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Published as doi: 10.1096/fj.06-7536com.
 (The FASEB Journal. 2007;21:3096-3106.)
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Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-ß: implications for diabetic atherosclerosis

Anne-Valerie Cantero*,1, Manuel Portero-Otín{dagger},1, Victòria Ayala{dagger}, Nathalie Auge*, Marie Sanson*, Meyer Elbaz*, Jean-Claude Thiers*, Reinald Pamplona{dagger}, Robert Salvayre*,2 and Anne Nègre-Salvayre*,2

* Inserm U-466 and Biochemistry Department, IFR 31, CHU Rangueil, University Paul Sabatier, Toulouse, France; and

{dagger} Metabolic Pathophysiology Research Group, Department of Basic Medical Sciences, University of Lleida, Spain

2Correspondence: INSERM U466, IFR 31, CHU Rangueil, Toulouse 31059 Cedex 9, TSA 50032, France. E-mail: anesalv{at}toulouse.inserm.fr or salvayre{at}toulouse.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Low molecular weight carbonyl compounds, such as the {alpha}-ketoaldehydes methylglyoxal (MGO) and glyoxal (GO), are formed under hyperglycemic conditions and behave as advanced glycation end product (AGE) precursors. They form adducts on proteins, thereby inducing cellular dysfunctions involved in chronic complications of diabetes. Methods and main findings: Nontoxic concentrations of GO or MGO altered the PDGF-induced PDGFRß-phosphorylation, ERK1/2-activation, and nuclear translocation, and the subsequent proliferation of mesenchymal cells (smooth muscle cells and skin fibroblasts). This resulted mainly from inhibition of the intrinsic tyrosine kinase of PDGFRß and in part from altered PDGF-BB binding to PDGFRß. Concomitantly, the formation of AGE adducts (N{epsilon}carboxymethyl-lysine and N{epsilon}carboxyethyl-lysine) was observed on immunoprecipitated PDGFRß. Arginine and aminoguanidine, used as carbonyl scavengers, reversed the inhibitory effect and the formation of AGE adducts on PDGFRß. AGE-PDGFRß adducts were also detected by anti-AGE antibodies in PDGFRß immunopurified from aortas of diabetic (streptozotocin-treated) compared to nondiabetic apolipoprotein E-null mice. Mass spectrometry analysis of aortas demonstrated increased AGE formation in diabetic specimens. Conclusions: these data indicate that MGO and GO induce desensitization of PDGFRß that helps to reduce mesenchymal cell proliferation.—Cantero, A.-V., Portero-Otín, M., Ayala, V., Auge, N., Sanson, M., Elbaz, M., Thiers, J-C., Pamplona, R., Salvayre, R., Nègre-Salvayre, A. Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-ß: implications for diabetic atherosclerosis.


Key Words: oxidative stress • growth arrest • MGO • wound healing • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE DICARBONYL COMPOUNDS METHYLGLYOXAL (MGO) and glyoxal (GO) are generated by cell metabolism, glucose autoxidation, and lipid peroxidation (1 , 2) . These compounds have been found in normal mammalian tissues, where they may be involved in a variety of detrimental processes in vivo, particularly under hyperglycemic conditions in diabetes (3 , 4) . Those highly reactive compounds may react with amino and sulfhydryl groups in proteins to form irreversible advanced glycation end products (AGE) (2 3 4 5) . N{epsilon}-(carboxymethyl)lysine (CML) and N{epsilon}-(carboxyethyl)lysine (CEL) are the major AGE adducts formed from GO and MGO, respectively (1 , 2) .

AGE formed on circulating proteins from diabetic patients (4) interact with specific RAGE receptors, which trigger cell activation and inflammatory response (5 6 7) . MGO and GO may also react with and impair the function of cellular proteins, such as HSP27 (8) , p38MAPK (9) , EGF receptor (10) , insulin receptor (11 , 12) , and transcriptional corepressor mSin3A (13) . Impaired cellular signaling may lead to inflammatory responses, growth arrest, and apoptosis (12 , 14) . In diabetes, such cellular dysfunction may contribute to impaired wound healing, accelerated atherosclerotic, and athero-thrombotic events (3 4 5 6) . Consistently, AGE scavenger drugs have been shown to reduce the progression of diabetes-accelerated atherosclerosis (14 15 16) .

Among the numerous growth factors, cytokines, and extracellular matrix components implicated in cell proliferation (17 , 18) , platelet-derived growth factor (PDGF) have important roles in embryonic development, as well as in wound healing and various pathophysiological conditions such atherosclerotic plaque formation, restenosis, and angiogenesis (19) . PDGF-BB and PDGFRß are expressed in atherosclerotic areas (19) , where they may stimulate SMC migration and proliferation leading to intimal hyperplasia, fibrous cap formation, and restenosis (17 18 19) . A defect in endothelial PDGF expression results in microaneurysm formation and microhemorrhage resembling late changes in diabetic microangiopathy (20) . Inhibition of PDGF signaling in apoE–/– mice delays SMC proliferation in atherosclerotic plaques (19) . Moreover, PDGF has been shown to help accelerate the healing of foot diabetic ulcer and to be useful in plastic surgery and bone fractures.

