| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
increases entry of macromolecules into
luminal endothelial cell glycocalyx
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22906
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The endothelial luminal glycocalyx has been largely
ignored as a target in vascular pathophysiology even though it occupies a key location. As a model of the inflammatory response, we tested the
hypothesis that tumor necrosis factor-
(TNF-) can alter the
properties of the endothelial apical glycocalyx. In the intact hamster
cremaster microcirculation, fluorescein isothiocyanate (FITC)-labeled
Dextrans 70, 580, and 2,000 kDa are excluded from a region extending
from the endothelial surface almost 0.5 µm into the lumen. This
exclusion zone defines the boundaries of the glycocalyx. Red blood
cells (RBC) under normal flow conditions are excluded from a region
extending even farther into the lumen. The cremaster microcirculation
was pretreated with topical or intrascrotal applications of TNF-.
After infusion of FITC-dextran, FITC-albumin, or FITC-immunoglubulin G
(IgG) via a femoral cannula, microvessels were observed with
bright-field and fluorescence microscopy to obtain estimates of the
anatomic diameters and the widths of fluorescent tracer columns and of
the RBC columns (means ± SE). After 2 h of intrascrotal
TNF- exposure, there was a significant increase in access of
FITC-Dextrans 70 and 580 to the space bounded by the apical glycocalyx
in arterioles, capillaries, and venules, but no significant change in
access of FITC-Dextran 2,000. The effects of TNF- could be observed
as early as 20 min after the onset of topical application. TNF-
treatment also significantly increased the penetration rate of
FITC-Dextran 40, FITC-albumin, and FITC-IgG into the glycocalyx and
caused a significant increase in the intraluminal volume occupied by
flowing RBC. White blood cell adhesion increased during TNF-
application, and we used the selectin antagonist fucoidan to attenuate
leukocyte adhesion during TNF- stimulation. This did not inhibit the
TNF--mediated increase in permeation of the glycocalyx. These
results show that proinflammatory cytokines can cause disruption of the
endothelial apical glycocalyx, leading to an increased macromolecular
permeation in the absence of an increase in leukocyte recruitment.
inflammation; intravital microscopy; fluorescein
isothiocyanate-dextrans; plasma proteins; leukocytes; tumor necrosis
factor-
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
THE VASCULAR
INFLAMMATORY response in vivo is characterized by endothelial
cell and leukocyte activation, leading to leukocyte adhesion and
extravasation with accompanying tissue edema. There is much evidence in
the literature implicating proinflammatory cytokines such as tumor
necrosis factor- (TNF-) as the initiating agents. The effects of
TNF- on vascular endothelium include a variety of events ranging
from nuclear changes to cell surface modifications (24).
There is emerging evidence of the importance of the endothelial cell
glycocalyx in vascular function, and, given its key location at the
interface between plasma and the endothelial interface, we thought it
probably plays a significant role in inflammatory responses. Although
the literature abounds with information on the recruitment and
extravasation of leukocytes during inflammation, little is known about
the response of the endothelial cell glycocalyx per se to
proinflammatory cytokines.
The glycocalyx on the luminal surface has recently been shown to be as much as 0.5 µm thick (29). It excludes anionic macromolecules the size of 70-kDa dextran and larger, reduces the functional diameter of the vessel by excluding red blood cells, and can be removed by light-dye treatment (29) or made more permeable to macromolecules by enzyme treatment (8). Substantial evidence supports a role for the glycocalyx as a dynamic component of endothelial cell function. The distribution of endothelial surface glycoconjugates can change with altered states of permeability, i.e., after cold-lesion injury to the brain (31). Also, frog mesenteric capillaries become more permeable when their glycocalyx is enzymatically digested (1).
During inflammation, adhesion molecules on leukocytes must make contact
with their ligands on the endothelium. Because these receptors are
presumably buried within the glycocalyx, it would be functional if this
thick surface layer were shed or modified during inflammation, thus
enabling easy access of leukocytes or blood-borne macromolecules to the
site of injury. The present study was undertaken to primarily examine
the hypothesis that TNF- can cause changes in the glycocalyx either
dependent or independent of leukocyte adhesion. In this paper, we
provide evidence that TNF- treatment causes modifications of the
endothelial luminal glycocalyx that result in increased solute
permeation and an increase in the intraluminal volume occupied by red
blood cells. These changes occurred while leukocyte adhesion was
blocked by the selectin antagonist fucoidan.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animal preparation. All of the reagents were obtained from Sigma Chemical (St. Louis, MO). All of the procedures and the care of animals were in accordance with institutional guidelines.
