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*New York Medical College, Valhalla, New York; Tel Aviv University, Israel; The University of Tokyo, Japan; The Catholic University of Korea, Seoul, Korea.
Correspondence to Dr. Byung-Soo Kim, Department of Medicine, New York Medical College, Valhalla, NY 10595. Phone: 914-594-4730; Fax: 914-594-4732; E-mail: kbsnep{at}yahoo.co.kr
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Introduction |
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Endothelial cells are the major target for all members of the VEGF family. VEGF-A is a potent endothelial-specific mitogen and a stimulator of angiogenesis. Furthermore, VEGF is a vascular permeability factor, possibly acting by promoting endothelial fenestration (8). The role of VEGF in the maintenance of highly permeable and extensive vasculature in different tumors and in endocrine organs has been established (9).
In the kidney, the peculiar anatomy of microvasculature is characterized by the first division of arterioles into glomerular capillaries, which is followed by the "rete mirabile" of the second subdivision of the efferent arterioles into peritubular capillaries. Both the glomerular and peritubular capillary endothelium is characterized by extensive fenestration and high permeability to solutes. This analogy with the microvasculature of endocrine organs and tumors provided an impetus for investigations into the production of VEGF in the different nephron segments of the kidney (9). VEGF has been found to be produced by the glomerular epithelial cells, podocytes, where it has been tentatively linked to the formation of fenestrae in glomerular endothelial cells. Little is known, however, about peritubular capillaries and the mechanisms of their extensive branching, maintenance, and fenestration. In the remnant kidney model, Kang et al. (10) observed a strong correlation between the number of peritubular capillaries and tubular VEGF expression. Changes in VEGF production have been also studied under hypoxic and diabetic conditions, yielding some controversial findings (11,12).
It is difficult, however, to examine the role of VEGF, especially in the presence of other growth factors, in the maintenance of glomerular and peritubular capillaries in vivo; furthermore, both heterozygous and homozygous VEGF knockout mice are not viable (13), which effectively limits investigative manipulation of VEGF production in vivo. For this reason, we elected to study epithelial-endothelial cell interactions in vitro in a co-culture system. Here, we demonstrate (1) that VEGF produced by glomerular epithelial or proximal tubular cells in culture is necessary and sufficient for branching angiogenesis under control conditions and (2) that hypoxic environment stimulates VEGF production by both epithelial cell types and augments branching angiogenesis, whereas (3) hyperglycemic microenvironment, although also stimulatory for VEGF production, fails to augment angiogenesis.
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Materials and Methods |
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Rat glomerular epithelial cells (GEC) were established by Dr. Esai Noiri (University of Tokyo, Japan), as (described previously 15). Briefly, 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 (Life Technologies, BRL, Gaithersburg, MD). After colonies were formed, the medium was aspirated and high-titer (108 virus/ml) wild-type simian virus-40 (SV-40) was added for 60 min. 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 (results not shown), confirming the adequacy of transfection. Several markers of GEC were expressed by these cells. The Wilms tumor protein has been detected in situ in podocytes, and immunocytochemical staining also showed the presence of this marker in GEC. Moreover, nephrin mRNA was detected in GEC. A monoclonal antibody GSA3 recognizing a cell-specific surface antigen on podocytes (19) showed positive immunostaining of GEC. Furthermore, puromycin exerted cytotoxic effect in GEC, and cells lacked markers characteristic of endothelial and mesangial cells—von Willebrand factor and Thy-1 antigen (not shown). These findings collectively 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. GEC were grown in K-1 medium (Nipro). At confluency, medium was replaced with the serum-free and growth factor-free medium (EBM-2, Clonetics). Cells were divided to five experimental groups similar to those described for the RPTEC.
Human umbilical vein endothelial cell (HUVEC) were obtained from Clonetics Corp and grown in endothelial basal medium-2 (EBM-2, Clonetics) as preiviously detailed (15).
