2025 Volume 48 Issue 3 Pages 262-266
Toxic advanced glycation end-products (TAGEs) are glyceraldehyde (GA)-derived AGEs with strong cytotoxic effects. TAGEs are also involved in lifestyle-related diseases. Notably, modification of TAGEs by GA causes protein dysfunction. As endothelial nitric oxide synthase (eNOS) is constitutively expressed in vascular endothelial cells and is a source of nitric oxide (NO), we focused on it as a TAGE modification-targeting protein. Our laboratory has reported that Ras guanyl nucleotide-releasing protein 2 (RasGRP2) activates Rap1 and R-Ras, among other small GTPases, and suppresses apoptosis and TAGE-induced vascular hyperpermeability in vascular endothelial cells. Therefore, in this study, we investigated the effects of RasGRP2 on cell death, TAGE formation, and TAGE modification of eNOS in vascular endothelial cells following GA treatment using RasGRP2-overexpressing (R) cells and mock (M) immortalized human umbilical vein endothelial cells. GA treatment decreases the viability of both cell types in a concentration-dependent manner. In M cells, GA treatment increased the formation of TAGEs and TAGE modification of eNOS in a concentration-dependent manner, but this increase was suppressed in R cells. Additionally, co-treatment with aminoguanidine, an inhibitor of AGEs formation, suppressed cell death and TAGE modification of eNOS induced by GA. These results indicate that GA induces cell death, the formation of TAGEs, and TAGE modification of eNOS in vascular endothelial cells. Additionally, RasGRP2 is a protective factor that suppresses TAGE formation.
Advanced glycation end-products (AGEs) are structures formed at the late stage of a nonenzymatic reaction (Maillard reaction) between proteins and reducing sugars such as glucose. Among them, glyceraldehyde (GA)-derived AGEs are cytotoxic and are classified as toxic advanced glycation end-products (TAGEs).1) Reportedly, TAGEs are involved in diabetic retinopathy and nephropathy caused by vascular endothelial disorders.2) The leakage of TAGEs formed inside cells to the outside affects the function of surrounding cells and increases serum TAGE concentration, which is considered to promote the development of lifestyle-related diseases.3) Notably, the activity of proteins is altered by TAGE modification,4,5) and not only the toxicity of TAGEs itself but also the functional changes in proteins caused by TAGE modification are attracting attention. However, the exploration of target proteins for TAGE modification in endothelial dysfunction is insufficient, and further research is required to examine the dysfunction of proteins caused by GA-induced TAGE formation.
Nitric oxide (NO) is a gaseous molecule involved in the relaxation of blood vessels and protection of vascular endothelial cells. NO is produced by NO synthase (NOS) during the conversion of l-arginine (Arg) to l-citrulline.6) Endothelial NOS (eNOS) is constitutively expressed in vascular endothelial cells and is a source of NO that produces physiological effects.7) Dimerization of eNOS is necessary for NO production by eNOS. Monomeric eNOS produces O2− that reacts with NO to form ONOO−, leading to the expansion of damage in the acute phase of cerebral ischemia.8) Reportedly, the expression level of eNOS protein is decreased in aging and arteriosclerosis,9,10) and eNOS proteins are monomerized in hyperglycemia.11) However, the detailed mechanism of the decrease in eNOS expression and dysfunction in the context of these diseases has not yet been clarified.
We have previously demonstrated that the guanine nucleotide exchange factor Ras guanyl nucleotide-releasing protein 2 (RasGRP2) is expressed in vascular endothelial cells. We also reported that RasGRP2 may act as a protective factor against vascular endothelial injury by activating the small G proteins Rap1 and R-Ras to suppress apoptosis and that RasGRP2 suppresses vascular hyperpermeability induced by TAGE treatment.12–15) However, the effect of RasGRP2 on the toxicity of intracellularly generated TAGEs remains unknown.
Therefore, in this study, we investigated the effects of RasGRP2 on cell death and TAGE modification of eNOS by GA-induced intracellular TAGE formation using human telomerase reverse transcriptase immortalized human umbilical vein endothelial cells (TERT HUVECs) stably overexpressing RasGRP2.
