Uncoupling of Vascular Endothelial Growth Factor with Nitric Oxide as a Mechanism for Diabetic Vasculopathy
Takahiko Nakagawa,
Waichi Sato,
Yuri Y. Sautin,
Olena Glushakova,
Byron Croker,
Mark A. Atkinson,
C. Craig Tisher and
Richard J. Johnson
Division of Nephrology, Hypertension and Transplantation, University of Florida, Gainesville, Florida
Address correspondence to: Dr. Takahiko Nakagawa, Division of Nephrology, Hypertension and Transplantation, University of Florida, PO Box 100224, Gainesville, FL 32610. Phone: 352-392-2448; Fax: 352-392-5465; E-mail: nakagt{at}medicine.ufl.edu
Received for publication July 22, 2005.
Accepted for publication December 6, 2005.
The role of VEGF in vascular disease is complicated. Vascularendothelial growth factor (VEGF) expression can be deleteriousin diabetic vasculopathy, especially in kidney and retina. Incontrast, VEGF seems to be renoprotective in nondiabetic renaldisease. VEGF exerts it biologic effects in association withnitric oxide (NO), yet it is known that NO bioavailability isreduced in diabetes. Thus, it was hypothesized that this diversebiologic effect of VEGF on diabetic vasculopathy is due to uncouplingof VEGF with NO. VEGF stimulated NO production in a dose-dependentmanner in bovine aortic endothelial cells (BAEC), and this wasinhibited by either high glucose or N-nitro-l-arginine methylester (L-NAME) treatment. Endothelial NO synthase phosphorylationby VEGF was also inhibited by high glucose. It is interestingthat both high glucose and L-NAME enhanced the proliferativeresponse of endothelial cells, which was prevented by an NOdonor. Furthermore, high glucose as well as L-NAME stimulatedVEGF and kinase-insert domain receptor (KDR) (VEGF receptor2) mRNA expression in BAEC. These data suggest that the uncouplingof VEGF with NO enhances endothelial cell proliferation viathe KDR pathway. Compatible with these findings, a KDR antagonistblocked this response. In addition, a VEGF mutant, which bindsonly KDR, induced extracellular signal–regulated kinase(ERK) activation, and inhibition of ERK completely blocked endothelialcell proliferation under this condition, suggesting a role ofthe KDR–ERK1/2 pathway on endothelial cell proliferation.In conclusion, high glucose causes an uncoupling of VEGF withNO, which enhances endothelial cell proliferation via activationof the KDR–ERK1/2 pathway. These results may provide newinsights into the understanding of the mechanism of diabeticvascular disease.
The role of vascular endothelial growth factor (VEGF) is complicatedin the diseased kidney. Recently, we and others demonstratedthat VEGF administration has a beneficial effect in both acuteand chronic nondiabetic renal disease (1–3). In progressiverenal diseases, such as the remnant kidney model and cyclosporinenephropathy, decreased VEGF expression was associated with renaldysfunction and capillary loss, and VEGF administration wasfound to stimulate capillary repair and improve renal function.In contrast, both circulating and local VEGF levels are highin diabetes, and the excessive VEGF has been shown to have arole in mediating glomerular hypertrophy, proteinuria, and retinopathy(4,5). Although the role of excessive angiogenesis induced byVEGF has been implicated most in diabetic retinopathy, abnormalangiogenesis also has been observed in diabetic nephropathy,where it has been linked with progression (6). Recently, newvessel formation at the vascular pole of the glomerulus wasdemonstrated in the diabetic kidney and was correlated withVEGF expression (7). Thus, a simple explanation for the paradoxof VEGF and kidney disease is that diabetic nephropathy involvesan excessive VEGF state, whereas most nondiabetic renal diseasesare low VEGF states. However, whether this is the precise mechanismremains unclear.
