the blood – brain barrier

M E T H O D S I N M O L E C U L A R M E D I C I N E
TM
Edited by
Sukriti Nag
The
Blood–Brain
Barrier
Biology and Research Protocols
Edited by
Sukriti Nag
The
Blood–Brain
Barrier
Biology and Research Protocols
3
1
Morphology and Molecular Properties of Cellular
Components of Normal Cerebral Vessels
Sukriti Nag
1. Introduction
The blood–brain barrier (BBB) includes anatomical, physicochemical, and bio-
chemical mechanisms that control the exchange of materials between blood and brain
and cerebrospinal fluid (CSF). Thus two distinct systems, the BBB and the blood–CSF
barrier systems, control cerebral homeostasis. However, both systems are unique, the
BBB having a 5000-fold greater surface area than the blood–CSF barrier (1,2). The con-
centrations of substances in brain interstitium, which is determined by transport through
the BBB, can differ markedly from concentrations in CSF, the composition of which is
determined by secretory processes in the choroid plexus epithelia (3). This review will
focus on cellular components of cerebral vessels with emphasis on endothelium, base-
ment membrane, and pericytes as well as the perivascular macrophage (Figs. 1 and 2A),
which in light of new information is distinct from pericytes. This review deals less with

pathogenesis and more with some of the molecules that have been discovered in these
cell types in the past decade. Although astrocytes invest 99% of the brain surface of
the capillary basement membrane and are important in induction and maintenance
of the BBB, this topic will not be discussed and readers are referred to reviews in the
literature (4–11).
2. Cerebral Endothelial Cells
Cerebral capillaries are continuous capillaries, their wall being composed of one or
more endothelial cells. The endothelial surface of 1 g of cerebral tissue has been cal-
culated to be approx 240 cm
2
(12). Cerebral endothelial cell surface properties such as
charge and lectin binding are discussed in Chapter 9. Some of the markers specific for
localization of cerebral endothelium and others that are ubiquitous, being present in
endothelia of non-neural vessels, are given in Table 1 (13–28).
2.1. Cerebral Endothelial Cells in Vasculogenesis and Angiogenesis
Vasculogenesis is the process whereby a primitive network is established during
embryogenesis from multipotential mesenchymal progenitors. This occurs in the rat by
embryonic d 10 after which the intraparenchymal network develops by sprouting from
preexisting vessels, a process termed angiogenesis. Research in the past decade has led
From: Methods in Molecular Medicine, vol. 89:
The Blood–Brain Barrier: Biology and Research Protocols
Edited by: S. Nag © Humana Press Inc., Totowa, NJ
to a greater understanding of cerebral angiogenesis during brain development (28,29)
and in pathological states, including neoplastic (28) and non-neoplastic conditions (30).
During angiogenesis, endothelial cells participate in proteolytic degradation of the base-
ment membrane and extracellular matrix and migrate with concomitant proliferation and
tube formation. Subsequent stages of angiogenesis involve increases in the length of indi-
vidual sprouts, the formation of lumens, and the anastomosis of adjacent sprouts to form
vascular loops and networks. An integral component of angiogenesis is microvascular
hyperpermeability, which results in the deposition of plasma proteins in the extracellu-

lar space forming a matrix that supports the ingrowth of new vessels (31,32).
Endothelial proliferation is tightly regulated during brain development (33). In the
mouse brain, for example, endothelial turnover and sprouting are maximal at postna-
tal d 6–8 (34). Proliferation then slows and the turnover is very low in the adult brain
(35). However, endothelial cells in the adult are not terminally differentiated and post-
mitotic cells and when stimulated such as occurs during wound healing or tumor growth,
they can rapidly resume cell proliferation giving rise to new capillaries.
4 Nag
Fig. 1. Segment of normal cerebral cortical capillary wall consists of endothelium (e) and a
pericyte (p) separated by basement membrane. This rat was injected with ionic lanthanum, which
has penetrated the interendothelial space upto the tight junction (arrowhead). ×70,000.
Fig. 2. (see facing page) (A) A cryostat section shows perivascular macrophages using anti-
ED2 antibody. The inset shows these cells at higher magnification. Note that these cells are asso-
ciated with vessels, which have the caliber of veins and not capillaries. (C,D) Merged confocal
images of normal rat brain dual labeled for Ang-1 and Ang-2 proteins. Normal vessels show
endothelial localization of Ang-1 (green) only in rat brain (B) and choroid plexuses (C) and there
is no detectable localization of Ang-2. Note the granular immunostaining in choroid plexus
epithelial cells indicating colocalization of Ang-1 and Ang-2 (yellow). (D) Cultured cells derived
from cerebral microvessels show adherance of antibody-coated ox red blood cells forming rosettes
indicating presence of Fc receptors. Note that many of these cells contain Factor VIII indicating
that they are endothelial cells (arrowheads). (E) Electron micrograph demonstrating that the
cells to which antibody-coated ox red cells have adhered also show cytoplasmic Factor VIII
immunostaining indicating its endothelial nature. Scale bar A–C = 50 µm; Inset = 25 µm;
D × 100; E × 8000.
Morphology and Molecular Properties 5
Morphologic studies have shown that brain capillaries are derived from endothelial
cells from outside the brain that invade the neuroectoderm and differentiate in response
to the neural environment (28). Using chick-quail transplantation experiments, con-
vincing evidence was presented that BBB characteristics could be induced in endothe-
lial cells, which invade brain transplants (36). Conversely, brain capillaries become

permeable after invasion of somite transplants (36). These results indicate that organ-
specific characteristics of endothelial cells may be induced and maintained by the local
environment. Janzer and Raff (5) provided direct evidence that when purified type I
astrocytes are transplanted into the rat anterior eye chamber or the chick chorioallan-
toic membrane, the astrocytes induce a permeability barrier in invading endothelial cells.
The specific mechanisms regulating angiogenesis are not fully understood but sev-
eral potential regulators of this process include fibroblast growth factor, epidermal
growth factor, transforming growth factors α and β, platelet-derived growth factor,
ephrins, the family of the vascular endothelial growth factors (VEGF) and the angiopoi-
etins (Ang). The VEGF family includes several members; VEGF or VEGF-A is best
characterized, and numerous studies indicate the importance of VEGF-A in vasculo-
genesis and angiogenesis during brain development (28,33,37). During embryonic
brain angiogenesis, VEGF-A is expressed in neuroectodermal cells of the subependy-
mal layer correlating with the invasion of endothelial cells from the perineural plexus
(33). The high affinity tyrosine kinase receptors that bind VEGF-A, VEGFR-1 (flt-1;
38) and VEGFR-2 (flk-1; 39,40) are highly expressed in invading and proliferating
6 Nag
Table 1
Markers for Localization of Normal Cerebral Endothelium
Markers Specific for Cerebral Endothelium
Glucose transporter-1 Kalaria et al. (13)
γ-glutamyl transpeptidase Albert et al. (14)
Neurothelin/HT7 protein (chick) Risau et al. (15); Schlosshauer and Herzog (16)
(human) Prat et al. (17)
OX-47 (Rat homologue of HT7) Fossum et al. (18)
Endothelial barrier antigen (only in rat) Sternberger & Sternberger (19); Cassella et al. (20)
Jefferies et al. (21)
Transferrin receptor Dermietzel and Krause (22)
Tight junction proteins: Liebner et al. (23)
ZO-1

Occludin
Markers Common to all Endothelia
Endoglin (CD 105) Personal Observation
Factor VIII Weber et al. (24)
Growth Factors:
VEGF-B Nag et al. (25)
Angiopoietin-1 Nourhaghighi et al. (26)
Lectin binding:
Ulex europaeus agglutinin I Weber et al. (24)
Enzymes: Endothelial nitric oxide synthase Nag et al. (27)
Platelet/endothelial cell adhesion Plate (28)
molecule-1, (PECAM-1, CD31)
endothelial cells during brain development. This suggests that VEGF-A may act as a
paracrine angiogenic factor. Both VEGF-A and its receptors are largely switched off in
the adult (37,39).
The importance of VEGF-A in cerebral angiogenesis has also been demonstrated in
central nervous system (CNS) neoplasia (28) and non-neoplastic conditions, such as
brain infarction (41–43) and brain injury (25,32). Recent studies demonstrate that
VEGF-B, another member of the VEGF family, is constitutively expressed in cerebral
endothelial cells (25). Angiogenesis following injury is also associated with increased
expression of VEGF-B at both the gene and protein level at the injury site during
angiogenesis (25).
Angiopoietin-1 and -2 constitute a novel family of endothelial growth factors that
function as ligands for the endothelial-specific receptor tyrosine kinase, Tie-2 (44).
Angiopoietins are involved in later stages of angiogenesis when vessel remodeling and
maturation takes place and have a role in the interaction of endothelial cells with
smooth muscle cells/pericytes (45). Recent studies show constitutive expression of
angiopoietin-1 protein in endothelium of normal cerebral vessels (Fig. 2B; 26,46). This
protein is not specific for cerebral endothelium because it is also present in endothe-
lium of choroid plexus and pituitary vessels (Fig. 2C). Increased angiopoietin-2 expres-

