The role of RAGE, MAPK and NF-κB pathway in the advanced glycation end-products induced HUVECs dysfunction

Xun Wang; Yi-Shu Wang; Qing-Long Zeng; Chen-Yang Qiu; Yang-Yan He; Zi-Heng Wu; Yun-Jun He; Tao Shang; Hong-Kun Zhang; Qian-Qian Zhu; Dong-Lin Li

doi:10.4103/2589-9686.360874

ORIGINAL ARTICLE

Year : 2022 | Volume : 5 | Issue : 3 | Page : 80-87

The role of RAGE, MAPK and NF-κB pathway in the advanced glycation end-products induced HUVECs dysfunction

Xun Wang, Yi-Shu Wang, Qing-Long Zeng, Chen-Yang Qiu, Yang-Yan He, Zi-Heng Wu, Yun-Jun He, Tao Shang, Hong-Kun Zhang, Qian-Qian Zhu, Dong-Lin Li
Department of Vascular Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

Date of Submission	23-Jan-2022
Date of Decision	20-Mar-2022
Date of Acceptance	24-Mar-2022
Date of Web Publication	10-Nov-2022

Correspondence Address:
Dr. Dong-Lin Li
Department of Vascular Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou
China
Dr. Qian-Qian Zhu
Department of Vascular Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2589-9686.360874

Abstract

OBJECTIVE: The objective of this study was to investigate how receptor for advanced glycation end-products–mitogen-activated protein kinase–nuclear factor-kappa B (MAPK-NF-κB) pathway is involved in advanced glycation end-product (AGE)-induced human umbilical venous endothelial cell (HUVEC) dysfunction.
MATERIALS AND METHODS: HUVECs were cultured with AGEs, anti-RAGE, inhibitors of MAPK or NF-κB respectively. Then we detected endothelial nitric oxide synthase (eNOS) activation, nitric oxide (NO) concentration, cell migration ability, and RAGE expression of HUVECs.
RESULTS: AGEs depressed eNOS activation, decreased NO concentration, impaired endothelial cell (EC) migration, and upregulated RAGE expression, which could be recovered by p38 inhibitor and extracellular regulated protein kinases (ERK) inhibitor. However, these effects could not be recovered by NF-κB inhibitor.
CONCLUSIONS: AGEs increase RAGE expression and decrease NO release and migration of HUVECs through RAGE-MAPK pathway, but not NF-κB pathway.

Keywords: Advanced glycation end-products, endothelial dysfunction, mitogen-activated protein kinase, nuclear factor-kappa B

How to cite this article:
Wang X, Wang YS, Zeng QL, Qiu CY, He YY, Wu ZH, He YJ, Shang T, Zhang HK, Zhu QQ, Li DL. The role of RAGE, MAPK and NF-κB pathway in the advanced glycation end-products induced HUVECs dysfunction. Vasc Invest Ther 2022;5:80-7

How to cite this URL:
Wang X, Wang YS, Zeng QL, Qiu CY, He YY, Wu ZH, He YJ, Shang T, Zhang HK, Zhu QQ, Li DL. The role of RAGE, MAPK and NF-κB pathway in the advanced glycation end-products induced HUVECs dysfunction. Vasc Invest Ther [serial online] 2022 [cited 2023 Jun 4];5:80-7. Available from: https://www.vitonline.org/text.asp?2022/5/3/80/360874

Introduction

Diabetes-associated microvascular and cardiovascular events are among the major causes of patient mortality.^[1] It has been shown that endothelial dysfunction contributes to diabetic atherosclerosis and cardiovascular damage.^[2] Advanced glycation end-products (AGEs) are produced by nonenzymatic glycation and oxidation of proteins, which have been recognized as an important pathogenesis in diabetic-associated endothelial dysfunction.^[3] AGEs impair the function of endothelial progenitor cells (EPCs)/endothelial cells (ECs) and accelerate the progression of atherosclerosis.^[4],[5] AGEs promote EPC/EC apoptosis, inhibit EPC migration and adhesion ability, and impair tube-forming ability.^[6],[7] One of its receptors, the receptor for AGEs (RAGE), mediates intracellular pathological signal transduction.^[8],[9]

