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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 TRPM7 siRNA on eNOS protein expression, NO and ROS generation in HG treated HUVECs.The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) Representative immunoblots showing eNOS protein expression level. (B) The corresponding bar graphs showing the relative expression of eNOS protein normalized to beta-actin. (C) The production of NO was determined by measurement of nitrite, a stable product of NO. (D) The production of intracellular ROS was assessed by the oxidation of 2’,7’-dichlorofluorescin diacetate to fluorescent 2’,7’-dichlorofluorescein. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=3 for immunoblotting , 5 for ROS generation assay and 6 for NO measurement.
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pone-0079540-g004: Effect of TRPM7 siRNA on eNOS protein expression, NO and ROS generation in HG treated HUVECs.The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) Representative immunoblots showing eNOS protein expression level. (B) The corresponding bar graphs showing the relative expression of eNOS protein normalized to beta-actin. (C) The production of NO was determined by measurement of nitrite, a stable product of NO. (D) The production of intracellular ROS was assessed by the oxidation of 2’,7’-dichlorofluorescin diacetate to fluorescent 2’,7’-dichlorofluorescein. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=3 for immunoblotting , 5 for ROS generation assay and 6 for NO measurement.

Mentions: As shown in Figure 4A and B, eNOS protein expression was down-regulated at 72 h following the exposure to HG (control: 100, HG: 73.2±5.1, p<0.01; n=3). Silencing TRPM7 ameliorated the reduction of eNOS protein expression induced by HG (control siRNA: 75.2±0.3, TRPM7 siRNA: 94.2±0.8, p<0.05, n=3). To investigate whether TRPM7 knockdown could prevent the reduction of NO production, we measured nitrite, a stable product of NO. As shown in Figure 4C, the production of nitrite was decreased by HG treatment (control: 100, HG: 65.58±6.56, p<0.01; n=6), and that silencing TRPM7 ameliorated the reduction of nitrite production (Figure 4C, control siRNA: 68.16±5.22, TRPM7 siRNA: 93.77±4.91, p<0.01, n=6).


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 TRPM7 siRNA on eNOS protein expression, NO and ROS generation in HG treated HUVECs.The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) Representative immunoblots showing eNOS protein expression level. (B) The corresponding bar graphs showing the relative expression of eNOS protein normalized to beta-actin. (C) The production of NO was determined by measurement of nitrite, a stable product of NO. (D) The production of intracellular ROS was assessed by the oxidation of 2’,7’-dichlorofluorescin diacetate to fluorescent 2’,7’-dichlorofluorescein. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=3 for immunoblotting , 5 for ROS generation assay and 6 for NO measurement.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815131&req=5

pone-0079540-g004: Effect of TRPM7 siRNA on eNOS protein expression, NO and ROS generation in HG treated HUVECs.The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) Representative immunoblots showing eNOS protein expression level. (B) The corresponding bar graphs showing the relative expression of eNOS protein normalized to beta-actin. (C) The production of NO was determined by measurement of nitrite, a stable product of NO. (D) The production of intracellular ROS was assessed by the oxidation of 2’,7’-dichlorofluorescin diacetate to fluorescent 2’,7’-dichlorofluorescein. **p<0.01 vs. control; ##p<0.01 vs. control siRNA. n=3 for immunoblotting , 5 for ROS generation assay and 6 for NO measurement.
Mentions: As shown in Figure 4A and B, eNOS protein expression was down-regulated at 72 h following the exposure to HG (control: 100, HG: 73.2±5.1, p<0.01; n=3). Silencing TRPM7 ameliorated the reduction of eNOS protein expression induced by HG (control siRNA: 75.2±0.3, TRPM7 siRNA: 94.2±0.8, p<0.05, n=3). To investigate whether TRPM7 knockdown could prevent the reduction of NO production, we measured nitrite, a stable product of NO. As shown in Figure 4C, the production of nitrite was decreased by HG treatment (control: 100, HG: 65.58±6.56, p<0.01; n=6), and that silencing TRPM7 ameliorated the reduction of nitrite production (Figure 4C, control siRNA: 68.16±5.22, TRPM7 siRNA: 93.77±4.91, p<0.01, n=6).

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