sion at both the gene and protein level occurs during angiogenesis after brain injury
(26,46),in cerebral tumors (47,48), and after infarction (43,49,50).
2.2. Properties of Cerebral Endothelial Cells
Features that distinguish cerebral endothelial cells from those of non-neural vessels
and form the structural basis of the BBB include the presence of tight junctions between
cerebral endothelial cells, reduced endothelial plasmalemmal vesicles or caveolae, and
increased numbers of mitochondria.
2.2.1. Endothelial Junctions
2.2.1.1 M
ORPHOLOGY
Transmission electron microscopy (TEM) shows that the junctions between adjacent
cerebral endothelial cells are characterized by fusion of the outer leaflets of adjacent
plasma membranes at intervals along the interendothelial space producing a penta-
laminar apperarance and forming tight or occluding junctions that prevent paracellular
diffusion of solutes via the intercellular route (see Figs. 3 and 4; 51–53). These tight
junctions form the most apical element of the junctional complex, which includes both
tight and adherens junctions. Subsequent studies using horseradish peroxidase as a tracer
suggested that tight junctions extend circumferentially around cerebral endothelial
cells; hence, their name zonula occludens (54,55). Permeability of these junctions to
protein and protein tracers is further discussed in Chapter 6. Tight junctions are also
present between arachnoidal cells located at the outer layers of the dura (56,57), and at
the apical ends of choroid plexus epithelial cells (52,58–60) and ependymal cells (61).
Certain areas of the brain, most of which are situated close to the ventricle and are
therefore called circumventricular organs, have endothelial cells that do not form tight
junctions. These areas include the hypothalamic median eminence, pituitary gland,
choroid plexus, pineal gland, subfornicial organs, the area postrema, and the organum
vasculosum of the lamina terminalis (60,62–64); together they comprise less than 1%
Morphology and Molecular Properties 7
of the brain. Endothelium in these areas is fenestrated with circular pores having a diam-
eter of 40–60 nm that are covered by diaphragms that are thinner than a plasma mem-

brane and of unknown composition. Fenestrations allow free exchange of molecules
between the blood and adjacent neurons. The epithelial cells, which delimit the cir-
cumventricular organs, however, impede diffusion into the rest of the brain and the CSF
(65). Therefore, substances that have entered these areas do not have unrestricted access
to the rest of the brain.
Freeze-fracture studies show that the tight junctions of cerebral endothelium of
mammalian species are characterized by the highest complexity of any other body ves-
sels (66). Eight to 12 parallel junctional strands having no discontinuities run in the lon-
gitudinal axis of the vessel, with numerous lateral anastomotic strands. This pattern
extends into the postcapillary venules, although in a less complex fashion (66). In cere-
bral arteries, tight junctions consist of simple networks of junctional strands, with occa-
sional discontinuities, whereas collecting veins, of which there are a few, have tight
junctional strands that are free-ending and widely discontinuous (66). Another feature
of cerebral endothelial tight junctions is the high association with the protoplasmic
(P)-face of the membrane leaflet, which is 55% as compared with endothelial cells of
non-neural blood vessels, which have a P-face association of only 10% (67). The tight
junctions of choroidal epithelium consist of four or more strands or fibrils, arranged in
parallel with few interconnections (68). In addition, focal discontinuities have been
noted in the junctional strands, which may represent hydrated channels, thus explain-
ing the leakiness of choroid plexus epithelium (69). Further details of freeze fracture
studies of endothelial tight junctions are discussed in Chapter 3 and in studies in the
literature (22,23,67,70–72).
2.2.1.2. TRANSENDOTHELIAL RESISTANCE
The physiologic correlate of tightness in epithelial membranes is transepithelial resis-
tance. Leaky epithelia generally exhibit electrical resistances between 100–200 Ω/cm
2
.
8 Nag
Fig. 3. (A) Segment of cortical arteriolar endothelium from a control rat injected intra-
venously with HRP showing tight junctions (arrowheads) and a zonula adherens (za) junction

along the intercellular space between two endothelial cells. Also present are cross sections of
actin filaments (ac) and microtubules (m) and two plasmalemmal vesicles (v). The vesicle at the
luminal plasma membrane contains HRP. × 132,000.
Cultured brain endothelial cells grown in the absence of astrocytes have an electrical
resistance of approximately 90 Ω/cm
2
(73). The latter is 100-fold less than the electri-
cal resistance across the BBB in vivo, which is estimated to be approx 4–8000 Ω/cm
2
(74,75). The electrical resistance of cultured endothelial cells can be increased to
400–1000 by using special substrata such as type IV collagen and fibronectin (76).
Co-culture of brain microvascular endothelial with astrocytes increases transendothe-
lial electrical resistance by 71% (77) and treatment with glial-derived neurotrophic
factor and cAMP increases transendothelial electrical resistance by approx 250%. (78).
The resistance of the isolated arachnoid membrane with its tight junctions is less than
that of intracerebral vessels being approx 2000 Ω/cm
2
(79),while the junctions of
Morphology and Molecular Properties 9
Fig. 4. Proposed locations of the major proteins associated with tight junctions (TJs) at the
BBB are shown. The tight junction is embedded in a cholesterol-enriched region of the plasma
membrane (shaded). Three integral proteins—claudin 1 and 2, occludin and junctional adhe-
sion molecule (JAM)—form the tight junction. Claudins make up the backbone of the TJ strands
forming dimers and bind homotypically to claudins on adjacent cells to produce the primary
seal of the TJ. Occludin functions as a dynamic regulatory protein, whose presence in the mem-
brane is correlated with increased electrical resistance across the membrane and decreased para-
cellular permeability. The tight junction also consists of several accessary proteins, which
contribute to its structural support. The zonula occludens proteins (ZO-1 to 3) serve as recog-
nition proteins for tight junctional placement and as a support structure for signal transduction
proteins. AF6 is a Ras effector molecule associated with ZO-1. 7H6 antigen is a phosphopro-

tein found at tight junctions impermeable to ions and molecules. Cingulin is a double-stranded
myosin-like protein that binds preferentially to ZO proteins at the globular head and to other
cingulin molecules at the globular tail. The primary cytoskeletal protein, actin, has known bind-
ing sites on all of the ZO proteins. (Modified from ref. 93.)
choroid plexus epithelial cells have a resistance of 73 Ω/cm
2
and hence are considered
to be leaky epithelia (80).
2.2.1.3. MOLECULAR STRUCTURE OF TIGHT JUNCTIONS
Research in the past decade has provided new information on the proteins
composing tight junctions using Madin Darby canine kidney epithelial cells, endothe-
lial cells of non-neural vessels, cerebral endothelial cells, and other cell types. Tight
junctions are composed of an intricate combination of transmembrane and cytoplasmic
proteins linked to an actin-based cytoskeleton that allows these junctions to form a seal
while remaining capable of rapid modulation and regulation (Fig. 4). Three integral pro-
teins—claudin 1 and 2 (81), occludin (82) and junction adhesion molecule (JAM)
(83)—form the tight junction. Claudins form dimers and bind homotypically to claudins
on adjacent endothelial cells to form the primary seal of the tight junction (84). Occludin
is a regulatory protein, whose presence at the BBB is correlated with increased elec-
trical resistance across the barrier and decreased paracellular permeability (85).
Occludin is not present in non-neural vessels thus differentiating the tight junctions of
cerebral and non-neural vessels (85). Junctional adhesion molecules are localized at the
tight junction and are members of the immunoglobulin superfamily, which can func-
tion in association with platelet endothelial cellular adhesion molecule 1 (PECAM) to
regulate leukocyte migration (83). Overexpression of JAM in cells that do not normally
form tight junctions increases their resistance to the diffusion of soluble tracers, sug-
gesting that JAM functionally contributes to permeability control (83).
Tight junctions are also made up of several accessory proteins that are necessary for
structural support such as ZO-1 to 3, AF-6, 7H6 and cingulin. The zonula occludens
(ZO) proteins 1–3 (86,87) belong to a family of proteins known as membrane-

associated guanylate kinase-like proteins (88),afamily of multidomain cytoplasmic
molecules involved in the coupling of transmembrane proteins to the cytoskeleton.
ZO-1 is a component of the human and rat BBB (89). The ALL-1 fusion partner from
chromosome 6 (AF-6) is associated with ZO-1 and serves as a scaffolding component
of tight junctional complexes by participating in regulation of cell–cell contacts via
interaction with ZO-1 at the N terminus Ras-binding domain (90). 7H6 antigen is a phos-
phoprotein found at tight junctions that are impermeable to ions and macromolecules
(91). A recent review suggests that 7H6 is sensitive to the functional state of the tight
junction (92). In response to cellular adenosine triphosphate (ATP) depletion, 7H6
reversibly disassociates from the tight junction while ZO-1 remains attached and there
is concurrent increase in paracellular permeability (93). Cingulin is a double-stranded
myosin-like protein localized at the tight junction and found in endothelial cells as well.
Recent in vitro studies have shown that ZO-1, ZO-2, ZO-3, myosin, JAM and A6 inter-
act with cingulin at the N-terminus, while myosin and ZO-3 bind at the C-terminus (94).
Thus, cingulin appears to serve as a scaffolding protein that links tight junction acces-
sory proteins to the cytoskeleton.
Availability of antibodies to some of the tight junction proteins has allowed local-
ization of these proteins in cerebral endothelium by immunohistochemistry. ZO-1
immunoreactivity occurs along the entire perimeter of cultured cerebral endothelial cells
(73) and endothelial cells in cryostat sections of human brain (95). Cryostat sections
10 Nag
of adult brain also show anti-ZO-1 immunoreactivity as fibrillar fluorescence along the
lateral aspects of brain microvessels (22) which constitute interendothelial tight junc-
tion domains. The ZO-1 protein occurs in approximately the same quantities (molecules
per micron) as the intramembranous particles that constitute the junctional fibrils in
freeze-fracture preparations (96,97). ZO-1 immunoreactivity is also observed in brain
endothelial cells of chick and rat along with immunoreactivity for occludin, claudin-1
and claudin-5 (23). ZO-1 immunoreactivity is also present at the other known sites of
tight junction locations within the CNS such as in the leptomeningeal layer, choroid
plexus epithelium, and the ependyma (22).

