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Originally published online as doi:10.2353/ajpath.2008.070416 on September 4, 2008

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(American Journal of Pathology. 2008;173:938-948.)
© 2008 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2008.070416

Vascular Endothelial Growth Factor-C, a Potential Paracrine Regulator of Glomerular Permeability, Increases Glomerular Endothelial Cell Monolayer Integrity and Intracellular Calcium

Rebecca R. Foster*, Sadie C. Slater*, Jaqualine Seckley*, Dontscho Kerjaschki{dagger}, David O. Bates{ddagger}, Peter W. Mathieson* and Simon C. Satchell*

From the Academic Renal Unit,* Southmead Hospital,{ddagger} and the Microvascular Research Laboratories, Department of Physiology, University of Bristol, Bristol, United Kingdom; and the Clinical Institute of Pathology,{dagger} Medical University of Vienna, Vienna, Austria


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
We have previously reported expression of vascular endothelial growth factor (VEGF)-A and -C in glomerular podocytes and actions of VEGF-A on glomerular endothelial cells (GEnC) that express VEGF receptor-2 (VEGFR-2). Here we define VEGFR-3 expression in GEnC and investigate the effects of the ligand VEGF-C. Renal cortex and cultured GEnC were examined by microscopy, and both cell and glomerular lysates were assessed by Western blotting. VEGF-C effects on trans-endothelial electrical resistance and albumin flux across GEnC monolayers were measured. The effects of VEGF-C156S, a VEGFR-3-specific agonist, and VEGF-A were also studied. VEGF-C effects on intracellular calcium ([Ca2+]i) were measured using a fluorescence technique, receptor phosphorylation was examined by immunoprecipitation assays, and phosphorylation of myosin light chain-2 and VE-cadherin was assessed by blotting with phospho-specific antibodies. GEnC expressed VEGFR-3 in tissue sections and culture, and VEGF-C increased trans-endothelial electrical resistance in a dose-dependent manner with a maximal effect at 120 minutes of 6.8 {Omega} whereas VEGF-C156S had no effect. VEGF-C reduced labeled albumin flux by 32.8%. VEGF-C and VEGF-A increased [Ca2+]i by 15% and 39%, respectively. VEGF-C phosphorylated VEGFR-2 but not VEGFR-3, myosin light chain-2, or VE-cadherin. VEGF-C increased GEnC monolayer integrity and increased [Ca2+]i, which may be related to VEGF-C-S particular receptor binding and phosphorylation induction characteristics. These observations suggest that podocytes direct GEnC behavior through both VEGF-C and VEGF-A.

Normal glomerular filtration depends on the combined properties of the three layers of the glomerular capillary wall; glomerular endothelial cells (GEnC), glomerular basement membrane, and glomerular epithelial cells or podocytes. Each of these layers has unique features essential for their function. GEnC, unlike the majority of endothelial cells, are highly fenestrated suggesting the action of particular local chemical or physical factors that induce this phenotype. Vascular endothelial growth factor (VEGF or VEGF-A) is a strong candidate molecule because it can be shown to induce fenestrations both in vivo and in vitro.1,2 VEGF-A is the prototypical VEGF family member and has powerful actions on endothelial cells including survival, proliferation, chemotaxis (all important in angiogenesis), and induction of permeability.3 These actions on endothelial cells are predominantly mediated through VEGF receptor 2 (VEGFR-2).4,5 VEGF-A also activates VEGFR-1 but not VEGFR-3. VEGF-C has many similar functions to VEGF-A, also acting via VEGFR-2, and can stimulate the proliferation and migration of endothelial cells and increase vascular permeability in vivo, but with potency four to five times less than VEGF-A.6,7 VEGF-C also has angiogenic properties8,9 and can induce fenestration formation10 but is a more potent inducer of lymphangiogenesis.11,12 Actions on lymphatic endothelium occur primarily via VEGFR-3.5,11,13,14 VEGF-C does not activate VEGFR-1.

VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels.15 Thereafter it is widely expressed in vascular and lymphatic endothelium during embryonic development13,16 and is also essential for normal lymphangiogenesis.17 After organogenesis it becomes primarily restricted to lymphatic endothelium13,16 but has been detected in some vascular endothelia in adult tissues notably in discontinuous endothelia of bone marrow and splenic sinusoids, some fenestrated endothelia, including that of endocrine organs,18,19 and in capillaries of various organs.20

We and others have shown that podocytes express VEGF-A and angiopoietin 1 (ang1, a member of another family of endothelium-regulating factors21 ) whereas the adjacent GEnC express the cognate receptors VEGFR-1, VEGFR-2 and Tie2.22,23 These observations suggest a relationship analogous to that seen between mural cells and endothelial cells in other vessels, where podocytes may influence GEnC phenotype and hence their contribution to the unique permeability characteristics of the glomerular capillary wall. Indeed VEGF-A and ang1 both have effects on barrier properties of GEnC in vitro.24 Further evidence in support of such an interaction is accumulating. Both experimental administration of neutralizing antibodies to VEGF-A and podocyte-specific reduction in VEGF-A production cause GEnC abnormalities including swelling, vacuolation, and detachment along with proteinuria.25-27

We have recently described the expression of VEGF-C by podocytes, both in vivo and in vitro, and have demonstrated autocrine actions on podocytes.28 Observations by other groups showing that GEnC express VEGFR-3 in vivo18,19 suggest that VEGF-C, along with VEGF-A and ang1, might also be a mediator of the podocyte’s ability to direct GEnC phenotype. We have now confirmed the expression of VEGFR-3 in isolated glomeruli and GEnC in vivo and in culture and have examined the actions of VEGF-C on GEnC, demonstrating effects, some of which are opposite to those seen with VEGF-A. Our results point more toward involvement of VEGFR-2, rather than VEGFR-3, in these effects on GEnC. These observations lend credence to a physiological role for VEGF-C in podocyte-GEnC interactions.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Nephrectomy Tissue

Human renal tissue was obtained, with consent and under ethical approval from the Southmead Hospital local research ethics committee, from patients undergoing nephrectomy for renal carcinoma. Within 30 minutes after nephrectomy, samples of cortex from the normal pole were taken for extraction of glomeruli by sieving.