Male Syrian golden hamsters weighing 110-160 were anesthetized with intraperitoneal sodium pentobarbital (70 mg/kg). The trachea was cannulated to ensure a patent airway, and the hamster spontaneously breathed room air. The left femoral vein was cannulated for fluid replacement and maintenance of anesthesia (0.5 ml/h) and for infusion of FITC-dextrans or other reagents. Throughout the surgical preparation and experimental protocol, esophageal temperature was maintained at 37oC. After we placed the hamster on a Plexiglas platter, the right cremaster muscle was prepared for visualization according to standard methodology (29). The tissue was continually superfused at 5 ml/min with a bicarbonate-buffered saline solution consisting of (in mM) 131.9 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, and 20 NaHCO3. Succinylcholine (10
5 M) was added to this solution to reduce spontaneous
muscle contractions. The pH of the solution was maintained at
7.35-7.45 by bubbling with 5% CO2-95%
N2, and the temperature was maintained at 34oC.
After at least 30 min of stabilization, when arteriolar tone was
evaluated, a bolus of 0.3 ml FITC-dextran (40, 70, 580, or 2,000 kDa;
20 mg/ml in saline), FITC-albumin, or FITC-IgG (20 mg/ml) was given via
the femoral cannula.
Intravital microscopy.
After FITC-dextran infusion, microvessels (capillaries, arterioles, and
venules; 7-15 µm in diameter) were observed at ×55 with a Leitz
water-immersion objective (numerical aperture = 0.84) on a Zeiss
intravital microscope equipped with a SIT 66 MTI video camera.
Transillumination light for bright-field measurements was obtained from
a 150-W xenon lamp, and measurements were made as described previously
(6). Light from a 75-W xenon lamp and epi-illuminator
equipped with a 450- to 490-nm excitation filter, a dichroic beam
splitter (FT 510), and a barrier filter (LP 520) was used for
epi-illumination of the fluorescent tracer (29). Epi-illumination was controlled by a manual shutter and limited to 
10
s per vessel to prevent light-dye injury to the endothelium (29). Transillumination was for 30 to 40 s. Images of
vessels were displayed on a Dage MTI video monitor and recorded on
S-VHS videotapes for subsequent image analysis.
Intrascrotal TNF- treatment and infusion of macromolecules.
To determine the effect of an inflammatory mediator on
macromolecular penetration into the glycocalyx of skeletal muscle
microvessels, recombinant murine TNF- (0.5 µg in 0.3 ml of saline)
was injected into the scrotal sac of 24 hamsters to induce a localized
inflammatory response. After 2 h, the cremaster muscle was
prepared for intravital microscopy. After stabilization, three hamsters
each received a 0.3-ml bolus of either FITC-Dextran 40, 70, 580, or
2,000 or of the physiologically relevant plasma proteins, FITC-albumin, or FITC-goat-IgG via the femoral cannula. We used IgG (~150 kDa) to
compare the effect of TNF- on penetration rates of plasma proteins
of different sizes because it is a major component of plasma and more
than twice the molecular weight of albumin (~67 kDa). Six hamsters
were pretreated with fucoidan before being injected with TNF-; three
received FITC-albumin and three received FITC-IgG. Three of the control
hamsters were injected with saline only before plasma protein infusion,
and three of the control hamsters were pretreated with fucoidan in
saline only (25 mg/kg iv) before plasma protein infusion. Images of
vessels were recorded for analysis as described above.
Inhibition of leukocyte rolling and adhesion. Numerous studies have shown that adherent leukocytes can directly injure the endothelium (3, 15, 17, 19). However, it is not known if an inflammatory mediator will induce changes in the endothelial glycocalyx independent of disruption by bound leukocytes. Leukocyte rolling and adhesion were determined offline in postcapillary venules of four hamsters. Leukocytes that moved at a velocity less than that of the red blood cells were defined as rolling. Those that remained stationary for at least 30 s were considered to be adherent. Rolling leukocytes that passed a line perpendicular to the longitudinal axis of the vessel were monitored, and their velocities were calculated. Assuming cylindrical geometry of the venules, the number of adherent leukocytes was expressed per unit of inner surface area.
To determine the response of the endothelium to TNF- in the absence
of increased leukocyte adhesion, the selectin-binding carbohydrate
fucoidan was used to block the initial capture and rolling of activated
leukocytes. Three hamsters were pretreated with a 0.3-ml bolus of
fucoidan (25 mg/kg iv in saline) before TNF- intrascrotal injection.
After 2 h, the cremaster was prepared for intravital microscopy,
and the permeation of FITC-Dextran 70 and leukocyte adhesion and
rolling velocity were studied as described above.
Topical application of TNF- to the cremaster.
To evaluate the time course of the onset of TNF--induced dextran
penetration and leukocyte adhesion, topical applications of the
cytokine were performed by micropipette in four hamsters. Three control
cremasters were superfused with buffer only. For each cremaster, a
microvascular unit consisting of a feed arteriole, capillaries,
postcapillary venules, and a collecting venule was selected.
