Journal of Neuropathology and Experimental Neurology
Copyright q 2002 by the American Association of Neuropathologists
Vol. 61, No. 8
August, 2002
pp. 725 735
Increased Endothelial Vesicular Transport Correlates with Increased Blood-Tumor Barrier
Permeability Induced by Bradykinin and Leukotriene C4
KAZUHIRO HASHIZUME, MD
AND
KEITH L. BLACK, MD
Abstract. Bradykinin and leukotriene C4 (LTC4) have been shown to increase molecular transport across the blood-tumor
barrier (BTB). The aim of this study was to quantitatively assess whether an increase in vesicular transport or opening of
tight junctions was responsible for this increase in permeability. Wistar rats bearing RG2 or C6 gliomas were infused with
bradykinin or LTC4 through the right carotid artery for 15 min and then perfused to achieve fixation. Prepared specimens
were observed using transmission electron microscopy. Infusion of either bradykinin or LTC4 resulted in significantly increased
vesicular density in capillary endothelial cells of the BTB but not in normal brain capillaries. The opening of tight junctions,
assessed by determining a cleft index, was found to be greater in tumor capillaries compared to normal controls. However,
neither bradykinin nor LTC4 produce variations in the cleft index. A significant accumulation of horseradish peroxidase was
seen in the intercellular peri-capillary spaces and in endothelial transport vesicles after infusion of bradykinin, demonstrating
that the formation of vesicles was associated with macromolecular transcytosis. These findings suggest that pinocytotic vesicular transport is the primary means by which luminal to abluminal transport occurs in response to vasomodulation with
bradykinin or LTC4.
Key Words:
Blood-brain barrier; Blood-tumor barrier; Bradykinin; Electron microscopy; Tight junction; Vesicle.
INTRODUCTION
To improve therapy of brain tumors we have focused
on methods that increase delivery of antitumor compounds to tumor tissue (1). Bradykinin and leukotriene
C4 (LTC4) selectively increase the transport of compounds to brain tumors (2–8). In studying possible mechanisms, we reasoned that nitric oxide (NO) and cyclic
GMP could play an important role in the pathways activated by bradykinin. It has been shown that endothelial
NO synthase (NOS) and neuronal NOS are expressed in
tumor cells and tumor microvessel endothelial cells and
that the selective permeability increase in brain tumor
microvessels after bradykinin infusion is mediated by NO
(9). Increased cyclic GMP in tumor microvessels can increase permeability in response to bradykinin (10). However, the process which facilitates the passage of molecules through the abnormal blood-brain barrier (BBB)
has not been defined even though bradykinin-induced increases in permeability have been demonstrated (6, 8)
and confirmed (11, 12).
The morphological basis of the BBB has been well
investigated by the application of transmission electron
microscopy (TEM). Generally, 3 different types of vascular pores allow movement of molecules across the vessel wall and transport across the BBB: 1) a paracellular
route between adjacent endothelial cells that is more or
less restricted by tight junctions; 2) vesicular pores
From the Maxine Dunitz Neurosurgical Institute (KH, KLB), Cedars
Sinai Medical Center, Los Angeles, California.
Correspondence to: Kazuhiro Hashizume, MD, Department of Neurosurgery, Yamanashi Medical University, 1110 Shimokato, Tamahotown, Nakakomagun, Yamanashi-pref. 409-3898 Japan.
This work was supported by NIH Grants #2RO1 NS32103-06 and
by Maxine Dunitz Neurosurgical Institute, Cedars Sinai Medical Center.
formed by endothelial vesicles; and 3) transcellular pores
located within fenestrations (13). In BBB capillaries, the
paracellular route is blocked by long, continuous tight
junctions, vesicles are present only in very low densities,
and fenestrations are absent except in small circumventricular areas where the capillaries are highly permeable.
To investigate how vasoactive treatments could influence the passage of molecules through the blood-tumor
barrier (BTB), we focused on the paracellular route and
vesicular transport. Opening of the tight junctions is one
possible alteration of the brain endothelium that will
compromise barrier function. The paracellular route has
been proposed to be a tortuous pathway formed by confluent channels, or clefts, between the bands of tight junctions (13, 14). To evaluate paracellular passage via tight
junctions by TEM, we quantified the proportion of profiles formed by clefts. This parameter, called the cleft
index, was shown by others to be higher in leaky capillaries compared to normal BBB capillaries (15).
