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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

SKF96365 and 2-APB reduce Ca2+ influx and HMGB1-induced permeability.EA.hy926 cells were preincubated with 1, 5, 10 μM SKF96365 (A), or 10, 30, 50 μM 2-APB(B) or vehicle (DMSO), then stimulated with 200 ng/ml HMGB1, followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer. The cells were preincubated with 1, 5, 10, 20 μM SKF96365 (C), or 10, 30, 50, 70 μM 2-APB (D) for 1 h, respectively. HMGB1 200 ng/ml was then added and the cells were incubated for an additional 24 h. After incubation, the integrity of the tight junctions was assessed by measuring the TER. 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.g004: SKF96365 and 2-APB reduce Ca2+ influx and HMGB1-induced permeability.EA.hy926 cells were preincubated with 1, 5, 10 μM SKF96365 (A), or 10, 30, 50 μM 2-APB(B) or vehicle (DMSO), then stimulated with 200 ng/ml HMGB1, followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer. The cells were preincubated with 1, 5, 10, 20 μM SKF96365 (C), or 10, 30, 50, 70 μM 2-APB (D) for 1 h, respectively. HMGB1 200 ng/ml was then added and the cells were incubated for an additional 24 h. After incubation, the integrity of the tight junctions was assessed by measuring the TER. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).

Mentions: Evidence suggests that regulation of endothelial permeability is a complex process regulated by SOCE [31]. We used the SOCE inhibitors SKF96365 and 2-APB to investigate the role of SOCE in HMGB1-induced changes in cytosolic Ca2+. HMGB1 caused an initial increase in cytosolic Ca2+, with a rapid Ca2+ influx into cells following the addition of extracellular Ca2+. The apparent Ca2+ influx was significantly reduced in cells pretreated with 5–10 μM SKF96365 and 50 μM 2-APB, respectively, without affecting Ca2+ store release(Fig 4A and 4B). We further investigated the effects of SKF96365 and 2-APB on HMGB1-induced permeability. Endothelial cells were exposed to 1–20 μM SKF96365 or 10–70 μM 2-APB prior to HMGB1 treatment. As shown in Fig 4C and 4D, both SKF96365 and 2-APB significantly decreased HMGB1-induced cell hyperpermeability at 5–20 μM and 50–70 μM, respectively (P<0.05). Because SOCE inhibitors decreased HMGB1-induced endothelial cell hyperpermeability, in which Src was activated, we determined if SKF96365 and 2-APB might inhibit cell hyperpermeability by regulating the Src signaling pathway. As shown in Fig 5A and 5B, both SKF96365 and 2-APB significantly inhibited Src phosphorylation at 5–20 μM and 50–100 μM, respectively (P<0.05).


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)

SKF96365 and 2-APB reduce Ca2+ influx and HMGB1-induced permeability.EA.hy926 cells were preincubated with 1, 5, 10 μM SKF96365 (A), or 10, 30, 50 μM 2-APB(B) or vehicle (DMSO), then stimulated with 200 ng/ml HMGB1, followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer. The cells were preincubated with 1, 5, 10, 20 μM SKF96365 (C), or 10, 30, 50, 70 μM 2-APB (D) for 1 h, respectively. HMGB1 200 ng/ml was then added and the cells were incubated for an additional 24 h. After incubation, the integrity of the tight junctions was assessed by measuring the TER. 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.g004: SKF96365 and 2-APB reduce Ca2+ influx and HMGB1-induced permeability.EA.hy926 cells were preincubated with 1, 5, 10 μM SKF96365 (A), or 10, 30, 50 μM 2-APB(B) or vehicle (DMSO), then stimulated with 200 ng/ml HMGB1, followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer. The cells were preincubated with 1, 5, 10, 20 μM SKF96365 (C), or 10, 30, 50, 70 μM 2-APB (D) for 1 h, respectively. HMGB1 200 ng/ml was then added and the cells were incubated for an additional 24 h. After incubation, the integrity of the tight junctions was assessed by measuring the TER. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
Mentions: Evidence suggests that regulation of endothelial permeability is a complex process regulated by SOCE [31]. We used the SOCE inhibitors SKF96365 and 2-APB to investigate the role of SOCE in HMGB1-induced changes in cytosolic Ca2+. HMGB1 caused an initial increase in cytosolic Ca2+, with a rapid Ca2+ influx into cells following the addition of extracellular Ca2+. The apparent Ca2+ influx was significantly reduced in cells pretreated with 5–10 μM SKF96365 and 50 μM 2-APB, respectively, without affecting Ca2+ store release(Fig 4A and 4B). We further investigated the effects of SKF96365 and 2-APB on HMGB1-induced permeability. Endothelial cells were exposed to 1–20 μM SKF96365 or 10–70 μM 2-APB prior to HMGB1 treatment. As shown in Fig 4C and 4D, both SKF96365 and 2-APB significantly decreased HMGB1-induced cell hyperpermeability at 5–20 μM and 50–70 μM, respectively (P<0.05). Because SOCE inhibitors decreased HMGB1-induced endothelial cell hyperpermeability, in which Src was activated, we determined if SKF96365 and 2-APB might inhibit cell hyperpermeability by regulating the Src signaling pathway. As shown in Fig 5A and 5B, both SKF96365 and 2-APB significantly inhibited Src phosphorylation at 5–20 μM and 50–100 μM, respectively (P<0.05).

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