VEGF-induced mobilization of caveolae and increase in
permeability of endothelial cells
Jun
Chen1,*,
Filip
Braet2,*,
Sergey
Brodsky1,
Talia
Weinstein3,
Victor
Romanov1,
Eisei
Noiri4, and
Michael S.
Goligorsky1
1 Departments of Medicine and Physiology, University
Microscopy Center, State University of New York, Stony Brook, New
York 11794-8152; 2 Department of Cell Biology and Histology,
Free University of Brussels, 1090 Brussels-Jette, Belgium;
3 Department of Medicine, Tel Aviv University, Tel Aviv
49372, Israel; and 4 Department of Medicine, The University of
Tokyo, Tokyo 113, Japan
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ABSTRACT |
Glomerular epithelial cells (GEC) are a
known site of vascular endothelial growth factor (VEGF) production. We
established immortalized rat GEC, which retained the ability to produce
VEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells cultured
on GEC-conditioned matrix, an indicator of the permeability of
monolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased by
VEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showed
that VEGF results in a rapid appearance of transcellular elongated
structures decorated with caveolin. Transmission electron microscopy of
endothelial cells showed that caveolae undergo rapid internalization
and fusion 30 min after application of VEGF-165. Later (36 h),
endothelial cells pretreated with VEGF developed fenestrae and showed a
decrease in electrical resistance. Immunoelectron microscopy of
glomeruli confirmed VEGF localization to podocytes and in the basement
membrane. In summary, immortalized GEC retain the ability to synthesize
VEGF. Matrix-deposited and soluble VEGF leads to the enhancement of
caveolae expression, their fission and fusion, formation of elongated
caveolin-decorated structures, and eventual formation of fenestrae,
both responsible for the increase in endothelial permeability.
vascular endothelial growth factor; podocyte; caveolin; fenestrae; endothelial permeability; green fluorescent protein
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INTRODUCTION |
TWO INTRACELLULAR
STRUCTURES are believed to regulate permeability of endothelial
cells (fenestrae and caveolae). Fenestration of endothelial cells is
known to take place mainly in endocrine glands, the choroid plexus, the
gastrointestinal tract, and the kidney (reviewed in Refs.
16 and 27). Roberts and Palade (20, 21) have
provided convincing evidence that vascular endothelial growth factor
(VEGF) is responsible for the fenestration of vascular endothelial
cells in several tumors that overproduce this growth factor and in
normal vascular beds pretreated with VEGF. Feng et al. (8)
and Vasile et al. (26) have demonstrated that VEGF
increases vascular permeability by increasing the density of clustered
caveolae, termed vesiculovacuolar organelles, in endothelial cells
(8, 26). Glomerular epithelial cells (GEC) have recently
been identified as the site of constitutive production of VEGF
(4, 11). It has been suggested, therefore, that VEGF produced by GEC may be responsible for the maintenance of the fenestrated phenotype of glomerular endothelial cells (4,
24), thus facilitating the high rate of glomerular
ultrafiltration. This view, however, requires reinforcement because of
the fact that hydrodynamics of fluxes in the glomerular capillary wall are unfavorable for such an upstream paracrine action.
The significance and potential implications of the above hypothesis
warrant extensive investigations of VEGF production by GEC and its
action on the endothelium. Unfortunately, cell culture models are
scarce, and in vivo studies present difficulties in interpreting the
results because of the circulating VEGF and other angiogenic/vasoactive
substances. Several investigators have previously reported a successful
isolation and culture of primary GEC (reviewed in Ref.
13); however, the procedure is tedious, cells rapidly dedifferentiate, and the properties of these primary cultures can
fluctuate. Attempts to immortalize these cells have been reported (1). In the present study, we established and
characterized a Simian virus (SV)-40-transformed GEC line and provide
evidence of VEGF synthesis by GEC. Furthermore, a coculture model
developed in this study yielded data on the effect of VEGF produced by
GEC on the permeability of endothelial cells in vitro. In addition, the
data obtained with green fluorescent protein (GFP)-caveolin and
supplemented with electron microscopic analysis of renal microvascular and human umbilical vein endothelial cells (RMVEC and HUVEC,
respectively) demonstrated that caveolin-decorated structures traverse
endothelial cells and elongate after application of VEGF.
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MATERIALS AND METHODS |
Cell cultures.