Another route for molecules to cross the BBB is via
endothelial vesicles. In normal BBB capillaries the vesicular densities are much lower than in some permeable
vessels (16–19). The presumed low level of vesicular
transport in the normal BBB is consistent with the observation of the small number of vesicles in brain capillaries. Under some conditions of compromised barrier
function the number of endothelial vesicles increases in
the leaking vascular segments, supporting the claim that
vesicles are involved in the vascular leakage.
Administration of vasoactive compounds may also
produce compromised barrier function. Intracarotid infusion of dibutyryl cyclic GMP was found to increase the
permeability of brain microvessels and the number of
transport vesicles (20). Thus, it is possible to induce increase in pinocytic activity of endothelial cells even in
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HASHIZUME AND BLACK
the normal BBB. We, therefore, postulated that the number of vesicles in endothelial cells may also be increased
by the intracarotid infusion of bradykinin, since bradykinin increases cyclic GMP in tumor tissue.
In the absence of comparative quantitative studies, it
remains unclear what kind of ultrastructural changes occur in the brain tumor capillary, and how molecules pass
through the BTB after administration of vasomodulators.
In the present study, we investigated the relative contributions of 2 alternative mechanisms (opening of the tight
junctions and increased vesicular transport) to increased
BTB permeability after intracarotid infusion of vasoactive compounds.
in PBS at pH 7.4 through the heart. After infusion-fixation the
brains were immediately removed and the regions of interests
were selected and cut into 1-mm3 fragments. The small samples
were immersion-fixed in the same fixative at 48C for 2 hours
(h) and rinsed in 5% sucrose in 0.1 M phosphate buffer (PB)
at pH 7.40 at 48C, overnight, with continuous swirling on a
rotary platform and changing the rinse solution 4–5 times. They
were postfixed for 2 h in 1% osmium tetroxide with 0.1 M PB,
dehydrated in an ascending series of ethanols ending with propylene oxide, and embedded in Epon. Semithin sections were
cut and stained using toluidine blue for selecting appropriate
blocks. Ultrathin sections were made from selected blocks,
mounted on formvar-coated slot grids, and double stained with
uranyl acetate and lead citrate for observation under a JEOL
electron microscope operating at 80 kV.
MATERIALS AND METHODS
Fifty-one female Wistar rats (Harlan, CA), weighing 180–
220 g and aged 6–8 wk, were used for this study. All animal
experimentation was conducted in accordance with policies set
by the Institutional Animal Care and Use Committee of CedarsSinai Medical Center and NIH guidelines.
Tumor Inoculation
The RG2 and C6 glioma cell lines were originally derived
from an ethylnitrosourea-induced rat glioma. RG2 and C6 glioma cells were kept frozen until use when they were thawed
and maintained in a monolayer culture in F12 medium with
10% calf serum. The rats were anesthetized using ketamine (50
mg/kg) and xylazine (5 mg/kg) intraperitoneally (i.p.). Glial tumors were stereotactically implanted into the right hemisphere
by intracerebral injection of 1 3 105 glioma cells in 5 ml of F12
medium with 1.2% methylcellulose using a Hamilton syringe
with 30-gauge needle. The coordinates used were 3 mm lateral
to the bregma and 4.5 mm deep to the dural surface.
Animal Preparation
Nine or 10 days after tumor implantation the rats were anesthetized with i.p. ketamine (50 mg/kg) and xylazine (5 mg/kg),
and a polyethylene catheter was inserted retrograde through the
external carotid artery to the common carotid artery bifurcation,
ipsilateral to the tumor. The right femoral artery and vein were
cannulated to monitor systemic blood pressure and to administer the tracer horseradish peroxidase (HRP) (Sigma Chemical
Co., St. Louis, MO), respectively. Body temperature was maintained at 378C, and arterial blood gases, blood pressure, and
hematocrit were monitored. The doses and infusion rates of
bradykinin (Sigma Chemical Co.), LTC4 (Cayman Chemical
Co., Ann Arbor, MI), and HRP are described elsewhere (4, 6).