RPTEC-HUVEC or GEC-HUVEC Sandwich Co-Cultures
To examine the effect of RPTEC or GEC on the phenotype of endothelial cells, we applied the co-culture model using these two cell lines. RPTEC or GEC were plated in 24-well clusters and cultured for 24 h in REBM or K-1, respectively. Cells were subjected to hypoxia, or 30 mM D-glucose or L-glucose or normoxia for 24 h, as described above. After 24 h under the above conditions, culture medium was aspirated, 100 ul of growth factor–depleted basement membrane matrix (Matrigel; Fisher Scientific, Pittsburgh, PA) diluted with growth factor-free EMB-2 was used as an overlay and was allowed to gel for 30 min in the incubator at 37°C. This step was followed by the addition of 1 ml of EBM-2 serum-free and growth factor–free medium. Thereafter, 30,000 HUVEC cells were added to each well to complete a sandwich co-culture, as described previously (15). Co-cultures were incubated for additional 24 to 96 h without addition of any growth factors or serum.
To study the role of VEGF in capillary tube formation, parallel cultures were treated with 1 µg/ml neutralizing anti-VEGF antibody (PeproTech, Rocky Hill, NJ), which was added at the time of HUVEC seeding.
Angiogenesis (capillary cord formation) was evaluated after 24, 48, and 96 h in co-culture. To quantify the rate of angiogenesis, digital images of 5 fields/well in each group were obtained under the low-power objective using an inverted Nikon microscope and a CoolPix camera (Nikon, Japan). Images were superimposed on a digital grid using Photoshop Pro 5 program (Adobe, Mountain View, CA). The number of capillaries crossing the grid was calculated for each image, and the average number per each field in the experimental group was calculated according to the previously described technique (16). We referred to this number as the angiogenesis score.
Transmission Immunoelectron Microscopy
To visualize the VEGF distribution in the kidney, immunoelectron microscopy of rat kidney sections was performed as previously detailed (15). Rat kidneys were fixed in 4% paraformaldehyde/0.5% glutaraldehyde (GA) in phosphate buffered saline (PBS), pH 7.4. For immunohistochemistry using electron microscopy (EM), 1-mm3 tissue blocks of GA-fixed kidneys were washed with PBS, dehydrated in ethanol, and embedded in LR-white resin (Polysciences, Washington, PA). For EM morphology, similar tissue blocks were post-fixed 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 approximately 60 nm were mounted on 200 mesh nickel grids, coated with formvar film, and impregnated with carbon. The sections were treated with 0.5 Tween 20 (blocking buffer) for 15 min, labeled with polyclonal anti-VEGF (Santa Cruz Biochemicals, Santa Cruz, CA) 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, England) 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.
Relative Quantitative Reverse Transcription-PCR (RT-PCR)
Oligonucleotide primers were designed to amplify VEGF mRNA from GEC and RPTEC cells to identify unique VEGF isoforms. The sequence of the sense primer was 5'-GGACATCTTCCAGGAGTACC-3', and the sequence of the antisense primer was 5'-GTTCCCGAAACCCTGAGG-3'. Total RNA was isolated from GEC and Clone 8 cells with Trizol total RNA isolation reagent (Life Technologies BRL), and the mRNA was then reverse transcribed to cDNA with AMV reverse transcriptase and amplified with expand high fidelity enzyme mix, which were provided in the Titan One Tube RT-PCR System (Boehringer Mannheim, Indianapolis, IN). Equal amounts of mRNA (0.5 µg) from different samples were used in one 25-µl reaction containing 1 x RT-PCR reaction buffer, 0.2 mM dNTP, 5 mM DTT, and 0.4 uM each primer. The RT-PCR profile consisted of a 30-min incubation at 50°C and 2-min denaturation at 94°C and was then followed by 10 s of denaturation at 94°C, 10 s of annealing at 55°C, and 20 s of elongation at 68°C. 18S mRNA internal control was also co-amplified by incorporate 18S/18S competition mRNA primer (Ambison, Austin, TX) in an another reaction tube. The appropriate 18S:18S Competimer ratio and optimal PCR cycles were selected for each cell line according to manufacture-suggested procedure. Products were analyzed by running 10% of the reaction mixture on a 2% agarose gel with ethidium bromide staining. The ratios of VEGF to 18 sRNA were obtained in at least four independent experiments. The sequences of primers of other VEGF which we used are as follows:
