Am J Physiol Cell Physiol Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 295: C1510-C1517, 2008. First published October 8, 2008; doi:10.1152/ajpcell.00252.2008
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/6/C1510    most recent
00252.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamawaki, H.
Right arrow Articles by Hara, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamawaki, H.
Right arrow Articles by Hara, Y.

VASCULAR BIOLOGY

Methylglyoxal mediates vascular inflammation via JNK and p38 in human endothelial cells

Hideyuki Yamawaki, Kazuaki Saito, Muneyoshi Okada, and Yukio Hara

Department of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan

Submitted 13 May 2008 ; accepted in final form 1 October 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Methylglyoxal (MGO) is a reactive metabolite of glucose. Since the plasma concentration of MGO is increased in diabetic patients, MGO is implicated in diabetes-associated vascular endothelial cells (ECs) injury, which might be responsible for atherosclerosis. In the present study, we examined effects of treatment of human umbilical vein ECs with MGO on EC morphology and inflammatory responses. MGO (24 h) induced cytotoxic morphological changes in a concentration-dependent manner (0–420 µM). MGO induced mRNA and protein expression of cyclooxygenase (COX)-2 in a concentration (0–420 µM)- and time (6–24 h)-dependent manner. COX-2 induction was associated with increased PGE2 release. Acute treatment with MGO (20 min) induced concentration-dependent (0–420 µM) activation of JNK and p38 MAP kinase but not ERK or NF-{kappa}B. Both the JNK inhibitor SP600125 and the p38 inhibitor SB203580 prevented the MGO induction of COX-2. However, inhibiting JNK and p38 or COX-2 was ineffective to the morphological damage by MGO (420 µM, 24 h). EUK134, a synthetic combined superoxide dismutase/catalase mimetic, had no effect on MGO-induced COX-2. Present results indicated that MGO mediates JNK- and p38-dependent EC inflammatory responses, which might be independent of oxidative stress. On the other hand, MGO-induced morphological cell damage seems unlikely to be associated with COX-2-PGE2.

diabetes; vascular endothelium; inflammation; signal transduction; mitogen-activated protein kinase

IT IS WELL KNOWN THAT diabetes mellitus (DM) is one of the major risk factors for atherosclerosis (7, 18). Vascular endothelial cells (ECs) inflammatory injury represents an important early pathogenic feature of atherosclerosis (17, 28). Although DM is often associated with ECs inflammatory injury (13), the underlying mechanisms are not fully clarified.

Since the plasma concentration of advanced glycation end-products (AGEs) is reported to be significantly elevated in diabetic patients (4, 15, 23), previous investigators have mainly explored the effects of AGEs as well as the high concentration of glucose on vascular endothelium. However, controversial results have been obtained (8, 14, 34, 42). On the other hand, we recently provided evidence that one of the glucose metabolites, glyoxal, is a more powerful inducer for EC inflammatory injury rather than glucose itself or AGEs (38).

Methylglyoxal (MGO) is a reactive metabolite of glucose and serves as an intermediate between glucose and AGEs (30). It is known that MGO can stimulate the cellular signal transduction (2, 3). MGO is produced from triose phosphates under hyperglycemic conditions and eventually metabolized into AGEs mainly by nonenzymatic glycation of protein (1, 25). A recent study (16) revealed that the plasma MGO level is significantly increased in diabetic patients. Furthermore, an increased MGO-derived AGEs level in diabetic patients seems to be associated with diabetic complications such as nephropathy (22) and retinopathy (10). In addition, recent studies (35, 37) indicated that MGO is also implicated in diabetes-related vascular disorders like hypertension. Therefore, we hypothesized that MGO could affect vascular ECs to induce inflammatory injury, which could be responsible for the DM-associated atherosclerosis. To prove this, we examined the effects of treatment with MGO on human vascular ECs by especially focusing on the inflammatory signal events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Reagent sources were as follows: MGO solution (Sigma-Aldrich, St. Louis, MO); SB203580 (Calbiochem, San Diego, CA); EUK134 and NS398 (Cayman, Ann Arbor, MI); SP600125 (Jena Bioscience, Jena, Germany); and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Antibody sources were as follows: phospho-p38, phospho-ERK, phospho-NF-{kappa}B p65 (Ser536), and total JNK2 (Cell Signaling, Beverly, MA); endothelial nitric oxide (NO) synthase (eNOS), cyclooxygenase (COX)-2, I{kappa}B-{alpha}, total p38, ERK 1, NF-{kappa}B p65, and actin (Santa Cruz Biotech, Santa Cruz, CA); and phospho-JNK (Promega, Madison, WI).

