BBB Anatomia

B RA I N R E SE A R CH RE V I EW S 64 ( 20 1 0 ) 3 2 83 6 3
available at www.sciencedirect.com
www.elsevier.com/locate/brainresrev
Review
Looking at the bloodbrain barrier: Molecular anatomy and

possible investigation approaches
Filipa Loureno Cardoso, Dora Brites, Maria Alexandra Brito
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
A R T I C LE I N FO
AB S T R A C T
Article history:
The bloodbrain barrier (BBB) is a dynamic and complex interface between blood and the
Accepted 19 May 2010
central nervous system that strictly controls the exchanges between the blood and brain
Available online 26 May 2010
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
Keywords:
microvascular endothelial cells and intercellular junctions are examined. The specific
Bloodbrain barrier
interactions between endothelial cells and basement membrane as well as neighboring
Brain microvascular endothelial cell
perivascular pericytes, glial cells and neurons, which altogether constitute the
Intercellular junction
neurovascular unit and play an essential role in both health and function of the central
Neurovascular unit
nervous system, are also explored. Some relevant pathways across the endothelium, as well
Signaling
as mechanisms involved in the regulation of BBB permeability, and the emerging role of the
Transport system
BBB as a signaling interface are addressed as well. Furthermore, we summarize some of the
Stem-cell niche
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.
2010 Elsevier B.V. All rights reserved.
Corresponding author. Faculdade de Farmcia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. Fax: +351
217946491.
E-mail address: abrito@ff.ul.pt (M.A. Brito).
Abbreviations: ABC, ATP-binding cassette transporter; AIDS, acquired immune deficiency syndrome; AJ, adherens junction; BBB, blood
brain barrier; BMVEC, brain microvascular endothelial cell; CAM, cell adhesion molecules; cAMP, cyclic AMP; cGMP, cyclic GMP; CNS, central
nervous system; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; ERK 1/2, extracellular signal-related kinases; GLUT-1, glucose
transporter-1; HBMVEC, human brain microvascular endothelial cell; HIV, human immunodeficiency virus; ICAM, intercellular adhesion
molecule; IL, interleukin; JAM, junctional adhesion molecule; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAGUK, membraneassociated guanlylate kinase; MAPK, mitogen-activated protein kinase; MLC, myosin light-chain; MLCK, myosin light-chain kinase;
MMP, matrix metalloproteinase; NO, nitric oxide; NVU, neurovascular unit; PI3K, phosphatidylinositol-3 kinase; P-gp, P-glycoprotein;
PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PKG, protein kinase G; PTK, protein tyrosine kinase; ROCK, Rho kinase;
ROS, reactive oxygen species; SEM, scanning electron microscopy; SFK, Src family of kinases; TEER, transendothelial electrical resistance;
TEM, transmission electron microscopy; TJ, tight junction; TNF-, tumor necrosis factor-alpha; VCAM, vascular CAM; VE, vascular
endothelial; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; ZO, zonula occludens
0165-0173/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainresrev.2010.05.003
329
B RA I N RE SE A R CH RE V I EW S 64 ( 20 1 0 ) 3 2 83 6 3
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
Neurovascular unit. . . . . . . . . . . . . . . . . . . . . . . .
2.1. Basement membrane . . . . . . . . . . . . . . . . . .
2.2. Neurons . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Microglia . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Pericytes . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Astrocytes. . . . . . . . . . . . . . . . . . . . . . . . .
2.6. Endothelial cells . . . . . . . . . . . . . . . . . . . . .
3.
Intercellular junctions . . . . . . . . . . . . . . . . . . . . .
3.1. Molecular constituents. . . . . . . . . . . . . . . . . .
3.1.1. Tight junctions . . . . . . . . . . . . . . . . .
3.1.2. Adherens junctions . . . . . . . . . . . . . . .
3.2. Signaling pathways involved in intercellular junctions
3.2.1. Protein kinases . . . . . . . . . . . . . . . . .
3.2.2. Mitogen-activated protein kinases . . . . . . .
3.2.3. G-proteins . . . . . . . . . . . . . . . . . . . .
3.2.4. Endothelial nitric oxide synthase . . . . . . .
3.2.5. Calcium . . . . . . . . . . . . . . . . . . . . .
3.2.6. Wnt/-catenin . . . . . . . . . . . . . . . . . .
4.
The blood-brain barrier as a signaling interface . . . . . . . . .
4.1. Signaling from periphery to brain. . . . . . . . . . . .
4.2. Signaling from brain to periphery. . . . . . . . . . . .
5.
Pathways across bloodbrain barrier . . . . . . . . . . . . . .
5.1. Caveolae . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Glucose transporter-1 . . . . . . . . . . . . . . . . . .
5.3. ATP-binding cassette transporters . . . . . . . . . . .
6.
Study approaches . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Experimental systems . . . . . . . . . . . . . . . . . .
6.1.1. In vivo . . . . . . . . . . . . . . . . . . . . . .
6.1.2. Ex vivo . . . . . . . . . . . . . . . . . . . . .
6.1.3. In vitro . . . . . . . . . . . . . . . . . . . . .
6.1.4. In silico . . . . . . . . . . . . . . . . . . . . .
6.2. Assessment of BBB structure and function . . . . . . .
6.2.1. Morphology . . . . . . . . . . . . . . . . . . .
6.2.2. Properties . . . . . . . . . . . . . . . . . . . .
7.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
Introduction
In 1885, Paul Ehrlich experimentally demonstrated that vital

dyes injected into the circulatory system stain all organs of the
mammalian body except the brain and spinal cord and
attributed this observation to a low affinity of nervous tissue
to the dye (Ehrlich, 1885, 1906). About 30 years later, an Ehrlich's
student, Edwin Goldmann, noticed the opposite phenomenon
by injecting trypan blue directly into the cerebro-spinal fluid,
which stained all central nervous system (CNS) and none of the
peripheral organs (Goldmann, 1913), suggesting the presence of
a barrier between the CNS and the circulation. The term
bluthirnschranke, bloodbrain barrier (BBB), was first used by
Lewandowsky (1900) while studying the limited permeation of
potassium ferrocyanate into the brain. Further studies showed
that endothelial tight junctional complexes physically limit
solute exchanges between the blood and the brain. This was
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achieved by injecting horseradish peroxidase intravascularly,

showing diffusion between endothelial cells (EC) lining skeletal
and cardiac vessels, thought it did not pass between EC in
cerebral microvasculature (Reese and Karnovsky, 1967) (Fig. 1).
Major BBB functions include: (1) maintenance of CNS homeostasis, (2) protection of brain from extracellular environment, (3)
constant supply of nutrients by specific transport systems and
(4) direct inflammatory cells to act in response to changes in
local environment (Petty and Lo, 2002; Lee et al., 2006; Persidsky
et al., 2006a). Maintenance of homeostasis is achieved through
the regulation of ion balance (Wolburg and Lippoldt, 2002;
Hawkins and Davis, 2005; Persidsky et al., 2006a) and of
compounds influx/efflux (Chaudhuri, 2000; Khan, 2005). This is
essential in protection against harmful substances, variations in
blood composition and breakdown of concentration gradients
(Kniesel and Wolburg, 2000; Petty and Lo, 2002; Wolburg and
Lippoldt, 2002).
330
Fig. 1 Injected horseradish peroxidase diffuses through

brain vasculature but does not pass through endothelial cells
to brain parenchyma. Designed based on original
observations of Reese and Karnovsky (1967).
The BBB is present in all brain regions, except in those
regulating autonomic nervous system and endocrine glands of
the body, where blood vessels permit diffusion of blood-borne
molecules across the vessel wall (Ballabh et al., 2004).
BBB's specific structural and biochemical properties arise
from existent layers between the blood and the brain and from
interactions between a large variety of cell types (Khan, 2005;
Hawkins et al., 2006), which will be explored in the next sections.
2.
Neurovascular unit
In the developing brain, capillaries are differentiated and

matured into the BBB (Lee et al., 2006). Only capillary vessels
have complete BBB properties, since leakiness increases as the
vessel diameter increases (Marchi et al., 2004; Hawkins and
Davis, 2005).
Although BBB permeability itself is controlled by the
biochemical properties of brain microvascular endothelial
cells (BMVEC) (Pardridge, 1999), brain microvascular biology
results overall from interactions of these cells with the
basement membrane and neighboring glial cells (Kaur and
Ling, 2008), such as microglia and astrocytes, as well as
neurons and perivascular pericytes (Wolburg and Lippoldt,
2002; Zlokovic, 2008; Zozulya et al., 2008). Altogether these
constitute the neurovascular unit (NVU) (Persidsky et al.,
2006a; Choi and Kim, 2008) (Fig. 2), essential for both health
and function of the CNS (Hawkins and Davis, 2005).
2.1.
Basement membrane
The basement membrane is an essential part of the BBB. It

surrounds BMVEC and engulfs pericytes, anchoring the cells in
place and establishing the connection with the surrounding
brain resident cells (Carvey et al., 2009). BMVEC, pericytes and
astrocytes all cooperate to generate and maintain the basement membrane that is constituted by 3 apposed laminas,
composed of different extracellular matrix classes of molecules (Persidsky et al., 2006a; Weiss et al., 2009). These are
structural proteins (collagen and elastin), specialized proteins
(fibronectin and laminin) and proteoglycans (Adibhatla and
Hatcher, 2008; Wolburg et al., 2009). The basement membrane
also includes matrix adhesion receptors, known as cell
Fig. 2 Schematic overview of the neurovascular unit

composed of endothelial cells, basement membrane,
astrocytes and microglia, neurons and pericytes. Adapted
from Abbott et al. (2006).
adhesion molecules (CAM), as well as signaling proteins,

which form an extensive and complex matrix (Carvey et al.,
2009). These molecules are expressed in the vascular cells,
neurons and supporting glial cells and are essential for maintenance of the BBB. Disruption of the basement membrane can
lead to alterations in the cytoskeleton of BMVEC, which in turn
affects tight junctions (TJ) proteins and barrier integrity. Matrix
metalloproteinases (MMP) are known to digest the basement
membrane, which leads to reduced anchoring of brain EC and
matrixEC signaling that affects TJ integrity and impairs the
barrier integrity (Carvey et al., 2009). Accordingly, disruption
of extracellular matrix has been strongly associated with increased BBB permeability in several pathological conditions
(Hawkins and Davis, 2005; Carvey et al., 2009; Zlokovic, 2008).
2.2.
Neurons
Little is known about the developmental role that neurons

have on the BBB phenotype. However, there is some evidence
that neurons can regulate the function of blood vessels in
response to metabolic requirements by inducing expression of
enzymes unique for BMVEC (Persidsky et al., 2006a). Also,
BMVEC and astrocytic processes are directly innervated by
noradrenergic, serotonergic, cholinergic, and GABA-ergic neurons, among others (Hawkins and Davis, 2005). Moreover,
mature endothelium has a reciprocal function in inducing a
stable brain microenvironment that enables proper neuronal
activity (Choi and Kim, 2008).
2.3.
Microglia
Microglia play a very important role in immune responses of

the CNS, surveying local microenvironment and changing the
phenotype in response to homeostatic disturbance of the CNS.
In fact, these cells present themselves in two forms: resting
and activated microglia. When resting, cells have small bodies
and long, thin processes; in contrast, activated microglia
assume a phagocytic morphology by shifting from long to
short processes (Zlokovic, 2008). In accordance with the
macrophage population in other organs, these cells have
Fig. 3 Pericytes have a close physical association with the

endothelium covering up to 32% of the capillaries.
diverse effector functions (Ransohoff and Perry, 2009). As

microglia are found in perivascular space, it is thought that
their interactions with BMVEC may contribute to the BBB
properties (Choi and Kim, 2008). Still, the exact mechanisms
on how this occurs remains unknown.
2.4.
Pericytes
Pericytes (also known as vascular smooth muscle cells, mural

cells, or myofibroblasts) are important cellular constituents of
the capillaries and post-capillary venules (Dore-Duffy, 2008),
having a close physical association with the endothelium.
They share the same basement membrane with the EC (Bagley
et al., 2005) (Fig. 2) and cover 22 to 32% of the capillaries (Kim et
al., 2006) (Fig. 3). The location of pericytes on the microvessel
and the degree of coverage varies considerably between
different microvessel types (Allt and Lawrenson, 2001),
seeming to correlate with the degree of tightness of the
interendothelial junctions (Lai and Kuo, 2005). The vascular
pericyte synthesizes most elements of the basement membrane including a number of proteoglycans. In addition,
pericyte synthesis and release of laminal proteins is thought
to be a critical step in the differentiation of the BBB (DoreDuffy et al., 2006).
Proper association of pericytes with microvascular endothelia is essential to maintain structural support and junctional integrity (Lai and Kuo, 2005; Braun et al., 2007). Pericytes
and EC communicate with each other through several
apparatuses such as gap junctions, TJ, adhesion plaques
(Allt and Lawrenson, 2001) and soluble factors (Bagley et al.,
2005).
The association of pericytes to blood vessels has been
suggested to regulate EC proliferation, migration and differentiation (Persidsky et al., 2006a). There is also evidence that
pericytes can induce and up-regulate P-glycoprotein (P-gp)
functional activity in these cells (Dohgu et al., 2005).
Furthermore, interactions between pericytes and BMVEC
are important for the remodeling and maintenance of the
vascular system via the secretion of growth factors or
modulation of the extracellular matrix (Allt and Lawrenson,
2001). There is evidence that pericytes are involved in the
transport across the BBB as well (Allt and Lawrenson, 2001).
Pericytes make regulatory adjustments in response to stress
stimuli, as during severe and prolonged O2 deprivation
(Al Ahmad et al., 2009).
331
Pericytes show rich contents of -smooth muscle actin,

which is a characteristic of the vascular smooth muscle cell,
suggesting a contractile ability of pericytes both in vitro (Kelley
et al., 1987; Shepro and Morel, 1993) and in vivo (Peppiatt et al.,
2006). The physical contacts of pericytes' cellular processes
over interendothelial junctions, coupled with the -smooth
muscle actin contents, point to a functional role of pericytes in
controlling blood flow (Peppiatt et al., 2006), as well as in
regulating junctional permeability (Edelman et al., 2006).
Pericytes have been shown to migrate away from brain
microvessels in rapid response to hypoxia and traumatic
brain injury. Therefore, both of these conditions are associated
with increased BBB permeability (Hawkins and Davis, 2005).
The disruption of BBB caused by the detachment of pericytes
reinforces the idea of pericytes' role in junctional permeability
(Nishioku et al., 2009).
2.5.
Astrocytes
Astrocytes are glial cells whose endfeet form a lacework of fine

lamellae closely apposed to the outer surface of the BBB
endothelium and respective basement membrane (Abbott,
2002) (Fig. 4). They play a major role in promoting proteoglycan
synthesis with a resultant increase in BMVEC charge selectivity
and playing an important role in the induction of BBB functions
(Yamagata et al., 1997; Bernoud et al., 1998). Astrocytes are also
important for proper neuronal function and the close proximity of neuronal cell bodies to brain capillaries suggests that
interactions between all these elements are essential for a
functional NVU (Persidsky et al., 2006a).
In some areas of the CNS the microvessels lack astrocytic
ensheathment but still exhibit some BBB features, which are
likely due to soluble factors acting from the glia limitans or the
subarachnoid cerebro-spinal fluid (Abbott, 2002). Subsequent
studies also showed loss and restoration of barrier integrity in
vivo following a temporary focal loss of astrocytes (Willis et al.,
2004; Persidsky et al., 2006a). Attempts to recover BBB
properties in BMVEC cultures have included co-culturing
BMVEC with astrocytes and/or astrocyte-conditioned medium
(Colgan et al., 2008). Astrocytes may therefore modulate the
BBB phenotype without being directly involved in the physical
BBB properties. Studies using non-contact co-cultivation with
Fig. 4 Astrocytes's endfeet form a fine lamellae closely

apposed to the outer surface of the capillary endothelium.
332
astrocytes revealed that the secretion of factors into the

medium contribute to the barrier properties of human BMVEC
(HBMVEC) by up-regulating the tight junctional proteins
zonula occludens (ZO)-1 and occludin, and reducing the transendothelial permeability across the HBMVEC (Siddharthan et
al., 2007; Colgan et al., 2008). Interestingly, astrocytes are also
able to endow non-neural EC with BBB properties (Hayashi et
al., 1997), whereas enteric glia, which share morphological,
biochemical, and functional properties with astrocytes, accelerates the formation of the characteristics of the BBB in spinal
cord capillaries (Jiang et al., 2005).
2.6.
Endothelial cells
EC were demonstrated to play a key role in BBB properties as it

was observed that horseradish peroxidase could not pass the
endothelial layer from either direction, and that brain
capillaries from amphibians have high electric impedance,
indicative of restriction to the movement of ions, despite the
absence of surrounding astrocytes (Hawkins et al., 2006). It is
now accepted that the cerebral endothelium forms the
anatomic basis of the BBB in higher animals (Hawkins et al.,
2006) and that the capillaries make up the primary part of the
BBB (Khan, 2005).
BMVEC interact intimately with other brain cells of the
NVU, and hence can act as mediators between blood and
brain (Calabria and Shusta, 2008) (Fig. 5). They regulate the
selective transport and metabolism of substances from blood
to brain as well as in the opposite direction from the parenchyma back to the systemic circulation (Zheng et al., 2003).
The BMVEC barrier line is the most critical for preventing
toxic substances from entering the brain (Ueno, 2007). Communication between EC and other surrounding cells (Fig. 2)
enhances the barrier functions consequently resulting in
maintenance and elaboration of proper brain homeostasis
(Choi and Kim, 2008).
The BMVEC lining the cerebral capillaries differs fundamentally from other vascular endothelia in their capacity to
regulate the passage of molecules and cells to and from the
neural parenchyma (Ge et al., 2005; Weksler et al., 2005). The
Fig. 5 Brain microvascular endothelial cells establish the

contour of blood vessels and interact intimately with the
basement membrane and cells of the neurovascular unit,
such as neurons, astrocytes, microglia and pericytes, hence
operating as mediators between blood and brain. Adapted
from Francis et al. (2003).
capillary endothelium in the brain is 50100 times tighter than

peripheral microvessels as a result of special properties that
cause severe restriction of the paracellular pathway for
diffusion of hydrophilic solutes (Abbott, 2002). The EC
cytoplasm has uniform thickness with no fenestrae, low
pinocytotic activity and a continuous basement membrane
(Chaudhuri, 2000; de Boer and Gaillard, 2006). In addition, the
BMVEC have a negative surface charge that repulses negatively charged compounds (de Boer and Gaillard, 2006). They
have a greater number and volume of mitochondria compared
with endothelium in other organs. These characteristics
enhance the energy potential (Persidsky et al., 2006a),
providing energy for enzymes to break down compounds
and allowing various selective transport systems to actively
transport nutrients and other compounds into and out of the
brain (de Boer and Gaillard, 2006).
BMVEC lining the vascular wall have narrow junctional
complexes that eliminate gaps or spaces between cells and
prevent any free diffusion of blood-borne substances into the
brain parenchymal space (Zlokovic, 2008; Weiss et al., 2009). In
fact, the cerebral microvasculature lining is characterized by the
presence of an elaborated junctional complex that includes
mainly TJ and adherens junction (AJ) proteins (Hawkins and
Davis, 2005) that will be addressed in more detail below. Gap
junctions have also been identified at the BBB, but their role in
the barrier function is not clear (Zlokovic, 2008).
Endothelial cells are tethered to the basement membrane
through focal adhesions, which consist mainly of transmembrane proteins (Kumar et al., 2009) that also participate in
intercellular adhesion (Wolburg et al., 2009). The transmembrane proteins have been classified into three families of CAM
according to their structure: selectins, immunoglobulin superfamily, and integrins (Lee and Benveniste, 1999). In EC,
integrins play an important role during angiogenesis and in
the maintenance of vascular integrity (Wolburg et al., 2009).
They function as adhesion receptors, in addition to transmitting chemical signals and mechanical forces between the
matrix and the cytoskeleton (Yuan, 2003). The focal adhesion
complex also contains a host of signaling molecules, such as
focal adhesion kinase, Src tyrosine kinases and Rho GTPases
that participate in integrin engagement and focal adhesion
assemblage; this way, the contractile and adhesive components interact with each other, playing a determinant role in
the assembly and disassembly of focal adhesions, which
allows a dynamic control of cellmatrix interactions, endothelial contractility and permeability properties (Yuan, 2003).
The brain endothelial cytoskeleton has a critical role in
establishing interendothelial junctional integrity and endothelialextracellular matrix adherence. As in other cell types,
the three primary elements of the cytoskeleton are actin
filaments, intermediate filaments and microtubules. Actin
filaments are composed of globular monomers of G-actin,
which polymerize to form helical and asymmetrical filaments
of F-actin that form contractile bundles and filamentous
networks, essential for the maintenance of cell shape and
integrity (Kierszenbaum, 2007). At the BMVEC, bands of F-actin
are anchored to proteins involved in the adhesion to
extracellular matrix (Kierszenbaum, 2007) and linked to
membrane and cytosolic proteins involved in TJ and AJ to
form a structure denoted as the actin-rich adhesion belt
(Stamatovic et al., 2008), sometimes also referred to as

perijunctional actin (Terry et al., 2010). According to functional
needs or depending on stimuli such as inflammatory mediators, actin filaments may interact with myosin, a protein that
drives contractile processes in non-muscle cells, leading to the
formation of contractile actinmyosin filaments, known as
stress fibers. These events are controlled by phosphorylation
of the myosin light-chain (MLC) present on the head of the
myosin molecule by the enzyme myosin light-chain kinase
(MLCK), which in turn is regulated by the Ca2+-binding protein
calmodulin (Yuan, 2003; Kierszenbaum, 2007). This way, actin
is intimately involved in EC tension force via MLC phosphorylation (Stamatovic et al., 2008), playing an active role in
regulating cellular functions rather than acting as a static
component of cellular structure (Lai et al., 2005). Vimentin is
the major intermediate filament in EC. Although its role on
cytoskeletal structure and properties of EC has been underevaluated, a previous report indicates that its link with an AJ
protein, vascular endothelial (VE)-cadherin, is affected by
histamine, a known inducer of microvascular hyperpermeability (Shasby et al., 2002). The microtubule system, formed
by polymers of - and -tubulin, participate in rapid assembly
of actin filaments and focal adhesion, cellular contraction
and/or increased transendothelial leukocyte migration (Statamatovic et al., 2008).
The BBB is more than an impermeable wall. EC at the BBB
have a unique pattern of receptors and specific transport systems that facilitate the uptake of important nutrients and
hormones, in addition to active pumps that help to regulate the
concentrations of ions, metabolites and xenobiotics in the brain
(Zheng et al., 2003; Weksler et al., 2005; Zlokovic, 2008), as
schematically represented in Fig. 6. The BMVEC are able to
efficiently supply the brain with the metabolites required while
contributing to the maintenance of the brain's ionic homeostasis
and protecting the CNS from a large variety of potentially harmful hydrophobic compounds (Betz, 1992; Choi and Kim, 2008).
BMVEC also have a fundamental role in neurogenic niches
(Fig. 7), the micro-anatomical units where neurogenesis
occurs in the adult mammalian brain. In these niches, mainly
located in the subventricular zone, progenitor cells are in close
proximity to the capillaries (Goldberg and Hirschi, 2009;
Tavazoie et al., 2008), which lack astrocyte endfeet and
pericyte coverage, giving them direct access to vascular and
blood-derived signals. Within this vascular niche, EC secret a
number of diffusible signals such as vascular endothelial
growth factor (VEGF), brain-derived neurotrophic factor, and
the pigment epithelium-derived factor, as well as unidentified
factors that affect neural precursors, promoting stem-cell
renewal and subverting stem-cell fate decisions (Shen et al.,
2004; Ramrez-Castillejo et al., 2006; Tavazoie et al., 2008).
Therefore, the vascular compartment within the niche has the
unique opportunity to regulate neural stem-cell behavior,
which is further influenced by ependymal cells and the
cerebro-spinal fluid (Tavazoie et al., 2008).
3.
Intercellular junctions
The EC lining at the BBB is stabilized by specialized cell

junctions between adjacent cells. Here, we will summarize the
333
molecular constituents of TJ and AJ, as well as the signaling

pathways involved in the regulation of their assembly.
3.1.
Molecular constituents
3.1.1.
Tight junctions
TJ are the main structures responsible for the barrier properties.