Primary rat GEC cultures were obtained and maintained according to the
previously published procedure (15). Primary GEC were
plated on collagen IV-coated 3-cm dishes and maintained in K-1 medium
(Nipro, Osaka, Japan) supplemented with 2% NuSerum I (Collaborative
Biomedical Products, Bedford, MA), insulin-transferrin-selenium (Collaborative Biomedical Products), 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO-BRL, Gaithersburg, MD). After colonies were formed, the medium was aspirated and high-titer (108 virus/ml) wild-type SV-40 was added for 60 min as
previously reported (25). GEC were isolated by limiting
cloning. To validate the authenticity of the immortalized GEC clone,
the expression of several markers of these cells and the lack of
markers characteristic of endothelial and mesangial cells were
examined. The selected clone of SV-40-transformed GEC expressed large-T
antigen (data not shown; Noiri, unpublished observation), confirming
the adequacy of transfection. Several markers of GEC were expressed by
these cells. The Wilm's tumor protein has been detected in situ in
podocytes, and immunocytochemical staining also showed the presence of
this marker in GEC (data not shown). Moreover, nephrin mRNA was
detected in GEC. A monoclonal antibody (GSA3) recognizing a
cell-specific surface antigen on podocytes (15) showed
positive immunostaining of GEC (data not shown). Furthermore, puromycin
exerted a cytotoxic effect in GEC, and cells lacked markers
characteristic of endothelial and mesangial cells (von Willebrand
factor and Thy-1 antigen; data not shown; Noiri, unpublished
observation). Collectively, these findings identify the clone of
SV-40-transformed GEC as podocytes and rule out any possible
contamination of the cells with other resident glomerular cells
(endothelial and mesangial cells).
Renal microvascular endothelial cells were previously established and
characterized by our laboratory; these SV-40-immortalized cells
established from explant cultures of microdissected rat renal
resistance arteries express receptors for acetylated low-density lipoprotein and immunodetectable von Willebrand antigen and are capable
of capillary tube formation (25). Cells were grown in gelatin-coated dishes in medium-199 (Mediatech, Washington, DC) supplemented with 5% FBS (HyClone Labs, Logan, UT), 100 U/ml
penicillin, and 100 µg/ml streptomycin (GIBCO-BRL).
GEC-endothelial cell coculture.
To examine the effects of GEC on the phenotype of endothelial cells,
GEC were grown on collagen IV-coated glass coverslips. At
subconfluence, cells were overlayed with Vitrogen 100 or matrigel, which formed a thin gel layer on the surface of GEC within 60 min of
incubation at 37°C. Endothelial cells were seeded atop to form a
"sandwich" and were allowed to coincubate for 4, 8, and 24 h.
Cells were fixed, critical point-dried, and studied using scanning
electron microscopy (EM). Alternatively, GEC cultured for 3 days on
glass coverslips were thoroughly removed using repeated cycles of
freezing-thawing, dishes were washed exhaustively with PBS until GEC
remnants were undetectable by light microscopy, and endothelial cells
were plated on GEC-conditioned extracellular matrix. In control
experiments, the same endothelial cells served as a feeder layer to
produce and condition the extracellular matrix.
Electrical resistance as an index of cell permeability to
solutes.
To examine the permeability of endothelial cells to solutes,
endothelial cells were grown to confluency on the GEC-conditioned extracellular matrix or in the sandwich configuration (see above) on
the microelectrodes of the epithelial cell impedence system. Each well
contained a gold microelectrode and a reference electrode, both
electroplated on the bottom of the well. Electrode units were placed in
an incubator and connected to a lock-in amplifier interfaced to a
computer registering electrical resistance and capacitance every
second. The amplifier measured the in- and out-of-phase (real and
imaginary) voltages across the small electrode, and these were
converted by the computer into a resistance and capacitance in series,
taking the external circuit into consideration. When cells are plated
on this surface, the electrical resistance initially reflects the
degree of cell adhesion and spreading and, upon reaching a confluent
monolayer, reports the permeability of cells to solutes. Electrical
impedance was monitored in real time for 10 h after addition of
1.0, 10.0, or 20.0 ng/ml human recombinant VEGF-165 (PeproTech, Rocky
Hill, NJ) or rabbit polyclonal neutralizing antibodies to VEGF
(PeproTech), as specified in RESULTS. To monitor the
long-term permeability change, HUVEC were seeded at a higher density on
a thin layer of matrigel-coated dishes, and electrical impedance
measurements were performed for up to 40 h. To ensure that
monolayers were unperturbed, only those wells showing high resistance
(>16 k
) and displaying no "gaps" under light microscopy were
selected for analyses.
RT-PCR and identification of VEGF isoforms.
Oligonucleotide primers flanking the insertion/deletion site of
VEGF-188 were designed to amplify VEGF mRNA from GEC-T cells to
identify the unique VEGF isoforms. The sequence of sense primer was
5'-GGACATCTTCCAGGAGTACC-3', and the antisense primer was 5'- GTTCCCGAAACCCTGAGG-3'. Total RNA was isolated from GEC-T cells with
Trizol total RNA isolation reagent (GIBCO-BRL), and the mRNA was then
reverse transcribed to cDNA with avian myeloblastosis virus reverse
transcriptase and amplified with expand high-fidelity enzyme mix that
was provided in the Titan One Tube RT-PCR System (Boehringer Mannheim,
Indianapolis, IN). About 10-100 ng of total RNA were used in a
50-µl reaction containing 1× RT-PCR reaction buffer, 0.2 mM dNTPs, 5 mM dithiothreitol, and 0.4 µM of each primer. The RT-PCR profile
consisted of a 30-min incubation at 50°C, 2 min denaturation at
94°C, followed by 35 cycles of 30 s of denaturation at 94°C,
30 s of annealing at 55°C, and 2 min elongation at 68°C, and
finally a 6-min extension at 68°C. Products were analyzed by running
10% of the reaction mixture on a 2% agarose gel. The bands that have
proper expected size were excised from the gel, recovered with a
QIAquick gel extraction kit (Qiagen, Valencia, CA), and then sequenced
with an ABI Prism BigDye Terminator Cycle Sequencing Kit (PE Applied
Biosystems, Foster, CA) directly or after cloning into PCR 2.1 plasmid
vector (Invitrogen, Carlsbad, CA). In the case of VEGF isoform 205, 20 cycles of secondary PCR reaction were carried out to enrich the cDNA
fragment that has the predicted size. After being cloned into PCR 2.1 vector, the insert cDNA fragments were then sequenced as described above.