Animal Methods for Morphometric Transmission
Electron Microscopy (TEM) Studies
Forty-seven rats were used for routine TEM analysis. The
rats were grouped by tumor types and infused drugs. After rat
preparation, phosphate buffered saline (PBS) as a control, bradykinin (10 mg/kg/min in PBS), or LTC4 (1.67 mg/kg/min in
5% ethanol) was injected into the right carotid artery at a rate
of 53.3 ml/min for 15 min. At the end of the injection the vasculature was perfused free of blood with approximately 10 ml
cold PBS, followed by fixation with 250 ml 1% glutaraldehyde
J Neuropathol Exp Neurol, Vol 61, August, 2002
Basic Features of the Capillaries
Ten profiles of capillaries sectioned transversely in each
group were selected and photographed at low magnification
(37,200) for evaluation of their general features. Printed micrographs were placed onto the surface of a digitizing screen
and structural features measured using Scan Pro 4TM, a computer-assisted image analysis system (Jandel Scientific, Corte
Madera, CA). Abluminal and luminal circumferences, areas of
endothelial cytoplasms excluding nuclei and vacuoles, and
mean thickness of endothelial cytoplasms were measured and
compared with those in the control group. The mean thickness
of endothelium was calculated by subtraction of luminal radius
from abluminal radius, which were obtained from the areas encircled by luminal and abluminal circumferences, respectively.
Evaluation of the Increase of Vesicles
To evaluate the density of vesicles, the measurement was
done by a third person who was blinded to treatment information regarding the samples. Only the microphotographs were
given to the blinded person who counted all the vesicles in the
endothelial cytoplasm.
Our approach has been to obtain a large number of blocks
from each animal and to select 1 or 2 blocks through the observation of semithin sections under light microscopy. For detailed observation with the TEM, we selected transversely sectioned capillary profiles that were defined as vessels having a
single, nearly round endothelial cell surrounded by basement
membrane. Four test zones of endothelial cytoplasm were photographed at higher magnification (329,000). They were chosen
randomly, typically examining 3, 6, 9, and 12 o’clock positions
on the capillary circumference. Preliminary analysis indicated
fewer vesicles were present between luminal and abluminal
membranes around the nucleus in both control and treatment
groups, and since some capillary sections did not include the
nucleus, this area was excluded from our analysis. Three vessels
were sampled from each rat and each treatment group contained
5–6 rats; therefore, each group consisted of 15–18 capillaries
and a population of 60–72 test zones. The number of vesicles
that was contained in each test zone was counted on the printed
micrograph. Intracytoplasmic vesicles were recognized as bilayered circular organelles having lighter electron density within, and all vesicles were counted. Invaginations of the luminal
or abluminal membrane were counted as vesicles if their neck
727
VESICLE TRANSPORT REGULATES BTB PERMEABILITY
TABLE
Morphological Characteristics of BBB and BTB Capillaries
Tissue
Infusion
Control
RG2 tumor
C6 tumor
PBS
BK
LTC4
PBS
BK
LTC4
PBS
BK
LTC4
Abluminal
circumference
(mm)
19.3
19.5
18.3
28.2
28.2
29.2
33.4
32.8
31.5
6
6
6
6
6
6
6
6
6
3.0
2.4
3.9
4.6
8.3
7.7
7.1
7.5
10.1
Luminal
circumference
(mm)
17.1
17.4
16.5
24.4
24.7
26.4
32.2
30.3
29.3
6
6
6
6
6
6
6
6
6
2.9
1.9
3.2
5.4
7.2
7.5
9.4
7.2
10.5
Cytoplasmic
area (mm2)
4.0
4.0
4.3
12.4
12.6
12.4
14.4
11.2
11.8
6
6
6
6
6
6
6
6
6
1.2
0.8
1.4
4.4
5.2
3.8
5.2
3.3
5.1
Mean wall
thickness (mm)
0.24
0.26
0.28
0.57
0.59
0.55
0.55
0.44
0.48
6
6
6
6
6
6
6
6
6
0.05
0.04
0.07
0.14
0.12
0.13
0.15
0.09
0.11
Values are means 6 standard deviation. Abbreviations: BBB 5 blood-brain barrier; BTB 5 blood-tumor barrier; PBS 5 phosphate
buffered saline; Control 5 ipsilateral basal ganglia in a non-tumor bearing rat; BK 5 bradykinin; LTC4 5 leukotriene C4.
was narrower than their maximum diameter. The cytoplasmic
areas of the test zones excluding nuclei and vacuoles were digitally measured on each 8 3 10 micrograph and vesicle density
was expressed as a number per mm2 of cytoplasm.