These are elaborate structures located on the apical region of EC
(Fig. 8). They function both as a seal that regulates lateral
diffusion between the apical and basolateral plasma membrane
domains, which enables asymmetric distribution of membrane
constituents, and as a limit to paracellular permeability (Ge et
al., 2005; Hawkins and Davis, 2005; Hawkins and Egleton, 2006;
Persidsky et al., 2006a). Studies using TJ from different tissues
with varying transendothelial and transepithelial electrical
resistances figured out a correlation between increased organization of cytoplasmic fibrils and decreased membrane permeability (Huber et al., 2001). The BBB TJ should not be regarded as
isolated barrier molecules, but as highly dynamic structures
that are under the close regulation of the brain microenvironment (Wolburg et al., 2009).
Recently, another role for TJ as dynamic heteromeric
signaling complexes has begun to be unraveled, involving
the control of gene expression, cell proliferation and differentiation (Gonzlez-Mariscal et al., 2008). The signaling at TJ is
bi-directional, so that signals are transmitted from the cell
interior to forming or existing TJ to regulate its assembly and
function, whereas TJ co-ordinately receive and transmit
information back to the cell interior to regulate gene expression and subsequent cellular responses (Terry et al., 2010).
The BBB TJ of mammalian species is characterized first of all
by the highest complexity in the vasculature of the body (Kniesel
and Wolburg, 2000). Structurally, TJ form an intricate complex of
parallel, interconnected, transmembrane and cytoplasmatic
strands of proteins arranged as a series of multiple barriers
(Wolburg and Lippoldt, 2002). The transmembrane proteins are
integral membrane proteins that interact with those of neighboring plasma membrane and form the TJ barrier (Petty and Lo,
2002). They are often grouped according to the number of times
they span the plasma membrane: there are single-pass
membrane proteins such as junctional adhesion molecules
(JAM), and four-pass transmembrane domains such as claudins
and occludin, which seem to be more directly linked to epithelial
barrier function (Terry et al., 2010). The cytoplasmic TJ accessory
proteins ZO, cingulin, 7H6 and AF-6 are involved in the
connection of integral TJ to the actin cytoskeleton (Lee et al.,
2004; Persidsky et al., 2006a; Petty and Lo, 2002; Weiss et al.,
2009). These scaffolding proteins recruit to the TJ several
signaling proteins and transcription factors (Terry et al., 2010).
More than 40 proteins, including transmembrane, scaffolding
and signaling proteins, have been found to be associated with TJ
(Gonzlez-Mariscal et al., 2003), forming multimeric protein
complexes that receive and send regulatory signals (Terry et al.,
2010). Next, we will review the literature regarding the most well
studied of these proteins.
3.1.1.1. Claudins. The claudins are a family of TJ proteins,

with 24 family members identified so far, which seem to fulfill
the task of establishing barrier properties (Ueno, 2007; Zlokovic,
2008). These proteins, with approximately 2027 kDa (Persidsky
334
Fig. 6 Endothelial cells comprise a large variety of specific transporters and receptors, which regulate brain concentrations of
nutrients, hormones, metabolites, and xenobiotics. For simplicity, only the glucose transporter-1, the insulin receptor and the
ABC transporters, as the multidrug resistance-associated protein and the P-glycoprotein, and respective substrates, are
represented.
et al., 2006a; Ueno, 2007; Zlokovic, 2008), are integral membrane

proteins that share the four transmembrane domains of
occludin, but do not contain any sequence homology to occludin
(Wolburg and Lippoldt, 2002). Therefore, claudins and occludins
are proteins formed by different aminoacids with similar
conformation, without sharing genetic information. Claudins
form dimers and bind homotypically to other claudin molecules
in adjacent BMVEC (Huber et al., 2001; Petty and Lo, 2002). These
proteins are believed to be responsible for permeability restriction (Tsukita et al., 2001). Although overexpression of claudins
can induce cell aggregation and formation of TJ-like structures
(Yamamoto et al., 2008), occludin expression does not result in
the TJ formation. Thus, it appears that claudins form the
primary seal of the TJ, and occludin acts as an additional support
structure (Persidsky et al., 2006a).
The claudins are not randomly distributed throughout
the organs, but instead follow certain rules that are not
completely understood. In fact, claudins appear to be

expressed in a tissue-specific manner and most cell types
express more than two claudin isoforms conferring different
size and charge selectivity qualities that result in cell- or
tissue-type specific barrier function. In mammalian BMVEC
it has been reported the presence of claudin-1, -3, -5, -12 and
-18 (Petty and Lo, 2002; Blanger et al., 2007; Krause et al.,
2008; Mahajan et al., 2008; Neuhaus et al., 2008) and, more
recently, of claudin-2 and -11 as well (Romanitan et al., 2009).
Claudin-1 and claudin-5 are associated with maintenance of
normal BBB function (Vorbrodt and Dobrogowska, 2003) and
appear to be important in angiogenesis and in disease
processes with increased vessel permeability (Wolburg and
Lippoldt, 2002). In line with the role of claudin-5 in
permeability, claudin-5 knockout mice are characterized
by a size-selective BBB defect (Nitta et al., 2003), whereas
down-regulation of claudin-5 expression correlated with
Fig. 7 Stem cell niches are micro-anatomical units where neural stem cells interact intimately with ependymal cells and
with capillaries, mainly within the subventricular zone. Neural stem cells receive signals from ependymal cells and the
cerebro-spinal fluid, as well as from capillaries, which lack astrocyte endfeet and pericyte coverage and therefore give them
direct access to vascular and blood-derived signals.
335
Fig. 8 Endothelial cells at the bloodbrain barrier present an elaborated junctional complex formed by tight junctions and
adherens junctions. A, Tight junctions are located on the apical region of endothelial cells and form an intricate complex of
parallel, interconnected, transmembrane and cytoplasmatic strands of proteins arranged as a series of multiple barriers.
B, Adherens junctions are located below the tight junctions and are composed of transmembrane glycoproteins linked to the
cytoskeleton by cytoplasmatic proteins, giving place to the adhesion belt.
breakdown of the BBB in an experimental mouse model

of autoimmune encephalomyelitis (Argaw et al., 2009).
Lately, new functions of claudin family proteins in response
to cellular stress (Romanitan et al., 2009), as well as in the
regulation of embryonic morphogenesis (Gupta and Ryan,
2010) were suggested.
3.1.1.2. Occludin. Occludin, a transmembranous TJ protein of

approximately 65 kDa, was the first tight junctional transmembrane molecule discovered (Furuse et al., 1993) and the bestknown one. It is highly expressed and consistently stains in a
distinct, continuous pattern along the cell margins in the
cerebral endothelium, whereas it is much more sparsely
336
distributed in non-neural endothelium (Hawkins and Davis,

2005). It has been shown that high levels of occludin ensure
decreased paracellular permeability (Huber et al., 2001) and high
electrical resistance of the BMVEC monolayers (Persidsky et al.,
2006a), having therefore an active role in BBB function
(Yamamoto et al., 2008). However, it must be seen more in a
regulatory context than as a major structural protein in the
establishment of the barrier properties (Wolburg and Lippoldt,
2002). Results of several knockout and knockdown experiments
indicate that occludin is not essential for the formation of TJ
(Hawkins and Davis, 2005) and that normal expression and
localization of other junctional proteins compensate for occludin loss (Zlokovic, 2008). Recently, there has been evidence that
occludin regulates epithelial cell differentiation (Zlokovic, 2008)
and plays a role in signal transduction (Erickson et al., 2007).
A feature of occludin is a Ca2+-independent adhesiveness,
which is mediated by the first extracellular loop of occludin
and depends on the presence of ZO-1 and AJ components
(Kniesel and Wolburg, 2000). The cytoplasmic C-terminal
domain provides the connection of occludin with the cytoskeleton via accessory proteins (Shimizu et al., 2008).
3.1.1.3. Junctional adhesion molecules. Adhesion molecules,

such as the JAM family and the newly discovered EC-selective
adhesion molecule are localized at TJ as well (Wolburg and
Lippoldt, 2002; Stamatovic et al., 2008). The JAM family
consists of JAM-1, JAM-2, and JAM-3, also designated as JAMA, JAM-B and JAM-C, respectively. These are 40-kDa proteins
from the IgG superfamily (Persidsky et al., 2006a; Bernacki et
al., 2008) composed by a single membrane-spanning chain
with a large extracellular domain (Hawkins and Davis, 2005)
that mediates homophilic and probably also heterophilic
interactions in the tight junctional region (Wolburg and
Lippoldt, 2002; Vorbrodt and Dobrogowska, 2003; Stamatovic
et al., 2008).
A tissue-specific distribution of JAM was observed, reflecting the involvement of these proteins in different interendothelial junctions. In fact, JAM-1 is predominantly expressed
in brain (Aurrand-Lions et al., 2001; Bernacki et al., 2008),
accounting for the network of tight junctional strands unique
to the brain vascular system, whereas JAM-2 is highly
expressed in EC of lymphatic organs and lymphatic EC, and
JAM-3 is not expressed in lymphatic sinuses but is found in
most endothelial contacts ranging from brain vasculature to
high endothelium venules (Aurrand-Lions et al., 2001). Interestingly, JAM are also expressed in circulating monocytes,
neutrophils, lymphocyte subsets, and platelets, as well as in
dendritic cells (Ogasawara et al., 2009; Ueno, 2007).
JAM-1 is involved in cell-to-cell adhesion, organizing the
tight junctional structure, and taking part in the formation of
TJ as an integral membrane protein together with occludin
and claudins (Vorbrodt and Dobrogowska, 2003). JAM also play
a role in developmental processes and regulate the transendothelial migration of leukocytes (Persidsky et al., 2006a;
Stamatovic et al., 2008). Although the functions of JAM are
still largely unknown in mature BBB, it has been suggested
that altered expression of JAM-A, in addition to affecting the
junctional tightness, may also disturb leukocyte trafficking,
with implications for immune status within the diseased CNS
(Zlokovic, 2008).
3.1.1.4. Cytoplasmatic proteins. Submembranous TJ-associated proteins such as ZO proteins (ZO-1, ZO-2 and ZO-3)
belong to the family of membrane-associated guanylate
kinase (MAGUK) proteins (Persidsky et al., 2006a), which
serve as recognition proteins for TJ placement and as a
support structure for signal transduction proteins (Huber
et al., 2001).
MAGUK proteins share defined core regions: 3 PDZ domains
(postsynaptic density-95, disc large, and zonula occludens-1
protein), a SH3 domain (SRC homology 3 domain), and a
guanylate kinase (GuK) domain (Erickson et al., 2007). The PDZ
domains mediate specific binding to carboxy-terminal cytoplasmic ends of transmembrane proteins; the SH3 domain
binds signaling proteins and cytoskeletal elements, and the
GuK catalyzes the ATP-dependent transformation of GMP to
GDP (Wolburg and Lippoldt, 2002). The SH3-GuK region is
further involved in binding to TJ and AJ proteins (Erickson et al.,
2007). ZO-1 acts as a central organizer of the TJ complex as its
carboxy-terminal region binds to actin, linking the TJ to the
cytoskeleton (Erickson et al., 2007).
ZO-1, the first TJ-associated protein identified and characterized, is a 220-kDa phosphoprotein mostly expressed in
endothelial and epithelial cells that normally form the TJ
assembly (Kniesel and Wolburg, 2000; Persidsky et al., 2006a).
Though it is also expressed in other cell types that do not form
TJ, there is no TJ without ZO-1. ZO-1 molecules are located in
the cytoplasmic side of the BMVEC plasma membranes
delimiting the interendothelial cleft (Vorbrodt and Dobrogowska, 2003) and connecting transmembranous TJ proteins
with the actin cytoskeleton. Loss or dissociation of ZO-1 from
the junctional complexes is associated with increased barrier
permeability (Choi and Kim, 2008). ZO-2, a 160-kDa phosphoprotein, has significant homology to ZO-1. It was thought that
ZO-2 was restricted exclusively to the TJ region (Kniesel and
Wolburg, 2000); however, it has also been found in non-TJcontaining tissues. Very much like 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
(Persidsky et al., 2006a). It is not only an extremely important
structural protein, but also a nuclear factor influencing gene
expression (Wolburg et al., 2009) and blocking cell cycle
progression (Gonzlez-Mariscal et al., 2009).
Initially simply known as a protein that co-precipitates
with the ZO-1/ZO-2 complex, ZO-3 has a close homology to
ZO-1 and ZO-2. There is evidence that ZO-3 binds occludin and
ZO-1 directly, but not ZO-2 (Kniesel and Wolburg, 2000).
The complex formed by ZO-1 protein together with its
supplementary components, ZO-2 and ZO-3, is considered to
be involved in cadherin-based cell adhesion through their
binding to -catenin and to actin filaments. Binding of ZO
proteins to actin suggests that one possible function of these
molecules is to form a scaffold to link TJ to the cytoskeleton.
The presence of ZO proteins in zonula adherens indicates the
involvement of these proteins in cell-to-cell signal transduction (Vorbrodt and Dobrogowska, 2003). Diverse kinases,
phosphatases, small G proteins and nuclear and transcriptional factors are clustered in the scaffold (Gonzlez-Mariscal
et al., 2008), accounting for the role of TJ in signaling pathways.
In adverse conditions, as chemical stress or mechanical injury,
ZO-1 and ZO-2 concentrate at the nucleus and associate with
proteins involved in the regulation of gene transcription and

cell proliferation (Gonzlez-Mariscal et al., 2008).
Other cytoplasmic proteins are cingulin, AF-6 and 7H6.
Phosphoprotein cingulin, with approximately 150 kDa, is located
at the cytoplasmatic side of TJ as well. It connects to ZO and JAM
proteins, AF-6 (180 kDa) and myosin, implying a role as a scaffold
between transmembrane proteins and the cytoskeleton (Cordedenonsi et al., 1999; Sandoval and Witt, 2008). There are studies
that also imply a role of cingulin in regulation of TJ permeability
(Cordenonsi, et al., 1999). 7H6 phosphoprotein (155 kDa) seems to
be correlated with TJ impermeability to ions and large molecules.
There is evidence that this protein may detach from TJ when ATP
levels decrease, resulting in increased paracellular permeability
(Mitic and Anderson, 1998; Bernacki et al., 2008).
3.1.2.
Adherens junctions
Below the TJ, in the basal region of lateral plasma membrane

there are AJ that give place to a continuous belt the adhesion
belt (Petty and Lo, 2002) (Fig. 8).
AJ mediate events such as the adhesion of BMVEC to each
other, the contact inhibition during vascular growth and
remodeling, the initiation of cell polarity, and the regulation
of paracellular permeability (Hawkins and Davis, 2005), in
addition to the contribution to the barrier function (Carvey et
al., 2009). Cellcell adhesion through actin filaments linking
(Cook et al., 2008) involves VE-cadherin and catenins, constituents of AJ (Perrire et al., 2007). The cadherin family and
their intracellularly associated catenin proteins form complexes of central importance to the sorting and morphogenic
processes of developing animal tissues and in maintaining the
integrity and identity of adult tissues (Tao et al., 1996).
Ca2+-dependent cellcell adhesion is dependent on a family of transmembrane glycoproteins named
cadherins (Navarro et al., 1998), which can also be defined as
major transmembrane components of AJ (Vorbrodt and Dobrogowska, 2003). Disruption of the AJ by removal of extracellular
Ca2+ leads to the opening of the TJ (Hirase et al., 1997).
Cadherins present a certain degree of cell type specificity.
VE-cadherin, also known as cadherin-5, is an integral membrane glycoprotein expressed exclusively in cells of vascular
epithelial origin, whereas neural (N)-cadherin is expressed in
cells of the nervous tissue, vascular smooth muscle cells, and
myocytes (Navarro et al., 1998).
VE-cadherin is an important determinant of microvascular
integrity both in vitro and in vivo (Vorbrodt and Dobrogowska,
2003). It clusters at cell junctions and mediates cell adhesion
in a Ca2+-dependent manner, inhibits cell proliferation, and
decreases cell permeability and migration when overexpressed in various cell types (Cook et al., 2008).
All cadherins contain a plasma membrane-spanning
domain and a cytoplasmic domain associated with catenins,
other molecular components of the junctional complex
(Vorbrodt and Dobrogowska, 2003). VE-cadherin's expression
at cell junctions, however, is independent of -catenin
binding, which appears to be required only for junction
stabilization (Cook et al., 2008).
3.1.2.1. Cadherins.
3.1.2.2. Catenins.
Catenins were first characterized as linking the cytoplasmic domains of cadherin cellcell adhesion
337
molecules to the cortical actin cytoskeleton. Catenins' major

role is to anchor the cadherin complex to the actin cytoskeleton but they also participate in cell and developmental
signaling pathways (Stamatovic et al., 2008).
There are four types of catenin proteins: -, -, - and catenin. - and -catenin are located in interendothelial
junctions of BBB-type brain capillaries and their expression
is required for cadherins to work as adhesion molecules
(Vorbrodt et al., 2008). -catenin is essential in endothelial
cells for normal vascular patterning. It is a structural protein
that participates in cellcell adhesion and is also involved in
the Wingless signaling pathway and in gene expression (Cook
et al., 2008; Vorbrodt et al., 2008), serving as a mediator in
regulation of P-gp and other multidrug efflux transporters in
brain vasculature (Lim et al., 2008). In addition, -catenin is
linked to the cell membrane in a complex with VE-cadherin
and plateletendothelial cell adhesion molecule. This adhesion molecule mediates homophilic adhesion (Wolburg et al.,
2009). Upon stimulation with growth factors this membraneassociated complex is dissociated, releasing another source of
-catenin for movement to the nucleus and transcription
(Petty and Lo, 2002). It is possible that the up-regulation of catenin accounts for the maintenance of TJ protein assembly
and barrier function (Vorbrodt et al., 2008). -catenin has been
implicated as a regulator of the NF-B transcription factor
(Perez-Moreno et al., 2006), while -catenin is closely related to
-catenin and can substitute it in the cadherincatenin
complex (Vorbrodt and Dobrogowska, 2003).
3.2.
Signaling pathways involved in intercellular junctions
regulation
TJ are highly dynamic structures that depending on physiological and pathological conditions undergo disintegration
and reassembly, which correspond to variable degrees of
sealing of the paracellular cleft (Gonzlez-Mariscal et al.,
2008; Deli, 2009). A relationship between TJ function and the
degree of protein phosphorylation was first demonstrated
based on the observation that the TJ protein ZO-1 is
significantly more phosphorylated in low resistance cells
than in high resistance monolayers (Stevenson et al., 1989).
From then on, phosphorylation over distinct aminoacid
residues of proteins by the action of different kinases has
been reported (Persidsky et al., 2006a,b; Drfel et al., 2009).
Moreover, the influence of stimuli such as oxidative stress
(e.g., the free radical nitric oxide, NO), vasogenic agents (e.g.,
VEGF), inflammatory and lipid mediators (e.g., tumor necrosis factor-, TNF-, and prostaglandin E2, respectively), as
well as infective agents (e.g., human immunodeficiency
virus, HIV) on TJ phosphorylation and dephosphorylation
status has been characterized (Persidsky et al., 2006a,b;
Stamatovic et al., 2008).
The signaling routes mainly involve protein kinases,
members of the mitogen-activated protein kinases (MAPK)
pathway, the endothelial nitric oxide synthase (eNOS), and
G-proteins (Fig. 9). The phosphorylation cascades trigger
biochemical and conformational changes in EC that ultimately lead to an opening of the paracellular pathway. Ca2+mediated signal transduction and internalization or degradation of the junctional molecules are further involved in
338
Fig. 9 Endothelial cell permeability is regulated by multiple signaling pathways. A, Endothelial cells are connected to each other
by tight junction (in green) and adherens junction (in blue) proteins. Both tight junctions and adherens junctions are composed of
transmembrane proteins that are anchored to the cytoskeletal actin (in brown) by cytosolic proteins (in orange and pink,
respectively). This endothelial lining is tethered to the extracellular matrix through focal adhesions mediated by transmembrane
integrins (in purple). A dynamic interaction among these structural elements controls the opening and closing of the paracellular
pathway for fluid, proteins and cells to move across the endothelium. B, Binding of an agonist (e.g. vasogenic agents,
inflammatory and lipid mediators, free radicals and infective agents) to its respective receptor expressed on the endothelial
surface initiates distinct signaling cascades, namely protein kinases, RhoA/Rho kinase (ROCK), and mitogen-activated protein
kinases (MAPK). Phosphorylation of tight junctions and adherens junctions proteins cause the junction complex to dissociate
from its cytoskeleton anchor, leading to a weakened cellcell adhesion. In parallel, there is a rise in intracellular Ca2+ levels and
activation of endothelial nitric oxide synthase (eNOS) and of myosin light-chain kinase (MLCK). The Ca2+/calmodulin-dependent
MLCK catalyzes phosphorylation of myosin light chains (red) triggering binding to actin and their cross-bridge movement. This
reaction promotes cytoskeleton contraction and cell retraction, further contributing to paracellular hyperpermeability. The
formation of stress fibers (in black) impairs cell attachment to the basement membrane (in yellow) aggravating the loss of barrier
properties. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the mechanisms that lead to paracellular hyperpermeability

(Kumar et al., 2009).
Presently, there is intensive research directed to the
clarification of the novel and multiple signals, how they
interact and affect the phosphorylation of junction proteins
and cytoskeleton assembly with the final goal of a better
understanding of BBB opening and modulation for therapeutic
purposes. Here, we will summarize the major ones.
3.2.1.
Protein kinases
Protein kinases are classified as serine/threonine kinases and

as tyrosine kinases based on their substrate specificity.
3.2.1.1. Protein kinase C. Protein kinase C (PKC), one of the

most studied regulators of TJ and TJ-mediated permeability,
are a family of serine/threonine kinases. PKC isoforms are
homologous enzymes in which the functional specificity is
determined by their subcellular localization (Dempsey et al.,
2000) and activation depends on both activator and EC type
(Stamatovic et al., 2008). There are 11 isozymes of PKC,
categorized based on their modes of activation: the conventional PKC (, I, II, ) require Ca2+, whereas the novel (, , , ,
), and the atypical (, ) PKC are Ca2+ independent (Hofmann,
1997), which were all demonstrated to be present in BBB endothelial cells in vitro (Fleegal et al., 2005).
PKC regulate a variety of cell functions. These include

proliferation, gene expression, cell cycle, differentiation,
cytoskeletal organization, cell migration and apoptosis
(Poole et al., 2004). They are also involved in hypoxia-induced
impairment in endothelial-dependent vasodilation (Numamaguchi et al., 1996), and in neuronal damage during cerebral ischemia (Aronowski and Labiche, 2003; Hamabe et al.,
2005).
PKC may alter BBB permeability by several mechanisms,
affecting different barrier-involved structures, i.e., cellcell
junctions, cellmatrix adhesions and cytoskeleton, either
directly or via activation of intracellular signaling cascades.
In fact, it was demonstrated that activation of PKC stimulates
occludin phosphorylation (Harhaj et al., 2006), leads to
disassembly of AJ, via VE-cadherin (Sandoval et al., 2001), and
is involved in intercellular adhesion molecule (ICAM)-1mediated phosphorylation of actin-associated proteins as
well as in cytoskeletal rearrangement in brain EC lines
(Etienne-Manneville et al., 2000), which result in increased
endothelial paracellular permeability. The downstream signals of PKC include the Rho family of GTPases and MAPK
(Kumar et al., 2009), as discussed below. The NO pathway has
also been increasingly recognized as a potential downstream
target of PKC, based on the enhanced NO production observed
in EC exposed to PKC activators (Yuan, 2003). PKC can further
directly stimulate the cytoskeletal contractile machinery by
phosphorylating MLCK, a key molecule triggering actin
myosin motor function (Kumar et al., 2009).
Despite the important regulatory role of PKC its activity is
in turn determined by other regulators, such as protein
kinases (Gonzlez-Mariscal et al., 2008) and Ca2+(Kumar et
al., 2009), whereas their activation is negatively regulated by
serine/threonine protein phosphatases (Yuan, 2003).
3.2.1.2. Protein kinases A and G. Protein kinase A (PKA)

appears to be involved in endothelial barrier integrity and
barrier function of TJ, upon activation with cyclic AMP (cAMP)
(Yuan, 2003; Gonzlez-Mariscal et al., 2008). In fact, elevation
of intracellular cAMP results in increased transendothelial
electric resistance (TEER) and decreased paracellular permeability through enhanced expression of claudin-5 (Krizbai and
Deli, 2003; Deli et al., 2005; Honda et al., 2006). The barrierprotection effect of PKA may further be related to its ability:
to stabilize endothelial cytoskeletal and adhesive structures;
to induce the dephosphorylation of MLC, the dissociation of
F-actin from myosin, and the strengthening of cellmatrix
adhesion; and to inhibit leukocyte adhesion and platelet
aggregation, which prevents damage of endothelium by
activated leukocytes and platelets (Yuan, 2003; Weis, 2008).
Protein kinase G (PKG) is activated by cyclic GMP (cGMP),
whose production is associated with increased NO levels and
guanylate cyclase activation. In contrast to the general
consensus on the barrier tightening role of PKA, there is
controversy as to whether PKG signaling induces a barrierprotective or permeability-increasing effects (Yuan, 2003;
Gonzlez-Mariscal et al., 2008). In fact, there are studies
relating a decreased permeability of HBMVEC monolayers
with cGMP-dependent protein kinase activation following
exposure to NO (Krizbai and Deli, 2003; Wong et al., 2004; Deli
et al., 2005), whereas others relate the hyperpermeability of
339
brain EC to rearrangement of endothelial junctional proteins

and activation of PKG induced by hypoxia, ischemia, VEGF or
excessive NO release (Krizbai and Deli, 2003; Fischer et al., 2004;
Deli et al., 2005).
3.2.1.3. Protein kinase B. Protein kinase B (PKB), also known

as Akt, is ubiquitously expressed in cells, namely EC.
Phosphorylation of its multiple targets in response to various
stimuli, including growth factors or cytokines, promotes cell
survival and inhibits apoptosis, being involved in tumor
progression (Somanath et al., 2006; Vichalkovski et al., 2010).
In EC, activation of Akt via phosphatidylinositol-3 kinase
(PI3K) was shown to involve activation of eNOS and increased
NO production, thus leading to vasodilation and increased
permeability (Cirino et al., 2003; Vogel et al., 2007). Accordingly, VEGF-induced brain EC barrier dysfunction and hyperpermeability was shown to be mediated by Akt/PI3K signaling,
and activation of nitric oxide synthase and PKG signaling
(Vogel et al., 2007). This pathway also up-regulates the
expression of the ICAM-1, resulting in brain microvessel EC
migration (Radisavljevic et al., 2000). In addition, PI3K/Akt
signaling was involved in the reduced expression of claudin-5
levels triggered by treatment with HIV-1 Tat protein (Andrs
et al., 2005). PKB was also shown to be involved in BBB disruption by reactive oxygen species (ROS), as indicated by the
decreased TEER and increased brain EC permeability, occurring either directly or indirectly via the actin cytoskeleton, and
causing dissociation and subsequent disappearance of the TJ
proteins claudin-5 and occludin. The importance of the PKB
pathway as a mediator of ROS-induced alterations in brain EC
was confirmed by the prevention of ROS-induced monocyte
migration across an in vitro model of the BBB upon inhibition
of PKB activity (Schreibelt et al., 2007). Despite the evidence of
an hyperpermeability-inducing effect of PI3K/Akt signaling,
the role of PKB in EC is complex due to the highly branched
signaling network leading to different and sometimes opposite effects upon activation (Somanath et al., 2006; GonzlezMariscal et al., 2008).
3.2.1.4. Protein tyrosine kinase. Phosphorylation of proteins
on serine/threonine residues is often initiated by or coupled
with tyrosine phosphorylation. Phosphorylation of tyrosine
residues is catalyzed by protein tyrosine kinases (PTK), which
are divided into two main classes: receptor PTK and cellular, or
non-receptor, PTK (al-Obeidi et al., 1998). Both classes
participate in the signal transduction that mediates changes
in endothelial barrier structure and function, therefore playing
an important role in regulation of brain endothelial barrier
permeability. Enhanced PTK activity has been observed in
several pathologies and is associated with increased endothelial permeability (al-Obeidi et al., 1998; Yuan, 2003).
Receptor PTK have extracellular domains with one or more
identifiable structural motifs, as well as binding sites for several
growth factors, such as epidermal growth factor receptor,
platelet-derived growth factor and VEGF (Stamatovic et al.,
2008). Activation of the corresponding receptors triggers activation and interaction with other signaling molecules like NO and
PKC, resulting in BBB hyperpermeability (Yuan, 2003; Stamatovic et al., 2008). VEGF receptors (VEGFR) are a family of closely
related receptor protein kinases consisting of VEGFR-1 (Flt-1),
340
VEGFR-2 (flk-1/KDR) and VEGFR-3 (Flt-4), of which VEGFR-2 is

expressed in vascular EC mediating the permeability actions of
VEGF, among other actions (Holmes et al., 2007). Upon ligand
binding, VEGFR-2 undergoes dimerization and phosphorylation
at multiple tyrosine residues, which provides the molecular
configuration that initiates signaling cascades involving Ca2+
release, NO production, and activation of kinases as PKC and
MAPK (Holmes et al., 2007; Gonzlez-Mariscal et al., 2008). In
addition to these pathways, VEGFR-2 triggers integrin signaling,
as well as the tyrosine phosphorylation of AJ proteins, VEcadherin and catenins (Kumar et al., 2009).
Axl is another member of the receptor PTK family. Its
activation results in autocatalytic tyrosine phosphorylation
and recruitment of different signaling molecules (Weinger et
al., 2008). It can rescue EC from apoptosis (D'Arcangelo et al.,
2006), and is also involved in vascular remodeling (Korshunov
et al., 2007) as well as in angiogenesis (Holland et al., 2005).
This kinase may also be a protective mechanism, playing an
important role in the adaptive response of cells to hyperosmotic stress (Wilhelm et al., 2008).
Cellular PTK are located in the cytoplasm, nucleus or
anchored to the inner leaflet of the plasma membrane and are
involved in several processes as cell growth, cell differentiation and cell adhesion (Stamatovic et al., 2008). Among the
multiple non-receptor PTK, there is the Src family of kinases
(SFK), which has been implicated in the barrier-opening action
of stimuli like inflammatory mediators (e.g. TNF) (Nwariaku et
al., 2002), oxidant species (e.g. H2O2) (Kevil et al., 2001) and
growth factors (e.g. VEGF) (Eliceiri et al., 1999). In EC, SFK
regulate endothelial architecture and cellular monolayer
permeability through mediation of tyrosine phosphorylation
of numerous molecules involved in cytoskeleton assembly,
junctional complexes and focal adhesions (Yuan, 2003; Kim
et al., 2009a; Kumar et al., 2009). In fact, SFK-mediated
phosphorylation and activation of MLCK initiates the crossbridge formation between myosin and actin, leading to
cytoskeleton contraction that results in morphologic changes,
intercellular gap formation and increased permeability (Garcia
et al., 1999; Birukov et al., 2001). VE-cadherin and -catenin are
also the subject of phosphorylation by SFK, which results in
the disruption of intracellular cadherinactin complexes and
of the protein complexes that bind EC together, thus leading to
alterations in endothelial permeability (Roura et al., 1999).
Finally, SFK induces the phosphorylation of focal adhesions,
thus compromising the EC attachment to the extracellular
matrix, and activates MMP, which impair endothelial basement membrane integrity (Haorah et al., 2008), further
contributing to hyperpermeability.
3.2.2.
Mitogen-activated protein kinases
MAPK are involved in multiple cascades of serine/threonine

and tyrosine phosphorylating reactions that mediate diverse
cellular responses to inflammatory mediators, oxidant species and growth factors (Yuan, 2003). A common characteristic of these protein kinases is their activation by a dual
phosphorylation on specific sites. When these sites are
phosphorylated, the kinases become active and, in turn,
phosphorylate their substrates (Miller et al., 2005). MAPK can
phosphorylate a large number of proteins that are located
both in the cytoplasm and the nucleus, including transcrip-
tion factors that regulate the transcription of diverse genes