The primary cytoskeletal protein, actin, has known binding sites on all ZO proteins,
and on claudin and occludin (98). Electron microscopy shows microfilaments having
the dimensions of actin grouped near the cytoplasmic margins in proximity to cell junc-
tions in cerebral endothelium (see Fig 3; 10,99,100) and actin has been localized to the
plasma membrane by molecular techniques (101). ZO-1 binds to actin filaments and
the C-terminus of occludin (98), which couples the structural and dynamic properties
of perijunctional actin to the paracellular barrier.
Tight junctions are localized at cholesterol-enriched regions along the plasma mem-
brane associated with caveolin-1 (102). Caveolin-1 interacts with and regulates the activ-
ity of several signal transduction pathways and downstream targets (103). Several
cytoplasmic signaling molecules are concentrated at tight junction complexes and are
involved in signaling cascades that control assembly and disassembly of tight junctions
(104). Regulation of tight junctions is discussed in previous reviews (67,93,104–106).
Adherens junctions are located near the basolateral side of endothelial cells (Fig. 3).
Adherens junction proteins include the E, P, and N cadherins, which are single-pass
transmembrane glycoproteins that interact homotypically in the presence of Ca
2+
(107).
These cadherins are not specific for cerebral endothelial junctions being present in
endothelium of non-neural blood vessels as well (108). Cadherins are linked intracel-
lularly to a group of proteins termed catenins (109). α-catenin is a vinculin homolog
that binds to β-catenin and probably links cadherins to the actin-based cytoskeleton and
to other signaling components. γ-catenin is related to β-catenin and can substitute for
it in the cadherin-catenin complex. Catenins are, thereby, part of the system by which
adherens and tight junctions communicate. All these molecules are expressed at junc-
tions in brain endothelial cells (110,111). The newer cadherin-associated protein, p120
and a related protein p100 are associated with the cadherin/catenin complex in both
epithelial and endothelial cells (112). The interaction of these proteins in junctional per-
meability has been recently reviewed (67,106).
2.2.2. Endothelial Plasmalemmal Vesicles or Caveolae

2.2.2.1. M
ORPHOLOGY
TEM studies show membrane-bound vesicles open to both the luminal and ablumi-
nal plasmalemma through a neck 10–40 nm in diameter and also free in the endothe-
lial cytoplasm of vessels of most organs (Fig. 3; Chapter 6, Fig. 1). These non-coated
structures referred to in the previous literature as pinocytotic vesicles are now gener-
ally referred to as plasmalemmal vesicles or caveolae. These vesicles are distinct
from clathrin-coated vesicles, which have an electron-dense coat and are involved in
Morphology and Molecular Properties 11
receptor-mediated endocytosis. Free cytoplasmic caveolae are spherical structures
having a mean diameter of approx 70 nm. Studies of frog mesenteric capillaries sug-
gest that caveolae are part of two racemose systems of invaginations of the luminal and
abluminal cell surfaces and not freely moving entities (113). There is considerable het-
erogeneity in endothelium in different parts of a single organ hence the findings in frog
mesenteric capillaries may not necessarily apply to all species or to brain capillaries.
Morphometric studies show that normal cerebral endothelium contains a mean
of 5 plasmalemmal vesicles/µm
2
in arteriolar (114) and capillary endothelium
(70,115–117). Other authors report higher values of 10–14 vesicles/µm
2
in capillary
endothelium of rats and mice (118,119),which they attribute to small sample size. As
compared with endothelium of non-neural vessels such as myocardial capillaries (120),
cerebral endothelium contains 14-fold fewer vesicles. The decreased number of vesi-
cles in cerebral endothelium implies limited transcellular traffic of solutes. In contrast,
capillaries in areas where a BBB is absent such as the subfornicial organ and area
postrema (63,118) and muscle capillaries (121) that are highly permeable have signif-
icantly higher numbers of endothelial vesicles. Theoretical models of vesicular trans-
port agree in predicting a transport time in the order of seconds (122).

2.2.2.2. FUNCTION
Endothelial caveolae are either endocytic or transcytotic (123). The permeant mol-
ecules can either be internalized within endothelial cells by endocytosis or may be
translocated across the cell to the interstitial fluid, a process termed transcytosis. Both
endocytosis and transcytosis may be receptor-mediated or fluid phase and require ATP
and can be inhibited by N-ethylmaleimide (NEM), an inhibitor of membrane fusion
(124). There is increasing evidence that in defined vascular beds, receptor-mediated
transcytosis of caveoli are involved in transport of low density lipoprotein (LDL),
β-very low density lipoprotein (VLDL), transferrin, insulin, albumin, ceruloplasmin,
and transcobalamin across the endothelium (125,126). Transcytosis is a known mech-
anism for passage of plasma solutes and macromolecules across endothelium of non-
neural vessels (127–129). Two distinct but not mutually exclusive mechanisms for
transendothelial transport by the caveolar system have been proposed and debated
without resolution in the past four decades. The oldest or “shuttle” hypothesis suggests
that single caveolae at the luminal or abluminal surfaces “bud off” to become free within
the cytoplasm carrying their molecular cargo across the cell to fuse with the opposite
plasma membrane. Second, caveolae are postulated to fuse to form a transendothelial
channel extending from the luminal to the abluminal plasma membrane, which allows
passage of substances from the blood to tissues or in the reverse direction (125,127,130).
Such channels have been demonstrated in non-neural vessels in steady states (125,130)
but not in normal cerebral endothelium either by freeze fracture (71), transmission (131),
or high voltage electron microscopy (132). The latter technique allows examination of
0.25-0.5 µm thick plastic sections and is further discussed in Chapter 4. Transendothelial
channels have been observed in cerebral endothelium following BBB breakdown as dis-
cussed in Chapter 6.
In addition to transcytosis of molecules across endothelial cells, caveolae have been
implicated in endocytosis, potocytosis, signal transduction, mechano-transduction in
endothelial cells and control of cholesterol trafficking (103,133–140).
12 Nag
2.2.2.3. MOLECULAR STRUCTURE OF CAVEOLAE

Studies of non-neural endothelial cells and other cell types have provided informa-
tion about the molecular structure of caveolae. The specific marker and major compo-
nent of caveolae is caveolin-1, an integral membrane protein (20–22 kDa) having both
amino and carboxyl ends exposed on the cytoplasmic aspect of the membrane (141).
Caveolae in most epithelial and endothelial cells contain caveolin-1 (142),which
belongs to a multigene family of caveolin-related proteins that show similarities in struc-
ture but differ in properties and distribution. Caveolin-2 has a similar distribution with
caveolin-1 (143) and endothelial cells express only caveolin-1 and -2 (144). Caveolae
have a unique lipid composition, of which cholesterol and sphingolipids (sphingomyelin
and glycosphingolipid) are the main components. The sphingolipids are substrates for
synthesis of a second intracellular messenger, the ceramides (145). Cholesterol may
create the frame in which all other caveolar elements are inserted.
Caveolin acts as a multivalent docking site for recruiting and sequestering signaling
molecules through the caveolin-scaffolding domain that recognizes a common sequence
motif within caveolin-binding signaling molecules (146). Signaling molecules found
in caveolar domains and form complexes with caveolin are: heterotrimer G protein, com-
ponents of the Ras-mitogen-activated protein kinase pathway, Src tyrosine kinase, pro-
tein kinase C, H-Ras, and endothelial nitric oxide synthase (139). Receptors having
caveolar localization are involved in transport and signaling and include growth factor
receptors like epidermal growth factor, insulin, and platelet derived growth factor recep-
tors, G-protein coupled receptors like PAR, mAcR, CCK-, and receptors for endothe-
lin, advanced glycation end products (RAGE), inositol triphosphate, CD36 (147),
albumin binding proteins (148), and albondin (149). Other antigens residing in endothe-
lial caveolae are PV-1, and the plasma membrane calcium ATPase (150–154). Other
molecules partially localized in caveolae include thrombomodulin, the functional throm-
bin receptor, GP85/115, heterotrimeric G proteins, and dynamin (155–159).
Caveoli contain the molecular machinery that promote vesicle formation, fission,
docking, and fusion with the target membrane. Isolated caveoli from lung capillaries
demonstrate vesicle-associated SNAP receptor (vSNARE), vesicle-associated mem-
brane protein-2 (VAMP-2) (160), monomeric and trimeric GTPases, annexins II and