Cell Culture

Human GEnC were obtained at passage 3 from the Applied Cell Biology Research Institute (Kirkland, WA) and were used for experiments up to passage 8. We have previously characterized these cells in detail including their expression of intercellular adhesion molecules, formation of monolayers in culture and responses to control mediators in barrier property assays.24 Human lung microvascular endothelial cell (LMVEC, derived from adult lung tissue; Clonetics Corp., San Diego, CA) were obtained at passage 3 and used for experiments up to passage 7. Human lymphatic microvascular endothelial cells (LEC, derived from adult dermal tissue; Clonetics) were obtained at passage 3 and used up to passage 5. All endothelial cells were cultured in EGM2-MV (endothelial growth medium 2, microvascular; Cambrex, Wokingham, UK), made up from EBM2 (endothelial basal medium 2, Cambrex) and fetal calf serum (5%), antimicrobial agents, and growth factors as supplied, excepting VEGF-A.

Primary and Secondary Antibodies

A goat polyclonal antibody to VEGFR-3 (AF349; R&D Systems, Minneapolis, MN) was used for confocal microscopy, Western blotting, and immunoprecipitation studies. A second VEGFR-3 antibody (mouse monoclonal anti-human VEGFR-3, 9D9, was a kind gift from Prof. K. Alitalo, Helsinki, Finland) was used for immunoelectron microscopy and for Western blotting and immunoprecipitation studies. For confocal microscopy an Alexa Fluor 633-conjugated secondary was used (Molecular Probes, Invitrogen, Paisley, UK). Two VEGFR-2 antibodies were used, one for immunoprecipitation (rabbit polyclonal, sc-504; Santa Cruz Biotechnology, Santa Cruz, CA) and one for Western blotting (mouse monoclonal antibody, 05-554; Upstate, Millipore, Billerica, MA). An anti-phosphotyrosine mouse monoclonal antibody (4G10, Cell Signaling Technology, Danvers, MA) was also used for Western blotting of immunoprecipitated proteins. Antibodies to phospho-VE cadherin (Tyr658, AB1955; Chemicon, Millipore) phospho-myosin light chain (MLC)-2 (Ser19, 3671s; Cell Signaling Technology), total VE-cadherin and total MLC-2 (F-8 and FL-172, respectively, both Santa Cruz Biotechnology) were used to examine downstream effector pathways by Western blotting.

Confocal and Immunoelectron Microscopy

GEnC grown to confluence on glass coverslips were fixed in 4% fresh paraformaldehyde in phosphate-buffered saline (PBS), permeabilized in 0.2% Triton X-100 in PBS and incubated with 0.1% sodium borohydride. Cells were then incubated with primary antibody in 1% bovine serum albumin (BSA) and secondary antibody in PBS. In the control samples, primary antibody was replaced with nonimmune immunoglobulin of the same species and concentration. Cells were co-stained with 5 µmol/L 4,6-diamidino-2-phenylindole before mounting in MOWIOL 4-88 (Calbiochem, San Diego, CA) containing 0.6% 1,4-diazabicyclo-(2.2.2)octane (Dabco; Sigma Chemical Co., St Louis, MO) as an anti-photobleaching agent. Confocal microscopy was performed using an AOBS SP confocal laser-scanning system (Leica, Wetzlar, Germany) attached to a Leica DM IRE2 inverted epifluorescence microscope. Immunoelectron microscopy, using an indirect immunogold procedure, was performed on Lowicryl ultra-thin human renal cortex sections, as described previously.29

Western Blotting

GEnC were lysed in Laemmli sample buffer and solubilized protein concentrations were determined (bicinchoninic acid assay; Pierce Chemical Co., Rockford, IL). Lysates of HMVECs, vascular smooth muscle cells (VSMC) (a gift from Dr. C. Shanahan, University of Cambridge, Cambridge, UK), LEC, and sieved glomeruli were used as controls. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and were blotted onto nitrocellulose membranes. The membranes were air-dried and blocked in 5% fat-free milk before incubation with antibodies to VEGFR-3 (as above) and actin (Sigma) to confirm loading of comparable amounts of protein in each lane. After incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz), bands were detected using the ECL chemiluminescence system (Amersham Biotech Ltd., Bucks, UK).