Microvessel units were then subjected to TNF- treatment for 2 h
by topical micropipette application. At 10-min intervals, the vessels
were viewed by trans- and epi-illumination, and images were recorded as
described above.
Data and statistical analyses.
Recorded video images of the microvessels were captured using Image-1
software (Universal Imaging) and a digital, time-base corrector. For
determining tracer penetration into the glycocalyx of arterioles and
venules, only vessels of 15 µm in diameter were used due to the
difficulty in focusing on the dextran column boundary in larger
vessels. For each vessel, the anatomic diameter, the width of the
tracer column, and the red blood cell column were measured with the use
of on-screen calipers that were calibrated with a vertical and
horizontal image of a stage micrometer (Graticules, Kent, UK).
Positioning of the calipers for anatomic measurements was done
according to a previously validated method (6). With a
comparison of in vivo and in vitro measurements, the accuracy in
measuring the width of the fluorescent tracer column has been demonstrated (29). Briefly, the vessel midplane was
brought into focus during transillumination, and the vessel was then
viewed under epi-illumination. One of the calipers was positioned
outside the dextran column with its luminal surface against the edge of the dextran column. The other caliper was positioned just inside the
dextran column with its outer surface against the edge of the dextran
column. Prior measurement established the accuracy of the endothelial
boundary by comparing the bright-field diameter measurement to peaks of
an intensity profile across a vessel labeled with a fluorescent
membrane dye in the endothelium (29).
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Intrascrotal injection of TNF-.
For the purposes of this study, microvessels were categorized into
capillaries (<7 µm diameter), small arterioles and postcapillary venules (7-10 µm diameter), and larger arterioles and
postcapillary venules (10-15 µm diameter). In control hamsters,
FITC-Dextran 70 had no access to a region extending ~0.4 µm from
the endothelial cell membrane of small arterioles and venules (Fig.
1A). In larger arterioles and
collecting venules, this region extended to ~0.5 µm. Restricted
access of dextran to this space was stable over a period of >2 h.
Treatment with TNF- induced a significant increase in permeation of
the glycocalyx of all vessels to FITC-Dextran 70. This increase in
permeation of the glycocalyx was evident even when leukocyte adhesion
was prevented by fucoidan. Immediately after infusion in the treated
tissues, FITC-Dextran 70 filled all but 0.6 µm of the vessel lumen
(Fig. 1B). Over the next 45 min, Dextran 70 penetrated the
space adjacent to the endothelial cell with a half-time of 10 min and
had reached the cell surface by 30 min.
|
treatment, the
region from which red blood cells were excluded had decreased to ~0.6
µm from the endothelial surface in all vessels, indicating a
significant increase in intraluminal volume available to the red blood
cells. Only a few small petechiae were observed. Fucoidan treatment did
not prevent the increase in intraluminal volume available to the red
blood cells.
|
treatment, there was a significant increase in permeation of
the capillary glycocalyx to FITC-Dextran 70, and the region from which
red blood cells were excluded had decreased to ~0.6 µm from the
endothelial surface, indicating a significant increase in intraluminal
volume available to the red blood cells.
|
-treated cremaster preparations were observed for an
additional 2 h after infusion of dextran. However, an increase in
background fluorescence occurred over time, which was attributed to
extravasation of dextran molecules in the surrounding interstitium. This made detection of the dye front within the vessels extremely difficult. Thus a reversal of the glycocalyx changes induced by TNF-
could not be adequately determined using the current methodology.
Effect of TNF- and fucoidan on leukocyte adhesion
and rolling.
In the controls, there were few adherent leukocytes in postcapillary
venules and collecting venules (Fig.
4A). As expected, TNF-
treatment caused a significant increase in the number of adherent
leukocytes. Leukocyte rolling velocity was significantly slower in
TNF--treated hamsters (8.2 ± 1.1 µm/s) compared with control
hamsters (28.6 ± 2.5 µm/s) and hamsters treated with both fucoidan and TNF- (38.6 ± 2.3 µm/s) (Fig. 4B).
The increase in leukocyte rolling velocity in the presence of fucoidan
was significantly greater compared with saline treatment.
|
alone.
Importantly, fucoidan pretreatment did not prevent the TNF--mediated
increase in FITC-Dextran 70 penetration into the glycocalyx (Fig.
1A, dark gray bars), although it prevented the increase in
leukocyte adhesion (Fig. 4A).
Effect of TNF- on penetration of albumin, IgG, and Dextrans 40, 580, and 2,000.