IV, N-ethyl maleimide-sensitive fusion factor (NSF), and soluble NSF attachment pro-
tein (SNAP) (161). These molecules interact in the stages of transcytosis as follows:
Caveoli form at the cell surface through ATP-, guanosine 5′-triphosphate (GTP)- and
Mg
2+
-polymerization of caveolin-1 and 2, a process stabilized by cholesterol (141).
Caveolin oligomers may also interact with glycosphingolipids (162); these protein-
protein and protein-lipid interactions are thought to be the driving force for caveoli for-
mation (163). A component of the caveolar fission machinery is the large GTPase,
dynamin, which oligomerises at the neck of caveolae and probably undergoes hydrol-
ysis for fission and release of caveolae so it becomes free in the cytoplasm (158,164).
The transcellular movement of caveolae is facilitated by association with the actin-
cytoskeleton related proteins, such as myosin HC, gelsolin, spectrin, and dystrophin
(165). Fusion at the abluminal membrane is aided by NSF, which interacts with solu-
ble attachment proteins (SNAPs) that can associate with complementary SNAP recep-
tors to form a functional SNARE fusion complex. Before fusion of the target and
vesicle membrane, v-SNARE (VAMP), the targeting receptor located on the vesicles,
Morphology and Molecular Properties 13
recognizes and docks with its cognate t-SNARE (syntaxin) on the target membrane
(166,167). Specific docking, is aided by endothelial VAMP-2 (160),which is localized
in caveolae.
The evidence thus far favors the hypothesis that caveolae are dynamic vesicular
carriers budding off from the plasma membrane to form free transport vesicles that
fuse with specific target membrane molecules as described in recent reviews
(125,126,168,169). Evidence that caveolae can traffic their cargo across cells (tran-
scytosis) has been recently shown (170). In addition, caveolin-1 gene knock-out mice
show defects in the uptake and transport of albumin in vivo (171). A new direction is
molecular mapping of the endothelium in its native state in tissue in order to identify
novel tissue-specific and disease-induced targets on the endothelial-cell surface and
within caveolae, which could be targeted by therapeutic strategies such as drug or gene

delivery in vivo (126,172).
2.2.2.4. ALBONDIN
Most of the molecular studies on caveolae have been done in non-neural endothe-
lium and other cell types. It would be interesting to know whether the caveolae in brain
endothelium are similar or whether tissue-specific differences exist. One difference that
has been identified thus far is the low expression of albondin in brain endothelium (149).
Albondin is a 60-kDa albumin-binding sialoglycoprotein that is expressed selectively
by vascular endothelium and is present on the luminal surface of continuous endothe-
lium in situ and in culture. It binds albumin apparently not only to initiate its transcy-
tosis via caveolae but also to increase capillary permselectivity (149). Microvascular
endothelial cells isolated from rat heart, lung and epididymal fat pad express albondin
and bind albumin whereas various nonendothelial cells do not. Low expression or lack
of expression of albondin in brain-derived microvascular endothelial cells accounts for
restricted albumin passage into brain in steady states.
2.2.3. Mitochondria
Most of the mitochondria in cerebral endothelium are located in the vicinity of the
nucleus, but occasional mitochondria occur throughout the cytoplasm and these tend
to be parallel to the cell surface. Murine cerebral endothelium contains greater num-
bers of mitochondria (173), with 10% (174) and 13.7% (175) of the endothelial volume
being occupied by mitochondria, which is greater than found in endothelia of other tis-
sues. Increased mitochondria in cerebral endothelium may provide the metabolic work
capacity for maintaining the ionic gradient across the BBB. Other studies have obtained
a lower value of 2–6% for the proportion of cerebral endothelial cytoplasmic volume
occupied by mitochondria in rat (116),chick (36), mouse (118), and human (117) cere-
bral capillaries. The different values obtained by both groups may be related to the dif-
ferent morphometric method used by each group. Endothelial mitochondrial density is
lower in capillaries of the subfornicial organ, which lies outside the BBB (63).
2.3. Endothelium in Inflammation
The brain exists in an immunologically privileged environment, however, this does
not exclude immune cells from reaching the brain under steady states (176). Many of

the same processes that are active in the beneficial monitoring also play key roles in
14 Nag
initiating and /or potentiating detrimental inflammatory reactions (177). Cerebral
endothelial cells, by virtue of their capacity to express adhesion molecules and
chemokines and their potential to act as antigen presenting cells and participate in death
receptor signaling are intricately involved in inflammatory processes. They also express
cytokines and represent a point of passage for immune cells present in the blood into
the extracellular environment of the brain as discussed in Chapter 6, Subheading 6.5.
2.3.1. Adhesion Molecule Expression
The interaction of leukocytes with endothelium is a sequential and multistep process
that involves leukocyte rolling on the endothelium mediated by adhesion molecules lead-
ing to firm adhesion. The latter process is mediated by interaction of the immunoglob-
ulin family (ICAM-1 and VCAM-1) or the selectin family (E-selectin and P-selectin)
of cellular adhesion molecules expressed by endothelial cells and their leukocyte lig-
ands. Immunohistochemical studies of frozen normal brain tissue show minimal to non-
detectable localization of various integrin adhesion molecules including ICAM-1,
VCAM-1 and PECAM-1 in endothelium (178–180) and in vitro studies show low
E-selectin (181) and P-selectin (182) localization on human microvascular endothelial
cells. Small numbers of immune cells are able to cross into the CNS using the minimal
level of these adhesion molecules that exist normally (177). The ICAM-1, VCAM-1
and E-selectin levels on brain microvascular endothelial cells are upregulated 3- to
15-fold in response to IFN-γ, TNF-α,and IL-1 treatment (181,183–186) while
P-selectin levels increase in response to histamine or thrombin (182).
2.3.2. Chemokine Production
Chemokines are a subgroup of small cytokines (8–10 kD) that are produced within
a target tissue and provide a mechanism by which specific populations of inflamma-
tory cells including Th1 and Th2 cells and monocytes are attracted to the target tissue
(187–191). Production of chemokines within the CNS is well documented; cellular
sources include glial and endothelial cells (192). The presence of MCP-1 and MIP-1β-
binding sites on human brain microvessels (193) suggests that chemokines produced

locally by perivascular astrocytes and microglial cells either diffuse or are transported
to the endothelial cell surface, where they are immobilized for presentation to leuko-
cytes. Such a process has been demonstrated in the periphery with the chemokine inter-
leukin (IL)-8 (194).
MCP-1 immunoreactivity can be detected in brain endothelial cells (195) at the
onset of inflammation and before clinical expression of disease during experimental
allergic encephalomyelitis. Human brain endothelial cells produce and secrete bioac-
tive IL-6 and MCP-1 (196–198) and can also express IP-10, MIP1β, and RANTES
(199,200). Prominent upregulation of these chemokines is observed in response to glial
cell-derived pro-inflammatory cytokines (200,201).
2.3.3. Antigen Presentation at the BBB
Although both neural antigen-specific and nonspecific T-cells can enter brain, only
those that recognize their specific antigen presented by a competant antigen present-
ing cell would persist and initiate an inflammatory reaction (176). Cerebral microvas-
cular endothelial cells are potentially significant antigen presenting cells because of their
Morphology and Molecular Properties 15
large cumulative surface area and unique anatomical location between circulating
T-cells and the extravascular sites of antigen exposure. Endothelial cells do not consti-
tutively express MHC class II molecules in vivo or in vitro but can be induced to do so
(179,202–204). Fc receptor for the constant region of immunoglobulin has also been
demonstrated on cerebral endothelial cells (Fig. 2D,E; 205,206).
2.3.4. Death Receptor Signaling
Recent evidence suggests that endothelial cells can induce immune cell death and
downregulate an ongoing immune cell-mediated inflammatory process. Resting human
brain endothelial cells produce minimal levels of Fas and TRAIL mRNA (199).
However, after stimulation with IFN-γ or IFN-β, up regulation of both Fas and TRAIL
mRNA occurs (199). Upon secretion/cleavage from the cell surface, TRAIL binds to
death receptors and induces the apoptotic death of the target cell. Conversely, Fas mol-
ecules expressed by human brain endothelial cells could be produced as soluble Fas,
secreted and act as antagonists to Fas ligand expressed by immune cells, thus inhibit-

ing death mediated signals.
Thus it appears that cerebral endothelium is equipped with the necessary molecules
to participate in immune responses. Most of the cited studies are in vitro studies using
cultured cerebral endothelial cells. Methods for conducting such studies are given in
part IV of this book. Further work is necessary to identify the molecular mechanisms
occurring in vivo that lead to the inflammatory response in various brain diseases.
3. Basement Membrane
The basement membrane is a specialized, extracellular matrix, which separates
endothelial cells and pericytes from the surrounding extracellular space (Fig. 1). The
basement membrane develops its “mature” form between postnatal d 21 and 28 by
apparent fusion and thickening of the lamellae from both endothelia and astroglia (207)
resulting in a change in both the quantity of the basement membrane, as measured by
the hydroxyproline content (208),and the quality as determined by immunohisto-
chemistry (209,210). In adults this membrane is 30–40 nm thick and is synthesized by
both astrocytes and endothelial cells which are connected with the basement membrane
via fine filaments. Ultrastructural studies show that the basement membrane has an inner
electron-dense layer called the lamina densa; and less electron-dense layers called the
laminae rarae (22). The basement membrane is composed of laminin (211), collagen
IV (212),proteoglycans, notably heparan sulphate (213,214),fibronectins (215), nido-
gen (216) and entactin (217). The chemical composition of these individual basement
membrane components differs among various organs (218).
The subendothelial basal lamina is no impediment to the extracellular flow of trac-
ers such as horseradish peroxidase. The basal lamina of capillaries shows strong label-
ing with cationic colloidal gold indicating that it forms a negatively charged screen or
filter controlling the movement of charged solutes between blood and the brain inter-
stitial fluid (219). Large, charged molecules such as ferritin do not cross the basal lamina
(220). The subendothelial basal lamina also serves as a repository for growth factors
such as basic fibroblast growth factor and heparin binding proteases and protease
inhibitors (214). Therefore regulated release of growth factors and proteases from the
basal lamina reservoir could play a role in angiogenesis and the invasion of the inter-