PDGF signal transduction is mediated by PDGF receptors (PDGFR) {alpha} and ß, which belong to the large family of receptor tyrosine kinases (21) . PDGFR{alpha} binds PDGF-A and PDGF-B isoforms, and is involved in SMC hypertrophy. PDGFRß, which binds only the PDGF-B chain, is implicated in cell migration and proliferation (22 , 23) . PDGF-BB binding triggers the activation of PDGFRß tyrosine kinase, which induces autophosphorylation of tyrosine residues of the receptor. Phosphotyrosines are docking sites for SH2 (src homology 2) domain and PTB (phosphotyrosine binding) domain-containing proteins (21 22 23) . Recently, we reported that PDGFRß acts as a sensor for oxidized lipids and that 4-hydroxynonenal (4HNE)-PDGFRß adduct formation is associated with either activation or inactivation of the receptor, depending on the concentration and time of contact with oxidized lipids (24 , 25) .

Atherosclerosis and subsequent cardiovascular complications are a leading cause of mortality in Western countries, and an increased incidence of cardiovascular diseases is observed in diabetes mellitus (26 , 27) . In addition to conventional cardiovascular risk factors, increasing evidence suggests that hyperglycemia associated with insulin resistance contributes to the onset and development of accelerated atherosclerosis (28) . During atherogenesis, the formation of fibro-atheroma plaques involves a complex sequence of events, including endothelial activation, lipoprotein oxidation and modification, local inflammatory response, migration of monocytes, lipid accumulation in macrophagic cells, and migration and proliferation of smooth muscle cell (SMC) (17 , 29) . A fibrous cap resulting from a local fibro-proliferative response covers atherosclerotic plaques. Excessive SMC proliferation and extracellular matrix biosynthesis may lead to intima thickening and arterial stenosis. In contrast, weak proliferation of SMC and/or an increased rate of SMC apoptosis and of ECM degradation may lead to "thin cap fibro-atheroma," which is associated with a lipid-rich core and inflammatory cells in vulnerable plaques prone to athero-thrombotic events (30) .

The aim of this work was to investigate whether GO or MGO induce PDGFRß modification and impair PDGF-BB-induced signaling and subsequent cell proliferation. It may be noted that we compared the effects of GO and MGO, and their presence in atherosclerotic lesions of nondiabetic and diabetic (STZ-treated) mice, because GO may be formed during glycoxidation and lipid peroxidation whereas MGO is mainly formed during glycoxidation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
[{gamma}-33P]ATP was obtained from ICN (Orsay, France), [125I]PDGF from PerkinElmer NEN Radiochemicals (Boston, MA, USA). [3H]Xuccinimidyl propionate and an ECL system were from Amersham-Pharmacia (Buckinghamshire, UK). Human recombinant PDGF was from PeproTech (Tebu, Le Perray, France); GO, MGO, and protein G-Sepharose were from Sigma (St. Louis, MO, USA). Anti-PDGFRß and anti-ERK1/2 were from Santa Cruz (Tebu, France), anti-phosphoMAPK was from Promega (Madison, WI, USA), and anti-CEL or anti-CML antibodies were from TransGenic, Inc. (Kumamoto, Japan)

Cell culture, dicarbonyl treatment, and determination of cell proliferation, migration, and viability
Rabbit femoral smooth muscle cells (SMC) (ATCC, Manassas, VA, USA) grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum were starved overnight in serum-free RPMI before the addition of GO or MGO at the times and concentrations indicated. To avoid potential interactions between GO or MGO with PDGF, the medium was removed before stimulation by PDGF and replaced by fresh RPMI. SMC were stimulated by adding 10 ng/ml PDGF at 37°C at the times indicated. Toxicity was evaluated using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test and apoptosis cells were counted after fluorescent labeling of cells by SYTO-13/IP (24) . DNA synthesis was evaluated by [3H]thymidine incorporation under previously used conditions (24) .

Cell migration assays were performed using a Boyden chamber according to the procedure of the manufacturer (Corning Life Sciences, Acton, MA, USA). Briefly, SMC or fibroblasts were seeded on Transwell inserts with 8 µ pore size membrane in RPMI medium supplemented with 10% FCS and preincubated overnight with GO (100 µM) or MGO (500 µM) in Hanks’ medium (added in the upper chamber). This medium was then discarded, PDGF (10 ng/ml) was added to the lower chamber (up to 400 µl), and cells were left to migrate for 8 h at 37°C. Nonmigrating cells were removed with a cotton swab and migrated cells were fixed with glutaraldehyde, stained with 0.5% crystal violet, eluted with acetic acid, and quantified by spectrophotometry (OD570 nm).

Animal studies
All experimental procedures were performed in accordance with the recommendations of the European Institute for Accreditation of Laboratory Animal Care. ApoE-null mice were purchased from Charles River (Les Oncins, France) and maintained on a 12-h light-dark cycle in a pathogen-free environment with free access to rodent chow and water. At 6 wk of age, mice were rendered diabetic by administration of 5 daily intraperitoneal injections of streptozotocin, 60 mg/kg in citrate buffer (0.05 mol/L; pH 4.5). Control mice were similarly treated with citrate buffer only. Serum glucose was measured weekly from tail vein blood using a glucometer: glycemia levels were 170 ± 25 mg/dl in control apoE–/– mice and 510 ± 65 mg/dl in 20-wk-old diabetic animals (high blood glucose subgroup). The mice were killed at 20 wk of age.