RT-PCR amplification of mRNA encoding for VEGF receptors 1 and 2 was performed following the above protocol. Sense and antisense primers used were as follows:
Immunoprecipitation
After exposure to different culture condition, media from both cell lines were collected and centrifuged for 5 min at 10,000 rpm to remove cell debris. Cells were harvested and lysed in lysis buffer (RIPA buffer containing 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 0.01 M Tris, pH 8.0, 0.14 M NaCl) containing proteinase cocktail (Roche, Mannheim, Germany) for 30 min at 4°C. The cell lysates were centrifuged at 10,000 rpm for 15 min, and supernatant fractions were collected. For detecting VEGF, media and cell lysates were subjected to immunoprecipitation. Briefly, protein concentration in each group of cell lysates was balanced according to the amount of protein in cell lysates. Protein-balanced medium and cell lysates were kept overnight at 4°C on a rocker with addition of 1 µg/ml rabbit anti-human VEGF polyclonal antibody (Santa Cruz Biochemicals). Next, 15 µl of GammaBind plus sepharose beads (Pharmacia, Uppsala, Sweden) were added for another 2 h. The sepharose beads were collected by centrifugation, washed two times in RIPA buffer and one time in TSA buffer (0.01 M Tris, 0.14 M NaCl, pH 8.0), and one additional wash was performed with 0.05 M Tris, pH 8.0. After boiling for 5 min in SDS gel loading buffer, supernatants were collected and divided into two portions: with and without addition of 2.5% 2-mercaptoethanol. Twenty-microliter samples were electrophoresed on a 4 to 20% SDS polyacrylamide gel. Separated proteins were blotted onto PVDF membranes (Millipore, Bedford, MA), blocked in PBS containing 1% casein for 60 min and incubated overnight at 4°C in 1:3000 dilution of primary mouse anti-human VEGF monoclonal antibodies (Santa Cruz Biochemicals). After intense washing, the membranes were incubated with 1:5000 dilution of secondary horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Life Science, Arlington Heights, IL) for 1 h at room temperature. The membranes were thereafter washed once again and incubated in enhanced chemiluminescence substrate reagent (Amersham) for 1 min. The blots were exposed to x-ray film for 1 to 5 min, and the molecular weight of immunodetected bands was compared with molecular weight standards (Norvex, CA). To further confirm the balanced loading of media and cell lysate for immunoprecipitation, Western blot analyses of 
-tubulin was performed. Results were quantitated by scanning films and determining band density by using Scion-image beta-3b software.
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Analysis of VEGF-B showed that 24-h hypoxia resulted in an almost 50% decrease of the mRNA expression in GEC (Figure 4). There was no change of in VEGF-C mRNA expression after hypoxia in GEC (data not shown).
When GEC were exposed to 30 mM D-glucose versus L-glucose and each group compared with control cells incubated in the presence of 5 mM D-glucose, there were no significant changes of mRNA expression in GEC. However, VEGF protein expression in GEC after D-glucose was increased 1.5-fold (P < 0.01). In RPTEC, there was a 1.9-fold increase of VEGF mRNA expression after the exposure to high D-glucose (P < 0.05). VEGF120 was increased significantly, but VEGF164 did not change. In addition, VEGF protein expression was increased 1.45-fold (P < 0.01) in 30 mM D-glucose group compared with control.
In GEC subjected to combined treatment with high D-glucose and hypoxia, VEGF protein abundance showed 1.3-fold increase without significant increase in mRNA expression. In RPTEC, VEGF mRNA expression significantly increased (4.7-fold) in combined treatment group, which was higher than in the 30 mM D-glucose group, but there was no additive increase compared with hypoxic group. VEGF120 and VEGF164 mRNA expression were also increased 4.6- and 4.9-fold, respectively, in RPTEC. Combining 30 mM D-glucose and hypoxia resulted in a 1.4-fold increase in VEGF protein expression (P < 0.01).
