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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

HMGB1 induces Src activation.A. Representative immunoblots showing HMGB1-induced Src activation. EA.hy926 cells were treated with 200 ng/ml HMGB1 for 1, 2, 3 and 4h, respectively. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. B. PP2 and CGP77675 inhibit HMGB1-induced permeability. 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, 2.5, 5, 10 or 20μM PP2 (upper) or 0.5, 1, 2.5, 5 or 10 μM CGP77675 (lower) 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. Representative immunoblots showing that PP2 (C) and CGP77675 (D) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 2.5, 5, or 10μM PP2 or 0.5, 1, 2.5 or 10 μM CGP77675 for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 24h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. GAPDH was used as a loading control. Western blots were quantified and analyzed statistically based on three independent experiments. 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.g003: HMGB1 induces Src activation.A. Representative immunoblots showing HMGB1-induced Src activation. EA.hy926 cells were treated with 200 ng/ml HMGB1 for 1, 2, 3 and 4h, respectively. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. B. PP2 and CGP77675 inhibit HMGB1-induced permeability. 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, 2.5, 5, 10 or 20μM PP2 (upper) or 0.5, 1, 2.5, 5 or 10 μM CGP77675 (lower) 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. Representative immunoblots showing that PP2 (C) and CGP77675 (D) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 2.5, 5, or 10μM PP2 or 0.5, 1, 2.5 or 10 μM CGP77675 for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 24h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. GAPDH was used as a loading control. Western blots were quantified and analyzed statistically based on three independent experiments. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).

Mentions: HMGB1-induced hyperpermeability has been reported to be mediated through the Src family of tyrosine kinase [13]. We therefore measured Src phosphorylation subsequent to HMGB1 treatment to determine if HMGB1 could activate this signaling pathway in EA.hy926 endothelial cells. Src was phosphorylated, with a peak at 2–3 h, followed by a decrease (Fig 3A). Moreover, we used the Src inhibitors PP2 and CGP77675 to investigate the role of Src in HMGB1-induced cell hyperpermeability. As shown in Fig 3B, both 10–20 μM PP2 and 1–10 μM CGP77675 significantly decreased HMGB1-induced cell hyperpermeability. Furtheremore, we detected whether the Src inhibitors PP2 and CGP77675 could inhibit the Src activation. Both 5–10 μM PP2 and 1–5 μM CGP77675 significantly blocked HMGB1-induced Src phosphorylation (Fig 3C and 3D).


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)

HMGB1 induces Src activation.A. Representative immunoblots showing HMGB1-induced Src activation. EA.hy926 cells were treated with 200 ng/ml HMGB1 for 1, 2, 3 and 4h, respectively. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. B. PP2 and CGP77675 inhibit HMGB1-induced permeability. 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, 2.5, 5, 10 or 20μM PP2 (upper) or 0.5, 1, 2.5, 5 or 10 μM CGP77675 (lower) 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. Representative immunoblots showing that PP2 (C) and CGP77675 (D) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 2.5, 5, or 10μM PP2 or 0.5, 1, 2.5 or 10 μM CGP77675 for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 24h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. GAPDH was used as a loading control. Western blots were quantified and analyzed statistically based on three independent experiments. 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.g003: HMGB1 induces Src activation.A. Representative immunoblots showing HMGB1-induced Src activation. EA.hy926 cells were treated with 200 ng/ml HMGB1 for 1, 2, 3 and 4h, respectively. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. B. PP2 and CGP77675 inhibit HMGB1-induced permeability. 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, 2.5, 5, 10 or 20μM PP2 (upper) or 0.5, 1, 2.5, 5 or 10 μM CGP77675 (lower) 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. Representative immunoblots showing that PP2 (C) and CGP77675 (D) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 2.5, 5, or 10μM PP2 or 0.5, 1, 2.5 or 10 μM CGP77675 for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 24h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. GAPDH was used as a loading control. Western blots were quantified and analyzed statistically based on three independent experiments. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
Mentions: HMGB1-induced hyperpermeability has been reported to be mediated through the Src family of tyrosine kinase [13]. We therefore measured Src phosphorylation subsequent to HMGB1 treatment to determine if HMGB1 could activate this signaling pathway in EA.hy926 endothelial cells. Src was phosphorylated, with a peak at 2–3 h, followed by a decrease (Fig 3A). Moreover, we used the Src inhibitors PP2 and CGP77675 to investigate the role of Src in HMGB1-induced cell hyperpermeability. As shown in Fig 3B, both 10–20 μM PP2 and 1–10 μM CGP77675 significantly decreased HMGB1-induced cell hyperpermeability. Furtheremore, we detected whether the Src inhibitors PP2 and CGP77675 could inhibit the Src activation. Both 5–10 μM PP2 and 1–5 μM CGP77675 significantly blocked HMGB1-induced Src phosphorylation (Fig 3C and 3D).

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