Apelin-13 Inhibits Methylglyoxal-Induced Unfolded Protein Responses and Endothelial Dysfunction via Regulating AMPK Pathway

Figures

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  Figure 1. MGO induces endothelial apoptosis via unfolded protein responses in HUVECs. HUVECs were stimulated with 100 µM MGO for the indicated time periods. Protein levels were analyzed by immunoblotting with specific antibodies as follows: (A) anti-phospho-PERK, anti-phospho-eIF2α, anti-ATF4, anti-CHOP, and anti-tubulin antibodies; (B) anti-phospho JNK, anti-JNK, and anti-tubulin antibodies; (C) anti-spliced XBP-1 and anti-tubulin antibodies; (D) anti-ATF6 and anti-tubulin antibodies. Bar graphs represent the densitometric results of Western blot bands. * p < 0.05 and ** p < 0.01 vs. vehicle control (n = 3). Results are representative of three independent experiments. (E) HUVECs were exposed to the indicated doses of MGO for 6 h and cell lysates were applied for immunoblotting using specific antibodies against ATF-4, CHOP, and tubulin. The graph shows the densitometric quantification of Western blot bands. * p < 0.05 and ** p < 0.01 vs. vehicle control (n = 3). Results are representative of three independent experiments. (F) HUVECs were exposed to vehicle or 100 µM MGO in the presence or absence of TUDCA (200 µM). Cells were lysed and then applied for Western blotting analysis. Protein…

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  Figure 2. Apelin-13 protects endothelial cells from MGO-induced UPR and apoptosis. HUVECs were pretreated with 1 µM apelin-13 for 1 h followed by exposure to 100 µM MGO for 6 or 24 h. (A) Cells were exposed to 100 µM MGO for 6 h in the presence or absence of apelin-13 (0, 0.2, 0.5, 1 µM). Amount of protein expression was determined by immunoblotting with specific antibodies against ATF4, CHOP, and tubulin. Bar graphs represent the densitometric results of Western blot bands. ** p < 0.01 vs. vehicle control, # p < 0.05; ## p < 0.01 vs. MGO-treated cells (n = 3). Results are representative of three independent experiments. (B) HUVECs were lysed and then applied for Western blotting analysis. Protein level amounts were determined by immunoblotting with anti-PARP-1 and anti-caspase-3 antibodies. The graph shows the densitometric quantification of Western blot bands. ** p < 0.01 vs. vehicle control, ## p < 0.01 vs. MGO-treated cells (n = 3). Results are representative of three independent experiments. (C) MTT assay was performed as described in the Materials and Methods section. Results are expressed as means ± SDs and are representative of three independent experiments. * p < 0.05; ** p < 0.01…

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  Figure 3. Apelin-13 protects aortic vessels from MGO-induced endothelial dysfunction. To determine the role of apelin-13 on MGO-induced cell apoptosis and endothelial dysfunction ex vivo, C57BL/6 mice were anesthetized and aorta was isolated. (A) Aorta treated with MGO (100 µM) for 24 h, with or without apelin-13 (1 µM) pretreatment was stained with anti-VE-cadherin antibody for endothelial cell junction. Apoptosis was measured by TUNEL staining using the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN) as described in the Materials and Methods section. Signals were observed under the confocal microscope (×400). Arrows indicate TUNEL positive cells which are apoptotic endothelial cells. Scale bars: 100 µm. Quantification of apoptosis is shown as the percentage of TUNEL positive cells. More than 200 cells were counted for each category. Data represent means ± SDs. * p < 0.05 (n = 5). (B,C) The effect of acetylcholine on phenylephrine-induced vessel contraction in mouse aortic rings. (B) Mouse aortic rings were preincubated with phenylephrine (10−7 M) and then exposed to acetylcholine (10−9–10−6 M). The results shown are representative of three independent experiments. * p < 0.05;…

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  Figure 4. The effect of NAC in MGO-induced UPR and apoptosis in HUVECs. HUVECs pretreated with 5 mM NAC for 1 h were exposed to 100 µM MGO for 24 h. (A) HUVECs were lysed and then applied for Western blotting analysis. Protein level amounts were determined by immunoblotting with anti-PARP-1 and anti-caspase-3 antibodies. The graph shows the densitometric quantification of Western blot bands. ** p < 0.01 vs. vehicle control, ## p < 0.01 vs. MGO-treated cells (n = 3). Results are representative of three independent experiments. (B) MTT assay was performed as described in the Materials and Methods section. Results are expressed as means ± SDs and are representative of three independent experiments. * p < 0.05, ** p < 0.01 (n = 3). (C) Cells were stimulated with 100 µM MGO for the indicated time periods (3 and 6 h) in the presence or absence of 5 mM NAC. Amount of protein expression was determined by immunoblotting with specific antibodies against anti-ATF4, and anti-CHOP and anti-tubulin. Bar graphs represent the densitometric results of Western blot bands. * p < 0.05 and ** p < 0.01 vs. vehicle control, # p < 0.05 and ## p < 0.01 vs. MGO-treated cells (3 h), † p < 0.05 and †† p < 0.01 vs.…

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  Figure 5. Apelin-13 ameliorates MGO-induced apoptosis through AMPK pathway in HUVECs. Cells pretreated with wortmannin (50 nM), U73122 (1 µM), and compound C (1 µM) were stimulated with 100 µM MGO for 24 h in the presence or absence of 1 µM apelin-13. (A) HUVECs were lysed and then applied for Western blotting analysis. Protein level amounts were determined by immunoblotting with anti-PARP-1 and anti-caspase-3 antibodies. The graph shows the densitometric quantification of Western blot bands. ** p < 0.01 vs. vehicle control, ## p < 0.01 vs. MGO-treated cells (n = 3). Results are representative of three independent experiments. (B) MTT assay was performed as described in the Materials and Methods section. Results are expressed as means ± SDs and are representative of three independent experiments. * p < 0.05, ** p < 0.01 (n = 3). (C,D) To examine the protective role of AMPK in MGO-induced apoptosis, HUVECs infected with Ad-LacZ or Ad-CA-AMPKα1 were exposed to 100 µM MGO for 24 h. (C) Cells were applied for Western blotting analysis. The graph shows the densitometric quantification of Western blot bands. * p < 0.05 vs. vehicle control, # p < 0.05 vs. MGO-treated cells (n = 3). Results are…

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  Figure 6. Molecular mechanisms involved in the protective effects of apelin-13 against methylglyoxal-induced endothelial dysfunction. NAC (ROS inhibitor) and TUDCA (ER stress inhibitor) inhibit MGO-induced ER stress and endothelial apoptosis. The activation of AMPK might be involved in the protective effects of apelin-13 against MGO-induced endothelial dysfunction.