VEGF Is a Necessary and Sufficient Growth Factor Supporting Capillary Formation under Control and Hypoxic, but Not Hyperglycemic, Conditions
To examine the functional role of VEGF produced by GEC and RPTEC, we next performed co-culture experiments using a sandwich epithelial-endothelial cell configuration separated by a growth-factor-depleted Matrigel. By the experimental design, any observed effects of epithelial cells on morphology of HUVEC in this system could be attributed to growth factors generated by the epithelium. In a separate series of experiments, neutralizing VEGF antibodies were introduced into co-cultures after 24 h to examine the role of VEGF among the secretory growth factors. Figure 6 shows representative images of capillary tube formation by HUVEC in co-culture system and grids that were used to quantify the angiogenesis score. The data demonstrated (Figures 6 and 7) that RPTEC supported capillary cord formation and sprouting of endothelial cell (HUVEC cultured alone do not survive for 24 h without serum or growth factors; not shown). This process was enhanced 1.2-fold by RPTEC exposed to hypoxia for 24 h before plating HUVEC (P < 0.05). When RPTEC were exposed to 30 mM D-glucose, significant decrease of capillary tube formation was detected, compared to control group after 24 h (P < 0.01). Defective angiogenic response could not be accounted for by decreased expression of VEGF receptors; in fact, both hypoxia and hyperglycemia resulted in the increased abundance of mRNA encoding for VEGFR1 and VEGFR2 (Figure 8). Under the conditions of combined hypoxia and 30 mM D-glucose, capillary tube formation was significantly decreased compared with the hypoxia group but not different from the control group. After administration of neutralizing VEGF antibodies, significant regression of capillary tube formation was observed in both the control and hypoxic groups. There was no significant change in capillary tube formation in high D-glucose or L-glucose groups. Under all these conditions, administration of the neutralizing VEGF antibodies, but not the irrelevant IgG (data not shown), resulted in a dramatic regression of endothelial capillaries. The above quantitative experimental data presented in Figures 2 through 8 are summarized qualitatively in Table 1 for clarity of presentation.
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Discussion |
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In the rat, normal kidney showed diffuse expression of VEGF in all tubules of the renal cortex and medulla (20). However, proximal tubules in the human kidney exhibit only faint, if any, labeling of VEGF mRNA and VEGF protein when studied by in situ hybridization and immunohistochemistry (19,20,21). Only one publication reported that human proximal tubular cells produced VEGF in culture (22). However, these researchers found no significant increase of VEGF mRNA expression after hypoxia. In contrast, we detected a robust constitutive VEGF expression in proximal tubular epithelium of the rat kidney using immunoelectron microscopy. We confirmed this finding of VEGF expression under normal condition in cultured human proximal tubular cells by RT-PCR and immunoprecipitation. These findings prompted us to search for the possible role of VEGF produced by proximal tubular epithelium in the maintenance of peritubular capillaries using an in vitro sandwich co-culture system.
As summarized in Table 1, we found that VEGF mRNA and protein expression after hypoxia were increased in both RPTEC and GEC. Hypoxia is a well-established potent stimulus for VEGF production in some cells, acting by inducing transcription of VEGF gene and stabilization of VEGF mRNA (23,24). Our results showed that VEGF164 was increased in both RPTEC and GEC after hypoxia, whereas VEGF120 was increased only in the RPTEC. The observed differences could be due to the cell type-specific variations in VEGF gene transcription, translation, or post-translational modifications and were beyond the scope of this study.
Previous studies showed that both the low and the high ambient glucose increase the production of VEGF in several cell types (25,26,27,28). There is little data, however, on the effect of high glucose on VEGF expression by renal epithelial cells. One study showed that high glucose induced upregulation of VEGF mRNA and protein expression in podocytes (29). In our experiments, GEC incubated in high glucose showed an increase of VEGF protein expression only, whereas there was an increase of both VEGF mRNA and protein expression in RPTEC.
When high D-glucose and hypoxia were combined, VEGF mRNA and protein expression was higher than control in both cell types (except that mRNA was unchanged in GEC) but there was no additivity of the effects of high D-glucose and hypoxia.
