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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 inhibit Src activation.Representative immunoblots showing that SKF96365 (A) and 2-APB (B) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 5, 10, 20 μM SKF96365, or 25, 50, 70, 100 μM 2-APB for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 2 h. 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 at least 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.g005: SKF96365 and 2-APB inhibit Src activation.Representative immunoblots showing that SKF96365 (A) and 2-APB (B) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 5, 10, 20 μM SKF96365, or 25, 50, 70, 100 μM 2-APB for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 2 h. 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 at least 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: 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 inhibit Src activation.Representative immunoblots showing that SKF96365 (A) and 2-APB (B) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 5, 10, 20 μM SKF96365, or 25, 50, 70, 100 μM 2-APB for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 2 h. 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 at least three independent experiments. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401536&req=5

pone.0123432.g005: SKF96365 and 2-APB inhibit Src activation.Representative immunoblots showing that SKF96365 (A) and 2-APB (B) decreased HMGB1-induced Src phosphorylation. Cells were preincubated with 1, 5, 10, 20 μM SKF96365, or 25, 50, 70, 100 μM 2-APB for 1 h, respectively. 200 ng/ml HMGB1 was then added and cells were incubated for an additional 2 h. 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 at least 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: 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