Effect of VEGF-C on trans-Endothelial Electrical Resistance (TEER) of GEnC Monolayers

Polycarbonate supports (0.4-µm pore size, 0.5 cm2 surface area) in tissue culture inserts (1 cm diameter; Nalge Nunc International, Rochester, NY) were seeded with GEnC at 100,000 cells/cm2. After 5 days the medium was replaced with serum-free medium (EBM2) and 1 hour later the TEER of cell monolayers was measured as described previously using an Endohm 12 electrode chamber and an EVOMx voltohmeter (World Precision Instruments, Sarasota, FL).24 Tissue culture inserts were placed sequentially in the chamber and the resistance recorded after 10 seconds. VEGF-C (R&D Systems), or control, was then added to give a final concentration of 1 or 10 nmol/L and TEER was remeasured at 60 and 120 minutes. Some inserts were also treated with 1 nmol/L VEGF-A (R&D Systems) as a control because the effect of VEGF-A in this assay is already established.24 In other experiments, VEGF-C was replaced with the mutant VEGF-C, VEGF-C156S (R&D Systems), a selective agonist of VEGFR-3 that does not bind VEGFR-2.5,7 VEGF-C156S was used at concentrations of 1, 10, and 50 nmol/L.

Effect of VEGF-C on Protein Passage across GEnC Monolayers

Trans-monolayer permeability to macromolecules was assessed by measuring passage of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA, Sigma) across the monolayer essentially as described previously.24 GEnC monolayers were prepared in tissue culture inserts and after 5 days the medium was replaced with EBM2 as above. After 1 hour VEGF-C was added to give a final concentration of 10 nmol/L. Simultaneously FITC-BSA was added to medium within the inserts to give a final concentration of 500 µg/ml and unlabeled BSA was added to the medium in the well to the same concentration. At 1, 2, and 3 hours 100-µl aliquots were removed and replaced with 100 µl of serum-free medium (SFM) containing unlabeled BSA. The fluorescence emission of the aliquots at 520 nm after excitation at 490 nm was measured on a Packard Instruments FluoroCount fluorospectro-photometer (PerkinElmer Life Sciences, Wellesley, MA) and the concentration of FITC-BSA calculated by reference to a set of standard dilutions.

Effects of VEGF-C on [Ca2+]i

GEnC grown to confluence on 22-mm diameter glass coverslips were incubated with the high-affinity, ratiometric, and UV light-excitable intracellular calcium indicator Fura 2-AM (10 µmol/L, Molecular Probes) for 90 minutes in EBM2 at 37°C, and each coverslip in turn was then placed in a coverslip holder. The holder was mounted on a rig consisting of an inverted fluorescence microscope (DM IRB, Leica) equipped with a UV source (Cairn Instruments, Faversham, UK) with filters for excitation at 340 and 380 nm. Fast switching was achieved using a rotary filter wheel at 50 Hz and a spectrophotometer for photometric measurement (Cairn Instruments). The spectrophotometer received emitted light via a 400-nm dichroic filter and a 510- to 530-nm barrier filter in front of the photometer. A Powerlab data acquisition unit and Chart 5 software (both from AD Instruments, Colorado Springs, CO) were used for analysis and graphic display. Experiments were conducted in Krebs-Ringer phosphate buffer (150 mmol/L NaCl, 6 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L D-glucose, and 10 mmol/L HEPES and CaCl2 1.5 mmol/L). Baseline recordings were established and Krebs-Ringer buffer was changed with that containing 10 nmol/L human recombinant VEGF-C (final concentration), 10 nmol/L VEGF-C156S, or 0.1% BSA vehicle and left to record for 20 minutes. One nmol/L VEGF-A was used in similar experiments as a positive control for the assay because it has been shown to increase [Ca2+]i in human umbilical vein endothelial cells (HUVEC).30 To ensure that [Ca2+]i was effectively measured, 10 µmol/L ionomycin was added to stimulate Ca2+ entry into the cells. One mmol/L of manganese chloride (MnCl2), in the continued presence of 10 µmol/L ionomycin, was then used to quench the Fura 2-AM to determine the background (Ca2+-independent) fluorescence signal. Emission fluorescent measurements (If) were taken four times a second. The ratio of the If measured during 340-nm excitation to that during 380-nm excitation (Rnorm), proportional to the calcium concentration, was calculated from Rnorm = Rexp/Rmin where Rexp = (If340 – B340)/(If380 – B380). If340 is the If measured during excitation at 340 nm, If380 is the If measured during excitation at 380 nm, and B340 and B380 are the background If values measured during excitations at 340 and 380 nm, respectively (measured as the If after Mn2+ quenching). Rmin is the in vitro ratio for zero [Ca2+]i. The use of this ratio facilitates the detection of small changes in [Ca2+]i, which are independent of the Fura 2-AM loading. The Rnorm was plotted against time and the peak to baseline ratios of Rnorm were calculated as a measure of the maximal effect on [Ca2+]i, and compared between treatment and control groups. The rate of rise of [Ca2+]i was also plotted as dRnorm/dt and compared between VEGF-A- and VEGF-C-treated cells.