stitium by tumor cells (214).
16 Nag
4. Pericytes
Some authors refer to perivascular cells in relation to all types of vessels whether
capillaries, arterioles and venules as pericytes (221,222). In this review pericytes will
refer to the cells, which form a discontinuous layer on the outer aspect of capillaries
and the small post-capillary venules, both of which are indistinguishable by electron
microscopy (Fig. 1). Pericyte coverage of microvessels varies and in rat capillaries cov-
erage is 22–32% (223). In isolated brain capillary preparations, there is approximately
one pericyte for every three endothelial cells (224). In the CNS, pericytes have an oval
to oblong cell body arranged parallel to the vessel long axis (225). The cell body of the
pericyte consists of a prominent nucleus with limited perinuclear cytoplasm from which
extend cytoplasmic processes that also run parallel to the long axis of the blood vessel;
orthogonally oriented secondary processes arise along the length of the primary process
and partially encircle the vascular wall. Pericytes may be “granular” or “agranular,”
depending on whether cytoplasmic lysosomes are abundant or sparse respectively (221).
A quantitative study shows that human cerebral pericytes are exclusively granular
(226). Cerebral pericytes are rich in cytoplasmic plasmalemmal vesicles.
Although pericytes are separated from endothelium by the basement membrane,
pericyte-endothelial cell gap junctions have been described in human cerebral capillar-
ies (227). Scanning electron microscopic studies show contacts between the pericyte and
endothelial cell which is made by cytoplasmic processes of the pericyte indenting the
endothelial cell and vice versa, forming so-called “peg-and-socket” contacts (228,229).
In brain and retina, the adhesive glycoprotein, fibronectin, has been characterized at junc-
tional sites between pericytes and endothelial cells adjacent to “adhesion plaques” at the
pericyte plasma membrane (230). The plaques suggest a mechanical linkage between
the two cells, which would permit contractions of the pericyte to be transmitted to the
endothelial cell resulting in the reduction of the microvessel diameter.
Pericytes share many markers with smooth muscle cells such as aminopeptidase N,
aminopeptidase A and nestin (Ta ble 2, 230–233). Angiopoietin-2 (26,44), and

γ-glutamyl transpeptidase, a frequently used endothelial marker is also present in brain
microvessel pericytes (234). Glutamic acid decarboxylase (235) and butyryl-
cholinesterase (236) have also been localized in pericytes. Although α-smooth muscle
actin protein and mRNA have been demonstrated in isolated cerebral pericytes (237),
immunohistochemistry at the light microscopical level does not show this protein in
paraffin sections of brain (personal observation) or in isolated bovine brain capillaries
(224). However, immunogold labeling of isolated cerebral vessels with anti-α-smooth
muscle actin shows smooth muscle cell labeling frequently aligned along the myofila-
ments and dense labeling of pericytes (233). These authors did not observe
luminal/abluminal polarity in the immunogold labeling in either cell.
Three major functional roles have been ascribed to pericytes associated with CNS
microvasculature. They include contractility, regulation of endothelial cell activity and
a role in inflammation.
4.1. Contractility
Contraction (and reciprocal relaxation) appears to be the way that pericytes regulate
microvascular blood flow, similar to the smooth muscle of larger vessels. The demon-
stration of contractile elements in pericytes support their contractile role. Actin and actin
Morphology and Molecular Properties 17
filaments (238,239) have been demonstrated in pericytes in vitro with a portion of this
actin corresponding to the α-isoform or muscle-specific form (233). These authors did
not observe α-smooth muscle actin in all CNS pericytes suggesting that contraction may
not be a universal property of all pericytes. Functional capability of pericyte-derived
actin was demonstrated in biochemical assays, where it was shown to activate myosin
Mg
2+
-ATPase (240). Tropomyosin and both muscle-specific and non-muscle isoforms
of myosin are present in pericytes (241,242) including cerebral pericytes (243), with
the muscle-specific myosin being distributed in the same location as α-actin. Further
support for the contractile function of pericytes comes from in vitro studies where their
contraction has been directly observed (244). A number of agents, which regulate con-

traction of pericytes such as histamine, serotonin, endothelin-1 and angiotensin II have
been reviewed previously (225).
4.2. Pericyte-Endothelial Interactions
Pericytes not only reside in proximity to vascular endothelial cells, but they also have
several different types of direct contact with these cells as described above. The com-
bination of intimate cellular contacts and their presence during development suggests
that they may regulate endothelia and vascular development (245,246). Pericytes can
inhibit endothelial cell proliferation and thereby stabilize developing microvessels (247).
18 Nag
Table 2
Markers for Cerebral Pericytes, Smooth Muscle Cells
and Perivascular Macrophages
Smooth Perivascular
Marker Pericyte muscle cell macrophage
Aminopeptidase A + + NK
Aminopeptidase N + + NK
Nestin + + NK
γ-glutamyl transpeptidase + + +
Angiopoietin-2 + – –
α-SM actin + + –
SM myosin – + –
β-actin + + NK
NM myosin + + NK
Vimentin + + +
Calponin – + NK
Desmin – + –
Skeletal/SM – + NK
CD11b + – +
ED-2 ± – +
CD45 – – +

MHC class II – – +
GSA – – +
SM = smooth muscle; NM = nonmuscle; GSA = Griffonia Simplicifolia Agglutinin; NK = not known.
References: Alliott et al. (231); Balabanov and Dore-Duffy (232); Bandopadhyay et al. (233).
In vitro studies demonstrate inhibition of endothelial cell growth and proliferation by
pericytes (248,249),which is mediated by transforming growth factor-β-1 only when
there is physical contact between these cells (45,248). On the other hand pericytes also
provide factors, which stimulate the growth and development of endothelial cells.
Pericytes have been implicated in all stages of angiogenesis including initiation, vas-
cular sprout extension and endothelial migration (250). In vitro studies demonstrate that
pericytes produce basic fibroblast growth factor (45,251), VEGF growth factor
(252,253), and angiopoietins (44), all of which are involved in neovascularization and
angiogenesis. Thus pericytes have both a negative and positive effect on endothelial cells.
Endothelial cells on the other hand have a reciprocal regulatory effect on pericytes.
Endothelial cells produce transforming growth factor (254), VEGF (255), endothelin-1
(256,257), and platelet-derived growth factor, all of which have been shown to influ-
ence pericyte function. Transforming growth factor inhibits pericyte growth, whereas
VEGF stimulates pericyte proliferation in vitro. Endothelin-1 stimulates pericyte con-
traction and is a pericyte mitogen in vitro. The origin of platelet-derived growth factor
from endothelial cells is less certain. However, in the absence of this agent, pericytes
do not develop (258), hence it is thought to serve as a migration and/or differentiation
signal (259,260).
4.3. Role in Inflammation
Pericytes are thought to serve as macrophages in the brain. Morphologic studies
demonstrate numerous cytoplasmic lysosomes that increase in size and number in con-
ditions associated with BBB breakdown to exogenously administered proteins such as
horseradish peroxidase (261–263). In this context, pericytes form a second line of
defence for the BBB by phagocytosing molecules that pass through endothelium.
Pericytes in situ contain the components recognized by the macrophage-selective mon-
oclonal antibodies EBM/11 (264) and ED2 (265,266), and the macrophage-specific pro-

tein class II major histocompatibility complex (267,268). In addition, they exhibit
phagocytosis (265,269). Thus there is significant evidence supporting the role of peri-
cytes as macrophages in the CNS.
CNS pericytes may be actively involved in the regulation of leukocyte transmigra-
tion, antigen presentation, and T-cell activation (270–272). Pericytes present antigen in
vitro and differentially activate Th1 and Th2 CD4+ lymphocytes (273). CNS pericytes
produce a number of immunoregulatory cytokines such as interleukin-1β, interleukin-6
and granulocyte-macrophage colony stimulatory factor (271).
For detailed discussions of pericyte functions, readers are referred to reviews in the
literature (221,225,232,274,275). Some of the studies described in this review have been
done in non-neural pericytes. The availability of a technique to isolate CNS pericytes
(see Chapter 25) provides a means to study the role of this cell in BBB biology.
5. Perivascular Macrophages
These are a heterogenous population found in the CNS and the peripheral nervous
system. Various names have been used to describe these cells, including perivascular
cells, perivascular macrophages, perivascular microglia, and fluorescent granular
perithelial cells (Mato cells), which reflect heterogeneity within this population as well
as different models of neuropathology, anatomic locations, and species studied (276).
Morphology and Molecular Properties 19
There is consensus regarding their location, phenotype, and putative immune functions.
Perivascular macrophages are a minor population in the CNS situated adjacent to
endothelial cells and are immediately beyond the basement membrane of small arteri-
oles and venules (Fig. 2 A; 10,267,277). Within the perivascular space, these cells abut
CNS endothelial cells and can extend long branching processes that enwrap the ves-
sels to which they are apposed (267,277). Under certain pathologic conditions, includ-
ing autoimmune inflammation and viral encephalitides, perivascular macrophages can
accumulate transiently and then disappear (262,263,278,279), or they can remain for
long periods (280). Perivascular macrophages are bone marrow derived and continu-
ously replaced by monocytes. Bone marrow chimera studies in rodents (277,281) and
transplantation studies in humans (282) show a steady rate of perivascular macrophage

