1 Department of Internal
Medicine, Vascular
endothelial growth factor (VEGF) is a key regulator of vasculo- and
angiogenesis. Earlier studies demonstrated a permeability-increasing
effect of VEGF in skin tests, leading to its other name,
vascular permeability factor. We wondered whether VEGF-induced
hyperpermeability was a direct effect of VEGF on endothelial cells and
studied the permeability of human and porcine endothelial cell
monolayers in a well-characterized in vitro system. VEGF increased the
hydraulic conductivity up to 20-fold and simultaneously decreased the
albumin reflection coefficient. This effect occurred after a delay of
150 min, although VEGF-induced early endothelial cell activation was
verified by enhanced inositol phosphate accumulation within 5 min and
increased P-selectin expression within 15 min. Platelet-derived growth
factor and granulocyte-macrophage colony-stimulating factor, two
endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF
increased endothelial permeability by a direct action on endothelial
cells. Based on the pattern of endothelial cell activation by growth
factors, VEGF appears to be a unique stimulus.
vascular endothelial growth factor; cultured human endothelial
cells; hydraulic conductivity; adhesion molecules; platelet-derived
growth factor; granulocyte-macrophage colony-stimulating factor
VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) represents a
paracrine signaling system crucial to the development, differentiation, and adaptation of the vascular system (14). This endothelial cell-specific mitogen is also active in several pathophysiologically important situations such as tumor growth, diabetic retinopathy, and
wound healing (14, 17). Ischemia and hypoxia appear to be the
strongest stimuli for VEGF production that finally results in
neovascularization of the affected vascular bed (28).
VEGF was originally described as a tumor-derived vascular permeability
factor that caused vascular leakage in vivo (21). On intradermal
injection or topical application to muscle preparations, VEGF indeed
caused a hyperpermeability that occurred within minutes (9, 18, 21). In
the case of sustained hypoxia, VEGF action, therefore, would be
twofold: a rapid increase in endothelial permeability for enhanced
substrate supply and, in the long run, formation of new capillaries.
VEGF165, the best-studied VEGF
variant, is secreted as a 46-kDa, heparin-binding homodimeric
glycoprotein by a variety of cells and acts almost exclusively on
endothelial cells by binding with high affinity to the cognate tyrosine
kinase receptors VEGFR-1 (f lt-1) and VEGFR-2 (KDR/f lk-1) (7,
14). At least four isoforms of human VEGF mRNA encoding VEGF proteins
of 121, 165, 189, and 206 amino acids are produced as a result of
alternative splicing from a single gene (26).
The endothelium provides a major permeability barrier of the vessel
wall. Under resting conditions, extravasation of macromolecules is
highly restricted, whereas small molecules use para- and transcellular pathways to cross capillaries rapidly. In inflammed tissues,
stimulation of endothelial cells with thrombin, hydrogen peroxide, or
bacterial toxins will result in cell retraction accompanied by opening
of intercellular gaps, thereby allowing enhanced paracellular fluid flux (15, 22).
The mechanisms that underlie VEGF-induced vascular hyperpermeability
are unclear. The in vivo studies mentioned did not differentiate between VEGF-induced alterations of the local hemodynamics, with possibly increased filtration pressures; VEGF-related activation of
inflammatory cells; and/or a direct effect of VEGF on the
endothelium itself. The demonstration of a rapid fenestration of the
endothelium in cremaster vessels as well as the recent description of
vesiculovacuolar organelles (VVO) in skin endothelial cells after VEGF
exposure points to the endothelium as a possible direct target of the
VEGF action in vivo (9, 18).
We therefore analyzed the effects of VEGF on the permeability of human
endothelial cell monolayers in a well-characterized in vitro system.
The results indicated that VEGF increased the hydraulic conductivity up
to 20-fold and simultaneously decreased the albumin reflection
coefficient (RC) of human endothelial monolayers, suggesting that VEGF
increases permeability by direct action on endothelial cells.