TERT HUVECs were provided by Dr. Kazuto Nishio (Kindai University, Osaka, Japan).15) TERT HUVECs were cultured in endothelial cell growth medium (C-22010, PromoCell, Heidelberg, Germany) at 5% CO2 and 37°C. A stable RasGRP2-overexpressing cell line was previously established.15) The amplified DNA fragment of rasgrp2 was transfected into TERT HUVECs using pEB Multi-Hyg (050-08121, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) as a vector and ViaFect Transfection Reagent (E4981, Promega, WI, U.S.A.) as a transfection reagent.
Cells were seeded into dishes (2.0 × 105 cells/mL) and incubated for 24 h before each experiment.
TreatmentCells were treated with GA (17014-81, NACALAI TESQUE, Kyoto, Japan) dissolved in phosphate-buffered saline (PBS) and added to the medium at a final concentration of 1, 2, or 4 mM for 6 h. The cells were also treated with aminoguanidine (AG, 328-26432, FUJIFILM Wako Pure Chemical Corporation) dissolved in PBS and added to the medium at the same time as GA at a final concentration of 1 or 5 mM for 6 h.
WST-8 AssayAfter each treatment, the entire volume of the culture medium was removed, and a mixture of Cell Counting Kit-8 (CK04, DOJINDO, Kumamoto, Japan) and Endothelial Cell Growth Medium (EGM) was added and incubated for 2 h. Absorbance was measured at 450 and 650 nm using a SPECTRAMAX 190 (Molecular Devices, San Jose, CA, U.S.A.).
Western Blot (WB)Cells were collected in 100 µL of Pierce® IP Lysis Buffer (87787, Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing Halt™ Protease Inhibitor (78442, Thermo Fisher Scientific) and Phosphatase Inhibitor (07575-51, NACALAI TESQUE). The collected cell lysate was centrifuged at 12000 × g at 4°C for 10 min, and the supernatant was collected. Electrophoresis was performed at 300 V and 30 mA for 50 min on 10% sodium dodecyl sulfate-polyacrylamide gel with 10 µL of sample. Transfer to an Immobilon®-P Membrane (IPVH00010, Millipore, Germany) was performed at 100 V and 2.00 A for 2 h. The membrane was incubated at 37°C for 1 h with a polyvinylidene difluoride (PVDF) Blocking Reagent for Can Get Signal® (NYPBR01, TOYOBO, Osaka, Japan). After blocking, the membrane was washed for 5 min with PBS containing 0.05% Tween-20 (PBS-T), reacted with a primary antibody specific for β-actin (1 : 10000; sc-4778, Santa Cruz Biotechnology, Dallas, TX, U.S.A.) or eNOS (1 : 1000; sc-376751, Santa Cruz Biotechnology), diluted with Can Get Signal Solution 1 (NKB-201, TOYOBO) for 1 h, rinsed with PBS-T, and washed three times for 5 min each wash. Next, the horseradish peroxidase (HRP) conjugated secondary antibody (anti-mouse immunoglobulin G (IgG), 1 : 5000; P0260, Dako, Denmark) diluted with Can Get Signal Solution 2 (NKB-301, TOYOBO) was reacted for 1 h, rinsed with PBS-T, and washed 4 times for 15 min each wash. The reacted membranes were incubated with ECL Chemiluminescence HRP (T7101B, TaKaRa, Shiga, Japan) or ECL Quant HPR (T7102B, TaKaRa), and immunoreactive bands were detected using an X-ray film (28906837, Cytiva, Tokyo, Japan).
Slot Blot (SB)The detection of intracellular TAGE levels was performed according to the method of Takata.16) Cells were collected in 100 µL of lysis buffer containing 30 mM Tris (206-07884, Wako, FUJIFILM Wako Pure Chemical Corporation), 2 M thiourea (204-01202, Wako), 7 M urea (43009-01, Kanto Chemical, Tokyo, Japan), 4% CHAPS (347-04723, DOJINDO), and 1 tablet or 10 mL of CompleteTM Mini EDTA-free Protease Inhibitor Cocktail (11836170001, Roche, Switzerland). The collected cell lysate was centrifuged at 10000 × g at 4°C for 15 min, and the supernatant was collected. Using a Biodot SF (1706542, Bio-Rad, Japan), 200 µL of sample (1 µg of protein) was added to each well and adsorbed on an Immobilon®-P Membrane. The adsorbed membrane was incubated at 37°C for 1 h with the PVDF Blocking Reagent for Can Get Signal. The blocked membrane was washed with PBS-T and then reacted at 4°C overnight with the anti-TAGE polyclonal antibody (1 : 1000, prepared as described in a previous study)17) diluted with Can Get Signal Solution 1. The anti-TAGE antibody was incubated with bovine serum albumin or TAGE standard protein (prepared as in a previous study)17) for 1 h at room temperature before reacting with the membrane. The antibody that reacted with TAGEs was used as the neutralizing antibody and washed 3 times for 15 min each with PBS-T. HRP-conjugated anti-rabbit IgG antibody (NB7179, Novus Biologicals, Centennial, CO, U.S.A.) diluted with Can Get Signal Solution 2 was allowed to react for 1 h and then washed 4 times for 15 min with PBS-T. The reacted membranes were incubated with ECL Chemiluminescence HRP, and immunoreactive bands were detected using an X-ray film.
