furuse et al. 1993). Multiple phosphorylation sites were identified on occludin serine and threonine residues, and the phosphorylation state of occludin regulates its association with the cell membrane and barrier permeability (Sakakibara et al. 1997; Wachtel et al. 1999; Hirase et al. 2001). The cytoplasmic C-terminal domain provides the connection of occludin with the cytoskeleton via accessory proteins, ZO-1 and ZO-2 (Fanning et al. 1998). Although several knockout and knockdown experiments provided evidence that occludin is not essential for the formation of TJ, diminished occludin expression was associated with BBB dysfunction in a number of disease states (Bolton et al. 1998; Dallasta et al. 1999; Pesidsky et al. 2006).
Claudins (20–24-kDa proteins) share very similar membrane locations with occludin without having any sequence homology (Morita et al. 1999). Up to 24 claudins sharing the high sequence homology in the first and fourth transmembranous domains and extracellular loops were identified in mammals (Furuse et al. 2002). The homophilic and heterophilic interactions between the extracellular loops of claudins ensure tight contacts of the cell monolayers (Morita et al. 2003). Although overexpression of claudins can induce cell aggregation and formation of TJ-like structures, occludin expression does not result in the TJ formation (Kubota et al. 1999). Claudins feature contiguous staining along endothelial cell borders in and outside CNS. Thus, it appears that claudins form the primary “makeup” of the TJ, and occludin further enhances TJ tightness. BMVEC express predominantly claudin-3 and claudin-5 (Nitta et al. 2003; Hawkins and Davis 2005). Significant disruption of claudin-5 was detected in HIV-1 encephalitis (HIVE), and monocyte coculture with BMVEC led to claudin-5 phosphorylation via activation of Rho and Rho kinase (Pesidsky et al. 2006).
Junctional adhesion molecules
JAM-1, a 40-kDa protein, belongs to the IgG superfamily. It mediates the early attachment of adjacent cell membranes via homophilic interactions of a single membrane-spanning chain with a large extracellular domain (Martin-Padura et al. 1998; Del Maschio et al. 1999). JAM-2 and JAM-3 are also present in endothelial cells of different organs (including lymphatics), but not epithelial cell linings (Aurrand-Lions et al. 2002). JAMs play a role in developmental processes, and they were shown to regulate the transendothelial migration of leukocytes in animal models (Del Maschio et al. 1999). The functions of JAMs are largely unknown in mature BBB.
Membrane-associated guanylate kinase – like proteins
Membrane-associated guanylate kinase (MAGUK) proteins are accessory elements for the transmembranous components of the TJ. MAGUK proteins possess multiple binding domains for protein–protein interactions (Gonzalez-Mariscal et al. 2000), enabling MAGUK proteins to form a clustering of protein complexes to the cell membrane (Gonzalez-Mariscal et al. 2000). Three MAGUK proteins associated with the TJ are ZO-1, ZO-2, and ZO-3.
ZO-1, a 220-kDa phosphoprotein, is mostly expressed in endothelial and epithelial cells that normally form the TJ assembly (Mitic and Anderson 1998); however, ZO-1 protein is also expressed in other cell types that do not form the TJ (Howarth et al. 1992). ZO-1 connects transmembranous TJ proteins with the actin cytoskeleton (Fanning et al. 1998). Loss or dissociation of ZO-1 from the junctional complexes is associated with increased barrier permeability (Mark and Davis 2002). It was suggested that ZO-1 could communicate the state of the TJ to the interior of the cell or vice versa.