Cell culture. Human umbilical vein ECs (HUVECs) were purchased from Kurabo (Osaka, Japan) and cultured in Medium 200 supplemented with low serum growth supplement (Cascade Biologics, Portland, OR) as described previously (38–40). Cells at passage 4 to 7 were used for experiments.

Morphological examination. HUVEC morphological changes were observed under light microscope (CKX31, Olympus, Tokyo, Japan) equipped with digital camera (SP-350, Olympus).

Cell counting. The living cell number was counted using a Cell Counting-8 kit (Dojindo Laboratories, Kumamoto, Japan) as described previously (39). After HUVECs were treated at ~90% confluence with MGO (0–420 µM) for 24 h, water-soluble tetrazolium salt (WST-8) was added for 1 h and the culture medium was collected. Conversion of WST-8 into formazan by living cells (active mitochondria) was measured using a standard 96-well plate reader at absorbance 490 nm. The total number of living cells was expressed as relative to untreated control samples (0 µM MGO).

Western blotting. Western blotting was performed as described previously (38–40). Protein lysates were obtained by homogenizing HUVECs with Triton-based lysis buffer (1% Triton X-100, 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 0.1% protease inhibitor mixture; Nacalai Tesque, Kyoto, Japan). Protein concentration was determined using the bicinchoninic acid method (Pierce, Rockford, IL). Equal amounts of proteins (10–15 µg) were separated by SDS-PAGE (7.5–10%) and transferred to a nitrocellulose membrane (Pall, Ann Arbor, MI). After being blocked with 3% BSA or 0.5% skim milk, membranes were incubated with primary antibody (1:500–1,000 dilution) at 4°C overnight, and membrane-bound antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution, 1 h) and the ECL-plus system (Amersham Biosciences, Buckinghamshire, UK). Equal loading of protein was confirmed by measuring total actin or eNOS expression. The resulting autoradiograms were analyzed using National Institutes of Health Image 1.63 software.

Quantitative determination of PGE2 release. The production of PGE2 from HUVECs was measured using an enzyme immunoassay kit (Cayman) in accordance with the instructions of the manufacturer. Briefly, after HUVECs were treated at ~90% confluence with MGO (420 µM) for 24 h, the culture medium was collected. PGE2 released into the culture medium was measured at absorbance 410 nm using a standard 96-well plate reader.

Immunofluorescence staining. Immunofluorescence staining was performed as described previously (5). Cells were fixed with 4% paraformaldehyde (pH 7.4) for 10 min at 4°C and permeabilized with 0.1% Triton X-100 for 15 min at room temperature. After being blocked with 5% normal goat serum for 60 min, cells were incubated with NF-{kappa}B p65 antibody (1:500 dilution) at 4°C over night followed by fluorescent-conjugated secondary antibody (Alexa Fluor 488; 1:500 dilution, 1 h, Invitrogen). Images were obtained using fluorescence microscope (BX-51, Olympus) equipped with cooled CCD camera (MicroPublisher 5.0 RTV, Roper Japan, Tokyo, Japan).

RT-PCR. Total RNA was extracted from HUVECs by using QuickGene SP kit (Fuji Film, Tokyo, Japan) according to the instructions of the manufacturer. The first strand of cDNA was synthesized using random hexamers and Superscript II RT at 65°C for 5 min, 25°C for 2 min, 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. PCR amplification was performed using HotStarTaq MasterMix kit (Qiagen, Valencia, CA). The oligonucleotide primers for COX-2 were GAG CTG TAT CCT GCC CTT CTG GTA (sense) and GGA AGA ACT TGC ATT GAT GGT GAC (antisense). The oligonucleotide primers for GAPDH were GCT GAT GCC CCC ATG TTT G (sense) and GGG TGG TGG ACC TCA TGG C (antisense). After initial activation at 95°C for 15 min, 28 cycles of amplifications at 94°C for 0.5 min, 52°C for 0.5 min, and 72°C for 1 min followed by final extension at 72°C for 10 min were done with a thermal cycler (PC707, ASTEC, Fukuoka, Japan). PCR products were electrophoresed on 2% agarose gel containing 0.1% ethidium bromide. Detectable fluorescent bands were visualized by an ultraviolet transilluminator and analyzed using CS Analyzer 3.0 software (ATTO, Tokyo, Japan).