Immunocytochemistry
Cells grown on uncoated glass coverslips were fixed in 3% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100, then incubated with the indicated antibodies, and finally examined by fluorescence microscopy, as used before (24 , 25) .

Quantification and characterization of atherosclerotic lesions
The lesions were estimated as described by Biucharelli et al. (31) . Hearts were washed in PBS, frozen on a cryostatmount with OCT compound (Tissue-Tek, Sakura, Japan), and stored at –80°C. Serial sections 10 µm-thick of aortic sinus were stained with Oil Red O, counterstained with hematoxylin-eosin, and evaluated for morphometric evaluation of the lesion size using a computerized Biocom morphometry system (San Diego, CA, USA). The mean lesion size in aortic sinus was expressed in µm2 ± SE. For biochemical studies, ascending aortas were directly frozen at –80°C until use.

[125I] PDGF binding assay
[125I]PDGF binding was performed according to Vindis et al. (25) . Control SMC and cells preincubated for 8 h with GO 500 µM or MGO 100 µM as indicated were incubated with [125I]PDGF (70,000 cpm/ml, 30 pmol/L), then washed in PBS containing 0.5% BSA, and cell-associated radioactivity was determined (Minaxi, Packard, Meriden, CT, USA). Nonspecific binding was determined on the basis of excess unlabeled PDGF (10 ng/ml).

Tyrosine kinase activity of PDGFR
PDGFR was immunoprecipitated from unstimulated SMC. PDGFR-IP were incubated in vitro for 4 h at 37°C with GO and MGO (500 µM) in 50 mM HEPES pH 7.5, as described (24 , 25) , then PDGFR tyrosine kinase activity was evaluated by measuring the phosphorylation of poly Glu-Tyr (10 µM) by PDGFR-IP in the presence of 20 µM ATP containing 5 µCi of [{gamma}-33P]ATP (3000 Ci/mmol, NEN) in phosphorylation buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MnCl2, 10 mM MgCl2, 10 µM NaVO4, 0.2% Triton X-100). After incubation (15 min at 37°C), the reaction was stopped by spotting an aliquot of the mixture on phosphocellulose membranes and the radioactivity was counted.

Western blot and immunoprecipitation analysis
Tissue fragments (from ascending aorta) and cultured SMC were homogenized in extraction buffer. An equal amount of protein was used for PDGFR immunoprecipitation and SDS-PAGE, then transferred onto nitrocellulose membranes, as described (25) .

Derivatization of the PDGFR-free amino groups
The free amino group content was evaluated on PDGFR immunoprecipitated from aortic tissues or from SMC preincubated for 18 h with 500 µM GO using an amine reactive probe, the [3H]succinimidyl propionate, as reported (24 , 25) .

Quantitation of AGE and oxidative protein modifications
The concentrations of AGE markers N{epsilon}-carboxymethyl-lysine and N{epsilon}-carboxyethyl-lysine, as well as protein oxidative (glutamic and aminoadipic semialdehydes) and lipoxidative (malondialdehyde-lysine) modifications in soluble proteins from aorta or SMC, were measured by gas chromatography/mass spectrometry (GC/MS) as reported (32) . Briefly, samples containing 0.1 mg of protein were delipidated using chloroform:methanol (2:1 v/v) and proteins were precipitated by adding 10% trichloroacetic acid (final concentration). Protein samples were reduced overnight with 500 mM NaBH4 (final concentration) in 0.2M borate buffer, pH 9.2, then reprecipitated by adding 1 ml of 20% trichloroacetic acid. Isotopically labeled internal standards were added: [2H8]Lysine (d8-Lys; CDN Isotopes, Pointe-Claire, QC, Canada), [2H4]CML (d4-CML), [2H4]CEL (d4-CEL), [2H8]malondialdehyde-lysine (d8-malondialdehyde-lysine), [2H5] 5-hydroxy-2-aminovaleric acid (for glutamic semialdehyde quantitation), and [2H4]6-hydroxy-2-aminocaproic acid (for aminoadipic semialdehyde quantitation) prepared as described (32 33 34) . The samples were hydrolyzed at 155°C for 30 min in 1 ml of 6N HCl and dried in vacuo. GC/MS analyses were carried out on a Hewlett-Packard model 6890 gas chromatograph equipped with an HP-5MS capillary column (30 mx0.25 mmx0.25 µm) coupled to a Hewlett-Packard model 5973A mass selective detector (Agilent, Barcelona, Spain). The amounts of the products were expressed as the ratio µmol glutamic semialdehyde, aminoadipic semialdehyde, CML, CEL, or malondialdehyde-lysine/mol lysine.