TNF- administration produced an increase in both the penetration
rate and the cutoff size for exclusion of macromolecules. In control
hamsters, FITC-albumin (~67 kDa) and FITC-IgG (~150 kDa) penetrated
the apical glycocalyx of the venules with a half-time of 40 min (Fig.
5, A and B). After
treatment with TNF-, both albumin and IgG penetrated the apical
glycocalyx with half-times of 15 min. The increase in permeation of
both plasma proteins was significant as early as 5 min after infusion.
Both macromolecules penetrated the full thickness of the glycocalyx
within 25 min. A similar increase in penetration was also observed
after TNF- in the presence of the selectin antagonist fucoidan.
Similar results were observed for the capillaries and arterioles. In
the presence of fucoidan alone, there was no significant difference in
penetration of the macromolecules compared with the controls.
|
on the
permeation of the glycocalyx to two larger dextrans and one smaller
dextran in the absence of leukocyte adhesion was also evaluated. In a
previous study, we observed that FITC-Dextrans 580 and 2,000 did not
penetrate the intact apical glycocalyx even after hyaluronidase
treatment (8). On the other hand, Dextran 40 has been
shown to penetrate the intact endothelial surface layer with a
half-time of 40 min (30). In this study, however, after
TNF- treatment, the dextran molecules occupied an increased intraluminal volume immediately after infusion with the dye fronts located ~0.3 µm from the endothelial surface (Fig.
6). We also observed even faster
penetration of FITC-Dextran 40 (half-time ~10 min). Given this rapid
penetration of Dextran 40, dextrans smaller than 40 kDa were excluded
from this study. The depth of penetration of FITC-Dextran 580 was
significant as early as 5 min after infusion. By 15 min after infusion,
FITC-Dextran 580 had penetrated the glycocalyx to within 0.1 µm of
the endothelial membrane. On the other hand, TNF- treatment did not
induce significant penetration of FITC-Dextran 2,000.
|
Topical TNF- treatment to a local area of
cremaster.
Topical microapplication of TNF- to selected microvascular
components caused a significant increase in penetration of FITC-Dextran 70 into the luminal glycocalyx of those vessels after 20 min of exposure compared with control hamsters (Fig.
7A). This showed that the
effects we observed were local and not due to other secondary systemic
effects of the TNF-. After 40 min of TNF- exposure, leukocyte
adhesion to the endothelium had increased significantly compared with
controls (Fig. 7B), and petechiae were observed at many
sites of extravasated leukocytes. This was taken as evidence of direct
injury to the endothelial layer by adherent leukocytes after TNF-
treatment.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
For decades, it has been known that the interaction of leukocytes and endothelium during inflammation can damage the endothelium and alter its permeability properties. However, nothing was known of the effect of a proinflammatory cytokine on penetration of macromolecules into the endothelial glycocalyx per se. Activation by cytokines results in upregulation of adhesion molecules on leukocytes and on the luminal surface of the endothelium (14). Thus leukocyte recruitment to sites of inflammation is facilitated.
Adherent leukocytes can directly injure the endothelium by releasing free radicals, proteases, and cationic proteins (3, 15, 19). An increase in extravasation of plasma macromolecules such as albumin leads to water retention in the interstitium, producing the edema that is characteristic of an inflammatory response. Much of the damage is attributed to release of proteases and free radicals by adherent leukocytes as they migrate through the endothelial wall (3, 10, 22, 23). For example, neutrophil-derived elastase degrades matrix constituents such as fibronectin and can contribute significantly to the increased permeability of macromolecules (17).
Previous findings from our laboratory showed that luminally applied
TNF- can increase arteriolar permeability to fluorescein in vitro,
suggesting an increased transport of small water-soluble reagents
(15). TNF- can also cause an increase in pulmonary vascular permeability independent of neutrophil adhesion
(11). These findings imply that TNF- can alter vascular
transport mechanisms independent of leukocyte involvement. In this
study, we have shown that TNF- also alters the endothelial surface
layer causing increased macromolecular penetration, and that leukocyte
adhesion is not a requirement for this response.
We therefore propose three mechanisms by which TNF- might affect the
properties of the apical glycocalyx (Fig.
8). Increased macromolecular penetration
of the glycocalyx might result from disruption by rolling or adherent
leukocytes after TNF- treatment. TNF- might activate passing
leukocytes to release free radicals and/or proteases that degrade
components of the glycocalyx. TNF- might also activate
endothelial-derived free radicals or surface-bound proteases or
phospholipases (2) that release syndecans or glypicans from the glycocalyx.
|
Once activated, leukocytes express selectins on the tips of their
microvilli and are captured by their ligands on the endothelium (14). This initiates the slow rolling phase of adhesion
and facilitates subsequent firm adhesion and extravasation by the integrins and Ig-type adhesion molecules. In our experiments, the
increase in the capture and rolling of leukocytes was significantly reduced by fucoidan, yet we still observed an increased penetration of
dextran and plasma proteins into the venular glycocalyx after TNF-
treatment. Furthermore, the capillaries and arterioles, which are not
normal binding sites for leukocytes, also had disrupted glycocalyces
after TNF- treatment in the presence of fucoidan. Thus we have
eliminated leukocyte adhesion as a necessary mechanism by which TNF-
increases penetration into the glycocalyx (Fig. 8).