Statistical analysis. Data are means ± SE. Statistical evaluations were performed using one-way ANOVA followed by Tukey's test. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of chronic treatment of HUVECs with MGO. We first examined morphological changes after chronic treatment of HUVECs with MGO. Treatment for 24 h with MGO induced a cytotoxic morphological change in a concentration-dependent manner (0–420 µM; Fig. 1A, a–e). Especially in cells treated with high concentration of MGO (420 µM), the cell density seemed decreased and spindle typed cells were often observed (Fig. 1A, f, g). Figure 1B showed that morphological damage by MGO was associated with decreased living cell number (at 420 µM MGO, 20.9 ± 5.3% decrease from 0 µM MGO; n = 6; P < 0.01).


Figure 1
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 1. A: representative photomicrographs of human umbilical vein endothelial cells (HUVECs) treated for 24 h with methylglyoxal (MGO); a, f: 0 µM MGO; b: 42 µM MGO; c: 140 µM MGO; d: 280 µM MGO; and e, g: 420 µM MGO. Magnification: x100 (a-e) and x200 (f, g). B: total number of living cells was counted using water-soluble tetrazolium salt. Results are shown as relative to 0 µM MGO (n = 6). **P < 0.01 vs. 0 µM MGO.

 
To examine whether the morphological changes are associated with vascular inflammatory states, expressions of inflammation-associated proteins were measured by Western blotting. Treatment of HUVECs with MGO (24 h) induced COX-2 protein expression in a concentration-dependent manner (0–420 µM; n = 4; Fig. 2A). On the other hand, induction of VCAM-1 protein was not detectable by the treatment with MGO (0–420 µM; n = 3; data not shown). COX-2 induction by MGO (420 µM) was observed after 6 h treatment (1.3 ± 0.8-fold relative to 0 µM MGO; n = 3), became maximum after 12 h (2.9 ± 0.7-fold; n = 3), and remained clearly detectable after 24 h (1.9 ± 0.4-fold; n = 3; Fig. 2B). Figure 2C showed that increased COX-2 expression by MGO (420 µM, 24 h) was associated with increased PGE2 release from HUVECs [2,037.0 ± 284.1 pg/mg protein in 420 µM MGO vs. 1,178.1 ± 141.4 pg/mg in 0 µM MGO (control); n = 15; P < 0.05].


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 2. A, B: concentration- and time-dependent effects of MGO on induction of cyclooxygenase (COX)-2 protein in HUVECs. After HUVECs at ~90% confluence were treated with MGO (A) at varying concentrations (0–420 µM, 24 h) or for varying times (B; 6–24 h, 420 µM), total cell lysates were harvested. Expression of COX-2 protein was determined by Western blotting. Equal protein loading was confirmed using endothelial nitric oxide synthase (eNOS) antibody (n = 3–4). COX-2 expression is shown as fold increase relative to 0 µM MGO. C: MGO-stimulated production of PGE2 from HUVECs. After HUVECs were treated with MGO (420 µM, 24 h), culture medium was collected. PGE2 released into the medium was measured with an enzyme immunoassay kit. Results are shown as pg/mg protein (n = 15). *P < 0.05 vs. 0 µM MGO.

 
Signaling transduction mechanisms relating to MGO-mediated COX-2 induction. To examine the signal mechanisms responsible for COX-2 induction by MGO, the activation of MAP kinases was measured by Western blotting using phosphorylation-specific antibodies. Acute treatment of HUVECs with MGO (20 min) induced activation of JNK and p38 but not ERK in a concentration-dependent manner (0–420 µM; Fig. 3A). Maximal phosphorylation of JNK or p38 was obtained by the treatment with 420 µM MGO (p-JNK1/2, 2.8 ± 0.5-fold relative to 0 µM MGO; n = 3; P < 0.05; Fig. 3B; and p-p38, 6.2 ± 0.8-fold; n = 3; P < 0.01; Fig. 3C). We confirmed that the total amount of these kinases was not changed by the treatment with MGO (0–420 µM, 24 h; Fig. 3D).


Figure 3
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 3. Acute effects of MGO on inflammatory signaling in HUVECs. After HUVECs were treated with MGO (0–420 µM) for 20 min, total cell lysates were harvested. A: activation of JNK, p38, and ERK was determined by Western blotting using phospho-specific antibodies (n = 3). Equal protein loading was confirmed using eNOS antibody. B, C: phosphorylation of JNK (B) or p38 (C) is shown as fold increase relative to 0 µM MGO. *P < 0.05, **P < 0.01 vs. 0 µM MGO. D: total amount of MAP kinases (JNK, p38, and ERK) and eNOS after MGO treatment (0–420 µM, 24 h) was shown. Equal protein loading was confirmed using total actin antibody.

