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Role of TRPM7 channels in hyperglycemia-mediated injury of vascular endothelial cells.

Sun H, Leng T, Zeng Z, Gao X, Inoue K, Xiong ZG - PLoS ONE (2013)

Bottom Line: Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72 h.In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect.HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China ; Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72 h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.

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Effect of U0126 on viability and phospho-ERK1/2 expression in HG treated HUVECs.The cells were preincubated with U0126 (10 μM) for 18h, then treated with TRPM7 siRNA for 48h, and finally stimulated with HG for 72h. (A) Cell viability was assessed by MTT assay. (B) Representative immunoblots showing phospho-ERK1/2 expression level. (C) The corresponding bar graphs showing the relative expression of phospho-ERK1/2 protein normalized to beta-actin. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=5 for immunoblotting.
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pone-0079540-g006: Effect of U0126 on viability and phospho-ERK1/2 expression in HG treated HUVECs.The cells were preincubated with U0126 (10 μM) for 18h, then treated with TRPM7 siRNA for 48h, and finally stimulated with HG for 72h. (A) Cell viability was assessed by MTT assay. (B) Representative immunoblots showing phospho-ERK1/2 expression level. (C) The corresponding bar graphs showing the relative expression of phospho-ERK1/2 protein normalized to beta-actin. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=5 for immunoblotting.

Mentions: MEK1/2 is an ERK kinase, which phosphorylates ERK resulting in its activation. To further elucidate the role of ERK pathway in the protective effect against HG by silencing TRPM7, U0126, an inhibitor of MEK1/2, was used. U0126 (at a concentration of 10 µM) was added to the culture medium. After 18h, cells were treated with TRPM7 siRNA or control siRNA and then stimulated with HG for 72h. As shown in Figure 6A and B, HG increased cell cytotoxicity, while silencing TRPM7 decreased cell cytotoxicity, which was consistent with the above mentioned result (e.g. Figure 3). Following the treatment of U0126, the protective effect by silencing TRPM7 in HG treated HUVECs was almost completely eliminated as demonstrated by MTT assay (Figure 6A). Consistent with the result above (Figure 5), HG decreased phospho-ERK1/2 protein expression while silencing TRPM7 increased phospho-ERK1/2 protein expression (Figure 6B and C). Pretreatment with U0126 dramatically decreased phospho-ERK1/2 protein expression in HUVECs (Figure 6B and C).


Role of TRPM7 channels in hyperglycemia-mediated injury of vascular endothelial cells.

Sun H, Leng T, Zeng Z, Gao X, Inoue K, Xiong ZG - PLoS ONE (2013)

Effect of U0126 on viability and phospho-ERK1/2 expression in HG treated HUVECs.The cells were preincubated with U0126 (10 μM) for 18h, then treated with TRPM7 siRNA for 48h, and finally stimulated with HG for 72h. (A) Cell viability was assessed by MTT assay. (B) Representative immunoblots showing phospho-ERK1/2 expression level. (C) The corresponding bar graphs showing the relative expression of phospho-ERK1/2 protein normalized to beta-actin. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=5 for immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815131&req=5

pone-0079540-g006: Effect of U0126 on viability and phospho-ERK1/2 expression in HG treated HUVECs.The cells were preincubated with U0126 (10 μM) for 18h, then treated with TRPM7 siRNA for 48h, and finally stimulated with HG for 72h. (A) Cell viability was assessed by MTT assay. (B) Representative immunoblots showing phospho-ERK1/2 expression level. (C) The corresponding bar graphs showing the relative expression of phospho-ERK1/2 protein normalized to beta-actin. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=5 for immunoblotting.
Mentions: MEK1/2 is an ERK kinase, which phosphorylates ERK resulting in its activation. To further elucidate the role of ERK pathway in the protective effect against HG by silencing TRPM7, U0126, an inhibitor of MEK1/2, was used. U0126 (at a concentration of 10 µM) was added to the culture medium. After 18h, cells were treated with TRPM7 siRNA or control siRNA and then stimulated with HG for 72h. As shown in Figure 6A and B, HG increased cell cytotoxicity, while silencing TRPM7 decreased cell cytotoxicity, which was consistent with the above mentioned result (e.g. Figure 3). Following the treatment of U0126, the protective effect by silencing TRPM7 in HG treated HUVECs was almost completely eliminated as demonstrated by MTT assay (Figure 6A). Consistent with the result above (Figure 5), HG decreased phospho-ERK1/2 protein expression while silencing TRPM7 increased phospho-ERK1/2 protein expression (Figure 6B and C). Pretreatment with U0126 dramatically decreased phospho-ERK1/2 protein expression in HUVECs (Figure 6B and C).

Bottom Line: Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72 h.In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect.HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China ; Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72 h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.

Show MeSH
Related in: MedlinePlus