Platelet-derived growth factor (PDGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), two endothelial cell nonspecific mitogens, were without effect in this system. Interestingly,
VEGF-related hyperpermeability occurred after a delay of 150 min. To
verify early endothelial cell activation, we also studied stimulation of phosphatidylinositol metabolism and P-selectin expression in cultured human endothelial cells.
Materials. Tissue culture
plasticware was obtained from Becton Dickinson (Heidelberg, Germany).
Medium 199, FCS, Hanks' balanced salt solution (HBSS), PBS,
trypsin-EDTA solution, Puck's A, HEPES, and antibiotics
were from GIBCO (Karlsruhe, Germany).
Excell 400 medium was from Biochrom (Munich, Germany). Collagenase (CLS
type II) was purchased from Worthington Biochemical (Freehold, NJ). Gelatin from porcine skin type I, glutaraldehyde grade II, sodium nitroprusside (SNP), borax
(Na2B4O7 · 10H2O),
thrombin, GM-CSF, PDGF-AB, and cholera toxin were purchased from Sigma
(Munich, Germany). Polycarbonate micropore filter membranes (25-mm
diameter, 5-µm pore size) were purchased from Nucleopore
(Tübingen, Germany). 3H2O
(1 mCi/mg),
[methyl-14C]albumin
(0.026 mCi/mg),
myo-[3H]inositol
(535 mCi/mg), and
Na251CrO4
(1 mCi/ml) were from Amersham Buchler (Braunschweig, Germany). All
other chemicals used were analytic grade and obtained from commercial
sources.
Purification of VEGF. Human
VEGF165 cDNA, expressed in the
Baculovirus transfer vector pVL 1393, was used to infect SF9 insect cells. Human recombinant VEGF was
purified from conditioned serum-free Excell 400 medium of the infected
SF9 cells as previously described (4). Briefly, 100 ml of conditioned
medium were applied on a heparin high trap (Pharmacia, Freiburg,
Germany) equilibrated with 50 mM phosphate buffer (pH 7.0) and 120 mM
NaCl. The column was eluted with high-performance liquid chromatography
(SmartSystem Pharmacia, Freiburg, Germany) with an ascending salt
gradient (0-1.5 M NaCl), and fractions were assessed for purity by
SDS-PAGE with the PhastGel apparatus (Pharmacia) and subsequent
staining as previously described (4). For final purification (a single band in SDS-PAGE), it was sufficient to repeat the liquid
chromatography one or two times.
Monoclonal antibodies. Purified
freeze-dried monoclonal antibodies (MAbs) directed against P-selectin
(CLB/thromb6) were obtained from Dianova (Hamburg, Germany), and MAbs
directed against intercellular adhesion molecule (ICAM-1; RR1/1) and
E-selectin (H18/7) were from Serva (Heidelberg, Germany). Horseradish
peroxidase-conjugated polyclonal sheep anti-mouse IgG antibodies were
purchased from Amersham (Dreieich, Germany). All antibodies used were
azide free. To further characterize the adhesion system, endothelial
cells were preincubated with 20 µg/ml of inhibitory MAb for 30 min.
Preparation of human umbilical cord vein endothelial
cells. Cells were isolated from umbilical cord veins
and identified as previously described (12). Isolated endothelial cells
were seeded in tissue culture flasks (80 cm2) or on 6-, 24-, or 96-well
plates (Becton Dickinson). Confluent primary cultures of human
umbilical cord vein endothelial cell (HUVEC) monolayers were used for
quantification of adhesion molecules (cell-surface ELISA),
polymorphonuclear neutrophil (PMN) adhesion assay, and determination of
inositol phosphates. Endothelial monolayer permeability (hydraulic
conductivity and albumin RC) was determined using HUVECs in their third
and, in selected experiments, first passages.
Preparation of porcine endothelial cell
monolayers. Endothelial cells were isolated from
pulmonary arteries of freshly slaughtered pigs by exposure to 0.1%
collagenase in Puck's saline for 12-15 min at 37°C. Cells
were dispersed, characterized, and maintained in medium 199-10%
FCS in a humified atmosphere (37°C, 5%
CO2) as previously described
(11, 22-25). Permeability studies performed were done using
confluent porcine endothelial cell monolayers in their third and, in
selected experiments, first passages.
