Top Med Chem (2014) 10: 120DOI: 10.10077355_2013_20Springer-Verlag Berlin Heidelberg 2013 [603023]
Top Med Chem (2014) 10: 1–20DOI: 10.1007/7355_2013_20©Springer-Verlag Berlin Heidelberg 2013
Published online: 13 December 2013
The Blood–Brain Barrier: An Introduction toIts Structure and Function
Anne Mahringer, Melanie Ott, and Gert FrickerAbstract The blood-brain barrier (BBB) formed by the brains microvascular system
is impermeable for most therapeutically us ed compounds and overcoming this barrier
remains to be one of the big challenges in modern medicine. It is composed of
highly specialized endothelial cells, whi ch are surrounded by pericytes and a basal
membrane. Together with nearby astrocytes and neurons they constitute the so-called
neurovascular unit, which r estricts substance transfe r from blood to brain and vice
versa and maintains the cereb ral ion homeostasis. Chapters of this book describe the
discovery of the BBB, its evolutionary development as well as the cellular and
molecular mechanisms, which underlay its st ructure and function in health and disease.
The organization of tight junctional compl exes or specific transport processes at the
BBB will be addressed as well as methods to investigate BBB function in vitro and invivo. Changes in the barrier function under several disea ses conditions such as stroke
or inflammation will be discussed as well a s approaches to overcome the barrier by
colloidal carriers or ultrasound.Keywords Blood–brain barrier, Neurovascular unit, Morphology, Transporter
Contents
1 The BBB: A Historical Perspective …………………………………………………. 2
2 Evolutionary Development of a BBB ………………………………………………. 5
3 Anatomic Principles of the BBB ……………………………………………………. 5
4 Pericytes ………………………………………………………………………….. 75 Astrocyte: Endothelium Interactions … …………………………………………….. 8
A. Mahringer (
*), M. Ott and G. Fricker
Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls University, Im NeuenheimerFeld 366, 69120 Heidelberg, Germanye-mail: [anonimizat] ;[anonimizat] ;[anonimizat]
6 Neurons ………………………………………………………………………….. 8
7 Basal Membrane …………………………………………………………………. 98 Junctional Complexes at the BBB …………………………………………………. 9
9 Transport Proteins at the BBB …………………………………………………….. 12
10 Cytotic Processes at the BBB ……………………………………………………… 14
11 Outlook ………. …………………………………………………………………. 15
References ……………………………………………………………………………. 16
The brain is the most critical organ in our body, which requires a very well-balanced ion homeostasis. It is extremely sensitive to a large variety of chemicals,which include potentially toxic metabolites or constituents of our daily food intakewithout being toxic to other parts of the body. Therefore, it is obvious that thecentral nervous system (CNS) needs special protection, which is set up by the braincapillaries, the so called blood–brain barrier (BBB). This microvessel networkoperates as a dynamic regulator of ion balance, a mediator of nutrient transport,and an impediment to harmful molecules. This barrier also represents a majorobstacle to the development of CNS drugs. Approximately 98% of small moleculeand all large molecule drugs, e.g., recombinant peptides or anti-sense-agents arenormally excluded from the brain [ 1,2]. Hence, the understanding of the morpho-
logy of the BBB as well as the molecular and cellular mechanisms that determine itsfunction is an inevitable prerequisite for successful drug delivery to the brain. Here,we review the BBB from a historical perspective and discuss the current knowledgeabout the components of that barrier and their integrated function.
1 The BBB: A Historical Perspective
The first experiments indicating the existence of the barrier were performed in 1885by the German immunologist Paul Ehrlich. He observed that a peripherallyadministered dye stained animal organs but failed to color brain tissue [ 3]. The
initial interpretation of this finding was based on different binding affinities [ 4].
Subsequent pharmacological studies by Bield and Kraus [ 5] and Lewandowsky
provoked the existence of a barrier at the level of cerebral vessels (1900), especiallywhen Lewandowsky was studying the limited permeation of potassium ferrocyanateinto the brain. This barrier was named “blood–brain barrier” by Goldmann [ 6].
Goldmann, a student of Ehrlich, also performed staining experiments with dogsand rabbits where he demonstrated a clear, exclusive staining of the choroid plexusafter injection of water soluble dyes into the peripheral circulation, whereas thesurrounding brain tissue and cerebrospinal fluid (CSF) remained colorless [ 7], thus
confirming observations of other scientists in the years before [ 5,8–10]. He also
found that after sub-arachnoidal injection the brain was stained except the choroidplexus and concluded that the plexus epithelium was the very barrier preventing thetransfer of dye into the brain [ 6]. However, the Russian physiologist Lina Stern
observed that some test compounds could be found selectively in the brain and in the2 A. Mahringer et al.