Previous reports has showed that AGE-RAGE binding effected diverse signaling pathways, such as p38 and ERK mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways.^{[10],[11],[12],[13]} AGE-induced MAPK activation was associated with reduced endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) release in EPCs.^[13],[14] Furthermore, AGE-induced NF-κB activation could be attenuated by various MAPK inhibitors,^[12] supporting the role of RAGE-MAPK-NF-κB pathway in the diabetic-associated endothelial dysfunction. However, the underlying mechanism has remained undefined.

In this study, we sought to investigate how RAGE-MAPK-NF-κB pathway is involved in AGE-induced HUVEC dysfunction, and to explore the relationship of these pathways.

Materials and Methods

Antibodies and reagents

AGEs were obtained from Sigma (St. Louis, MO, USA). Anti-eNOS, anti-phosphorylated-eNOS (p-eNOS), anti-RAGE, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ERK1/2 MAPK inhibitor (PD98059), p38 MAPK inhibitor (SB202190), and NF-κB inhibitor (helenalin) were purchased from Stressgen Bioreagents (Ann Arbor, MI, USA). Human serum albumin (HSA) was purchased from Kangchen Biotechnology (Shanghai, China). A protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), an electrochemiluminescence kit, and a total NO assay kit were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Radio-immunosuppression (RIPA) buffer was obtained from Kangchen Biotechnology, and matrix gel from BD Biosciences (Bedford, MA, USA).

Cell culture

HUVECs were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle medium (DMEM) and Opti-MEM were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS, USA). The HUVECs were propagated in DMEM supplemented with 10% FBS and incubated at 37°C in a humidified chamber of 95% air–5% CO₂. The medium was refreshed every other day.

Advanced glycation end-product treatment

HUVECs in growth medium were seeded equally into culture plates. When reaching 80%–90% confluence, the cells were pretreated for 1 h with 50 mg/L anti-RAGE, or 50 μmol/L PD98059 (ERK1/2 MAPK inhibitor), or 10 μmol/L SB202190 (p38 MAPK inhibitor), or 50 μmol/L helenalin (NF-κB inhibitor), or without anything. Then, the cells were stimulated with AGEs (200 mg/L) or HSA (200 mg/L).

Western blot analysis

After washing with PBS, HUVECs were lysed with RIPA buffer which contained PMSF. The lysates were centrifuged at 12,000 × g and 4°C for 20 min. Supernatants were harvested and total protein was quantified by a bicinchoninic acid assay. Equal amounts of protein (40 μg) were separated through 3%–8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. Membranes were blocked in Tris-buffered saline (TBS) containing 5% nonfat dry milk powder, and incubated overnight at 4°C with anti-RAGE (at 1:1000 dilution), anti-eNOS (at 1:200 dilution), anti-p-eNOS (at 1:200 dilution), or anti-GAPDH (at 1:2000 dilution). The membranes were then washed with TBS with 0.1% Tween 20 and exposed to secondary antibody conjugated to HRP (at 1:2000 dilution) for 1 h at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (Amersham Biosciences, Piscataway, NJ, USA). The signal intensity of blotting was normalized to the Western blot signal of the corresponding total protein. Relative intensities of protein bands were calculated by ImageJ software v2.0 (NIH, Bethesda, MD, USA).

Measurement of nitric oxide concentration

The NO level in the HUVEC culture supernatant was evaluated by using a total NO assay kit to measure nitrite, the oxidation product of NO according to the manufacturer's specifications.