A second possible explanation for the increased dextran and protein
penetration and increase in intraluminal volume after exposure to
TNF- is that the localized pool of cytokine in the cremaster
activates passing leukocytes that then release factors to disrupt the
glycocalyx (Fig. 8). A third mechanism is that TNF- directly
stimulates the endothelium to either shed some constituents of the
glycocalyx, release free radicals, or activate surface-bound proteases
or phospholipases that degrade it (Fig. 8). Endothelial-derived
metalloproteases are upregulated by TNF- and have been implicated in
changes in endothelial morphology (7, 21). Recent studies
have shown changes in endothelial cAMP and protein kinase expression
induced by TNF- (12, 13), as well as changes in
endothelial tight junctions, actin cytoskeletal changes, and increased
permeability (32). In addition, endothelial cells
internalize and degrade thrombomodulin (18) and shed
plasminogen activator inhibitor-1 and tie receptor from their surfaces
(26, 33). However, the degree to which these
molecules might contribute to forming the glycocalyx is unknown.
TNF- treatment causes the endothelium to release superoxide
(5). Thus it is reasonable to propose that TNF-
activation might also cause endothelial cell changes involving the glycocalyx.
To detect changes in the glycocalyx, our laboratory devised a sophisticated method of in vivo measurements of dextran penetration (6, 29). This required making sure that vessels were not underfocused or overfocused during transillumination or epi-illumination so that the boundary of the vessel (anatomic diameter) could be visualized as a thin black line. This works well for smaller vessels (up to ~15 µm), but, in larger vessels, edge detection becomes more dificult. For measuring the anatomic diameter of each vessel, precise placement of the calipers was achieved by positioning one caliper outside the thin black line, indicating the vessel wall with its luminal surface against the edge of the black line. The other caliper was positioned just inside the thin black line, indicating the vessel wall with its outer surface against the black line. Similarly, to measure the fluorescent tracer column, one caliper was positioned outside the tracer column with its luminal surface against the edge of the tracer column. The other caliper was positioned just inside the tracer column with its outer surface against the edge of the tracer column. By calculating the depth of tracer penetration as one-half of the difference between the bright-field anatomic diameter and the tracer column diameter, we obtain not the absolute value of tracer column dimensions but rather the difference in perceived boundaries of tracers within the vessel lumen. When measurements are made in this way, the results are consistent.
The release of endothelial superoxide on cytokine stimulation
(5) is likely to contribute to degradation of constituents of the glycocalyx. Hyaluronan, a component of the glycocalyx, is
particularly sensitive to superoxide cleavage, and depolymerization of
hyaluronan has often been used as an assay for the presence of free
radicals (4). A preliminary study in our laboratory showed
that treatment with exogenous hyaluronan or free radical scavengers
protected the endothelium from light-dye injury, suggesting involvement
of free radicals in degradation of the hyaluronan content of the
glycocalyx (C. Henry and B. Duling, unpublished results).
Results from a follow-up study in our laboratory also suggest that free
radicals and proteases are involved in degradation of the glycocalyx
after TNF- exposure without a concurrent increased leukocyte
adhesion (9).
Here, we provide evidence that TNF- significantly changes the
penetration rate of the major plasma macromolecules albumin and IgG
into the glycocalyx. In the intact glycocalyx, albumin (67 kDa)
penetrates with a half-time of 40 min (30). We observed that in control hamsters, IgG, although more than twice the size of
albumin, penetrates the glycocalyx at the same rate. Vink and Duling
(30) observed identical penetration rates of two proteins of vastly different molecular weights and suggested that mechanisms other than simple diffusion are involved in protein penetration into
the endothelial surface layer. After TNF- treatment, the penetration
of both albumin and IgG increases by the same rate, suggesting a
specific interaction between proteins and the apical glycocalyx that is
disrupted by inflammatory stimuli.