These data collectively established the fact of VEGF production and hypoxic and hyperglycemic regulation of this process in proximal tubular epithelium. Its functional role was approached using a co-culture system, consisting of proximal tubular epithelial cells and endothelial cells. Our zero-hypothesis was that, if the epithelial cells produce VEGF, this growth factor could support the angiogenic phenotype of endothelial cells in the absence of any other added growth factors. We observed that capillary tube formation occurred after 24-h co-culture under normoxic conditions. When RPTEC were exposed to hypoxia before the initiation of co-culture, the capillary tube formation was significantly increased. This finding was consistent with the observed hypoxic induction of VEGF and suggested that it was this VEGF induction that enhanced capillary formation. Under both circumstances, capillary pruning occurred after application of neutralizing VEGF antibodies, further confirming the primary role of VEGF in the in vitro angiogenesis, when all other growth factors have been intentionally depleted from the medium.
There were no significant differences in angiogenesis of L-glucose–treated cells compared with control. In spite of the fact that VEGF production in high D-glucose–treated epithelial cells was increased, capillary tube formation was significantly reduced compared with euglycemic control. In addition, we observed that capillary tube formation was decreased in the group with combined hypoxia and high D-glucose compared with hypoxia alone. The possible causes for suppressed angiogenic competence of HUVEC in hyperglycemic environment, though not examined in this study, have been the focus of previous work (30,31). It has been demonstrated that VEGF receptor expression was depressed in patients with diabetes mellitus, inhibiting neovascularization of transplanted islets (32). This was not the case in our HUVEC, which showed a modest increase in mRNA for VEGF receptors 1 and 2, suggesting that the short-term hyperglycemia does not downregulate these receptors. Furthermore, it has been reported that diabetic environment delayed angiogenic response to HGF, uPA, and uPAR (32) due to development of endothelial dysfunction (33,34). Potential changes in the expression of glucose transporters, although not studied by our group, have been examined previously (35). It has been demonstrated that endothelial cells (except retinal endothelium) are unable to downregulate the expression of glucose transporters in hyperglycemia and therefore remain continuously exposed to the elevated glucose influx and oxidant stress (36,37,38). These data collectively indicate that preexisting endothelial dysfunction may modify cell responsiveness to VEGF and that VEGF under these circumstances becomes insufficient for angiogenesis. We and others have previously demonstrated that endothelial generation of nitric oxide is a prerequisite for cell migration in response to VEGF or endothelin-1 and that responsiveness of endothelial cell to motogenic stimuli may become aberrant in hyperglycemic states or upon exposure to glycated matrix proteins (30,34,39–43). It should be emphasized, however, that any extrapolations of data from our experiments in a model system to the whole animal or humans with diabetes mellitus require most judicious precaution.
In addition to the above changes in VEGF-A mRNA and protein expression, mRNA for VEGF-B was significantly decreased after the exposure to high glucose and hypoxia compared with control. There were no detectable changes in the abundance of VEGF-C mRNA, effectively eliminating the potential contribution of these factors to our data. These results and the clear-cut effect of anti-VEGF antibodies definitively attribute the observed co-culture angiogenic phenomena to changes in VEGF-A produced by epithelial cells. Table 1 summarizes comparative characteristics of RPTEC and GEC under basal and stress conditions.
Although the function of VEGF in the glomerulus is thought to be related to the fenestration of glomerular endothelium and increased permeability, thus facilitating glomerular filtration, the role of VEGF produced by the proximal tubular epithelial cells has been enigmatic. Our study shows (1) that proximal tubular epithelial cells constitutively produce VEGF, (2) that its production is dramatically upregulated by hypoxia and by high glucose, (3) that VEGF secreted by proximal tubular epithelial cells support angiogenesis in culture, and (4) that hyperglycemic environment suppresses hypoxia-induced angiogenesis. We hypothesize that the regulated production of VEGF by the proximal tubular epithelium may play a profound role in the regulation of peritubular capillary network.
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in hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis. Nature 394: 485–490, 1998[CrossRef][Medline]
stimulate vascular endothelial growth factor production in human proximal tubular cells. Kidney Int 58: 43–50, 2000[CrossRef][Medline]
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