(Gonzlez-Mariscal et al., 2008).
The most studied MAPK in mammalian cells are p42/p44
extracellular signal-related kinases (ERK 1/2), c-Jun N-terminal
kinases (JNK) and p38 proteins (Cross et al., 2000). MAPK
signaling pathway modulates TJ paracellular transport by upand down-regulating the expression of several TJ proteins in
different cell types, hence altering the molecular composition
within TJ complexes and participating in the regulation of the
barrier function (Gonzlez-Mariscal et al., 2008; Deli, 2009).
Upon exposure to insults, MAPK activation has been consistently associated with a down-regulation or redistribution of
TJ proteins and increased permeability in different BBB
models. In fact, increased paracellular permeability in porcine-brain-derived microvascular EC by H2O2 was shown to be
mediated by elevation of intracellular Ca2+ levels, p44/42
MAPK activation and changes in the distribution of the TJ
proteins occludin, ZO-1 and ZO-2 (Fischer et al., 2005).
Increased permeability by oxidative stress further involves
occludin down-regulation, as well as disruption of the
cadherin--catenin assembly and thus of the junctional
complex, mediated by ERK 1/2 activation (Krizbai et al.,
2005). Activation of the p42/44 MAPK was also observed to
mediate transcytosis induced by TNF- in an in vitro BBB
model (Miller et al., 2005). Moreover, activation of all MAPK
were involved in increased TJ protein phosphorylation and
permeability induced by lipopolysaccharide (LPS) in rats
(Singh et al., 2007), as well as in HIV-1 penetration in BMVEC
(Liu et al., 2002). More recently, it was reported that amyloid
peptide 140 induces a marked increase in the permeability of
the HBMVEC line hCMEC/D3, which was associated with a
specific decrease in the TJ protein occludin, mediated by JNK
and p38MAPK pathways (Tai et al., 2009).
3.2.3.
G-proteins
G-proteins play an essential role in the establishment and

modulation of TJ, thus maintaining the barrier integrity
(Persidsky et al., 2006a). The G-proteins involved in that
process are the classical heterotrimeric G-proteins and the
small GTPases (Ras superfamily) (Wolburg and Lippoldt, 2002).
The RhoGTPases are a family of small GTPases with
profound actions on the actin cytoskeleton of the cells,
regulating cell shape, cell contraction and cell adhesion (van
Hinsberg and van Niew Amerongen, 2002), thereby providing a
major regulation of TJ formation, maintenance and disruption
(Stamatovic et al., 2008). In addition, they are important
regulators of gene expression and contribute to MAPK
cascades and inflammatory signaling (Terry et al., 2010). The
Rho proteins act as a molecular switch, cycling between the
active GTP-bound state and inactive GDP-bound state, under
the regulation of guanine nucleotide exchange factors and
GTPase activating proteins; when activated, the RhoGTPase
undergoes a conformational change, enabling the recruitment
of effector proteins that mediate downstream effects (Terry et
al., 2010).
In mammals, the Rho family of GTPases contains 20
members (Etienne-Manneville and Hall, 2002). RhoA, Rac and
Cdc42 are family members with distinct subcellular targeting
and functions that have been characterized: RhoA promotes
the formation of stress fibers, cell contractility and focal
adhesion, whereas Cdc42 and Rac1 are major players in cell

membrane protrusion and cell migration (van Hinsberg and
van Niew Amerongen, 2002; Kumar et al., 2009). Accordingly,
RhoA activation is associated with hyperpermeability, whereas Cdc42 and Rac1 have generally been considered as barrier
protectors (Kumar et al., 2009), stabilizing microvascular
endothelial barrier functions in vitro and in vivo, likely by
increasing the junction-associated actin cytoskeleton (Waschke
et al., 2006). Nevertheless, a recent study suggests that Rac1
contributes to VEGF-induced hyperpermeability by mediating
disassembly of intercellular junctions (Gavard and Gutkind,
2006). Interestingly, it appears that Rho GTPases, rather than
having opposite effects, are coordinated (Wojciak-Stothard et
al., 2005) and that a fine balance is needed for optimal junction
integrity (Terry et al., 2010).
Stimuli like VEGF, histamine and thrombin, induce the
activation of RhoA and its effector kinase Rho kinase (ROCK),
in a Ca2+/calmodulin-mediated way. This activation, together
with the inhibition of MLC phosphatase, leads to MLC
phosphorylation and actinmyosin contraction, therefore
impairing the interaction between the cytoskeleton and
junctional proteins and increasing endothelial permeability
(van Hinsberg and van Niew Amerongen, 2002; Kumar et al.,
2009). Moreover, activation of RhoA/ROCK pathway leads to
direct phosphorylation of occludin and claudin-5 in BMVEC
exposed to HIV-1-infected monocytes, and the increased
phosphorylation was related with a decreased barrier tightness (Persidsky et al., 2006a,b; Yamamoto et al., 2008).
Therefore, Rho signaling appears to interfere with paracellular
permeability by different molecular mechanisms, ranging
from regulation of actinomyosin contraction to direct regulation of junctional membrane proteins.
Although the importance of RhoGTPases in the assembly
and function of TJ is recognized, the mechanisms underlying
its regulation are still unclear. The G proteins may represent
the functional link, since they have been implicated in Rho
activation and in the regulation of TJ (Terry et al., 2010).
3.2.4.
Endothelial nitric oxide synthase
In EC, NO production is catalyzed by the constitutive enzyme

eNOS. NO functions as a messenger in the nervous system,
cardiovascular system, and immune system, reacting with a
diverse number of cellular targets with multiple purposes. EC
produce small amounts of NO, which are released to control the
local blood flow and are thought to have a protective effect by
allowing vasodilation (Thiel and Audus, 2001). This is the basis
for the therapeutic use of statins in conditions such as acute
ischemic stroke in order to improve cerebral blood flow (Sawada
and Liao, 2009).
eNOS is a Ca2+/calmodulin-dependent enzyme, which
renders eNOS very sensitive to changes in intracellular Ca2+
levels (Thiel and Audus, 2001). In addition to calmodulin,
caveolins, the resident scaffolding proteins of caveolae, also
regulate eNOS activity. In fact, calmodulin and caveolins
undergo a reciprocal Ca2+-dependent association and dissociation with eNOS in the caveolar membrane that activates
(calmodulin) and inhibits (caveolins) eNOS activity (Kone,
2000). Upon eNOS stimulation, NO released from EC activates
the guanylate cyclase signaling cascade (Hobbs et al., 1999).
The soluble form of guanylate cyclase converts GTP to cGMP,
341
which in turn is required for PKC activation as described

above.
The role of NO on BBB permeability is still unclear as there are
studies showing that NO decreases the permeability of the
human BBB (Wong et al., 2004), whereas others show an
hyperpermeability of the BBB (Shukla et al., 1996; Mayhan,
2000; Yamauchi et al., 2007). Studies performed by Yamagata
et al. (2004) related NO-mediated increased permeability with
changes in the expression of TJ proteins and a redistribution of
AJ proteins, pointing to a role of NO in signal transduction
cascades induced by hypoxia that lead to junctional disorganization and BBB breakdown.
Moreover, treatment of BMVEC with NO donors was shown
to activate MLCK, leading to phosphorylation of MLC and of
the TJ proteins occludin, claudin-5, and ZO-1 in BMVEC
monolayers (Haorah et al., 2005). These alterations lead to TJ
and cytoskeletal rearrangements, ultimately compromising
BBB integrity and properties.
Another possible mechanism linking eNOS-derived NO to
BBB disruption involves MMP. MMP are a family of zincdependent endopeptidases that are secreted as zymogens and
must be cleaved to be fully active, which digest almost all
extracellular matrix molecules (Bauer et al., 2010). MMP are
well known mediators of BBB disruption, playing a key role in
the degradation of vascular extracellular matrix and brain
blood microvessels, featuring leukocyte infiltration and activation of microglia (Rosell et al., 2006; Rosenberg and Yang,
2007) as part of the neuroinflammatory response in ischemia,
multiple sclerosis, and infection (Cunningham et al., 2005).
The role of oxidative stress in mediating activation of MMP
via PTK pathway was also demonstrated in BMVEC exposed to
NO donors (Haorah et al., 2007a). NO was further related with
Src signaling, thus pointing to its role in redox-linked signal
transduction pathways of PTK (Nakashima et al., 2002).
3.2.5.
Calcium
Ca2+ is a potent second messenger. Therefore, intracellular

Ca2+ concentration is strictly regulated by membrane Ca2+
channels and Ca2+ pumps that remove Ca2+ from the cytoplasm
and return it to the extracellular space or intracellular storage
(Brown and Davis, 2002). Both elevation and depletion of
intracellular Ca2+ may result in disruption of intercellular
junctions, attesting the importance of Ca2+ homeostasis on
the regulation of BBB opening (Persidsky et al., 2006a).
Activation of Ca2+ signaling pathways occurs after a rise in
the ion intracellular levels. Elevated intracellular Ca2+ may be
triggered by binding of an agonist to its respective receptor on
the endothelial surface, by influx through Ca2+ channels, or
result from the ion release from intracellular stores (Persidsky
et al., 2006a; Kumar et al., 2009). Accumulated Ca2+ can target
either cytoskeleton proteins or junction structures that
determine permeability properties (Brown and Davis, 2002;
Persidsky et al., 2006a; Kumar et al., 2009). Accordingly,
increased Ca2+ levels were shown to affect BBB permeability
either by disruption of TJ and AJ formation or by altering their
protein expression. At the cytoskeleton, Ca2+ activates Ca2+/
calmodulin-dependent MLCK, which phosphorylates MLC and
promotes a cross-bridge movement between actin and
myosin. As mentioned above, this interaction generates a
contractile force that leads to cell retraction, weakening cell
342
cell adhesion and pulling neighboring cells apart from each

other that result in intercellular gap formation. The Ca2+mediated activation of MLCK further causes phosphorylation
of the TJ proteins occludin and claudin-5, known to impair BBB
integrity (Haorah et al., 2007b). In parallel, Ca2+ stimulates
eNOS leading to the production of NO and subsequently of
cGMP, which activates the cGMP-dependent protein kinase,
PKG, that mediates structural and functional responses at the
endothelial barrier (Kumar et al., 2009). In addition, other
cascades as those modulated by PKC and MAPK are also
regulated by Ca2+ (Brown and Davis, 2002; Kumar et al., 2009).
The Ca2+-induced altered activity of signaling cascades concur
to the changes in TJ and AJ protein expression that compromise the barrier function (Brown and Davis, 2002).
The exposure of epithelial cells to low Ca2+ levels, by using
2+
Ca -free medium or Ca2+-chelating agents, also results in
increased permeability and barrier dysfunction due to loss of
TJ proteins, as well as to conformational changes at TJ complexes that compromise TJ assembly (Bowman et al., 1983;
Ma et al., 2000). Therefore, not only elevation but also
depletion of intracellular Ca2+ may result in disruption of
intercellular junctions via decreased expression and/or disruption of proteinprotein interactions in BBB models.
3.2.6.
Wnt/-catenin
The canonical Wnt/wingless pathway, which acts via catenin stabilization and is also known as Wnt/-catenin
pathway, is a well-defined transcriptionally active signaling
cascade, acting as a major regulator of brain development
(Chenn, 2008; Schller and Rowitch, 2007). It favors translocation of -catenin to the nucleus where it binds to transcription
factors and, thus, modulates gene transcription (Moon, 2005).
The Wnt signaling regulates cellular proliferation and polarity,
apoptosis, branching morphogenesis, and maintenance of
stem cells in an undifferentiated, proliferative state (Zerlin et
al., 2008).
Wnts are a family of secreted glycoproteins that accumulate in the extracellular matrix to activate pathways in
adjacent cells. Wnt ligands trigger these pathways by binding
an appropriate frizzled receptor, which belongs to a family of
seven-pass transmembrane proteins (Zerlin et al., 2008).
Frizzleds are G-protein-coupled receptors (Slusarski et al.,
1997; Liu et al., 2001), and Wnt binding to frizzleds can activate
distinct branches of the Wnt signaling cascade, namely the
canonical pathway. Through this pathway, the default
mechanisms that normally prevent accumulation of -catenin in the cytoplasm of normal cells are interrupted and the
Ca2+ ionic influx is activated. The Wnt-induced accumulation
of -catenin in the cytoplasm promotes its entry into the
nucleus and the transcription of genes implicated in cell
growth regulation (Zerlin et al., 2008).
Recently, Liebner et al. (2008) added the Wnt/-catenin to the
list of regulators of TJ proteins (e.g., protein kinases, G-proteins,
etc.). In fact, they demonstrated that Wnt signaling plays an
active role in the development of the BBB by regulating
expression of key protein constituents of the TJ. Particularly,
they showed that claudin-3 protein and mRNA expression
increases in response to Wnt in a -catenin-dependent manner.
In addition to mediating the transcriptional output from Wnt
signaling, -catenin also functions in cellcell adhesion through
its interaction with cadherins at the AJ. Therefore, any resulting

alteration to the AJ complex could indirectly impart its close
neighbor, the TJ. Accordingly, ablation of endothelial -catenin
led to deficient cellcell contacts and increased paracellular
permeability (Cattelino et al., 2003). In contrast, elevated catenin transcriptional activity induces BBB properties in
nonbrain-derived EC, providing evidence for the role of the
canonical Wnt pathway as a key regulator of the BBB phenotype
in EC (Liebner et al., 2008).
The new findings by Liebner et al. (2008) suggest that
activation of Wnt signaling in the brain may constitute a
therapeutic approach to reinforce the TJ barrier in order to
prevent or retard the passage of leukocytes and water-soluble
plasma components into the brain in degenerative or inflammatory diseases of the CNS; conversely, transient inhibition of
Wnt signaling and the ensuing breakdown of the TJ could
enable access of therapeutics normally denied by the BBB
(Polakis, 2008).
4.
The blood-brain barrier as a signaling interface
The BBB EC produce mediators like cytokines and NO. These

substances may be constitutively expressed or induced by
stimulation as exemplified by the increased production of
interleukin (IL)-8 in acquired immune deficiency syndrome
(AIDS), of TNF- by cocaine exposure and of NO in Alzheimer's
disease (Banks and Erickson, 2010). Because of the bipolar
nature of the BBB these mediators may be secreted into either
the CNS compartment or the blood. Therefore, an emerging
role of BMVEC as a communication interface between the
peripheral and CNS compartments has been revealed, so that
nowadays the BBB is viewed as a relay station (Quan, 2008;
Carvey et al., 2009). In fact, vascular signals cause the release of
cytokines by BBB EC into the brain parenchyma, which
determine brain cells' action, whereas signaling from brain
parenchyma to the periphery via EC also occurs (Carvey et al.,
2009).
4.1.
Signaling from periphery to brain
During systemic inflammation, activation of BBB-dependent

pathways is critical for CNS-controlled responses for generalized neuroimmune effects (Quan, 2008). One mechanism by
which blood-borne cytokines might affect the function of the
CNS is by crossing BBB for direct interaction with brain tissue
(Banks et al., 1995). Saturable transport systems at the BBB
have initially been described for IL-1, IL-1, IL-6, and TNF-
(Banks et al., 1995; Pan and Kastin, 2002). Along the last
decade, the number of cytokines found to cross the BBB
expanded significantly (Quan, 2008). The study of the mechanism underlying the transport of TNF- across the BBB
revealed to be a more complicated process than simple
receptor-mediated endocytosis (Pan and Kastin, 2002). It is
nowadays accepted that EC have cytokine binding sites, which
alter intracellular function (receptors) or convey the cytokine
across the BBB (transporter), appearing that in some cases the
transporter and the receptor are the same, whereas in other
cases, as for IL-1, the transporter is distinct from the cytokine
receptor (Banks and Erickson, 2010).
In addition to the role of the EC lining in transporting

peripheral cytokines into the brain, BMVEC also have an active
role in their production. In fact, brain EC monolayers
stimulated by LPS produce different cytokines, as IL-1 and
TNF (Banks, 2006), and stimulation by cytokines can induce
their own release (Banks and Erickson, 2010). Moreover,
secretion can be stimulated from one side of the BBB with
release into the other side. For example, application of LPS to
the abluminal (brain side) surface of brain EC increases the
release of IL-6 from its luminal (blood side) by about 8-fold
(Banks, 2006). On the other hand, BMVEC may modulate the
secretions into the CNS as indicated by the fact that
adiponectin, a protein that affects feeding, inhibits the release
of the centrally active IL-6 from brain EC, a phenomenon that
was paralleled by a similar trend of other pro-inflammatory
cytokines (Spranger et al., 2006).
The strategic position of EC further allows the transfer of
information from the blood to the CNS. Therefore, the vascular
system plays a key role in immune-CNS communication,
responsible for activation of brain circuits and key autonomic
functions during systemic immune challenges. Although the
signaling pathway mediating these functions are not completely clarified, it was shown that systemic treatment with IL1 stimulates vascular cells of the brain to produce prostaglandin E2, which acts as the main endogenous brain ligand
not only to trigger the fever pathways, but also those
participating in the activation of the hypothalamicpituitaryadrenal axis (Gosselin and Rivest, 2008), thus inducing
CNS-mediated effects.
Interestingly, it was proposed that in addition to BBBdependent pathways, there are also BBB-independent pathways, which use neuronal routes that bypass the BBB and
elicits early and integrated responses from the CNS, during a
time-period when BBB-dependent pathways are not yet active
(Quan, 2008).
4.2.
Signaling from brain to periphery
The normal brain undergoes routine, but limited, immune

surveillance; therefore, extravasation of blood leukocytes
into the brain is critical for brain protection (Carvey et al.,
2009). This process is signaled by pro-inflammatory mediators, including cytokines, which activate EC at post-capillary venules to which many properties of the BBB extend
(Engelhardt, 2009), therefore leading to increased expression
of CAM of the E-selectin subfamily. These selectins bind to
the glycosylated ligand integrins on monocytes causing
monocytes to slow and roll along the luminal surface of the
vessel, a process called tethering. The binding of monocyte
integrin to EC selectins signals the EC to increase the
expression of other CAM, as vascular CAM (VCAM)-1 and
ICAM-1, which form into clusters that further increase
binding affinity for monocytes. Slowed by tethering, monocytes roll down the luminal wall to paracellular junctions
and transmigrate into the brain (Carvey et al., 2009). In
addition to paracellular migration, leukocytes may also
enter the brain by transcellular migration (Carman
and Springer, 2004). This process also requires the involvement of MMP to digest the basement membrane (Man et al.,
2007).
5.
343
Pathways across bloodbrain barrier
Passage of molecules across the EC of the BBB can occur

between adjacent cells (the paracellular pathway) or through
the cells (the transcellular pathway) (Pardridge, 1999). In the
endothelium, the relationship of paracellular and transcellular permeability is of crucial importance for the regulation of
overall transendothelial permeability (Wolburg et al., 2009).
When it comes to the paracellular passage, ions and solutes
diffuse between adjacent cells according to their concentration gradient (Petty and Lo, 2002). As for the transcellular
pathway, it involves different mechanisms including passive
diffusion of lipophilic compounds, receptor-mediated shuttling and transcytosis (Fig. 10). Small lipophilic molecules,
such as oxygen, CO2 and ethanol (Abbott, 2002), can pass the
BBB freely by diffusion (Zheng et al., 2003), whereas hydrophilic molecules, such as peptides and proteins, may enter the
brain through specific transport mechanisms (Norsted et al.,
2008). For that, an elaborate system of transport proteins such
as the glucose transporter-1 (GLUT-1) and ATP-binding
cassette (ABC) transporters, among others, is expressed on
the luminal and abluminal EC membranes (Hawkins et al.,
2002) (Fig. 6). Polar and lipid-insoluble molecules do not cross
the BBB (Scherrmann, 2002).
5.1.
Caveolae
Caveolae are sites of endothelial transcytosis, endocytosis,

and signal transduction, and as docking sites for glycolipids
and gycosylphosphatidylinositol-linked proteins (Simionescu
et al., 2002; Wolburg et al., 2009).
Caveolae are dynamic pieces of membrane that are either
opened for receiving and releasing material or closed for
processing, storage, and delivery to the cell (Anderson, 1993).
These invaginations of the plasma membrane are rich in the
structural and functional coat protein caveolin (Fig. 11). Whilst
caveolin is the distinct biochemical marker within caveolae
domains, caveolae are also rich in cholesterol and sphingolipids (Smith and Gumbleton, 2006). Caveolin-1 is the major
structural protein of caveolae and is involved in various
aspects of vesicular trafficking and signal transduction pathways (Huber et al., 2001). In the BBB, the presence of both
caveolin-1 and -2 in microvessels was demonstrated for
BMVEC of rats, rhesus monkeys, porcine and of normal
human brain samples (Virgintino et al., 2002). In resting EC,
the V-ATPase complex was identified as the major caveolar
protein in addition to the resident proteins related to structure,
as caveolin-1 (Sprenger et al., 2006). The molecular composition of caveolae also indicates that they have the capability to
store and process messengers such as Ca2+ and to initiate
phosphorylation cascades, as those involving non-receptor
tyrosine kinases and eNOS (Anderson, 1993; Sprenger et al.,
2006). On the other hand, caveolae formation and their
detachment from the plasma membrane as endocytic vesicles
are regulated through the tyrosine phosphorylation of caveolin-1 by Src; the caveolae are then transported through the
intracellular space and fuse with the basal endothelial surface,
releasing their contents into the interstitial space (Kim et al.,
2009a). Caveolin-1 appears to act not as a determinant of
344
Fig. 10 The transcellular pathway: passage of molecules across the endothelial cells of the bloodbrain barrier can occur
through different mechanisms such as passive diffusion of lipophilic compounds or receptor-mediated shuttling. Polar
molecules do not cross the blood-brain barrier. Adapted from Abbott et al. (2006).
caveolae invagination and internalization but rather as a

regulator that stabilizes caveolae at the plasma membrane
and reduces the endocytic potential of caveolae domains (Nabi
and Le, 2003).
Caveolin-1 also appears to constitute an early and critical
modulator that controls signaling pathways leading to the
disruption of TJ proteins (Zhong et al., 2008) as indicated by
the localization of caveolae close to junctional cell contacts
and the fact that both occludin and ZO-1 are organized within TJ by association with caveolin-1 (Smith and Gumbleton,
2006).
5.2.
Glucose transporter-1
GLUT-1 ensures nutrient delivery, supplying glucose for the

brain (Persidsky et al., 2006a), which is its main energy source.
The 55-kDa form of GLUT-1 is highly restricted to the capillary
EC in the brain (Wolburg et al., 2009). The density of GLUT-1
at the abluminal membrane is higher than at the luminal.
The asymmetrical distribution provides homeostatic control
for glucose influx into the brain by preventing accumulation
at levels higher than those in the blood (Zlokovic, 2008).
Interestingly, GLUT-1 may also be present at peripheral nerve
Fig. 11 Caveolin-1 is the major structural protein of caveolae

and is involved in various aspects of vesicular trafficking and
signal transduction pathways.
pericytes transporting D-glucose from the circulating blood

into the brain and peripheral nervous parenchyma in cooperation with that on the EC (Shimizu et al., 2008).
5.3.
ATP-binding cassette transporters
ABC transporters, as the P-gp and the multidrug resistanceassociated proteins (Fig. 6), have the potential to reduce the
penetration of many drugs into the brain and increase their
efflux from the brain. Therefore, they are the focus of
intensive research on drug delivery to the brain.
P-gp is a transporter for the acquisition of the multidrug
resistance phenotype, as it prevents the entry of blood-borne
substances into the brain and facilitates their transport out of
brain parenchyma (Choi and Kim, 2008). The expression of this
efflux transporter among bloodbrain interface is still under
investigation. In fact, although P-gp in the brain has been
thought to be primarily located in the apical or luminal
membranes of EC (Virgintino et al., 2002; Gazzin et al., 2008),
it was also reported that such protein localizes to both the
luminal and the abluminal membranes of the BBB capillary
EC (Bendayan et al., 2006). These apparently contradictory
findings can be reconciled by the recent studies of Tai et al.
(2009), which showed that the transporter is present in both
membranes but P-gp molecules associated with the apical
membrane are nearly 3 times greater than that on the basolateral membrane. The localization of the transporter in both
poles would permit that the efflux of molecules from the brain
endothelium can be initiated at the luminal membrane,
whereas the elimination of drugs from the brain occurs due
to the abluminal membrane P-gp that acts in concert with the
luminal transporter.
It appears to exist an association between P-gp and
caveolin-1, as the down-regulation of caveolin-1 enhances
the transport activity of P-gp (Demeule et al., 2000; Barakat et
al., 2007). P-gp is also expressed at the endothelial subcellular
level along the nuclear envelope and in caveolae, cytoplasmic
vesicles, Golgi complex, and rough endoplasmic reticulum
(Bendayan et al., 2006; Tai et al., 2009). In addition to EC, P-gp
is also expressed at the plasma membrane of both pericytes
and astrocytes, suggesting that this glycoprotein may regulate
drug transport processes in the entire CNS BBB (Bendayan et
al., 2006).
Multidrug resistance-associated proteins are a family of
transporters, where the breast cancer resistance protein, the
organic anion transporting polypeptide family and the organic