Immunoprecipitation and Western blot analysis.
After being washed with ice-cold PBS, cells were lysed in 200 µl of
SDS gel-loading buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and
0.001% bromphenol blue) containing 2.5% 2-mercaptoethanol. After
being boiled for 10 min, samples were sonicated on ice and centrifuged
for 10 min at 10,000 rpm. Supernatants were collected, and 20-µl
samples were electrophoresed on 4-20% SDS polyacrylamide gel. For
detecting VEGF secreted in the culture medium, samples were subjected
to immunoprecipitation. Briefly, 1.4 ml conditioned culture medium were
kept overnight at 4°C on a rocker with the addition of 1 µg/ml
rabbit anti-human VEGF polyclonal antibody (Santa Cruz). Next, 15 µl
GammaBind plus Sepharose beads (Pharmacia, Uppsala, Sweden) were added
for another 2 h. The Sepharose beads were then collected by
centrifugation, washed two times in 0.01 M Tris (pH 8.0), 0.14 M NaCl,
and 0.025% NaN3 (TSA) containing 0.1% Triton X-100, one
time in TSA buffer alone, and one additional time in 0.05 M Tris, pH
6.8. After being boiled for 5 min in 1× SDS gel-loading buffer,
supernatant was transferred to two tubes with or without 2.5%
2-mercaptoethanol, boiled for an additional 5 min, and electrophoresed
on a 4-20% SDS polyacrylamide gel. Separated proteins were
blotted on polyvinylidene difluoride membranes (Millipore), blocked in
PBS containing 1% casein for 60 min, and incubated overnight at 4°C
in 1:100-200 diluted primary antibodies (rabbit anti-human VEGF
polyclonal and mouse anti-human VEGF monoclonal antibody for cell
lysate samples and mouse anti-human VEGF monoclonal antibody for
immunoprecipitation samples; Santa Cruz). After intense washing, the
membranes were incubated with 1:2,000 diluted secondary horseradish
peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse IgG
(Amersham Life Sciences, Arlington Heights, IL) for 30 min at room
temperature. Thereafter, the membranes were washed one time again and
incubated in enhanced chemiluminescence substrate reagent (Amersham)
for 1 min. The blots were exposed to X-ray film for 5-30 s, and
the molecular weight of the immunodetected bands was compared with
molecular weight standards (Novex).
Caveolin-1-GFP expression vector.
In preliminary studies, the following two constructs were
generated: caveolin-1-GFP and GFP-caveolin-1; studies presented herein
utilized the first construct, as previously reported (12). The full open-reading frame of the human caveolin-1 (nucleotides 35-571) was cloned from the HUVEC
11phage cDNA library by PCR using appropriate primers containing Xho I and
BamH I restriction sites at 5' and 3' with the stop codon
mutated. cDNA was digested with Xho I and BamH I
and ligated in sense orientation at the appropriate cloning site of the
pEGFP-N1 plasmid (Clonetech) using a rapid DNA Ligation Kit (Boehringer
Mannheim). Ligated plasmids were used to transform One Shot
INValphaF'cells (Invitrogen). Transformed cells were selected for
kanamycin resistance, propagated, and isolated with Maxi-Prep
(Quiagen). The construct was sequenced using a Dye Terminator kit and a
377 DNA automated sequencer (Applied Biosystems), and the authenticity
of the product was confirmed.
HUVEC were incubated in endothelial basal medium (EBM)-2 basal medium
(Clonetics) for 5 h. The caveolin-GFP fusion constructs (2 µg)
were used in conjunction with the FuGENE 6 transfection reagent
(Boehringer Mannheim), according to the manufacturer's instructions.
The transfection was carried out in EBM-2 media. The cells were used in
the experiments 24-48 h after transfection. In a series of
preliminary experiments, transfection of HUVEC with the caveolin
cassette with GFP fluorescent tag resulted in an appropriately
localized and functionally competent protein (12);
therefore, this construct was used in all reported experiments.
Transmission electron microscopy and confocal fluorescence
microscopy.