VEGF exerts its pleiotropic effects on endothelial cells bybinding to and activating VEGF receptor 1 (VEGFR-1; Flt-1) andVEGFR-2 (kinase-insert domain receptor [KDR]) (8). It is widelyknown that the beneficial effects of VEGF are mediated in partby its ability to stimulate endothelial nitric oxide (NO) releaseand production (9–12). In other words, when VEGF coupleswith NO, it maintains endothelial cell function and structure.However, the bioavailability of endothelial NO is severely reducedin diabetes (13–18). Given the increased expression ofVEGF in diabetes, the reduced NO bioavailability theoreticallycould result in an uncoupling of VEGF with NO. It is interestingthat there is evidence that blockade of endothelial NO resultsin a compensatory increase in VEGF (19), which engages an NO-independentpathway to stimulate endothelial cell proliferation (20). Thismay result in deleterious as opposed to beneficial effects,such as unopposed angiogenesis (via KDR) (21–24). Indeed,we have shown that long-term inhibition of NO synthase (NOS)resulted in excessive endothelial cell proliferation in preglomerularvessels along with de novo VEGF expression in a model of progressiverenal disease (25). Moreover, it has been reported that an increasedVEGF under NOS inhibition was a major factor responsible forvascular injury in coronary artery disease (26). We thus hypothesizethat reduced endothelial NO bioavailability in diabetes resultsin uncoupling of the VEGF–NO axis and that this resultsin VEGF causing diverse biologic effects that could contributeto diabetic nephropathy. Herein, we examined (1) the effectsof glucose on endothelial cell proliferation as well as on NOproduction in response to VEGF and (2) the mechanism by whichuncoupling of VEGF with NO mediates endothelial cell proliferationunder high glucose (HG) conditions.
VEGF (Peprotech, Rocky Hill, NJ), basic fibroblast growth factor(bFGF) (Peprotech), NONOate (NOC18; Sigma, St. Louis, MO), andd-mannitol (Sigma) were used. A mouse mAb to phosphorylatedextracellular signal–regulated kinase (ERK) Ab and polyclonalAb to phosphorylated endothelial NOS (eNOS)-Ser1177, total eNOS,and total p44/42 kinase Ab were obtained from Cell Signaling(Beverly, MA). Uo126 (Cell Signaling) or SU1498 (Calbiochem,San Diego, CA) was used to inhibit the activity of ERK1/2 andKDR, respectively. A plasmid with a VEGF mutant that selectivelybinds to KDR (KDR-sel) was provided by N. Ferrara (Genentech,South California, CA).
Cell Culture
Bovine aortic endothelial cells (BAEC; Cambrex, Walkersville,MD) were cultured in endothelial basal media (EBM) using theBullet kit (Cambrex), which contained FBS, bovine brain extract,hydrocortisone, EGF, and gentamicin/amphotericin B. After confluence,cells were stimulated with various compounds in EBM to examineNO production at 24 h, eNOS activation at 2.5 to 30 min, endothelialcell proliferation at 24 or 48 h, and ERK1/2 activation at 10min to 24 h. Cells between passages 4 and 6 were used for experiments.All experiments were repeated at least three times.
Measurement of NO in Culture Medium
BAEC were seeded at 100,000 cells/well in 24-well plates andgrown to confluence, which rendered them quiescent in EBM withBullet kit. Twenty-four hours later, cells were stimulated withincreasing concentrations of VEGF165 (10 to 50 ng/ml) in EBMthat contained 5 mM glucose (low glucose [LG]), 30 mM glucose(HG), 1 mM N-nitro-l-arginine methyl ester (L-NAME), or 30 mMmannitol. Reactions were terminated by removal of the supernatantthat was subsequently centrifuged and stored at –80°Cfor NO analysis. Levels of total NO were measured in the gasphase using a standardized Seivers NOA 280 chemiluminescenceanalyzer (Analytix, Durham, UK). Results were corrected forbackground levels of NO present in culture medium alone andwere expressed as nM/µg (NO/total protein).
Endothelial Cell Proliferation Assay [3H]Thymidine Incorporation.
