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Store-operated Ca2+ entry plays a role in HMGB1-induced vascular endothelial cell hyperpermeability.

Zou M, Dong H, Meng X, Cai C, Li C, Cai S, Xue Y - PLoS ONE (2015)

Bottom Line: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment.Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability.Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.

ABSTRACT

Aims: Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and results: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions: These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca2+ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.

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Related in: MedlinePlus

Orai1 knockdown decreases HMGB1-induced permeability.A. Orai1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-Orai1 antibodies, with anti-GAPDH antibodies as a loading control. Orai1 expression was quantified and analyzed statistically based on three independent experiments. B. HMGB1-induced permeability was inhibited by Orai1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
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pone.0123432.g007: Orai1 knockdown decreases HMGB1-induced permeability.A. Orai1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-Orai1 antibodies, with anti-GAPDH antibodies as a loading control. Orai1 expression was quantified and analyzed statistically based on three independent experiments. B. HMGB1-induced permeability was inhibited by Orai1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).

Mentions: Since STIM1 may promote endothelial permeability independently of Ca2+ entry[32], Orai1, one of key molecules in SOCE[33], was downregulated by siRNA to confirm crucial role of calcium entry in the HMGB1-induced cell hyperpermeability. HMGB1-induced endothelial cell hyperpermeability was significantly inhibited by knockdown of Orai1 (Fig 7). As shown in Fig 7A, transfection of EA.hy926 endothelial cells with two Orai1 siRNAs, but not with scrambled siRNA, significantly blocked the expression of Orai1 protein. Orai1 protein expression levels were reduced by approximately 85% in cells treated with the Orai1 siRNAs, compared with cells treated with scrambled siRNA. We further investigated the effects of Orai1 siRNA on HMGB1-induced permeability. siRNA knockdown of Orai1 had no effect on endothelial cell permeability, but HMGB1-induced endothelial cell hyperpermeability was significantly suppressed (Fig 7B).


Store-operated Ca2+ entry plays a role in HMGB1-induced vascular endothelial cell hyperpermeability.

Zou M, Dong H, Meng X, Cai C, Li C, Cai S, Xue Y - PLoS ONE (2015)

Orai1 knockdown decreases HMGB1-induced permeability.A. Orai1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-Orai1 antibodies, with anti-GAPDH antibodies as a loading control. Orai1 expression was quantified and analyzed statistically based on three independent experiments. B. HMGB1-induced permeability was inhibited by Orai1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
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Related In: Results  -  Collection

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pone.0123432.g007: Orai1 knockdown decreases HMGB1-induced permeability.A. Orai1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-Orai1 antibodies, with anti-GAPDH antibodies as a loading control. Orai1 expression was quantified and analyzed statistically based on three independent experiments. B. HMGB1-induced permeability was inhibited by Orai1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with Orai1 siRNA-1, Orai1 siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
Mentions: Since STIM1 may promote endothelial permeability independently of Ca2+ entry[32], Orai1, one of key molecules in SOCE[33], was downregulated by siRNA to confirm crucial role of calcium entry in the HMGB1-induced cell hyperpermeability. HMGB1-induced endothelial cell hyperpermeability was significantly inhibited by knockdown of Orai1 (Fig 7). As shown in Fig 7A, transfection of EA.hy926 endothelial cells with two Orai1 siRNAs, but not with scrambled siRNA, significantly blocked the expression of Orai1 protein. Orai1 protein expression levels were reduced by approximately 85% in cells treated with the Orai1 siRNAs, compared with cells treated with scrambled siRNA. We further investigated the effects of Orai1 siRNA on HMGB1-induced permeability. siRNA knockdown of Orai1 had no effect on endothelial cell permeability, but HMGB1-induced endothelial cell hyperpermeability was significantly suppressed (Fig 7B).

Bottom Line: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment.Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability.Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.

ABSTRACT

Aims: Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and results: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions: These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca2+ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.

Show MeSH
Related in: MedlinePlus