The cultured cells were rinsed two times with PBS and fixed with 2%
glutaraldehyde in sodium cacodylate buffer (0.1 M cacodylate and 0.1 M
sucrose) at pH 7.4 for 12 h. Cells were subsequently postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate at pH
7.4 for 1 h. Samples were further dehydrated in graded alcohol solutions and embedded in epon. After hardening of the embedding medium, the culture dishes were broken using liquid nitrogen. Transverse sections of 60 nm were cut with a diamond knife, stained first with uranyl acetate and subsequently with lead citrate, and
examined under a Philips Tecnai 10 transmission electron microscope at
80 kV. Morphometric analysis was performed on randomly acquired digitized images (MegaView II camera connected to the microscope operated with the analysis 3.0 software) at magnifications of ×2,900
or ×5,000, calibrated with a Polaron cross-grating replica (Polaron
54,800 lines/in. grating). Subsequently, the UTHSCSA Image Tool 2.0 software was used to trace the number and diameter of uncoated
vesicular organelles. Caveolae and uncoated vesicles were discriminated
from coated vesicles and vacuoles based on their morphology and size,
as described previously (9). For each experiment, five
cells were randomly selected, and images were obtained at both
magnifications. All experiments were repeated three times, and data
were expressed as means ± SE. Statistical analysis was performed
with the Mann-Whitney two-tailed U-test.
In a separate series of experiments, immunoelectron microscopy of rat
kidney sections was performed to visualize the distribution of VEGF.
Slices of each kidney were fixed in 0.5% glutaraldehyde in PBS, pH
7.4. For immunohistochemistry using EM, 1-mm3 tissue blocks
of glutaraldehyde-fixed kidneys were washed with PBS, dehydrated in
ethanol, and embedded in London Resin (LR)-white resin (Polysciences,
Washington, PA). For EM morphology, similar tissue blocks were
postfixed with 1% OsO4 in veronal-acetate buffer, pH 7.4, for 1 h at 4°C, dehydrated in ethanol and propylene oxide, and
embedded in araldite (Polysciences). For EM morphology, ultrathin araldite sections were mounted on naked 400-mesh grids, stained with
uranyl acetate and lead citrate, and coated with carbon. For EM
immunohistochemistry, ultrathin LR-white sections of ~60 nm were
mounted on 200-mesh nickel grids, coated with Formvar film, and
impregnated with carbon. The sections were treated with 1% BSA-0.05%
Tween 20-BSA (blocking buffer) for 15 min, labeled with polyclonal
anti-VEGF (Santa Cruz) diluted 1:50 in blocking buffer for 2 h,
rinsed five times in PBS, and incubated for 1 h with goat
anti-rabbit IgG conjugated to 15 nm gold (Biocell) diluted 1:50 in
blocking buffer. This was followed by five rinses in PBS, a rinse with
a stream of distilled water, and staining for 5 min with saturated
uranyl acetate in 50% ethanol. Examination of all sections was carried
out using a JEOL-100B electron microscope at 80 kV.
Fluorescence confocal microscopy was performed on fixed cells or
intravitally with the distance between focal planes 0.2-0.5 µm,
as specified in RESULTS, using a real time laser system
(Odyssey; Noran Instruments, Middleton, WI). Images were analyzed with
MetaMorph software (Universal Imaging) using a Silicon Graphic system.
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RESULTS |
VEGF expression and distribution.
The staining of GEC with polyclonal antibodies to VEGF (Santa Cruz)
revealed that the cells expressed immunodetectable VEGF (Fig.
1, A-C). These data indicated
that immortalized GEC preserved the ability to produce VEGF. Further
confirmation was obtained in studies of splice variants of VEGF, as
described below.

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Fig. 1.
Immunocytochemical detection of
vascular endothelial growth factor (VEGF) distribution in glomerular
epithelial cells (GEC). A-C: representative field of GEC
stained with anti-VEGF. A: rhodamine phalloidin staining;
B: anti-VEGF antibody; C: merged
images.
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The VEGF gene contains eight exons, and several splicing variants
exist. VEGF-188 has all eight exons, VEGF-205 has an insert, VEGF-164
lacks exon 6, and VEGF-120 lacks exons 6 and
7. Using primers that flank the common insertion and
deletion site, we expected to amplify all VEGF isoforms with PCR and
then distinguish them from one another by the predicted size of the
amplified cDNA, as well as analysis of nucleic acid sequences. After 35 PCR cycles, four distinct cDNA bands were seen on agarose gels, which
had the expected size for different rat VEGF isoforms as follows: 306 bp for VEGF-120, 438 bp for VEGF-164, 510 bp for VEGF-188 (these three
are readily detectable after 20 PCR cycles), and 564 bp for VEGF-205
(Fig. 2, A and B).
Sequence analysis confirmed the identity of the transcripts as
representing VEGF-120, VEGF-164, VEGF-188, and VEGF-205. (An additional
band above VEGF-205 is the result of a nonspecific amplification, and
its sequence showed no homology with VEGF.) Hence, the established
immortalized GEC cell line maintains the ability of producing four
different VEGF isoforms, and among them VEGF-120 and -164 are the two
abundant isoforms, and VEGF-188 and, especially, -205 are less
abundant.

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Fig. 2.