Isolation and labeling of human PMNs.
Heparinized human donor blood was centrifuged in a discontinuous
Percoll gradient to yield a PMN fraction of >97% purity as
previously described (12). Freshly isolated neutrophils were
radiolabeled with
Na251CrO4
(1 mCi/ml). Briefly, after isolation, PMNs were incubated with 100 µCi of 51Cr at 37°C for 1 h
in RPMI 1640 medium containing 10% FCS. Subsequently, the cells were
washed twice in HBSS (with calcium, without magnesium) to remove
unincorporated 51Cr.
Determination of hydraulic conductivity and albumin
RC. Endothelial cell monolayers were grown on
polycarbonate filter membranes as previously described (11,
22-25). A confluent monolayer on a filter membrane was mounted in
a modified chemotaxis chamber, and a hydrostatic pressure of 10 cmH2O was applied to the
"luminal" side of the cell monolayer. The filtration rate across
the endothelial monolayer was continuously determined, and the
hydraulic conductivity was calculated and expressed as
10 Experimental protocol. Only monolayers
that showed a final hydraulic conductivity of <0.5 × 10 PMN adhesion to HUVEC monolayer. The
medium was aspirated, and the endothelial cells were washed two times
with HBSS. 51Cr-labeled PMNs (1 × 106 in 1 ml of buffer)
were added to each well (24-well plate). The cell mixture was
stimulated with VEGF or thrombin for 3-30 min. Subsequently,
unbound PMNs were removed by gentle aspiration, and each well was
washed two times with HBSS. Adherent PMNs and endothelial cells were
lysed with 2 M
H2SO4
for 30 min. Radioactivity of the lysate was quantitated with a
Cell-surface ELISA for P-selectin, ICAM-1, and
E-selectin expression on HUVECs. Expression of adhesion
molecules on monolayers of human endothelial cells was determined with
a cell-surface ELISA technique (12). Briefly, confluent HUVEC
monolayers in 96-well flat-bottom microtiter plates were washed;
stimulated with VEGF, GM-CSF, PDGF, thrombin, or lipopolysaccharide as
indicated; and finally fixed with 4% paraformaldehyde. Human Ig was
used to reduce nonspecific binding, and primary antibodies were added for 30 min. Thereafter, the cells were washed three times and exposed
to a horseradish peroxidase-conjugated rabbit anti-mouse Ig antibody
for 30 min. After the cells were washed,
o-phenylenediamine was
added for 5 min. Data are given as optical density at 492 nm.
Total inositol phosphates. The sum of
inositol mono-, di-, and trisphosphate was determined, with minor
modifications, as previously described (10). Briefly, confluent primary
cultures of HUVECs were incubated with 10 µCi/ml of
D-myo-[3H]inositol
in medium 199 overnight. Before experimental use, the cells were washed
three times with HBSS and incubated for 30 min in the presence of 10 mM
LiCl. At different times after stimulus application, the reaction was
stopped by rapid aspiration of HBSS and addition of ice-cold 0.5 M
HClO4. The cell monolayers were scraped, and the cell suspension was centrifuged at 1,300 g for 10 min at 4°C. The
supernatants were neutralized with 0.72 M KOH in 0.6 M
K2CO3,
added to 20 mM myo-inositol, and
processed to separate the inositol phosphates on Dowex anion-exchange
columns as previously described (10). For determination of total
inositol phosphates, columns were eluted with 1 M ammonium formiate-0.1 M formic acid directly into liquid scintillation vials.
Statistical methods. Depending on the
number of groups (A) and the number
of different time points studied
(B), the data in Figs. 1-5 were
analyzed by an A × B ANOVA. A one-way ANOVA was used for
the data in Tables 1 and 2. The main effects were then compared by an
F probability test (8).