ZO-2, a 160-kDa phosphoprotein, has significant homology to ZO-1 (Itoh et al. 1999), and it was also found in non-TJ-containing tissues (Betanzos et al. 2004). Similar to ZO-1, ZO-2 binds to transmembranous proteins of the TJ and transcription factors, and it is localized in the nucleus during stress and proliferation (Islas et al. 2002; Traweger et al. 2003). ZO-2 function is less studied compared with ZO-1 (Hawkins and Davis 2005). Interestingly, ZO-2 may function somewhat redundantly with ZO-1, replacing it and facilitating formation of morphologically normal TJ in cultured epithelia lacking ZO-1 (Umeda et al. 2004). Immunofluorescence microscopy studies showed that ZO-3 was concentrated at the TJ of epithelial cells, but not in endothelial cardiac muscle cells (Inoko et al. 2003).
Intracellular signaling regulating TJ function
During physiologic and pathologic changes, TJ structure and function responds quickly to intracellular signaling events modulating the TJ complexes. Although little is known about the pathways leading to the TJ regulation, TJ appears to be highly dynamic structures. TJ proteins undergo rapid changes in expression, subcellular redistribution, and posttranslational modifications, which in turn affect protein–protein interactions (Huber et al. 2001; Feldman et al. 2005). In BMVEC, one of the most studied mediators relaying molecular cues to downstream effectors is Ca2+ signaling.
Earlier works focusing on BBB permeability demonstrated the importance of this second messenger on the regulation of the BBB opening. Extracellular calcium was first shown to be a critical component of TJ regulation in models of Ca2+ addition/depletion (Palant et al. 1983; Gonzalez-Mariscal et al. 1985). The exposure of epithelial cells to low Ca2+ levels, by using calcium-free medium containing calcium-chelating agent, compromised the TJ assembly leading to increased permeability and decreased transepithelial resistance (Balda et al. 1991; Said and Ma 1994). The process is reversible because reintroduction of calcium after its removal restored normal barrier function (Shasby and Shasby 1986). It has been proposed that in this context, removal of extracellular calcium prevents its binding to calcium-binding sites on cadherin extracellular domains (Alexander et al. 1998), and these conformational changes at the TJ complexes resulted in barrier dysfunction. Another possibility is that calcium-free conditions initiate signaling cascades that lead to disassembly and redistribution of ZO-1 and occludin away from apical–lateral borders (Ma et al. 2000). Moreover, this effect on distribution appeared to be mediated through activation of myosin light chain kinase (MLCK), which was blocked by the MLCK inhibitor (Ma et al. 2000). Under similar calcium-absent conditions, mammary epithelial cells (EpH4) showed membrane loss of ZO-1, ZO-2, and occludin (Klingler et al. 2000). However, in the presence of protein kinase A inhibitors, normal barrier properties were preserved (Klingler et al. 2000). The protein kinase C (PKC) was also implicated in the modulation of TJ assembly (Balda et al. 1993; Etienne-Manneville et al. 2000). In fact, the deleterious effect of low calcium in the extracellular environment can be overcome by application of the PKC activator, 1,2-diactanoylglycerol (Balda et al. 1993). Intracellular Ca2+ is also central to the regulation of TJ integrity (Abbott 2000). Elevation of intracellular Ca2+ (via calcium ionophores, or release from intracellular stores) cannot only activate signaling cascades that directly alter posttranslational TJ distribution, but also transcriptionally regulating TJ expression (Brown and Davis 2002). It was demonstrated that alcohol metabolism in BMVEC stimulate intracellular Ca2+ release resulting in activation of MLCK, causing phosphorylation of TJ proteins (occludin, claudin-5) and diminished BBB integrity (Haorah et al. 2005a).
Although it is known that some disease states interrupt calcium homeostasis, the emerging picture from calcium studies in models of the BBB suggest that both abnormally high and very low (Ye et al. 1999; Beyenbach 2003) intracellular Ca2+ results in disruption of TJ via decreased expression and/or disruption of protein–protein interactions; however, the triggers that cause increases/decreases in Ca2+ are still an open question.