VEGF Receptor Phosphorylation Assays and Immunoprecipitation

To determine the effect of exogenous VEGF-C on VEGFR-2 and VEGFR-3 phosphorylation in GEnC, cells were serum-starved for 4 hours and then treated with 10 nmol/L VEGF-C for 2, 5 ,7, 10, 30, or 90 minutes or with vehicle control. LEC were used as a positive control for VEGFR-3 phosphorylation. Effects of VEGF-C on VEGFR-2 phosphorylation were compared with those of VEGF-A (1 nmol/L, 2 minutes). Cells were then lysed, cleared, and protein was quantified as described above. Lysate from each sample was incubated with 4 µg of primary antibody (anti-VEGFR-2 or anti-VEGFR-3, 9D9) per 1000 µg of total protein overnight at 4°C with gentle agitation. Sixty µl of washed A/G agarose beads per 500 µl of lysate was then added to each of the samples and left to incubate for at least 4 hours, rocking at 4°C. Samples were then centrifuged for 3 minutes at 13,000 rpm at 4°C, and the supernatant was removed and retained and the precipitate was washed three times with lysis buffer. Equal quantities of supernatant and total lysate, or the entire precipitate of each protein sample lysate were mixed with 3x SDS loading buffer (100 mmol/L Tris, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, and 0.2% bromophenol blue) and boiled for 5 minutes. The precipitate samples were vortexed, then centrifuged for a further 30 seconds at 13,000 rpm. All samples were run on a 7.5% SDS-PAGE gel at 100 V and were then transferred to nitrocellulose membranes for 120 minutes at 60 V. The membrane was probed with an anti-phosphotyrosine antibody, then stripped and reprobed for total VEGFR-2 or VEGFR-3 (AF349). The membranes were blocked in 5% fat-free milk (or 3% BSA/PBS-Tween for the anti-phospho-tyrosine antibody) before incubation with antibodies described above. After incubation with horseradish peroxidase-conjugated secondary antibodies, bands were detected using the ECL chemiluminescence system or the SuperSignal West Femto maximum sensitivity substrate (Pierce).

Analysis of Downstream Effector Molecules

To further investigate the differential effects of VEGF-A and VEGF-C on barrier properties, we examined phosphorylation of the downstream effector molecules VE-cadherin and MLC-2. GEnC were serum-starved for 4 hours, then stimulated with 1 nmol/L VEGF-A or 10 nmol/L VEGF-C for 10 (MLC-2) or 30 minutes (VE-cadherin) before lysis and analysis by Western blotting. Resulting membranes were immunoblotted for phospho-VE-cadherin or phospho-MLC-2 then stripped and reprobed for total protein.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism (GraphPad Prism Software Inc., San Diego, CA), SPSS 11.0 (SPSS, Chicago, IL), or Excel. Comparisons were made by Student’s t-test or analysis of variance. Results shown are representative of, or a combination of, at least three separate experiments as indicated in the figure legends. Results are presented as mean ± SE.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
VEGFR-3 Expression

Immunoelectron microscopy showed strong expression of VEGFR-3 on GEnC in human renal cortex using a second VEGFR-3 antibody (Figure 1) . Minimal podocyte staining was seen with this antibody (9D9) and technique. Western blotting of sieved glomeruli and Western blotting and confocal microscopy of cultured GEnC confirmed the presence of VEGFR-3 in sieved glomeruli and in cultured GEnC (Figure 2) . The 130-kDa proteolytically cleaved and 170-kDa unglycosylated forms31 were clearly identified in GEnC. The larger glycosylated form (195 kDa) was more faintly detected. LMVEC expressed high levels of VEGFR-3, consistent with evidence that primary culture microvascular endothelial cells contain a mixture of lymphatic and vascular endothelial cells.5 (This is not for the case for GEnC because glomeruli do not contain lymphatics.) Similarly, LEC expressed high levels of VEGFR-3 with bands corresponding to those in GEnC and LMVEC in Western blots using both AF349 and 9D9 antibodies.


Figure 1
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  		Figure 1. Glomerular VEGFR-3 expression by immunoelectron microscopy on human renal cortex tissue sections using a mouse monoclonal antibody (9D9). Gold particles can be clearly seen labeling the glomerular endothelium indicating VEGFR-3 expression (arrows). The bottom image includes a grazing section of the glomerular endothelium with fenestrations visible en face. Minimal podocyte labeling was seen. Scale bars = 100 nm.

 

Figure 2
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  		Figure 2. A: Confocal microscopy showing expression of VEGFR-3 by human GEnC cultured on coverslips. B: Western blotting analyses of protein extracted from GEnC, human LMVEC, vascular smooth muscle cells (VSMC), glomeruli sieved from normal renal cortex (Gloms), and LEC using goat polyclonal (AF349) and mouse monoclonal (9D9) antibodies. LMVEC and LEC are used as positive controls, VSMC as negative controls. The blots confirm expression of VEGFR-3 in GEnC (albeit at a lower level than in LMVEC and LEC) and in human glomeruli. The 130-kDa proteolytically cleaved and 170-kDa unglycosylated forms are clearly identified in GEnC. The larger glycosylated form (195 kDa) is more faintly detected. Actin bands confirm the loading of comparable amounts of protein. Numbers on left and right indicate expected molecular weight of bands and molecular weights of marker proteins, respectively.

 
Effects of VEGF-C on GEnC Monolayer Barrier Properties

VEGF-C caused an increase in TEER of GEnC monolayers and this effect was dose-dependent with a maximal effect of 10 nmol/L VEGF-C at 120 minutes of 6.8 ± 0.5{Omega} (Figure 3) . Importantly the direction of this effect is opposite to that seen with VEGF-A, which, as we have reported previously causes a reduction in GEnC monolayer TEER.24 In contrast, VEGF-C156S in concentrations up to 50 nmol/L had no effect on TEER of GEnC monolayers (not shown). Consistent with the increase in TEER, VEGF-C also reduced passage of labeled albumin across GEnC monolayers throughout 3 hours by 32.8% (Figure 4) .


Figure 3
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  		Figure 3. The effects of VEGF-C (1 or 10 nmol/L) and VEGF-A (1 nmol/L) on TEER of GEnC monolayers over time. Bars show mean ± SE normalized to control 0, n = 6, P < 0.0001 overall by repeated measures (rm) analysis of variance. Post hoc comparisons (Bonferroni), 1 and 10 nmol/L VEGF-C versus control both P < 0.01. Ten nmol/L VEGF-C increased TEER by a maximum of 6.8 ± 0.5{Omega} at 120 minutes. Results shown are representative of five separate experiments.