cerebrospinal fluid after i.v. administration in contrast to others and called thisphenomenon “barrie `re he´matoence ´phalique“ [ 11]. Some years later Spatz and
colleagues suggested the concept of two separate CNS barriers: the BBB and theblood–liquor barrier [ 12–15]. In 1929, H. Foertig wrote the first scientific paper
entitled “Die Bluthirnschranke” or “the blood–brain barrier,” which was a ratherprovocative term at that time [ 16]. Broman [ 17] also argued that the barrier function
of the BBB was localized to the capillary endothelial cells but not to the astrocyticend feet. He also claimed that the BBB showed defects in brain diseases anddemonstrated a transient opening or disruption of the BBB after intracarotid arterialadministration of hypertonic solutions [ 17]. Friedemann postulated in 1942 that
electrochemical properties of injected compounds influence the distribution behaviorwithin the CNS. Accordingly, capillaries are permeable for uncharged and positivelycharged compounds, but impermeable for negatively charged compounds [ 18].
In 1946, August Krogh speculated about active transport mechanisms when he wasthinking of the presence of nutrient supply across the endothelial cells or of the BBBas a selective impermeable obstacle [ 19]. Even though the exact nature of the barrier
was subject to many controversies until the 1960s, the introduction of the electronmicroscope revealed the presence of extracellular fluid in the cortex [ 20] and the
localization of the barrier function within the endothelial cells of the brain capillaries[21,22] confirming that the endothelium is indeed the principal anatomical site of the
barrier (Fig. 1).
In 1971, Oldendorf demonstrated BBB permeability to sugars, amines, amino
acids, and neurotransmitters by the use of radiolabeled substances [ 24]. Freeze
fracture analysis indicated that the tight junctions between endothelial cells formcomplex net-like anastomoses around the endothelial cells, which restrict the passage
Fig. 1 Diffusion of the dye
Trypan blue from thecerebrospinal fluid (CSF) intothe brain. Trypan blue wasinjected into the blood andinto the CSF, respectively.Brain, CSF, and blood wereanalyzed (modified from [ 23])The Blood–Brain Barrier: An Introduction to Its Structure and Function 3
of macromolecules and also of low-molecular-weight substances down to a diameterof 10–15 A ˚[25,26]. In addition, this cell layer exhibits a very high transendothelial
electrical resistance between approximately 1,400 and 1,900 Ohm /C2cm
2[27,28].
Furthermore, the endothelial cells are surrounded by pericytes, astrocytic foot-processes, and a basal membrane. In the late 1980s molecular biology techniquesemerged and first studies at the BBB were performed, resulting in cloning andsequencing of the glucose transporter gene Glut1 [ 29]. Table 1gives a short summary
about the development in the past 100 years of BBB research.Table 1 Milestones in the development of blood–brain barrier research
Year Discoveries and concepts1885 Systemic application of a blue dye stained all organs except the brain and the
spinal cord [ 3]
1898 Systemically administered bile acids were not neurotoxic but intracerebrally injected
bile acids showed neurotoxicity [ 5]
1900 Postulation of a barrier between blood circulation and neural tissue to describe the
phenomenon [ 8]
1913 Intrathecal administration of trypan blue results in staining of the brain tissue,
whereas intravenous application does not. Definition of the concept of theblood–brain barrier [ 6]
1921–1922 “Barrie `re he´matoence ´phalique” was characterized as a cerebral blood vessel
compartment, whereas the choroid plexus epithelium was semipermeable,facilitating the flow of substances from the blood into the CSF [ 30,31]
1941 Intracarotid arterial administration of hypertonic solutions caused a transient opening
or disruption of the blood–brain barrier explaining the mechanisms behindobserved defects at the blood–brain barrier in brain diseases [ 17]
1942 Friedemann postulated in 1942 that electrochemical properties of injected
compounds influence the distribution behavior within the CNS. Thereby,capillaries would be permeable for uncharged and positively charged compounds,but impermeable for negatively charged compounds [ 18]
1950s Electron microscopy could not detect an extracellular fluid compartment in the gray
matter, which was considered as an explanation for the failure of tracers to enterthe brain. Later, this turned out to be an artifact in 1960s
1960s The presence of extracellular fluid in the cortex was determined by further electron
microscopy studies on “freeze-substituted” tissue [ 20]
1967 Fine structural localization of the blood–brain barrier, demonstration of tight
junctions [ 22]
1969 Visual proof of junctions between endothelial cells [ 21]
1971 Blood–brain barrier permeability to sugars, amines, amino acids, and
neurotransmitters proven by radiolabeled substances [ 24]
1978 Description of the passage of substances in extracellular fluids from brain to CSF
along the CSF “bulk flow” gradient; “sink effect” that removes substances fromthe brain
1982 Observation of extremely high transendothelial electrical resistances [ 28]
1980s Studies in molecular biology of the blood–brain barrier. Cloning and sequencing of
glucose transporter gene [ 29]
1990s Importance of ABC transporters for barrier function becomes obvious [ 32]
2000s Signaling cascades of transporters [ 33]4 A. Mahringer et al.