Statistical AnalysisAll experiments were performed at least 3 times. Data are presented as mean ± standard deviation. Differences between the groups were analyzed using 1-way ANOVA and Fisher's least significant difference post hoc tests. The statistical significance was set at p < 0.05.
To evaluate the toxicity of GA, each cell was treated with 0, 1, 2, or 4 mM GA for 6 h, and cell viability was evaluated using the WST-8 assay. GA treatment for 6 h significantly decreased the viability of both cell types in a GA concentration-dependent manner. Compared to M cells, R cells significantly suppressed the GA-induced decrease in cell viability; however, the difference was slight (Fig. 1).
Assessment of cell viability using the Cell Counting Kit-8 assay. Data are presented as the mean (percentage relative to each GA 0 mM) ± S.D. from 3 samples; statistical significance is denoted by p-values (Fisher’s LSD tests).
The amount of intracellular TAGEs formed by GA treatment was examined by the SB method using the anti-TAGE antibody. Additionally, nonspecific adsorption of the anti-TAGE antibody onto the membrane was evaluated by performing experiments using a neutralized anti-TAGE antibody in the same manner as the anti-TAGE antibody. GA increased the band intensity in a concentration-dependent manner in the anti-TAGE antibody analysis. In the analysis using neutralizing antibodies, nonspecific adsorption was observed in both cell types (Fig. 2a). Correcting for effects due to nonspecific adsorption, treatment with 2 mM GA for 6 h significantly increased intracellular TAGE amounts, whereas no significant increase was observed in R cells (Fig. 2b).
Assessment of intracellular TAGE formation using the SB method. (a) The SB image (upper: anti-TAGE antibody; lower: neutralized anti-TAGE antibody); (b) TAGE formation is indicated as the difference between each intensity of the band for anti-TAGE antibody and neutralized anti-TAGE antibody. Data are presented as the mean ± S.D. from 3 independent experiments; statistical significance is denoted by p-values (Fisher’s LSD tests).
The bands of eNOS were detected with a shift to a higher molecular weight than the band near 135 kDa as a monomer and were smeared by GA treatment in WB (Fig. 3a). The higher molecular shift of the band and smearing significantly increased in a GA concentration-dependent manner in both cell types. However, in R cells, the shift to a higher molecular weight and smearing of the eNOS band by 2 and 4 mM GA was lower than that in M cells (Fig. 3b).
Detection of the eNOS shift to a higher molecular weight using WB analysis. (a) The WB images (upper: eNOS; lower: β-actin). (b) The amount of shifted eNOS was analyzed by ImageJ. Data are presented as the mean (relative to GA 0 mM in the M cell) ± S.D. from 3 independent experiment; statistical significance is denoted by p-values (Fisher’s LSD tests).
As AG is an AGEs and TAGE formation inhibitor that reacts with intermediates in the Maillard reaction,18) we investigated whether the toxicity of GA was inhibited by AG. The effect of co-treatment with GA and the glycation inhibitor AG on cell viability was investigated. Co-treatment with GA and AG significantly inhibited the decrease in cell viability observed with GA alone. Compared to M cells, treatment with GA 2 mM and AG 5 mM significantly attenuated the decrease in cell viability of R cells. However, the difference was small (Fig. 4). Co-treatment with GA and AG reduced the shift to a higher molecular weight and the smearing of the eNOS bands (Fig. 5a). Co-treatment with GA and AG also significantly reduced TAGE-modified eNOS to a similar extent in both cell types compared to that in response to treatment with GA alone (Fig. 5b).