 
The activation of NF-{kappa}B pathway was next examined. Phosphorylation of NF-{kappa}B p65 at Ser536 was not induced by the treatment of HUVECs with MGO (0–420 µM, 20 min; Fig. 4A; n = 4) in contrast to TNF-{alpha} treatment (10 ng/ml, 5 min). Consistently, degradation of I{kappa}B-{alpha} protein was not caused by the MGO treatment (Fig. 4A; n = 4). We also observed that nuclear translocation of NF-{kappa}B p65 was not induced by MGO (420 µM, 15–60 min; n = 3; Fig. 4B), suggesting that the NF-{kappa}B pathway is not involved in the COX-2 induction by MGO.


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 4. MGO had no effects on NF-{kappa}B pathway. A: after HUVECs were treated with MGO (0–420 µM) for 20 min, total cell lysates were harvested. Activation of NF-{kappa}B was determined by measuring phosphorylation of NF-{kappa}B p65 at Ser536 as well as degradation of I{kappa}B-{alpha} protein. TNF-{alpha} (10 ng/ml, 5 min) was used as positive control. Equal protein loading was confirmed using total actin antibody (n = 4). B: nuclear translocation of NF-{kappa}B p65 was examined by immunofluorescence staining after HUVECs were treated with MGO (420 µM; a: 0 min; b: 15 min; and c: 60 min; n = 3). TNF-{alpha} (10 ng/ml, 15 min) was used as positive control (d).

 
To clarify whether activation of JNK and/or p38 is responsible for the COX-2 induction by MGO, HUVECs were pretreated with a specific inhibitor of JNK or p38 (30 min) before MGO stimulation (420 µM, 24 h). Pretreatment with the specific JNK inhibitor SP600125 (3 µM) significantly suppressed the COX-2 induction by MGO (COX-2 expression in MGO + SP600125, 1.1 ± 0.1-fold relative to 0 µM MGO, n = 8, P < 0.01 vs. 420 µM MGO; Fig. 5A). We confirmed that SP600125 significantly inhibited the MGO (420 µM, 20 min)-mediated JNK phosphorylation (n = 6; P < 0.01; Fig. 5B). Figure 6A showed that pretreatment of HUVECs with the specific p38 inhibitor SB203580 (100 µM, 30 min) significantly inhibited the MGO (420 µM, 20 min)-mediated p38 phosphorylation (n = 5; P < 0.05). We observed that SB203580 at <100 µM was ineffective to MGO-induced p38 phosphorylation (n = 4; data not shown). However, this concentration of SB203580 (100 µM) was toxic to HUVECs if treated chronically (24 h). Therefore, SB203580 was washed before MGO (420 µM, 24 h) stimulation according to the methods previously described (11). Figure 6B showed that pretreatment of HUVECs with SB203580 (100 µM, 30 min) significantly suppressed the COX-2 induction by MGO (n = 5; P < 0.01).


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 5. Effect of a JNK inhibitor on MGO-mediated COX-2 induction in HUVECs. After HUVECs were treated with 420 µM MGO for 24 h (A) or 20 min (B) in the absence or presence of SP600125 (3 µM, pretreatment for 30 min), total cell lysates were harvested. Expression of COX-2 (A; n = 6–8) and phosphorylation of JNK (B; n = 6–7) were determined by Western blotting. Equal protein loading was confirmed using actin antibody. COX-2 expression (A) or JNK phosphorylation (B) is shown as fold increase relative to 0 µM MGO. **P < 0.01 vs. 0 µM MGO; ##P < 0.01 vs. 420 µM MGO.

 

Figure 6
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 6. Effect of a p38 MAP kinase inhibitor on MGO-mediated COX-2 induction in HUVECs. After HUVECs were treated with 420 µM MGO for 20 min (A) or 24 h (B) in the absence or presence of SB203580 (100 µM, pretreatment for 30 min), total cell lysates were harvested. Phosphorylation of p38 (A; n = 5) and expression of COX-2 (B; n = 5) were determined by Western blotting. Equal protein loading was confirmed using eNOS antibody. Phosphorylation of p38 (A) or COX-2 (B) expression is shown as fold increase relative to 0 µM MGO. *P < 0.05, **P < 0.01 vs. 0 µM MGO; #P < 0.05, ##P < 0.01 vs. 420 µM MGO.