The cumulative area of all vesicles contained in the cytoplasm was also measured to compare with that of the cytoplasm. The proportion of total vesicular area to the cytoplasmic
area was expressed as a percentage in order to derive another
parameter to characterize vesicular transport.
All data were expressed as means 6 SD. The effect of intracarotid infusion of bradykinin or LTC4 was compared with that
of saline infusions by statistical analysis of the vesicular density
and proportion of cumulative vesicular area using analysis of
variance (ANOVA) followed by unpaired parametric analysis
of Student t-test or Welch’s t-test and also by a post hoc intergroup multiple comparison procedure, Bonferroni/Dunn test.
Evaluation of Tight Junctional Integrity
Thirty junctional profiles were randomly selected from each
group under TEM observation. Each could be traced all the way
from the luminal side to abluminal side. They were evaluated by
measuring the total length of the junctional profile and the cumulative length and area of the junctional clefts—areas where
the outer leaflets of apposing membranes were not fused. To
quantify the opening degree of tight junctions we determined 2
indices: a cleft index and a cleft area index. A cleft index was
defined as the proportion of the junctional profile that was composed of clefts and was expressed as a percentage of the cumulated length of clefts to the total length of junctional profile
(15). The cleft area is a translucent zone located between 2 electron-dense tight junctions and regions of adjacent capillary membranes that run between the luminal and abluminal surfaces. A
cleft area index, which we derived to identify enlarged clefts,
was defined as the proportion of the cumulative area of the clefts
to the total length of the junctional profile. We tested for a significant difference in junctional cleft index among tissue types
and between treatment groups. Statistical analysis of cleft index
and cleft area index was performed as described above.
TEM Examination with HRP
Four rats were used for this study using HRP as a tracer.
After rat preparation, bradykinin (10 mg/kg/min in PBS; n 5 2)
or saline (control; n 5 2) was injected as described above. Five
min after the start of the intracarotid infusion, 200 mg/kg HRP
was injected by an intravenous bolus through the right femoral
vein (21). Ten min after HRP injection, rats were perfused with
a mixture of 2% glutaraldehyde and 2% paraformaldehyde in
0.1 M sodium phosphate buffer solution at pH 7.4 through the
heart. After fixation, the brains were removed and cut into 40mm sections using a vibratome. The sections were preincubated
for 30 min at room temperature in a medium consisting of 5
ml 0.06 M Tris buffer, 3,39-diaminobenzidine tetrahydrochloride (0.7 mg/ml) without hydrogen peroxide followed by the
same medium with the addition of hydrogen peroxide (0.2 mg/
ml) for 2–3 min. Sections were trimmed to the areas of interest
and postfixed for 2 h in 2% osmium tetroxide with 0.1 M PB.
The ensuring embedding procedures were performed as detailed
above. Extravasation of the HRP tracer was visualized by DAB/
peroxide treatment.
RESULTS
Basic Features of the Capillaries
Physiological parameters, which included systemic
blood pressure, arterial blood gas, and hematocrit, were
not significantly changed by intracarotid bradykinin or
LTC4 infusion at a rate of 10 mg/kg/min or 1.67 mg/kg/
min, respectively, during the experiments (data not
shown).
All of the vessel profiles examined were capillaries in
which the vessel wall consisted of an endothelial cell, a
basement membrane, and occasional pericyte. The values
of general capillary features, including luminal and abluminal circumferences, cytoplasmic areas of the capillaries, and mean capillary thickness, were obtained. Although RG2 and C6 tumor capillaries are larger than
those of normal brain, the intracarotid infusion of bradykinin or LTC4 did not alter their morphological features (Table). Perfusion-fixed BBB capillaries are significantly smaller than rat model BTB capillaries. When
normal control basal ganglia capillaries were compared
with both tumor groups, the endothelial circumferences,
J Neuropathol Exp Neurol, Vol 61, August, 2002
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HASHIZUME AND BLACK
Fig. 1. Representative electron microscopic photographs of endothelial cells in the normal ipsilateral basal ganglia BBB in
non-tumor bearing rats, showing transport vesicles in the endothelial cytoplasm after infusion of saline (A), bradykinin (B), or
LTC4 (C). Note the paucity of pinocytotic vesicles (arrowheads) in the normal BBB, regardless of infusion with saline, bradykinin,
or LTC4. Original magnification, 379,900; scale bar 5 0.5 mm.
areas, and thicknesses of RG2 and C6 tumors were significantly higher than the control group.