P < 0.05 was considered significant.
Sealed endothelial cell monolayers of human and porcine origin
displayed a hydraulic conductivity of <0.5 × 105
cm · s
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
5 cm per second per
centimeter of water. For calculation of the selectivity of the
endothelial monolayer,
3H2O
(1 mCi/g) and
[methyl-14C]albumin
(0.026 mCi/mg) were added to the upper compartment. The amount of
3H2O
and
[methyl-14C]albumin
in the lower compartment was continuously measured with a Ramona LS-5
radioactivity monitor (Raytest, Heidelberg, Germany). The albumin RC
was calculated on the basis of
3H2O
and
[methyl-14C]albumin
in the upper and lower compartments of the filter system as previously
described (11, 22-25).
5
cm · s
1 · cmH2O
1
in the presence of a hydrostatic pressure of 10 cmH2O were used ("sealed"
filters; see Ref. 24 for details). In all experiments, VEGF was added
as a bolus into the upper compartment at time
0. SNP was applied as a bolus at time point
5
min. SNP was also added to the fluid reservoir that provided the
hydrostatic pressure to the upper compartment. Thus fluid filtrated
from the upper into the lower compartment was replaced by
SNP-containing buffer from the reservoir. Cholera toxin was given 60 min before the addition of VEGF.
-counter (Cobra Autogamma B5003, Canberra Packard, Frankfurt,
Germany). The percentage of PMN adhesion was calculated as the
51Cr fraction in the lysate in
relation to the total radioactivity added (12).
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
1 · cmH2O
1
and an albumin RC of >0.75. These in vitro data compare very well to
corresponding values in the microcirculation (16). A bolus addition of
VEGF (0.1-100 pg/ml) to HUVECs time and dose dependently increased
the hydraulic conductivity of the cell monolayers up to 20-fold. The
effect occurred after a delay of ~150 min. In contrast, thrombin (1 U/ml) increased the permeability of HUVEC monolayers maximally within
30 min (Fig. 1). Control cell monolayers were stable throughout the experimental period and reacted promptly on
addition of staphylococcal
-toxin (10 µg/ml), a well-known permeability-increasing agent (23, 24) (Fig. 1). VEGF-induced hyperpermeability was accompanied by a reduction in the selectivity of
the HUVEC monolayer as indicated by the drop in the albumin RC from 0.8 to 0.4 in the presence of 100 pg/ml of VEGF (Fig. 2). In control monolayers, this parameter
was stable for at least 5 h. VEGF (and thrombin) also increased the
hydraulic conductivity of porcine pulmonary artery endothelial cell
monolayers with similar kinetics, although the VEGF (and thrombin)
dose-response curve was shifted to the right in the porcine system
(Table 1).
View larger version (28K):
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Fig. 1.
Vascular endothelial growth factor (VEGF) time and dose dependently
increased hydraulic conductivity (used as a measure of monolayer
permeability) of cultured human umbilical venous endothelial cell
(HUVEC) monolayers. VEGF effects are shown in comparison to thrombin.
Stimuli (VEGF and thrombin) were added as a bolus at
time 0 (arrow). Control monolayers
were stable throughout experimental period and responded promptly on
addition of staphylococcal -toxin, an established
permeability-increasing agent. Data are means ± SE of 4 separate
experiments.
View larger version (33K):
[in a new window]
Fig. 2.
VEGF decreased selectivity of endothelial cell monolayers. Resting
(sealed) cell monolayers displayed a hydraulic conductivity (HC) of
<0.5 × 10 5
cm · s
1 · cmH2O
1
and an albumin reflection coefficient (RC) of >0.8. VEGF increased HC
substantially and decreased RC to 0.4. Both permeability parameters
were determined simultaneously on the same endothelial monolayer. Data
are means ± SE of 4 separate experiments.
Table 1.