turnover in the normal noninflamed CNS. Approximately 30% of perivascular
macrophages are replaced during a 3-mo period in rats (283).
5.1. Immunophenotype of Perivascular Macrophages
ED2 (Table 2; 266,277,284) is a universally accepted marker of perivascular cells
along with CD45 (266,278,285), CD4 (286) and OX-42 (266,277,278), although, pop-
ulations of these cells are identified that are OX-42 and GSA-I-B4 isolectin negative
(266). Fc receptor for the constant region of immunoglobulin is also found on perivas-
cular macrophages (287–290). Rodent perivascular macrophages have constitutive
major histocompatibility class II (MHC II) expression that is further augmented after
exposure to interferon-γ/tumor necrosis factor-α (291), and in experimental allergic
encephalomyelitis (277,278), and neuronal damage (292,293).
Perivascular macrophages in human and nonhuman primates are immunoreactive
with CD14, CD45 (276,289,290,294) and esterase antibodies (289,294). They also
have constitutive MHCII and CD4 expression (267,295,296). Basal levels of MHC II
antigens, B7 and CD40, and Fc receptor are increased on perivascular macrophages in
inflammatory CNS conditions including multiple sclerosis (289,290,296–299).
Numerous immune functions have been ascribed to perivascular macrophages based
solely on the expression of immune molecules. They have been implicated as the resi-
dent CNS antigen presenting cells (277), they undergo immune activation in response
to inflammation or neuronal injury/death (292,293,300) and can perform phagocytosis
and pinocytosis or respond to cytokines and LPS in the peripheral blood (280,301,302).
In conclusion, an attempt has been made to describe some of the molecules that have
been discovered in cerebral vessels in the past decade. It is hoped that the methods
described in this book will aid researchers in the isolation of molecules not described
thus far and in increasing our understanding of how these molecules interact at the BBB
to maintain cerebral homeostasis as well as the mechanisms that result in BBB break-
down. A greater understanding of the molecular mechanisms occurring at the BBB in
diseases is necessary in order to identify substances/molecules that can be targeted for
pharmacological manipulation and/or gene therapy.
Acknowledgments

Grant support from the Heart and Stroke Foundation of Ontario for the period 1978
to 2002 is gratefully acknowledged. Thanks are expressed to Drs. David Robertson, Jim
Eubanks, and Cynthia Hawkins for their helpful suggestions during the preparation of
this manuscript.
20 Nag
References
1. Crone, C. (1971) The blood-brain barrier—facts and questions. In Ion Homeostasis of the
Brain. (Siesjo, B. K., and Sorensen, S. C., eds.) Munksgaard, Copenhagen, pp. 52–62.
2. Pardridge, W. M., Frank, H. J. L., Cornford, E. M., Braun, L. D., Crane, P. D., and Oldendorf,
W. H. (1981) Neuropeptides and the blood-brain barrier. In Neurosecretion and Brain
Peptides. (Martin, J. B., Reichlin, S., and Bick, K. L., eds.) Raven, New York,
pp. 321–328.
3. Hutson, P. H., Sarna, G. S., Katananeni, B. D., and Curzon, G. (1985) Monitoring effects
of tryptophan load on endometabolism in freely moving rats by simultaneous cerebrospinal
fluid sampling and brain dialysis. J. Neurochem. 44, 1266–1273.
4. DeBault, L., and Cancilla, P. A. (1980) γ-glutamyl transpeptidase in isolated brain endothe-
lial cells: induction by glial cells in vitro. Science 207, 635–645.
5. Janzer, R. C., and Raff, M. C. (1987) Astrocytes induce blood-brain properties in endothe-
lial cells. Nature 325, 253–257.
6. Kacem, K., LaCombe, P., Seylaz, J., and Bonvento, G. (1998) Structural organization of the
perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter:
a confocal microscopy study. Glia 23, 1–10.
7. Laterra, J., Guerin, C., and Goldstein, G. W. (1990) Astrocytes induce neural micro-
vascular endothelial cells to form capillary-like structures in vitro. J. Cell Physiol. 144,
204–215.
8. Pardridge, W. M. (1995) The Blood-Brain Barrier. Cellular and Molecular Biology. Raven,
New York, pp. 137–164.
9. Pardridge, W. M. (1998) Introduction to the Blood-Brain Barrier. Methodology, Biology and
Pathology. Cambridge University Press, Cambridge, UK.
10. Peters, A., Palay, S. L., and DeF Webster, H. (1976) The Fine Structure of the Nervous

System: The Neurons and Supporting Cells. W.B. Saunders Co., Philadelphia.
11. Raub, T. J., Kuentzel, S. L., and Sawada, G. A. (1992) Permeability of bovine brain microves-
sel endothelial cells in vitro: barrier tightening by a factor released from astroglioma cells.
Exp. Cell Res. 199, 330–340.
12. Crone, C. (1963) The permeability of capillaries in various organs as determined by use of
the ‘indicator diffusion’ method. Acta Physiol. Scand. 58, 292–305.
13. Kalaria, R. N., Gravina, S. A., Schmidley, J. W., Perry, G., and Harik, S. I. (1988) The glu-
cose transporter of the human brain and blood-brain barrier. Ann. Neurol. 24, 757–764.
14. Albert, Z., Orlowski, M., Rzucidlo, Z., and Orlowska, J. (1966) Studies on γ-glutamyl
transpeptidase activity and its histochemical localization in the central nervous system of
man and different species. Acta Histochem. 25, 312–320.
15. Risau, W., Hallmann, R., Albrecht, U., and Henke-Fahle, S. (1986) Brain induces the expres-
sion of an early cell surface marker for blood-brain barrier-specific endothelium. EMBO J.
5, 3179–3183.
16. Schlosshauer, B., and Herzog, K H. (1990) Neurothelin: inducible cell surface glycopro-
tein on blood-brain barrier-specific endothelial cells and distinct neurons. J. Cell Biol. 110,
1261–1274.
17. Prat, A., Biernacki, K., Pouly, S., Nalbantoglu, J., Couture, R., and Antel, J. P. (2000) Kinin
B1 receptor expression and function on human brain endothelial cells. J. Neuropathol. Exp.
Neurol. 59, 896–906.
18. Fossum, S., Mallett, S., and Barclay, A. N. (1991) The MRC OX-47 antigen is a member
of the immunoglobulin superfamily with an unusual transmembrane sequence. Eur.
J. Immunol. 21, 671–679.
19. Sternberger, N. H., and Sternberger, L. A. (1987) Blood-brain barrier protein recognized
by monoclonal antibody. Proc. Natl. Acad. Sci. USA 84, 8169–8173.
Morphology and Molecular Properties 21
20. Cassella, J. P., Lawrenson, J. G., Lawrence, L., and Firth, J. A. (1997) Differential distrib-
ution of an endothelial barrier antigen between the pial and cortical microvessels of the rat.
Brain Res. 744, 335–338.
21. Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C., and Mason, D.

Y. (1984) Transferrin receptor on endothelium of brain capillaries. Nature 312, 162–163.
22. Dermietzel, R., and Krause, D. (1991) Molecular anatomy of the blood-brain barrier as
defined by immunocytochemistry. Int. Rev. Cytol. 127, 57–109.
23. Liebner, S., Kniesel, U., Kalbacher, H., and Hartwig, W. (2000) Correlation of tight junc-
tion morphology with the expression of tight junction proteins in blood-brain barrier
endothelial cells. Eur. J. Cell Biol. 79, 707–717.
24. Weber, T., Seitz, R. J., Liebert, U. G., Gallasch, E., and Wechsler, W. (1995) Affinity cyto-
chemistry of vascular endothelia in brain tumors by biotinylated Ulex europaeus type I lectin
(UEA I). Acta Neuropathol. (Berl.) 67, 128–135.
25. Nag, S., Eskandarian, M. R., Davis, J., and Stewart, D. J. (2002) Differential expression of
vascular endothelial growth factor A and B after brain injury. J. Neuropathol. Exp. Neurol.
61, 778–788.
26. Nourhaghighi, N., Stewart, D. J., and Nag, S. (2000) Immunohistochemical characteriza-
tion of Angiopoietin-1, Angiopoietin-2, and Tie-2 in an in vivo model of cerebral trauma
and angiogenesis. Brain Pathol. 10, 555.
27. Nag, S., Picard, P., and Stewart, D. J. (2001) Expression of nitric oxide synthases and
nitrotyrosine during blood-brain barrier breakdown and repair after cold injury. Lab. Invest.
81, 41–49.
28. Plate, K. H. (1999) Mechanisms of angiogenesis in the brain. J. Neuropathol. Exp. Neurol.
58, 313–320.
29. Risau, W. (1997) Mechanisms of angiogenesis. Nature 386, 671–674.
30. Nag, S. (2002) The blood-brain barrier and cerebral angiogenesis: lessons from the cold-
injury model. Trends Mol. Med. 8, 38–44.
31. Dvorak, H. F., Nagy, J. A., Feng, D., Brown, L. F., and Dvorak, A. M. (1999) Vascular per-
meability factor/vascular endothelial growth factor and the significance of microvascular
hyperpermeability in angiogenesis. Curr. Top. Microbiol. Immunol. 237, 97–132.
32. Nag, S., Takahashi, J. T., and Kilty, D. (1997) Role of vascular endothelial growth factor in
blood-brain barrier breakdown and angiogenesis in brain trauma. J. Neuropathol. Exp.
Neurol. 56, 912–921.
33. Risau, W. (1994) Molecular biology of blood-brain barrier ontogenesis and function. Acta