2 Evolutionary Development of a BBB
The BBB developed during evolution with the increasing complexity of neuraltissue. Many invertebrates do not have a distinct barrier but only a leaky endo-thelium. Insects, crustaceans and cephalopods have a glial BBB [ 34].
A careful examination of the BBB of different species of evolutionary old fish
gives evidence that ancestral vertebrates also had a glial barrier. About 400–500million years ago apparently all vertebrates had a glial barrier, which has repeatedlybeen replaced by an endothelial barrier during evolution [ 35]. In elasmobranches
(sharks, skates, rays) and sturgeons the BBB is set up by perivascular astrocytes. Inmost vertebrates including tetrapods (amphibia, reptiles, birds, and mammals), thebarrier is formed by the brain microvessel endothelium [ 36], suggesting that once
the neural tissue became larger and more complex during evolution the endo-thelium became tight enough to take on the barrier role (Fig. 2).
3 Anatomic Principles of the BBB
The primary element of the barrier is formed by the endothelial cells of brainmicrovessels, which pervade the brain with a total length of approximately 600 km,a mean distance of 40 μm and a capillary surface area available for molecular
transport of about 20 m
2[37]. It has been suggested that nearly every neuron in
human brain is supplied by its own capillary [ 38]. A morphometric analysis of the
mouse cortical vasculature indicates that perfused capillaries (4–8 μm in diameter)
and small arterioles and venules (10–60 μm in diameter) occupy between 3–4% and
4–6% of the brain volume, respectively (Fig. 3). This correlates well with in vivo
Fig. 2 Phylogenetic
development of theblood–brain barrier ( red:
blood–endothelial brainbarrier; blue: blood–glial
barrier; from [ 35])The Blood–Brain Barrier: An Introduction to Its Structure and Function 5
measurements of the blood volume in the gray matter in human brain determined bymagnetic resonance imaging [ 39].
Brain capillaries exhibit some fundamental differences compared to peripheral
capillaries. Whereas peripheral capillaries are fenestrated with gaps up to 50 nmwide the endothelial cells of brain capillaries are closely connected to each other bytight junctions and zonulae occludentes [ 40,41]. In addition, the number of
mitochondria is about five to ten times higher than in cells of peripheral microvessels,
Fig. 4 Cross-section of a brain microvessel: endothelial cells surround the blood lumen and are
ensheathed by the basal lamina containing pericytes. Astrocytic perivascular endfeet are attachedto the basal lamina and are in contact with microglia and neuronal brain tissue
Fig. 3 Brain capillary network. ( a) Plastinate of the blood–brain barrier network isolated from an
adult human brain (adapted from [ 46]). (b) Electron microscope picture showing blood vessels in
adult human cortex: pial vessels ( 1), long ( 2) and middle ( 3) cortical arteries, superficial ( 4),
middle ( 5), and deep ( 6) capillary zone, scale bar 0.86 mm (adapted from [ 47]). (c) Confocal
microscope picture of an isolated porcine brain capillary ( insert ) and picture of a monolayer of
cultured porcine brain capillary endothelial cells (PBCECs)6 A. Mahringer et al.
indicating a high metabolic activity [ 42–44]. Furthermore, the cells exhibit a very low
pinocytotic activity [ 45].
Five major components form the barrier – brain capillary endothelial cells, which
make the actual barrier, pericytes, and the foot processes of astrocytes. Endothelialcells and pericytes are embedded into and surrounded by a basal membrane, and allthese components are in close interaction with neurons (Fig. 4). Together the whole
morphological framework is named the “neurovascular unit”. In the following thedistinct components of this unit will be discussed in more detail.