BAEC were plated in 24-well plates in growth medium (EBM withBullet kit) at a density of 100,000 cells/well. VEGF in variousconcentrations was added in the presence of 30 mM glucose (HG)or 1 mM L-NAME in serum-free EBM medium. After 48 h of incubation,cells were labeled with 1 µCi/ml [3H] thymidine (AmershamInternational, Bucks, UK). Six hours later, cells were washedwith PBS and fixed in 5% ice-cold TCA. Cells were lysed in 0.5ml of 0.5 N NaOH, and the incorporated [3H]thymidine was measuredin a liquid scintillation counter.
Methylthiazoletetrazolium Assay.
To examine endothelial cell number, we performed the methylthiazoletetrazolium(MTT) assay (MTT assay kit; Sigma). After cells were stimulatedwith various stimuli for 48 h in 96-well plates, 150 µlof MTT solution (5 mg/ml) was added. Two hours later, the formazancrystals that formed were solubilized in MTT lysis solution(10% Triton 0.1 N HCl in isopropanol), and the absorbance wasmeasured in a 96-well plate reader at 570 to 690 nm.
Cell Number.
Alternatively, cell number was determined by measuring of DNAcontent using the CyQuant cell proliferation assay kit accordingto the manufacturer’s protocol (Molecular Probes, Eugene,OR). Data are expressed as a mean ± SD of three independentexperiments.
ERK1/2 Activation with KDR-Sel
Transient transfection of the expression vectors for KDR-seland VEGF was performed in NRK cells (ATCC, Manassas, VA) using2.0 µg of DNA with the Lipofectamine Plus Reagent kit(Life Technologies BRL, Grand Island, NY). Twenty-four hoursafter transfection, cells were maintained in DMEM with 10% serum.VEGF concentration in culture medium of NRK cells was examinedwith the human VEGF ELISA kit (R&D Systems, Minneapolis,MN). Then, confluent BAEC were stimulated with NRK culture mediumthat contained KDR-sel or VEGF.
Real-Time PCR
To quantify mRNA expression for VEGF or KDR, we performed real-timePCR as described previously (27). Briefly, after 1 µgof total RNA was converted to cDNA with Platinum PCR supermix(Bio-Rad, Hercules, CA), PCR was performed as follows: 94°Cfor 5 min, then 35 cycles of denaturation at 94°C for 30s, annealing at 61°C for 1 min, and extension at 72°Cfor 90 s. The sizes of amplicons were 149 bp (bovine VEGF),231 bp (bovine KDR), 165 bp (bovine Flt-1), and 145 bp (bovineglyceraldehyde-3-phosphate dehydrogenase [GAPDH]). Reactionspecificity was confirmed by electrophoretic analysis of productsin 2% agarose gel before real-time reverse transcription–PCR,and bands of expected size were detected. Ratios to GAPDH mRNAwere calculated for each sample and expressed as mean ±SD.
Western Blot Analysis
As described previously (27), 20 µg of cell protein sampleswas resolved on NuPAGE Bis-Tris Gel (4 to 12%) and transferredto polyvinylidene difluoride membranes by electroblotting. Eachprimary antibody was incubated at 4°C overnight. After washingwith TBST, membrane was rocked with secondary Ab (anti-mouseIgG or anti-rabbit IgG, horseradish peroxidase–linkedantibody [Cell Signaling]). The blot then was developed usingthe ECL detection kit (Amersham International).
Inhibition Studies
The MEK inhibitor that blocks ERK1/2 (Uo 126) or the specificVEGFR-2 (KDR) inhibitor (SU1498) was incubated with the BAECfor 30 min before VEGF stimulation. Cell viability in each experimentalcondition was examined by lactate dehydrogenase assay usingthe TOX-7 LDH assay kit (Sigma).
Statistical Analysis
All values presented are expressed as mean ± SD. ANOVAfollowed by Bonferroni correction (ANOVA) or t test was used.Significance was defined as P < 0.05.