Increased BTB Vesicular Formations
To summarize our TEM studies of capillary microvessels, we observed that endothelial vesicular density was
unchanged in the normal BBB either by infusion of bradykinin or LTC4 compared with saline-infused control
group (Fig. 1). In contrast, vasoactive treatments dramatically increased vesicular formation in the BTB. Bradykinin and LTC4 treatment significantly increased endothelial vesicular density and the cumulative area of
vesicles in both RG2 and C6 tumors (Figs. 2, 3).
Among continuous capillaries, the density of vesicles
after infusion of these vasoactive compounds in RG2 and
C6 tumor tissues correlates reasonably well with our previous results obtained using quantitative autoradiography
(4, 6, 8). Figures 1, 2, and 3 highlight the changes in
vesicular density. Vesicular density was significantly increased in RG2 tumor after bradykinin (n 5 6) and LTC4
(n 5 6) infusion compared with control (n 5 5). In C6
tumor, bradykinin (n 5 5) and LTC4 (n 5 5) infusion
also caused a significant increase of vesicular density
compared with control (n 5 5). Figure 4A demonstrates
that bradykinin and LTC4 treatments produce a 2–3-fold
increase in vesicular density and that the effect is selective for the BTB, but not the normal BBB.
The mean vesicular:cytoplasmic area ratio was also
significantly increased approximately 2–3-fold by both
J Neuropathol Exp Neurol, Vol 61, August, 2002
treatments in the BTB (Fig. 4B). Bradykinin (n 5 6) and
LTC4 (n 5 6) infusion in RG2 tumor-bearing rats increased the relative vesicular areas compared with controls (n 5 5). Likewise, in C6 tumor, bradykinin (n 5 5)
and LTC4 (n 5 5) infusion caused an increase in the
vesicular area compared with controls (n 5 5). In normal
brain, both vasoactive compounds did not increase vesicular density or vesicular area compared with saline infusion.
When vesicular areas are expressed as a function of
cytoplasmic area, to account for the larger cytoplasmic
area seen in tumor capillaries, significant increases in vesicular areas are only apparent in tumor endothelia. This
suggests that the action of these vasoactive agents is selectively expressed in tumor, but not normal endothelial
cells.
Each vesicular size was essentially unchanged by any
of the 3 treatment infusions (saline, bradykinin, or LTC4).
All of the measured vesicular diameters in the present
study were compatible with the accepted range of values
(50–100 nm) determined previously (22).
Opening of Tight Junctions
The cleft indices and cleft area indices of RG2 tumor
capillaries did not show any significant differences between the treatment subgroups, i.e. saline-, bradykinin-,
and LTC4-treated groups. The same results were seen in
C6 tumor capillaries (Fig. 5A, B). Thus, within 15 min
VESICLE TRANSPORT REGULATES BTB PERMEABILITY
729
Fig. 2. Representative electron microscopic photographs of endothelial cells in RG2 glioma BTB, showing transport vesicles
after infusion of saline (A), bradykinin (B), or LTC4 (C). The endothelial cells in RG2 tumor tissue have thick walls and appear
edematous. Although saline infusion fails to increase formation of vesicles, intracarotid infusion of bradykinin and LTC4 causes
a significant increase of transport vesicles in the endothelial cytoplasm (arrowheads). Original magnification, 379,900; scale bar
5 0.5 mm.
J Neuropathol Exp Neurol, Vol 61, August, 2002
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Fig. 3. Representative electron microscopic photographs of endothelial cells in C6 glioma BTB, showing transport vesicles
after infusion of saline (A), bradykinin (B), or LTC4 (C). The number of endothelial vesicles does not increase after saline
infusion. In contrast, both bradykinin and LTC4 infusion significantly increase the number of vesicles in the endothelial cytoplasm
(arrowheads). Original magnification, 379,900; scale bar 5 0.5 mm.
J Neuropathol Exp Neurol, Vol 61, August, 2002
VESICLE TRANSPORT REGULATES BTB PERMEABILITY
Fig. 4. Evaluation of the vesicular population in the endothelial cytoplasm in each tissue and in each treatment. Columns,
mean; bars, SD. A: Vesicular density of the endothelial cytoplasm (number of vesicles/mm2). Note that both vesicular density is significantly increased after bradykinin and LTC4 infusion and that this effect is selective for BTB capillaries, but not
normal BBB capillaries. B: The proportion of total vesicular
area to the cytoplasmic area in which they are contained (%).