Agarose gel electrophoresis of RT-PCR products of VEGF
mRNA in GEC cells. A: primers were designed based on the
common sequences among different VEGF isoforms that flank the common
insertion (between exons 6 and 7 for VEGF-205 ),
full-length (VEGF-188), or deletion (exon 6 for VEGF-164,
exons 6 and 7 for VEGF-120) sites in the VEGF
mRNAs. B: sizes expected for VEGF-120, VEGF-164, VEGF-188,
and VEGF-205 are 306, 438, 510, and 564 bp, respectively. After 35 cycles of amplification, four bands with the expected size were
detected in GEC mRNA. Based on the electrophoretic mobility and
additional sequencing results of each band (data not shown), it was
confirmed that they represent VEGF-120, VEGF-164, VEGF-188, and
VEGF-205, respectively. It appears that GEC in culture retain the
ability to produce four different VEGF isoforms. Among them, VEGF-120
and -164 are the two abundant isoforms, and VEGF-188 and -205 are less
expressed. C: Western blot analysis of GEC lysates
immunoblotted with poly- and monoclonal antibodies (note the expression
of three isoforms, except for VEGF-205). D: VEGF in
GEC-conditioned culture medium, under reducing and nonreducing
conditions, detected using immunoprecipitation (IP) with the polyclonal
antibody, followed by blotting (IB) with a monoclonal antibody. Ab,
antibody.
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Western blot analysis of cell lysates and conditioned medium under
reducing conditions (Fig. 2, C and D) revealed
three bands at 17, 22, and 27 kDa, which correspond to the predicted
molecular size for 120, 164, and 188 amino acid VEGF isoforms (using
polyclonal and monoclonal antibodies). Under nonreducing conditions, we
identified a band near 45 kDa present in the conditioned medium, which
corresponds to the VEGF-164 homodimer. The lower molecular weight band
seen under nonreducing conditions may represent a nondimerized
VEGF-164. VEGF-205 was undetectable with this technique.
Effects of VEGF, GEC, and GEC-conditioned extracellular matrix on
the permeability of endothelial cells.
Transmission electron microscopy (TEM) of HUVEC and RMVEC treated with
VEGF for various periods of time revealed an increase in the number of
caveolae and an increase in the diameter of caveolae (Fig.
3 and Table
1). The increased number of
uncoated vesicular organelles was detectable within
10-30 min, and these organelles exhibited multiple contacts, fused
and formed vesiculovacuolar-like structures 10-30 min after VEGF
application (Fig. 3, C-E). After 60 min, the burst of
caveolae formation has subsided, and internalized uncoated vesicular
structures were observed within the cytoplasm (Fig. 3F).
Moreover, morphometry revealed a twofold increase in the number of
uncoated vesicular organelles 60 min after application of VEGF, and the
size of these uncoated vesicular structures, as measured by average
diameters, was also enlarged (Fig. 3F and Table 1).
Representative images in Fig. 3 were taken from HUVEC; similar results
were also obtained with RMVEC (data not shown).

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Fig. 3.
Transmission electron micrographs (TEM) of control
(A-B) and VEGF-treated (C-F) human umbilical
vein endothelial cells (HUVEC). A: low magnification showing
the cell nucleus (N) and surrounding cytoplasm. Bar, 1 µm.
B: high magnification showing caveolae at the basal plasma
membrane (large arrowhead) and cytoplasm (small arrowhead). Bar, 200 nm. C: as early as 10 min after VEGF treatment, an increase
in the number and accumulation of caveolar structures at the basal
cytoplasm is observed (arrowheads). Bar, 200 nm. D: after 30 min of VEGF treatment, a higher number of caveolae at the basal plasma
membrane (large arrowhead) and accumulation of fused caveolae within
the cytoplasm (small arrowheads) are observed. Bar, 200 nm.
E: moreover, around the perinuclear area, a group of
high-density caveolae could be noticed, and some of these organelles
make contacts, fuse, and form tubulovesicular-like structures
(arrowheads). Bar, 200 nm. F: after 60 min of VEGF
treatment, the burst of caveolae formation has subsided, and
internalized uncoated vesicular structures are observed (arrowheads).
Bar, 200 nm.
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Next, we argued that, if VEGF induces the caveolae-enriched phenotype
of endothelial cells, the electrical resistance of cell monolayers
should serve as a convenient reporter of any changes in cell
permeability, whereas changes in the electrical capacitance should
reflect the state of the lipid membrane convolution. To accomplish
this, cells were cultured in specially designed five-well plates with a
miniature gold electrode and a large reference electrode electrosprayed
on the bottom of the wells (14). As shown in Fig.
4A, HUVEC cultured on
vitrogen-coated electrodes responded to blocking anti-VEGF antibodies
with a rise in electrical resistance; in contrast, addition of VEGF
decreased the electrical resistance, and this phenomenon did not occur
when cells were pretreated with an inhibitor of endothelial nitric
oxide synthase (nitro-L-arginine methyl ester), consistent
with the previous data on nitric oxide production in response to VEGF
(14). In coculture experiments, GEC, 48 h after
plating, were removed by repeated cycles of freezing-thawing, and rat
renal microvascular endothelial cells were plated on the GEC-conditioned extracellular matrix. In control experiments, endothelial cells were plated directly on the endothelial
cell-conditioned matrix (Fig. 4B). Application of 1-10
ng/ml VEGF-165 to the endothelial cell monolayers, kept in a VEGF-free
medium for 12 h, resulted in the decline of the electrical
resistance. Impedance analysis of endothelial cells grown on
GEC-conditioned extracellular matrix (similar results were obtained in
coculture) showed that, when neutralizing antibodies against VEGF were
added and their effect was compared with that of VEGF-165, a sharp
dissociation of curves occurred at 45 min and reached the plateau
2-4 h after application of these agents; the resistance of
monolayers treated with the neutralizing antibody showed a gradual
increase by 20 ± 4%, whereas that of VEGF-treated cultures
showed a decrease in electrical resistance by 17 ± 5%
(n = 3 each in triplicate; P < 0.05;
Fig. 4B). Changes in the capacitance of these cells followed
a mirror pattern; it decreased after treatment with the neutralizing
antibodies and increased after administration of VEGF-165 (Fig.
