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Mechanisms of endothelial response to oxidative aggression: protective role of autologous VEGF and induction of VEGFR2 by H₂O₂

¹Laboratorio de Nefrología-Hipertensión, Fundación Jiménez Díaz; and ²Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain

Submitted 5 December 2005 ; accepted in final form 18 April 2006

	ABSTRACT

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The defense mechanisms of endothelial cells (EC) against reactive oxygen species (ROS) are insufficiently characterized. We have addressed the hypothesis that vascular endothelial growth factor (VEGF) and its receptors are relevant elements in this response. Cell viability, VEGF and VEGF receptor (VEGFR1 and VEGFR2) expression, and transcription factor activation were studied on transient exposure of monolayer EC to H₂O₂. Wild-type and mutant inhibitors of B (IB) constructions were used to further assess the role of NF-B in the induction of VEGFR2 expression. A concentration of H₂O₂ 60 µM elicited clear-cut damaging effects on EC, whereas lower concentrations (2–4 µM) were cytoprotective. The cytoprotective effect was shifted to an EC-damaging pattern by means of specific VEGF blockade, therefore revealing a major role of autologous VEGF. Exposure to H₂O₂ increased VEGF and VEGFR2 mRNA and protein in EC, without affecting VEGFR1 expression. Also, H₂O₂ challenge was accompanied by increased NF-B, activator protein-1, and specific protein-1 nuclear binding. A role of NF-B as the mediator of the H₂O₂ induction of VEGFR2 mRNA expression was supported by inhibition by the ROS scavenger pyrrolidine dithiocarbamate and by the blocking effect of transfected IB. Exposure to exogenous VEGF also increased VEGFR2 and induced NF-B in EC. In summary, autologous VEGF is instrumental for EC protection induced by low concentrations of ROS. ROS induce expression not only of VEGF but also of VEGFR2. VEGFR2 increase by ROS is mainly driven through a NF-B-dependent pathway.

cytoprotection; reactive oxygen species; vascular endothelial growth factor receptor; nuclear factor-b

Most of the studies dealing with the effects of ROS on endothelial cells (EC) have been devoted to examining their injuring capability (18). On the other hand, the mechanisms of vascular defense against ROS have received considerably less attention. In fact, at moderate, nontoxic concentrations, ROS act as physiological signal transduction messengers (18). In the same line of evidence, a variety of natural stimuli works by changing the cellular redox state through ROS formation as a part of the normal intracellular signaling network (18). In practical terms, the characterization of the factors involved in the ability of the vessel wall to mount an efficient protective response against ROS-mediated injury has outstanding pathophysiological and therapeutic implications.

Among the factors putatively involved in the response of the endothelium to oxidative challenge is VEGF, the importance of which is emphasized by recent data. The VEGFs and, in particular, VEGF-A (hereinafter referred to as VEGF) have a critical role in angiogenesis and vascular permeability. VEGF signal in arterial and venous EC is transduced through two main receptors with tyrosine kinase activity, namely, VEGFR1 and VEGFR2 (6). Although the initial investigations on the role of VEGF were mainly focused on its angiogenic and permeabilizing properties, a number of studies, including ours, have given support to outstanding properties of VEGF as a mediator of EC survival (3, 7).

Endothelial and vascular smooth muscle cells are both capable of producing VEGF under diverse stimuli (3, 5, 6, 8, 15). More specifically for the present study, a stimulatory effect of H₂O₂ on VEGF expression by EC and vascular smooth muscle cells has been described, although without characterizing other VEGF-related responses, e.g., VEGF receptor expression or the actual functional role of autologous VEGF in the cellular response against oxidation (5, 15). The fact that the expression of VEGF receptors, particularly VEGFR2, is stimulated in conditions coincident with VEGF induction, e.g., tumor growth, suggests that the exposure to H₂O₂ may induce changes not only on VEGF but also on its receptors, which can be putatively related to EC defense mechanisms. Moreover, the possibility that endogenous VEGF is involved in endothelial protective actions during oxidative stress has not been specifically addressed. The present study, therefore, has been aimed at examining the effects of ROS challenge on the survival properties of EC and in the regulation of VEGF and VEGF receptor expression. Experiments on mechanisms were more specifically focused on the role of the transcription factor NF-B, for its particular importance in the response to oxidative challenge and its critical role on VEGFR2 expression.