This parameter increased in parallel with the vesicular density
after infusion of bradykinin and LTC4. Number of rats (n) 5
5–6 for each mean. Control 5 ipsilateral normal basal ganglia
in non-tumor bearing rat; PBS 5 phosphate buffered saline; BK
5 bradykinin; LTC4 5 leukotriene C4. *Significantly different
from saline-infused tumor at p , 0.01.
of intracarotid infusion, neither bradykinin nor LTC4 affected the morphology of the junctional profiles. In the
comparison among each tissue type, RG2 and C6 tumor
capillaries had higher values in both cleft and cleft area
indices compared to normal brain capillaries. Figure 6A
compares a typical BBB tight junction with a larger cleft
731
Fig. 5. Morphometric evaluation of the opening degree of
the tight junctions in each tissue and in each treatment. Columns, mean; bars, SD. A: Cleft index (%) was devised to evaluate the facilitation of material passage through the paracellular
route. Note that cleft morphology is altered in BTB capillaries
in both RG2 and C6 tumors compared with BBB capillaries,
but is not changed as a result of either bradykinin or LTC4
infusion. Significant differences in cleft index are seen between
the control group and RG2 or C6 glioma group, respectively.
B: Cleft area index (‰) was developed in order to evaluate the
effect of wide gaps in tight junctions. It shows the same tendency as the cleft index and may show the distinct difference
between BBB capillaries and BTB capillaries. Significant differences in cleft area indices are seen between the control group
and RG2 or C6 glioma group, respectively. Derivations of the
cleft index and cleft area index are detailed in the methods.
Number of rats (n) 5 5–6 for each mean. Control 5 ipsilateral
basal ganglia in non-tumor bearing rat; PBS 5 phosphate buffered saline; BK 5 bradykinin; LTC4 5 leukotriene C4.
J Neuropathol Exp Neurol, Vol 61, August, 2002
732
HASHIZUME AND BLACK
Fig. 6. Representative electron microscopic photographs of tight junctions in normal brain, RG2 tumor, and C6 tumor. The
normal tight junction has a long, sealed portion and low value of cleft index and cleft area index (A). In contrast, both of RG2
tumor (B) and C6 tumor (C) have leaky tight junctions in which the cumulative unsealed segments (arrowheads) occupy a large
part of the whole junctional profile. This contributes to a high value of cleft index. Also some of the clefts have wider areas,
which contributes to a high value of cleft area index. Intracarotid infusion of bradykinin or LTC4 did not affect the nature of the
tight junctions. Note also that BTB capillaries of RG2 tumors (B) and C6 tumors (C) are significantly thicker than control (A)
BBB endothelia. Original magnification, 379,900; scale bar 5 0.1 mm.
area in the RG2 glioma (Fig. 6B). The enlarged cleft area
is also apparent in abutting endothelia of the C6 glioma
(Fig. 6C).
Penetration of the Tracer HRP
TEM observation using the tracer HRP was performed
to test whether capillary and tumor cell vesicles were
actually carrying blood-borne materials in RG2 tumors.
In the control, little HRP staining was seen around the
capillary by PBS infusion (Fig. 7A). In contrast, there
was significant HRP staining in the abluminal side of the
capillary and in the intercellular spaces after bradykinin
infusion (Fig. 7B). There were also HRP-containing vesicles in the endothelial cell, especially near the abluminal
membrane, some of which were fused together or attached to the abluminal cell membrane (Fig. 7C). HRP
reaction was also seen in the tumor cells adjacent to the
capillary. There was significant HRP staining on the surface of the tumor cell membrane and within pinocytic
vesicles of the tumor cytoplasm (Fig. 7D). These observations strongly indicate that blood-borne proteins pass
through the BTB and enter the tumor cells after bradykinin infusion.
DISCUSSION
It is commonly accepted that BBB capillaries have
very low permeability to most hydrophilic substances.