Although TNF- treatment disrupts the barrier properties of the
luminal glycocalyx and causes some degradation, it does not cause total
removal of the matrix. The continued size dependence of dextran
penetration for up to 3 h after TNF- exposure is evidence of
this. Although all dextrans used in this study were anionic, the
observed penetration rates in control hamsters reflect variations in
both size and charge. It is unlikely that osmotic effects could produce
these differences in penetration rates because the final blood
concentration of each dextran was small (<104 M). The
FITC-dextran molecules we used are labeled with an efficiency of about
0.01 FITC-molecules/glucose (Sigma, personal communication). Thus the
net charge would be proportional to the molecular weight. If the
glycocalyx had been totally lost after TNF- treatment, we would have
expected to see no difference in penetration rate of the dextrans, and
we should have detected a change in the exclusion zone between the red
blood cells and the endothelial surface. Therefore, we conclude that
certain constituents of the surface coat remained intact and that these
are sufficient to restrict free movement of the dextrans.
Taken together, these findings support our earlier data on the
heterogeneous composition of the glycocalyx (8). In that study, we showed that cleavage of cell surface hyaluronan by the enzyme
hyaluronidase, resulted in increased penetration of FITC-Dextrans 70 and 145 without increasing the penetration of FITC-Dextrans 580 and
2,000 or the intravascular volume available for the red blood cells.
Here, we now show that the TNF- response also involves disruption of
glycocalyx constituents resulting in increased penetration of
FITC-Dextran 70 and plasma proteins. However, there are important differences. TNF- treatment produced a significant increase in the
intravascular volume available to red blood cells, as well as an
increase in penetration of FITC-Dextran 580. Hyaluronidase treatment
did not produce these effects. Thus the degradation induced by TNF-
appears to be more extensive than that produced by hyaluronidase.
Other inflammatory mediators and vasoactive molecules may have a
similar effect on the glycocalyx. We observed increased penetration of
FITC-Dextran 70 into the luminal glycocalyx of hamster cremaster microvessels after topical adenosine or histamine application (C. Henry
and B. Duling, unpublished results). Histamine can also cause
selectin-mediated leukocyte adhesion and leukocyte-independent extravasation of plasma macromolecules in rat mesenteric venules (28). We cannot ignore the possibility that other
endogenous cytokines were induced by TNF- in our experiments. For
example, interferon-
has been shown to be upregulated by TNF- and
then to act synergistically with it in enhancing the inflammatory
response (27). Endothelial activation by TNF- can also
upregulate the production of interleukin-1, another proinflammatory
cytokine (20).
In conclusion, we have shown that TNF- treatment significantly
alters the permeation properties of the endothelial luminal glycocalyx
independent of leukocyte adhesion. This supports the idea that the
glycocalyx is a significant site of cytokine-mediated injury. Taken
together with evidence in the literature, our findings lead us to
further hypothesize that initial disruption of the matrix by
proinflammatory cytokines may result in loss of anticoagulant surface
molecules and predispose the endothelium to exposure to blood
components. Coupled with leukocyte adhesion and extravasation, as well
as disruption of intercellular junctions, these processes might lead to
increased microvascular dysfunction.
| |
ACKNOWLEDGEMENTS |
|---|
B. R. Duling was supported in part by National Institutes of Health Grant HL-12792, and C. B. S. Henry was supported by American Heart Association Fellowship 9920377U.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: B. R. Duling, Dept. of Molecular Physiology and Biological Physics, Univ. of Virginia, PO Box 800736, Charlottesville, VA 22908-0736 (E-mail: brd{at}virginia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 November 1999; accepted in final form 14 July 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Adamson, RH.
Permeability of frog mesenteric capillaries after partial pronase digestion of the endothelial glycocalyx.
J Physiol (Lond)
428:
1-13,
1990
2.
Bashkin, P,
Neufeld G,
Gitay-Goren H,
and
Vlodavsky I.
Release of cell surface-associated basic fibroblast growth factor by glycosylphosphatidylinositol-specific phospholipase C.
J Cell Physiol
151:
126-137,
1992[Web of Science][Medline].
3.
Carden, DL,
Smith JK,
and
Korthuis RJ.
Neutrophil-mediated microvascular dysfunction in postischemic canine skeletal muscle. Role of granulocyte adherence.
Circ Res
66:
1436-1444,
1990
4.
Del Maestro, RF,
Arfors K-E,
and
Lindblom R.
Free radical depolymerization of hyaluronic acid. Influence of scavenger substances.
Bib Anat
18:
132-135,
1979.
5.
Friedl, HP,
Till GO,
Ryan US,
and
Ward PA.
Mediator-induced activation of xanthine oxidase in endothelial cells.
FASEB J
3:
2512-2518,
1989[Abstract].
6.
Gretz, JE.
Measurement uncertainties associated with the use of bright-field and fluorescence microscopy in the microcirculation.
Microvasc Res
49:
134-140,
1995[Web of Science][Medline].
7.
Hanemaaijer, R,
Koolwijk P,
le Clercq L,
de Vree WJ,
and
van Hinsbergh VW.
Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Effects of tumour necrosis factor alpha, interleukin-1 and phorbol ester.
Biochem J
296:
803-809,
1993.