4 Pericytes
About 20% of the endothelial cells are directly covered by pericytes at their abluminalmembrane [ 48,49]. These cells, which are also named Rouget-Cells [ 50], belong to the
vascular smooth muscle cell (VSMC) lineage [ 51]. They are contractile, responding to
several vasoactive stimuli [ 52,53] and appear to regulate brain capillary blood flow
through contraction and relaxation [ 53]. It has been suggested that pericytes encircle
30–70% of the capillary wall. They are linked to the endothelial cells by gap junctions,focal adhesion plaques, and the so-called peg-and-socket-invaginations, exhibitmacrophage-like activity [ 54], and help to regulate the endothelial cells [ 55]. Pericyte
cytoplasma contains a relatively high number of lysosomes and the cells are able totake up macromolecular compounds, which are otherwise degraded by macrophages[56,57]. Recent studies demonstrated that pericytes release various growth factors and
angiogenic molecules, which regulate microvascular permeability and angiogenesis[58]. The interaction between pericytes and endothelial cells appears to be modulated
by several ligand-receptor systems [ 59]. Hori et al. [ 60] showed that Angiopoietin-1
released from pericytes induces occludin expression via the Tie-2 receptor. The cellsmay also be implicated in endothelial differentiation by TGF β, S1P (sphingosine-1-
phosphate) or PDGF release via the respective receptors as well as anti-apoptoticmechanisms [ 61,62]. Recent findings suggest that pericytes may be involved in the
development of neuropathological alterations in several CNS diseases such as hyper-tension, diabetes, multiple sclerosis, CNS tumor formation, Alzheimer´s disease, orcentral nervous infections [ 51,59,63–66]. For example, it was postulated [ 67]t h a t
pericytes are more permissive for human cytomegalovirus replication compared toendothelial cells and that pericytes could serve as amplification reservoirs for HCMV.Recently, Yemisci et al. [ 68] demonstrated that pericytes contracted during acute
ischemia and remained unchanged despite a reopening of the artery in a mouse focalischemia model. Contracted pericytes induced narrowing of capillary lumen, whichentrapped erythrocytes and clogged microcirculation. Thus, ischemia/reperfusion-induced injury to pericytes may be a major mechanism that negatively affects tissuesurvival by limiting oxygen and substrate delivery. Amyloid deposits have beendetected within degenerating pericytes in the brains of patients with Alzheimer’sdisease [ 69,70]. LRP (low density lipoprotein-related receptor)-mediated degradation
of Amyloid- β(Aβ) in pericytes lowers A βlevels in perivascular spaces ([ 71,72],
reviewed by [ 73]). Thus, it may be speculated that pericyte dysfunction plays also aThe Blood–Brain Barrier: An Introduction to Its Structure and Function 7
role in impaired A β-peptide clearance in Alzheimer’s disease and initiates secondary
neurodegenerative changes [ 74].
5 Astrocyte: Endothelium Interactions
Astrocytes appear to be an important component in the development and/or mainte-nance of BBB characteristics [ 75]. Co-culture of brain endothelial cells with
astrocytes [ 76,77] or with astrocyte-conditioned media [ 78] has been demonstrated
to improve BBB characteristics in vitro. These observations are supported by in vivostudies showing loss and restoration of barrier integrity after a temporary focal lossof astrocytes [ 79]. Further on, a dynamic bidirectional Ca
2+-signaling occurs
between neurons, astrocytes, and the endothelium, for which two mechanismshave been proposed – an intracellular IP3 (inositol-trisphosphate)- and gapjunction-dependent pathway and a pathway involving extracellular diffusion viagap junctions and purinergic transmission [ 80–82] – which might play a role in the
regulation of microvascular permeability [ 83]. Co-culture experiments with endo-
thelial cells and astrocytes showed that TGF βproduced by astrocytes downregulates
tissue plasminogen activator (tPA) and anticoagulant thrombo-modulin (TM)expression in cerebral endothelial cells [ 84], which might be relevant at intracerebral
bleeding or intraventricular hemorrhage. Glial cell-derived neurotrophic growthfactor (GDNF), a member of the TGF βgroup, seems to be involved in postnatal
maturation of brain microvessels [ 85].
Vice versa, endothelial cells produce leukemia-inhibiting factor (LIF), which
plays a role in the induction of astrocyte differentiation [ 86]. When neonatal mouse
astrocytes were co-cultured with a mouse endothelial cell line an alteration of theastrocytes from confluent monolayers to elongated multicellular columns occurred[87]. In addition, aquaporin-4 expression was upregulated in astrocytes under co-
culture conditions [ 88].
6 Neurons
It is obvious that the cerebral microcirculation needs to be responsive to the nearbybrain tissue. Very early reports suggest that brain activity imposes the transfer ofoxygen and nutrients from the circulation into activated regions through a “neuro-vascular coupling” process [ 89]. Although the intracellular pathways involved in
neurovascular coupling are not fully understood, a large number of data indicatethat diverse mediators released in response to neuronal glutamate influence themicrocirculation. A recent study shows that neuronal activity drives localizedtransport of serum insulin-like growth factor-I across the BBB (which may helpto explain distinct observations such as proneurogenic effects of epileptic seizures,rehabilitation upon neuronal stimulation, and modulation of blood flow in responseto brain activity) [ 90]. Transplantation experiments gave strong evidence that BBB
characteristics of capillary endothelial cells depend on their neural environment8 A. Mahringer et al.
[91]. Although neurons are not directly structurally involved in the formation of the
BBB, there is evidence that microvascular endothelium and/or associated astrocyticfoot processes underlie innervation by noradrenergic [ 92,93], serotonergic [ 94],
cholinergic [ 95,96], GABA-ergic [ 97], and other neurons [ 98]. In Alzheimer´
s disease a significant loss of cholinergic innervation of cortical microvessels hasbeen observed, which might explain why the disease is associated with an impairedcerebrovascular function [ 95].