Phosphorylation regulates function of transmembrane and accessory proteins of TJ (Sakakibara et al. 1997). Occludin phosphorylation occurs at serine and threonine residues, which control intracellular distribution of this TJ protein and subsequent barrier properties (Andreeva et al. 2001). For example, Wong (1997) reported that hyperphosphorylated occludin was found primarily at intercellular junctions and that this form appeared necessary for maintaining normal barrier function. Moreover, decreased phosphorylation status of occludin, by inhibition of PKC, was shown to correlate with a rapid fall in transendothelial resistance (Clarke et al. 2000). Phosphorylation of the claudins was implicated in the regulation of paracellular permeability. Increases in claudin-4 phosphorylation at threonine residues in epithelial cell line was demonstrated to alter TJ properties (Le Moellic et al. 2005). Claudin-5 could be phosphorylated at threonine residues, and its phosphorylation at threonine 207 seems to mediate permeability of small molecules (5 kDa) but not those of larger size (182 kDa), indicating further regulatory complexity resulting from phosphorylation of TJ proteins (Soma et al. 2004).
Tight junction phosphorylation and dephosphorylation status depends on the type of stimulus (e.g., inflammatory cytokines, oxidative stress, or growth factors) and the amino acid residues where phosphorylation occurs (serine, threonine, or tyrosine). Vascular endothelial growth factor (VEGF) caused an increase of BBB permeability accompanied by TJ phosphorylation (Antonetti et al. 1999) and dexamethasone decreased both occludin phosphorylation and BBB permeability (Antonetti et al. 2002). On the other hand, during calcium depletion, phorbol ester treatment, and bacterial infection, occludin undergoes dephosphorylation paralleling TJ disruption (Denker and Nigam 1998; Tsukamoto and Nigam 1999; Pedram et al. 2002). Stamatovic et al. (2006) demonstrated that β chemokine, monocyte chemoattractive protein 1 (MCP-1), acts in a manner similar to VEGF, inducing phosphorylation of serine and threonine residues of occludin, ZO-1, ZO-2, and claudin-5. Interactions between monocytes and brain endothelium also result in phosphorylation of TJ proteins on the same residues (Pesidsky et al. 2006). To date, no clear-cut pattern has been established between the site of phosphorylation and TJ function because multiple sites for phosphorylation were found in claudin-5, ZO-1, and occludin (Sakakibara et al. 1997). Alcohol metabolism in BMVEC, or the oxidative stress associated with it, led to phosphorylation of occludin and claudin-5 (Haorah et al. 2005a) and redistribution of TJ protein staining in brain endothelium in vitro (Haorah et al. 2005b). Hawkins and Davis (2005) suggested that most probably phosphorylation of distinct serine, tyrosine, and threonine residues has distinct structural and functional effects.
G-proteins play a role in the establishment and modulation of TJ (Hopkins et al. 2000). Importantly, they coordinate BMVEC responses during migration of T-lymphocytes and neutrophils into the brain (Adamson et al. 1999). The Rho family of GTPases alters BBB permeability by interactions with the cytoskeletal proteins, acting downstream of G-protein-coupled receptors for lysophosphatidic acid (Schulze et al. 1997), prostaglandins (Schmeck et al. 2003), MCP-1 (Stamatovic et al. 2006), or HIV-1 protein gp120 via β-chemokine receptor, CCR5, or α-chemokine receptor, CXCR4 (Kanmogne et al. 2005).
In addition to GTPases (like Rho), Stamatovic et al. (2006) found that two specific PKC isoforms, PKCα and PKCζ, were activated by MCP-1 leading to increased BBB permeability. PKC plays an important role in endothelial TJ complex assembly/disassembly. Two classic forms of PKC, PKCα and PKCβ (activated by hydrogen peroxide, thrombin, and glucose), as well as atypical forms of PKC (PKCζ and PKCλ) are thought to be mostly involved in TJ disassembly (Chen et al. 2002; Nunbhakdi-Craig et al. 2002). The inhibition of PKCα and activation of PKCζ prevented MCP-1-induced increase in BBB permeability (Stamatovic et al. 2006). HIV-1 gp120 was shown to activate three PKC isoforms in BMVEC [PKC-α/βII, PKC(pan)-βII, and PKC-ζ/λ] via chemokine receptors (Kanmogne et al. 2006). PKC inhibitors (acting at the ATP-binding and calcium release site) blocked gp120-induced PKC activation and prevented increase in BBB permeability, supporting the biological significance of these results.