HG Inhibits eNOS Activation and NO Production in Response to VEGF in BAEC
First we confirmed that VEGF dose-dependently stimulates NOrelease into the supernatant of culture medium of BAEC at 24h (Figure 1A). Whereas 10 ng/ml VEGF stimulates NO productionunder LG conditions (5 mM glucose), HG (30 mM glucose) blockedNO production. Conversely, 30 mM mannitol had no effect, suggestingthat the effect was not due to a change in osmolarity (Figure 1B).VEGF induced eNOS-Ser1177 phosphorylation as early as 2.5min (Figure 1C) and remained positive for 10 min. Peak activationwas observed at 2.5 to 5 min (4.5-fold increase by densitometrycompared with control). HG partially inhibited the VEGF-inducedeNOS activation. In addition, acute stimulation with VEGF andglucose did not change total amount of eNOS protein in BAEC(Figure 1C).
Figure 1. Nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). (A) The level of NO, which was converted from nitrite in supernatant of culture media, was examined. Vascular endothelial growth factor (VEGF) dose-dependently stimulated NO release from BAEC at 24 h. (B) High glucose (HG; 30 mM) reduced the NO level in response to 10 ng/ml VEGF in BAEC. Mannitol (30 mM) does not inhibit NO production that is induced by VEGF. (C) VEGF (10 ng/ml) induced endothelial NO synthase (eNOS) phosphorylation at 2.5 min, which was partially prevented by HG. LG, low glucose (5 mM).
HG Enhances BAEC Proliferation in Response to VEGF
To examine the role of HG on BAEC proliferation in responseto VEGF, we used thymidine incorporation (Figure 2A), cell numberwith DNA content (Figure 2B), and MTT assay (Figure 2C). HGenhanced thymidine incorporation, DNA content, and viabilityin response to VEGF (Figure 2, A through C). In contrast, 30mM mannitol did not have any effect on VEGF-induced proliferation(Figure 2C). These data demonstrate that VEGF-induced BAEC proliferationis enhanced by HG.
Figure 2. HG enhances endothelial cell proliferation in response to VEGF. (A) Cell proliferation was examined by [3H]Thymidine incorporation, DNA content, and methylthiazoletetrazolium (MTT) assay. Under LG conditions (5 mM), VEGF (10 ng/ml) stimulated [3H]Thymidine incorporation (A) and DNA content (B), which were enhanced by HG (30 mM). (C) The effect of HG or mannitol on MTT reduction (a reflection of total cell number) is shown. HG increased MTT reduction compared with LG or mannitol in the absence of VEGF (a, P < 0.01). In the presence of VEGF (10 ng/ml), an increased proliferative response was observed in normal (LG) conditions (b, P < 0.01). HG further increased the MTT reduction in response to VEGF (10 ng/ml) (c, P < 0.01 versus VEGF in LG).
Blocking of NO Mediates BAEC Proliferation in Response to VEGF
Because HG blocks NO production, we hypothesized that a similarincrease in BAEC proliferation would be observed with L-NAME,which blocks NO synthases. L-NAME (1 mM) completely blockedNO production by VEGF in BAEC at 24 h (Figure 3A). L-NAME treatmentalso enhanced thymidine incorporation in BAEC in response toVEGF (Figure 3B), similar to what had been observed with HG(Figure 2). Conversely, administration of NO by NONOate (10–5to 10–4 M) partially suppressed VEGF-induced BAEC proliferationwith L-NAME treatment (Figure 3C), which is compatible witha previous report (20). Under HG conditions, the NO donor alsoprevented BAEC proliferation in response to VEGF (Figure 3D).These data suggest that NO negatively regulates BAEC proliferation.