7 Basal Membrane
Endothelial cells and pericytes are embedded into the basal membrane consistingmainly of laminin, collagen type IV, proteoglycans, heparan sulfate, fibronectin, andother extracellular matrix proteins [ 99]. This membrane, which is 30–40 nm thick,
appears to have a direct impact on the endothelium via interaction of laminin andother matrix proteins with endothelial integrin receptors [ 100] and the regulation of
endothelial tight junction protein expression by matrix proteins [ 101,102]. Conse-
quently, disruption of this extracellular matrix is strongly associated with increasedBBB permeability in pathological states [ 103,104]. In addition, cell–matrix
interactions can stimulate a number of intracellular signaling pathways (reviewedin [105]).
8 Junctional Complexes at the BBB
The BBB is characterized by exceptionally high electrical resistances being indicative
for very tight intercellular c onnections. Three types of junctions are found: Adherens
junctions [ 106], tight junctions [ 41,107,108], and possibly gap junctions [ 80,109–111].
Adherens junctions mediate the mutual adhesion of endothelial cells and play a
role in setting up cellular polarity and contact inhibition during vascular growth[109,112]. They are formed by vascular endothelial (VE)-cadherin, a Ca
2+-dependent
protein that mediates cell–cell adhesion [ 113]. At its intracellular site VE-cadherin
binds to β-catenin and plakoglobin, which is then linked via α-catenin, α-actinin, and
vinculin to the actin cytoskeleton [ 114–116].
Closely associated with the adherens junction proteins are those of the tight
junctions suggesting that both junctional types are interspersed at the BBB. Tightjunctions are mainly responsible for the high transendothelial resistances at theBBB. They are also composed of transmembrane proteins that form the primarylining linked via accessory proteins to the actin cytoskeleton [ 107]. Tight junction
proteins include occludin, junctional adhesion molecule (JAM)-1, and the claudins.
Occludin is a 60 kDA transmembrane protein, which spans the cell membrane
four times with a short cytoplasmic N-terminus and a long carboxy-terminal
cytoplasmic domain. It is highly expressed in tight junctions of the BBB, but notin endothelial tight junctions of non-neuronal tissues [ 117,118]. Its role in
tight junction formation and maintenance has not been completely clarified.The Blood–Brain Barrier: An Introduction to Its Structure and Function 9
For example, transfection of insect cells devoid of endogenous tight junctions with
occludin cDNA demonstrated that occludin is not sufficient to form tight junction
strands, suggesting that occludin is rather needed for regulation than for
establishing BBB properties [ 41]. However, occludin seems to interact with
claudins in a heterophilic manner and is recruited into the long strands formed by
coexpression of claudin-1 and claudin-2 [ 119,120]. A consistent staining for
occludin along cell–cell contacts of porcine (PBC) and rat brain capillaries
(RBC) as well as of cultured brain endothelial cells is seen in Fig. 5.
JAM-1 is a member of the IgG superfam ily and appears to mediate the early
attachment of adjacent cell membranes [ 121]. It is a transmembrane immunoglobulin-
like molecule composed of a single membrane- spanning chain with a large extracellular
domain [ 122], which co-distributes with tight ju nctions components. JAM-2 and JAM-3
being related to JAM-1 are also present in e ndothelial tissues and lymphatic cells, but
not epithelia [ 123,124]. Interestingly, in West Nile vi rus infections endocytosis of
JAM-1 occurs, which ultimately results in lysosomal degradation of the protein.
Understanding this process might offer a basis for revealing the mechanism of viral
neuroinvasion [ 125]. Apparently it is also involved in leukocyte extravasation during
acute inflammation [ 126].
Amongst 24 known claudins [ 123,124] claudin-1, claudin-3, claudin-5, and
claudin-12 are expressed at the BBB. They appear to be essential for barrier
formation and maintenance (Fig. 6). A comparison of microvessels from different
human glioblastoma multiforme showed a loss of claudin-1 expression in most of
the tumor tissues, suggesting that the increase in microvascular permeability in
human gliomas, which contribute to the symptoms of brain edema, is a result of a
dysregulation of junctional proteins [ 127]. However, other studies failed to detect
claudin-1 at the BBB [ 128,129]. On the other hand, claudin-1, which was integrated
into BBB tight junctions by transient transfection of endothelial cells, reduced BBB
leakiness for both a small molecular tracer as well as endogenous plasma proteins.
Claudin-1 induced sealing of BBB tight junctions during experimental autoimmune
Fig. 5 Immunostaining for occludin in PBCs ( a), PBCECs ( b), and RBCs ( c). (a) Occludin
staining of a PBC shows localization along cell–cell contacts ( green ), nuclei ( blue) were
counterstained with DAPI. ( b) Occludin revealed a belt-like staining along PBCEC contacts in
cell culture confirming its membrane localization ( green ). (c) RBC stained for occludin ( green )
demonstrates expression along the plasma membrane of adherent cells (insert with transmitted
light picture and stained nuclei)10 A. Mahringer et al.
encephalomyelitis (EAE; a model for multiple sclerosis) and was found to signifi-
cantly ameliorate the chronic phase of EAE in two independent transgenic mouse
lines [ 130].