Statistical analysis
Data are given as mean ± SE. Estimates of statistical significance were performed by Anova (Tukey test, SigmaStat software), values of P < 0.05 being considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MGO and GO desensitize PDGF-BB signaling and proliferation of SMC and fibroblasts
In SMC and fibroblasts preincubated for 16 h with increasing concentrations of GO or MGO, the mitogenic response to PDGF (10 ng/ml) was progressively lost, as shown by the decrease in DNA synthesis (Fig. 1 A). Time course experiments of preincubation with GO (500 µM) or MGO (100 µM) showed that the inhibition of PDGF-BB-induced DNA began after 8 h and was almost complete after 24 h of preincubation in SMC and fibroblasts (Fig. 1B, C ). In addition, GO and MGO strongly inhibited the PDGF-induced migration of SMC and fibroblasts (Fig. 1D, E ). It may be noted that the concentrations of GO and MGO (500 µM and 100 µM, respectively) were chosen to induce a major inhibition of DNA synthesis and migration (Fig. 1A-E ) but no toxicity, as assessed by MTT assay and syto13/IP labeling (Fig. 1F, G ).


Figure 1
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  		Figure 1. GO and MGO inhibit PDGF-stimulated SMC proliferation and migration. SMC in serum-free RPMI were pretreated with GO (hatched bars) or MGO (filled bars), then medium was replaced by fresh RPMI and PDGF (10 ng/ml) was added. DNA synthesis was evaluated by [3H]thymidine incorporation and cell migration was evaluated using a Boyden chamber under the conditions indicated in Materials and Methods. A–C) GO and MGO inhibit the PDGF-induced DNA synthesis in SMC (A, B) and fibroblasts (C). A) Dose-response of GO or MGO pretreated for 16 h with SMC before incubation with PDGF and determination of DNA synthesis. B) Effect of preincubation time of SMC with GO (500 µM) or MGO (100 µM) before stimulating cells with PDGF and DNA synthesis. C) Effect of GO (500 µM) or MGO (100 µM) preincubated for 16 h with fibroblasts before stimulation by PDGF and determination of DNA synthesis. D, E) GO (500 µM) and MGO (100 µM) preincubated for 16 h with cells inhibit PDGF-induced migration of SMC and fibroblasts. F) Evaluation of the cytotoxic effect of variable concentrations of GO or MGO on SMC (24 h incubation) and of a fixed concentration of GO or MGO (500 µM and 100 µM, respectively, for 24 h) on fibroblasts using the MTT assay. Quantitative data are represented as means ± SE of 4 experiments (*P<0.05). G) Fluorescence microscopy of cells, untreated (control) or treated by GO (500 µM), MGO (100 µM), and taxol (10 µM) for 16 h, as positive control of toxicity and stained by SYTO-13/IP. Representative of 3 separate experiments.

In SMC preincubated for 16 h with GO (500 µM) or MGO (100 µM), PDGF signaling was severely impaired, as shown by the inhibition of PDGF-induced PDGFRß autophosphorylation (Fig. 2 A). Subsequently, PDGF-induced activation of ERK1/2 was severely inhibited by preincubation with GO (Fig. 2B ) and MGO (Fig. 2C ). Nuclear translocation of ERK1/2 triggered by PDGF was also inhibited in cells preincubated with GO and MGO (Fig. 2D ). Altogether, these data show that GO and MGO block all steps of the PDGF-induced mitogenic signaling. As expected, similar results were obtained on cultured skin fibroblasts (Fig. 2C ), thus demonstrating that MGO- and GO-induced dysfunction of PDGF signaling is not restricted to vascular cells, but probably is a general mechanism.


Figure 2
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  		Figure 2. GO inhibits PDGFR and ERK1/2 phosphorylation induced by PDGF. SMC (A–D) and fibroblasts (D, right panel) were preincubated for 16 h with GO (500 µM) or MGO (100 µM), then stimulated by PDGF (10 ng/ml) for 15 min or for the time indicated. A) Western blots of PDGFR immunoprecipated from cells treated by GO or MGO, as indicated in Materials and Methods and revealed by antiphosphotyrosine and anti-PDGFR antibodies. B, C) Western blots of cell treated by GO (B) or MGO (C) labeled by antiphosphotyrosine, anti-PDGFR, anti-activated ERK1/2, and anti-total ERK2 antibodies. D) Nuclear translocation of ERK1/2 induced by PDGF and inhibition by GO or MGO. Immunocytochemistry representative of 4 separate experiments.

It may be noted that, in our experiments, inhibition of PDGF signaling by GO or MGO did not result from PDGF modification and inactivation by these carbonyl compounds, since cells were preincubated with carbonyl compounds (the preincubation medium containing GO or MGO was removed before addition of PDGF). This led us to investigate whether PDGFRß itself was a target for MGO and GO.