Figure 3. NO inhibition induces BAEC proliferation. (A) N-nitro-l-arginine methyl ester (L-NAME) (1 mM) blocked NO release in BAEC at 24 h in response to VEGF. (B) A total of 10 ng/ml VEGF increased [3H]Thymidine incorporation, which was enhanced further by L-NAME in BAEC at 48 h. (C) NONOate (NONO; 10–5 to 10–4 M) reduced VEGF-induced BAEC proliferation, which was enhanced by L-NAME at 24 h. a, P < 0.01 versus L-NAME alone; b, P < 0.05 versus VEGF+L-NAME; c, P < 0.01 versus VEGF+L-NAME. (D) BAEC proliferation in response to VEGF was partially inhibited by NONOate (10–5 to 10–4 M) under HG condition. a, P < 0.01 versus HG alone; b, P < 0.05 versus VEGF+HG; c, P < 0.01 versus VEGF+HG. Control, serum-free and LG medium.
Expression of VEGF or KDR under HG or L-NAME Treatment
To examine the mechanism of endothelial proliferation observedwith uncoupling of VEGF with NO, we examined the expressionof VEGF, kinase-insert domain receptor (KDR), and Flt-1 mRNAin BAEC by PCR. Specific primers were designed as shown in Table 1.The expected sizes of amplicons, 149 bp for bovine VEGF,231 bp for bovine KDR, 165 bp for bovine Flt-1, and 145 bp forGAPDH were obtained (Figure 4A). Real-time PCR showed that eitherHG or L-NAME treatment stimulated the expression of VEGF aswell as KDR at 24 h (Figure 4, B and C). However, Flt-1 expressionwas induced only by HG, whereas L-NAME treatment did not increaseFlt-1 expression (Figure 4D).
Figure 4. mRNA expression for bovine VEGF, kinase-insert domain receptor (KDR), and Flt-1. (A) Amplicons of PCR for bovine VEGF, KDR, Flt-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in 2% agarose gel: 1, VEGF 149 bp; 2, KDR 231 bp; 3, Flt-1 165 bp; and 4, GAPDH 145 bp. (B and C) Real-time PCR for VEGF (B) and KDR (C). HG (30 mM) as well as L-NAME (1 mM) induced VEGF and KDR mRNA expression at 24 h in BAEC. Flt-1 expression is induced by only HG but not L-NAME (D). a, P < 0.01 versus LG.
KDR Mediates BAEC Proliferation in the Setting of Uncoupling of the VEGF–NO Axis but not NO Production
Next, the role of KDR was examined using the MTT assay. VEGFinduced BAEC proliferation under both LG and HG conditions,and this was completely inhibited by SU1498, a KDR-specificinhibitor (Figure 5A). Similarly, BAEC proliferation inducedby VEGF with L-NAME was also blocked by SU1498 (Figure 5B).In contrast, blocking of KDR failed to inhibit NO productionin response to VEGF in BAEC (Figure 5C). This finding was alsosupported by the observation that SU1498 did not block the activationof eNOS in response to VEGF (Figure 5D). Furthermore, to confirmthe role of KDR on eNOS activation, we used a KDR-selectiveVEGF mutant (KDR-sel) that was derived from culture medium oftransfected NRK cells and selectively binds KDR (28). As shownin Figure 5D, KDR-sel (100 pg/ml) did not stimulate eNOS phosphorylation,suggesting that KDR mediates cell proliferation but not NO productionin BAEC.
Figure 5. KDR mediates BAEC proliferation under uncoupling of VEGF with NO, but NO production is not regulated by KDR. (A) By MTT assay, BAEC proliferation was stimulated by VEGF under either LG (5 mM) or HG (30 mM) at 48 h. The endothelial cell proliferation was blocked by SU1498 (SU; 5 µM). (B) L-NAME (1 mM) stimulated BAEC proliferation, which was blocked by SU1498 at 48 h. (C) The measurement of NO in supernatant of culture medium. SU1498 did not block NO production in response to VEGF at 24 h. (D) Western blotting for eNOS-Ser1177. eNOS activation in response to 10 ng/ml VEGF was not blocked by 5 µM SU1498. A total of 100 pg/ml KDR-sel did not induce eNOS phosphorylation. a, P < 0.01 versus control. Control, serum-free and LG medium.