	MATERIALS AND METHODS

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Cell culture and experimental maneuvers. Bovine aorta EC were cultured and characterized as previously described (1, 3). Challenge with H₂O₂ (30 min) used a range of concentrations involving those reported in normal in vivo conditions (12). A specific anti-VEGF MAb (1 µg/ml; Sigma-Aldrich, Madrid, Spain) was used to inhibit VEGF effects. This antibody has been extensively tested in our laboratory and blocks the effects of VEGF₁₆₅ within a range of physiological and supraphysiological concentrations, i.e., up to 10^–9 M (1, 3). Phorbol myristate acetate (200 nM, Sigma-Aldrich) was employed as positive control for the stimulation of expression of both VEGF and VEGF receptors, and pyrrolidine dithiocarbamate (PDTC) (Sigma-Aldrich) was used as ROS scavenger and inhibitor of NF-B activation.

Citotoxicity assay. Confluent EC were preincubated as previously described (3) for 18 h in MEM D-Val containing 20% FBS and 0.5 µCi/ml of sodium ⁵¹Cr (Amersham Biosciences, Barcelona, Spain). EC were exposed to ROS or vehicle and incubated for an additional 24 h in MEM D-Val with 0.5% FBS and without ⁵¹Cr. Supernatant fractions (200 µl) were sampled and counted at different times, and ⁵¹Cr-release was calculated with respect to total ⁵¹Cr content. Flow cytometry was used as a confirmatory method, as previously described (1, 3, 4). The length of ROS exposure (30 min) was designed to resemble a transient challenge, as occurs in episodes of limited in vivo ischemia-reperfusion, e.g., in revascularization techniques.

Northern blot analysis, RT-PCR, and real-time quantitative PCR. Total RNA was extracted from confluent EC at 0, 6, and 24 h after different treatments. Northern blot analysis and routine RT-PCR were carried out with the use of a probe and specific primers of bovine VEGF, VEGFR1, and VEGFR2, based on previously described sequences [National Center for Biotechnology Information GenBank (1, 3, 4)]. For real-time quantitative PCR, primers and TaqMan MGB probe were designed using the Primer Express software (Applied Biosystems, Foster City, CA) from VEGFR2 sequence: forward, 5'-TCT CCG TTA TTG CTT CTG TTA G-3'; reverse, 5'-GTG ATA CCT TGC ACA GAG TGA CAC-3'; and TaqMan MGB probe, 5'-ACA AAA AAC AAA ACT GAC AC-3' were used. cDNA was synthesized from 2 µg of total RNA, and real-time quantitative PCR was carried out with the ABI PRISM 7700 Systems (Applied Biosystems), following the manufacturer's instructions. PCR amplification of 18S rRNA was done for each sample as loading control and to allow normalization between samples. The mRNA fold changes were calculated on four triplicate experiments by using the comparative critical threshold cycle (CT) 2^–CT value. Results were expressed as fold changes relative to unstimulated cells, after normalization against 18S rRNA, as previously described (20).

Protein extraction and Western blot analysis. Proteins were extracted in lysis buffer [50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% (vol/vol) Igepal, 0.5% (wt/vol) deoxycholic acid, and 0.1% (wt/vol) SDS] and processed as previously described (1, 8). Proteins (50 µg) were electrophoretically separated by 6% SDS-PAGE and transferred to nitrocellulose (Bio-Rad, Madrid, Spain). Blocked membranes were incubated with blocking buffer containing rabbit polyclonal IgG anti-VEGFR2 (1:1,000, Calbiochem, La Jolla, CA) at 4°C overnight and then with blocking buffer containing goat anti-rabbit IgG-horseradish peroxidase conjugated (1:6,000, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. Membranes were developed by using the ECL system and exposed to Hyperfilm ECL (Amersham Biosciences). Mouse IgG MAb anti--tubulin (1:2,000; Sigma-Aldrich) and goat MAb anti-mouse IgG-horseradish peroxidase conjugated (1:6,000; Bio-Rad) were respectively used as loading controls.