4C). The observed VEGF-induced increase in the capacitance
of endothelial monolayers is well correlated with the data presented in
the serial TEM performed at different times after application of
VEGF-165 (Fig. 3, C-F, and Table 1). All of these events
associated with the amplification of internalized membranes explain the
observed increase in capacitance of endothelial monolayers.

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Fig. 4.
Electrical resistance and capacitance of endothelial
cells. A: typical changes in the electrical resistance of
HUVEC monolayers cultured on vitrogen-coated microelectrodes after
application of blocking anti-VEGF antibody, VEGF-165, or
nitro-L-arginine methyl ester (L-NAME). Arrow
depicts the time of additions. A/b, antibody. B: normalized
impedance of renal microvascular vein endothelial cells (RMVEC),
cultured on GEC-conditioned matrix, increased after application of
neutralizing VEGF antibodies and decreased after addition of 10 ng/ml
VEGF-165. Untreated cells showed no changes in electrical resistance.
Arrow depicts the time of VEGF additions. The same results were
obtained in 3 separate experiments. C: changes in electrical
capacitance of RMVEC treated with 10 ng/ml VEGF-165 (recorded
simultaneously with electrical resistance, shown in B). Note
the VEGF-induced increase in capacitance, consistent with the observed
amplification of the cellular area of the lipid bilayers.
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Intravital imaging of GFP-caveolin-1 and the distribution of
caveolae.
To visualize the dynamics of caveolae, we constructed a chimeric
caveolin-1-green fluorescent protein vector and transiently transfected
endothelial cells with this construct. Using this approach, treatment
of HUVEC and RMVEC with VEGF-165 (10 nM) did not affect the intensity
of fluorescence and its planar distribution, but the three-dimensional
distribution of GFP-caveolin underwent a striking reorganization.
Z-reconstruction of confocal images showed that VEGF resulted in a
reversible formation and elongation of cell-spanning structures
oriented between the apical and basal cell surfaces, resembling
previously described vesiculovacuolar organelles (Fig.
5, A-C). Cell-spanning GFP-
and caveolin-1-decorated structures appeared hollow, and their average
length increased twofold as early as 10 min after the application of
VEGF-165. Incubation of transfected HUVEC with Texas red-conjugated
horseradish peroxidase (HRP) showed no significant incorporation of the
tagged probe into HUVEC (data not shown). After application of 10 ng/ml VEGF (30 min), this fluorescent probe was found entangled in the network of GFP-caveolin, as demonstrated by intravital confocal fluorescence microscopy of dual-labeled cells (Fig.
6, A and B). These
data indicate that HRP is readily incorporated into the vesiculovacuolar structures decorated with GFP-caveolin after stimulation of endothelial cells with VEGF, strongly suggesting that
these convoluted channel-like structures are permeable to the
macromolecules.

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Fig. 5.
Three-dimensional reconstruction of intravital confocal
fluorescence microscopy images of GFP- and caveolin-transfected HUVEC
treated with VEGF. A: center, plane view of a
transfected cell. Dynamics of three-dimensional images before and 10, 30, and 60 min after the addition of 10 ng/ml VEGF-165 is depicted in
boxes. Bar = 10 µm. B: average length of
vesiculovacuolar-like organelles decorated by GFP-caveolin.
*P < 0.05 vs. control. C: reconstruction of
a single vesiculovacuolar organelle demonstrating a hollow,
elongated transcellularly oriented GFP- and caveolin-labeled
structure. Top, planar view. Bottom, transverse
view.
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Fig. 6.
Serial confocal images of a HUVEC expressing GFP-caveolin-1 in the
presence of Texas red-horseradish peroxidase (HRP). Application
of VEGF to HUVEC transfected with GFP-caveolin-1 results in the gradual
incorporation of Texas red-HRP into tubulovesicular organelles.
Confocal microscopy was performed as described in MATERIALS AND
METHODS. Images were obtained from the same visual field 30 min
after VEGF stimulation with 0.2-µm distances between each consecutive
frame. No incorporation was detectable in the absence of VEGF (data not
shown). Green, GFP-caveolin-1; red, HRP. A and B,
planar and transverse optical sections, respectively. White line shows
the site of transverse optical sectioning.
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When microscopic analysis of endothelial cells grown on matrigel was
performed 36 h after VEGF-165 treatment or when endothelial cells
were cocultured with GEC, two distinct patterns were observed, i.e.,
VEGF-treated HUVEC showed an elaborate capillary-like network at the
light microscopic level (Fig.