In brains of mice with EAE a selective loss of claudin-3 immunostaining from tight
junctions of venules was seen, whereas the localization of the other tight junction
proteins remained unchanged [ 129]. A similar finding was made in altered cerebral
microvessels of human glioblastoma multiforme resulting in a compromised BBB.
From these observations it was concluded that claudin-3 is a central component
determining the integrity of BBB tight junctions in vivo.
Further on, in the brains of claudin-5-defi cient mice a size-selective loosing for
molecules <800 Da was observed [ 131]. In immortalized mouse brain capillary
endothelial cells a significant decrease of c laudin-12 expression was determined when
cells were exposed to pathophysiologicall y high concentrations of ammonia, which is a
key neurotoxin involved in neurological c omplications of acute liver failure [ 132].
The tight junctional proteins are linked to the cytoskeleton by the submembranous
components ZO-1, ZO-2, ZO-3/p130 and the peripherally tight junction associated
proteins 7H6 and cingulin (for a detailed review, see [ 108]).
Although not traditionally considered as a tight junction protein, the actin cyto-
skeleton in brain endothelial cells pl ays also a critical role in modulating BBB
permeability [ 133].
A group of proteins, which have considerable impact on tight junctional integrity
in diverse disease states, are matrix metalloproteinases (MMPs). Under normal
conditions the expression of MMPs in the adult brain is very low. However, clinical
and experimental studies give evidence that several MMPs such as MMP-2, MMP-3,
MMP-7, or MMP-9 are upregulated and activated after ischemic stroke and neuro-
degenerative disorders (for review, see [ 134,135]). They are expressed by various
cell types including endothelial cells, microglia, neurons, and astrocytes and are
synthesized and secreted as inactive pro-enzymes that subsequently are proteo-
lytically cleaved and activated.
Fig. 6 Western blot for claudin-1 in rat brain homogenate, rat brain capillaries, and rat brain
capillary membrane fraction as well as in brain, brain capillaries, membrane fraction of brain
capillaries and in the membrane fraction of cultured endothelial cells derived from pig. It shows
enhanced expression of the tight junction protein in the plasma membrane of brain endothelial
cells in rat and pig. In vitro cell culture conditions did not influence expression levelsThe Blood–Brain Barrier: An Introduction to Its Structure and Function 11
9 Transport Proteins at the BBB
Due to its tight barrier properties the BBB has to be passed via the transcellularroute. Only few small polar compounds including water, glycerol, or urea diffuseacross tight junctions.
However, lipophilicity hardly correlates with BBB permeability. More than 98%
of small molecules do not cross the barrier nor do large molecules includingrecombinant proteins or monoclonal antibodies [ 1,2]. Some cerebral nutrients
such as glucose or amino acids pass the BBB via carrier-mediated mechanismssuch as facilitated diffusion or active transport processes. Glucose is transportedfollowing its concentration gradient by the GLUT1transporter. Other transporters inthe BBB belong to the family of solute carrier proteins (SLC), such as themonocarboxylate transporters MCT-1 and MCT-2 (SLC16a1/2), which transportshort-chain monocarboxylic acids (e.g., lactate, pyruvate or mevalonate). SLC7transports cationic amino acids (arginine, lysin, and ornithine). For mice it has beenshown that the thyroid-transporters SLC16a2 and SLCO1c1, the sulfate transporterSLC13a4, the
L-ascorbic acid transporter SLC23a2, the amino acid transporter SLC
38a3, and the folate transporter SLC19a1 are also highly expressed in the BBB [ 136].
The most interesting export proteins for drug transport across the BBB are the
primary active, ATP-dependent ones, which represent a major defense mechanismof the brain: P-Glycoprotein (P-gp, ABCB1), the Mdr1 gene product, was the first ofthese export pumps being identified at the BBB [ 137,138]. It is of particular
relevance, since it recognizes a multitude of diverse substrates and it is subject ofcomplex signaling cascades ([ 123,124,139,140]) regulating its expression and
function. One cascade is triggered by tumor necrosis factor- α, which signals through
TNF-R1 (tumor necrosis factor- αreceptor 1) resulting in the release of endothelin-1.
Endothelin-1 itself signals through the ET
Breceptor which alters P-gp expression
and function through nitric oxide synthase and protein kinase C βI[123,124,
141–144]. A second pathway is activated by glutamate, which acts via the N-
methyl- D-aspartate receptor, cyclooxygenase-2, and the prostaglandin E2 receptor
EP1 to up-regulate P-gp expression and activity [ 145–149]. A third type of cascades
involves activation of orphan or nuclear receptors including pregnane xenobioticreceptor (PXR), aryl hydrocarbon receptor (AhR), the glucocorticoid receptor (GR),and the constitutive androstane receptor (CAR) to regulate expression of xenobioticeliminating systems [ 123,124,150–155].
Recent studies using a fluorescent labeled construct of P-gp indicate that the export
pump is not organized as a single molecule within the endothelial membrane butforms clusters of several proteins close together [ 156]. Interestingly, expression and
function of P-gp may be altered at pathological conditions, e.g., Alzheimer´s diseasesor drug resistant epilepsy [ 157–160].