Immunocytochemistry. Confluent EC grown on glass chamber slides (Labclinics, Barcelona, Spain) were submitted to the different experimental maneuvers, washed with PBS, and fixed in 95% ethanol. Immunostaining was performed by a modification of the avidin-biotin-peroxidase complex method, using a specific anti-VEGF antibody raised in our institution, as previously reported (3). The antibody was prepared by rabbit immunization with the peptide APMAEGGGQNHHEVVKFM coupled to keyhole limpet hemocyanin. The sequence was chosen from a common region for both human and bovine. The antibody was affinity purified by a peptide-bound thiopropyl-Sepharose column (Pharmacia Biotech, Uppsala, Sweden). The specificity of the anti-VEGF polyclonal antibody was checked against commercial VEGF (Sigma-Aldrich). Parallel cultures were treated with nonimmune rabbit IgG as negative controls. Cells were incubated with biotinylated swine anti-rabbit IgG (DAKO, Glostrup, Denmark, 30 min), sequentially followed by avidin-biotin-peroxidase complex (DAKO) and 3,3 9-diaminobenzidine (Sigma-Aldrich).

Plasmids and transfections. Overexpression of NF-B regulatory subunits was used for blocking NF-B activation. Because phosphorylation (Ser32 and Ser36) of inhibitors of B (IBs) by IB kinase is necessary for their release from NF-B and subsequent degradation, two vectors, namely, pcDNA3-IB-wt and pcDNA3-IB-mt, were transfected and overexpressed. The former leads to competitive blockade of phosphorylation due to excess IB; the latter has mutated Ser36 for alanine, therefore impeding phosphorylation and degradation; this leads to a persistent binding of the inhibitory subunit to NF-B. Both constructions were kindly provided by Dr. J. Alcamí (Centro Nacional de Microbiología, Instituto de Salud Carlos III). Transient transfection of EC was carried out with the use of FuGENE 6 transfection reagent (Roche Diagnostics, Barcelona, Spain) according to the manufacturer's protocol. The optimal transfection of plasmid DNA was achieved at a 6:2 (vol/wt) ratio of transfection reagent to DNA complex, with a transfection efficiency of 36.9 ± 2%. Flow cytometry was used to evaluate the transfection efficiency with pEGFP-N1 vector (BD, Madrid, Spain). A pcDNA3-empty vector was used as control of nonspecific effects of the transfection (data not shown).

Nuclear extraction and electrophoretic mobility shift assay. Nuclear proteins were extracted as previously described (1) at 0, 1, 5, 10, and 180 min after H₂O₂ challenge (2 and 250 µM, 30 min). For the case of NF-B, a specific concentration-response curve was also performed 6 h after exposure to H₂O₂. Nuclear proteins of untreated EC were used as controls. Electrophoretic mobility shift assay was performed by using nuclear proteins (6 µg) and different consensus oligonucleotide sequences (dsDNA, 0.5 ng) labeled with [-³²P]dATP in 1x binding buffer. Poly(dI-dC)·Poly(dI-dC) was included as a competitive DNA. After incubation, samples were separated by electrophoresis in 6% acrylamide-bisacrylamide gels and exposed to X-Omat films. Commercial oligonucleotides (Santa Cruz Biotechnology) were used for activator protein-1 (AP-1), specific protein-1 (Sp-1), and NF-B electrophoretic mobility shift assays. Supershift with specific antibodies (anti-c-Fos, anti-c-Jun, anti-p65, and anti-p50) and competitive assays in the presence of consensus cold oligonucleotides (50x) were performed to assess the specificity of the bands. The usefulness of the antibodies in the present setting had been probed in previous studies.

Statistics. Data are shown as means (SD) and, unless stated otherwise, correspond to at least four triplicate experiments. Paired and unpaired Student's t-tests were used when appropriate. Multiple comparisons were done by one-way ANOVA and Scheffé's tests (StatView and SPPS 10.0 packages, Jandel, San Rafael, CA; and Windows). A P value <0.05 was considered significant.

	RESULTS

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Role of autocrine VEGF on EC viability under oxidative stress. Exposure to H₂O₂ elicited a dual response on EC; namely, at concentrations from 60 µM to 1 mM, a cytotoxic effect was evident. On the contrary, at lower concentrations (2–4 µM), cytotoxicity was no longer evident; instead, a moderate, albeit consistent, cytoprotective effect was observed (significant decrease with respect to basal conditions; Fig. 1A). As a confirmation of these results, a similar concentration-response pattern was observed by means of flow cytometry analysis of cell death (data not shown). To examine the role of VEGF in the observed responses, similar experiments were conducted in the presence of a specific blocking anti-VEGF MAb. These experiments resulted in a clear-cut shifting from protective or neutral effects to cell-damaging effects of H₂O₂ (Fig. 1B). However, at a higher H₂O₂ concentration (250 µM), no further increase in ⁵¹Cr-release was induced in the presence of anti-VEGF MAb.