Effect of VEGF, GM-CSF, and PDGF on hydraulic conductivity of
human and porcine endothelial cell monolayers
Studies applying electron microscopy revealed the presence of VVO and fenestrae in VEGF-treated HUVECs on filter membranes after 150 min of exposure to 100 ng/ml of VEGF (S. Hippenstiel, H. Wolburg, and N. Suttorp, unpublished observations).
We wished to extend the observations made to endothelial cell nonspecific growth factors and exposed endothelial cell monolayers to GM-CSF (10 pg/ml to 100 ng/ml) and PDGF (10 pg/ml to 100 ng/ml). Both mitogens had no effect on endothelial monolayer permeability within the time frame tested (5 h; Table 1).
Previous studies (23, 25) have shown that endothelial
hyperpermeability provoked by thrombin, hydrogen peroxide, or bacterial toxins was blocked by increased endothelial nucleotide levels. In
detail, activation of adenylyl or guanylyl cyclase with cholera toxin
or nitric oxide, respectively, proved to be a very effective measure to
antagonize enhanced endothelial monolayer permeability (23, 25).
Similarly, pretreatment of HUVEC monolayers with 109 M cholera toxin or
10
6 M SNP abolished
VEGF-induced endothelial hyperpermeability (Fig. 3). These pretreated cell monolayers still
reacted promptly on addition of 10 µg/ml of staphylococcal
-toxin,
indicating the viability of the endothelial preparation and confirming
the specificity of the approach (Fig. 3).
|
As a second endothelial function and as a measure of early endothelial cell activation, we analyzed the expression of adhesion molecules in VEGF-stimulated HUVEC monolayers and noted a rapid upregulation (3-15 min) of P-selectin in the presence of 0.1-25 pg/ml of VEGF (Fig. 4, left). Increased P-selectin expression was accompanied by enhanced PMN adhesion to VEGF-stimulated HUVEC monolayers, an effect that was suppressed by anti-P-selectin antibodies (Fig. 4, right). Overall with respect to P-selectin, VEGF turned out to be as potent as thrombin (Fig. 4). Exposure of HUVECs to VEGF for 4 and 16 h had no effect on the expression of E-selectin and ICAM-1; in these experiments, lipopolysaccharide (10 ng/ml)-stimulated cells served as positive controls (Table 2). Similar to VEGF, GM-CSF and PDGF also upregulated P-selectin in HUVECs rapidly, whereas E-selectin and ICAM-1 expression were unchanged (Table 2).
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To confirm rapid activation of endothelial cells by VEGF, the accumulation of total inositol phosphates was analyzed. Although less potent than thrombin, VEGF exposure clearly resulted in a substantial accumulation of these products (Fig. 5). This VEGF effect was not specific because GM-CSF and PDGF also stimulated phosphatidylinositol metabolism (Fig. 5).
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DISCUSSION |
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The results presented indicate that VEGF increased the permeability of endothelial monolayers by a direct action of VEGF on human and porcine endothelial cells. VEGF was a strong stimulus as indicated by the 20-fold increase in hydraulic conductivity and the simultaneous drop in albumin RC. The effect of VEGF was specific in the sense that two other mitogens, PDGF and GM-CSF, did not increase endothelial permeability, although they acted on endothelial cells as evidenced by an enhanced phosphatidylinositol metabolism and an increased P-selectin expression.
Earlier studies (9, 18, 21) have demonstrated a permeability-increasing effect of VEGF (therefore, its other name, vascular permeability factor) in skin tests or after topical application to different preparations. These in vivo approaches cannot clarify underlying mechanisms of enhanced permeability. VEGF may alter local hemodynamics by arteriolar dilatation and/or venoconstriction, with subsequently increased capillary filtration pressures. Recent evidence (13), indeed, indicated that VEGF caused relaxation of arteries via nitric oxide liberation. In addition, the here-described VEGF-mediated expression of P-selectin could impair endothelial barrier function indirectly by initiating neutrophil adhesion to the endothelium. Finally, VEGF also acts on monocytes, which, in turn, could affect vascular permeability (4).