GO and MGO inhibit the binding of [125I]PDGF and tyrosine kinase activity of PDGFR
First, we investigated whether MGO and GO altered the expression or function of PDGFRß. Under the conditions used, exposure of SMC to GO or MGO (500 µM or 100 µM, for 16 h) elicited no notable change in the whole cellular content of PDGFRß, as assessed by Western blot experiments (Fig. 3 A) and in the level of PDGFRß expressed at the cell surface, as indicated by flow cytometry experiments (Fig. 3B ). As these data show that the dicarbonyl compounds do not alter quantitatively PDGFRß, we investigated whether they may alter the properties of PDGFRß. Experiments using [125I]PDGF-BB (30 pmol/L) showed that, in cells preincubated with GO (500 µM) or MGO (100 µM), [125I]PDGF-BB binding was reduced by 24 ± 5% (Fig. 3C ). These data suggest that the carbonyl compounds alter the binding site of PDGFRß for PDGF-BB. In addition, MGO and GO inhibited in vitro the activation of PDGFRß-associated tyrosine kinase. The in vitro preincubation of PDGFRß (immunoprecipitated from unstimulated SMC) with GO and MGO (4 h preincubation, 500 and 100 µM final concentration, respectively), followed by PDGF-BB stimulation, led to a strong inhibition of the PDGFRß-tyrosine kinase activity (Fig. 3D ), independent of any cellular activity.


Figure 3
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  		Figure 3. Effect of GO and MGO on PDGFR expression, [125I]PDGF binding, and PDGFR tyrosine kinase activity. A) Western blot showing the expression of PDGFR in SMC control and preincubated with MGO at the time indicated, and labeled by anti-PDGFR and anti-ß-actin as control. B) Evaluation of PDGFRß level exposed at the cell surface. Cells were preincubated 16 h with MGO (100 µM), GO (500 µM), or PDGF, harvested by trypsin/EDTA treatment, labeled with anti-PDGFR primary antibody (and FITC-labeled secondary antibody) and propidium iodide (only intact cells, i.e., non-IP labeled, were taken into account), and analyzed by flow cytometry. C) SMC control (circles) or preincubated for 16 h with GO (500 µM, triangles) or MGO (100 µM, squares) were incubated in the presence of a tracer amount of [125I]PDGF. Cell-associated radioactivity was evaluated (up to 30 min) as indicated in text. Mean ± SE of 3 separate experiments. D) PolyGluTyr phosphorylation elicited by PDGFR immunoprecipitates isolated from unstimulated SMC and incubated for 4 h with GO (500 µM) or MGO (100 µM), then stimulated by PDGF (10 ng/ml, 15 min) in the presence of 5 µM [33P]ATP, as reported in Materials and Methods. Mean ± SE of 3 separate experiments.

Altogether, these data suggest that dicarbonyls MGO and GO inhibit PDGFRß activity by at least two mechanisms: 1) by impairing the binding of PDGF-BB to PDGFRß, and 2) by inhibiting PDGFRß-tyrosine kinase. The direct alteration of PDGFRß functions by GO and MGO suggests that these dicarbonyls may induce structural modifications of PDGFRß by forming AGE-PDGFRß adducts.

GO and MGO form adducts on PDGFRß
AGE precursors such as MGO and GO are known to react with free amino groups and thiols to form AGE-protein adducts, thereby altering protein functionality (4 , 35) . This led us to investigate whether GO/MGO-induced PDGFRß dysfunction was associated with the formation of AGE-PDGFRß adducts, more precisely CML and CEL adducts, which are the major adducts formed from GO and MGO, respectively (4) .

Preincubation of SMC with GO or MGO (500 µM and 100 µM, respectively, for 16 h) resulted in a decrease of the free amino group content of PDGFRß, as determined on PDGFRß immunoprecipitates (Fig. 4 A). These preincubation experiments demonstrate that GO or MGO induce PDGFRß modification. As CML and CEL adducts are known to be the major adducts formed from GO and MGO with lysine, respectively (4) , we investigated whether these AGE-protein adducts were formed. As expected, under the experimental conditions used, GO and MGO preincubation induced the formation of CML- and CEL-PDGFR adducts, respectively, as shown on Western blots of immunoprecipitated PDGFRß (Fig. 4B, C ).


Figure 4
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  		Figure 4. Inhibitory effect of GO/MGO is associated with AGE-PDGFR adducts formation. A) Free amino groups content was determined on PDGFR immunoprecipitated from control SMC or SMC preincubated for 16 h with GO (500 µM) or MGO (100 µM) and stimulated or not by PDGF (10 ng/ml). Cell extracts were labeled with [3H]NSP and resolved by SDS-PAGE. Radioactivity of the 180 kDa band corresponding to PDGFR was then counted as reported in Materials and Methods. Mean values ± SE of 3 separate experiments (P<0.02). B) Detection of CML adducts on PDGFR immunoprecipitated from SMC preincubated for 16 h with GO (500 µM). Western blots revealed using anti-CML and anti-PDGFR antibodies and ß-actin. C) Detection of CEL adducts on PDGFR immunoprecipitated from SMC preincubated for 16 h with GO (500 µM) or MGO (100 µM). Western blots show use of anti-CEL and anti-PDGFR antibodies.

Altogether, these data indicate that pretreatment of SMC by AGE precursors GO or MGO induces the formation of AGE-PDGFRß adducts concomitant (and probably causally related) with the impairment of PDGF signaling.