View larger version (13K): [in this window] [in a new window]   Fig. 1. A: effect of different concentrations of H2O2 on 51Cr release by endothelial cells (EC). 51Cr release (24 h) was used for evaluation of cytotoxicity, as previously described (3). Concentration-response curve extends from 0 (basal conditions, no exogenous H2O2 added) to 1 mM H2O2. *P < 0.05, **P < 0.01, #P < 0.005 with respect to basal conditions. Data are expressed as means (SD) of percentage of 51Cr release with respect to basal conditions. Unless stated otherwise, in this and all the other experiments, each value corresponds to a minimum of 4 experiments done by triplicate. Note that the bars are represented with respect to baseline 51Cr release, which is situated at the x-axis; therefore, values above or below baseline are consistent with increased or decreased cell damage, respectively. B: effect of VEGF blockade on cytoprotection by H2O2: changes in percentage of 51Cr release in the presence of anti-VEGF MAb (1 µg/ml). *P < 0.05 and **P < 0.01 between cells incubated with (solid bars) or without (shaded bars) anti-VEGF MAb. Controls used identical conditions in the presence of a nonspecific IgG antibody (1 µg/ml).

Induction of VEGF and VEGFR2 mRNA and protein expression on H₂O₂ stimulation. Exposure of EC to H₂O₂ increased VEGF mRNA expression in a concentration-dependent manner between 0.5 and 10 µM (Fig. 2A); this stimulatory effect was not evident at higher H₂O₂ concentrations (100–250 µM). In a time-response curve (2 µM H₂O₂, n = 2), the expression of VEGF was significantly increased from 3 h (P < 0.05), was maximal at 6 h (P < 0.01), and returned to baseline [P = not significant (NS) with respect to time 0] at 24 h (data not shown). VEGF protein markedly increased with 2 µM H₂O₂ (Fig. 2B, 6 h); however, the immunocytochemical signal for VEGF became faint with 250 µM H₂O₂ (image not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 2. Effects of H2O2 on mRNA expression of VEGF. A: Northern blot analysis. Induction of the expression (6 h) of VEGF (top lane) by H2O2 is shown (0.5–250 µM, 30 min); housekeeping 28S was used for comparison (bottom lane). This experiment is representative of 3 experiments, giving similar results. Mean values of the 3 experiments are shown in bar diagram. *P < 0.05, **P < 0.01 with respect to baseline. B: VEGF immunocytochemistry (magnification x100). Increased VEGF immune signal (6 h) as induced by 30 min exposure to 2 µM H2O2. Inset: nonstimulated cells.

View larger version (15K): [in this window] [in a new window]   Fig. 3. A: real-time quantitative PCR. Induction of the expression of VEGF receptor 2 (VEGFR2) at 2 and 250 µM H2O2 (6 h after 30 min exposure). *P < 0.05, **P < 0.001 with respect to baseline. Concentrations were chosen on the basis of results shown in Fig. 1. B: Western blot analysis of VEFGR2. EC were exposed to 2 and 250 µM H2O2 (24 h after 30-min exposure). Top lane: VEGFR2 protein. Bottom lane: -tubulin. Phorbol myristate acetate (PMA) (200 nM) was used as control of VEGFR2 protein synthesis. C: VEGFR2 expression response to H2O2 in the presence and absence of anti-VEGF MAb. EC were exposed to 2 and 250 µM H2O2 (24 h after 30 min exposure). Top lane: VEGFR2 mRNA. Bottom lane: GAPDH, used for standardization. This figure shows result of 1 out of 3 experiments with similar results.

View larger version (18K): [in this window] [in a new window]   Fig. 4. A: VEGFR2 mRNA expression response to exogenous VEGF. EC were exposed to exogenous VEGF (20 ng/ml, 6 and 24 h). Top lane: VEGFR2 mRNA. Bottom lane: GAPDH. PMA was used as control. B: Western blot of VEGFR2. EC were exposed to exogenous VEGF (20 ng/ml, 24 h) and 2 and 250 µM H2O2 (24 h after 30 min exposure) in the presence or absence of anti-VEGF MAb (1 µg/ml). Top lane: VEGFR2 protein. Bottom lane: -tubulin. Pyrrolidine dithiocarbamate (PDTC) (25 µM) was used as reactive oxygen species scavenger.