7A), and TEM examination in
these areas of attenuated cytoplasm revealed diaphragmed fenestrae (Fig. 7B). These findings were reproducible in RMVEC treated
with VEGF-165 (data not shown). Long-term electrophysiological studies revealed that, 24-36 h after VEGF treatment, the electrical
resistance of HUVEC grown on matrigel-coated microelectrodes decreases
with the concomitant increase in the capacitance of confluent
monolayers (Fig. 7, C and D), both findings
consistent with the observed morphological changes. In the coculture
system, vacuolar structures and diaphragmed fenestrae were detectable
in RMVEC (Fig. 8).

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Fig. 7.
Long-term effect of VEGF resulted in the formation of fenestration
of endothelial cells. A: HUVEC were cultured on matrigel for
36 h in the presence of 10 ng/ml VEGF-165. Light microscopic image
shows an elaborate capillary-like network formed under these
conditions. B: TEM micrograph shows diaphragmed fenestrae
(arrowheads). C and D: changes in electrical
resistance and capacitance, respectively, in HUVEC treated with 10 ng/ml VEGF-165 (control cells were deprived of VEGF), demonstrating
concomitant decrease in resistance and increase in capacitance compared
with control. One-way ANOVA of experimental and control data in
B and C showed that these curves are
significantly different at P < 0.05.
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Fig. 8.
Long-term sandwich coculture of RMVEC with GEC: TEM
characteristics. A: typical TEM composite image of RMVEC
monolayer. B and C: RMVEC were treated with 10 ng/ml VEGF-165. Note that tight junctions are preserved (B),
and rare fenestrae appear (arrowheads in C). D:
RMVEC (EC) were cocultured with GEC (sandwich culture, see
MATERIALS AND METHODS for details) for 36 h in the
absence of exogenous VEGF. Note formation of vacuoles and fenestrae in
endothelial cells.
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Possible relevance to the ultrastructure of glomerular endothelial
cells.
To compare the above observations made in cell culture with the
ultrastructure of normal glomerular endothelial cells, normal rat
kidney sections were examined using EM in conjunction with immunogold
labeling of VEGF. As shown in Fig. 9,
gold-labeled VEGF was detectable in the podocytes and the glomerular
basement membrane, further strengthening the idea of a paracrine action of the GEC-produced and matrix-deposited VEGF on glomerular endothelial cells.

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Fig. 9.
Immunoelectron microscopy of VEGF distribution in the glomerulus of
rat kidney. A typical image of a glomerular tuft showing podocytes (P),
basement membrane (GBM), and endothelial cells. Gold-labeled anti-VEGF
was conspicuous in podocytes and in the basement membrane. Endothelial
cells showed no immunodetectable VEGF, thus arguing that VEGF in the
basement membrane was not blood borne but secreted by podocytes.
Magnification, ×20,000.
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|
 |
DISCUSSION |
Previous immunohistochemical studies have provided solid evidence
of VEGF production by the podocytes (4, 7, 11, 24). The
functional role of this phenomenon, however, remained obscure. Considering the intensity of ultrafiltration taking place in the glomerular capillaries, it was difficult to reconcile it with the
possible action of VEGF, produced by podocytes, on the target endothelial cells located upstream; the direction of flow should have
made such a paracrine activity a futile one. Two sets of observations
made in the cultured cells and in the rat kidney serve to reconcile
this controversy. First, the production of all four splice variants of
VEGF, three of which are heparan sulfate-binding, by cultured GEC
suggests the possibility of VEGF deposition into the basement membrane.
Second, immunoelectron microscopy of VEGF distribution in the rat
glomerulus showed gold labeling in association with the podocytes and
glomerular basement membrane.
Rat VEGF gene contains eight exons, and VEGF-188 incorporates all eight
exons, VEGF-164 lacks exon 6, and VEGF-120 lacks exons 6 and 7. The major known functional difference among
the various VEGF isoforms is their ability to bind to heparin and
heparan sulfate proteoglycans distributed on cellular surfaces and
within extracellular matrixes and basement membranes, and it is
believed that this ability is imparted mainly by exon 6.
Addition of the highly cationic 24-amino-acid residue sequence encoded
by this exon promotes even tighter binding of VEGF-188 to these
endogenous polyanions. The fact that GEC express mRNA for the soluble
secretory form VEGF-120, and for the soluble matrix-associable VEGF-164 and insoluble, heparin-binding matrix-associated VEGF-188 and VEGF-205
suggests that glomerular basement membrane may be the site of VEGF
accumulation and storage. Furthermore, at least the VEGF-188 isoform
requires urokinase for full activation, thus making the regulation of
this potential paracrine mechanism even more complex (17).
If VEGF-188 is indeed deposited in the glomerular basement membrane,
its activation should occur in the vicinity of capillary endothelial
cells producing urokinase, thus providing further spatial selectivity
of VEGF action. The latter observation on the diversity of GEC-produced
VEGF isoforms is not limited to the cell culture system; recent RT-PCR
findings by Kretzler et al. (11) revealed the similar
profile of VEGF splice variants in single aspirated podocytes obtained
from microdissected mouse glomeruli. This imparts further benefits to
the established immortalized GEC as a model to study VEGF production
and its regulation.