Migration assay

The migratory function of HUVECs was evaluated using a Millicell-PCF chamber (Millipore, Billerica, MA, USA) assay. In brief, a total of 4 × 10⁴ HUVECs were placed in the upper chamber, and culture medium with 20% FBS was placed in the lower chamber (as a chemoattractant). After incubating for 24 h at 37°C in a CO_{2 (}5%) equilibrated incubator, the membrane was then washed gently with PBS and fixed with methanol. Nonmigrating HUVECs were gently removed with cotton balls from the upper side of the membrane. The membrane of Transwell filter was stained using crystal violet solution. The migration of HUVECs was evaluated by counting the migrated cells in six random microscopic fields (×200 magnification).

Data Statistics

All experiments were repeated at least three times with triplicates. Results are expressed as mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance for multiple comparisons, and pairwise comparisons were performed by post hoc tests using the Fisher's least significant difference method. P < 0.05 was considered statistically significant. All analyses were performed by SPSS v18.0 (SPSS Inc., Chicago, IL, USA).

Results

Advanced glycation end-products and receptor for advanced glycation end-products–mitogen-activated protein kinase–nuclear factor-kappa B pathway

Phosphorylation of ERK1/2 and p38 in HUVECs was measured by Western blot. Phosphorylated ERK1/2 and p38 MAPK obviously increased in HUVECs when treated with AGEs. However, upregulated phosphorylated ERK1/2 and p38 could be inhibited when RAGE was blocked [Figure 1]a, [Figure 1]b, [Figure 1]d, and [Figure 1]e. Similarly, AGEs could activate the expression of phosphorylated NF-κB in HUVECs and the effect was inhibited when RAGE was blocked [Figure 1]c and [Figure 1]f. These results indicated that AGEs could activate the ERK and p38 MAPK-NF-κB signaling pathways in HUVECs through its receptor-RAGE.

Figure 1: AGEs activated ERK and P38 MAPK-NF-κB signal pathway by RAGE in HUVECs. (a-c) Effects of the AGEs and AGEs + anti-RAGE on ERK1/2 (a), p38 (b), and NF-κB (c) proteins in HUVECs. The HUVECs were treated with 200 mg/L HSA, 200 mg/L AGEs, and 50 mg/L anti-RAGE + 200 mg/L AGEs. ERK1/2, pERK1/2, p38, pp38, NF-κB p65, and pp65 proteins were detected by Western blotting. (d-f) Densitometry analysis of pERK1/2, pp38, and NF-κB pp65 is shown. AGEs: Advanced glycation end-products, RAGE: Receptor for advanced glycation end-products, HUVECs: Human umbilical venous endothelial cells, NF-κB: Nuclear factor-kappa B, MAPK: Mitogen-activated protein kinase

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Expression of receptor for advanced glycation end-products and mitogen-activated protein kinase–nuclear factor-kappa B pathway

We evaluated the effect of AGEs on the expression of RAGE in HUVECs. The result revealed that AGEs significantly upregulated the transcription and translation level of RAGE [Figure 2]a and [Figure 2]b while blocking RAGE could suppress the upregulation [Figure 2]b. The above data indicated that AGEs could induce the expression of RAGE on HUVECs mainly through its receptor-RAGE.

Figure 2: AGEs could induce the expression of RAGE on HUVECs through RAGE. (a) Expression of RAGE protein in HAS (200 mg/L, above)- and AGE (200 mg/L, below)-treated HUVECs. The expression of RAGE was measured by flow cytometer analyzer. (b) Effects of AGEs on RAGE gene expression in HUVECs. HUVECs were treated with medium, 200 mg/L HSA, 200 mg/L AGEs, and 50 mg/L anti-RAGE + 200 mg/L AGEs. The levels of RAGE gene transcripts were detected by real-time PCR. AGEs: Advanced glycation end-products, RAGE: Receptor for advanced glycation end-products, HUVECs: Human umbilical venous endothelial cells, NF-κB: Nuclear factor-kappa B, MAPK: Mitogen-activated protein kinase, PCR: Polymerase chain reaction

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Besides, we analyzed the expression of RAGE in HUVECs treated with AGEs, AGEs + PD98059, AGEs + SB202190, and AGEs + helenalin, respectively. The proportion of RAGE + HUVECs in the control, NF-κB, ERK, and P38 groups was 42.09%, 47.96%, 15.17%, and 16.42%, respectively [Figure 3]. These results indicate that AGEs can activate ERK and p38 signal pathways in HUVECs to induce the expression of RAGE. However, the NF-κB signal pathway does not play a role in AGE-induced RAGE expression.

