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

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.

Fig 1. HMGB1 increases endothelial cell permeability.

A. HMGB1-induced TEER decrease. EA.hy926 cells cultured on transwell filters were incubated for 3, 6, 12, 24, 36 and 48h, respectively, with or without 50, 100, 200 and 400 ng/ml HMGB1. The integrity of the tight junctions was assessed by measuring the TER. B. Cell viability in the cells treated by HMGB1. EA.hy926 cells were treated with 50, 100, 200, 400 and 800ng/ml HMGB1, respectively, for 24 h. The cell viability was measured by CCK-8 assay. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).

Fig 2. 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.

Fig 3. 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).

Fig 4. 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).

Fig 5. 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).

Fig 6. STIM1 knockdown decreases Ca2+ influx, HMGB1-induced permeability and Src phosphorylation.

A. STIM1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-STIM1 antibodies, with anti-GAPDH antibodies as a loading control. STIM1 expression was quantified and analyzed statistically based on three independent experiments. Transfected cells were also stimulated with 200 ng/ml HMGB1 (B) or 1 μM TG (C), 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. D. HMGB1-induced permeability was inhibited by STIM1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with STIM1 siRNA-1, 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. E. Representative immunoblots showing that STIM1 knockdown inhibits Src activation. Transfected cells were treated with or without 200 ng/ml HMGB1 for 2 h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).

Fig 7. 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).