ERK1/2 Is Regulated by KDR, which Mediates BAEC Proliferation under Uncoupling of VEGF with NO
To explore further the mechanism of the VEGF-mediated proliferationunder HG conditions, we examined the role of ERK1/2. Blockingof ERK1/2 with Uo126 inhibited BAEC proliferation under eitherHG or L-NAME treatment (Figure 6A). VEGF activated ERK1/2 asearly as 10 min (Figure 6B), and SU1498 blocked ERK1/2 activationin response to VEGF. Similarly, KDR-sel stimulated ERK1/2 activation,which was inhibited by SU1498. Furthermore, HG as well as L-NAMEtreatment further increased ERK activation in response to VEGFat 15 min (data not shown) as well as at 8 h (Figure 6C). Thesedata suggest that KDR regulates ERK1/2 phosphorylation, whichcan be activated further after uncoupling of VEGF with NO.
Figure 6. Role of extracellular signal–regulated kinase (ERK) on the BAEC proliferation under uncoupling of VEGF with NO. (A) Inhibition of ERK1/2 by 5 µM Uo126 (Uo) blocked BAEC proliferation under VEGF (10 ng/ml) stimulation with either HG or L-NAME at 48 h. (B) Both VEGF (10 ng/ml) and KDR-sel (100 pg/ml) stimulated ERK1/2 activation at 10 min, both of which were blocked by 5 µM SU 1498. (C) HG as well as L-NAME enhanced ERK1/2 phosphorylation in response to VEGF at 8 h. a, P < 0.05 versus LG alone; b, P < 0.01 versus LG alone; c, P < 0.01 versus VEGF alone. Control, serum-free and LG medium.
Enhancement of BAEC Proliferation Could Be Specific to VEGF under HG Conditions
To examine whether this enhancement of endothelial cell proliferationby VEGF under low NO conditions is specific, we stimulated BAECby 10 ng/ml bFGF in the presence of L-NAME or HG. As shown inFigure 7, endothelial cell proliferation was induced by bFGF,but neither HG nor L-NAME treatment enhanced BAEC proliferationin response to bFGF.
Figure 7. Basic fibroblast growth factor (bFGF) does not enhance endothelial cell proliferation under NO inhibition. HG (30 mM) and L-NAME (1 mM) treatment did not enhance BAEC proliferation in response to 10 ng/ml bFGF at 48 h.
Our question is why VEGF is deleterious to diabetic nephropathyin contrast to nondiabetic renal disease. Although this differencein VEGF levels likely accounts for much of the differences betweendiabetic and nondiabetic disease, another important questionrelates to whether VEGF-mediated effects may be altered underthe diabetic condition. In this regard, many of the effectsof VEGF are linked with stimulation of endothelial NO production(9–12), yet, in diabetes, endothelial NO bioavailabilityis low (13–18). This raised the hypothesis that uncouplingof the VEGF–endothelial NO axis in diabetes may resultin altered endothelial responses to VEGF.
To test this hypothesis, we examined the effect of the combinationof VEGF with HG or LG on endothelial cell NO production andcell proliferation. Although VEGF stimulates endothelial NOproduction, in the setting of HG concentration, this responsewas inhibited. The effect was not due to the higher osmolarityassociated with HG because mannitol was unable to block VEGF-inducedNO release. We further demonstrated that the presence of HGalso enhanced the proliferative response to VEGF via a KDR-and ERK1/2-mediated pathway. Parallel findings in which endothelialNO release was blocked by L-NAME were shown. In addition, thisenhancement of cell proliferation was not observed in responseto the growth factor bFGF, suggesting that the effect is specificfor VEGF. To our knowledge, this provides the first demonstrationthat VEGF-NO uncoupling could provide for a mechanism for enhancedendothelial cell proliferation in the diabetic condition.