Figure 3: Effects of NF-κB, ERK, and P38 inhibitors on AGE-induced RAGE upregulation. The expression of RAGE was measured by flow cytometer analyzer. AGEs: Advanced glycation end-products, RAGE: Receptor for advanced glycation end-products, NF-κB: Nuclear factor-kappa B, MAPK: Mitogen-activated protein kinase

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Nitric oxide secretion/endothelial nitric oxide synthase activation and receptor for advanced glycation end-products–mitogen-activated protein kinase–nuclear factor-kappa B pathway

HUVECs were incubated with medium, HAS, AGEs, and AGEs + anti-RAGE antibody, respectively. The concentration of NO obviously decreased in the AGE group (11.9 ± 0.56 umol/L) than in the control groups [24.8 ± 0.77 umol/L in the medium group and 24.9 ± 1.2 umol/L in the HAS group; P < 0.01; [Figure 4]a. Furthermore, blocking RAGE-ERK and p38 signal pathway other than RAGE-NF-κB could recover the synthesis of NO [12.0 ± 0.56 umol/L in the AGE group, 12.48 ± 0.83 umol/L in the NF-κB group, 19.33 ± 1.21 umol/L in the p38 MAPK inhibitor group, and 16.63 ± 0.57 umol/L in the ERK1/2 MAPK inhibitor group; P < 0.01; [Figure 4]b. Meanwhile, we found that AGEs could inhibit eNOS phosphorylation [P < 0.01; [Figure 5]a and [Figure 5]c and the activation of eNOS was attenuated in HUVECs after exposure to ERK and p38 inhibitors other than NF-κB inhibitors [Figure 5]b and [Figure 5]d. These results indicated that AGEs could inhibit the eNOS activation and NO synthesis of HUVECs through RAGE-ERK and p38 signaling pathways, but not NF-κB pathway.

Figure 4: AGEs could induce NO synthesis through RAGE-ERK and p38 signaling pathways. Effects of NF-κB, ERK, and P38 inhibitors on AGE-induced NO reduction. NO concentration of HUVEC culture supernatant was measured by a total NO assay kit. (a) The HUVECs were pretreated with medium, HSA, AGEs, and AGEs + anti-RAGE, respectively. (b) The HUVECs were pretreated with AGEs, AGEs + NF-κB inhibitor (helenalin), AGEs + p38 MAPK inhibitor (SB202190), and AGEs + ERK1/2 MAPK inhibitor (PD98059), respectively. AGEs: Advanced glycation end-products, RAGE: Receptor for advanced glycation end-products, HUVECs: Human umbilical venous endothelial cells, NF-κB: Nuclear factor-kappa B, MAPK: Mitogen-activated protein kinase, PCR: Polymerase chain reaction

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Figure 5: AGEs could inhibit eNOS phosphorylation through RAGE-MAPK signaling pathways but NF-κB pathway not involved (effects of NF-κB, ERK, and P38 inhibitors on AGE-induced eNOS phosphorylation decrease). (a and b) The proteins eNOS and p-eNOS were detected by Western blotting. (c and d) Densitometry analysis of eNOS and p-eNOS is shown. AGEs suppressed the activation of eNOS through RAGE, and the effect was inhibited by blocking ERK (PD98059) or p38 MAPK pathway (SB202190), but not inhibited by blocking NF-κB pathway (helenalin). AGEs: Advanced glycation end-products, RAGE: Receptor for advanced glycation end-products, HUVECs: Human umbilical venous endothelial cells, NF-κB: Nuclear factor-kappa B, MAPK: Mitogen-activated protein kinase, PCR: Polymerase chain reaction, eNOS: Endothelial nitric oxide synthase