8.
Henry, CBS,
and
Duling BR.
Permeation of the luminal capillary glycocalyx is determined by hyaluronan.
Am J Physiol Heart Circ Physiol
277:
H508-H514,
1999
9.
Henry, CBS,
and
Duling BR.
TNF--induced permeation of the endothelial glycocalyx is mediated by proteases and free radicals (Abstract).
FASEB J
14:
A686,
2000.
10.
Hoover, RL,
Briggs RT,
and
Karnovsky MJ.
The adhesive interaction between polymorphonuclear leukocytes and endothelial cells in vitro.
Cell
14:
423-428,
1978[Web of Science][Medline].
11.
Horvath, CJ,
Ferro TJ,
Jesmok G,
and
Malik AB.
Recombinant tumor recrosis factor increases pulmonary vascular permeability independent of neutrophils.
Proc Natl Acad Sci USA
85:
9219-9223,
1988
12.
Koga, S,
Morris S,
Ogawa S,
Liao H,
Bilezikian JP,
Chen G,
Thompson WJ,
Ashikaga T,
Brett J,
Stern DM,
and
Pinsky DJ.
TNF modulates endothelial properties by decreasing cAMP.
Am J Physiol Cell Physiol
268:
C1104-C1113,
1995
13.
Laird, SM,
Graham A,
Paul A,
Gould GW,
Kennedy C,
and
Plevin R.
Tumour necrosis factor stimulates stress-activated protein kinases and the inhibition of DNA synthesis in cultures of bovine aortic endothelial cells.
Cell Signal
10:
473-480,
1998[Web of Science][Medline].
14.
Ley, K,
and
Tedder TF.
Leukocyte interactions with vascular endothelium: new insights into selectin-mediated attachment and rolling.
J Immunol
155:
525-528,
1995[Abstract].
15.
MacGregor, IR,
Perrie AM,
Donnelly SC,
and
Haslett C.
Modulation of human endothelial thrombomodulin by neutrophils and their release products.
Am J Respir Crit Care Med
155:
47-52,
1997[Abstract].
16.
Matsuki, T,
Beach JM,
Klindt RL,
and
Duling BR.
Modification of vascular reactivity by alteration of intimal permeability: effect of TNF-.
Am J Physiol Heart Circ Physiol
264:
H1847-H1853,
1993
17.
McDonald, JA,
and
Kelly DG.
Degradation of fibronectin by human leukocyte elastase.
J Biol Chem
225:
8848-8858,
1980.
18.
Moore, KL,
Emson CT,
and
Emson NL.
Tumor necrosis factor leads to internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture.
Blood
73:
159-165,
1989
19.
Morita Clemens, YMG,
Miller LS,
Rangan U,
Kondo S,
Miyasaka M,
Yoshikawa T,
and
Bulkley GB.
Reactive oxidants mediate TNF--induced leukocyte adhesion to rat mesenteric venular endothelium.
Am J Physiol Heart Circ Physiol
269:
H1833-H1842,
1995
20.
Nawroth, PP,
Bank I,
Handley D,
Cassimeris J,
Chess L,
and
Stern D.
Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin-1.
J Exp Med
163:
740-745,
1986
21.
Nelimarkka, LO,
Nikkari ST,
Ravanti LS,
Kahari VM,
and
Jarvelainen HT.
Collagenase-1, stromelysin-1 and 92 kDa gelatinase are associated with tumor necrosis factor-alpha induced morphological change of human endothelial cells in vitro.
Matrix Biol
17:
293-304,
1998[Web of Science][Medline].
22.
Peterson, MW.
Neutrophil cathepsin G increases transendothelial albumin flux.
J Lab Clin Med
113:
297-308,
1989[Web of Science][Medline].
23.
Peterson, MW,
Stone P,
and
Shasby M.
Cationic neutrophil proteins increase transendothelial albumin movement.
J Appl Physiol
62:
1521-1530,
1987
24.
Pober, JS.
Effects of tumor necrosis factor and related cytokines on vascular endothelial cells.
In: Tumor Necrosis Factor and Related Cytokines. Ciba Foundation Symposium. Chichester, UK: Wiley, 1987, p. 170-184.
25.
Ryan, US,
and
Ryan JW.
The ultrastructural basis of endothelial cell surface functions.
Biorheology
21:
155-170,
1984[Web of Science][Medline].
26.
Soeda, S,
Tsunoda T,
Kurokawa Y,
and
Shimeno H.
Tumor necrosis factor-alpha-induced release of plasminogen activator inhibitor-1 from human umbilical vein endothelial cells: involvement of intracellular ceramide signaling event.
Biochim Biophys Acta
1448:
37-45,
1998[Medline].
27.
Stolpen, AH,
Golan DE,
and
Pober JS.