Abnormal angiogenesis plays a pathophysiologic role in the developmentof diabetic complications. Although neovascularization is prominentin the diabetic retina, it has been reported that new vesselformation is also observed in periglomerular arterioles andglomerular capillaries in the diabetic kidney (6,29). In theglomerulus, capillary number as well as its length is increasedin the early phase of diabetes (30). Many of these capillarieshave a thin wall, suggesting neovascularization (29). Moreover,new vessels adjacent to Bowman’s capsule were detectedin early diabetes (29). It is interesting that the developmentof small vessels at the vascular pole of glomeruli in the diabetickidney (6) correlated with VEGF expression (7).
The deleterious role of this uncoupling condition on the vasculardisease has been shown in several nondiabetic animal models.Long-term inhibition of NOS with L-NAME induced coronary arterydisease along with increased VEGF expression, and the vascularinjury was prevented by blocking VEGF with sFlt-1 treatment(a circulating VEGF inhibitor) (26). In the rat remnant kidneymodel, L-NAME treatment induced de novo VEGF expression in vascularsmooth muscle cells along with endothelial cell proliferationin arterioles and arteries (25). These data suggest that uncouplingof VEGF with NO causes vascular injury, which is associatedwith excessive endothelial cell proliferation. These findingswere supported by in vitro studies demonstrating that NO negativelyregulates human umbilical venous endothelial cell proliferationin response to VEGF (20).
Expression of both VEGF and KDR is increased in the kidneysof individuals with type 1 (31) and type 2 (32) diabetes. Reportedstimuli for VEGF expression in diabetes include HG (33), angiotensinII (34), advanced glycation end products (35), and TGF- (27,36).The increase in VEGF has been implicated in early diabetic nephropathy,and blocking VEGF prevented glomerular hypertrophy, reducedproteinuria, and partially prevented the glomerular hyperfiltrationin animal models of both type 1 and type 2 diabetes (4,5). VEGFis also a critical mediator of diabetic retinopathy (37).
In contrast, several mechanisms lead to low endothelial NO levelsin diabetes. First, oxidative stress may result in oxidationof tetrahydrobiopterin (BH4), which is required by eNOS forthe generation of NO (38–40). Second, whereas both mRNAand protein expression of eNOS were induced by conditions ofHG, the activation of eNOS was impaired (14,15), related toan increase in O-linked N-acetylglucosamine modification ofeNOS (15). Third, the increase in oxidants that are generatedin diabetes could result in the reaction of superoxide anionwith NO to form peroxynitrite, thereby scavenging NO and causingvascular injury (16,17). Finally, recent studies suggest thaturic acid, which is elevated in the metabolic syndrome and asubset of individuals with diabetes, also can reduce endothelialNO bioavailability (41). In this study, HG partially blockedeNOS phosphorylation in response to VEGF but had a more dramaticeffect on blocking the increase in NO measured in the culturesupernatants. This suggests that HG may lower endothelial NOby effects on eNOS activation as well as by a mechanism thatinvolves scavenging of NO.
In this study, we demonstrate a potential mechanism by whichuncoupling of VEGF with NO may result in vascular injury. NOdeficiency induced the compensatory increase in VEGF and KDRmRNA expression under either HG or L-NAME treatment, resultingin the activation of VEGF–KDR pathways, finally leadingto endothelial cell proliferation. It is interesting that anincrease in VEGF as well as KDR expression was also observedin early phase of diabetic nephropathy (31). Therefore, earlyactivation of VEGF-KDR in diabetes could play a role in aberrantangiogenesis in diabetic nephropathy. In contrast, it is ofinterest that Flt-1 mRNA expression is also regulated by HGbut not by inhibition of NO synthesis. However, the activationof Flt-1 by HG does not result in an increase in NO production.This observation may be explained by scavenging NO or inactivationof eNOS by HG (Figure 1).