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Human umbilical venous endothelial cell migration and receptor for advanced glycation end-products–mitogen-activated protein kinase–nuclear factor-kappa B pathway

Our research demonstrated that AGEs could inhibit the migration of HUVECs through its receptor-RAGE [Figure 6]a and [Figure 6]c. Meanwhile, we investigated the effect of ERK, P38, and NF-κB signal pathways on AGE-induced HUVEC migration decline. We added ERK inhibitor (PD98059), P38 inhibitor (SB202190), and NF-κB inhibitor (helenalin) to HUVECs which were previously treated with AGEs. The results showed that the number of migrated cells was higher in the ERK and P38 groups than in the control group [821.0 ± 43.1 in the ERK group, 872 ± 41.7 in the P38, and 341.0 ± 29.9 in the control group; P < 0.001; [Figure 6]b and [Figure 6]d. Yet, the number of migrated cells in the NF-κB group and the control group was similar [345.0 ± 22.9 in the NF-κB group and 341.0 ± 29.9 in the control group; P > 0.05; [Figure 6]b and [Figure 6]d. These results reveal that AGEs can inhibit the migration of HUVECs through ERK and p38 signal pathways rather than NF-κB.

Figure 6: Comparison of migrated HUVECs among different groups. (a and c) The HUVECs were incubated with HSA, AGEs, and AGEs + anti-RAGE, respectively. (b and d) HUVECs were incubated with AGEs, AGEs + helenalin, AGEs + SB202190, and AGEs + PD98059, respectively. AGEs suppressed the migration of HUVECs through RAGE, and the effect was inhibited by blocking ERK (PD98059) or p38 MAPK pathway (SB202190), but not inhibited by blocking NF-κB pathway (helenalin). AGEs: Advanced glycation end-products, RAGE: Receptor for advanced glycation end-products, HUVECs: Human umbilical venous endothelial cells, NF-κB: Nuclear factor-kappa B, MAPK: Mitogen-activated protein kinase, PCR: Polymerase chain reaction

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Discussion

AGEs accumulate in diverse biological settings such as diabetes, renal failure, cardiovascular injury, aging, and inflammation. It may affect the metabolism of diverse target proteins and induce pathophysiological changes through RAGE and downstream signal pathway activation. Chen et al. demonstrated that AGEs regulated the metabolism of collagen I in vaginal fibroblasts from patients with pelvic organ prolapse through RAGE-MAPK-NF-κB pathway.^[15] Feng et al. found that compound 4,4′-diphenylmethane-bis (methyl) carbamate attenuated high-mobility group box-1-mediated endothelial activation by ameliorating inflammation and oxidant stress responses via RAGE/ERK1/2 MAPK/NF-κB pathway.^[16] However, during the impairment of endothelial dysfunction due to AGEs, the role of RAGE-MAPK-NF-κB pathway has not been fully clarified. The current research studied the effects of AGEs on HUVEC dysfunction, such as depressed eNOS activation, decreased NO concentration, and defective EC migration. AGE-induced HUVEC apoptosis is mediated by p38 and ERK MAPK pathways, and NF-κB pathway might also play a part in this process.^[12] To explore the role and the mechanisms of RAGE-MAPK-NF-κB pathway in AGE-induced HUVEC dysfunction, we investigated the relationship of RAGE, MAPK pathways, and NF-κB pathway with the impairment. The results showed that AGEs depressed eNOS activation, decreased NO concentration, impaired EC migration, and upregulated RAGE expression, which could be attenuated by anti-RAGE, p38 inhibitor (SB202190), and ERK inhibitor (PD98059). However, these effects could not be attenuated by NF-κB inhibitor (helenalin). Our results indicated that the AGEs impair the endothelial function such as eNOS activation, NO secretion, and EC migration dependently through RAGE-MAPK pathway. However, this endothelial impairment was not NF-κB pathway dependent.