Rab proteins (regulators of vesicle trafficking) direct TJ proteins such as ZO-1 to the junction complex (Stevenson 1999). AF-6, one of the TJ accessory proteins interacting with ZO-1, is a target for Ras (Yamamoto et al. 1999). Ras activation paralleled disruption of cell–cell contacts, ZO-1 loss from the junction complexes, and decrease of BBB tightness.
Changes of tight junctions in disease states
Hypoxia and ischemia
Cerebral ischemia leads to disruption of blood flow, increased BBB permeability, and is associated with rapid depletion of essential nutrients and oxygen (del Zoppo and Hallenbeck 2000; Petty and Wettstein 2001). Using in vitro models of BBB, Mark and Davis (2002) demonstrated that hypoxia and reoxygenation increased BBB permeability and were associated with TJ impairment. Modeling of ischemia in vitro indicated that a number of transcriptional factors (nuclear factor-κB and hypoxia-inducible factor-1) were activated under these conditions (Witt et al. 2005). VEGF and nitric oxide (NO) mediate in part TJ alterations caused by hypoxia (Mark and Davis 2002). VEGF blocking diminished postischemic edema and tissue damage in vivo (van Bruggen et al. 1999), indicating that TJ disruption is likely involved in the progression of ischemic brain injury.
In Alzheimer's disease, activated microglial and astrocytes stimulated by β-amyloid produce proinflammatory factors [including interleukin-1β, tumor necrosis factor, (TNF-α), complement activation, transforming growth factor E, or ROS; McGeer et al. 2005]. Alternatively, β-amyloid deposition around microvessels can cause direct toxicity to BMVEC (Zlokovic 2002). It was suggested that impaired clearance of β-amyloid across the BBB, aberrant angiogenesis, and senescence of the cerebrovascular system could initiate neurovascular uncoupling, brain hypoperfusion, and neurovascular inflammation (Zlokovic 2005). The signal transduction pathway that mediates BBB changes in Alzheimer' disease await further investigation.
Multiple sclerosis is an autoimmune condition characterized by myelin sheath destruction by reactive T lymphocytes (Lassmann 2004). Reactive T cells interact with brain macrophages and microglial serving as antigen-presenting cells (Grigoriadis et al. 2006). Activated macrophages synthesize and produce cytokines (interferon-γ, TNF-α), MMPs, and NO (Raivich and Banati 2004). BBB injury precedes massive infiltration of T lymphocytes that attack myelin sheath–forming demyelinating foci (plaques; Minagar and Alexander 2003). TJ may play a role in other diseases. Polymorphism in claudin-5 was recently linked to the development of schizophrenia and BBB dysfunction (Sun et al. 2004).
The BBB is compromised in brain tumors, leading to increased vascular permeability (Groothuis et al. 1991). The expression of the TJ proteins either decreased (claudin-5) or was completely lost (claudin-1, occludin) in primary brain or metastatic tumors (Liebner et al. 2000; Papadopoulos et al. 2001). VEGF, cytokines, and growth factors secreted by tumor cells were implicated as a mechanism of TJ down-regulation and edema (Lamszus et al. 1999).