anion transporters are included, which mediates the efflux of
anionic compounds. These transporters are mostly located at
the luminal membrane, although also present at the abluminal side, and work together to reduce penetration of many
drugs into the brain and increase their efflux from the brain
(Zlokovic, 2008).
6.
Study approaches
BBB dysfunction has been described in a variety of neurological

diseases not only as a late event but as putatively involved in the
early steps of disease progression (Weiss et al., 2009). In fact,
recent data showed a faulty BBB clearance of potential brain
toxins in Alzheimer's and Parkinson's diseases, inefficient
clearance of excitotoxins across the BBB after an ischemic insult
or traumatic brain injury, increased transport of leukocytes
across the activated BBB in AIDS dementia and BBB breakdown
in epilepsy (Zlokovic, 2008; van Vliet et al., 2007a). BBB
impairment may thus initiate and/or contribute to progressive
nerve cell dysfunction in such disorders through a vicious
circle. Novel therapeutic approaches are required to abrogate
such disease processes, namely by modulation of TJ, or of the
transport systems. On the other hand, due to the unique
properties of the BBB, around 98% of drugs are not able to
cross the bloodbrain interface (Tosi et al., 2008), thus leaving
only a limited number of therapeutics capable of reaching the
CNS. These facts, together with the estimation that 1.5 billion
people are suffering from CNS disorders and that 50% of the total
worldwide population will show Alzheimer's symptoms by the
end of the 21st century (Tosi et al., 2008), have attracted a great
attention to the study of the BBB. Several approaches have been
used, depending on the purposes of the study and on the
expertise and resources of the researchers and laboratory where
the study is to be performed. Here, we will point to some
experimental systems, imaging techniques and functional
biomarkers that can be used to assess BBB properties in health
and disease, as well as to test the ability of therapeutic
compounds to reach the brain and improve CNS homeostasis.
6.1.
Experimental systems
The experimental systems that can be used to study BBB

basically rely on in vivo, ex vivo, in vitro and in silico studies. It
is generally accepted that the similarity of an experimental
system to the in vivo situation is directly related to its degree of
complexity (Silva et al., 2006). However, it is important to have
in mind that each category of experimental system, ranging
from whole animals used for in vivo studies to cell cultures
used for in vitro studies, have advantages and disadvantages
as well as specific applications, as exemplified in Table 1.
6.1.1.
In vivo
In vivo studies provide the most reliable reference information

for testing and validating other models. They take into
account not only a section but the whole brain microenvironment and biological processes in live animals. Properties can
be assessed using non-invasive methods for imaging,
addressed below. Since BBB perturbation is likely to be subtle,
345
studies require large sample sizes and appropriate controls to

detect modest but clinically relevant BBB changes in early
stages of cerebral microvascular disease and any subsequent
progression (Farrall and Wardlaw, 2009). In vivo studies have
been used to assess brain metabolism (Hasselbalch et al.,
2001), BBB disruption (Reinhard et al., 2006) and transport
(Kannan et al., 2009), as well as neurological disease progression as Parkinson's disease (Bartels et al., 2008), among others
(Farrall and Wardlaw, 2009).
In vivo studies however are not all as less invasive as
above. Hence, the largest majority of studies are performed on
animals such as mice (Abulrob et al., 2008; Coisne et al., 2009)
and primates (Astradsson et al., 2009). Using animal models it
is possible to use cranial window preparations to weigh up the
site and severity of disruption of the BBB by injury such as
acute hypertension (Mayhan, 1991). Based on in vivo studies it
has also been assessed the phosphorylation of the TJ proteins
claudin-5 and occludin (Yamamoto et al., 2008) and the
activity of P-gp, particularly in epileptic animals (van Vliet et
al., 2007b). Moreover, the use of animal models has been
helpful to study TJ functions and permeability properties, as
well as behavior patterns, in knockout animals for specific BBB
proteins (Gow et al., 1999; Furuse, 2009). However, the
differences observed between human and rodents (Alanne et
al., 2009) require caution when translating the results of
animal models to human diseases. On the other hand, in vivo
studies may also require expensive equipment (Cohen-Kashi
Malina et al., 2009) that may not be available in all laboratories.
Since these studies are highly demanding in technical and
economic terms, other alternatives such as ex vivo studies
have been used with success.
6.1.2.
Ex vivo
Ex vivo studies are performed on living tissue in an artificial

environment outside the organism with minimum alteration of
natural conditions. This allows experimentation under highly
controlled conditions impossible in the intact organism. One of
the most common ex vivo methods for brain studies are slices,
where interactions between all the components of the NVU are
preserved in a fashion close to the in vivo situation. In slices, the
cytoarchitecture of the tissue is retained and the original in vivo
biology is maintained, thus rendering them an appropriate tool
for research aiming to establish clear correlations between
structure and function. Two main types of slices are currently in
use: the acute slices (often simply referred to as slices), which
are short-living preparations usually amenable for experiments
in the range of a few hours, and the organotypic cultures that
can be followed over several weeks in vitro (Lossi et al., 2009).
Since slices are easy to prepare, provide good experimental
access and allow precise control of extracellular environments,
they permit the dissection of molecular pathways and the study
of their modulation with pharmacological agents, as well as the
screening of therapeutic molecules or novel genes, without
resource to whole animals (Cho et al., 2007; Lossi et al., 2009).
Moreover, slices can be easily obtained from brain specific
regions, allowing the study of regional-dependent patterns
associated with some neurologic and neurodegenerative
diseases.
A major advantage of using ex vivo tissues is the ability to
perform impossible or unethical studies in living subjects, and
346
Table 1 Experimental systems used to study the bloodbrain barrier: advantages, disadvantages and examples of
applications.
Experimental
system
Advantages
Disadvantages
Applications
In vivo
Allows analysis of whole

brain
Reliable
Frequent necessity of large amounts of live

animals
Requires extrapolation among species
Expensive
Limited application in humans
Not available in every laboratory
Brain metabolism (Hasselbalch et al., 2001)

Disruption assessment (Reinhard et al., 2006)
Transport (Kannan et al., 2009)
Neurological disease progression (Bartels et al.,
2008)
Drug biodistribution (Mano et al., 2002)
Ex vivo
Living tissue
Availability of autopsy
material
Controlled environment
Whole organs or systems
Applicable in humans
Rare human living samples; allows shortterm experiences only; artificial

environment
Post-mortem delay
Neurological diseases (Ebert and Svendsen,

2005; Baskin et al., 2008)
Infectious diseases (Persidsky et al., 2006b)
Neurovascular homeostasis (Mobley et al., 2009)
In silico
Cheaper
Reduction in animals and
reagents
Large numbers of compounds
can be screened rapidly
Based on prediction models

Does not take into account differences
between cells
Conformation of molecules might lead to
false result
Permeability (Chen et al., 2009)

Prediction of permeability (Garg and Verma,
2006)
Transporter properties (Demel et al., 2008)
Toxicity evaluation (Piotrowski et al., 2007)
In vitro
Simplified model
Need of less equipment
Allows the study of a variety
of properties and functions
Applicable in human (mainly
cell lines)
Similar phenotype (primary
cultures)
Might consider cell
interactions (co-cultures)
Does not take microenvironment into

account
Few models consider interactions with other
cells
Difficult to reproduce
Necessity of animals (primary cultures)
Limited passaging (primary cultures)
Differences in phenotype (cell lines)
Permeability (Hawkins et al., 2006; Cowan and

Easton, 2010; Li et al., 2010; Tai et al., 2009)
Transport (Cura and Carruthers, 2010; Poller et
al., 2010)
Disruption (Boveri et al., 2006; Kuhlmann et al.,
2008)
Protein expression (Frster et al., 2008; Perloff et
al., 2007)
Proteomics (Ricardo-Dukelow et al., 2007)
Inflammation (Silwedel and Frster, 2006;
Tweedie et al., 2009)
Toxicity (dos Santos et al., 2010)
Therapeutics (Morofuji et al., 2010)
Hypoxia (Peng et al., 2009)

Evaluation of brain capillaries permeability and
expression of efflux proteins (Boer et al., 2008)
Ex vivo gene therapy (Ebert and Svendsen, 2005)
allowing at the same time to study whole organs or organ

systems. All together these properties make them an attractive alternative/complement to experimentation in vivo and
single cell studies. Ex vivo studies have enabled so far insight
on a large variety of subjects such as Alzheimer's disease and
other neurological diseases (Ebert and Svendsen, 2005; Baskin
et al., 2008), retroviral infection (Afonso et al., 2008), neural
stem-cell therapeutic effects (Kim et al., 2009b), neurovascular
homeostasis (Mobley et al., 2009) and hypoxia (Peng et al.,
2009).
Ex vivo studies may also be performed on post-mortem
tissues. The use of tissue collected at autopsy has been widely
used to circumvent the limited availability of human brain
tissue. Study of TJ (Ballabh et al., 2005), basement membrane
assembly and MMP expression (Bttner et al., 2005; Sulik and
Chyczewski, 2008), as well as evaluation of brain capillary
permeability and expression of efflux proteins (Boer et al.,
2008) have been performed to assess BBB integrity and
function. The usefulness of post-mortem brain tissue is
attested by the number of studies addressing conditions as
neurologic (Harris et al., 2008) and neurodegenerative dis-
orders (Zlokovic, 2008; Weiss et al., 2009), infectious diseases

(Persidsky et al., 2006b; Sulik and Chyczewski, 2008), drug
abuse (Bttner et al., 2005), and angiogenesis (Rigau et al.,
2007), among others. However, the collection and distribution
of high quality human brain tissue from autopsy specimens is
a key component for successful studies since proteins undergo
degradation as a function of the post-mortem interval and
storage temperature. In fact, analysis of the human brain
proteome of prefrontal cortex samples highlighted the quantitative changes occurring in a variety of functional groups,
including metabolic, structural, stress response, and antioxidant proteins as the most vulnerable to degradation (Crecelius
et al., 2008). Therefore, the researcher must be aware of the
limitations concerning the use of post-mortem material as
such changes may render difficult the establishment of
conclusive results.
6.1.3.
In vitro
In vitro modeling of the BBB is a simplification of the in vivo

situation, that allows investigations difficult, or even impossible, to be carried out in vivo (Cecchelli et al., 2007).
Therefore, there is an ongoing pursuit for a faithful in vitro

model of the BBB. Such model should be simple, reproducible,
and mimic as closely as possible the in vivo barrier either
functionally or anatomically in order to allow the study of
BBB-related issues in normal and pathological states, as well
as drug delivery to the CNS. In particular, the cell model must
display a restrictive paracellular pathway, possess a realistic
cell architecture, display functional expression of transporter
mechanisms and cell cultures should be easy (Gumbleton
and Audus, 2001).
Recent improvements in cell cultures made available
several models of the BBB that are tight enough to study the
modulation of transendothelial permeability (Abbott, 2000),
despite the less elaborate TJ between EC in vitro as compared
to those in vivo, where brain microenvironment influences
the molecular composition of TJ (Wolburg et al., 2009). Even
though the in vitro models of the BBB so far available continue
to be labor-intensive and difficult to reproduce, they have
been used to mimic the BBB microenvironment while studying
a large range of properties and functions (Aschner et al., 2006),
as well as to perform studies in different animal species. Most
reports fall upon brain tissue from rat (Perrire et al., 2007),
mouse (Coisne et al., 2005), feline (Fletcher et al., 2009), porcine
(Zhang et al., 2006) and bovine (Zhang et al., 2009a). However,
the results obtained must take into account the differences
between species (Alanne et al., 2009) and care should be taken
in the interpretation of correlations between in vitro and in
vivo approaches as there can be divergent results (Cecchelli et
al., 2007).
In vitro models of the BBB essential rely on cell cultures of
EC as these cells are considered to be the anatomic basis of the
BBB. Although limited by the absence of in vivo signaling and
intercellular contacts, and subjected to the in vitro differentiation and phenotypic modification occurring when cells are
isolated and kept in culture, cell culture systems are still
privileged systems. In fact, they allow the assessment of a
large number of cell functions, biologic processes and disease
mechanisms, and may constitute the first approach in routine
toxicity and pharmacological testing, thus reducing the
number of animals used.
Different categories of brain EC cultures, comprising
primary cell cultures and cell lines, can be used to analyze
features such as cell morphology, energy metabolism and
receptor interaction under the direct effect of the substances
of interest. Primary cell cultures more likely represent a
simplified model of the in vivo condition but require
considerable technical resources and are more time consuming. On the other hand, cell lines are the less complex system
but are those that less resemble the in vivo condition (Thti et
al., 1995). Nevertheless, cell lines of HBMVEC became the most
obvious alternative to perform studies in human BBB models,
due to the rarity of samples from living individuals.
6.1.3.1. Cell lines. Cell lines are generated by immortalization

of BMVEC. They have the advantage of achieving confluence in a
few days, allowing a faster and more frequent access to BMVEC,
as well as a larger amount of cells per experience. Transformation of BMVEC from several species by different immortalizing
genes has originated a number of cell lines (Gumbleton and
Audus, 2001). Cell lines have been generated mostly by SV40 T
347
antigen (Greenwood et al., 1996; Stins et al., 1997), but also by

human papilloma E6E7 gene (Prudhomme et al., 1996), E1A
adenovirus gene (Roux et al., 1994), and Rous sarcoma virus
(Mooradian and Diglio, 1991), as well as by incorporating human
telomerase (Weksler et al., 2005). The rat RBE4 cell line,
originated by E1A adenovirus gene transformation (Roux et al.,
1994), has been extensively used in different type of studies,
namely of transport (Faria et al., 2010) and subcellular localization of transporters (Babakhanian et al., 2007), signaling pathways (Zhang et al., 2009a) and inflammatory cascades (Pan et al.,
2007), toxicity (dos Santos et al., 2010) and toxicity modulation
(Marreilha dos Santos et al., 2008), among others. The mice cell
line bEND3, established using the polyoma middle T-oncogen
(Williams et al., 1989), is commercially available and have also
been used in several studies, as exemplified by those involving
oxidative stress (Betzen et al., 2009), ionizing radiation (BanazYaar et al., 2005), and complement activation (Jacob et al., 2010).
BBB studies in human species profit from the existence of cell
lines obtained from human individuals (Stins et al., 2001;
Weksler et al., 2005), and have been performed in areas such
as infectious diseases (Teng et al., 2010; Dittmar et al., 2008),
neurodegenerative disorders (Bahbouhi et al., 2009) and angiogenesis (Avouac et al., 2008). Despite all the usefulness of cell
lines, they are usually characterized by a poorly differentiated
phenotype compared to their in vivo siblings. Moreover, they
usually present leaky intercellular junctions and lack paracellular barrier properties, which limit their effective use as a
robust in vitro BBB model, particularly when transendothelial
permeability screening is to be performed (Gumbleton and
Audus, 2001). Moreover, it was recently reported complex
karyotype changes, which renders genetic testing of cell lines
mandatory prior to their application in in vitro studies
(Mkrtchyan et al., 2009). Therefore, whenever possible, the use
of primary cultures offers a greater potential as a cell-based in
vitro model of the BBB.
6.1.3.2. Primary cultures. Primary cell cultures of BMVEC,

though technically challenging to isolate and maintain,
represent the closest possible phenotype to the in vivo BBB,
providing a convenient model and the optimal choice for BBB
research (Smith et al., 2007). Since Jo and Karnushina (1973)
successfully isolated viable microvessels and generated the
first in vitro BBB model system from rat brain in 1973, several
attempts to develop in vitro models of the BBB have been
reported, using BMVEC from different species (Smith et al.,
2007; Zenker et al., 2003), namely human (Siddharthan et al.,
2007; Bernas et al., 2010). However, the limited availability of
human brain tissue for culture hampers the utilization of
HBMVEC, the reason why studies with human material are
usually not considered a feasible option; in contrast, bovine
and porcine tissues have received most attention as a source
for brain endothelial cells due to brain size and availability
(Cecchelli et al., 2007).
Although primary cultures of BMVEC have shown to retain
some phenotypic characteristics of brain endothelium, many
problems have been encountered. Some relate to the difficulty
to obtain pure endothelial cultures, as the basal membrane
surrounding the microvascular endothelium also encloses
pericytes that are difficult to remove from the endothelial
fraction (Calabria et al., 2006). Therefore, these cells often
348
contaminate BMVEC cultures. Another problem relates to loss

of many of the functions observed in vivo, assigned to the
removal of BMVEC from local brain microenvironment (Calababria et al., 2006). They rapidly lose their specific characteristics in culture (Nakagawa et al., 2009) undergoing
dedifferentiation and senescence even upon limited passaging, thus hampering usefulness as in vitro models of the
human BBB (Weksler et al., 2005). Part of these inconvenience
may be surpassed by the new and simplified method to obtain
cells for an in vitro model of the human BBB that we have
developed. Cultures obtained are highly pure and reproducible, display characteristic brain endothelial markers and
robust expression of TJ and AJ proteins, as well as of
caveolin-1 and efflux protein P-glycoprotein. Moreover, confluent monolayers present characteristic high TEER and can be
passaged 34 times before phenotypic, cell culture purity,
and barrier properties are affected (Bernas et al., 2010). The
HBMVEC generated from this procedure have been used for in
vitro modeling of the BBB for studies of neuroinflammation
and disease processes (including HIV-1 and Alzheimer's
disease), substance abuse research (alcohol and methamphetamine), and CNS drug delivery (Liu et al., 2007; Haorah et al.,
2008; Ramirez et al., 2008, 2009; Zaghi et al., 2009).
6.1.3.3. Co-cultures. The recognition of the importance of

astrocytes to the induction of BBB properties, as well as of the
interplay between different cellular components of the NVU
on BBB function (Abbott et al., 2006; Cecchelli et al., 2007;
Weiss et al., 2009) has led to the establishment of more
complex in vitro models. Since 1986, research on BBB
functionality/recovery has been very much enhanced by the
availability of in vitro BBB co-culture systems (Beck et al., 1986;
Ramsauer et al., 2002; de Boer and Gaillard, 2006) (Fig. 12). The
discovery of cell-culture inserts with porous filter membranes
allowed the use of BMVEC for permeability studies in vitro
(Nakagawa et al., 2007). In addition, the use of such systems
allows the study in detail of BBB-related phenomena at the
subcellular level in the absence of feedback systems from the
rest of the body (Megard et al., 2002; de Boer and Gaillard,
2006). With co-cultures it has also been possible to evaluate
effects on BMVEC in the presence of other types of cells, as
astrocytes (Ghazanfari and Stewart, 2001; Gaillard et al., 2001;
Jeliazkova-Mecheva and Bobilya, 2003; Perrire et al., 2007; Al
Ahmad et al., 2009), pericytes (Kim et al., 2006; Al Ahmad et al.,
2009), neurons (Cestelli et al., 2001) and more recently

microglia (Sumi et al., 2010). Up until now, studies with
HBMVEC are performed by co-culturing with non-human
astrocytes (Santaguida et al., 2006). Most recently, there have
also been studies with triple cultures of BMVEC, astrocytes and
pericytes (Nakagawa et al., 2007, 2009), as well as with BMVEC,
astrocytes and neurons (Schiera et al., 2005), though all
extracted from rat brain. By incorporating the cross-talk
between BMVEC and neighboring elements of the NVU these
double and triple co-culture systems allow the closest
reproduction of the in vivo condition. However, its complexity
limits the wide utilization of such in vitro models of the BBB.
Co-cultures have brought a new light into the role of EC as
a critical component of the neural stem-cell niche. In fact,
studies performed by Shen et al. (2004) provided evidence that
BMVEC release soluble factors that stimulate neural stem-cell
self-renewal, inhibit their differentiation and enhance neuron
production
6.1.3.4. Dynamic in vitro models. In vivo, BMVEC interact

with astrocytes and are exposed to shear stress through blood
flow, which may influence the high tightness of the BBB in
vivo that has been difficult to reproduce in vitro. This has led
to the generation of more dynamic in vitro models where
these components are also included in order to more closely
mimic the in vivo condition.
The flow-based in vitro BBB model constructed by Siddharthan et al. (2007) consists in co-cultures of HBMVEC and
astrocytes, where inserts are housed in a chamber subjected to
flow. In these conditions, a tighter monolayer than that of
HBMVEC static monocultures is achieved, as revealed by the
increased expression of the TJ protein ZO-1 and the decreased
permeability. Therefore, the functional differentiation of brain
EC by flow-induced forces as well as by the presence of glial
cells or of factors released by them improves the barrier
properties of HBMVEC and approaches the tightness of the EC
monolayer to that existing in vivo, thus rendering this setup a
better model of the human BBB.
Janigro and colleagues (Stanness et al., 1996, 1997; Janigro et
al., 1999; Cucullo et al., 2008) developed an in vitro model of the
BBB characterized by a tridimensional architecture. The model
comprises a hollow-fiber tube or scaffold and incorporates
fluid flow pathways. EC are seeded intraluminally in the
hollow-fiber apparatus, exposed to flow conditions, whereas
Fig. 12 Schematic representation of a co-culture system. A, Double co-culture system where endothelial cells are cultured on
an insert and astrocytes at the bottom of a culture plate well; B, triple co-culture system where endothelial cells and pericytes
are cultured on the top and bottom of an insert, respectively, and astrocytes grow on the bottom of a culture plate well. Adapted
from Nakagawa et al. (2009).
astrocytes are cultured on the extraluminal surface of the tube.

This model displays polarized functional expression of transporters and allows the generation of a restrictive paracellular
pathway. In fact, the values obtained for the permeability and
TEER (Cucullo et al., 2007, 2008), widely used indicators of the
barrier tightness that will be addressed below, are similar to
the corresponding values in vivo (Gumbleton and Audus, 2001).
Moreover, by performing co-cultures of HBMVEC and astrocytes from brain tissue obtained from epileptic patients, it was
possible to mimick an antiepileptic drug-resistant BBB phenotype (Cucullo et al., 2007). Although this is a reliable and
promising in vitro model of the BBB, the required technical
demands hamper its wide use at present.
6.1.4.
In silico
In silico prediction methods are cheaper and less limited than

obtaining experimental data through other methods. Computational models of bloodbrain distribution of drugs are
typically based on in vivo experimental data. The increasing
computational possibilities and development of sophisticated
modeling algorithms have resulted in robust and especially
accurate predictions, with little need to recur to animals (Ekins
et al., 2007; Vastag and Keseru, 2009). Artificial neural network,
or neural network, is one of the novel approaches that have
shown its promising ability in different modeling processes
that can be used in the prediction of BBB permeability
(Mehdipour and Hamidi, 2009; Chen et al., 2009; Cohen-Kashi
Malina et al., 2009; Mensch et al., 2009). They try to predict the
brain permeability to drugs based on a range of physicochemical descriptors resulting from structures of the corresponding
compounds (Mehdipour and Hamidi, 2009). They are also very
useful in prediction of transporter properties (Demel et al.,
2008), channels (Hynna and Boahen, 2009; Huber et al., 2009),
protein activity (Maruszak et al., 2009), and toxicity evaluation
(Piotrowski et al., 2007), between others. In general, such
descriptors are seen to correlate well with in vivo brain drug
penetration (Gumbleton and Audus, 2001). However, and
despite several efforts, most of the models still have some
disadvantages. The major defect relies on the use of only total
concentrations for modeling, thus ignoring other determinants of the permeability process. Moreover, the role of
transporters and of brain microvascular EC metabolism is
underestimated (Gumbleton and Audus, 2001). In addition, it
is neglected the complex nature of BBB, which, collectively,
leads to uncertain results and impairs the establishment of
solid conclusions (Mehdipour and Hamidi, 2009).
6.2.
Assessment of BBB structure and function
There are plenty of approaches to study the BBB. Here, we will

mainly focus on microscopy analysis, which has been extensively used to assess morphology and structure in studies
performed in cell cultures, as well as on fixed cell or tissue
samples. We will also point to the indicators of BBB properties
that are widely used to ascertain BBB function and dysfunction.
6.2.1.
Morphology
Microscopy constitutes a widely used tool in BBB studies,

with a vast application spectrum that depends on the purpose of the analysis. So, from the visible and phase contrast to
349
the electron microscopes there is an array of different

equipments with a variable degree of sophistication and
usefulness.
6.2.1.1. Phase-contrast microscopy. The phase-contrast microscope is an essential equipment to monitor a cell-culture
progression since the isolation until confluence achievement.
For an experienced researcher, it provides important information on the purity of the cell culture, on the presence of
contaminations and on the appropriate timing to use cells for
experiments. A representative phase contrast microscopy
image of a primary culture of HBMEVC is shown in Fig. 13,
where the characteristic cobblestone appearance that confirms
the endothelial nature of the cells is visible. Phase-contrast
microscopy analysis can be extended to other applications such
as the monitorization of EC morphology upon exposure to an
insult as cytokines or oxygen deprivation (Al Ahmad et al., 2009;
Man et al., 2009), or the study of leukocyteendothelial
interactions following an inflammatory stimuli along time in
a dynamic in vitro model of the BBB by capturing microscopic
images on a CCD camera (Man et al., 2009).
6.2.1.2. Visible microscopy. Conventional visible microscopy
also provides an important contribution to BBB studies. For
example, albumin immunoreactivity around blood vessels in
brain paraffin sections allows the detection of focal changes in
BBB permeability (Boer et al., 2008), whereas immunohistochemical analysis of brain tissue permits the detection of
alterations on the expression of relevant proteins for BBB
integrity, as the skeleton protein F-actin and the TJ protein
occludin (Song et al., 2008).
6.2.1.3. Fluorescence microscopy. In what concerns to fluorescence microscopy, it has a privileged role in BBB studies.
In fact, most of the BBB-related proteins can be observed by
immunocytochemical analysis and a fluorescent microscope is
an equipment usually available in a standard laboratory.
Therefore, immunofluorescence analysis of a cell culture allows
its characterization in terms of EC markers, as well as of potential contaminant cells. Moreover, a huge number of publications
can be found in the literature showing immunofluorescence
images of proteins involved in TJ and AJ, CAM, cytoskeleton,
transporters, vesicular trafficking, etc, in several cellular models
of the BBB. Representative immunofluorescence images of
some of these proteins are shown in Fig. 13, where it is possible
to identify individual cells through the blue staining of the
nuclei with Hoechst 33258 dye. Examples provided are for von
Willebrand factor, a widely used marker of endothelial cells;
GLUT-1, the glucose transporter-1 that is currently used to
confirm the presence of BMVEC; and the -smooth muscle actin,
used to detect the presence of pericytes. Furthermore, the stainings for ZO-1 and -catenin, that represent TJ and AJ, respectively, as well as for the marker of the cell vesicular transport
machinery caveolin-1, and the efflux protein P-gp are also
shown. Fluorescence microscopy analysis has contributed to
relevant knowledge on BBB properties and modulation, its disruption by a number of insults and in several pathologies, as
well as its protection by specific agents, and also on the phosphorylation status of some proteins and the activation of signaling cascades, among others (Yamamoto et al., 2008; Al Ahmad et
350
Fig. 13 Morphological features of human brain microvascular endothelial cells observed by phase-contrast microscopy and by
fluorescence microscopy. Confluent monolayer observed by phase-contrast microscopy (A) shows the typical cobblestone
appearance (original magnification: 100). Immunofluorescence staining for glucose transporter-1 (B), von Willebrand factor
(C), -smooth muscle actin (D), zonula occludens-1 (E), -catenin (F), caveolin-1 (G) and p-glycoprotein (H). Nuclei were stained
with Hoechst 33258 dye (B-H). Scale bar = 40 m.
al., 2009; An and Xue, 2009; Siddharthan et al., 2007; Bernas et al.,
2010; Sumi et al., 2010; Tai et al., 2009; Zhong et al., 2008).
6.2.1.4. Confocal laser-scanning microscopy. Though not so