 

Figure 4
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  		Figure 4. The effect of VEGF-C (10 nmol/L) on passage of FITC-BSA across GEnC monolayers throughout time. Bars show mean ± SE, n = 5 to 8, P < 0.05 by repeated measures analysis of variance. VEGF-C reduced the amount of FITC-BSA passing through GEnC monolayers throughout 3 hours by 32.8%. Results shown are representative of three separate experiments.

 
Effects of VEGF-A and VEGF-C on [Ca2+]i

VEGF-A (1 nmol/L) caused an increase in Rnorm of 1.39 ± 0.02 (P < 0.001) indicating an increase in [Ca2+]i in GEnC (Figure 5) . This is consistent with previous observations in HUVEC.30 VEGF-C (10 nmol/L) also increased Rnorm, although to a lesser degree, by 1.15 ± 0.03 (P < 0.01). VEGF-C156S (10 nmol/L), which activates only VEGFR-3, did not increase [Ca2+]i (Rnorm 1.02 ± 0.01).


Figure 5
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  		Figure 5. The effects of VEGF-A (1 nmol/L) and VEGF-C (10 nmol/L) on the change in [Ca2+]i in GEnC incubated in Krebs-Ringer phosphate buffer. GEnC were serum-starved and loaded with fura-2-AM. VEGF-A, VEGF-C, or C156S was then added to the cells, in the presence of Krebs-Ringer. Fluorescence intensity changes were recorded and used to calculate the Rnorm, which represents changes in [Ca2+]i. Changes in the Rnorm in GEnC treated with VEGF-A were compared to effects of VEGF-C. A: An example of the effect of VEGF-A on GEnC, showing an increase in Rnorm. B: An example of the effect of VEGF-C on GEnC, showing an increase in Rnorm. C: An example of VEGF-C156S, showing no change in Rnorm. D–F: Data expressed as mean ± SE of the ratio of treatment/baseline Rnorm for VEGF-A, VEGF-C, and VEGF-C156S, respectively, compared to vehicle. The Rnorm was increased, corresponding to an increase in [Ca2+]i, in GEnC treated with both 1 nmol/L VEGF-A and 10 nmol/L VEGF-C (P < 0.05 in each case), but not 10 nmol/L VEGF-C156S. Composite data (D–F) are derived from 5, 12, and 7 experiments, respectively.

 
Analysis of the rate of Ca2+ flux (dRnorm/dt) revealed no significant difference between VEGF-A and VEGF-C treatment (Figure 6) . There was considerable variability in dRnorm/dt induced by VEGF-A that may have obscured a significant effect. However the profile of the response to the two mediators was different with the time to peak rate of Ca2+ flux occurring significantly later with VEGF-C (VEGF-A induced peak flux: 122.4 ± 13.0 seconds, VEGF-C induced peak flux: 415.7 ± 118.4 seconds, P < 0.05).


Figure 6
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  		Figure 6. The effects of VEGF-A and VEGF-C on the rate of calcium flux (dRnorm/dt) and the time taken to reach dRnorm/dt. A: An example demonstrating the comparison between Rnorm and dRnorm/dt in response to VEGF-A. B: An example demonstrating the comparison between Rnorm and dRnorm/dt in response to VEGF-C. C: Data expressed as mean ± SE of the peak dRnorm/dt for VEGF-A, VEGF-C. D: Data expressed as mean ± SE time to peak dRnorm/dt for VEGF-A and VEGF-C. VEGF-A-induced peak Ca2+ flux: 122.35 ± 13.02 seconds; VEGF-C: 415.65 ± 118.4 seconds, P < 0.05. Composite data (C and D) are derived from 5 and 12 experiments, respectively.

 
Effects of VEGF-C on VEGF Receptor Phosphorylation

VEGF-A induced the phosphorylation of VEGFR-2 at 2 minutes (Figure 7) . VEGF-C-induced phosphorylation of VEGFR-2 was not detectable at 2, 5, or 7 minutes (not shown) but was apparent at 10 minutes, maximal at 30 minutes (although to a lesser extent than with VEGF-A), and diminished by 90 minutes (Figure 8) . This is consistent with previous observations of VEGFR-2 phosphorylation by VEGF-D, which induced lesser phosphorylation and peaked at 30 minutes, in contrast to VEGF-A that induced a peak effect before 5 minutes.30 We could not demonstrate the phosphorylation of VEGFR-3 by VEGF-C or VEGF-C156S in GEnC after 5 minutes of exposure (Figure 8) or any other time point tested (not shown). Phosphorylation of VEGFR-3 in LEC by both molecules confirmed the ability of the assay to detect VEGFR-3 phosphorylation as well as the activity of the VEGF-C156S mutant. However, it is likely that the lower levels of VEGFR-3 expression in GEnC, compared with LEC, rendered phosphorylation more difficult to detect and we do not exclude the possibility that some phosphorylation has occurred, below the sensitivity of the assay.