BBB transport systems
In addition to structural elements assuring tightness of the BBB, transport systems and drug-metabolizing enzymes (including cytochrome P450 hemoproteins and uridine diphosphate glucuronosyltransferases) provide an enzymatic barrier (Lee et al. 2001; Leslie et al. 2005). Most of the BBB transporters are from the superfamily of ATP-binding cassette (ABC) proteins that mediate cellular extrusion of many therapeutic drugs with diverse structures and clinical applications. BMVEC possesses the so-called multidrug resistance (MDR) proteins, a phenotype first described for chemotherapy-resistant cancer cells that overexpressed one of these efflux transporters, P-gp (Schinkel and Jonker 2003).
Modern understanding of the transporter role and their contribution to the BBB was acquired in studies of P-gp (Fricker and Miller 2004). P-gp is present in several cell types, including BMVEC, astrocytes, and microglia (Lee and Bendayan 2004). P-gp expression in the lumenal plasma membrane of BMVEC prevents the passage of drugs and toxins across BBB into the brain and may facilitate their transport from brain to blood (Fricker and Miller 2004). The functional importance of P-gp in BBB was characterized through studies using Mdr1a(−/−) /Mdr1b(−/−) knockout mice. The Mdr1a isoform is primarily found in BMVEC, whereas Mdr1b is mainly detected in cells of brain parenchyma. Mdr1a-deficient animals showed more than 10 times increase of brain concentrations of P-gp substrates, ivermectin and vinblastine, and these mice are 3–100-fold and are much more sensitive to these compounds as compared with normal animals (Schinkel et al. 1994). A similar pattern was shown for other drugs that are P-gp substrates, including the peripheral analgesic asimadoline, HIV-1 protease inhibitors, and the antidiarrheal opiate loperamide (Graff and Pollack 2004). P-gp effects hamper significant penetration of drugs to the brain and are thought to contribute to resistance of modern therapies against HIV-1 CNS infection or epilepsy among others.
Similar to P-gp, the breast cancer resistance protein (BCRP, also known as ABCG2) is expressed predominantly at the luminal surface of brain capillaries (Zhang et al. 2003; Cisternino et al. 2004). A compensatory up-regulation of Bcrp1/Abcg2 in the absence of Mdr1a was found in Mdr1a(−/−)versus wild-type mice (Cisternino et al. 2004). Thus, there is convincing evidence that the Bcrp1/Abcg2 present in the mouse BBB is functional.
A number of neurological disorders are associated with altered transport expression that could play a role in their pathogenesis. P-gp expression was shown to correlate inversely with deposition of β-amyloid in Alzheimer's disease (Cirrito et al. 2005). Overexpression of transporters on BBB was described in epilepsy, and it is still unclear whether this is a result of chronic exposure to antiseizure drugs, P-gp substrates, or part of epilepsy pathogenetic mechanism (Marroni et al. 2003; Volk et al. 2005). P-gp was up-regulated on brain capillary endothelium after focal cerebral ischemia (Spudich et al. 2006). Furthermore, P-gp inhibition by pharmacological inhibition or genetic knockout preferably enhances the accumulation and efficacy of two neuroprotectants known as P-gp substrates in the ischemic brain. It was suggested that P-gp could play a role in mood disorders and schizophrenia (Loscher and Potschka 2005a). Decrease of functional activity of P-gp was demonstrated in human brain tissue in Parkinson's disease (Kortekaas et al. 2005).
Other important transport systems (like GLUT1 providing glucose supply for the brain) are also affected by different disease states like brain trauma, epilepsy, ischemia, and Alzheimer's disease (Guo et al. 2005). Mankowski et al. (1999) demonstrated an inverse relationship between severity of encephalitis induced by simian immunodeficiency virus and expression of the GLUT1 at the BBB in cortical gray matter, caudate nucleus, and cerebellum. The BBB possesses an ability to transport certain peptides (including cytokines and leptin among others) into the brain (Banks and Lebel 2002; Banks 2005).