The data obtained with human and porcine endothelial cell monolayers in our well-characterized in vitro system now clearly established the capability of VEGF to increase endothelial monolayer permeability by a direct action on endothelial cells. It is important to point out that our in vitro assay determines the permeability of sealed endothelial cell monolayers under convective conditions (22). A study (16) in isolated, ventilated, and perfused rabbit lungs indicated an RC of 0.7 for albumin in the pulmonary microvasculature, a value similar to our in vitro data (Fig. 2). Hydraulic conductivity in our permeability system also compares very well with corresponding values in the microcirculation in situ.
The mechanisms of the VEGF effect at the cellular level are unclear. One interesting feature was the pronounced delay of VEGF-related hyperpermeability. Compared with the fast thrombin effect at 15 min, VEGF-induced hyperpermeability occurred after 150 min as the earliest time point. This discrepancy was noted, although similarly rapid cell activation could be demonstrated after both stimuli as indicated by the prompt phosphoinositol accumulation and P-selectin expression.
A pronounced delay in VEGF-related hyperpermeability was also observed in cultured brain microvessel endothelial cells (29). In these cell preparations, VEGF increased permeability after 5-50 h of VEGF incubation (29). Bates and Curry (2) studied frog mesenteric microvessels using the Landis technique and on VEGF application noted a rapid increase in permeability within 30 s and a second substantial permeability peak after 24-48 h. Taken together, VEGF appears to be a unique stimulus with respect to the induction of endothelial permeability by growth factors.
Only little is known about VEGF-related signal transduction. VEGF acts
by binding with high affinity to the receptors VEGFR-1 (flt-1)
and VEGFR-2 (KDR/flk-1) (7, 14). Receptor occupation results in
dimer formation, activation of the tyrosine kinase domains, and
autophosphorylation of the receptors (6). Multiple proteins such as
p125FAK and paxillin are tyrosine phosphorylated (1). Phospholipase
C- is also tyrosine phosphorylated and activated, leading to
inositol phosphate formation, an intracellular Ca2+ rise, and protein kinase C
activation (3, 30). VEGF also increased phosphatidylinositol 3-kinase
activity (30). Moreover, activation of a 44- and 42-kDa
mitogen-activated protein kinase was recently demonstrated in
VEGF-stimulated bovine brain capillary endothelial cells (6). Finally,
p38 mitogen-activated protein kinase activation and subsequent actin
reorganization was noted in human endothelial cells after VEGF
stimulation (19).
The signals generated after VEGF addition will ultimately result in enhanced permeability, although the individual steps involved remain to be established. In this context, the inhibition of VEGF-induced hyperpermeability by increasing intracellular cyclic nucleotide levels is of interest. Previous studies (23,25) have shown that endothelial hyperpermeability provoked by thrombin, hydrogen peroxide, or bacterial toxins was blocked by high endothelial nucleotide levels. Activation of adenylyl or guanylyl cyclase with cholera toxin or nitric oxide, respectively, turned out to be a very effective measure to antagonize enhanced endothelial monolayer permeability (23, 25). Protein kinases A and G are important participants in the continuous cross talk between different second messenger systems, and it is therefore conceivable that adenylyl or guanylyl cyclase activation can interfere with VEGF-related signal transduction pathways. Consistent with this notion, a protein kinase A-related inhibition of the mitogenic action of VEGF in capillary endothelial cells as well as a reciprocal relationship between VEGF and nitric oxide in the regulation of endothelial integrity was reported very recently (5, 27).
The mechanisms that finally induce enhanced fluid flux across the endothelium remain unclear. In previous studies (11, 24), disruption of the endothelial cell microfilament system, provoked by two highly selective tools, Clostridium botulinum C2 toxin and C. difficile B toxin, resulted in endothelial cell retraction, thereby opening intercellular gaps that facilitated increased paracellular permeability. These alterations were unchanged by increased endothelial nucleotide levels (11), and these considerations, therefore, do not apply for the VEGF study presented.