The pathways by which VEGF regulates endothelial NO productionand release remain controversial. We demonstrated that KDR didnot regulate NO production or eNOS activation, suggesting thatNO is regulated by Flt-1, which is compatible with studies byothers (20). However, some investigators have reported thatKDR could be responsible for NO production in endothelial cells(12). This discrepancy may be accounted for by the time courseof eNOS expression. We detected an increase in NO productionin response to VEGF within 24 h, whereas the level of eNOS proteinexpression was unchanged. However, an increase in eNOS proteinexpression 2 to 4 d after VEGF exposure, which was mediatedby KDR (12), has been reported. These data suggest that theacute phase of NO production in response to VEGF was regulatedby Flt-1, whereas KDR may be responsible for a later increasein eNOS protein.
The role of NO is complicated in VEGF-induced endothelial cellproliferation because NO positively or negatively regulatesendothelial cell proliferation in response to VEGF (10,11,20).Gooch et al. (42) examined the effect of modulating NO levelson endothelial cells and reported that low concentrations ofNO stimulated whereas higher NO levels inhibited endothelialcell proliferation. In our preliminary experiments, NO productionis higher in BAEC, especially under low passage conditions ascompared with human umbilical vein endothelial cells. This observationmay explain why endogenous NO production in BAEC that is inducedby VEGF in this study might be sufficient to inhibit endothelialcell proliferation.
Gene targeting studies have demonstrated that the lethal phenotypeof Flt–/– mice is associated with an excessive proliferationof angioblasts (43). In human umbilical vein endothelial cells,engagement of Flt-1 inhibited KDR-dependent endothelial cells(44). These data suggest that Flt-1 can work as a negative regulatorof KDR. Given that NO production is mediated by Flt-1 (20),the excessive angiogenesis in diabetes could be accounted forby the activation of the KDR pathway, which is not counteredby the normal balance of Flt-1 activation because of a lossof endothelial NO bioavailability.
We present a novel mechanism for the neovascularization in diabeticvasculopathy. Our studies suggest that the normal coupling ofVEGF with endothelial NO production provides a mechanism ofautoregulation in which the production of NO feeds back to controlVEGF-stimulated endothelial cell proliferation. When this processis uncoupled, such as occurs in diabetes, there is increasedVEGF expression, increased KDR expression, and an enhanced endothelialproliferative response to VEGF, shifting the balance to excessiveendothelial proliferation. The dysregulation of this endothelialproliferative balance shifts VEGF from having a role in healthyangiogenesis to the dysregulated angiogenic state that is presentin patients with diabetic vascular and renal disease. Thesedata thus provide a mechanism to explain the development ofthese important diabetic complications.
Acknowledgments
This study was supported by a Program Project from the JuvenileDiabetes Research Foundation, by support from the National Instituteof Diabetes and Digestive and Kidney Diseases (DK-52121), andby generous funds from Gatorade.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
-nitro-l-arginine methyl ester (L-NAME) treatment. Endothelial NO synthase phosphorylation by VEGF was also inhibited by high glucose. It is interesting that both high glucose and L-NAME enhanced the proliferative response of endothelial cells, which was prevented by an NO donor. Furthermore, high glucose as well as L-NAME stimulated VEGF and kinase-insert domain receptor (KDR) (VEGF receptor 2) mRNA expression in BAEC. These data suggest that the uncoupling of VEGF with NO enhances endothelial cell proliferation via the KDR pathway. Compatible with these findings, a KDR antagonist blocked this response. In addition, a VEGF mutant, which binds only KDR, induced extracellular signal–regulated kinase (ERK) activation, and inhibition of ERK completely blocked endothelial cell proliferation under this condition, suggesting a role of the KDR–ERK1/2 pathway on endothelial cell proliferation. In conclusion, high glucose causes an uncoupling of VEGF with NO, which enhances endothelial cell proliferation via activation of the KDR–ERK1/2 pathway. These results may provide new insights into the understanding of the mechanism of diabetic vascular disease. 
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