Neurochir. (Suppl.) 60, 109–112.
34. Robertson, P. L., Du Bois, M., Bowman, P. D., and Goldstein, G. W. (1985) Angiogenesis
in developing rat brain: an in vivo and in vitro study. Dev. Brain Res. 23, 219–223.
35. Engerman, R. L., Pfaffenbach, D., and Davis, M. D., (1967) Cell turnover of capillaries.
Lab. Invest. 17, 738–743.
36. Stewart, P. A., and Wiley, M. J. (1981) Developing nervous tissue induces formation of
blood-brain barrier characteristics in invading endothelial cells: a study using quail-chick
transplantation chimeras. Dev. Biol. 84, 183–192.
37. Breier, G., Albrecht, U., Sterrer, S., and Risau, W. (1992) Expression of vascular endothe-
lial growth-factor during embryonic angiogenesis and endothelial-cell differentiation.
Development 114, 521–532.
38. Devries, C., Escobedo, J. A., Ueno, H., Houck, K., Ferrara, N., and Williams, L. T. (1992)
The fms-like tyrosine kinase, a receptor for vascular endothelial growth-factor. Science 255,
989–991.
39. Millauer, B., Wizigmann-Voos, S., Schnürch, H., et al. (1993) High affinity VEGF binding
and developmental expression suggests Flk-1 as a major regulator of vasculogenesis and
angiogenesis. Cell 72, 835–846.
22 Nag
40. Risau, W., and Wolburg, H. (1991) The importance of the blood-brain barrier in fetuses and
embryos—reply. TINS 14, 14–15.
41. Croll, S. D., and Wiegand, S. J. (2001) Vascular growth factors in cerebral ischemia. Mol.
Neurobiol. 23, 121–135.
42. Plate, K. H., Beck, H., Danner, S., Allegrini, P. R., and Wiessner, C. (1999) Cell type spe-
cific upregulation of vascular endothelial growth factor in a MCA-occlusion model of cere-
bral infarct. J. Neuropathol. Exp. Neurol. 58, 654–666.
43. Mandriota, S. J., Pyke, C., Di Sanza, C., Quindoz, P., Pittet, B., and Pepper, M. S. (2000)
Hypoxia-inducible angiopoietin-2 expression is mimicked by iodonium compounds and
occurs in the rat brain and skin in response to systemic hypoxia and tissue ischemia. Amer.
J. Pathol. 156, 2077–2089.
44. Maisonpierre, P. C., Suri, C., Jones, P. F., et al. (1997) Angiopoietin-2, a natural antagonist

for Tie-2 that disrupts in vivo angiogenesis. Science 277, 55–60.
45. Folkman, J., and D′Amore, P. A. (1996) Blood vessel formation: What is its molecular basis?
Cell 87, 1153–1155.
46. Nag, S., Nourhaghighi, N., Teichert-Kuliszewska, K., Davis, J., Papneja, T., and Stewart,
D. J. (2002) Altered expression of angiopoietins during blood-brain barrier breakdown and
angiogenesis. J. Neuropathol. Exp. Neurol. 61, 453.
47. Stratmann, A., Risau, W., and Plate, K. H. (1998) Cell type-specific expression of
angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis.
Am. J. Pathol. 153, 1459–1466.
48. Zagzag, D., Hooper, A., Friedlander, D. R., et al. (1999) In situ expression of angiopoietins
in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis. Exp.
Neurol. 159, 391–400.
49. Beck, H., Acker, T., Wiessner, C., Allergrini, P. R., and Plate, K. (2000) Expression of
Angiopoietin-1 and Angiopoietin-2, and Tie-2 receptors after middle cerebral artery occlu-
sion in the rat. Am. J. Pathol. 157, 1473–1483.
50. Lin, T. N., Wang, C K., Cheung, W M., and Hsu, C Y. (2000) Induction of angiopoietin
and Tie Receptor mRNA expression after cerebral ischemia-reperfusion. J. Cereb. Blood
Flow Metab. 20, 387–395.
51. Muir, A. R, and Peters, A. (1962) Quintuple-layered membrane junctions at terminal bars
between endothelial cells. J. Cell Biol. 12, 443–447.
52. Brightman, M. W., and Reese, T. S. (1969) Junctions between intimately apposed cell mem-
branes in the vertebrate brain. J. Cell Biol. 40, 648–677.
53. Nag, S., Dinsdale, H. B., and Robertson, D. M. (1977) Cerebral cortical changes in acute
experimental hypertension. An ultrastructural study. Lab. Invest. 36, 150–161.
54. Reese, T. S., and Karnovsky, M. J. (1967) Fine structural localization of a blood-brain bar-
rier to exogenous peroxidase. J. Cell. Biol. 34, 207–217.
55. Feder, N., Reese, T. S., and Brightman, M (1969) Microperoxidase, a new tracer of low mol-
ecular weight. A study of the interstitial compartments of the mouse brain. J. Cell Biol. 43,
35a–36a.
56. Dermietzel, R. (1975) Junctions in the central nervous system of the cat. V. The junctional

complex of the pia-arachnoid membrane. Cell Tissue Res. 164, 309–329.
57. Nabeshima, S., Reese, T. S., Landis, D. M. D., and Brightman, M. W. (1975) Junctions in
the meninges and marginal glia. J. Comp. Neurol. 164, 127–169.
58. Dermietzel, R., Meller, N., Tetzlaff, W., and Waelsch, M. (1977) In vivo and in vitro for-
mation of the junctional complex in choroid epithelium. A freeze-etching study. Cell Tissue
Res. 181, 427–441.
59. van Deurs, B. (1980) Structural aspects of brain barriers, with special reference to the
permeability of the cerebral endothelium and choroidal epithelium. Int. Rev. Cytol. 65,
117–191.
Morphology and Molecular Properties 23
60. Nag, S. (1991) Effect of atrial natriuretic factor on permeability of the blood-cerebrospinal
fluid barrier. Acta Neuropathol. (Berl.) 82, 274–279.
61. Brightman, M. W., and Palay, S. L. (1963) The fine structure of ependyma in the brain of
the rat. J. Cell Biol. 19, 415–439.
62. Bouchaud, C., and Bosler, O. (1986) The circumventricular organs of the mammalian brain
with special reference to monoaminergic innervation. Int. Rev. Cytol. 105, 283–327.
63. Gross, P. M., Sposito, N. M., Pettersen, S. E., and Fenstermacher, J. D. (1986) Differences
in function and structure of the capillary endothelium in gray matter, white matter and a
circumventricular organ of rat brain. Blood Vessels 23, 261–270.
64. Weindl, A. (1973) Neuroendocrine aspects of circumventricular organs. In Frontiers in
Neuroendocrinology. (Ganong, W. F., and Martin, L., eds), Oxford University Press, New
York, pp. 3–32.
65. Krisch, B., and Leonhardt, H. (1989) Relations between leptomeningeal compartments and
the neurohemal regions of circumventricular organs. Biomed. Res. 10, 155–168.
66. Nagy, Z., Peters, H., and Hüttner, I. (1984) Fracture faces of cell junctions in cerebral
endothelium during normal and hyperosmotic conditions. Lab. Invest. 50, 313–322.
67. Kniesel, U., and Wolburg, H. (2000) Tight junctions of the blood-brain barrier. Cell Mol.
Neurobiol. 20, 57–74.
68. Brightman, M. W., and Tao-Cheng, J. H. (1993) Tight junctions of brain endothelium and
epithelium. In The Blood-Brain Barrier. Cellular and Molecular Biology. (Pardridge, W.

M., ed.), Raven, New York, pp. 107–125.
69. van Deurs, B., and Koehler, J. K. (1979) Tight junctions in the choroid plexus epithelium,
a freeze fracture study including complementary replicas. J. Cell Biol. 80, 662–673.
70. Connell, C. J., and Mercer, K. L. (1974) Freeze-fracture appearance of the capillary endothe-
lium in the cerebral cortex of mouse brain. Am. J. Anat. 140, 595–599.
71. Farrell, C. L., and Shivers, R. R. (1984) Capillary junctions of the rat are not affected by
osmotic opening of the blood-brain barrier. Acta Neuropathol. (Berl.) 63, 179–189.
72. Shivers, R. R., Edmonds, C. L., and Del Maestro, R. F. (1984) Microvascular permeability
in induced astrocytomas and peritumor neuropil of rat brain. A high-voltage electron micro-
scope-protein tracer study. Acta Neuropathol. (Berl.) 64, 192–202.
73. Krause, D., Mischeck, U., Galla, H. J., and Dermietzel, R. (1991) Correlation of zonula
occludens ZO-1 antigen and transendothelial resistance in porcine and rat cultured cerebral
endothelial cells. Neurosci. Letts. 128, 301–304.
74. Crone, C., and Olesen, S. P. (1982) Electrical resistance of brain microvascular endothe-
lium. Brain Res. 241, 49–55.
75. Smith, Q. R., and Rapoport, S. I. (1986) Cerebrovascular permeability coefficients to
sodium, potassium and chloride. J. Neurochem. 46, 1732–1742.
76. Tilling, T., Korte, D., Hoheisel, D., and Galla, H. J. (1998) Basement membrane
proteins influence brain capillary endothelial barrier function in vitro. J. Neurochem. 71,
1151–1157.
77. Kondo, T., Kinouchi, H., Kawase, M., and Yoshimoto, T. (1996) Astroglial cells inhibit
the increasing permeability of brain endothelial cell monolayer following hypoxia/
reoxygenation. Neurosci. Letts. 208, 101–104.
78. Igarashi, Y., Utsumi, H., Chiba, H., et al. (1999) Glial cell line-derived neurotrophic factor
induces barrier function of endothelial cells forming the blood-brain barrier. Biochem.
Biophys. Res. Commun. 261, 108–112.
79. Perez-Gomez, J., Bindslev, N., Orkand, P. M., and Wright, E. M. (1976) Electrical proper-
ties and structure of the frog arachnoid membrane. J. Neurobiol. 7, 259–270.
80. Frömter, E., and Diamond, J. (1972) Route of passive ion permeation in epithelia. Nature
235, 9–13.