Tumor necrosis factor and immune interferon act in concert to slow the lateral diffusion of proteins and lipids in human endothelial cell membranes.
J Cell Biol
107:
781-789,
1988
28.
Valeski, JE,
and
Baldwin AL.
Effect of early transient adherent leukocytes on venular permeability and endothelial actin cytoskeleton.
Am J Physiol Heart Circ Physiol
277:
H569-H575,
1999
29.
Vink, H,
and
Duling BR.
Identification of distinct luminal domains for macromolecules, erythrocytes and leukocytes within mammalian capillaries.
Circ Res
79:
581-589,
1996
30.
Vink, H,
and
Duling BR.
Capillary endothelial surface layer selectively reduces plasma solute distribution volume.
Am J Physiol Heart Circ Physiol
278:
H285-H289,
2000
31.
Vorbrodt, AW.
Changes in the distribution of endothelial surface glycoconjugates associated with altered permeability of brain microvessels.
Acta Neuropathol (Berl)
70:
103-111,
1986[Medline].
32.
Wong, RK,
Baldwin AL,
and
Heimark RL.
Cadherin-5 redistribution at sites of TNF-- and IFN--induced permeability in mesenteric venules.
Am J Physiol Heart Circ Physiol
276:
H736-H748,
1999
33.
Yabkowitz, R,
Meyer S,
Black T,
Elliott G,
Merewether LA,
and
Yamane HK.
Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.
Blood
93:
1969-1979,
1999
This article has been cited by other articles:
|
|
 |
|
  D. Chappell, M. Jacob, K. Hofmann-Kiefer, M. Rehm, U. Welsch, P. Conzen, and B. F. Becker Antithrombin reduces shedding of the endothelial glycocalyx following ischaemia/reperfusion Cardiovasc Res, July 15, 2009; 83(2): 388 - 396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Meuwese, H. L. Mooij, M. Nieuwdorp, B. van Lith, R. Marck, H. Vink, J. J. P. Kastelein, and E. S. G. Stroes Partial recovery of the endothelial glycocalyx upon rosuvastatin therapy in patients with heterozygous familial hypercholesterolemia J. Lipid Res., January 1, 2009; 50(1): 148 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. G. E. VanTeeffelen, A. A. Constantinescu, J. Brands, J. A. E. Spaan, and H. Vink Bradykinin- and sodium nitroprusside-induced increases in capillary tube haematocrit in mouse cremaster muscle are associated with impaired glycocalyx barrier properties J. Physiol., July 1, 2008; 586(13): 3207 - 3218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Svennevig, T. Hoel, A. Thiara, S. Kolset, A Castelheim, T. Mollnes, F Brosstad, E Fosse, and J. Svennevig Syndecan-1 plasma levels during coronary artery bypass surgery with and without cardiopulmonary bypass Perfusion, May 1, 2008; 23(3): 165 - 171. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Singh, S. C. Satchell, C. R. Neal, E. A. McKenzie, J. E. Tooke, and P. W. Mathieson Glomerular Endothelial Glycocalyx Constitutes a Barrier to Protein Permeability J. Am. Soc. Nephrol., November 1, 2007; 18(11): 2885 - 2893. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Stevens, V. Hlady, and R. O. Dull Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L328 - L335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. van den Berg and H. Vink Glycocalyx perturbation: cause or consequence of damage to the vasculature? Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2174 - H2175. [Full Text] [PDF]
|
|
|
|
|
|
I. Rubio-Gayosso, S. H. Platts, and B. R. Duling Reactive oxygen species mediate modification of glycocalyx during ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2247 - H2256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. van den Berg, J. A. E. Spaan, T. M. Rolf, and H. Vink Atherogenic region and diet diminish glycocalyx dimension and increase intima-to-media ratios at murine carotid artery bifurcation Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H915 - H920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bruegger, M. Jacob, M. Rehm, M. Loetsch, U. Welsch, P. Conzen, and B. F. Becker Atrial natriuretic peptide induces shedding of endothelial glycocalyx in coronary vascular bed of guinea pig hearts Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1993 - H1999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Flessner The transport barrier in intraperitoneal therapy Am J Physiol Renal Physiol, March 1, 2005; 288(3): F433 - F442. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Platts and B. R. Duling Adenosine A3 Receptor Activation Modulates the Capillary Endothelial Glycocalyx Circ. Res., January 9, 2004; 94(1): 77 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. A. van Haaren, E. VanBavel, H. Vink, and J. A. E. Spaan Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2848 - H2856. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Weinbaum, X. Zhang, Y. Han, H. Vink, and S. C. Cowin Inaugural Article: Mechanotransduction and flow across the endothelial glycocalyx PNAS, June 24, 2003; 100(13): 7988 - 7995. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
