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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

Disruption of VE-cadherin and intercellular gap formation in HMGB1-treated EA.hy926 cells.A. Representative immunoblots showing reduced expression of VE-cadherin protein by HMGB1. Total and cell membrane VE-cadherin protein levels were measured by western blotting in EA.hy926 cells treated with HMGB1 for 6, 12, 24 and 48 h, respectively. GAPDH and Na,K-ATPase α1 were used as loading controls for intact cells and plasma membranes, respectively. Western blots were quantified and analyzed statistically based on three independent experiments. *Indicates significant difference compared with wild-type group (P<0.05). B. HMGB1 increased intercellular gap formation. EA.hy926 cells were plated onto a Petri dish until the formation of a tight monolayer then treated with 200 ng/ml HMGB1 for 6, 12 and 24 h, respectively. The cells were fixed and distribution of VE-cadherin was detected using rabbit anti-human VE-cadherin antibody and FITC-labeled goat anti-rabbit antibody. Nuclei were stained with DAPI. Red arrows indicate intercellular gaps. A merged picture is shown for each condition. A representative field for each condition was captured using an Olympus FV1000 confocal microscope. Scale bar = 10 μm.
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pone.0123432.g002: Disruption of VE-cadherin and intercellular gap formation in HMGB1-treated EA.hy926 cells.A. Representative immunoblots showing reduced expression of VE-cadherin protein by HMGB1. Total and cell membrane VE-cadherin protein levels were measured by western blotting in EA.hy926 cells treated with HMGB1 for 6, 12, 24 and 48 h, respectively. GAPDH and Na,K-ATPase α1 were used as loading controls for intact cells and plasma membranes, respectively. Western blots were quantified and analyzed statistically based on three independent experiments. *Indicates significant difference compared with wild-type group (P<0.05). B. HMGB1 increased intercellular gap formation. EA.hy926 cells were plated onto a Petri dish until the formation of a tight monolayer then treated with 200 ng/ml HMGB1 for 6, 12 and 24 h, respectively. The cells were fixed and distribution of VE-cadherin was detected using rabbit anti-human VE-cadherin antibody and FITC-labeled goat anti-rabbit antibody. Nuclei were stained with DAPI. Red arrows indicate intercellular gaps. A merged picture is shown for each condition. A representative field for each condition was captured using an Olympus FV1000 confocal microscope. Scale bar = 10 μm.

Mentions: VE-cadherin is associated with atherosclerosis, and its dysfunction has a proatherogenic effect on vessels [30]. We investigated changes in VE-cadherin protein expression and morphology in HMGB1-treated vascular endothelial cells. VE-cadherin expression was unaffected at 6 and 12 h of treatment, but significant decreases in both total VE-cadherin and cleavage of VE-cadherin were observed after 24 h treatment of HMGB1 (Fig 2A). VE-cadherin plays a key role in endothelial barrier function at the plasma membrane. VE-cadherin expression at the plasma membrane was dramatically decreased by 24 and 48 h treatment with HMGB1 (Fig 2A). Immunofluorescence staining for VE-cadherin showed that VE-cadherin was mostly localized to the lateral cell surfaces in control endothelial cell monolayers, which contained continuous VE-cadherin and tight cell-to-cell contacts without intercellular gaps. Immunofluorescence staining for membrane-associated VE-cadherin showed no difference at 6 and 12 h of treatment, but decreased immunofluorescence was found in endothelial cells exposed to 200 ng/ml HMGB1 for 24 h. Intercellular gap formation was slightly disrupted starting from 6 h of treatment, and there was considerable morphologic diversity among adherens junctions at 24 h of treatment (Fig 2B).


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)

Disruption of VE-cadherin and intercellular gap formation in HMGB1-treated EA.hy926 cells.A. Representative immunoblots showing reduced expression of VE-cadherin protein by HMGB1. Total and cell membrane VE-cadherin protein levels were measured by western blotting in EA.hy926 cells treated with HMGB1 for 6, 12, 24 and 48 h, respectively. GAPDH and Na,K-ATPase α1 were used as loading controls for intact cells and plasma membranes, respectively. Western blots were quantified and analyzed statistically based on three independent experiments. *Indicates significant difference compared with wild-type group (P<0.05). B. HMGB1 increased intercellular gap formation. EA.hy926 cells were plated onto a Petri dish until the formation of a tight monolayer then treated with 200 ng/ml HMGB1 for 6, 12 and 24 h, respectively. The cells were fixed and distribution of VE-cadherin was detected using rabbit anti-human VE-cadherin antibody and FITC-labeled goat anti-rabbit antibody. Nuclei were stained with DAPI. Red arrows indicate intercellular gaps. A merged picture is shown for each condition. A representative field for each condition was captured using an Olympus FV1000 confocal microscope. Scale bar = 10 μm.
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Related In: Results  -  Collection

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pone.0123432.g002: Disruption of VE-cadherin and intercellular gap formation in HMGB1-treated EA.hy926 cells.A. Representative immunoblots showing reduced expression of VE-cadherin protein by HMGB1. Total and cell membrane VE-cadherin protein levels were measured by western blotting in EA.hy926 cells treated with HMGB1 for 6, 12, 24 and 48 h, respectively. GAPDH and Na,K-ATPase α1 were used as loading controls for intact cells and plasma membranes, respectively. Western blots were quantified and analyzed statistically based on three independent experiments. *Indicates significant difference compared with wild-type group (P<0.05). B. HMGB1 increased intercellular gap formation. EA.hy926 cells were plated onto a Petri dish until the formation of a tight monolayer then treated with 200 ng/ml HMGB1 for 6, 12 and 24 h, respectively. The cells were fixed and distribution of VE-cadherin was detected using rabbit anti-human VE-cadherin antibody and FITC-labeled goat anti-rabbit antibody. Nuclei were stained with DAPI. Red arrows indicate intercellular gaps. A merged picture is shown for each condition. A representative field for each condition was captured using an Olympus FV1000 confocal microscope. Scale bar = 10 μm.
Mentions: VE-cadherin is associated with atherosclerosis, and its dysfunction has a proatherogenic effect on vessels [30]. We investigated changes in VE-cadherin protein expression and morphology in HMGB1-treated vascular endothelial cells. VE-cadherin expression was unaffected at 6 and 12 h of treatment, but significant decreases in both total VE-cadherin and cleavage of VE-cadherin were observed after 24 h treatment of HMGB1 (Fig 2A). VE-cadherin plays a key role in endothelial barrier function at the plasma membrane. VE-cadherin expression at the plasma membrane was dramatically decreased by 24 and 48 h treatment with HMGB1 (Fig 2A). Immunofluorescence staining for VE-cadherin showed that VE-cadherin was mostly localized to the lateral cell surfaces in control endothelial cell monolayers, which contained continuous VE-cadherin and tight cell-to-cell contacts without intercellular gaps. Immunofluorescence staining for membrane-associated VE-cadherin showed no difference at 6 and 12 h of treatment, but decreased immunofluorescence was found in endothelial cells exposed to 200 ng/ml HMGB1 for 24 h. Intercellular gap formation was slightly disrupted starting from 6 h of treatment, and there was considerable morphologic diversity among adherens junctions at 24 h of treatment (Fig 2B).

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