Yet, P-gp is not the only important contrib utor to the selective barrier: Breast cancer
resistance protein (Bcrp; ABCG2) is another efflux pump at the BBB [ 161,162], which
has a partially overlapping sub strate specificity with P-gp and also significantly restricts
xenobiotic permeability in th e brain. It is also target of several signaling pathways,12 A. Mahringer et al.
e.g., 17 β-estradiol induces the down-regulation o f Bcrp on transcriptional and transla-
tional levels via the activa tion of the estrogen receptor βin the BBB [ 163]. Moreover,
– similar to P-gp – its expression is induced b y activation of nuclear receptors such as
CAR, PXR or AhR [ 154,155].
In addition to P-gp and Bcrp multidrug resistance related proteins, Mrps, are
expressed at the BBB. However, there is still considerable discussion about theextent of expression, involvement in drug transport across the BBB and subcellularlocalization [ 164]. Mrp1, Mrp2, Mrp4, and Mrp5 appear to be localized at the
luminal surface of the BBB, although significant species differences have beenobserved. MRP1/Mrp1 has been detected in cow and human, Mrp2 has beenobserved in rat, but not in cow or human species, MRP4/Mrp4 was seen in mouse,cow, and human and MRP5/Mrp5 has been detected in human and cow [ 165–170].
Mrp1, Mrp3, Mrp4, and Mrp5 are also found on microglia and astrocytes.
ABCA1 and ABCA2 appear to be involved in lipid and cholesterol homeostasis
in the brain and in brain capillary endothelial cells [ 171,172]. Recently it has been
suggested that at least ABCA1 may play a role in the cerebral clearance of AmyloidB and is thus likely to be involved in the pathogenesis of Alzheimer’s disease, too(for review, see [ 173]).
A comprehensive discussion about expression, signaling cascades and function
of the ABC efflux pumps is found in the chapter by D. Miller (ABC Transporter) ofthis book.
Members of the above-mentioned solute carrier family (SLC) include also
transporters for organic cations (OCTs/ SLC22A1-3 , OCTNs/ SLC22A4-5 ) and
organic anions (organic anion transporters, OAT/ SLC22A6-8, 11, and organic
anion transporting polypeptides, OATP/ SLCO/SLC21 ).
From the SLC22 family OAT3, OCTN2, and RST are expressed in the BBB.
Oat3 recognizes a broad variety of substrates, including amphiphilic organic anionssuch as estradiol-17 β-glucuronide (E217 βG), estrone sulfate, and dehydroepian-
drosterone sulfate, hydrophilic organic anions, such as benzylpenicillin, indoxyl-sulfate, homovanillic acid or PAH (para aminohippuric acid), and the organiccations ranitidine and cimetidine (for review, see [ 174]).
Renal-specific transporter (RST) is a mouse homolog of the human urate transporter
(URAT1) with 74% identity at the amino aci d level and has been identified in brain
capillary endothelial cells. However, its pr ecise localization remains to be clarified.
Octn2/OCTN2 has been characterized as a so dium-dependent carnitine transporter.
It is involved in brain uptake of carnitine, an d it was found that functional loss of Octn2
is associated with a decreased brai n concentration of acetyl-carnitine [ 175].
The SLCO/SLC21 family comprises 14 members in human and rodents, whereof
Oatp1a4, Oatp1a5, and Oapt1c1 are expressed in brain capillaries [ 174]. Immuno-
fluorescence studies indicate that Oatp1a4 is expressed both on the luminal andabluminal membrane of brain capillaries [ 176]. It also recognizes multiple
substrates including cardiac glycosides (digoxin, ouabain), bile acids, steroidconjugates, some peptides, as well as some cations (e.g., N-(4,4-azo-n-pentyl)-21-
deoxyajmalinium, N-methyl-quinidine, N-methyl-quinine and rocuronium; for
review, see [ 174]). RT-PCR studies indicate also expression of Oatp1a5 in theThe Blood–Brain Barrier: An Introduction to Its Structure and Function 13
BBB [ 177]. The human OATP1A2 has structural similarity to the rodent Oatp1a4
and Oatp1a5 and exhibits also a very broad substrate specificity, but its precisemembrane localization at the human BBB is yet unclear [ 178].
24S-Hydroxycholesterol (24S-OH-chol) is a major cerebral cholesterol meta-
bolite and the elimination mechanism of 24S-OH-chol from the brain is one of thekey issues for understanding cerebral cholesterol homeostasis (Fig. 7). Studies with
Xenopus laevis oocytes expressing rat Oatp2 exhibited significant transport of [
(3)H]
24S-OH-chol suggesting that Oatp2 might be responsible for the 24S-OH-cholelimination from brain to blood [ 179]. Moreover, other relevant transporters include
equilibrative (es, ei) and concentrative (N2, N3) nucleoside transporters (ENT,CNT)that cover the demands of cerebral nucleosides needed as precursors of nucleic acidsynthesis [ 181].