Arginine and aminoguanidine prevent MGO- and GO-induced PDGFRß modification and dysfunction
To confirm the relationship between modification and dysfunction of PDGFRß, we examined whether carbonyl scavengers may prevent both PDGFRß modification and the loss of function of PDGFRß induced by GO or MGO. We used arginine and aminoguanidine as nontoxic carbonyl scavengers that react readily with GO/MGO through the guanidino group (2 , 35 , 36) .

As shown in Fig. 5 , arginine added simultaneously with GO during preincubation (Arg/GO, 1/1, mol/mol) prevented both structural modification and dysfunction of PDGFRß. Arginine relieved GO-induced CML-protein adduct formation (Fig. 5A, B ) as well as inhibition of PDGF signaling (Fig. 5C ). Similarly, arginine was able to prevent the MGO-induced PDGFRß modification and desensitization (data not shown). In the same way, aminoguanidine added simultaneously with MGO (AG/MGO, 1/1 mol/mol) relieved PDGFRß modification and dysfunction induced by MGO, as well as DNA synthesis (Fig. 5D, E ).


Figure 5
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  		Figure 5. Arginine and aminoguanidine prevent the inhibitory effect of GO and MGO on PDGFR. A, B) Quantification of CML adducts by GC/MS in SMC preincubated with or without GO and/or arginine (500 µM each, 16 h). C) Western blots of PDGFR and ERK1/2 activation in SMC treated with or without GO and/or arginine before stimulation by PDGF. D) Western blots of PDGFR in SMC treated with or without MGO and/or aminoguanidine (100 µM each, 16 h), stimulated by PDGF, and blotted by antiphosphotyrosine, anti-CEL, and anti-PDGFR antibodies. E) PDGF-induced DNA synthesis in SMC preincubated with or without MGO and arginine (arg) or aminoguanidine (Am) before stimulation by PDGF.

Altogether, these data suggest that 1) modification of PDGFRß adduct formation is causally related to PDGFR impairment; and 2) both structural modification and dysfunction of PDGFRß can be prevented by the carbonyl scavengers arginine and aminoguanidine

AGE-PDGFRß adducts in atherosclerotic lesions of diabetic apoE–/– mice
We investigated the in vivo relevance of PDGFRß modification by GO and MGO in aortas of apoE–/– mice rendered diabetic by streptozotocin (Stz) treatment.

In agreement with earlier reports (31) , the analysis of Oil Red O-stained cross sections of aortic sinus revealed that 20-wk-old Stz-treated apoE–/– mice displayed larger atherosclerotic lesions than did nondiabetic apoE–/– mice (Fig. 6 A). Biochemical analysis of aortic extracts from diabetic Stz-treated apoE–/– compared to nondiabetic apoE–/– mice showed that the free reactive amino group content was significantly lower in Stz-treated apoE–/– than in apoE–/– (Fig. 6B ). Conversely, we observed higher levels of oxidation, lipoxidation, and glycoxidation markers (determined by GC/MS) in aortas from Stz-treated apoE–/–(Fig. 6C ). The two prevailing adducts were glutamic semialdehyde (GSA, 36036±1936 µmol/mol lysine) and CML (7027±56 µmol/mol lysine). In Stz-treated apoE–/– aortas, the rise of GSA, aminoadipic semialdehyde (AASA), CEL, malondialdehyde-lysine (MDAL) was between 50 and 60%, whereas the increase in CML was less (~+20%) (Fig. 6C ). These data correlated with the high levels of CEL adducts in proximal aorta of Stz-treated apoE–/– compared to nondiabetic apoE–/–, as shown by immuno-histochemical detection (Fig. 6D, E ). Finally, we evaluated the presence of CEL and CML adducts on PDGFRß immunoprecipitated from aortas of untreated apoE–/– and Stz-treated apoE–/– mice. Western blots showed that labeling of PDGFRß by anti-CEL was higher in extracts from ascending aorta from Stz-treated apoE–/– than in extracts from nondiabetic apoE–/– (Fig. 6F ). In contrast, no clear difference was found with anti-CML antibodies between aortic extracts from apoE–/– and Stz-treated apoE–/– mice (data not shown). These data are consistent with the view that CEL may be generated during both glycoxidation and lipid peroxidation, whereas CML accumulation in atherosclerotic lesions results mainly from lipid oxidation and is independent of diabetes, in agreement with Baynes et al. (1) .


Figure 6
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  		Figure 6. AGE content and CEL-PDGFR adducts from aortas of STZ-treated apoE null mice. A) Aortic atherosclerosis in apoE–/– (E) or Stz-treated apoE–/– (E/S) mice. Determination of atherosclerotic areas in aortic sinus. Mean values ± SE of from 10 animals in each group: (E) and (E/S) (P<0.01). B) Free amino group content in aortic tissue extracts from control, apoE–/– (E), and STZ-treated (ES) mice. C) Increases in glutamic and aminoadipic semialdehydes content, and in AGE content in aortic extracts from untreated apoE–/– and STZ-treated apoE–/– mice. Data are expressed as % changes of mean ± SE over values of untreated apoE–/– (glutamic semialdehyde: 36036±1936 µmol/mol lysine; aminoadipic semialdehyde: 1461±82 µmol/mol lysine; CML: 7027±56 µmol/mol lysine; CEL: 1802±23 µmol/mol lysine; malondialdehyde-lysine (MDAL): 1802±23 µmol/mol lysine). *P < 0.05. A–C) Mean values ± SE of 3 separate experiments (P<0.05). D, E) Immuno-histochemistry of CEL adducts in aortic atherosclerotic plaques of apoE–/– and STZ-treated mice. Panels D, E are representative of 3 separate experiments. F) Detection of CEL adducts on PDGFR immunoprecipitated from E and E/S mice aortas. G–I) Immuno-histochemistry of {alpha}-actin (G), PDGFR (H), and CEL adducts (I) in aortic atherosclerotic plaques of STZ-treated apoE–/– mice. Representative of 3 separate experiments.