 
It was also confirmed that COX-2 mRNA was significantly increased by the treatment of HUVECs with MGO (420 µM, 6 h, P < 0.01 vs. 0 µM MGO; n = 3) and that the COX-2 induction was significantly prevented by the pharmacological inhibition of JNK (3 µM SP600125, 30 min, P < 0.01 vs. 420 µM MGO; n = 3) or p38 (100 µM SB203580, 30 min, P < 0.05 vs. 420 µM MGO; n = 3; Fig. 7).


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 7. MGO induced mRNA for COX-2 in HUVECs, which is due to JNK and p38 activation. After HUVECs were treated for 6 h with MGO (420 µM), total RNA was harvested. A: expression of COX-2 mRNA was determined by RT-PCR analysis using the gene-specific primers to human COX-2. Equal loading of template cDNA was confirmed by ensuring the GAPDH expression (n = 3). B: expression is shown as fold increase relative to 0 µM MGO. **P < 0.01 vs. 0 µM MGO; #P < 0.05, ##P < 0.01 vs. 420 µM MGO.

 
MGO-induced COX-2-PGE2 is dissociating from EC morphological damage. We next examined whether MGO-induced COX-2 is associated with EC morphological damage. Treatment of HUVECs with SP600125 (3 µM), SB203580 (100 µM), or the specific COX-2 inhibitor NS398 (1–10 µM) did not prevent MGO (420 µM, 24 h)-induced morphological damage (Fig. 8), suggesting that MGO-induced COX-2-PGE2 is dissociating from morphological damage.


Figure 8
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 8. Representative photomicrographs of HUVECs treated for 24 h with MGO (A: 0 µM; B–E: 420 µM) in the presence of SP600125 (C: 3 µM, pretreatment for 30 min), SB203580 (D: 100 µM), SP600125 + SB203580 (E), or NS398 (F: 10 µM) are shown. Magnification: x100.

 
Effects of anti-oxidant on MGO-mediated EC inflammation. To finally examine whether EC inflammation by MGO is associated with increased oxidative stress, HUVECs were pretreated with EUK134 (3 µM, 30 min) before MGO stimulation (420 µM, 24 h). EUK134 is a synthetic superoxide dismutase/catalase mimetic (27, 29). Treatment of HUVECs with EUK134 had no effect on both EC morphology (data not shown) and MGO-mediated COX-2 induction (Fig. 9; n = 3), suggesting that the effect of MGO is independent of reactive oxygen species.


Figure 9
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 9. Effect of antioxidant on MGO-induced COX-2. HUVECs were treated with MGO (420 µM, 24 h) in the absence or presence of EUK134 (3 µM, pretreatment for 30 min). Effect of EUK134 on COX-2 expression was determined by Western blotting. COX-2 expression is shown as fold change relative to 0 µM MGO (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of the present study are that MGO, a metabolite of glucose induced inflammatory responses as determined by COX-2 induction and PGE2 production in human vascular ECs (Fig. 10). It was clarified that induction of COX-2 was mediated via activating the cellular signal transduction of JNK and p38 MAP kinase but not reactive oxygen species production. We also found that MGO induced cytotoxic morphological damage in ECs, which is independent of COX-2-PGE2. Since ECs inflammatory response and morphologic damage are early pathogenic steps of atherosclerosis, our results suggest that MGO might play a significant role in diabetes-related atherosclerosis.


Figure 10
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 10. Proposed pathways leading to MGO-mediated endothelial cell inflammation and morphological injury, which might be responsible for the diabetes-associated atherosclerosis.

 
The present study used 56–420 µM of MGO for in vitro experiments, and this concentration of MGO caused ECs inflammatory injury in a concentration-dependent manner. There is a report (16) showing that plasma MGO concentration in poorly controlled human diabetic patients is ~400 µM, whereas others (20, 33) demonstrated that it was much less. It has been suggested that the local MGO concentration in tissues is much higher than the plasma level (26). It was also reported that cultured cells may produce larger amounts of MGO (as much as 310 µM; Ref. 9). Nevertheless, determination of the chronic effects of a lower concentration of MGO especially in blood vessels and whole animals is necessary to validate the present in vitro results.

In the present study, we determined that COX-2 induction by MGO was mediated via activating JNK and p38 MAP kinase. It is well known that COX-2 induction by various stimuli is mainly regulated by the MAP kinase family (ERK, JNK, and p38; Refs. 11, 24, 36) and/or NF-{kappa}B (31), supporting our results. In contrast, our recent results (38) showed that glyoxal, one of the metabolites of glucose, induced COX-2 protein but failed to activate JNK and p38. MGO is different from glyoxal only at the methyl group, and we could not explain why such differences occurred in the cellular signal transduction. It is speculated that another target rather than protein kinases might also exist for the effects of glucose metabolites. The recent report (41) that MGO modifies nuclear transcriptional regulatory proteins might support the concept.