10 Cytotic Processes at the BBB
The BBB contains several receptors being responsible for the passage of largemolecules, such as the transferrin receptor (TfR), insulin receptor (IR), insulin-likegrowth factor 1 receptor (IGF1R), LDL receptor, leptin receptor (OBR), lowdensity lipoprotein-related receptor 1 (LRP1), or the receptor of advanced glycationend products (RAGE). The latter two are of particular interest in the pathogenesis ofAlzheimer‘s disease as they are involved in the cerebral homeostasis and clearanceof Aβ[182]. In general, these receptors may provide targets for the brain directed
delivery of drugs, which under normal circumstances do not cross the BBB,including large biopharmaceuticals. Recombinant proteins, enzymes, and mono-clonal antibodies can be re-engineered for transport across the human BBB with themolecular Trojan horse technology either by direct coupling to antibodies versus adistinct receptor or by packing them into a colloidal carrier, such as nanoparticles or
Fig. 7 Localization of transport proteins at the blood–brain barrier. Transporters for amino acids
as well as small inorganic molecules are not shown. P-gp (p-glycoprotein, Abcb1), Bcrp (Breastcancer resistance protein, Abcg2), Mrp (Multidrug resistance protein, Abcc1/2/4/5), Oatp (Organicanion transporting polypeptide, Slco1a4/1a5/1c1), Octn (Organic cation transporter, Slc22a5),Glut (glucose transporter, Slc2a1), Cnt (Concentrative nucleoside transporter, Slc28a2), Ent(Equilibrative nucleoside transporter, Slc29a1/2), Rlip (Ral-binding protein) [ 180]14 A. Mahringer et al.
liposomes, which are surface modified with receptor-directed antibodies or anti-body fragments. A detailed discussion of delivery options and targeted receptors isgiven by Jones and Shusta [ 183].
In addition to specific receptor internalization there are two other pathways across
the BBB mediated by caveolae or plasmalemmal vesicles and clathrin-coated pits/vesicles (for review, see [ 184]). The caveolae-mediated permeation across endo-
thelial cells is also known as bulk-phase or fluid-phase transcytosis, which is inde-pendent of interactions between the transported molecules and the caveolar vesiclemembrane. It is under debate to what extent this mechanism plays a role at the BBBbecause of the relatively low occurrence of caveolae in brain capillaries [ 185]. The
density of clathrin-coated pits/vesicles at the BBB appears to be much higher [ 186].
Because of the negative surface charge of the clathrin-coated pits, only very few ofthe plasma proteins can be transcytosed randomly within the fluid phase of clathrin-coated vesicles. However, this pathway is of interest for transport of positivelycharged molecules including artificially cationized proteins, such as albumin [ 187],
when electrostatic interactions occur between the positively charged moieties ofthe proteins and negatively charged membrane surface regions on the endothelialcells [ 188].
Another option for drug delivery to the CNS offer cell-penetrating peptides,
which are quite heterogeneous in size (10–27 amino acid residues), but they allpossess positive charges. Cell-penetrating peptides derived from natural proteinsinclude the transcription-activating factor Tat, penetratin, and the so-called Syn-Bvectors as well as engineered short peptides like the homoarginine vectors,transportan or sequence signal-based peptide (SBP) and fusion sequence-basedpeptide (FBP) [ 184]. The exact mechanisms, by which these peptides are
internalized and carry their payload, are still under discussion and may be differentfor the distinct peptides, but several studies indicate a crucial role of basic residuesin the translocating ability of these molecules [ 189–192].
11 Outlook
Since its discovery about 100 years ago the BBB has become immensely importantand rapidly experiences increasing attention from different scientific disciplines. Inorder to proceed it is important to better understand the communication betweencells of the neurovascular framework under various physiological and pathophysio-logical conditions and to explore how distinct components of the BBB are linked toeach other and how their expression and function is regulated. Although significantachievements have been made in the past 10 years, a lot of open questions remain tobe answered. For example, it is still not yet completely clear how tight junctionmolecules assemble, how they are regulated in health and CNS diseases, and howthey interact with several mediators, neurotransmitters, or medications. In addition,transporters and receptors including their signaling cascades become more andmore interesting as targets to ameliorate CNS drug delivery and brain protection.Besides, drug delivery systems which are able to pass the BBB and to release theirThe Blood–Brain Barrier: An Introduction to Its Structure and Function 15
load within the CNS for treatment of neurological diseases have been developedand proven to be successful in animal studies. Nevertheless, further research of thebasic mechanisms underlying the BBB should help to identify new approaches tothe rational treatment of CNS-related diseases.
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Acest articol: Top Med Chem (2014) 10: 120DOI: 10.10077355_2013_20Springer-Verlag Berlin Heidelberg 2013 [603023] (ID: 603023)
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