J Neuropathol Exp Neurol, Vol 61, August, 2002
The striking features of normal brain capillaries are that
they are unfenestrated, that continuous tight junctions
seal the paracellular route, and that there is a low level
of vesicular transport (18). To develop a treatment strategy for effective drug delivery to the malignant glioma,
we have been investigating mechanisms for increasing
BTB permeability. Our previous studies evaluating the
regional permeabilities by the quantitative autoradiographic technique have shown that intracarotid administration of certain vasoactive compounds selectively increases the permeability in tumors but not in normal brain
(6, 8). With regard to the mechanism by which bradykinin produces such effects, the NO and cyclic GMP pathways are presumed to mediate signal transduction triggered by B2 receptor activation with bradykinin (7, 9,
10). However, the morphological changes of tumor capillary endothelial cells having increased permeability following infusion of vasoactive compounds have not previously been defined. With respect to the cellular
mechanism, either the activation of pinocytic vesicles in
the endothelial cytoplasm or the opening of the tight
junctions have been proposed as means by which certain
substances can enter the brain from the blood circulation
(13). The possible contribution of capillary fenestrations
to the increased vascular permeability is considered unlikely because fenestrations are rarely seen in either normal brain capillaries or glioma capillaries (13, 23, 24).
VESICLE TRANSPORT REGULATES BTB PERMEABILITY
733
Fig. 7. Representative electron microscopic photographs of RG2 tumor after infusion of bradykinin or saline using HRP as
the tracer. HRP reaction was hardly seen on the abluminal side of the capillary in the tumor tissue with infusion of saline (A).
In contrast, there was significant HRP staining surrounding the capillaries and in the intercellular spaces between tumor cells
following bradykinin infusion (B). HRP reaction was also observed in the endothelial vesicles, which sometimes fused together
and made a chain in the same case as panel B. Arrowheads indicate the abluminal membrane (C). HRP staining was also observed
on the surface of cell membrane (arrowheads) and in the vesicles in tumor cells, which means HRP was taken into the tumor
cells by pinocytosis (D). Original magnifications: A, D, 338,600; B, 316,000; C, 379,900; scale bars: A, C, D 5 0.5 mm; B,
1.0 mm.
Our present studies showed a significant increase in
vesicular density of the endothelial cytoplasm of both
RG2 and C6 gliomas after intracarotid infusion of either
bradykinin or LTC4. These treatments did not affect vesicular density in the normal brain. The cleft index and
cleft area index, which indicate compromised function of
tight junctions, were higher in both RG2 and C6 gliomas
than in normal brain. However, we saw no significant
differences in these indices between saline, bradykinin,
and LTC4 infusions within each tissue type. This result
suggests that within 15 min of treatment with vasomodulators, changes in the morphology of tight junctions
are not apparent. Our results, therefore, suggest that the
formation of an increased number of vesicles plays a critical role in vasomodulator-mediated transport through the
BTB. Increased pinocytotic activity may be an acute response, occurring within 15 min of bradykinin or LTC4
treatment. In contrast, morphological changes in tight
junctional clefts may be a more longstanding, chronic
response of BTB capillaries.
Previous TEM studies have shown that increased numbers of vesicles were observed in the cerebral endothelium under various pathological conditions (25), such as
stab injury (26), hypertension (27, 28), radiation (29),
mechanical injuries (30), seizure (31), and human brain
tumors (32). Increased vesicles have been interpreted to
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represent enhanced vesicular transport through the capillary endothelium. The hypothesis that a low permeability in normal BBB depends on a low level of vesicular
transport is consistent with the observation of very few
endothelial vesicles in brain endothelium, whereas muscle capillaries have high permeability and correspondingly larger numbers of vesicles in the endothelial cytoplasm
(16, 17, 19). Recent evidence shows that the transcytotic
pathway involves both movement of vesicles within the
brain capillary cell and a series of fusions and fissions of
the vesicular and cellular membranes (22). On the other
hand, increased vesicular density does not always correlate with increased permeability. Stewart and Hayakawa
(33) pointed out the discrepancy between low density of
vesicles and high vascular permeability in fetal mouse
brain. Other reports also show that the vesicular density
is low in some brain tumor endothelia in spite of increased vascular permeability (21, 34).