Leukocyte migration across BBB
Well-controlled leukocyte migration is a key physiologic event in immune surveillance, acute self-limiting inflammation, and antigen recognition. Uninhibited transendothelial migration and tissue accumulation of leukocyte is a sign of chronic pathologic inflammatory processes (i.e., multiple sclerosis, encephalitis). BBB impairment during CNS inflammation is believed to result from disruption of junction complexes between BMVEC with subsequent formation of a paracellular route that facilitates entry of leukocytes into the brain parenchyma. Although other pathways for leukocyte migration were proposed (e.g., transcytosis), both experimental and clinical observations point to the importance of the paracellular route in leukocyte entry and across BBB during CNS inflammation (Schenkel et al. 2004). Understanding the complex multistep process of leukocyte migration is crucial for the development of therapeutics aimed at amelioration of chronic destructive CNS inflammatory responses. Migration is mediated by leukocyte–endothelial cell [through integrins and intercellular adhesion molecules (ICAM)] and endothelial–endothelial cell interactions (via various small GTPases), where endothelial cells are active participants of this process (Cullere et al. 2005; Yang et al. 2005).
As discussed above, BMVEC are connected by TJ and AJ, and modifications of TJ/AJ are required during paracellular leukocyte migration. Rho GTPases are potent modulators of actin cytoskeleton and BMVEC adhesion (Burridge and Wennerberg 2004). It was shown that neutrophil adhesion caused intracellular Ca2+ from endothelial cells, and blockage of calcium release prevented leukocyte migration (Wittchen et al. 2005b). Subsequently, it was discovered that intracellular Ca2+ release activates Rac1 (Price et al. 2003). Inhibition of Rac1 diminished leukocyte migration across endothelium (van Wetering et al. 2002) most likely because of Rac1 contribution to TJ disassembly (van Wetering et al. 2002). Increased Ca2+ flux leads to increase in isometric tension in endothelial cells. It is suggested that activation by cross-linking signals and/or soluble activators from the leukocyte stimulates a transient increase in BMVEC intracellular free Ca2+ (Muller 2003). This activates the calmodulin-dependent enzyme MLCK by phosphorylation, thereby causing a conformational change in myosin II, facilitating contraction of actin–myosin bundles. This would put tension on actin filaments near the cell border enabling retraction of the endothelial cells at their borders and facilitating leukocyte passage.
Inactivation of endothelial Rho by Clostridium limosum exoenzyme C led to a diminished rate of monocyte transmigration across a monolayer of dermal microvascular endothelial cells devoid of TJ (Strey et al. 2002). The authors assumed that Rho may stabilize the cytoskeleton because the absence of stress fiber formation was demonstrated by F-actin staining. These data suggest that endothelial Rho and RhoK regulate transendothelial leukocyte passage by modulating the cytoskeletal events. Stamatovic et al. (2003, 2005) demonstrated that Rho and RhoK are involved in redistribution of TJ proteins and increased permeability of monolayer of mouse brain endothelial cells treated with MCP-1.
The importance of Rho in leukocyte migration through endothelium was shown via cross-linking of ICAM-1 (recapitulating leukocyte–endothelial cell interactions), which resulted in Rho-dependent cytoskeleton reorganization in BMVEC, and barrier disruption (Etienne et al. 1998). Blockage of Rho prevented clustering of adhesion molecules [ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin] and decreased monocyte adhesion to endothelial cells (Wojciak-Stothard et al. 1999). Application of the Rho inhibitor to BMVEC significantly decreased T-lymphocyte migration and endothelial actin reorganization, without altering lymphocyte BMVEC adhesion (Adamson et al. 1999). These studies demonstrate that BMVEC actively participate in leukocyte migration through the BBB and that this process requires functional Rho (Wittchen et al. 2005a). However, the precise role of Rho in endothelial TJ regulation during leukocyte migration is currently unknown.