To investigate the potential for paracrine VEGF signaling, we have
analyzed the following two coculture systems: a sandwich GEC-collagen-RMVEC system and RMVEC plated on the GEC-deposited and
conditioned extracellular matrix. Endothelial cell permeability was
studied directly using a highly sensitive measurement of electrical resistance. These studies showed that the application of the
neutralizing anti-VEGF antibodies increases the resistance of
endothelial monolayers grown either in the sandwich configuration or on
the surface of GEC-conditioned matrix, whereas the addition of VEGF to
renal microvascular endothelial cells cultured in the absence of this growth factor resulted in the decline of electrical resistance. These
data are consistent with VEGF or GEC-conditioned extracellular matrix
serving to increase the permeability of endothelial cells.
The morphological route(s) for the VEGF-induced increase in
endothelial permeability has been suggested (6, 8, 20, 21). Palade and colleagues (16) consider caveolae
as plausible structures involved in the increase in endothelial
permeability. Indeed, some investigators argued that caveolae, if
studied by serial sectioning, extend far beyond the plasmalemmal
vesicles (5) to form extensive invaginations. However,
differences in techniques for serial sectioning and the choice of
fixation protocols have been incriminated in the variability of
findings (22, 23). In an attempt to resolve some of the
existing problems in reconstructing the three-dimensional organization
of caveolae, we have generated a GFP-caveolin-1 vector to enable
intravital microscopy of endothelial cells subjected to VEGF. Although
fluorescence microscopy of transfected endothelial cells did not reveal
significant changes in the distribution of GFP-caveolin, confocal
microscopy disclosed that the probe is decorating transcellular
channel-like structures that become conspicuous after exposure to VEGF.
These data demonstrate, for the first time in vivo, that caveolin is
organized into elongated cell-spanning structures in cells exposed to
VEGF. EM studies confirmed and further extended these observations by
demonstrating the enrichment in caveolae, their fission, and fusion
after application of VEGF. An alternative route for increased
permeability via fenestrae could not be detected in HUVEC or RMVEC at
early times after application of VEGF. However, 36 h after
addition of VEGF-165, HUVEC and RMVEC exhibited diaphragmed fenestrae.
Furthermore, RMVEC cocultured with GEC (sandwich culture), in the
absence of exogenous VEGF, showed vacuolation and fenestration,
phenomena that have recently been associated with capillary remodeling
and lumen formation (3). In a coculture model of
adrenal capillary endothelial cells and choroid plexus
epithelium, as well as in endothelial cells treated with 50-100
ng/ml VEGF-165, Esser and coworkers (7) were able to
detect fenestrae only 24 h after the treatment. The same authors
consistently observed fission and fusion of caveolae shortly after VEGF
treatment. Vasile and coauthors (26) have recently
provided additional evidence of VEGF-induced clustering of caveolae,
resulting in formation of vesiculovacuolar organelles in bovine
microvascular endothelial cells cultured on floating matrigel-collagen
gels. It is conceivable that VEGF elicits a rapid increase in vascular
permeability via mobilization of caveolae, whereas the long-term effect
requires formation of fenestrae. Recent demonstration of two VEGF
receptors, neuropilin-1 and fetal liver kinase-1, in developing and
mature glomerular capillaries further supports the idea of paracrine
signaling from GEC to endothelial cells (18, 19).
Collectively, the development of GEC-endothelial cell coculture systems
and data obtained using intravital confocal microscopy techniques
support the hypothesis that VEGF deposited in the basement membrane
immediately acts upon endothelial cells by remodeling caveolae,
elongating vesiculovacuolar structures, and increasing endothelial
permeability. The long-term effect of VEGF, however, results in the
formation of fenestrae. The observed time course of VEGF action on
endothelial cells may explain why caveolae are so sparse in glomerular
endothelial cells in vivo. On the other hand, these data suggest that
the unique ultrastructure of these cells is determined by their
microenvironment rather than by the inherent propensity of the
glomerular endothelial cells to form diaphragmed fenestrae. This
particular feature of the endothelium may relate to changes in
glomerular permeability after damage to podocytes (2, 10).
 |
ACKNOWLEDGEMENTS |
We are grateful to D. Colflesh for help with confocal microscopy
and R. De Zanger for statistical analysis. M. Baekeland and D. Blijweert provided excellent technical assistance.
 |
FOOTNOTES |
*
J. Chen and F. Braet contributed equally to this work.
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-45462 and DK-54602 (to M. S. Goligorsky). F. Braet is a postdoctoral fellow of the Fund of
Scientific Research, Flanders.
Address for reprint requests and other correspondence: M. S. Goligorsky, Dept. of Medicine, SUNY, Stony Brook, NY 11794-8152 (E-mail: mgoligorsky{at}mail.som.sunysb.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.
First published December 19, 2001;10.1152/ajpcell.00292.2001
Received 28 May 2001; accepted in final form 8 December 2001.
 |
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