In other studies (15, 22, 23, 25) using bacterial exotoxins, thrombin, hydrogen peroxide, or a calcium ionophore, we and others obtained evidence for endothelial cell retraction. In subsequent experiments performed in the presence of high endothelial nucleotide levels, enhanced permeability was reversed, suggesting that endothelial cell relaxation can counteract hyperpermeability (23, 25). Thus there is a parallelism to the VEGF study presented, but morphological information obtained by light microscopy did not provide evidence for VEGF-induced endothelial cell retraction with the occurrence of large gaps between endothelial cells.
A third possibility relates to the formation of small transient pores that are not readily resolved by light microscopy as described by Schaeffer et al. (20) for bradykinin as the stimulus. Moreover, VEGF-related induction of rapid fenestration of the endothelium was shown by Roberts and Palade (18). Topical application or intradermal injection of VEGF increased microvascular permeability and induced endothelial fenestration of postcapillary venules and capillaries in the cremaster muscle and skin (18).
Finally, the recent description of VVO in skin endothelial cells after VEGF injection appears to identify a fundamental mechanism that may be instrumental in understanding the increased permeability (9). According to this study, VVO, which can occupy up to 18% of the endothelial cytoplasma, represent an efficient shuttle system across the endothelium. Initial studies applying electron microscopy revealed the presence of VVO as well as fenestrae in VEGF-treated HUVECs on filter membranes after 150 min of exposure to 100 ng/ml of VEGF, suggesting that these structures provide the basis for VEGF-induced enhanced endothelial monolayer permeability (S. Hippenstiel, H. Wolburg, and N. Suttorp, unpublished observations).
The interpretation of our study is limited because cultured HUVECs and porcine pulmonary artery endothelial cells were used. For an exact analysis of the alterations of endothelial barrier function in clinical disorders, it would be desirable to study human endothelial cells of different vascular levels (arteriole, capillary, and venule) and of different organs. The culture of human pulmonary microvascular endothelium is difficult, and, therefore, the applicability of the data presented to human pulmonary disease is not clear.
In this context, the pronounced temporal discrepancy between in vivo (within 30 min) and in vitro effects (after 150 min) of VEGF on endothelial permeability may be related to differences between endothelial cells of micro- and macrovessels. On the other hand, in vivo systems are complex, and VEGF-induced hyperpermeability may be a combined effect of VEGF action on the endothelium, blood cells, and humoral factors (2, 4, 9, 13, 18, 21).
In conclusion, VEGF, a key regulator of vasculo- and angiogenesis, induced endothelial hyperpermeability by a direct action on endothelial cells. Compared with other growth factors, VEGF appears to be a unique stimulus.
Hypoxia is the strongest stimulus for VEGF synthesis and VEGF-receptor expression as demonstrated in rat lungs exposed to acute and chronic hypoxia (28). Subsequently, VEGF may increase endothelial permeability that enhances substrate supply, which, in turn, provides the basis for vascular remodeling, a process that represents a common feature of many chronic lung diseases.
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ACKNOWLEDGEMENTS |
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The technical assistance of S. Tannert-Otto and H. Geisel is greatly appreciated. We are greateful to Dr. H. Weich for providing recombinant vascular endothelial growth factor (VEGF) and supernatants from VEGF-producing insect cells. We thank the staff of the Delivery Services of the Krankenhaus Lich and Dept. of Gynecology, University of Giessen (Germany) for invaluable help in collecting umbilical cords.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB 547/B2 to N. Suttorp and SFB547/C5 to W. Risau and M. Clauss) and the German Federal Research Ministry (Bundesministerium für Bildung und Forschung) (to N. Suttorp) and JUGEN (to W. Risau).
N. Suttorp is a recipient of a Hermann and Lilly Schilling Professorship. Parts of this work will be included in the MD thesis of A. Ikemann.
Address for reprint requests: N. Suttorp, Dept. of Internal Medicine, Justus-Liebig-Univ., Klinikstrasse 36, 35392 Giessen, Germany.
Received 2 December 1996; accepted in final form 23 January 1998.
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