accessible to any laboratory, the confocal laser-scanning
microscope greatly improved the resolution and selectivity
of imaging particular structures by the use of a laser beam, and
has been increasingly used in BBB studies (Man et al., 2009;
Argaw et al., 2009; Nakagawa et al., 2009; Schreibelt et al., 2007;
Song et al., 2007; Sprenger et al., 2006; Virgintino et al., 2002).
6.2.1.5. Electron microscopy. Electron microscopy uses a

beam of electrons to illuminate the specimen and create a
magnified image of it. The microscope has a greater resolving
power because it uses electrons that have wavelengths about
100,000 times shorter than visible light. They can achieve
magnifications of up to 1,000,000 and allow resolution of
structures as small as 1 nm, thus permitting the study of
subcellular morphology. The two main types of electron
microscopy, transmission electron microscopy (TEM) and
scanning electron microscopy (SEM), have been used to
observe a wide range of biological specimens including biopsy

samples, cells and large molecules, among others (Collan et
al., 2005; Filioreanu et al., 2009).
In TEM, the electrons pass through a very thin section of
fixed tissue, some components of which absorb all the
electrons (electron dense), whereas others allow the passage
of all electrons through the other side of the tissue section
(electron lucent). Due to its high resolution, TEM has been
widely used in ultrastructural studies where TJ appear as sites
where the intercellular space between neighboring cells is
obliterated and the adjoining membranes appear to fuse
(Gonzlez-Mariscal et al., 2008). In what concerns to BBB
studies, it can be used to examine TJ and AJ between BMVEC
(Weiss et al., 2009) and the several components of the NVU
(Wolburg et al., 2009), as well as to analyze the TJ assembly in
a triple co-culture in vitro BBB model (Nakagawa et al., 2009)
and the opening of BMVEC TJ upon exposure to insults (Zhang
et al., 2009b). Furthermore, TEM has been used to study
receptor-mediated transcitosis of blood-borne ligands (Ueno,
2007) and to look at the role of caveolae in the transmigration
pathway of Candida albicans through HBMVEC (Lossinsky et al.,
2006).
Immunoelectron TEM, particularly using gold markers, has
undergone a steady development over the past years because
it uniquely provides the ability to quantitatively establish
protein expression at specific tissue, cellular and subcellular
locations. It has also been used to reveal antigens that may be
present in low or trace amounts, and thus contributed to a
greater understanding of cell mechanisms and functional
specialization domains within tissues. Immunoelectron TEM
has shown that ZO-1 is located at the cytoplasmic side of
interendothelial junctions and that occludin, claudin-5 and
JAM are located inside or at close proximity to the interendothelial cleft (Vorbrodt and Dobrogowska, 2003). It has also
shown that - and -catenins are scattered along the
interendothelial junction cleft in the cytoplasmic side of the
plasma membranes and that VE-cadherin is associated with
the EC plasma membrane, inside the interendothelial cleft
(Vorbrodt and Dobrogowska, 2003). Moreover, the quantitative
assessment of gold particles on luminal and abluminal
membranes within the same cell has precisely identified the
amount of immunoreactive epitopes within these two
domains for transporters such as GLUT-1 (Cornford and
Hyman, 2005) and P-gp (Tai et al., 2009).
Since the pioneer studies of Staehelin (1973) and of Wade
and Karnovsky (1974), the freeze-fracturing technique has
been the method of choice to study the structure of TJ. Freezefracturing technique, also referred as cryofracture, relies on
the fact that when living cells are frozen and then fractured
there is a tendency for the fractures to open cells along
membranes and distinct planes, which can then be studied
using the transmission electron microscope. This technique
provides information about the surface features of cell
membranes, where TJ are detected as a network of strands
that encircles the cell bellow the apical surface (GonzlezMariscal et al., 2008). It has been used in BBB studies, not only
to analyze the complexity of junction strands networks in TJ
(Lippoldt et al., 2000; Liebner et al., 2008), but also to visualize
the close apposition of astrocytic endfoot, basal lamina and
brain EC within the NVU (Wolburg et al., 2009).
351
SEM uses solid pieces of tissue rather than ultrathin tissue

sections, and allows perception of three-dimensional views of
the surface of the cells, tissues and subcellular structures. In
SEM, samples are coated with a thin layer of metal such as
gold, and the electron beam scans the specimen in a square
raster pattern. As a result of the interaction of incident
electrons with atoms in the surface layer, low-energy secondary electrons are produced and are converted in a fine threedimensional representation of the surface. SEM has provided a
valuable contribution regarding the characterization of novel
formulations designed to deliver drugs into the CNS. In fact,
there has been dedicated a great effort in the improvement of
the access into the brain of therapeutic agents that do not
cross the BBB. The particulate drug carrier systems composed
of various recipes and manufactured under various conditions
enclosing the therapeutic agents have to be examined for their
properties, such as size, shape and aggregation phenomena,
for which SEM has been used (Feng et al., 2004; Ranganath et
al., 2009; Wilson et al., 2009).
Other types of SEM are further available and have proven to
be useful in BBB studies. One example is the environmental
scanning electron microscopy that allowed the characterization
of a novel flow-based hollow-fiber BBB in vitro model (Neuhaus
et al., 2006). With this technique it is possible to scan biological
material in native and wet status, with samples staying
turgescent during the measurement and shrinking effects due
to preparation hardly occur. Thus, this technique allowed to
prove cell attachment, cell growth and to show BBB characteristic formation of endothelial monolayers and astrocyte endfoot
in the in vitro model, mimicking part of a functional BBB gliovascular unit in vivo.
6.2.1.6. Other types of microscopy. The need to enhance the

resolution and to assess increasingly smaller subcellular
compartments and constituents, as well as to allow the
observation of cellular dynamics and to evaluate real time in
vivo molecular imaging, led to the development of a series of
sophisticated methodologies that also require special preparation of the samples and specialized personnel, are expensive
and are only available in a few laboratories. Some examples
are the atomic force microscopy (Obataya et al., 2005), the
combined video fluorescence and 3D electron microscopy
(Mironov et al., 2008), the atomic force microscopy and
confocal laser-scanning microscope hybrid instrument (Doak
et al., 2008), the multifocal fluorescence fluctuation microscopy (Heuvelman et al., 2009), and the miniaturized confocal laser-scanning fluorescence microscopy (Fottner et al.,
2010).
6.2.2.
Properties
6.2.2.1. Transendothelial electric resistance. TEER is a useful

indicator of paracellular ion flux as it reflects the impedance to
the passage of small ions through a physiological barrier. TEER
has been considered one of the most accurate and sensitive
measures of BBB integrity and barrier function, as a decrease
in TEER reflects an increase in permeability and a loss of
barrier function (Calabria et al., 2006; Cohen-Kashi Malina et
al., 2009). There are several methods to measure TEER: some
are simple and accessible to most laboratories, as those using a
352
voltohmmeter coupled to a pair of electrodes (Fig. 14), whereas

others require more sophisticated equipment connected to a
computer that continuously monitors TEER, as the Electrical
Cell-Substrate Impedance Sensing (ECIS) system from Applied
Biophysics. This system works by using the free ions in the
culture media, which are then used to generate an AC current
flow between an electrode and counter-electrode located in
specialized tissue culture arrays. The instrument measures
continuously the complex impedance, providing readouts for
impedance, resistance and capacitance. TEER readings from
cultures of HBMVEC on collagen-coated electrode arrays can be
monitored until monolayer confluence and barrier formation
occurs (Bernas et al., 2010). Once the steady state TEER is
reached, the barrier can be evaluated in its response to a particular stimulus or modulator and the change from normalized
resistance values of HBMVEC can be recorded.
It has been hard to reproduce in cultured cells the high
TEER normally found across BBB in situ (Santaguida et al.,
2006), and in pial vessels in vivo (Butt, 1995; Revest et al., 1994)
as TEER in BMVEC monocultures is often 100 cm2 or lower
(Calabria and Shusta, 2008). On the other hand, it is difficult to
compare or repeat the obtained results in other laboratories
(Deli et al., 2005). Therefore, improved protocols to fully mimic
and assess in vitro the properties of the BBB in vivo are still to
be developed. The use of dynamic in vitro models, as that
developed by Cucullo et al. (2007, 2008) mentioned above,
appears to be a promising approach once the difficulty in its
implementation in any laboratory is surpassed.
6.2.2.2. Permeability. Elevated permeability of the normally

highly restrictive BBB accompanies a variety of CNS afflictions,
including inflammation, infection, ischemia, stroke, seizures,
and trauma (de Boer and Gaillard, 2006; Song et al., 2007;
Sandoval and Witt, 2008).
Transcellular permeability to small molecule tracers can
also yield valuable information regarding barrier integrity
(Calabria and Shusta, 2008). Most tracers are labeled by a
fluorescent dye or isotope that helps the quantification of the
molecule. Albumin (a 67-kDa protein), for example, is a marker
of transendothelial permeability and it has been observed
in endothelial vesicles (Abbott, 2000; Deli et al., 2005). To
determine the limiting size for permeability, different molec-
Fig. 14 Schematic representation of the transendothelial

electrical resistance measurement with electrodes.
ular weight tracers can be used, such as fluorescent-conjugated dextran (20-,10- and 4-kDa FITC-dextran), propidium iodide
(668 Da), and sodium fluorescein (376 Da) (Ikenouchi et al.,
2005; Krug et al., 2009; Siddharthan et al., 2007; Nakagawa et al.,
2009). In addition to fluorescent labels, the permeability can
also be measured by the use of radioactive labels such as [3H]sucrose (Cucullo et al., 2007) or [3H]-mannitol (Kovac et al.,
2009). Similarly to TEER, the tightness of the barrier and permeability to polar molecules is less stringent in vitro than in
vivo, allowing compounds that would normally poorly penetrate across BBB in vivo to readily diffuse across the endothelial
monolayer in the static model (Santaguida et al., 2006). Also as
for TEER, the in vitro dynamic model (Cucullo et al., 2007, 2008)
appears to be able to approach the in vivo condition.
Evans blue, a dye with high affinity to serum albumin that is
not expected to permeate through the BBB, has been used as an
indicator of BBB permeability, particularly in in vivo studies
(Hawkins and Egleton, 2006; Bellavance et al., 2008; Ding et al.,
2010; Mullier et al., 2010). Following injection in an animal, the
staining at the surface of the brain is evaluated macroscopically against an arbitrary staining scale, providing a qualitative
evaluation of the BBB permeability. Although this approach
has several pitfalls as evaluation is inherently subjective and
only the cortical surface is surveyed (Bellavance et al., 2008), it
is still used nowadays, at least as a first approach to assess BBB
permeability in vivo. Such assessment can be improved by
semiquantitative analysis of brain slices by fluorescence
microscopy (Kozler and Pokorn, 2003) or by quantitative
determination of the dye in brain homogenates (Ay et al., 2008).
The integrity of BBB can also be assessed in in vivo studies
using an electron-dense compound as a flux tracer and TEM
analysis. As an example, lanthanum nitrate can be used whose
appearance in brain parenchyma indicates its extravasation
from capillaries by the interendothelial pathway (Ding et al.,
2010).
6.2.2.3. Intercellular junctions. The main features of the BBB

are the presence of tight intercellular junctions, which strictly
limit the diffusion of blood-borne solutes and cells into the
brain. Several studies have been performed regarding these
junctions, based on commonly used methodologies such as
Western blot and PCR, as well as several types of microscopy, as
addressed above. Using such approaches it was possible to study
the relation between -catenin expression and Down syndrome
in a mouse model (Vorbrodt et al., 2008), the disorganization of
ZO-1 during hypoxia (Lu et al., 2009), the ischemic preconditioning effects on TJ and cell adhesion of BMVEC (An and Xue, 2009),
the caveolae-mediated internalization of occludin and claudin-5
during TJ remodeling in BMVEC (Stamatovic et al., 2009), and the
depletion of intercellular junctions at the site of bacteriahost
cell interaction (Coureuil et al., 2009), among others. In addition
to the evaluation of the expression and distribution of intercellular junctions, the phosphorylation status of such proteins
have been examined and correlated with the barrier function
and monocyte migration across the BBB in encephalitic brain
tissue (Yamamoto et al., 2008).
6.2.2.4. Transport across the BBB. Membrane transporters
and vesicular mechanisms protect the healthy brain,
shielding it from toxic substances and allowing the entrance
of others which are necessary. Some of the most recent

studies regarding this barrier include the regulation of major
efflux transporters under inflammatory conditions (von
Wedel-Parlow et al., 2009), the differential expression and
function of ABC transporters during development and aging
(Bojanic et al., 2010), as well as their regulation by cytokines
(Poller et al., 2010). Also related to ABC transporters,
comparative gene expression profiles have been done in
BMVEC and brain in five species, including the human
(Warren et al., 2009).
The impermeable properties of the BBB constitute an
obstacle to drug delivery to the CNS, essential to treat brain
disorders. Therefore, transport of therapeutic agents across
the BBB is an area of intensive research. The different
ongoing approaches include: the molecular Trojan horses
that use either monoclonal antibodies against BBB receptors
(e.g., insulin and transferrin) through which the therapeutic
molecule passes through the endothelium; the encapsulation
of the drug in nanoparticles conjugated to ligands for BBB
receptors (e.g., LDL apoproteins) to carry drugs across the BBB
via the BBB LDL receptor; delivery via the BBB receptors that
are particularly expressed in disease states (e.g., RAGE in
Alzheimer's disease or stroke); and modulation of TJ to
reversibly open the BBB (Zlokovic, 2008; Deli, 2009).
7.
Concluding remarks
In summary, the anatomic basis of the BBB is composed of a

tightly sealed monolayer of BMVEC, characterized by the
absence of fenestrations, the low number of pinocytic vesicles,
and the elaborated junctional complex formed by TJ and AJ. Due
to the paracellular impermeability of the brain endothelium, the
bi-directional transport of hydrophilic molecules occurs
through BBB transport systems, as the GLUT-1, which transports
glucose and other hexoses into the brain. The members of the
ABC family, P-gp and the multidrug resistance-associated
proteins, which transport several molecules, in particular
xenobiotics, out of the brain, as well as the vesicular trafficking
across the endothelium, as that mediated by caveolae, are also
important in BBB function. Signaling pathways at the EC lining
of brain capillaries have been unraveled and shown to be
implicated in phosphorylation of junction proteins and/or actin
cytoskeletal remodeling, which may lead to BBB breakdown.
Moreover, the BBB involvement in the neuroimmune system is
attested by the transport and secretion of cytokines across the
barrier endothelium, as well as the trafficking of immune cells
through the bloodbrain interface. Finally, several investigation
approaches that have proven to be useful are summarized.
Among these are the in vitro studies relying on double and, more
recently, triple co-cultures with EC, astrocytes, and pericytes or
neurons, as well as the dynamic models where flow is also
included, which appear promising to further explore BBB
properties and dysfunction. Collectively, the most relevant
knowledge about the anatomy of the BBB is put together,
whereas the topics deserving further clarification, as the role of
microglia and neurons in the NVU and the signaling pathways
involved in the regulation of BBB permeability, are raised.
Moreover, advantages and disadvantages of some of the
possible experimental models to study BBB are discussed.
353
Acknowledgments
This work was supported by grant FCT-PTDC/SAU-FCF/68819/
2006 of the Fundao para a Cincia e a Tecnologia, Lisbon,
Portugal.
REFERENCES
Abbott, N.J., 2000. Inflammatory mediators and modulation of

bloodbrain barrier permeability. Cell. Mol. Neurobiol. 20, 131147.
Abbott, N.J., 2002. Astrocyteendothelial interactions and
bloodbrain barrier permeability. J. Anat. 200, 629638.
Abbott, N.J., Ronnback, L., Hansson, E., 2006. Astrocyteendothelial
interactions at the bloodbrain barrier. Nat. Rev. Neurosci. 7,
4153.
Abulrob, A., Brunette, E., Slinn, J., Baumann, E., Stanimirovic, D.,
2008. Dynamic analysis of the bloodbrain barrier disruption in
experimental stroke using time domain in vivo fluorescence
imaging. Mol. Imaging 7, 248262.
Adibhatla, R.M., Hatcher, J.F., 2008. Tissue plasminogen activator
(tPA) and matrix metalloproteinases in the pathogenesis of
stroke: therapeutic strategies. CNS Neurol. Disord. Drug
Targets 7, 243253.
Afonso, P.V., Ozden, S., Cumont, M.-C., Seilhean, D., Cartier, L.,
Rezaie, P., Mason, S., Lambert, S., Huerre, M., Gessain, A.,
Couraud, P.-O., Pique, C., Ceccaldi, P.-E., Romero, I.A., 2008.
Alteration of bloodbrain barrier integrity by retroviral infection.
PLoS Pathog. 4, e1000205.
Al Ahmad, A., Gassmann, M., Ogunshola, O.O., 2009. Maintaining
bloodbrain barrier integrity: pericytes perform better than
astrocytes during prolonged oxygen deprivation. J. Cell.
Physiol. 218, 612622.
Alanne, M.H., Pummi, K., Heape, A.M., Grnman, R., Peltonen, J.,
Peltonen, S., 2009. Tight junction proteins in human Schwann
cell autotypic junctions. J. Histochem. Cytochem. 57, 523529.
Allt, G., Lawrenson, J.G., 2001. Pericytes: cell biology and pathology.
Cells Tissues Organs 169, 111.
al-Obeidi, F.A., Wu, J.J., Lam, K.S., 1998. Protein tyrosine kinases:
structure, substrate specificity, and drug discovery.
Biopolymers 47, 197223.
An, P., Xue, Y.X., 2009. Effects of preconditioning on tight junction
and cell adhesion of cerebral endothelial cells. Brain Res. 1272,
8188.
Anderson, R.G., 1993. Caveolae: where incoming and outgoing
messengers meet. Proc. Natl. Acad. Sci. USA. 90, 1090910913.
Andrs, I.E., Pu, H., Tian, J., Deli, M.A., Nath, A., Hennig, B., Toborek,
M., 2005. Signaling mechanisms of HIV-1 Tat-induced
alterations of claudin-5 expression in brain endothelial cells.
J. Cereb. Blood Flow Metab. 25, 11591170.
Argaw, A.T., Gurfein, B.T., Zhang, Y., Zameer, A., John, G.R., 2009.
VEGF-mediated disruption of endothelial CLN-5 promotes
bloodbrain barrier breakdown. Proc. Natl. Acad. Sci. USA. 106,
19771982.
Aronowski, J., Labiche, L.A., 2003. Perspectives on
reperfusion-induced damage in rodent models of
experimental focal ischemia and role of gamma-protein
kinase C. ILAR J. 44, 105109.
Aschner, M., Fitsanakis, V.A., dos Santos, A.P., Olivi, L., Bressler, J.P.,
2006. Bloodbrain barrier and cellcell interactions: methods for
establishing in vitro models of the bloodbrain barrier and
transport measurements. Methods Mol. Biol. 341, 115.
Astradsson, A., Jenkins, B.G., Choi, J.K., Hallett, P.J., Levesque, M.A.,
McDowell, J.S., Brownell, A.L., Spealman, R.D., Isacson, O., 2009.
The bloodbrain barrier is intact after levodopa-induced
dyskinesias in parkinsonian primatesevidence from in vivo
neuroimaging studies. Neurobiol. Dis. 35, 348351.
354
Aurrand-Lions, M., Johnson-Leger, C., Wong, C., Du Pasquier, L.,

Imhof, B.A., 2001. Heterogeneity of endothelial junctions is
reflected by differential expression and specific subcellular
localization of the three JAM family members. Blood 98,
36993707.
Avouac, J., Wipff, J., Goldman, O., Ruiz, B., Couraud, P.O., Chiocchia,
G., Kahan, A., Boileau, C., Uzan, G., Allanore, Y., 2008.
Angiogenesis in systemic sclerosis: impaired expression of
vascular endothelial growth factor receptor 1 in endothelial
progenitor-derived cells under hypoxic conditions. Arthritis
Rheum. 58, 35503561.
Ay, I., Francis, J.W., Brown, R.H. Jr., 2008. VEGF increases
blood-brain barrier permeability to Evans blue dye and tetanus
toxin fragment C but not adeno-associated virus in ALS mice.
Brain Res. 1234, 198205.
Babakhanian, K., Bendayan, M., Bendayan, R., 2007. Localization of
P-glycoprotein at the nuclear envelope of rat brain cells.
Biochem. Biophys. Res. Commun. 361, 301306.
Bagley, R.G., Weber, W., Rouleau, C., Teicher, B.A., 2005. Pericytes
and endothelial precursor cells: cellular interactions and
contributions to malignancy. Cancer Res. 65, 97419750.
Bahbouhi, B., Berthelot, L., Pettr, S., Michel, L., Wiertlewski, S.,
Weksler, B., Romero, I.A., Miller, F., Couraud, P.O., Brouard, S.,
Laplaud, D.A., Soulillou, J.P., 2009. Peripheral blood CD4+ T
lymphocytes from multiple sclerosis patients are characterized
by higher PSGL-1 expression and transmigration capacity
across a human bloodbrain barrier-derived endothelial cell
line. J. Leukoc. Biol. 86, 10491063.
Ballabh, P., Braun, A., Nedergaard, M., 2004. The bloodbrain
barrier: an overview: structure, regulation, and clinical
implications. Neurobiol. Dis. 16, 113.
Ballabh, P., Hu, F., Kumarasiri, M., Braun, A., Nedergaard, M., 2005.
Development of tight junction molecules in blood vessels of
germinal matrix, cerebral cortex, and white matter. Pediatr.
Res. 58, 791798.
Banaz-Yaar, F., Tischka, R., Iliakis, G., Winterhager, E., Gellhaus,
A., 2005. Cell line specific modulation of connexin43
expression after exposure to ionizing radiation. Cell Commun.
Adhes. 12, 249259.
Banks, W.A., 2006. The bloodbrain barrier as a regulatory
interface in the gut-brain axes. Physiol. Behav. 89, 472476.
Banks, W.A., Erickson, M.A., 2010. The bloodbrain barrier and
immune function and dysfunction. Neurobiol. Dis. 37,
2632.
Banks, W.A., Kastin, A.J., Broadwell, R.D., 1995. Passage of cytokines
across the bloodbrain barrier. Neuroimmunomodulation 2,
241248.
Barakat, S., Demeule, M., Pilorget, A., Regina, A., Gingras, D.,
Baggetto, L.G., Beliveau, R., 2007. Modulation of p-glycoprotein
function by caveolin-1 phosphorylation. J. Neurochem. 101, 18.
Bartels, A.L., Willemsen, A.T., Kortekaas, R., de Jong, B.M., de Vries,
R., de Klerk, O., van Oostrom, J.C., Portman, A., Leenders, K.L.,
2008. Decreased bloodbrain barrier P-glycoprotein function in
the progression of Parkinson's disease, PSP and MSA. J. Neural
Transm. 115, 10011009.
Baskin, L., Urschel, S., Eiberg, B., 2008. A novel ex-vivo application
of RNAi for neuroscience. Biotechniques 45, 338339.
Bauer, A.T., Brgers, H.F., Rabie, T., Marti, H.H., 2010. Matrix
metalloproteinase-9 mediates hypoxia-induced vascular
leakage in the brain via tight junction rearrangement. J. Cereb.
Blood Flow Metab. 30, 837848.
Beck, D.W., Roberts, R.L., Olson, J.J., 1986. Glial cells influence
membrane-associated enzyme activity at the bloodbrain
barrier. Brain Res. 381, 131137.
Blanger, M., Asashima, T., Ohtsuki, S., Yamaguchi, H., Ito, S.,
Terasaki, T., 2007. Hyperammonemia induces transport of
taurine and creatine and suppresses claudin-12 gene
expression in brain capillary endothelial cells in vitro.
Neurochem. Int. 50, 95101.
Bellavance, M.A., Blanchette, M., Fortin, D., 2008. Recent advances

in bloodbrain barrier disruption as a CNS delivery strategy.
AAPS J. 10, 166177.
Bendayan, R., Ronaldson, P.T., Gingras, D., Bendayan, M., 2006. In
situ localization of P-glycoprotein (ABCB1) in human and rat
brain. J. Histochem. Cytochem. 54, 11591167.
Bernacki, J., Dobrowolska, A., Nierwinska, K., Malecki, A., 2008.
Physiology and pharmacological role of the bloodbrain
barrier. Pharmacol. Rep. 60, 600622.
Bernas, M.J., Cardoso, F.L., Daley, S.K., Weinand, M.E., Campos, A.R.,
Ferreira, A.J.G., Hoying, J.B., Witte, M.H., Brites, D., Persidsky, Y.,
Ramirez, S.H., Brito, M.A., 2010. Establishment of primary
cultures of human brain microvascular endothelial cells to
provide an in vitro cellular model of the bloodbrain barrier. Nat.
Protoc. 5, 12471254.
Bernoud, N., Fenart, L., Benistant, C., Pageaux, J.F., Dehouck, M.P.,
Moliere, P., Lagarde, M., Cecchelli, R., Lecerf, J., 1998. Astrocytes
are mainly responsible for the polyunsaturated fatty acid
enrichment in bloodbrain barrier endothelial cells in vitro.
J. Lipid Res. 39, 18161824.
Betz, A.L., 1992. An overview of the multiple functions of the
bloodbrain barrier. NIDA Res. Monogr. 120, 5472.
Betzen, C., White, R., Zehendner, C.M., Pietrowski, E., Bender, B.,
Luhmann, H.J., Kuhlmann, C.R., 2009. Oxidative stress
upregulates the NMDA receptor on cerebrovascular
endothelium. Free Radic. Biol. Med. 47, 12121220.
Birukov, K.G., Csortos, C., Marzilli, L., Dudek, S., Ma, S.F., Bresnick,
A.R., Verin, A.D., Cotter, R.J., Garcia, J.G., 2001. Differential
regulation of alternatively spliced endothelial cell myosin light
chain kinase isoforms by p60(Src). J. Biol. Chem. 276, 85678573.
Boer, K., Troost, D., Jansen, F., Nellist, M., van den Ouweland, A.M.,
Geurts, J.J., Spliet, W.G., Crino, P., Aronica, E., 2008.
Clinicopathological and immunohistochemical findings in an
autopsy case of tuberous sclerosis complex. Neuropathology
28, 577590.
Bojanic, D.D., Tarr, P.T., Gale, G.D., Smith, D.J., Bok, D., Chen, B.,
Nusinowitz, S., Lovgren-Sandblom, A., Bjorkhem, I., Edwards,
P.A., 2010. Differential expression and function of ABCG1 and
ABCG4 during development and aging. J. Lipid Res. 51, 169181.
Boveri, M., Kinsner, A., Berezowski, V., Lenfant, A.M., Draing, C.,
Cecchelli, R., Dehouck, M.P., Hartung, T., Prieto, P., Bal-Price, A.,
2006. Highly purified lipoteichoic acid from gram-positive
bacteria induces in vitro bloodbrain barrier disruption
through glia activation: role of pro-inflammatory cytokines
and nitric oxide. Neuroscience 137, 11931209.
Bowman, P.D., Ennis, S.R., Rarey, K.E., Betz, A.L., Goldstein, G.W.,
1983. Brain microvessel endothelial cells in tissue culture: a
model for study of bloodbrain barrier permeability. Ann.
Neurol. 14, 396402.
Braun, A., Xu, H., Hu, F., Kocherlakota, P., Siegel, D., Chander, P.,
Ungvari, Z., Csiszar, A., Nedergaard, M., Ballabh, P., 2007.
Paucity of pericytes in germinal matrix vasculature of
premature infants. J. Neurosci. 27, 1201212024.
Brown, R.C., Davis, T.P., 2002. Calcium modulation of adherens and
tight junction function: a potential mechanism for
bloodbrain barrier disruption after stroke. Stroke 33, 17061711.
Butt, A.M., 1995. Effect of inflammatory agents on electrical
resistance across the bloodbrain barrier in pial microvessels
of anaesthetized rats. Brain Res. 696, 145150.
Bttner, A., Kroehling, C., Mall, G., Penning, R., Weis, S., 2005.
Alterations of the vascular basal lamina in the cerebral cortex
in drug abuse: a combined morphometric and
immunohistochemical investigation. Drug Alcohol Depend. 79,
6370.
Calabria, A.R., Shusta, E.V., 2008. A genomic comparison of in vivo
and in vitro brain microvascular endothelial cells. J. Cereb.
Calabria, A.R., Weidenfeller, C., Jones, A.R., de Vries, H.E., Shusta, E.V.,
2006. Puromycin-purified rat brain microvascular endothelial cell
cultures exhibit improved barrier properties in response to