Activation of transcription factors: role of NF-B. The transcription factors analyzed were selected on the basis of previously available information on the regulation of VEGF and VEGFR2 expression (6). The electrophoretic mobility shift assays of the transcription factors analyzed revealed different individual responses (Fig. 5) as follows: 1) a brief AP-1 activation appeared with both H₂O₂ concentrations; this increase was detected at 10 min of incubation but was no longer evident at 3 h (Fig. 5A); 2) Sp-1 was rapidly (1 min) activated by the smaller (2 µM), but not by the higher (250 µM), H₂O₂ concentration, and this activation persisted at a high level until 10 min and at a lower but persistent level until 3 h (Fig. 5B); and 3) NF-B has a short-lived, bimodal (1 and 10 min) activation with 2 µM H₂O₂, which did not persist at 3 h. However, due to the potential importance of this factor, additional experiments were done to further trace activation up to 6 h after exposure to H₂O₂; at this time, no changes were observed with 2 µM H₂O₂, but a significant activation of NF-B was present in EC treated with the higher H₂O₂ concentration (250 µM) (Fig. 5C). A consistent activation of NF-B was also obtained by exposure of the EC to VEGF, which increased nuclear NF-B in a concentration-dependent manner (Fig. 6A). The effect of VEGF was already detected at 3 h and persisted up to the longest incubation time (24 h, Fig. 6B).

View larger version (26K): [in this window] [in a new window]   Fig. 5. Measurement of transcription factor activation by electrophoretic mobility shift assay. Retardation gel with nuclear proteins obtained from EC extracted after stimulation with H2O2 (2 and 250 µM) and extracted at different times. Experiments shown are representative of 3 to 5 studies yielding similar results. A: activator protein-1 (AP-1). B: specific protein-1 (Sp-1). C: NF-B. n.s., Nonspecific; NE, nuclear extract.

View larger version (14K): [in this window] [in a new window]   Fig. 6. A: concentration-dependent NF-B activation by exogenous VEGF. Retardation gel with nuclear proteins obtained from EC extracted at 6 h after stimulation with different VEGF concentrations. B: time-dependent response. EC were stimulated with VEGF (20 ng/ml), and nuclear proteins were extracted at indicated times. C: absence of effect of PDTC as a blocker of VEGF-induced increase of NF-B. VEGF-dependent NF-B activation was not blocked by PDTC (25 µM).

View larger version (13K): [in this window] [in a new window]   Fig. 7. A: real-time quantitative PCR. Induction of the expression of VEGFR2 at 250 µM H2O2 (6 h after 30 min of exposure) in the presence or absence of PDTC (25 µM). *P < 0.001 with respect to baseline; **P < 0.05 with respect to the other three conditions. B: real-time quantitative PCR. Effects of inhibitors of B (IB) transfection on induction of VEGFR2 mRNA expression by 250 µM H2O2 (6 h after 30 min of exposure). *P < 0.001 with respect to the other conditions. mt, Mutant; wt, wild-type.

	DISCUSSION

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In terms of mechanism, even though the induction of the expression of its own VEGFR2 receptor by VEGF has been described in different circumstances (4, 19), our results with a blocking anti-VEGF antibody reveal that this type of regulation is not relevant in the case of challenge with H₂O₂. Therefore, in the present experimental conditions, VEGFR2 upregulation appears to be mostly dependent on a direct effect of H₂O₂. Accordingly, the possibility was raised, as previously described (2), of a stimulation of transcriptional elements by redox changes.

The data on transcription factors activation add some clues to the interpretation of the results. The factors activated correspond to those involved in VEGF and/or VEGFR2 transactivation (9, 10, 14). At least three transcription factors, namely, AP-1, Sp-1, and NF-B, were activated by oxidative challenge, as predicted according to previous descriptions (16, 17, 21). Because of its particular importance in the response to oxidative challenge and its critical role in VEGFR2 expression, major interest was focused on NF-B. The pattern of activation of NF-B demonstrated a short-lived peak with the smaller (2 µM) concentration and a longer activation with the higher (250 µM) concentration. The latter was coincident with stimulation of VEGFR2 mRNA and protein expression. Of additional interest, we have found that VEGF per se activates NF-B in a time- and concentration-dependent manner. This result adds further information to data previously published by other groups (13).