Figure 7
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  		Figure 7. The effects of VEGF-A and VEGF-C on phosphorylation of VEGFR-2 in cultured GEnC. GEnC were serum-starved, then treated with 1 nmol/L VEGF-A, 10 nmol/L VEGF-C, or vehicle for varying lengths of time before cells were lysed and protein immunoprecipitated with VEGFR-2 antibodies. Immunoprecipitated protein plus lysate and total protein was run on a SDS-PAGE and Western blotting was performed using either a phosphotyrosine (PY) antibody or an antibody to VEGFR-2 to demonstrate total protein. A: VEGF-A at 2 minutes induced a much more intense phosphotyrosine band corresponding to VEGFR-2 than VEGF-C at 10 minutes. B: VEGF-C induced maximal VEGFR-2 phosphorylation at 30 minutes, which was reduced by 90 minutes. Numbers indicate position of molecular weight markers of corresponding size. Arrows indicate position of bands corresponding to VEGFR-2. Blots shown are representative of five separate experiments.

 

Figure 8
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  		Figure 8. The effect of VEGF-C and VEGF-C156S on phosphorylation of VEGFR-3 in cultured GEnC (A) and LEC used here as a positive control (B). A: GEnC were serum-starved, then treated with 10 nmol/L VEGF-C, 10 nmol/L VEGF-C156S, or vehicle for 5 minutes before cells were lysed and protein immunoprecipitated with VEGFR-3 (9D9) antibodies. Immunoprecipitated protein was run on a SDS-PAGE and Western blotting was performed using antibodies to either phosphotyrosine (PY) or to VEGFR-3 (AF349) to demonstrate total protein. The positions of the 130-, 170-, and 195-kDa VEGFR-3 bands are indicated in the total protein blot. Neither VEGF-C nor VEGF-C156S induced VEGFR-3 phosphorylation. Blots shown are representative of three separate experiments. B: LEC were treated as for GEnC. Phosphorylation of VEGFR-3 was not detected in the control lane, but was up-regulated by both VEGF-C and the VEGF-C156S mutant as expected. Phosphorylation is most clearly seen in the 130-kDa band. These results confirm the activity of VEGF-C and VEGF-C156S and the ability of this assay to detect VEGFR-3 phosphorylation. Arrows indicate the position of bands corresponding to VEGFR-3. These results confirm the activity of VEGF-C and VEGF-C156S and the ability of this assay to detect VEGFR-3 phosphorylation.

 
Analysis of Downstream Effector Molecules

VEGF-A induced phosphorylation of VE-cadherin and showed a trend toward increased phosphorylation of MLC-2 (Figure 9) as anticipated from studies in other endothelial cells. However, despite phosphorylation of VEGFR-2, VEGF-C did not induce phosphorylation of either molecule.


Figure 9
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  		Figure 9. The effect of VEGF-A and VEGF-C on phosphorylation of VE-cadherin (A) and MLC-2 (B) by Western blotting. GEnC were serum-starved for 4 hours, then treated with 1 nmol/L VEGF-A or 10 nmol/L VEGF-C for 30 minutes (VE-cadherin) or 10 minutes (MLC-2). Membranes were probed for phospho-VE-cadherin and phospho-MLC-2, respectively, then stripped and reprobed for total protein. Blots are representative of three and five separate experiments, respectively. A: Densitometry shows that VEGF-A caused a 2.2 ± 0.12-fold increase in VE-cadherin phosphorylation, whereas VEGF-C had a minimal increase (1.2 ± 0.2). Overall P = 0.016 by analysis of variance, post hoc (Bonferroni) vehicle versus VEGF-A: P = 0.023; vehicle versus VEGF-C: ns. B: Densitometry shows a trend toward up-regulation of MLC-2 phosphorylation in response to VEGF-A (1.8 ± 0.3-fold increase) whereas VEGF-C had a minimal effect (1.2 ± 0.2). Overall P = 0.10 by analysis of variance.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Expression of VEGFR-3 in Glomeruli and Cultured GEnC

Demonstration of the expression of VEGFR-3 in glomeruli is consistent with previous reports.18,19 Using immunoelectron microscopy we have shown that this expression is localized to the glomerular endothelium. Because VEGFR-3 is expressed only by some blood vessel endothelial cells, in particular in fenestrated capillaries, it is likely that VEGFR-3 is important in some, as yet undefined way, in maintenance of this endothelial cell phenotype. In addition to its physiological role in lymphatics, VEGFR-3 may be switched on in tumor neovasculature where it appears to be involved in promoting integrity of the endothelial lining.32 This action may be important in GEnC, which, like tumor vascular endothelial cells, are exposed to high concentrations of VEGF-A (derived from podocytes in the glomerulus). Confocal microscopy, Western blotting, and immunoprecipitation studies confirmed the continued expression of VEGFR-3 by cultured GEnC, in addition to VEGFR-1 and VEGFR-2 as we have previously shown.24

Effects of VEGF-C on GEnC Monolayer Barrier Properties

VEGF-C caused an increase in TEER of GEnC monolayers. TEER is a measure of ion flux and is inversely related to the fractional area of pathways open to water and small molecules across a cell monolayer. An increase in resistance suggests a reduction in such pathways. The observed maximal increase of 6.8{Omega} is similar to the response to ang1 (5.7{Omega}), one of few other mediators known to reduce endothelial permeability.24 VEGF-C caused a corresponding reduction in FITC-BSA passage; the 32.8% reduction in FITC-BSA passage is similar to the 45.2% reduction induced by ang1.24 VEGF-C thus has comparable effects in these respects to ang1 but opposite effects to those of VEGF-A. As we have shown previously VEGF-A reduces GEnC TEER (confirmed here) and increases passage of labeled albumin.24

This interesting observation raises the question of why two members of the VEGF family should have opposite effects on GEnC barrier properties. These in vitro results also contrast with in vivo effects of VEGF-C on frog mesenteric capillaries where VEGF-C increases hydraulic conductivity, at least in a proportion of vessels,33 and increases leakage of Evans blue dye in the Miles assay.7 Explanations for these apparent contradictions may lie in the important differences between VEGF-A and VEGF-C in their receptor-binding specificities and in the expression of VEGFR-3 in GEnC but not in systemic vessels.