glucocorticoid induction. J. Neurochem. 97, 922933.
Carman, C.V., Springer, T.A., 2004. A transmigratory cup in
leukocyte diapedesis both through individual vascular
endothelial cells and between them. J. Cell Biol. 167,
377388.
Carvey, P.M., Hendey, B., Monahan, A.J., 2009. The bloodbrain
barrier in neurodegenerative disease: a rhetorical perspective.
J. Neurochem. 111, 291314.
Cattelino, A., Liebner, S., Gallini, R., Zanetti, A., Balconi, G., Corsi, A.,
Bianco, P., Wolburg, H., Moore, R., Oreda, B., Kemler, R., Dejana,
E., 2003. The conditional inactivation of the -catenin gene in
endothelial cells causes a defective vascular pattern and
increased vascular fragility. J. Cell Biol. 162, 11111122.
Cecchelli, R., Berezowski, V., Lundquist, S., Culot, M., Renftel, M.,
Dehouck, M.P., Fenart, L., 2007. Modelling of the bloodbrain
barrier in drug discovery and development. Nat. Rev. Drug
Discov. 6, 650661.
Cestelli, A., Catania, C., D'Agostino, S., Di Liegro, I., Licata, L.,
Schiera, G., Pitarresi, G.L., Savettieri, G., De Caro, V., Giandalia,
G., Giannola, L.I., 2001. Functional feature of a novel model of
blood brain barrier: studies on permeation of test compounds.
J. Control. Release 76, 139147.
Chaudhuri, J.D., 2000. Blood brain barrier and infection. Med. Sci.
Monit. 6, 12131222.
Chen, Y., Zhu, Q.J., Pan, J., Yang, Y., Wu, X.P., 2009. A prediction model
for bloodbrain barrier permeation and analysis on its parameter
biologically. Comput. Meth. Programs Biomed. 95, 280287.
Chenn, A., 2008. beta-catenin/-catenin signaling in cerebral
cortical development. Organogenesis 4, 7680.
Cho, S., Wood, A., Bowlby, M.R., 2007. Brain slices as models for
neurodegenerative disease and screening platforms to identify
novel therapeutics. Curr. Neuropharmacol. 5, 1933.
Choi, Y.K., Kim, K.W., 2008. Bloodneural barrier: its diversity and
coordinated cell-to-cell communication. BMB Rep. 41, 345352.
Cirino, G., Fiorucci, S., Sessa, W.C., 2003. Endothelial nitric oxide
synthase: the Cinderella of inflammation? Trends Pharmacol.
Sci. 24, 9195.
Cohen-Kashi Malina, K.C., Cooper, I., Teichberg, V.I., 2009. Closing
the gap between the in-vivo and in-vitro bloodbrain barrier
tightness. Brain Res. 1284, 1221.
Coisne, C., Dehouck, L., Faveeuw, C., Delplace, Y., Miller, F., Landry,
C., Morissette, C., Fenart, L., Cecchelli, R., Tremblay, P.,
Dehouck, B., 2005. Mouse syngenic in vitro bloodbrain barrier
model: a new tool to examine inflammatory events in cerebral
endothelium. Lab. Invest. 85, 734746.
Coisne, C., Mao, W., Engelhardt, B., 2009. Cutting edge:
Natalizumab blocks adhesion but not initial contact of human
T cells to the bloodbrain barrier in vivo in an animal model of
multiple sclerosis. J. Immunol. 182, 59095913.
Colgan, O.C., Collins, N.T., Ferguson, G., Murphy, R.P., Birney, Y.A.,
Cahill, P.A., Cummins, P.M., 2008. Influence of basolateral
condition on the regulation of brain microvascular endothelial
tight junction properties and barrier function. Brain Res. 1193,
8492.
Collan, Y., Hirsimki, P., Aho, H., Wuorela, M., Sundstrm, J., Tertti,
R., Metsrinne, K., 2005. Value of electron microscopy in kidney
biopsy diagnosis. Ultrastruct. Pathol. 29, 461468.
Cook, B.D., Ferrari, G., Pintucci, G., Mignatti, P., 2008. TGF-1
induces rearrangement of FLK-1VE-cadherin-catenin
complex at the adherens junction through VEGF-mediated
signalling. J. Cell. Biochem. 105, 13671373.
Cordenonsi, M., D'Atri, F., Hammar, E., Parry, D.A., Kendrick-Jones,
J., Shore, D., Citi, S., 1999. Cingulin contains globular and
coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3 and
myosin. J. Cell Biol. 147, 15691582.
Cornford, E.M., Hyman, S., 2005. Localization of brain endothelial
luminal and abluminal transporters with immunogold electron
microscopy. NeuroRx. 2, 2743.
355
Coureuil, M., Mikaty, G., Miller, F., Lecuyer, H., Bernard, C.,
Bourdoulous, S., Dumenil, G., Mege, R.M., Weksler, B.B.,
Romero, I.A., Couraud, P.O., Nassif, X., 2009. Meningococcal
type IV pili recruit the polarity complex to cross the brain
endothelium. Science 325, 8387.
Cowan, K.M., Easton, A.S., 2010. Neutrophils block permeability
increases induced by oxygen glucose deprivation in a culture
model of the human model bloodbrain barrier. Brain Res.
1332, 2031.
Crecelius, A., Gtz, A., Arzberger, T., Arnold, G.J., Ferrer, I.,
Kretzschmar, H.A., 2008. Assessing quantitative post-mortem
changes in the gray matter of the human frontal cortex
proteome by 2-D DIGE. Proteomics 8, 12761291.
Cross, T.G., Scheel-Toellner, D., Henriquez, N.V., Deacon, E.,
Salmon, M., Lord, J.M., 2000. Serine/threonine protein kinases
and apoptosis. Exp. Cell Res. 256, 3441.
Cucullo, L., Hossain, M., Rapp, E., Manders, T., Marchi, N., Janigro,
D., 2007. Development of a humanized in vitro bloodbrain
barrier model to screen for brain penetration of antiepileptic
drugs. Epilepsia 48, 505516.
Cucullo, L., Couraud, P.O., Weksler, B., Romero, I.A., Hossain, M.,
Rapp, E., Janigro, D., 2008. Immortalized human brain
endothelial cells and flow-based vascular modeling: a marriage
of convenience for rational neurovascular studies. J. Cereb.
Cunningham, L.A., Wetzel, M., Rosenberg, G.A., 2005. Multiple
roles for MMPs and TIMPs in cerebral ischemia. Glia 50,
329339.
Cura, A.J., Carruthers, A., 2010. Acute modulation of sugar
transport in brain capillary endothelial cell cultures during
activation of the metabolic stress pathway. J. Biol. Chem. 285,
1543015439.
D'Arcangelo, D., Ambrosino, V., Giannuzzo, M., Gaetano, C.,
Capogrossi, M.C., 2006. Axl receptor activation mediates
laminar shear stress anti-apoptotic effects in human
endothelial cells. Cardiovasc. Res. 71, 754763.
de Boer, A.G., Gaillard, P.J., 2006. Bloodbrain barrier dysfunction
and recovery. J. Neural Transm. 113, 455462.
Deli, M.A., 2009. Potential use of tight junction modulators to
reversibly open membranous barriers and improve drug
delivery. Biochim. Biophys. Acta 1788, 892910.
Deli, M.A., brahm, C.S., Kataoka, Y., Niwa, M., 2005. Permeability
studies on in vitro bloodbrain barrier models: physiology,
pathology, and pharmacology. Cell. Mol. Neurobiol. 25, 59127.
Demel, M.A., Schwaha, R., Krmer, O., Ettmayer, P., Haaksma, E.E.J.,
Ecker, G.F., 2008. In silico prediction of substrate properties for
ABC-multidrug transporters. Expert Opin. Drug Metab. Toxicol.
4, 11671180.
Demeule, M., Jodoin, J., Gingras, D., Beliveau, R., 2000. P-glycoprotein
is localized in caveolae in resistant cells and in brain capillaries.
FEBS Lett. 466, 219224.
Dempsey, E.C., Newton, A.C., Mochly-Rosen, D., Fields, A.P.,
Reyland, M.E., Insel, P.A., Messing, R.O., 2000. Protein kinase C
isozymes and the regulation of diverse cell responses. Am. J.
Physiol. Lung Cell. Mol. Physiol. 279, L429L438.
Ding, G.R., Qiu, L.B., Wang, X.W., Li, K.C., Zhou, Y.C., Zhou, Y.,
Zhang, J., Zhou, J.X., Li, Y.R., Guo, G.Z., 2010. EMP-induced
alterations of tight junction protein expression and disruption
of the bloodbrain barrier. Toxicol. Lett. 196, 154160.
Dittmar, S., Harms, H., Runkler, N., Maisner, A., Kim, K.S.,
Schneider-Schaulies, J., 2008. Measles virus-induced block of
transendothelial migration of T lymphocytes and
infection-mediated virus spread across endothelial cell
barriers. J. Virol. 82, 1127311282.
Doak, S.H., Rogers, D., Jones, B., Francis, L., Conlan, R.S., Wright, C.,
2008. High-resolution imaging using a novel atomic force
microscope and confocal laser scanning microscope hybrid
instrument: essential sample preparation aspects. Histochem.
Cell Biol. 130, 909916.
356
Dohgu, S., Takata, F., Yamauchi, A., Nakagawa, S., Egawa, T., Naito,
M., Tsuruo, T., Sawada, Y., Niwa, M., Kataoka, Y., 2005. Brain
pericytes contribute to the induction and up-regulation of
bloodbrain barrier functions through transforming growth
factor- production. Brain Res. 1038, 208215.
Dore-Duffy, P., 2008. Pericytes: pluripotent cells of the blood brain
barrier. Curr. Pharm. Des. 14, 15811593.
Dore-Duffy, P., Katychev, A., Wang, X., Van Buren, E., 2006. CNS
microvascular pericytes exhibit multipotential stem cell
activity. J. Cereb. Blood Flow Metab. 26, 613624.
Drfel, M.J., Westphal, J.K., Huber, O., 2009. Differential
phosphorylation of occludin and tricellulin by CK2 and CK1.
Ann. NY Acad. Sci. 165, 6973.
dos Santos, A.P.M., Milatovic, D., Au, C., Yin, Z., Batoreu, M.C.C.,
Aschner, M., 2010. Rat brain endothelial cells are a target of
manganese toxicity. Brain Res. 1326, 152161.
Ebert, A.D., Svendsen, C.N., 2005. A new tool in the battle against
Alzheimer's disease and aging: ex vivo gene therapy.
Rejuvenation Res. 8, 131134.
Edelman, D.A., Jiang, Y., Tyburski, J., Wilson, R.F., Steffes, C., 2006.
Pericytes and their role in microvasculature homeostasis.
J. Surg. Res. 135, 305311.
Ehrlich, P., 1885. Das sauerstoff-bedrfnis des organismus. Eine
Farbenanalytische Studie. Habilitation thesis, Berlin.
Ehrlich, P., 1906. Ueber die beziehungen von chemischer
constitution, vertheilung, und pharmakologischen wirkung.
Collected Studies on Immunity. Wiley, Berlin, pp. 404442.
Ekins, S., Mestres, J., Testa, B., 2007. In silico pharmacology for drug
discovery: applications to targets and beyond. Br. J. Pharmacol.
152, 2137.
Eliceiri, B.P., Paul, R., Schwartzberg, P.L., Hood, J.D., Leng, J.,
Cheresh, D.A., 1999. Selective requirement for Src kinases
during VEGF-induced angiogenesis and vascular permeability.
Mol. Cell 4, 915924.
Engelhardt, B., 2009. PSGL-1the hidden player in T cell trafficking
into the brain in multiple sclerosis? J. Leukoc. Biol. 86,
10231025.
Erickson, K.K., Sundstrom, J.M., Antonetti, D.A., 2007. Vascular
permeability in ocular disease and the role of tight junctions.
Angiogenesis 10, 103117.
Etienne-Manneville, S., Hall, A., 2002. Rho GTPases in cell biology.
Nature 420, 629635.
Etienne-Manneville, S., Manneville, J.B., Adamson, P., Wilbourn, B.,
Greenwood, J., Couraud, P.O., 2000. ICAM-1-coupled
cytoskeletal rearrangements and transendothelial lymphocyte
migration involve intracellular calcium signaling in brain
endothelial cell lines. J. Immunol. 165, 33753383.
Faria, A., Pestana, D., Teixeira, D., Azevedo, J., De Freitas, V., Mateus,
N., Calhau, C., 2010. Flavonoid transport across RBE4 cells: a
bloodbrain barrier model. Cell. Mol. Biol. Lett. 15, 234241.
Farrall, A.J., Wardlaw, J.M., 2009. Bloodbrain barrier: ageing and
microvascular diseasesystematic review and meta-analysis.
Neurobiol. Aging 30, 337352.
Feng, S.S., Mu, L., Win, K.Y., Huang, G., 2004. Nanoparticles of
biodegradable polymers for clinical administration of
paclitaxel. Curr. Med. Chem. 11, 413424.
Filioreanu, A.M., Popescu, E., Cotrutz, C., Cotrutz, C.E., 2009.
Immunohistochemical and transmission electron microscopy
study regarding myofibroblasts in fibroinflammatory epulis
and giant cell peripheral granuloma. Rom. J. Morphol. Embryol.
50, 363368.
Fischer, S., Wiesnet, M., Marti, H.H., Renz, D., Schaper, W., 2004.
Simultaneous activation of several second messengers in
hypoxia-induced hyperpermeability of brain derived
endothelial cells. J. Cell. Physiol. 198, 359369.
Fischer, S., Wiesnet, M., Renz, D., Schaper, W., 2005. H2O2 induces
paracellular permeability of porcine brain-derived
microvascular endothelial cells by activation of the p44/42 MAP
kinase pathway. Eur. J. Cell Biol. 84, 687697.
Fleegal, M.A., Hom, S., Borg, L.K., Davis, T.P., 2005. Activation of
PKC modulates bloodbrain barrier endothelial cell
permeability changes induced by hypoxia and posthypoxic
reoxygenation. Am. J. Physiol. Heart Circ. Physiol. 289,
H2012H2019.
Fletcher, N.F., Bexiga, M.G., Brayden, D.J., Brankin, B., Willett, B.J.,
Hosie, M.J., Jacque, J.M., Callanan, J.J., 2009. Lymphocyte
migration through the blood brain barrier (BBB) in feline
immunodeficiency virus infection is significantly influenced
by the pre-existence of virus and tumour necrosis factor
(TNF)- within the central nervous system (CNS): studies using
an in vitro feline BBB model. Neuropathol. Appl. Neurobiol. 35,
592602.
Frster, C., Burek, M., Romero, I.A., Weksler, B., Couraud, P.O.,
Drenckhahn, D., 2008. Differential effects of hydrocortisone
and TNF on tight junction proteins in an in vitro model of the
human bloodbrain barrier. J. Physiol. 586, 19371949.
Fottner, C., Mettler, E., Goetz, M., Schirrmacher, E., Anlauf, M.,
Strand, D., Schirrmacher, R., Klppel, G., Delaney, P.,
Schreckenberger, M., Galle, P.R., Neurath, M.F., Kiesslich, R.,
Weber, M.M., 2010. In vivo molecular imaging of somatostatin
receptors in pancreatic islet cells and neuroendocrine tumors
by miniaturized confocal laser-scanning fluorescence
microscopy. Endocrinology 151, 21792188.
Francis, K., van Beek, J., Canova, C., Neal, J.W., Gasque, P., 2003.
Innate immunity and brain inflammation: the key role of
complement. Expert Rev. Mol. Med. 5, 119.
Furuse, M., 2009. Knockout animals and natural mutations as
experimental and diagnostic tool for studying tight junction
functions in vivo. Biochim. Biophys. Acta 788, 813819.
Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S.,
Tsukita, S., Tsukita, S., 1993. Occludin: a novel integral
membrane protein localizing at tight junctions. J. Cell Biol. 123,
17771788.
Gaillard, P.J., Voorwinden, L.H., Nielsen, J.L., Ivanov, A., Atsumi, R.,
Engman, H., Ringbom, C., de Boer, A.G., Breimer, D.D., 2001.
Establishment and functional characterization of an in vitro
model of the bloodbrain barrier, comprising a co-culture of
brain capillary endothelial cells and astrocytes. Eur. J. Pharm.
Sci. 12, 215222.
Garcia, J.G., Verin, A.D., Schaphorst, K., Siddiqui, R., Patterson, C.E.,
Csortos, C., Natarajan, V., 1999. Regulation of endothelial cell
myosin light chain kinase by Rho, cortactin, and p60(src). Am. J.
Physiol. 276, L989L998.
Garg, P., Verma, J., 2006. In silico prediction of blood brain barrier
permeability: an Artificial Neural Network model. J. Chem. Inf.
Model. 46, 289297.
Gavard, J., Gutkind, J.S., 2006. VEGF controls endothelial-cell
permeability by promoting the -arrestin-dependent
endocytosis of VE-cadherin. Nat. Cell Biol. 8, 12231234.
Gazzin, S., Strazielle, N., Schmitt, C., Fevre-Montange, M., Ostrow,
J.D., Tiribelli, C., Ghersi-Egea, J.F., 2008. Differential expression
of the multidrug resistance-related proteins ABCb1 and
ABCc1 between bloodbrain interfaces. J. Comp. Neurol. 510,
497507.
Ge, S., Song, L., Pachter, J.S., 2005. Where is the bloodbrain
barrier ... really? J. Neurosci. Res. 79, 421427.
Ghazanfari, F.A., Stewart, R.R., 2001. Characteristics of endothelial
cells derived from the bloodbrain barrier and of astrocytes in
culture. Brain Res. 890, 4965.
Goldberg, J.S., Hirschi, K.K., 2009. Diverse roles of the vasculature
within the neural stem cell niche. Regen. Med. 4, 879897.
Goldmann, E., 1913. Vitalfarbung am zentralnervensystem.
Abhandl Konigl preuss Akad Wiss. 1, 160.
Gonzlez-Mariscal, L., Betanzos, A., Nava, P., Jaramillo, B.E., 2003.
Tight junction proteins. Prog. Biophys. Mol. Biol. 81, 144.
Gonzlez-Mariscal, L., Tapia, R., Chamorro, D., 2008. Crosstalk of
tight junction components with signaling pathways. Biochim.
Biophys. Acta 1778, 729756.
Gonzlez-Mariscal, L., Tapia, R., Huerta, M., Lopez-Bayghen, E.,

2009. The tight junction protein ZO-2 blocks cell cycle
progression and inhibits cyclin D1 expression. Ann. NY Acad.
Sci. 1165, 121125.
Gosselin, D., Rivest, S., 2008. MyD88 signaling in brain endothelial
cells is essential for the neuronal activity and glucocorticoid
release during systemic inflammation. Mol. Psychiatry 13,
480497.
Gow, A., Southwood, C.M., Li, J.S., Pariali, M., Riordan, G.P., Brodie,
S.E., Danias, J., Bronstein, J.M., Kachar, B., Lazzarini, R.A., 1999.
CNS myelin and sertoli cell tight junction strands are absent in
Osp/claudin-11 null mice. Cell 99, 649659.
Greenwood, J., Pryce, G., Devine, L., Male, D.K., dos Santos, W.L.,
Calder, V.L., Adamson, P., 1996. SV40 large T immortalised cell
lines of the rat bloodbrain and bloodretinal barriers retain
their phenotypic and immunological characteristics. J.
Neuroimmunol. 71, 5163.
Gumbleton, M., Audus, K.L., 2001. Progress and limitations in the
use of in vitro cell cultures to serve as a permeability screen for
the bloodbrain barrier. J. Pharm. Sci. 90, 16811698.
Gupta, I.R., Ryan, A.K., 2010. Claudins: unlocking the code to tight
junction function during embryogenesis and in disease. Clin.
Genet. 77, 314325.
Hamabe, W., Fujita, R., Ueda, H., 2005. Insulin receptor-protein
kinase C- signaling mediates inhibition of
hypoxia-induced necrosis of cortical neurons. J. Pharmacol.
Exp. Ther. 313, 10271034.
Haorah, J., Knipe, B., Leibhart, J., Ghorpade, A., Persidsky, Y., 2005.
Alcohol-induced oxidative stress in brain endothelial cells
causes bloodbrain barrier dysfunction. J. Leukoc. Biol. 78,
12231232.
Haorah, J., Ramirez, S.H., Schall, K., Smith, D., Pandya, R.,
Persidsky, Y., 2007a. Oxidative stress activates protein tyrosine
kinase and matrix metalloproteinases leading to bloodbrain
barrier dysfunction. J. Neurochem. 101, 566576.
Haorah, J., Knipe, B., Gorantla, S., Zheng, J., Persidsky, Y., 2007b.
Alcohol-induced bloodbrain barrier dysfunction is mediated
via inositol 1, 4, 5-triphosphate receptor (IP3R)-gated
intracellular calcium release. J. Neurochem. 100, 324336.
Haorah, J., Schall, K., Ramirez, S.H., Persidsky, Y., 2008. Activation of
protein tyrosine kinases and matrix metalloproteinases causes
bloodbrain barrier injury: novel mechanism for
neurodegeneration associated with alcohol abuse. Glia 56, 7888.
Harhaj, N.S., Felinski, E.A., Wolpert, E.B., Sundstrom, J.M., Gardner,
T.W., Antonetti, D.A., 2006. VEGF activation of protein kinase C
stimulates occludin phosphorylation and contributes to
endothelial permeability. Invest Ophthalmol. Vis. Sci. 47,
51065115.
Harris, L.W., Wayland, M., Lan, M., Ryan, M., Giger, T., Lockstone,
H., Wuethrich, I., Mimmack, M., Wang, L., Kotter, M., Craddock,
R., Bahn, S., 2008. The cerebral microvasculature in
schizophrenia: a laser capture microdissection study. PLoS
ONE 3, e3964.
Hasselbalch, S.G., Knudsen, G.M., Capaldo, B., Postiglione, A.,
Paulson, O.B., 2001. Bloodbrain barrier transport and brain
metabolism of glucose during acute hyperglycemia in humans.
J. Clin. Endocrinol. Metab. 86, 19861990.
Hawkins, B.T., Davis, T.P., 2005. The bloodbrain
barrier/neurovascular unit in health and disease. Pharmacol.
Rev. 57, 173185.
Hawkins, B.T., Egleton, R.D., 2006. Fluorescence imaging of
bloodbrain barrier disruption. J. Neurosci. Methods 151,
262267.
Hawkins, R.A., Peterson, D.R., Vina, J.R., 2002. The complementary
membranes forming the bloodbrain barrier. IUBMB Life 54,
101107.
Hawkins, R.A., O'Kane, R.L., Simpson, I.A., Vina, J.R., 2006.
Structure of the bloodbrain barrier and its role in the transport
of amino acids. J. Nutr. 136, 218S226S.
357
Hayashi, Y., Nomura, M., Yamagishi, S., Harada, S., Yamashita, J.,
Yamamoto, H., 1997. Induction of various bloodbrain barrier
properties in non-neural endothelial cells by close apposition
to co-cultured astrocytes. Glia 19, 1326.
Heuvelman, G., Erdel, F., Wachsmuth, M., Rippe, K., 2009. Analysis
of protein mobilities and interactions in living cells by
multifocal fluorescence fluctuation microscopy. Eur. Biophys. J.
38, 813828.
Hirase, T., Staddon, J.M., Saitou, M., Ando-Akatsuka, Y., Itoh, M.,
Furuse, M., Fujimoto, K., Tsukita, S., Rubin, L.L., 1997. Occludin
as a possible determinant of tight junction permeability in
endothelial cells. J. Cell Sci. 110, 16031613.
Hobbs, A.J., Higgs, A., Moncada, S., 1999. Inhibition of nitric oxide
synthase as a potential therapeutic target. Annu. Rev.
Pharmacol. Toxicol. 39, 191220.
Hofmann, J., 1997. The potential for isoenzyme-selective
modulation of protein kinase C. FASEB J. 11, 649669.
Holland, S.J., Powell, M.J., Franci, C., Chan, E.W., Friera, A.M.,
Atchison, R.E., McLaughlin, J., Swift, S.E., Pali, E.S., Yam, G.,
Wong, S., Lasaga, J., Shen, M.R., Yu, S., Xu, W., Hitoshi, Y.,
Bogenberger, J., Nr, J.E., Payan, D.G., Lorens, J.B., 2005. Multiple
roles for the receptor tyrosine kinase axl in tumor formation.
Cancer Res. 65, 92949303.
Holmes, K., Roberts, O.L., Thomas, A.M., Cross, M.J., 2007. Vascular
endothelial growth factor receptor-2: structure, function,
intracellular signalling and therapeutic inhibition. Cell. Signal.
19, 20032012.
Honda, M., Nakagawa, S., Hayashi, K., Kitagawa, N., Tsutsumi, K.,
Nagata, I., Niwa, M., 2006. Adrenomedullin improves the
bloodbrain barrier function through the expression of
claudin-5. Cell. Mol. Neurobiol. 26, 109118.
Huber, J.D., Egleton, R.D., Davis, T.P., 2001. Molecular physiology
and pathophysiology of tight junctions in the bloodbrain
barrier. Trends Neurosci. 24, 719725.
Huber, V.J., Tsujita, M., Nakada, T., 2009. Identification of
aquaporin 4 inhibitors using in vitro and in silico methods.
Bioorg. Med. Chem. 17, 411417.
Hynna, K.M., Boahen, K.A., 2009. Nonlinear influence of T-channels
in an in silico relay neuron. IEEE Trans. Biomed. Eng. 56,
17341743.
Ikenouchi, J., Furuse, M., Furuse, K., Sasaki, H., Tsukita, S., Tsukita,
S., 2005. Tricellulin constitutes a novel barrier at tricellular
contacts of epithelial cells. J. Cell Biol. 171, 939945.
Jacob, A., Hack, B., Chiang, E., Garcia, J.G., Quigg, R.J., Alexander, J.J.,
2010. C5a alters bloodbrain barrier integrity in experimental
lupus. FASEB J. 24, 16821688.
Janigro, D., Leaman, S.M., Stanness, K.A., 1999. Dynamic modeling
of the bloodbrain barrier: a novel tool for studies of drug
delivery to the brain. Pharm. Sci. Technol. Today 2, 712.
Jeliazkova-Mecheva, V.V., Bobilya, D.J., 2003. A porcine
astrocyte/endothelial cell co-culture model of the bloodbrain
barrier. Brain Res. Protoc. 12, 9198.
Jiang, S., Khan, M.I., Lu, Y., Rathbone, M.P., 2005. Acceleration of
bloodbrain barrier formation after transplantation of enteric
glia into spinal cords of rats. Exp. Brain Res. 162, 5662.
Jo, F., Karnushina, I., 1973. A procedure for the isolation of
capillaries from rat brain. Cytobios 8, 4148.
Kannan, P., John, C., Zoghbi, S.S., Halldin, C., Gottesman, M.M.,
Innis, R.B., Hall, M.D., 2009. Imaging the function of
P-glycoprotein with radiotracers: pharmacokinetics and in vivo
applications. Clin. Pharmacol. Ther. 86, 368377.
Kaur, C., Ling, E.A., 2008. Blood brain barrier in hypoxicischemic
conditions. Curr. Neurovasc. Res. 5, 7181.
Kelley, C., D'Amore, P., Hechtman, H.B., Shepro, D., 1987.
Microvascular pericyte contractility in vitro: comparison
with other cells of the vascular wall. J. Cell Biol. 104,
483490.
Kevil, C.G., Okayama, N., Alexander, J.S., 2001. H2O2-mediated
permeability II: importance of tyrosine phosphatase and
358
kinase activity. Am. J. Physiol. Cell Physiol. 281,