As shown in Fig. 6G , the neointimal hyperplasia contained SMC (labeled by anti-{alpha}-actin antibody, Fig. 6G ), which expressed a high level of PDGFRß (Fig. 6H ). CEL were detected in the same area of atherosclerotic lesions (Fig. 6I ), suggesting that cellular proteins (among them PDGFRß) of the atherosclerotic neointima of diabetic mice are modified. These data agree with those of Fig. 6F .

As summarized in Fig. 7 , the reported data indicate that PDGFRß is a target for AGE formation in vitro (after incubation of cells with GO and MGO) and in vivo in aortas from diabetic animals. Such PDGFRß modification is associated in cultured SMC with impaired mitogenic signaling of PDGFRß and can be prevented by carbonyl scavengers.


Figure 7
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  		Figure 7. Schemas showing the mechanism of PDGFR inhibition elicited by AGE formation after contact with AGE precursors GO and MGO. Under standard conditions of cell culture or in normoglycemic animals, only low modification of PDGFR is observed (A). GO/MGO treatment in vitro or diabetes in Stz-treated mice induces AGE-PDGFR adducts formation. This PDGFR modification reduces PDGF-BB binding and severely inhibits the tyrosine kinase activity of PDGFR (red crosses, B), thereby inhibiting PDGFR activation (autophosphorylation) and subsequent mitogenic signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The molecular and cellular mechanisms leading to vascular complications of diabetes, including atherosclerosis, are thought to involve hyperglycemia, oxidative stress, and formation of AGE-protein adducts (1 , 4) . PDGFRß inhibition has been shown to induce growth arrest and apoptosis of SMC in the fibrous cap, thereby increasing the risk of plaque rupture (37) .

Our data show that high concentrations of GO or MGO, such as found in diabetic condition, induce the modification and alter the mitogenic function of PDGFRß in vitro, and that similar modification of PDGFRß is found in atherosclerosis lesions of diabetic apoE–/– mice, suggesting that a similar dysfunction may occur in vivo and may lead to fragile thin cap in atherosclerotic lesion. Proliferation of SMC and secretion of extracellular matrix play an important role in the progression of atherosclerotic lesions and in the formation of thick fibrous cap, thereby preventing plaque rupture and athero-thrombotic events (17 , 37) . PDGF-BB and PDGFRß are highly expressed in atherosclerotic lesions and are involved in the proliferation of SMC and fibrous cap formation (19) . During diabetes, the increased formation of reactive carbonyl compounds leads to enhanced formation of AGE-protein adducts that alter protein functions, affect intracellular signaling, and induce growth inhibition, mutagenesis, and apoptosis (4 5 6) .

We report here for the first time that GO and MGO pretreatment of mesenchymal cells, SMC, and fibroblasts results in enhanced AGE-PDGFRß adduct formation and altered mitogenic signaling in response to PDGF-BB. The presence of AGE-PDGFRß adducts in atherosclerotic aortas from diabetic (STZ-treated) apoE–/– mice suggests that PDGFRß is also in vivo a target for AGE precursors. The data establish a mechanistic link between AGE precursors formed during diabetes and PDGFRß dysfunction, which may contribute to vulnerable atherosclerotic plaque and impaired impaired tissue repair in diabetic patients. This hypothesis is consistent with our immunohistological data, which showed that CEL adducts were higher in atherosclerotic plaque of diabetic apoE–/– mice and were located in neointimal areas, where SMC migrate and proliferate and where PDGFRß expression was up-regulated.

A first observation is that preincubation of SMC with GO and MGO induces PDGFR modification, as assessed by the loss of free amino groups and the formation of CML- and CEL-PDGFRß adducts. GO and MGO promote a time- and dose-dependent inhibition of PDGFRß tyrosine phosphorylation, ERK1/2 activation, and SMC proliferation. This inhibitory effect results from two additional mechanisms: reduced PDGF-BB binding to PDGFRß and inhibition of the PDGFRß-intrinsic tyrosine kinase. The loss of free amino groups observed in PDGFRß immunoprecipitated from SMC pretreated with AGE precursors and from aorta extracts of STZ-treated apoE–/– mice is consistent with the known reactivity of dicarbonyl compounds with free amino, guanidino, or thiol groups of proteins usually associated with a loss of function of proteins (38 39 40 41) . Thus, inhibition of PDGFRß kinase could result from a modification of Lys residue in the active site, which is critical for the activity of PDGFRß intrinsic tyrosine kinase (42) . The decreased PDGF-BB binding could also result from modification of Lys and Arg residues in the Ig-like domains or of Cys involved in PDGFR dimerization (42) . It may be noted that a loss of PDGFRß was excluded, since we observed neither a decreased level of PDGFRß protein (as evidenced on Western blot experiments) nor reduced expression of PDGFRß at the plasma membrane (data not shown). Finally, although PDGF-BB may be modified by aldehydes, this was probably not the case in our experiments, since free GO and MGO were removed before adding PDGF to the culture medium.