On the other hand, we observed that MGO-induced ECs morphological damage was not associated with increased COX-2-PGE2. There are two possible explanations. MGO-induced COX-2-PGE2 was related to other ECs inflammatory states such as hyperpermeability (6, 19) and/or increased leukocyte-ECs interaction (12, 21) rather than direct ECs morphological damage. Alternatively, MGO might directly interact with DNA and damage the cell, since it is suggested that MGO mediates DNA glycation, which may be associated with mutation, DNA strand breaks, and cytotoxicity (32).

Whether the effects of MGO are due to extracellular effects or intracellular effects seems an important issue, since MGO is produced in the cytosol under hyperglycemic condition (30). Furthermore, whether the present results are caused directly via MGO or indirectly via MGO-derived AGEs is also an important issue. We postulate that the results obtained here might be mediated mainly via extracellular MGO itself but not AGEs by the following reasons. 1) Our preliminary data showed that MGO-derived AGEs was not detectable even 6 h after treatment of HUVECs with MGO (420 µM) as measured by anti-N{varepsilon}-(carboxyethyl) lysine (CEL), a specific MGO-derived AGEs Western blotting. 2) In our former study (38), we provided evidence that the pathological concentration of AGEs (100–500 µM, 24 h) had no effect on both COX-2 induction and morphology in HUVECs. However, it could not be ruled out that exogenously applied MGO might move inside the cells and cause cytotoxic injury by unknown mechanisms. Our preliminary results that a higher concentration of MGO (560 µM, 6–12 h), which is able to induce apoptosis in HUVECs (5), produced AGEs (CEL) specifically in the nuclei (as determined by immunofluorescence) might support the concept.

In summary, we demonstrated in cultured human vascular ECs that MGO, a reactive metabolite of glucose, could be a stimulant for vascular inflammatory responses. EC inflammation by MGO was mediated via the activation of JNK and p38 MAP kinase. Further investigations in blood vessels and whole animals might contribute to obtain further mechanistic insights into the diabetes-associated atherosclerosis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Grant-in-Aid for Scientific Research #19790195 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and Research Grants from the Sumitomo Foundation, the Takeda Science Foundation, and the Uehara Memorial Foundation (to Dr. H. Yamawaki).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Yamawaki, Dept. of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato Univ., Towada, Aomori 034-8628, Japan (e-mail: yamawaki{at}vmas.kitasato-u.ac.jp)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Ahmed MU, Brinkmann Frye E, Degenhardt TP, Thorpe SR, Baynes JW. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem J 324: 565–570, 1997.[Web of Science][Medline]

2. Akhand AA, Hossain K, Kato M, Miyata T, Du J, Suzuki H, Kurokawa K, Nakashima I. Glyoxal and methylglyoxal induce lyoxal and methyglyoxal induce aggregation and inactivation of ERK in human endothelial cells. Free Radic Biol Med 31: 1228–1235, 2001.[CrossRef][Web of Science][Medline]

3. Akhand AA, Hossain K, Mitsui H, Kato M, Miyata T, Inagi R, Du J, Takeda K, Kawamoto Y, Suzuki H, Kurokawa K, Nakashima I. Glyoxal and methylglyoxal trigger distinct signals for MAP family kinases and caspase activation in human endothelial cells. Free Radic Biol Med 31: 20–30, 2001.[CrossRef][Web of Science][Medline]

4. Aso Y, Inukai T, Tayama K, Takemura Y. Serum concentrations of advanced glycation endproducts are associated with the development of atherosclerosis as well as diabetic microangiopathy in patients with type 2 diabetes. Acta Diabetol 37: 87–92, 2000.[CrossRef][Web of Science][Medline]

5. Baden T, Yamawaki H, Saito K, Mukohda M, Okada M, Hara Y. Telmisartan inhibits methylglyoxal-mediated cell death in human vascular endothelium. Biochem Biophys Res Commun 373: 253–257, 2008.