C1940C1947.
Khan, E., 2005. An examination of the bloodbrain barrier in health
and disease. Br. J. Nurs. 14, 509513.
Kierszenbaum, A.L., 2007. Histology and Cell Biology: An
Introduction to Pathology. Mosby Elsevier, Canada.
Kim, J.A., Tran, N.D., Li, Z., Yang, F., Zhou, W., Fisher, M.J., 2006.
Brain endothelial hemostasis regulation by pericytes. J. Cereb.
Kim, M.P., Park, S.I., Kopetz, S., Gallick, G.E., 2009a. Src family
kinases as mediators of endothelial permeability: effects on
inflammation and metastasis. Cell Tissue Res. 335, 249259.
Kim, H.M., Hwang, D.H., Lee, J.E., Kim, S.U., Kim, B.G., 2009b. Ex
vivo VEGF delivery by neural stem cells enhances proliferation
of glial progenitors, angiogenesis, and tissue sparing after
spinal cord injury. PLoS ONE 4, e4987.
Kniesel, U., Wolburg, H., 2000. Tight junctions of the bloodbrain
barrier. Cell. Mol. Neurobiol. 20, 5776.
Kone, B.C., 2000. Proteinprotein interactions controlling nitric
oxide synthases. Acta Physiol. Scand. 168, 2731.
Korshunov, V.A., Daul, M., Massett, M.P., Berk, B.C., 2007. Axl
mediates vascular remodeling induced by deoxycorticosterone
acetate salt hypertension. Hypertension 50, 10571062.
Kovac, A., Zilkova, M., Deli, M.A., Zilka, N., Novak, M., 2009. Human
truncated tau is using a different mechanism from
amyloid-beta to damage the bloodbrain barrier. J. Alzheimers
Dis. 18, 897906.
Kozler, P., Pokorn, J. 2003. Altered blood-brain barrier
permeability and its effect on the distribution of Evans blue
and sodium fluorescein in the rat brain applied by intracarotid
injection. Physiol. Res. 52, 60714
Krause, G., Winkler, L., Mueller, S.L., Haseloff, R.F., Piontek, J.,
Blasig, I.E., 2008. Structure and function of claudins. Biochim.
Biophys. Acta 1778, 631645.
Krizbai, I.A., Deli, M.A., 2003. Signalling pathways regulating the
tight junction permeability in the bloodbrain barrier. Cell Mol.
Biol. 49, 2331.
Krizbai, I.A., Bauer, H., Bresgen, N., Eckl, P.M., Farkas, A., Szatmri,
E., Traweger, A., Wejksza, K., Bauer, H.C., 2005. Effect of
oxidative stress on the junctional proteins of cultured cerebral
endothelial cells. Cell. Mol. Neurobiol. 25, 129139.
Krug, S.M., Amasheh, S., Richter, J.F., Milatz, S., Gnzel, D.,
Westphal, J.K., Huber, O., Schulzke, J.D., Fromm, M., 2009.
Tricellulin forms a barrier to macromolecules in tricellular
tight junctions without affecting ion permeability. Mol. Biol.
Cell 20, 37133724.
Kuhlmann, C.R.W., Gerigk, M., Bender, B., Closhen, D., Lessman, V.,
Luhmann, H.J., 2008. Fluvastatin prevents glutamate-induced
bloodbrain barrier disruption in vitro. Life Sci. 82, 12811287.
Kumar, P., Shen, Q., Pivetti, C.D., Lee, E.S., Wu, M.H., Yuan, S.Y.,
2009. Molecular mechanisms of endothelial hyperpermeability:
implications in inflammation. Expert Rev. Mol. Med. 30, 11:e19.
Lai, C.H., Kuo, K.H., 2005. The critical component to establish in
vitro BBB model: pericyte. Brain Res. Brain Res. Rev. 50, 258265.
Lai, C.H., Kuo, K.H., Leo, J.M., 2005. Critical role of actin in
modulating BBB permeability. Brain Res. Brain Res. Rev. 50,
713.
Lee, S.J., Benveniste, E.N., 1999. Adhesion molecule expression
and regulation on cells of the central nervous system. J.
Lee, H.S., Namkoong, K., Kim, D.H., Kim, K.J., Cheong, Y.H., Kim, S.S.,
Lee, W.B., Kim, K.Y., 2004. Hydrogen peroxide-induced
alterations of tight junction proteins in bovine brain
microvascular endothelial cells. Microvasc. Res. 68, 231238.
Lee, S.W., Kim, W.J., Park, J.A., Choi, Y.K., Kwon, Y.W., Kim, K.W.,
2006. Bloodbrain barrier interfaces and brain tumors. Arch.
Pharm. Res. 29, 265275.
Lewandowsky, M., 1900. Zur lehre von der cerebrospinalflussigkeit.
Z. Klin. Med. 40, 480494.
Li, G., Simon, M.J., Cancel, L.M., Ji, X., Tarbell, J.M., Morrison III, B.,
Fu, B.M., 2010. Permeability of endothelial and astrocyte
cocultures: in vitro bloodbrain barrier models for drug delivery
studies. Ann. Biomed. Eng. 38, 24992511.
Liebner, S., Corada, M., Bangsow, T., Babbage, J., Taddei, A., Czupalla,
C.J., Reis, M., Felici, A., Wolburg, H., Fruttiger, M., Taketo, M.M.,
von Melchner, H., Plate, K.H., Gerhardt, H., Dejana, E., 2008.
Wnt/-catenin signaling controls development of the
bloodbrain barrier. J. Cell Biol. 183, 409417.
Lim, J.C., Kania, K.D., Wijesuriya, H., Chawla, S., Sethi, J.K., Pulaski,
L., Romero, I.A., Couraud, P.O., Weksler, B.B., Hladky, S.B.,
Barrand, M.A., 2008. Activation of -catenin signalling by GSK-3
inhibition increases p-glycoprotein expression in brain
endothelial cells. J. Neurochem. 106, 18551865.
Lippoldt, A., Kniesel, U., Liebner, S., Kalbacher, H., Kirsch, T.,
Wolburg, H., Haller, H., 2000. Structural alterations of tight
junctions are associated with loss of polarity in stroke-prone
spontaneously hypertensive rat bloodbrain barrier
endothelial cells. Brain Res. 885, 251261.
Liu, T., DeCostanzo, A.J., Liu, X., Wang, H., Hallagan, S., Moon, R.T.,
Malbon, C.C., 2001. G protein signaling from activated rat
frizzled-1 to the -cateninLefTcf pathway. Science 292,
17181722.
Liu, N.Q., Lossinsky, A.S., Popik, W., Li, X., Gujuluva, C., Kriederman,
B., Roberts, J., Pushkarsky, T., Bukrinsky, M., Witte, M.,
Weinand, M., Fiala, M., 2002. Human immunodeficiency virus
type 1 enters brain microvascular endothelia by
macropinocytosis dependent on lipid rafts and the
mitogen-activated protein kinase signaling pathway. J. Virol.
76, 66896700.
Liu, J., Johnson, T.V., Lin, J., Ramirez, S.H., Bronich, T.K., Caplan, S.,
Persidsky, Y., Gendelman, H.E., Kipnis, J., 2007. T cell
independent mechanism for copolymer-1-induced
neuroprotection. Eur. J. Immunol. 37, 31433154.
Lossi, L., Alasia, S., Salio, C., Merighi, A., 2009. Cell death and
proliferation in acute slices and organotypic cultures of
mammalian CNS. Prog. Neurobiol. 88, 221245.
Lossinsky, A.S., Jong, A., Fiala, M., Mukhtar, M., Buttle, K.F., Ingram,
M., 2006. The histopathology of Candida albicans invasion in
neonatal rat tissues and in the human bloodbrain barrier in
culture revealed by light, scanning, transmission and
immunoelectron microscopy. Histol. Histopathol. 21,
10291041.
Lu, D.Y., Yu, W.H., Yeh, W.L., Tang, C.H., Leung, Y.M., Wong, K.L.,
Chen, Y.F., Lai, C.H., Fu, W.M., 2009. Hypoxia-induced matrix
metalloproteinase-13 expression in astrocytes enhances
permeability of brain endothelial cells. J. Cell. Physiol. 220,
163173.
Ma, T.Y., Tran, D., Hoa, N., Nguyen, D., Merryfield, M., Tarnawski,
A., 2000. Mechanism of extracellular calcium regulation of
intestinal epithelial tight junction permeability: role of
cytoskeletal involvement. Microsc. Res. Tech.
51, 156168.
Mahajan, S.D., Aalinkeel, R., Sykes, D.E., Reynolds, J.L., Bindukumar,
B., Adal, A., Qi, M., Toh, J., Xu, G., Prasad, P.N., Schwartz, S.A., 2008.
Methamphetamine alters blood brain barrier permeability via
the modulation of tight junction expression: implication for
HIV-1 neuropathogenesis in the context of drug abuse. Brain Res.
1203, 133148.
Man, S., Ubogu, E.E., Ransohoff, R.M., 2007. Inflammatory cell
migration into the central nervous system: a few new twists on
an old tale. Brain Pathol. 17, 243250.
Man, S., Tucky, B., Bagheri, N., Li, X., Kochar, R., Ransohoff, R.M.,
2009. 4 Integrin/FN-CS1 mediated leukocyte adhesion to brain
microvascular endothelial cells under flow conditions. J.
Mano, Y., Higuchi, S., Kamimura, H., 2002. Investigation of the high
partition of YM992, a novel antidepressant, in rat brain in
vitro and in vivo evidence for the high binding in brain and the
high permeability at the BBB. Biopharm. Drug Dispos. 23,

351360.
Marchi, N., Cavaglia, M., Fazio, V., Bhudia, S., Hallene, K., Janigro,
D., 2004. Peripheral markers of bloodbrain barrier damage.
Clin. Chim. Acta 342, 112.
Marreilha dos Santos, A.P., Santos, D., Au, C., Milatovic, D.,
Aschner, M., Batoru, M.C., 2008. Antioxidants prevent the
cytotoxicity of manganese in RBE4 cells. Brain Res. 1236,
200205.
Maruszak, A., Safranow, K., Gustaw, K., Kijanowska-Haadyna, B.,
Jakubowska, K., Olszewska, M., Styczyska, M., Berdyski, M.,
Tysarowski, A., Chlubek, D., Siedlecki, J., Barcikowska, M.,
Zekanowski, C., 2009. PIN1 gene variants in Alzheimer's
disease. BMC Med. Genet. 10, 115 doi:10.1186/1471-2350-10-115
Mayhan, W.G., 1991. Disruption of the bloodbrain barrier in open
and closed cranial window preparations in rats. Stroke 22,
10591063.
Mayhan, W.G., 2000. Nitric oxide donor-induced increase in
permeability of the blood-brain barrier. Brain Res. 866, 101108.
Megard, I., Garrigues, A., Orlowski, S., Jorajuria, S., Clayette, P.,
Ezan, E., Mabondzo, A., 2002. A co-culture-based model of
human blood-brain barrier: application to active transport of
indinavir and in vivo-in vitro correlation. Brain Res. 927,
153167.
Mehdipour, A.R., Hamidi, M., 2009. Brain drug targeting: a
computational approach for overcoming bloodbrain barrier.
Drug Discov. Today 14, 10301036.
Mensch, J., Oyarzabal, J., Mackie, C., Augustijns, P., 2009. In vivo, in
vitro and in silico methods for small molecule transfer across
the BBB. J. Pharm. Sci. 98, 44294468.
Miller, F., Fenart, L., Landry, V., Coisne, C., Cecchelli, R., Dehouck,
M.P., Bue-Scherrer, V., 2005. The MAP kinase pathway
mediates transcytosis induced by TNF- in an in vitro
bloodbrain barrier model. Eur. J. Neurosci. 22, 835844.
Mironov, A.A., Polishchuk, R.S., Beznoussenko, G.V., 2008.
Combined video fluorescence and 3D electron microscopy.
Methods Cell Biol. 88, 8395.
Mitic, L.L., Anderson, J.M., 1998. Molecular architecture of tight
junctions. Annu. Rev. Physiol. 60, 121142.
Mkrtchyan, H., Scheler, S., Klein, I., Fahr, A., Couraud, P.O., Romero,
I.A., Weksler, B., Liehr, T., 2009. Molecular cytogenetic
characterization of the human cerebral microvessel endothelial
cell line hCMEC/D3. Cytogenet. Genome Res. 126, 313317.
Mobley, A.K., Tchaicha, J.H., Shin, J., Hossain, M.G., McCarty, J.H.,
2009. 8 integrin regulates neurogenesis and neurovascular
homeostasis in the adult brain. J. Cell Sci. 122, 18421851.
Moon, R.T. 2005. Wnt/beta-catenin pathway. Sci STKE. 2005, cm1.
Mooradian, D.L., Diglio, C.A., 1991. Production of a transforming
growth factor-beta-like growth factor by RSV-transformed rat
cerebral microvascular endothelial cells. Tumour Biol. 12,
171183.
Morofuji, Y., Nakagawa, S., So, G., Hiu, T., Horai, S., Hayashi, K.,
Tanaka, K., Suyama, K., Deli, M.A., Nagata, I., Niwa, M., 2010.
Pitavastatin strengthens the barrier integrity in primary
cultures of rat brain endothelial cells. Cell. Mol. Neurobiol. 30,
727735.
Mullier, A., Bouret, S.G., Prevot, V., Dehouck, B., 2010. Differential
distribution of tight junction proteins suggests a role for
tanycytes in bloodhypothalamus barrier regulation in the
adult mouse brain. J. Comp. Neurol. 518, 943962.
Nabi, I.R., Le, P.U., 2003. Caveolae/raft-dependent endocytosis. J.
Cell Biol. 161, 673677.
Nakagawa, S., Deli, M.A., Nakao, S., Honda, M., Hayashi, K.,
Nakaoke, R., Kataoka, Y., Niwa, M., 2007. Pericytes from brain
microvessels strengthen the barrier integrity in primary
cultures of rat brain endothelial cells. Cell. Mol. Neurobiol. 27,
687694.
Nakagawa, S., Deli, M.A., Kawaguchi, H., Shimizudani, T., Shimono,
T., Kittel, A., Tanaka, K., Niwa, M., 2009. A new bloodbrain
359
barrier model using primary rat brain endothelial cells,

pericytes and astrocytes. Neurochem. Int. 54, 253263.
Nakashima, I., Kato, M., Akhand, A.A., Suzuki, H., Takeda, K.,
Hossain, K., Kawamoto, Y., 2002. Redox-linked signal
transduction pathways for protein tyrosine kinase activation.
Antioxid. Redox Signal. 4, 517531.
Navarro, P., Ruco, L., Dejana, E., 1998. Differential localization of
VE- and N-cadherins in human endothelial cells: VE-cadherin
competes with N-cadherin for junctional localization. J. Cell
Biol. 140, 14751484.
Neuhaus, W., Lauer, R., Oelzant, S., Fringeli, U.P., Ecker, G.F., Noe,
C.R., 2006. A novel flow based hollow-fiber bloodbrain barrier
in vitro model with immortalised cell line PBMEC/C12. J.
Biotechnol. 125, 127141.
Neuhaus, W., Wirth, M., Plattner, V.E., Germann, B., Gabor, F., Noe,
C.R., 2008. Expression of Claudin-1, Claudin-3 and Claudin-5 in
human bloodbrain barrier mimicking cell line ECV304 is
inducible by glioma-conditioned media. Neurosci. Lett. 446,
5964.
Nishioku, T., Dohgu, S., Takata, F., Eto, T., Ishikawa, N., Kodama,
K.B., Nakagawa, S., Yamauchi, A., Kataoka, Y., 2009.
Detachment of brain pericytes from the basal lamina is
involved in disruption of the bloodbrain barrier caused by
lipopolysaccharide-induced sepsis in mice. Cell. Mol.
Neurobiol. 29, 309316.
Nitta, T., Hata, M., Gotoh, S., Seo, Y., Sasaki, H., Hashimoto, N.,
Furuse, M., Tsukita, S., 2003. Size-selective loosening of the
bloodbrain barrier in claudin-5-deficient mice. J. Cell Biol. 161,
653660.
Norsted, E., Gomuc, B., Meister, B., 2008. Protein components of the
bloodbrain barrier (BBB) in the mediobasal hypothalamus. J.
Chem. Neuroanat. 36, 107121.
Numaguchi, K., Shimokawa, H., Nakaike, R., Egashira, K., Takeshita,
A., 1996. PKC inhibitors prevent endothelial dysfunction after
myocardial ischemiareperfusion in rats. Am. J. Physiol. 270,
H1634H1639.
Nwariaku, F.E., Liu, Z., Zhu, X., Turnage, R.H., Sarosi, G.A., Terada,
L.S., 2002. Tyrosine phosphorylation of vascular endothelial
cadherin and the regulation of microvascular permeability.
Surgery 132, 180185.
Obataya, I., Nakamura, C., Han, S., Nakamura, N., Miyake, J., 2005.
Nanoscale operation of a living cell using an atomic force
microscope with a nanoneedle. Nano Lett. 5, 2730.
Ogasawara, N., Kojima, T., Go, M., Fuchimoto, J., Kamekura, R.,
Koizumi, J., Ohkuni, T., Masaki, T., Murata, M., Tanaka, S.,
Ichimiya, S., Himi, T., Sawada, N., 2009. Induction of JAM-A
during differentiation of human THP-1 dendritic cells. Biochem.
Biophys. Res. Commun. 389, 543549.
Pan, W., Kastin, A.J., 2002. TNF transport across the bloodbrain
barrier is abolished in receptor knockout mice. Exp. Neurol.
174, 193200.
Pan, W., Kastin, A.J., Daniel, J., Yu, C., Baryshnikova, L.M., von
Bartheld, C.S., 2007. TNF trafficking in cerebral vascular
endothelial cells. J. Neuroimmunol. 185, 4756.
Pardridge, W.M., 1999. Bloodbrain barrier biology and methodology.
J. Neurovirol. 5, 556569.
Peng, Y.J., Nanduri, J., Yuan, G., Wang, N., Deneris, E., Pendyala, S.,
Natarajan, V., Kumar, G.K., Prabhakar, N.R., 2009. NADPH
oxidase is required for the sensory plasticity of the carotid
body by chronic intermittent hypoxia. J. Neurosci. 29,
49034910.
Peppiatt, C.M., Howarth, C., Mobbs, P., Attwell, D., 2006.
Bidirectional control of CNS capillary diameter by pericytes.
Nature 443, 700704.
Perez-Moreno, M., Davis, M.A., Wong, E., Pasolli, H.A., Reynolds, A.B.,
Fuchs, E., 2006. p120-catenin mediates inflammatory responses
in the skin. Cell 124, 631644.
Perloff, M.D., von Moltke, L.L., Fahey, J.M., Greenblatt, D.J., 2007.
Induction of P-glycoprotein expression and activity by ritonavir
360
in bovine brain microvessel endothelial cells. J. Pharm.

Pharmacol. 59, 947953.
Perrire, N., Yousif, S., Cazaubon, S., Chaverot, N., Bourasset, F.,
Cisternino, S., Declves, X., Hori, S., Terasaki, T., Deli, M.,
Scherrmann, J.M., Temsamani, J., Roux, F., Couraud, P.O., 2007.
A functional in vitro model of rat bloodbrain barrier for
molecular analysis of efflux transporters. Brain Res.
1150, 113.
Persidsky, Y., Ramirez, S.H., Haorah, J., Kanmogne, G.D., 2006a.
Blood-brain barrier: structural components and function under
physiologic and pathologic conditions. J. Neuroimmune
Persidsky, Y., Heilman, D., Haorah, J., Zelivyanskaya, M., Persidsky,
R., Weber, G.A., Shimokawa, H., Kaibuchi, K., Ikezu, T., 2006b.
Rho-mediated regulation of tight junctions during monocyte
migration across the bloodbrain barrier in HIV-1 encephalitis
(HIVE). Blood 107, 47704780.
Petty, M.A., Lo, E.H., 2002. Junctional complexes of the bloodbrain
barrier: permeability changes in neuroinflammation. Prog.
Neurobiol. 68, 311323.
Piotrowski, P.L., Sumpter, B.G., Malling, H.V., Wassom, J.S., Lu, P.Y.,
Brothers, R.A., Sega, G.A., Martin, S.A., Parang, M., 2007. A
toxicity evaluation and predictive system based on neural
networks and wavelets. J. Chem. Inf. Model. 47, 676685.
Polakis, P., 2008. Formation of the bloodbrain barrier: Wnt
signaling seals the deal. J. Cell Biol. 183, 371373.
Poller, B., Drewe, J., Krahenbuhl, S., Huwyler, J., Gutmann, H., 2010.
Regulation of BCRP (ABCG2) and P-glycoprotein (ABCB1) by
cytokines in a model of the human bloodbrain barrier. Cell.
Mol. Neurobiol. 30, 6370.
Poole, A.W., Pula, G., Hers, I., Crosby, D., Jones, M.L., 2004.
PKC-interacting proteins: from function to pharmacology.
Trends Pharmacol. Sci. 25, 528535.
Prudhomme, J.G., Sherman, I.W., Land, K.M., Moses, A.V., Stenglein,
S., Nelson, J.A., 1996. Studies of Plasmodium falciparum
cytoadherence using immortalized human brain capillary
endothelial cells. Int. J. Parasitol. 26, 647655.
Quan, N., 2008. Immune-to-brain signaling: how important are the
bloodbrain barrier-independent pathways? Mol. Neurobiol.
37, 142152.
Radisavljevic, Z., Avraham, H., Avraham, S., 2000. Vascular
endothelial growth factor up-regulates ICAM-1 expression via
the phosphatidylinositol 3 OH-kinase/AKT/Nitric oxide pathway
and modulates migration of brain microvascular endothelial
cells. J. Biol. Chem. 275, 2077020774.
Ramirez, S.H., Heilman, D., Morsey, B., Potula, R., Haorah, J.,
Persidsky, Y., 2008. Activation of peroxisome
proliferator-activated receptor gamma (PPARgamma)
suppresses Rho GTPases in human brain microvascular
endothelial cells and inhibits adhesion and transendothelial
migration of HIV-1 infected monocytes. J. Immunol. 180,
18541865.
Ramirez, S.H., Potula, R., Fan, S., Eidem, T., Papugani, A.,
Reichenbach, N., Dykstra, H., Weksler, B.B., Romero, I.A.,
Couraud, P.O., Persidsky, Y., 2009. Methamphetamine disrupts
bloodbrain barrier function by induction of oxidative stress in
brain endothelial cells. J. Cereb. Blood Flow Metab. 29,
19331945.
Ramrez-Castillejo, C., Snchez-Snchez, F., Andreu-Agull, C.,
Frron, S.R., Aroca-Aguilar, J.D., Sanchz, P., Mira, H., Escribano,
J., Farias, I., 2006. Pigment epithelium-derived factor is a niche
signal for neural stem cell renewal. Nat. Neurosci. 9,
331339.
Ramsauer, M., Krause, D., Dermietzel, R., 2002. Angiogenesis of the
bloodbrain barrier in vitro and the function of cerebral
pericytes. FASEB J. 16, 12741276.
Ranganath, S.H., Kee, I., Krantz, W.B., Chow, P.K., Wang, C.H., 2009.
Hydrogel matrix entrapping PLGA-paclitaxel microspheres:
drug delivery with near zero-order release and implantability
advantages for malignant brain tumour chemotherapy. Pharm.