Effects of VEGF-C on [Ca2+]i

In view of opposite effects of VEGF-A and VEGF-C on barrier properties of GEnC, we went on to compare the [Ca2+]i responses to VEGF-C. VEGF-A is known to cause an increase in [Ca2+]i in other endothelial cell types (both in vitro in HUVEC,30,34 and in vivo in frog mesenteric vessels35 ). After VEGF-A or other stimuli, an increase in [Ca2+]i is usually associated with an increase in permeability. In contrast to the barrier property assays, VEGF-C here behaved similarly to VEGF-A, causing an increase in [Ca2+]i. Another group has shown that VEGF-D increases [Ca2+]i in HUVEC,30 and our novel finding of an increase in [Ca2+]i in response to VEGF-C is hence consistent with effects of other VEGF family members. However, the finding is surprising in the context of evidence of reduced permeability.

An explanation may lie in accumulating evidence showing that microvascular endothelial cells are relatively resistance to permeability increases as a consequence of raised [Ca2+]i because of differences from macrovascular endothelial cells in regulation of store-operated calcium entry.36 Furthermore the increase in [Ca2+]i in response to VEGF-C was smaller than that to VEGF-A, suggesting that there may be a threshold level of [Ca2+]i required to increase permeability, as previously shown for mesenteric microvessels.37 In view of recent evidence showing that in vivo permeability changes depend also on the rate of change of [Ca2+]i,38 we also determined the peak rate of change of [Ca2+]i. Although we could not demonstrate a difference in this measure per se, the timing of the peak rate of change of [Ca2+]i was much later in cells treated with VEGF-C confirming a delayed response profile, which may have implications for downstream signaling events.

Lack of Effect of VEGF-C156S and Receptor Phosphorylation Studies

To further investigate this differential effect of VEGF-A and VEGF-C, we sought to determine through which receptor VEGF-C was exerting its effects. VEGF-C156S, which binds to VEGFR-3 but not VEGFR-2, had no effect on GEnC TEER or on [Ca2+]i. Differences in VEGFR-3 binding affinity and hence activation between wild-type VEGF-C and VEGF-C156S suggest a possible explanation for the lack of effect of VEGF-C156S.5 However, at concentrations used, it would be expected that VEGFR-3 would be saturated and in lymphangiogenesis assays, VEGF-C and VEGF-C156S can be shown to be equipotent.14 Hence it is more likely that the absence of VEGFR-2 binding is the reason for the absence of an effect of VEGF-C156S.39

To determine whether wild-type VEGF-C was acting via VEGFR-2 or VEGFR-3, we studied receptor phosphorylation. VEGF-C phosphorylated VEGFR-2 to a lesser extent and with a delayed effect when compared to VEGF-A, as seen with the effect on [Ca2+]i and as has been shown for VEGF-D.30 Neither VEGF-C, nor the VEGFR-3-specific mutant VEGF-C156S, could be shown to phosphorylate VEGFR-3 in GEnC. Taken together with the absence of effects of VEGF-C156S on TEER or [Ca2+]i, and the phosphorylation of VEGFR-2 in response to VEGF-C, these results suggest the relative unimportance of VEGFR-3 in the effects of VEGF-C on GEnC barrier properties and [Ca2+]i but do not exclude a role for it. We should note that our results are inconsistent with previous reports showing phosphorylation of VEGFR-3 by VEGF-C in LEC, as repeated here, and in blood vascular endothelial cells.40 However, LEC natively express high levels of VEGFR-3 and where this effect has been shown in blood vascular endothelial cells, it has been in cells overexpressing VEGFR-3, rather than in primary culture cells constitutively expressing both VEGFR-2 and VEGFR-3. It may be that either VEGFR-3 behaves differently in response to VEGF-C when artificially overexpressed or more likely that overexpression allows detection of a low level of phosphorylation that cannot be detected in GEnC expressing normal VEGFR-3 levels. Similarly VEGF-C does not phosphorylate VEGFR-3 in cultured podocytes, which also constitutively express the receptor.28

Analysis of Downstream Effector Molecules

The intracellular pathways by which VEGF-A acts to increase endothelial cell permeability are complex and incompletely understood.3 However, effects on intercellular junctions and the actin cytoskeleton have been identified as important in transducing permeability effects. VE-cadherin is an endothelial cell-specific adhesion molecule with important roles in adherens junctions. VEGFR-2 phosphorylation leads to phosphorylation of VE-cadherin, disassembly of intercellular junctions, and hence to an increase in permeability.41 VEGFR-2 phosphorylation also leads to MLC-2 phosphorylation including via raised intracellular calcium but also through other signaling intermediaries. MLC-2 phosphorylation leads to actin-myosin cross-bridging and a cytoskeletal contractive force causing endothelial cell retraction and again an increase in permeability.42

Hence we studied VE-cadherin and MLC-2 as examples of effector molecules implicated in VEGF-A-induced increases in endothelial permeability. As anticipated from work in other types of endothelial cell, VEGF-A increased phosphorylation of both VE-cadherin and MLC-2 (trend only) in GEnC. More interestingly, however, VEGF-C did not lead to phosphorylation of either of these proteins. These observations confirm that in these studies VEGFR-2 phosphorylation and raised [Ca2+]i in response to VEGF-C did not lead to the usual downstream effector activation and hence provide a mechanistic explanation of why VEGF-C did not increase GEnC permeability. Either the time course and degree of VEGFR-2 phosphorylation or the rise in [Ca2+]i are insufficient to trigger activation of these downstream effectors or the pathways are subject to extrinsic modifiers, for example Rho family GTPases.