6. Barbieri SS, Weksler BB. Tobacco smoke cooperates with interleukin-1β to alter β-catenin trafficking in vascular endothelium resulting in increased permeability and induction of cyclooxygenase-2 expression in vitro and in vivo. FASEB J 21: 1831–1843, 2007.[Abstract/Free Full Text]

7. Bartnik M, Norhammar A, Ryden L. Hyperglycaemia and cardiovascular disease. J Intern Med 262: 145–156, 2007.[CrossRef][Web of Science][Medline]

8. Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, Kislinger T, Stern DM, Schmidt AM, De Caterina R. Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation 105: 816–822, 2002.[Abstract/Free Full Text]

9. Chaplen FW, Fahl WE, Cameron DC. Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells. Proc Natl Acad Sci USA 95: 5533–5538, 1998.[Abstract/Free Full Text]

10. Fosmark DS, Torjesen PA, Kilhovd BK, Berg TJ, Sandvik L, Hanssen KF, Agardh CD, Agardh E. Increased serum levels of the specific advanced glycation end product methylglyoxal-derived hydroimidazolone are associated with retinopathy in patients with type 2 diabetes mellitus. Metabolism 55: 232–236, 2006.[CrossRef][Web of Science][Medline]

11. Gallicchio M, Rosa AC, Benetti E, Collino M, Dianzani C, Fantozzi R. Substance P-induced cyclooxygenase-2 expression in human umbilical vein endothelial cells. Br J Pharmacol 147: 681–689, 2006.[CrossRef][Medline]

12. Hailer NP, Oppermann E, Leckel K, Cinatl J, Markus BH, Blaheta RA. Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells. Transplantation 70: 236–240, 2000.[Web of Science][Medline]

13. Hartge MM, Unger T, Kintscher U. The endothelium and vascular inflammation in diabetes. Diab Vasc Dis Res 4: 84–88, 2007.[Abstract/Free Full Text]

14. Ho FM, Lin WW, Chen BC, Chao CM, Yang CR, Lin LY, Lai CC, Liu SH, Liau CS. High glucose-induced apoptosis in human vascular endothelial cells is mediated through NF-kappaB and c-Jun NH2-terminal kinase pathway and prevented by PI3K/Akt/eNOS pathway. Cell Signal 18: 391–399, 2006.[CrossRef][Web of Science][Medline]

15. Kilhovd BK, Berg TJ, Birkeland KI, Thorsby P, Hanssen KF. Serum levels of advanced glycation end products are increased in patients with type 2 diabetes and coronary heart disease. Diabetes Care 22: 1543–1548, 1999.[Abstract/Free Full Text]

16. Lapolla A, Flamini R, Dalla Vedova A, Senesi A, Reitano R, Fedele D, Basso E, Seraglia R, Traldi P. Glyoxal and methylglyoxal levels in diabetic patients: quantitative determination by a new GC/MS method. Clin Chem Lab Med 41: 1166–1173, 2003.[CrossRef][Medline]

17. Libby P. Inflammation in atherosclerosis. Nature 420: 868–874, 2002.[CrossRef][Web of Science][Medline]

18. Maiti R, Agrawal NK. Atherosclerosis in diabetes mellitus: role of inflammation. Indian J Med Sci 61: 292–306, 2007.[Medline]

19. Mark KS, Trickler WJ, Miller DW. Tumor necrosis factor-alpha induces cyclooxygenase-2 expression and prostaglandin release in brain microvessel endothelial cells. J Pharmacol Exp Ther 297: 1051–1058, 2001.[Abstract/Free Full Text]

20. McLellan AC, Thornalley PJ, Benn J, Sonksen PH. Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin Sci (Lond) 87: 21–29, 1994.[Medline]

21. Moreno JJ. Antiflammin-2 prevents HL-60 adhesion to endothelial cells and prostanoid production induced by lipopolysaccharides. J Pharmacol Exp Ther 296: 884–889, 2001.[Abstract/Free Full Text]

22. Mostafa AA, Randell EW, Vasdev SC, Gill VD, Han Y, Gadag V, Raouf AA, El Said H. Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes. Mol Cell Biochem 302: 35–42, 2007.[CrossRef][Medline]

23. Ono Y, Aoki S, Ohnishi K, Yasuda T, Kawano K, Tsukada Y. Increased serum levels of advanced glycation end-products and diabetic complications. Diabetes Res Clin Pract 41: 131–137, 1998.[CrossRef][Web of Science][Medline]

24. Park YS, Kim J, Misonou Y, Takamiya R, Takahashi M, Freeman MR, Taniguchi N. Acrolein induces cyclooxygenase-2 and prostaglandin production in human umbilical vein endothelial cells: roles of p38 MAP kinase. Arterioscler Thromb Vasc Biol 27: 1319–1325, 2007.[Abstract/Free Full Text]