Res. 26, 21012114.
Ransohoff, R.M., Perry, V.H., 2009. Microglial physiology: unique
stimuli, specialized responses. Annu. Rev. Immunol. 27,
119145.
Reese, T.S., Karnovsky, M.J., 1967. Fine structural localization of a
bloodbrain barrier to exogenous peroxidase. J. Cell Biol. 34,
207217.
Reinhard, M., Hetzel, A., Krger, S., Talazko, J., Ziyeh, S., Weber, J.,
Els, T., 2006. Bloodbrain barrier disruption by low-frequency
ultrasound. Stroke 37, 15461548.
Revest, P.A., Jones, H.C., Abbott, N.J., 1994. Transendothelial
electrical potential across pial vessels in anaesthetised rats: a
study of ion permeability and transport at the bloodbrain
barrier. Brain Res. 652, 7682.
Ricardo-Dukelow, M., Kadiu, I., Rozek, W., Schlautman, J., Persidsky,
Y., Ciborowski, P., Kanmogne, G.D., Gendelman, H.E., 2007. HIV-1
infected monocyte-derived macrophages affect the human brain
microvascular endothelial cell proteome: new insights into
bloodbrain barrier dysfunction for HIV-1-associated dementia.
J. Neuroimmunol. 185, 3746.
Rigau, V., Morin, M., Rousset, M.C., de Bock, F., Lebrun, A., Coubes,
P., Picot, M.C., Baldy-Moulinier, M., Bockaert, J., Crespel, A.,
Lerner-Natoli, M., 2007. Angiogenesis is associated with
bloodbrain barrier permeability in temporal lobe epilepsy.
Brain 130, 19421956.
Romanitan, M.O., Popescu, B.O., Spulber, S., Bajenaru, O., Popescu,
L.M., Winblad, B., Bogdanovic, N., 2009. Altered expression of
claudin family proteins in Alzheimer's disease and vascular
dementia brains. J. Cell. Mol. Med. 14, 10881100.
Rosell, A., Ortega-Aznar, A., Alvarez-Sabin, J., Fernandez-Cadenas,
I., Ribo, M., Molina, C.A., Lo, E.H., Montaner, J., 2006. Increased
brain expression of matrix metalloproteinase-9 after ischemic
and hemorrhagic human stroke. Stroke 37, 13991406.
Rosenberg, G.A., Yang, Y., 2007. Vasogenic edema due to tight
junction disruption by matrix metalloproteinases in cerebral
ischemia. Neurosurg. Focus 22, E4.
Roura, S., Miravet, S., Piedra, J., Garca de Herreros, A., Duach, M.,
1999. Regulation of E-cadherin/catenin association by tyrosine
phosphorylation. J. Biol. Chem. 274, 3673436740.
Roux, F., Durieu-Trautmann, O., Chaverot, N., Claire, M., Mailly, P.,
Bourre, J.M., Strosberg, A.D., Couraud, P.O., 1994. Regulation of
gamma-glutamyl transpeptidase and alkaline phosphatase
activities in immortalized rat brain microvessel endothelial
cells. J. Cell. Physiol. 159, 101113.
Sandoval, K.E., Witt, K.A., 2008. Bloodbrain barrier tight
junction permeability and ischemic stroke. Neurobiol. Dis. 32,
200219.
Sandoval, R., Malik, A.B., Minshall, R.D., Kouklis, P., Ellis, C.A.,
Tiruppathi, C., 2001. Ca2+ signalling and PKC activate
increased endothelial permeability by disassembly of
VE-cadherin junctions. J. Physiol. 533, 433445.
Santaguida, S., Janigro, D., Hossain, M., Oby, E., Rapp, E., Cucullo, L.,
2006. Side by side comparison between dynamic versus static
models of bloodbrain barrier in vitro: a permeability study.
Brain Res. 1109, 113.
Sawada, N., Liao, J.K., 2009. Targeting eNOS and beyond: emerging
heterogeneity of the role of endothelial Rho proteins in stroke
protection. Expert Rev. Neurother. 9, 11711186.
Scherrmann, J.M., 2002. Drug delivery to brain via the bloodbrain
barrier. Vascul. Pharmacol. 38, 349354.
Schiera, G., Sala, S., Gallo, A., Raffa, M.P., Pitarresi, G.L., Savettieri, G.,
Di Liegro, I., 2005. Permeability properties of a three-cell type
in vitro model of bloodbrain barrier. J. Cell. Mol. Med. 9,
373379.
Schreibelt, G., Kooij, G., Reijerkerk, A., van Doorn, R., Gringhuis, S.I.,
van der Pol, S., Weksler, B.B., Romero, I.A., Couraud, P.O.,
Piontek, J., Blasig, I.E., Dijkstra, C.D., Ronken, E., de Vries, H.E.,
2007. Reactive oxygen species alter brain endothelial tight
junction dynamics via RhoA, PI3 kinase, and PKB signaling.

FASEB J. 21, 36663676.
Schller, U., Rowitch, D.H., 2007. -catenin function is required for
cerebellar morphogenesis. Brain Res. 1140, 161169.
Shasby, D.M., Ries, D.R., Shasby, S.S., Winter, M.C., 2002. Histamine
stimulates phosphorylation of adherens junction proteins and
alters their link to vimentin. Am. J. Physiol. Lung Cell. Mol.
Physiol. 282, L1330L1338.
Shen, Q., Goderie, S.K., Jin, L., Karanth, N., Sun, Y., Abramova, N.,
Vincent, P., Pumiglia, K., Temple, S., 2004. Endothelial cells
stimulate self-renewal and expand neurogenesis of neural
stem cells. Science 304, 13381340.
Shepro, D., Morel, N.M.L., 1993. Pericyte physiology. FASEB J. 7,
10311038.
Shimizu, F., Sano, Y., Maeda, T., Abe, M.A., Nakayama, H.,
Takahashi, R., Ueda, M., Ohtsuki, S., Terasaki, T., Obinata, M.,
Kanda, T., 2008. Peripheral nerve pericytes originating from the
bloodnerve barrier expresses tight junctional molecules and
transporters as barrier-forming cells. J. Cell. Physiol. 217,
388399.
Shukla, A., Dikshit, M., Srimal, R.C., 1996. Nitric oxide-dependent
blood-brain barrier permeability alteration in the rat brain.
Experientia 52, 136140.
Siddharthan, V., Kim, Y.V., Liu, S., Kim, K.S., 2007. Human
astrocytes/astrocyte-conditioned medium and shear stress
enhance the barrier properties of human brain microvascular
endothelial cells. Brain Res. 1147, 3950.
Silva, R.F.M., Falco, A.S., Fernandes, A., Gordo, A.C., Brito, M.A.,
Brites, D., 2006. Dissociated primary nerve cell cultures as
models for assessment of neurotoxicity. Toxicol. Lett. 163, 19.
Silwedel, C., Frster, C., 2006. Differential susceptibility of cerebral
and cerebellar murine brain microvascular endothelial cells to
loss of barrier properties in response to inflammatory stimuli.
J. Neuroimmunol. 179, 3745.
Simionescu, M., Gafencu, A., Antohe, F., 2002. Transcytosis of
plasma macromolecules in endothelial cells: a cell biological
survey. Microsc. Res. Tech. 57, 269288.
Singh, A.K., Jiang, Y., Gupta, S., Benlhabib, E., 2007. Effects of
chronic ethanol drinking on the blood brain barrier and
ensuing neuronal toxicity in alcohol-preferring rats
subjected to intraperitoneal LPS injection. Alcohol Alcohol. 42,
385399.
Slusarski, D.C., Corces, V.G., Moon, R.T., 1997. Interaction of Wnt
and a Frizzled homologue triggers G-protein-linked
phosphatidylinositol signalling. Nature 390, 410413.
Smith, M.W., Gumbleton, M., 2006. Endocytosis at the bloodbrain
barrier: from basic understanding to drug delivery strategies. J.
Drug Target. 14, 191214.
Smith, M., Omidi, Y., Gumbleton, M., 2007. Primary porcine brain
microvascular endothelial cells: biochemical and functional
characterisation as a model for drug transport and targeting. J.
Drug Target. 15, 253268.
Somanath, P.R., Razorenova, O.V., Chen, J., Byzova, T.V., 2006. Akt1
in endothelial cell and angiogenesis. Cell Cycle 5, 512518.
Song, L., Ge, S., Pachter, J.S., 2007. Caveolin-1 regulates expression
of junction-associated proteins in brain microvascular
endothelial cells. Blood 109, 15151523.
Song, Y., Xue, Y., Liu, X., Wang, P., Liu, L., 2008. Effects of acute
exposure to aluminum on bloodbrain barrier and the
protection of zinc. Neurosci. Lett. 445, 4246.
Spranger, J., Verma, S., Ghring, I., Bobbert, T., Seifert, J., Sindler,
A.L., Pfeiffer, A., Hileman, S.M., Tschp, M., Banks, W.A., 2006.
Adiponectin does not cross the bloodbrain barrier but
modifies cytokine expression of brain endothelial cells.
Diabetes 55, 141147.
Sprenger, R.R., Fontijn, R.D., van Marle, J., Pannekoek, H.,
Horrevoets, A.J., 2006. Spatial segregation of transport and
signalling functions between human endothelial caveolae and
lipid raft proteomes. Biochem. J. 400, 401410.
361
Staehelin, L.A., 1973. Further observations on the fine structure of

freeze-cleaved tight junctions. J. Cell Sci. 13, 763786.
Stamatovic, S.M., Keep, R.F., Andjelkovic, A.V., 2008. Brain
endothelial cellcell junctions: how to open the blood brain
barrier. Curr. Neuropharmacol. 6, 179192.
Stamatovic, S.M., Keep, R.F., Wang, M.M., Jankovic, I., Andjelkovic,
A.V., 2009. Caveolae-mediated internalization of occludin and
claudin-5 during CCL2-induced tight junction remodeling in
brain endothelial cells. J. Biol. Chem. 284, 1905319066.
Stanness, K.A., Guatteo, E., Janigro, D., 1996. A dynamic model of
the bloodbrain barrier in vitro. Neurotoxicology 17, 481496.
Stanness, K.A., Westrum, L.E., Fornaciari, E., Mascagni, P., Nelson,
J.A., Stenglein, S.G., Myers, T., Janigro, D., 1997. Morphological
and functional characterization of an in vitro bloodbrain
barrier model. Brain Res. 771, 329342.
Stevenson, B.R., Anderson, J.M., Braun, I.D., Mooseker, M.S., 1989.
Phosphorylation of the tight-junction protein ZO-1 in two
strains of MadinDarby canine kidney cells which differ in
transepithelial resistance. Biochem. J. 263, 597599.
Stins, M.F., Prasadarao, N.V., Zhou, J., Arditi, M., Kim, K.S., 1997.
Bovine brain microvascular endothelial cells transfected with
SV40-large T antigen: development of an immortalized cell line
to study pathophysiology of CNS disease. In Vitro Cell. Dev.
Biol. Anim. 33, 243247.
Stins, M.F., Badger, J., Kim, K.S., 2001. Bacterial invasion and
transcytosis in transfected human brain microvascular
endothelial cells. Microb. Pathog. 30, 1928.
Sulik, A., Chyczewski, L., 2008. Immunohistochemical analysis of
MMP-9, MMP-2 and TIMP-1, TIMP-2 expression in the central
nervous system following infection with viral and bacterial
meningitis. Folia Histochem. Cytobiol. 46, 437442.
Sumi, N., Nishioku, T., Takata, F., Matsumoto, J., Watanabe, T.,
Shuto, H., Yamauchi, A., Dohgu, S., Kataoka, Y., 2010.
Lipopolysaccharide-activated microglia induce dysfunction of
the bloodbrain barrier in rat microvascular endothelial cells
co-cultured with microglia. Cell. Mol. Neurobiol. 30, 247253.
Thti, H., Vaalavirta, L., Toimela, T., 1995. In vitro methods as a
tool in neurotoxicity studies. Altern. Lab. Anim. 23,
491496.
Tai, L.M., Reddy, P.S., Lopez-Ramirez, M.A., Davies, H.A., Male, D.K.,
Loughlin, A.J., Romero, I.A., 2009. Polarized P-glycoprotein
expression by the immortalised human brain endothelial cell
line, hCMEC/D3, restricts apical-to-basolateral permeability to
rhodamine 123. Brain Res. 1292, 1424.
Tao, Y.S., Edwards, R.A., Tubb, B., Wang, S., Bryan, J., McCrea, P.D.,
1996. -Catenin associates with the actin-bundling
protein fascin in a noncadherin complex. J. Cell Biol. 134,
12711281.
Tavazoie, M., Van derVeken, L., Silva-Vargas, V., Louissaint, M.,
Colonna, L., Zaidi, B., Garcia-Verdugo, J.M., Doetsch, F., 2008. A
specialized vascular niche for adult neural stem cells. Cell
Stem Cell 3, 279288.
Teng, C.H., Tseng, Y.T., Maruvada, R., Pearce, D., Xie, Y., PaulSatyaseela, M., Kim, K.S., 2010. NlpI contributes to Escherichia
coli K1 strain RS218 interaction with human brain
microvascular endothelial cells. Infect. Immun. 78, 30903096.
Terry, S., Nie, M., Matter, K., Balda, M.S., 2010. Rho signaling and
tight junction functions. Physiology 25, 1626.
Thiel, V.E., Audus, K.L., 2001. Nitric oxide and bloodbrain barrier
integrity. Antioxid. Redox Signal. 3, 273278.
Tosi, G., Costantino, L., Ruozi, B., Forni, F., Vandelli, M.A., 2008.
Polymeric nanoparticles for the drug delivery to the central
nervous system. Expert Opin. Drug Deliv. 5, 155174.
Tsukita, S., Furuse, M., Itoh, M., 2001. Multifunctional strands in
tight junctions. Nat. Rev. Mol. Cell Biol. 2, 285293.
Tweedie, D., Luo, W., Short, R.G., Brossi, A., Holloway, H.W., Li, Y.,
Yu, Q.S., Greig, N.H., 2009. A cellular model of inflammation for
identifying TNF- synthesis inhibitors. J. Neurosci. Methods
183, 182187.
362
Ueno, M., 2007. Molecular anatomy of the brain endothelial

barrier: an overview of the distributional features. Curr. Med.
Chem. 14, 11991206.
van Hinsbergh, V.W., van Nieuw Amerongen, G.P., 2002.
Intracellular signalling involved in modulating human
endothelial barrier function. J. Anat. 200, 549560.
van Vliet, E.A., da Costa Arajo, S., Redeker, S., van Schaik, R.,
Aronica, E., Gorter, J.A., 2007a. Bloodbrain barrier leakage may
lead to progression of temporal lobe epilepsy. Brain 130,
521534.
van Vliet, E.A., van Schaik, R., Edelbroek, P.M., Voskuyl, R.A.,
Redeker, S., Aronica, E., Wadman, W.J., Gorter, J.A., 2007b.
Region-specific overexpression of P-glycoprotein at the
bloodbrain barrier affects brain uptake of phenytoin in
epileptic rats. J. Pharmacol. Exp. Ther. 322, 141147.
Vastag, M., Keseru, G.M., 2009. Current In Vitro and In Silico
Models of the BloodBrain Barrier Penetration: A Practical
View. Curr. Opin. Drug Discov. Devel. 12, 115125.
Vichalkovski, A., Gresko, E., Hess, D., Restuccia, D.F., Hemmings,
B.A., 2010. PKB/AKT phosphorylation of the transcription
factor Twist-1 at Ser42 inhibits p53 activity in response
to DNA damage. Oncogene. 29, 35543565.
Virgintino, D., Robertson, D., Errede, M., Benagiano, V., Tauer, U.,
Roncali, L., Bertossi, M., 2002. Expression of caveolin-1 in
human brain microvessels. Neuroscience 115, 145152.
Vogel, C., Bauer, A., Wiesnet, M., Preissner, K.T., Schaper, W.,
Marti, H.H., Fischer, S., 2007. Flt-1, but not Flk-1 mediates
hyperpermeability through activation of the PI3-K/Akt
pathway. J. Cell. Physiol. 212, 236243.
von Wedel-Parlow, M., Wlte, P., Galla, H.J., 2009. Regulation of
major efflux transporters under inflammatory conditions at
the bloodbrain barrier in vitro. J. Neurochem. 111, 111118.
Vorbrodt, A.W., Dobrogowska, D.H., 2003. Molecular anatomy of
intercellular junctions in brain endothelial and epithelial
barriers: electron microscopist's view. Brain Res. Brain Res.
Rev. 42, 221242.
Vorbrodt, A.W., Li, S., Brown, W.T., Ramakrishna, N., 2008.
Increased expression of -catenin in brain microvessels of a
segmentally trisomic (Ts65Dn) mouse model of Down
syndrome. Brain Cell Biol. 36, 203211.
Wade, J.B., Karnovsky, M.J., 1974. The structure of the zonula
occludens. A single fibril model based on freeze-fracture. J. Cell
Biol. 60, 168180.
Warren, M.S., Zerangue, N., Woodford, K., Roberts, L.M., Tate, E.H.,
Feng, B., Li, C., Feuerstein, T.J., Gibbs, J., Smith, B., de Morais,
S.M., Dower, W.J., Koller, K.J., 2009. Comparative gene
expression profiles of ABC transporters in brain microvessel
endothelial cells and brain in five species including human.
Pharmacol. Res. 59, 404413.
Waschke, J., Burger, S., Curry, F.R., Drenckhahn, D., Adamson, R.H.,
2006. Activation of Rac-1 and Cdc42 stabilizes the
microvascular endothelial barrier. Histochem. Cell Biol. 125,
397406.
Weinger, J.G., Gohari, P., Yan, Y., Backer, J.M., Varnum, B.,
Shafit-Zagardo, B., 2008. In brain, Axl recruits Grb2 and the p85
regulatory subunit of PI3 kinase; in vitro mutagenesis defines
the requisite binding sites for downstream Akt activation. J.
Neurochem. 106, 134146.
Weis, S.M., 2008. Vascular permeability in cardiovascular disease
and cancer. Curr. Opin. Hematol. 15, 243249.
Weiss, N., Miller, F., Cazaubon, S., Couraud, P.O., 2009. The
bloodbrain barrier in brain homeostasis and neurological
diseases. Biochim. Biophys. Acta 1788, 842857.
Weksler, B.B., Subileau, E.A., Perrire, N., Charneau, P., Holloway,
K., Leveque, M., Tricoire-Leignel, H., Nicotra, A., Bourdoulous,
S., Turowski, P., Male, D.K., Roux, F., Greenwood, J., Romero,
I.A., Couraud, P.O., 2005. Bloodbrain barrier-specific properties
of a human adult brain endothelial cell line. FASEB J. 19,
18721874.
Wilhelm, I., Nagyszi, P., Farkas, A.E., Courad, P.O., Romero, I.A.,
Weksler, B., Fasakas, C., Dung, N.T.K., Bottka, S., Bauer, H.,
Bauer, H.C., Krizbai, I.A., 2008. Hyperosmotic stress induces Axl
activation and cleavage in cerebral endothelial cells. J.
Neurochem. 107, 116126.
Williams, R.L., Risau, W., Zerwes, H.G., Drexler, H., Aguzzi, A.,
Wagner, E.F., 1989. Endothelioma cells expressing the polyoma
middle T oncogene induce hemangiomas by host cell
recruitment. Cell 57, 10531063.
Willis, C.L., Nolan, C.C., Reith, S.N., Lister, T., Prior, M.J., Guerin, C.J.,
Mavroudis, G., Ray, D.E., 2004. Focal astrocyte loss is followed
by microvascular damage, with subsequent repair of the
bloodbrain barrier in the apparent absence of direct astrocytic
contact. Glia 45, 325337.
Wilson, B., Samanta, M.K., Santhi, K., Sampath Kumar, K.P.,
Ramasamy, M., Suresh, B., 2009. Significant delivery of tacrine
into the brain using magnetic chitosan microparticles for
treating Alzheimer's disease. J. Neurosci. Methods 177,
427433.
Wojciak-Stothard, B., Tsang, L.Y., Haworth, S.G., 2005. Rac and Rho
play opposing roles in the regulation of hypoxia/reoxygenationinduced permeability changes in pulmonary artery
endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 288,
L749L760.
Wolburg, H., Lippoldt, A., 2002. Tight junctions of the bloodbrain
barrier: development, composition and regulation. Vascul.
Wolburg, H., Noell, S., Mack, A., Wolburg-Buchholz, K.,
Fallier-Becker, P., 2009. Brain endothelial cells and the
glio-vascular complex. Cell Tissue Res. 335, 7596.
Wong, D., Dorovini-Zis, K., Vincent, S.R., 2004. Cytokines, nitric
oxide, and cGMP modulate the permeability of an in vitro
model of the human bloodbrain barrier. Exp. Neurol. 190,
446455.
Yamagata, K., Tagami, M., Nara, Y., Mitani, M., Kubota, A., Fujino, H.,
Numano, F., Kato, T., Yamori, Y., 1997. Astrocyte-conditioned
medium induces bloodbrain barrier properties in endothelial
cells. Clin. Exp. Pharmacol. Physiol. 24, 710713.
Yamagata, K., Tagami, M., Takenaga, F., Yamori, Y., Itoh, S., 2004.
Hypoxia-induced changes in tight junction permeability of
brain capillary endothelial cells are associated with IL-1beta
and nitric oxide. Neurobiol. Dis. 17, 491499.
Yamamoto, M., Ramirez, S.H., Sato, S., Kiyota, T., Cerny, R.L.,
Kaibuchi, K., Persidsky, Y., Ikezu, T., 2008. Phosphorylation of
claudin-5 and occludin by rho kinase in brain endothelial cells.
Am. J. Pathol. 172, 521533.
Yamauchi, A., Dohgu, S., Nishioku, T., Shuto, H., Naito, M., Tsuruo, T.,
Sawada, Y., Kataoka, Y., 2007. An inhibitory role of nitric oxide in
the dynamic regulation of the blood-brain barrier function. Cell
Mol. Neurobiol. 27, 263270.
Yuan, S.Y., 2003. Protein kinase signaling in the modulation of
microvascular permeability. Vascul. Pharmacol. 39,
213223.
Zaghi, J., Goldenson, B., Inayathullah, M., Lossinsky, A.S.,
Masoumi, A., Avagyan, H., Mahanian, M., Bernas, M., Weinand,
M., Rosenthal, M.J., Espinosa-Jeffrey, A., de Vellis, J., Teplow,
D.B., Fiala, M., 2009. Alzheimer disease macrophages
shuttle amyloid-beta from neurons to vessels, contributing
to amyloid angiopathy. Acta Neuropathol. 117,
111124.
Zenker, D., Begley, D., Bratzke, H., Rbsamen-Waigmann, H.,
von Briesen, H., 2003. Human blood-derived macrophages
enhance barrier function of cultured primary bovine and
human brain capillary endothelial cells. J. Physiol. 551,
10231032.
Zerlin, M., Julius, M.A., Kitajewski, J., 2008. Wnt/Frizzled signaling
in angiogenesis. Angiogenesis 11, 6369.
Zhang, Y., Li, C.S., Ye, Y., Johnson, K., Poe, J., Johnson, S.,
Bobrowski, W., Garrido, R., Madhu, C., 2006. Porcine brain
microvessel endothelial cells as an in vitro model to predict in

vivo bloodbrain barrier permeability. Drug Metab. Dispos. 34,
19351943.
Zhang, X., Alakhova, D.Y., Batrakova, E.V., Li, S., Yang, Z., Li, Y.,
Kabanov, A.V., 2009a. Effect of pluronic p85 on amino acid
transport in bovine brain microvessel endothelial cells. J.
Neuroimmune Pharmacol. 4, 3546.
Zhang, Z., Xia, C., Xue, Y., Liu, Y., 2009b. Synergistic effect of
low-frequency ultrasound and low-dose bradykinin on
increasing permeability of the bloodtumor barrier
by opening tight junction. J. Neurosci. Res. 87,
22822289.
363
Zheng, W., Aschner, M., Ghersi-Egea, J.F., 2003. Brain barrier

systems: a new frontier in metal neurotoxicological research.
Toxicol. Appl. Pharmacol. 192, 111.
Zhong, Y., Smart, E.J., Weksler, B., Couraud, P.O., Henning, B.,
Toborek, M., 2008. Caveolin-1 regulates HIV-1 Tat-induced
alterations of tight junction protein expression via
modulation of the Ras signaling. J. Neurosci. 28, 77887796.
Zlokovic, B.V., 2008. The blood-brain barrier in health and chronic
neurodegenerative disorders. Neuron 57, 178201.
Zozulya, A., Weidenfeller, C., Galla, H.J., 2008. Pericyteendothelial
cell interaction increases MMP-9 secretion at the bloodbrain
barrier in vitro. Brain Res. 1189, 111.

BBB Anatomia

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B RA I N R E SE A R CH RE V I EW S 64 ( 20 1 0 ) 3 2 83 6 3

Looking at the bloodbrain barrier: Molecular anatomy and

Accepted 19 May 2010

Available online 26 May 2010

interactions between endothelial cells and basement membrane as well as neighboring

Brain microvascular endothelial cell

In 1885, Paul Ehrlich experimentally demonstrated that vital

achieved by injecting horseradish peroxidase intravascularly,

Fig. 1 Injected horseradish peroxidase diffuses through

In the developing brain, capillaries are differentiated and

The basement membrane is an essential part of the BBB. It

Fig. 2 Schematic overview of the neurovascular unit

adhesion molecules (CAM), as well as signaling proteins,

Little is known about the developmental role that neurons

Microglia play a very important role in immune responses of

Fig. 3 Pericytes have a close physical association with the

diverse effector functions (Ransohoff and Perry, 2009). As

Pericytes (also known as vascular smooth muscle cells, mural

Pericytes show rich contents of -smooth muscle actin,

Astrocytes are glial cells whose endfeet form a lacework of fine

Fig. 4 Astrocytes's endfeet form a fine lamellae closely

astrocytes revealed that the secretion of factors into the

EC were demonstrated to play a key role in BBB properties as it

Fig. 5 Brain microvascular endothelial cells establish the

capillary endothelium in the brain is 50100 times tighter than

(Stamatovic et al., 2008), sometimes also referred to as

The EC lining at the BBB is stabilized by specialized cell

molecular constituents of TJ and AJ, as well as the signaling

TJ are the main structures responsible for the barrier properties.

3.1.1.1. Claudins. The claudins are a family of TJ proteins,

et al., 2006a; Ueno, 2007; Zlokovic, 2008), are integral membrane

completely understood. In fact, claudins appear to be

breakdown of the BBB in an experimental mouse model

3.1.1.2. Occludin. Occludin, a transmembranous TJ protein of

distributed in non-neural endothelium (Hawkins and Davis,

3.1.1.3. Junctional adhesion molecules. Adhesion molecules,

proteins involved in the regulation of gene transcription and

Below the TJ, in the basal region of lateral plasma membrane

molecules to the cortical actin cytoskeleton. Catenins' major

the mechanisms that lead to paracellular hyperpermeability

Protein kinases are classified as serine/threonine kinases and

3.2.1.1. Protein kinase C. Protein kinase C (PKC), one of the

PKC regulate a variety of cell functions. These include

3.2.1.2. Protein kinases A and G. Protein kinase A (PKA)

brain EC to rearrangement of endothelial junctional proteins

3.2.1.3. Protein kinase B. Protein kinase B (PKB), also known

VEGFR-2 (flk-1/KDR) and VEGFR-3 (Flt-4), of which VEGFR-2 is

Mitogen-activated protein kinases

MAPK are involved in multiple cascades of serine/threonine

tion factors that regulate the transcription of diverse genes

G-proteins play an essential role in the establishment and

adhesion, whereas Cdc42 and Rac1 are major players in cell

Endothelial nitric oxide synthase

In EC, NO production is catalyzed by the constitutive enzyme

which in turn is required for PKC activation as described

Ca2+ is a potent second messenger. Therefore, intracellular

cell adhesion and pulling neighboring cells apart from each

its interaction with cadherins at the AJ. Therefore, any resulting

The blood-brain barrier as a signaling interface

The BBB EC produce mediators like cytokines and NO. These

Signaling from periphery to brain

During systemic inflammation, activation of BBB-dependent

In addition to the role of the EC lining in transporting