Summary of Experimental Results

We have confirmed the expression of VEGFR-3 on GEnC both in vivo and in vitro and we have described for the first time the effects of VEGF-C on barrier properties and [Ca2+]i in cultured endothelial cells of any origin. VEGF-C increases TEER and reduces protein passage across GEnC monolayers whilst also increasing [Ca2+]i. It does not phosphorylate the downstream effectors of VEGF-A, VE-cadherin, and MLC-2. Hence in these cells the rise in [Ca2+]i is dissociated from its usual effect of increasing permeability.

Implications and Conclusions

What are the possible explanations for the differential effects of VEGF-A and VEGF-C on GEnC barrier properties (despite both activating VEGFR-2) and what might be the role of VEGFR-3 in GEnC in the absence of demonstrable phosphorylation? Firstly, receptor tyrosine kinases, including VEGFRs, require ligand-induced dimerization and trans-phosphorylation for activation. VEGF-C can induce both VEGFR-3 homodimerization and heterodimerization with VEGFR-2. Different patterns of phosphorylation of tyrosyl phosphorylation sites within the intracytoplasmic C-terminal portion of VEGFRs may result from hetero- or homodimer formation.39 Downstream effects of such differences are yet to be elucidated but this provides a potential mechanism whereby VEGF-C-induced VEGFR-2/3 heterodimerization could modulate downstream events in the absence of VEGFR-3 phosphorylation. The co-expression of VEGFR-3 and VEGFR-2 in certain blood vessels also suggests that heterodimerization may occur in vivo and these differences in phosphorylation may be important.

Secondly, binding of different ligands to the same VEGFR can induce different phosphorylation patterns and downstream events30,43 suggesting another potential explanation for the differences in both VEGFR-2 phosphorylation profiles and in observed effects on barrier properties and [Ca2+]i. The third possible explanation lies in recent evidence that permeability-inducing effects of VEGFs depend on VEGFR-1 binding.44 Hence because VEGF-C does not activate VEGFR-1 it does not increase permeability but does induce VEGFR-2-dependent events including raised intracellular calcium.44

Our results support the hypothesis that VEGF-C produced by podocytes has effects on the function of GEnC. VEGF-C is thus added to the list of mediators, which already includes VEGF-A,24 VEGF-A165b,45 and ang1,24 through which the podocyte may direct the GEnC phenotype. Apart from VEGF-A, this list includes mediators that tend to block or antagonize some of the effects of VEGF-A. As such they may help to explain why GEnC can be exposed to high levels of VEGF-A and yet contribute to the permeability barrier and resist the pro-angiogenic stimulus, at least in the normal state. This combination of growth factors may induce fenestrations while retaining GEnC in a quiescent state. The concurrence of VEGFR-3 with VEGFR-2 expression in fenestrated endothelia in other organs further points to the importance of VEGFR-3 in determining the phenotype of fenestrated endothelia. Other observations support this idea, suggesting that VEGFR-3 modulates signaling via VEGFR-2 to stabilize the endothelium32,46 and regulates embryonic vasculogenesis.47 In a recent study in adult animals, Kamba and colleagues19 showed that some capillaries in various organs regress on systematic VEGF-A blockade. Those vessels that regressed (ie, were VEGF-A-dependent) not only had relatively high levels of VEGFR-2, but also expressed VEGFR-3, providing further evidence for the importance of VEGFR-3 in modulating VEGFR-2 responses.

The data described here add to the increasing complexity of signaling pathways that may maintain the unique phenotypes of glomerular cells that in turn are essential for their filtration function. Alterations in levels of expression of mediators involved in such pathways have been identified in various glomerular diseases and in recovery from them.21 In some situations, recovery from glomerular injury can be shown to be dependent on restoration of the normal glomerular endothelium.2 Exogenous administration of VEGF-A has been shown to hasten recovery in experimental glomerulonephritis48 and VEGF-A is already in use in clinical trials in peripheral and cardiovascular disease.49 Further elucidation of these glomerular pathways will inform development of therapeutic approaches involving the manipulation of various mediators, tailored according to the particular pathophysiological process.


   Acknowledgements
 
We thank Dr. Henry Mellor and Dr. Stephanie Pellegrin, University of Bristol, for assistance with confocal microscopy; and Dr. Kari Alitalo, University of Helsinki, for help with immunostaining and immunoblotting through provision of the 9D9 antibody.


   Footnotes
 
Address reprint requests to Dr. S.C. Satchell, Academic Renal Unit, Paul O'Gorman Lifeline Centre, Southmead Hospital, Bristol, BS10 5NB, UK. E-mail: s.c.satchell{at}bristol.ac.uk

Supported by the Medical Research Council (grant G0500053, ID 73430), Kidney Research UK (grant R22/1/2003), and the Wellcome Trust (fellowship 075731 to S.C.S.).

Accepted for publication June 26, 2008.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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