25. Phillips SA, Thornalley PJ. The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. Eur J Biochem 212: 101–105, 1993.[Web of Science][Medline]

26. Randell EW, Vasdev S, Gill V. Measurement of methylglyoxal in rat tissues by electrospray ionization mass spectrometry and liquid chromatography. J Pharmacol Toxicol Methods 51: 153–157, 2005.[CrossRef][Medline]

27. Rong Y, Doctrow SR, Tocco G, Baudry M. EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology. Proc Natl Acad Sci USA 96: 9897–9902, 1999.[Abstract/Free Full Text]

28. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med 340: 115–126, 1999.[Free Full Text]

29. Samai M, Sharpe MA, Gard PR, Chatterjee PK. Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity. Free Radic Biol Med 43: 528–534, 2007.[CrossRef][Medline]

30. Shinohara M, Thornalley PJ, Giardino I, Beisswenger P, Thorpe SR, Onorato J, Brownlee M. Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis. J Clin Invest 101: 1142–1147, 1998.[Web of Science][Medline]

31. Syeda F, Grosjean J, Houliston RA, Keogh RJ, Carter TD, Paleolog E, Wheeler-Jones CP. Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways. J Biol Chem 281: 11792–11804, 2006.[Abstract/Free Full Text]

32. Thornalley PJ. Protecting the genome: defence against nucleotide glycation and emerging role of glyoxalase I overexpression in multidrug resistance in cancer chemotherapy. Biochem Soc Trans 31: 1372–1377, 2003.[Medline]

33. Thornalley PJ, Hooper NI, Jennings PE, Florkowski CM, Jones AF, Lunec J, Barnett AH. The human red blood cell glyoxalase system in diabetes mellitus. Diabetes Res Clin Pract 7: 115–120, 1989.[CrossRef][Medline]

34. Valencia JV, Mone M, Koehne C, Rediske J, Hughes TE. Binding of receptor for advanced glycation end products (RAGE) ligands is not sufficient to induce inflammatory signals: lack of activity of endotoxin-free albumin-derived advanced glycation end products. Diabetologia 47: 844–852, 2004.[CrossRef][Web of Science][Medline]

35. Wang X, Desai K, Chang T, Wu L. Vascular methylglyoxal metabolism and the development of hypertension. J Hypertens 23: 1565–1573, 2005.[Medline]

36. Wu G, Luo J, Rana JS, Laham R, Sellke FW, Li J. Involvement of COX-2 in VEGF-induced angiogenesis via P38 and JNK pathways in vascular endothelial cells. Cardiovasc Res 69: 512–519, 2006.[Abstract/Free Full Text]

37. Wu L. Is methylglyoxal a causative factor for hypertension development? Can J Physiol Pharmacol 84: 129–139, 2006.[CrossRef][Medline]

38. Yamawaki H, Hara Y. Glyoxal causes inflammatory injury in human vascular endothelial cells. Biochem Biophys Res Commun 369: 1155–1159, 2008.[Medline]

39. Yamawaki H, Iwai N. Cytotoxicity of water-soluble fullerene in vascular endothelial cells. Am J Physiol Cell Physiol 290: C1495–C1502, 2006.[Abstract/Free Full Text]

40. Yamawaki H, Pan S, Lee RT, Berk BC. Fluid shear stress inhibits vascular inflammation by decreasing thioredoxin-interacting protein in endothelial cells. J Clin Invest 115: 733–738, 2005.[CrossRef][Web of Science][Medline]

41. Yao D, Taguchi T, Matsumura T, Pestell R, Edelstein D, Giardino I, Suske G, Rabbani N, Thornalley PJ, Sarthy VP, Hammes HP, Brownlee M. High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A. J Biol Chem 282: 31038–31045, 2007.[Abstract/Free Full Text]

42. Zhang L, Zalewski A, Liu Y, Mazurek T, Cowan S, Martin JL, Hofmann SM, Vlassara H, Shi Y. Diabetes-induced oxidative stress and low-grade inflammation in porcine coronary arteries. Circulation 108: 472–478, 2003.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
E. A. Jaimes, P. Hua, R.-X. Tian, and L. Raij
Human glomerular endothelium: interplay among glucose, free fatty acids, angiotensin II, and oxidative stress
Am J Physiol Renal Physiol, January 1, 2010; 298(1): F125 - F132.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/6/C1510    most recent
00252.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamawaki, H.
Right arrow Articles by Hara, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamawaki, H.
Right arrow Articles by Hara, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.