The endothelium has been recognized as an important homeostatic part in regulating vasodilatation and vasoconstriction.^[17] Endothelial dysfunction contributes to atherosclerosis and increases the risk of diabetic vascular events.^[18] Furthermore, AGEs were reported to be one of the important pathogenic factors associated with diabetic endothelial dysfunction.^[19] RAGE, a multi-ligand member of the immunoglobulin superfamily of cell-surface molecules, is the best characterized AGE receptor and responsible for most of the harmful effects of AGEs.^[20] Our results confirmed that AGE-induced MAPK pathway activation, NF-κB pathway activation, and endothelial dysfunction were all attenuated by anti-RAGE neutralizing antibody. Moreover, it has been illustrated that the AGE-RAGE interaction would lead to a positive feedback activation, which further increases RAGE expression.^[21] The current study confirmed that results and further demonstrated that the positive feedback activation of RAGE expression was mediated by MAPK pathway, but not NF-κB pathway-dependent.

MAPK pathway was reported to be involved in AGE-induced diverse pathophysiological changes. Ho et al. proved that AGE-induced apoptosis in HUVECs could be attenuated by specific MAPK inhibitors and NF-κB inhibitor.^[22] Sun et al. showed that AGE-induced EPC dysfunction and EPC apoptosis were triggered by upregulated RAGE expression through MAPK pathway.^[12] They further demonstrated that AGE-induced NF-κB activation could also be attenuated by MAPK inhibitors. Our results showed that AGEs could depress eNOS activation, decrease NO release, impair EC migration, and increase RAGE expression through activation of MAPK pathway, which could be reversed by p38 MAPK inhibitor (SB202190) and ERK MAPK inhibitor (PD98059). This finding is in line with previous studies showing that ERK and p38 inhibition protected EPCs from apoptosis induced by AGEs^[11],[24] and proves the important role of RAGE-MAPK pathway during AGE-induced endothelial dysfunction.

The activation of NF-κB pathway was important in the pathomechanism of diabetic vascular events.^[25],[26] It is also often involved in the AGE-induced diverse endothelial impairment.^[27] Furthermore, the NF-κB activation could be significantly inhibited by blocking AGE-RAGE interaction^[28] and blocking MAPK pathway,^[23] supporting the possible role of RAGE-MAPK-NF-κB pathway during the endothelial dysfunction due to AGE impairment. It is reported that the MAPK pathway and NF-κB pathway are both required in the RAGE-mediated oxidant stress responses and cytokine secretion, which proved the existence of RAGE-MAPK-NF-κB pathway.^[16],[29] However, the current study found that the RAGE-MAPK pathway was involved in AGE-induced RAGE expression and impairment of the endothelial function such as eNOS activation, NO secretion, and EC migration, but the NF-κB pathway was not exclusively required. The limitation of our study is that we analyzed only eNOS activation, NO secretion, and EC migration when evaluating the endothelial function, which includes diverse effects. The results of current study do not deny the important role of NF-κB pathway in other pathophysiological effects induced by AGEs.

Conclusions

AGEs could induce RAGE expression, depress eNOS activation, reduce NO production, and impair EC migration. The RAGE-MAPK pathway was involved in the specific impairment, but the NF-κB pathway was not exclusively required.

Acknowledgment

The authors would like to acknowledge all members of the Department of Vascular Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine.

Financial support and sponsorship

This study was sponsored by the Program of Medical Science and Technology of Zhejiang Province (Grant number 2018256953), the Key Program of Natural Science Foundation of Zhejiang Province (LZ21H020001) and Youth Program of National Natural Science Foundation of China (Grant number 81700420).

Conflicts of interest

There are no conflicts of interest.

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