Assessment of cell viability using the Cell Counting Kit-8 assay. Data are presented as the mean (percentage relative to each GA 0 mM) ± S.D. from 3 samples; statistical significance is denoted by p-values (Fisher’s LSD tests).
Assessment of TAGE modification of eNOS using WB analysis. (a) The WB images (upper: eNOS; lower: β-actin). (b) The amount of shifted eNOS was analyzed by ImageJ. Data are presented as the mean (relative to GA 0 mM in the M cell) ± S.D. from 3 independent experiments; statistical significance is denoted by p-values (Fisher’s LSD tests).
In the cell viability assay, GA treatment for 6 h caused a significant decrease in the viability of both cell types in a GA concentration-dependent manner. Compared to M cells, R cells significantly inhibited the decrease in cell viability induced by treatment with GA; however, the inhibitory effect was slight (Fig. 1). GA is known to react with proteins to form TAGEs and cause cell death.3) Notably, necrosis is caused by the formation and accumulation of intracellular TAGEs.19) We have previously reported that RasGRP2 exerts an anti-apoptotic effect13,14); however, in this study, the effect of RasGRP2 on cell viability was slight. Therefore, the decrease in cell viability caused by GA treatment in this study was considered to be due to non-apoptotic cell death such as necrosis.
In the analysis of the amount of intracellular TAGEs formed by the SB method, the amount of TAGEs was significantly decreased in R cells compared to that in M cells (Fig. 2). Furthermore, the shift of eNOS bands to higher molecular weights caused by GA was decreased in R cells (Fig. 3). Additionally, RasGRP2 decreased the shifted band and smearing of eNOS in a manner similar to that of AG, which inhibits AGEs formation20) (Fig. 5). It has been reported that reactive oxygen species (ROS) promote the formation of AGEs.21) RasGRP2 has been reported to suppress ROS production via Rap1.13) Thus, intracellular TAGE formation is considered to be inhibited by RasGRP2 via alleviating oxidative stress.
In protein analysis by WB, a shift of eNOS bands to high molecular weights was detected after GA treatment. It was reported that GA reacts with the amino group in amino acid residues of the protein to form the structure of TAGEs in which 2 or 3 proteins are cross-linked through the 1,4-dihydropyrazine structure or intramolecular cross-linked structure through multistep reactions such as Schiff base formation and Amadori transition. Additionally, it has been reported that GA exhibits a higher reactivity with lysine (Lys) than with Arg during TAGE formation.1) In addition to eNOS, the binding of calmodulin (CaM) to the CaM binding site containing Lys residues causes electron transfer that is necessary for NO production, and the acetylation of this Lys residue inhibits CaM binding and prevents NO production.22,23) Furthermore, eNOS loses its ability to produce NO and enhances ROS production in monomeric form.24,25) Therefore, it is suggested that GA may induce the functional impairment of eNOS by TAGE modification of Lys residues in the CaM binding site of eNOS to prevent functional dimer formation or electron transfer. Furthermore, WB analysis results revealed that RasGRP2 decreased the high molecular weight shift of eNOS (Fig. 3), suggesting the possibility that RasGRP2 protected against eNOS dysfunction induced by TAGE modification.
RasGRP2, similar to AG, suppressed TAGE formation and prevented the high molecular weight shift of eNOS. However, it was not able to suppress the decrease in cell viability caused by GA to the same extent as AG (Figs. 2 and 4). AG has been reported to inhibit AGEs formation by trapping nonenzymatic glycation reaction intermediates.21) Therefore, RasGRP2 indirectly suppressed TAGE formation through ROS suppression and did not induce sufficient improvement in cell viability compared to AG, which directly suppressed the TAGE formation reaction in the high-concentration short-term GA treatment in this study. This may also explain the lack of an additive effect by AG and RasGRP2 for suppressing TAGE formation. However, RasGRP2 has been suggested to be an important factor in suppressing intracellular TAGE formation.
In conclusion, GA decreases the cell viability and TAGE modification of eNOS in vascular endothelial cells. Additionally, RasGRP2 acts as a protective factor against GA-induced TAGE formation in endothelial cells and may be useful for the treatment of vascular disorders such as diabetes by protecting eNOS function from TAGE modification.
This research was supported by JSPS KAKENHI (Grant Numbers: 21H04865 and 22K17789).
The authors declare no conflict of interest.