To further ascertain the mechanisms of the effect of H₂O₂, additional experiments were carried out by using the cell-permeable ROS scavenger PDTC (2). The inhibition of VEGFR2 expression by PDTC suggested that the effect of exogenous H₂O₂ on VEGFR2 expression was mediated by intracellular ROS, which are scavenged by this agent, e.g., ·OH radical. This result could be interpreted either as a direct effect on ROS or by the NF-B inhibitory effect of PDTC. This specific issue was addressed by transfecting the EC with IB (2). These studies demonstrated an inhibition of the VEGFR2 stimulation by IB, therefore supporting the significance of the role of NF-B in the effect of H₂O₂. Of interest, the effect of VEGF on VEGFR2 expression was not inhibited by PDTC. Because in these conditions VEGF-induced NF-B was not blocked by PDTC, this indicates that relevant differences do exist between the mechanisms involved in NF-B activation by H₂O₂ and VEGF. Albeit of considerable interest, the complete analysis of the molecular basis of these differences is beyond the scope of the present study.

As a complementary remark, AP-1 and Sp-1 activation could have a role in the induction of VEGFR2 expression, but further studies need to be performed to clarify this point. No experiments were done to examine the role of NF-B in the induction of VEGF, due to the existence of previous studies demonstrating such a relationship (6). In the same regard, no in-depth studies were performed on VEGFR1, due to the absence of change in its expression with the experimental maneuvers, with the exception of the previously known stimulation by phorbol myristate acetate (6).

Beyond the description of the mechanisms of combined increase of VEGF and VEGFR2, our results illustrate its functional significance in terms of EC damage and protection. In particular, the present results demonstrate that autologous VEGF has a significant role in the survival of EC in conditions of oxidative aggression; the H₂O₂ concentrations affording EC protection were within a relevant in vivo range. Of importance, the protective concentrations of H₂O₂ were coincident with those eliciting VEGF expression. This finding supports the role of VEGF as a principal component of a protective loop. Even though the action of VEGF in endothelial cytoprotection is known (3), no specific information was available up to date to indicate such a role during oxidative injury. Of particular interest, our main data were obtained in the absence of exogenously added VEGF and are, therefore, more directly related to in vivo effects. Because the effects of H₂O₂ on VEGFR2 span within the damaging range of H₂O₂ concentrations, the increased expression of VEGFR2 might be traced to extreme, albeit functionally ineffective, stimulation of defense mechanisms in a background of severe cellular injury. The results on VEGFR2 protein are further revealing of the actual functioning of this mechanism; i.e., the marked increase in VEGFR2 mRNA with high H₂O₂ concentrations did not result in a proportional increase in VEGFR2 protein. This can be either due to a submaximal stimulation of protein translation or increased protein catabolism, due to oxidative activation of proteases.

In summary, our results add elements of interpretation to the effect of mild oxidative stress as a protective mechanism in EC, indicating that autologous VEGF-related mechanisms are implicated. In other words, our data show that autocrine VEGF makes EC more resistant to injury by oxidative agents; on the contrary, VEGF blockade favors injuring effects by H₂O₂. These effects involve significant changes in VEGF and VEGFR2 gene expression. The latter is first described on oxidative challenge and appears to involve a NF-B-dependent mechanism.

	GRANTS

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This study was supported by grants from Fondo de Investigaciones Sanitarias (FIS 01/0345 and FIS 03/0888), Red Cardiovascular (RECAVA, ISCIII) Comunidad Autónoma de Madrid (CAM 08.3/10/2000 and 10/2002), and Instituto Reina Sofía de Investigación Nefrológica (Entrecanales trust). F. R. González-Pacheco is a postdoctoral investigator of CAM. M. Victoria Álvarez-Arroyo, J. J. P. Deudero, and S. Yagüe are a senior researcher and fellows of ISCIII, respectively. M. Angeles Castilla is a postdoctoral investigator of FIS.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Manuel Ortiz de Landázuri for assistance with the quantitative PCR studies and Dr. José Alcamí [Centro Nacional de Microbiología, Instituto de Salud Carlos III (ISCIII)] for the IB constructions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: C. Caramelo, Laboratorio de Nefrología-Hipertensión, Instituto de Investigaciones Médicas, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Av. Reyes Católicos 2, E-28040 Madrid, Spain (e-mail: ccaramelo{at}fjd.es)

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.

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