Several groups independently showed that leukocyte adhesion to endothelial cells is required for degradation of junction components via signaling mechanisms that enhance leukocyte migration and monolayer permeability (Del Maschio et al. 1996; Allport et al. 2000; Xu et al. 2005). Shaw et al. (2001) demonstrated that transmigrating monocytes cause a focal and reversible disruption/dispersion of the AJ (cadherin complex) in human umbilical endothelial cells. The authors speculated that transmigration is a sequential multistep process that involves active participation of both leukocytes and the endothelium, similar to the multistep adhesion cascade model. Wittchen et al. (2005a) presented evidence of association between enhanced TJ expression, increased tightness of endothelial cell monolayer, and decreased leukocyte migration across the BBB. Exact intracellular signals and their integration with functional changes of both cell types will require further studies.
Rho and RhoK in BMVEC play a role in TJ disassembly and monocyte migration across the BBB during HIVE (Pesidsky et al. 2006). Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. Loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVEC could be an underlying cause of BBB impairment during HIVE.
A number of studies support the idea that leukocyte-induced changes in endothelial cell TJ and cytoskeletal organization facilitates leukocyte migration. Adhesion molecules, such as E-selectin, VCAM-1, and ICAM-1, were proposed as the key molecules that affect the signal transduction pathway in endothelial cells and induce the functional changes of endothelial cells in the interaction with leukocytes (Wojciak-Stothard et al. 1999). These adhesion molecules were up-regulated on BMVEC in HIVE (Nottet et al. 1996) and on the BBB model during migration of virus-infected and activated macrophages (Persidsky et al. 1997).
Leukocyte adhesion transmits signal to endothelial cells via VCAM-1, causing its clustering, subsequent activation of Rac1, and formation of so-called “docking structure” (Barreiro et al. 2002). Docking structures involve formation of microvilli-like projections that embrace adherent leukocyte (Carman et al. 2003) and express clustered VCAM-1, ICAM-1, and proteins erzin and moesin (Barreiro et al. 2002). Downstream effects of Rac1 activation appear to be mediated by ROS (Khanday et al. 2006). When VCAM-1 was cross-linked to mimic endothelial–leukocyte interactions, Rac1 was activated and this was accompanied by ROS production and barrier disruption (van Buul and Hordijk 2004). It is assumed that Rac1 mediated ROS generation under a number of pathologic conditions associated with decrease of BBB tightness and enhanced leukocyte migration (Wittchen et al. 2005a).
Increased expression of adhesion molecules on endothelial cells and their respective ligands on monocyte–macrophages occurs shortly after exposure to oxidative stress or lipid oxidation products can be modulated by antioxidants (Blouin et al. 1999; Kunsch et al. 2004; Pratico 2005). Leukocyte–endothelial cell interactions via adhesion molecules lead to ROS generation and disruption of junction complexes (Cook-Mills 2002). ROS scavenging inhibits these effects on junctions and transendothelial leukocyte migration (Matheny et al. 2000). Leukocyte adhesion to endothelium induces ROS production, which in turn might transmit signals into the endothelium to facilitate leukocyte passage (van Buul and Hordijk 2004). Haorah et al. (2005a) provided evidence that by yet unknown mechanism, oxidative stress activated MLCK in BMVEC that phosphorylated cytoskeletal and TJ proteins lead to diminished integrity of BMVEC monolayer and enhanced monocyte migration across BBB in vitro.
The neurovascular unit performs numerous important functions ranging from structural integrity to transport of diverse substances in and out of the brain and leukocyte migration. Impairment of BBB functions is clearly linked to many pathologic conditions in the human CNS. The BBB is a target for a variety of neurotherapeutic interventions aiming to decrease pathology or enhance the delivery of drugs to the brain.
We thank Ms. Robin Taylor and Ms. Debra Baer for excellent administrative support. The NIH National NeuroAIDS Consortium and the CNND brain bank are acknowledged for brain tissue specimens used in this study. This work was supported in part by research grants by the National Institutes of Health: PO1 NS043985, RO1 AA015913, and RO1 MH65151 (Y.P.).
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