The impairment of PDGFRß is consistent with previous observations on the EGF receptor (10) , but differs from a recent report showing that short-term incubation with MGO did not inhibit the tyrosine phosphorylation of the insulin receptor (IR) after insulin stimulation in L6 cells (12) . In this case, MGO pretreatment of L6 cells resulted in a decrease in free amino group content in IRS-1 associated with a strong reduction in IRS-1 phosphorylation, PI3K activity, PKB activation, and glucose transport (43) . Moreover, a decrease in IR-tyrosine kinase activity induced by MGO pretreatment cannot be excluded in this model and may explain the reduced phosphorylation of IRS-1 (43) .

The experimental conditions used here (preincubation of cells with 500 µM GO and 100 µM MGO, for 16 h) were chosen to obtain an acute model for SMC proliferation without or with only minor cytotoxic effect. These dicarbonyl concentrations are relevant to plasma concentrations of GO and MGO, which range from 0.15–1 µM in normal subjects to 400 µM in diabetic patients (44) . Note that Passarelli et al. (45) utilized similar carbonyl concentrations to study the damages induced by GO on ABCA1, whereas Riboulet-Chavey et al. (12) used higher concentration (2.5 mM) for short-term experiments on insulin signaling. These concentrations are probably relevant to tissular or cellular concentrations of dicarbonyl compounds in diabetes, since MGO levels in rat tissues (particularly in the aorta) are an order of magnitude higher than in plasma, according Randell et al. (46) using solid-phase extraction, liquid chromatography, and electrospray ionization mass spectrometry. Finally, the in vivo formation of AGE- PDGFRß adducts is probably a slow but cumulative process, where local oxidative stress occurring in atherosclerotic lesions can generate additional reactive carbonyls that can contribute to adduct formation and subsequent pathogenic events, as reported (3) and as shown by the significant increase in MDAL-modified proteins, concomitant with CEL and CML adduct formation in vessels from diabetic mice. GO may be formed during glycoxidation and lipid peroxidation, whereas MGO is formed mainly during glycoxidation. This may explain why the level of CML adducts is much higher than that of CEL adducts in both apoE–/– and diabetic (STZ-treated) apoE–/– and that the relative difference is higher for CEL adducts than for CML adducts.

It may be noted that, in contrast to the data reported here, AGE-modified serum albumin stimulated SMC proliferation without any inhibition of PDGFRß autophosphorylation (47) . This was due to AGE/RAGE interactions, which elicit various signaling responses including oxidative stress, inflammatory events linked to NF-{kappa}B activation, and MAPK phosphorylation (5 6 7) . In our experimental conditions, neither oxidative stress (no effect of antioxidants) nor AGE-RAGE interactions were involved since GO and MGO reacted directly with PDGFRß. From a pathophysiological point of view, both mechanisms (circulating AGE interacting with RAGE and direct modification of signaling proteins such as PDGFRß) can coexist in atherosclerotic arteries in diabetic conditions, the balance between SMC proliferation, growth arrest, and apoptosis being modulated by the local concentration in glucose and glucose metabolites and by lipid peroxidation products present in atherosclerotic lesions (25) . The data reported here suggest that growth arrest induced by AGE formation on PDGFRß associated with the other properties of AGEs (inflammation, apoptosis) may alter plaque stability by inhibiting SMC proliferation, thus further contributing to plaque rupture and subsequent thrombotic events.

In addition, as growth factors and particularly PDGF are known to play a role in wound healing in diabetes (48 , 49) , it can be hypothesized that PDGFRß modification by AGE precursors in fibroblasts may participate in the mechanisms leading to impaired wound healing in diabetic patients.


   ACKNOWLEDGMENTS
 
This work was supported in part by grants from INSERM and University Toulouse-3 to UMR-466 and I2MR, from Ministère de la Recherche (ANR 05-PCOD-019–01 LiSA), from the Spanish Ministry of Health (FIS 05–2241, 04–0355), from La Caixa Foundation, from the Generalitat de Catalunya (2005SGR001101), and from the Spanish Ministry of Science and Technology (BFI 2003–01287, BFU 2006–14495) to the Metabolic Pathophysiology Research Group in Lleida. Supported also by the EU COST (Action B-35 Lipid Peroxidation Associated Disorders). M.S. is the recipient of a fellowship from "Groupe de Reflexion sur la Recherche Cardiovasculaire."


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication January 15, 2007. Accepted for publication April 12, 2007.


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 DISCUSSION
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