24 Nag
81. Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K., and Tsukita, S. (1998) Claudin-1 and -2:
Novel integral membrane proteins localizing at tight junctions. J. Cell Biol. 141, 1539–1550.
82. Furuse, M., Hirase, T., Ito, M., et al. (1993) Occludin: a novel integral membrane protein
localizing at tight junctions. J. Cell Biol. 123, 1777–1788.
83. Martìn-Padura, I., Lostaglio, S., Schneemann, M., et al. (1998) Junctional adhesion mole-
cule, a novel member of the immunoglobulin superfamily that distributes at intercellular
junctions and modulates monocyte transmigration. J. Cell Biol. 142, 117–127.
84. Furuse, M., Sasaki, H., and Tsukita, S. (1999) Manner of interaction of heterogenous
claudin species within and between tight junction strands. J. Cell Biol. 147, 891–903.
85. Hirase, T., Staddon, J. M., Saitou, M., et al. (1997) Occludin as a possible determinant of
tight junction permeability in endothelial cells. J. Cell Sci. 110, 1603–1613.
86. Haskins, J., Gu, L., Wittchen, E. S., Hibbard, J., and Stevenson, B. R. (1998) ZO-3, a novel
member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and
occludin. J. Cell Biol. 141, 199–208.
87. Stevenson, B. R., Siliciano, J. D., Mooseker, M. S., and Goodenough, D. A. (1986)
Identification of ZO-1: a high molecular weight polypeptide associated with the tight junc-
tion (zonula occludens) in a variety of epithelia. J. Cell Biol. 103, 755–766.
88. Anderson, J. M. (1996) Cell signalling: MAGUK magic. Curr. Biol. 6, 382–384.
89. Watson, P. M. M., Anderson, J., VanItallie C. M., and Doctrow, S. R. (1991) The tight junc-
tion-specific protein ZO-1 is a component of the human and rat blood-brain barrier. Neurosci.
Letts. 129, 6–10.
90. Yamamoto, T., Harada, N., Kano, K., et al. (1997) The Ras target AF-6 interacts with ZO-
1 and serves as a peripheral component of tight junctions in epithelial cells. J. Cell Biol.
139, 785–795.
91. Satoh, H., Zhong, Y., Isomura, H., et al. Saitoh, M., Enomoto, K., Sawada, N., (1996)
Localization of 7H6 tight junction-associated antigen along the cell border of vascular
endothelial cells correlates with paracellular barrier function against ions, large molecules,
and cancer cells. Exp. Cell Res. 222, 269–274.
92. Denker, B. M., and Nigam, S. K. (1998) Molecular structure and assembly of the tight junc-

tion. Am. J. Physiol. 274, F1–F9.
93. Huber, J. D., Egleton, R. D., and Davis, T. P. (2001) Molecular physiology and pathophys-
iology of tight junctions in the blood-brain barrier. TINS, 24, 719–725.
94. Cordenonsi, M., D’Atri, F., Hammar, E., Parry, D. A., Kendrick-Jones, J., Shore, D., and
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, 1569–1582.
95. Kuruganti, P. A., Hinojoza, J. R., Eaton, M. J., Ehmann, U. K., and Sobel, R. A. (2002)
Interferon-β counteracts inflammatory mediator-induced effects on brain endothelial tight
junction molecules—Implications for multiple sclerosis. J. Neuropathol. Exp. Neurol. 61,
710–724.
96. Anderson, J. M., Stevenson, B. R., Jesaitis, L. A., Goodenough, D. A., and Mooseker,
M. S. (1988) Characterization of ZO-1, a protein component of the tight junction from mouse
liver and Madin-Darby canine kidney cells. J. Cell Biol. 106, 1141–1149.
97. Stevenson, B. R., Anderson, J. M., Goodenough, D. A., and Mooseker, M. S. (1988) Tight
junction structure and ZO-1 content are identical in two strains of Madin-Darby canine
kidney cells which differ in transepithelial resistance. J. Cell Biol. 107, 2401–2408.
98. Itoh, M., Furuse, M., Morita, K., Kubota, K., Saitou, M., and Tsukita, S. (1999) Direct bind-
ing of three tight junction associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH
termini of claudins. J. Cell Biol. 147, 1351–1363.
99. Nag, S., Dinsdale, H. B., and Robertson, D. M. (1978) Cytoplasmic filaments in intracere-
bral cortical vessels. Ann. Neurol. 3, 555–559.
Morphology and Molecular Properties 25
100. Nag, S. (1995) Role of the endothelial cytoskeleton in blood-brain barrier permeability to
proteins. Acta Neuropathol. (Berl.) 90, 454–460.
101. Pardridge, W. M., Nowlin, D. M., Choi, T. B., Yang, J., Calaycay, J., and Shively, J. E.
(1989) Brain capillary 46,000 Dalton protein is cytoplasmic actin and is localized to
endothelial plasma membrane. J. Cereb. Blood Flow Metab. 9, 675–680.
102. Nusrat, A., Parkos, C. A., Verkade, P., et al. (2000) Tight junctions are membrane
microdomains. J. Cell Sci. 113, 1771–1781.
103. Schlegel, A., and Lisanti, M. P. (2001) The caveolin triad: caveolae biogenesis, choles-

terol trafficking, and signal transduction. Cytokine Growth Factor Rev. 12, 41–51.
104. Madara, J. L., Parkos, C., Colgan, S., Nusrat,A., Atisook, K., and Kaoutzani, P. (1992) The
movement of solutes and cells across tight junctions. Ann. New York Acad. Sci. 664, 47–60.
105. Gloor, S. M., Wachtel, M., Bolliger, M. F., Ishihara, H., Landmann, R., and Frei, K. (2001)
Molecular and cellular permeability control at the blood-brain barrier. Brain Res. Rev. 36,
258–264.
106. Rubin, L. L., and Staddon, J. M. (1999) The cell biology of the blood-brain barrier. Annu.
Rev. Neurosci. 22, 11–28.
107. Takeichi, M. (1995) Morphogenetic roles of classic cadherins. Curr. Biol. 7, 619–627.
108. Lampugnani, M. G., and Dejana, E. (1997) Interendothelial junctions: structure, sig-
nalling and functional roles. Curr. Opin. Cell Biol. 9, 674–682.
109. Gumbiner, B. M. (1996) Cell adhesion: the molecular basis of tissue architecture and mor-
phogenesis. Cell 84, 345–357.
110. Staddon, J. M., Herrenknecht, K., Smales, C., and Rubin, L. L. (1995) Evidence that tyro-
sine phosphorylation may increase tight junction permeability. J. Cell Sci. 108, 606–619.
111. Schultze, C., Smales, C., Rubin, L. L., and Staddon, J. M. (1997) Lysophosphatidic acid
increases tight junction permeability in cultured brain endothelial cells. J. Neurochem. 86,
991–1000.
112. Staddon, J. M., Smales, C., Schulze, C., Esch, F. S., and Rubin, L. L. (1995) p120, a p-
120 related protein (p100) and the cadherin/catenin complex. J. Cell Biol. 130, 369–381.
113. Frøkjaer-Jensen, J. (1980) Three-dimensional organization of plasmalemmal vesicles in
endothelial cells. An analysis by serial sectioning of frog mesenteric capillaries.
J. Ultrastruc. Res. 73, 9–20.
114. Nag, S., Robertson, D. M., and Dinsdale, H. B. (1979) Quantitative estimate of pinocy-
tosis in experimental acute hypertension. Acta Neuropathol. (Berl.) 46, 107–116.
115. Dux, E., and Joó, F. (1982) Effects of histamine on brain capillaries: fine structural and
immunohistochemical studies after intracarotid infusion. Exp. Brain Res. 47, 252–258.
116. Stewart, P. A., Hayakawa, K., Farrell, C. L., and Del Maestro, R. F. (1985) A quantitative
study of blood-brain barrier permeability ultrastructure in a new rat glioma model. Acta
Neuropathol. (Berl.) 67, 96–102.

117. Stewart, P. A., Magliocco, M., Hayakawa, K., et al. (1987) A quantitative analysis of blood-
brain barrier ultrastructure in the aging human. Microvasc. Res. 33, 270–282.
118. Coomber, B. L., and Stewart, P. A. (1985) Morphometric analysis of CNS microvascular
endothelium. Microvasc. Res. 30, 99–115.
119. Stewart, P. A. (2000) Endothelial vesicles in the blood-brain barrier: are they related to
permeability? Cell. Mol. Neurobiol. 20, 149–163.
120. Simionescu, N., Simionescu, M., and Palade, G. E. (1974) Morphometric data on the
endothelium of blood capillaries. J. Cell Biol. 60, 128–152.
121. Coomber, B. L., Stewart, P. A., Hayakawa, E. M., Farrell, C. L., and Del Maestro, R. F.
(1988) A quantitative assessment of microvessel ultrastructure in C6 astrocytoma spher-
oids transplanted to brain and muscle. J. Neuropath. Exp. Neurol. 47, 29–40.
122. Shea, S. M., and Raskova, J. (1983) Vesicular diffusion and thermal forces. Fed. Proc. 42,
2431–2434.
26 Nag