Attempts to simply correlate the population of endothelial vesicles to actual vascular permeability may be an
oversimplification. Recent studies suggest selective functional mechanisms produce vesicles involved in transcytosis of specific molecules (35, 36). Bendayan and Rasio
(35) proposed the existence of distinct populations of vesicles incorporating specific binding proteins that have a
transport function that is confined to substances such as
insulin and albumin. It is, therefore, postulated that the
predominant receptor proteins bound to their membranes
define the functions of individual vesicles, and it has been
shown that certain proteins in the bloodstream can pass
through the BBB by means of receptor-mediated transcytosis. Accordingly, one theory has been offered suggesting that the low level of protein transport in BBB capillaries is due not to a low level of vesicular transport, but
rather to a reduced expression of specific protein-binding
receptors that define pinocytotic functions (22). However,
bradykinin and LTC4–induced vesicular formation appears to facilitate BTB penetration of a wide variety of
compounds, from low molecular weight a-aminoisobutyric acid (AIB; MW 103) (6) to HRP (MW 44,000) (Fig.
7).
Recent evidence suggests that both bradykinin and
LTC4 may activate K1 channels in tumor microvessel
endothelial cells, hyperpolarizing the endothelial cell
membrane (unpublished observation). One speculation is
that this membrane hyperpolarization may trigger vesicular formation in endothelial cells.
Tight junctions can be disrupted by exposure of cells
to hyperosmotic solutions, which makes vascular endothelia shrink transiently. However, the subtle, transient
changes responsible for a possible regulation of a tight
junction’s permeability in the BBB are difficult to discern
morphologically (25, 37). Previous studies have correlated the uptake of radionuclides with the length of endothelial tight junctions in human gliomas (38). In one
J Neuropathol Exp Neurol, Vol 61, August, 2002
study, ultrathin serial sections showed that the clefts
were, in fact, confluent and that they form continuous
channels that connect the lumen with the abluminal perivascular space (14). The cleft index, therefore, was devised to evaluate the facilitation of material passage
through the paracellular route (15, 39). The index was
reported to be higher in peritumoral brain capillary than
in the normal BBB, although considerable variation was
seen (15). In order to quantify size changes in tight junctional gaps we developed the cleft area index. This index
increased in parallel with the cleft index and also suggests
that tight junctional function is altered in glioma capillaries.
There have been few studies relating the morphometric
analysis of brain tumor capillaries involving a vesicular
population and change of tight junctions using an experimental design in which administration of vasoactive
compounds is employed to selectively increase BTB permeability. Our results indicate that significantly increased
vesicular transport is more likely to contribute to the increased vascular permeability than opening of the tight
junctions. Changes that are seen in tight junctions of tumor capillaries seem to be attributable to tumor growth
processes. Alterations in cleft morphology may be further
evidence that the tumor capillary per se is leakier than
normal brain capillary. No significant difference was seen
in the analysis of tight junction opening between bradykinin- and LTC4-treated groups and saline infusions within each glioma. However, it does not appear that the
transportation of molecules in blood serum can be explained simply by vesicular density alone. As mentioned
above, additional functional mechanisms seem to exist in
order to activate the formation of vesicles. As the endothelial vesicles in normal brain capillary do not increase
after treatment with vasomodulators, brain tumor capillaries would appear to have some specific characteristics
that render them responsive to vasomodulators. One possible characteristic of glioma tissue is their high expression of NOS, cyclic GMP, and K1 channels, which presumably mediate the signal pathways initiated by
bradykinin or LTC4 treatments.
In conclusion, we make the following observations. 1)
Bradykinin and LTC4-induced increases in BTB permeability are coincident with a 2–3-fold increase in the
number of BTB endothelial vesicles. 2) Increased BTB
vesicle-transit of intravenously administered HRP was
also demonstrated after bradykinin treatment. 3) We
found no evidence for paracellular BTB transit at the endothelial tight junctions in bradykinin- or LTC4-treated
rat gliomas. We conclude vesicular transport is the primary mechanism contributing to selective opening of the
BTB in bradykinin- or LTC4-treated RG2 or C6 gliomas.
ACKNOWLEDGMENTS
We thank Dr. Eain M. Cornford (Veterans Administration West Los
Angeles Medical Center, Los Angeles, CA) and Shigeyo Hyman
VESICLE TRANSPORT REGULATES BTB PERMEABILITY
(UCLA, Los Angeles, CA) for critical comments. We also appreciate
the technical assistance of Birgitta Sjostrand (UCLA, Los Angeles, CA)
and Dr. Ken Samoto (Cedars Sinai Medical Center, Los Angeles, CA).
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Received January 4, 2002
Revision received April 24, 2002
Accepted May 2, 2002
J Neuropathol Exp Neurol, Vol 61, August, 2002