Journal: PLoS ONE

Article Title: Store-Operated Ca 2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

doi: 10.1371/journal.pone.0123432

Figure Lengend Snippet: 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).

Article Snippet: The human umbilical vein endothelial cell line EA.hy926 (provided by China Center for Type Culture Collection, Shanghai, China) was maintained in DMEM with 10% FBS with antibiotics.

Techniques: Cell Culture, Incubation, CCK-8 Assay, Control

Journal: PLoS ONE

Article Title: Store-Operated Ca 2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

doi: 10.1371/journal.pone.0123432

Figure Lengend Snippet: 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.

Article Snippet: The human umbilical vein endothelial cell line EA.hy926 (provided by China Center for Type Culture Collection, Shanghai, China) was maintained in DMEM with 10% FBS with antibiotics.

Techniques: Western Blot, Expressing, Membrane, Clinical Proteomics, Labeling, Staining, Microscopy

Journal: PLoS ONE

Article Title: Store-Operated Ca 2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

doi: 10.1371/journal.pone.0123432

Figure Lengend Snippet: 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).

Article Snippet: The human umbilical vein endothelial cell line EA.hy926 (provided by China Center for Type Culture Collection, Shanghai, China) was maintained in DMEM with 10% FBS with antibiotics.

Techniques: Western Blot, Activation Assay, SDS Page, Permeability, Incubation, Phospho-proteomics, Control

Journal: PLoS ONE

Article Title: Store-Operated Ca 2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

doi: 10.1371/journal.pone.0123432

Figure Lengend Snippet: 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).

Article Snippet: The human umbilical vein endothelial cell line EA.hy926 (provided by China Center for Type Culture Collection, Shanghai, China) was maintained in DMEM with 10% FBS with antibiotics.

Techniques: Microscopy, Incubation, Control

Journal: PLoS ONE

Article Title: Store-Operated Ca 2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

doi: 10.1371/journal.pone.0123432

Figure Lengend Snippet: 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).

Article Snippet: The human umbilical vein endothelial cell line EA.hy926 (provided by China Center for Type Culture Collection, Shanghai, China) was maintained in DMEM with 10% FBS with antibiotics.

Techniques: Expressing, Transfection, Control, Western Blot, Microscopy, Permeability, Knockdown, Incubation, Activation Assay, SDS Page

Journal: PLoS ONE

Article Title: Store-Operated Ca 2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

doi: 10.1371/journal.pone.0123432

Figure Lengend Snippet: 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).

Article Snippet: The human umbilical vein endothelial cell line EA.hy926 (provided by China Center for Type Culture Collection, Shanghai, China) was maintained in DMEM with 10% FBS with antibiotics.

Techniques: Expressing, Transfection, Control, Western Blot, Permeability, Knockdown, Incubation

Journal: Redox Biology

Article Title: Overview on hydrogen sulfide-mediated suppression of vascular calcification and hemoglobin/heme-mediated vascular damage in atherosclerosis

doi: 10.1016/j.redox.2022.102504

Figure Lengend Snippet: Proposed protective pathways of H 2 S on vascular plaque development provoked by hemoglobin and heme. Modification of lipid oxidation and vascular inflammation Infiltration of red blood cells (RBCs) into atheromatous plaques is common during atherosclerosis progression. Hemoglobin (Hb) released from RBCs readily oxidizes to met- (Fe 3+ ) and ferryl-Hb (Fe 4+ ), followed by release of heme from globin and iron from heme moiety. Hb oxidation facilitates protein radical formation damaging the globin structure that yields globin-globin crosslinking, Hb dimers, tetramers, multimers, and globin-derived peptides. Free heme and iron catalyze the oxidation of low-density lipoprotein (LDL) and plaque lipids resulting in oxidized LDL (LDLox) and the formation of reactive lipid mediator (RLM), releasing reactive oxygen species (ROS). Both ferryl-Hb and globin-derived peptides provoke endothelial cell activation and induce adhesion molecule expressions, such as vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), leading to increased endothelial cell permeability. Notably, ferryl-Hb and globin-derived peptides trigger inflammatory response via nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Ferryl-Hb also drives the pro-atherogenic polarization of macrophages in the vessel wall, thereby affecting diverse processes, among them inflammation, calcification, angiogenesis, cell damage, and tissue remodeling. Heme, oxidized lipid mediators, and ferryl-Hb induce cystathionine γ-lyase (CSE) expression in the resident cells of atherosclerosis. Significantly, H2S inhibits the oxidation of Hb and plaque lipids and prevents LDLox-induced cell death. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: In immortalized human vascular endothelial cells (EA.hy926), HIF-1α accumulation, in response to hypoxia (1% O2) and hypoxia-mimetics, is reversed by NaHS administration via enhancing eIF2α phosphorylation.

Techniques: Modification, Derivative Assay, Activation Assay, Permeability, Expressing

Journal: Redox Biology

Article Title: Overview on hydrogen sulfide-mediated suppression of vascular calcification and hemoglobin/heme-mediated vascular damage in atherosclerosis

doi: 10.1016/j.redox.2022.102504

Figure Lengend Snippet: Cross-talk between hypoxia and H 2 S/endogenous H 2 S synthesis Hypoxia and hypoxia-mimetic drugs induce the accumulation and nuclear translocation of hypoxia-inducible factor-1α (HIF-1α) followed by an increase in the expression of hypoxia-responsive genes, among them vascular endothelial growth factor (VEGF), which promotes neovascularization. In endothelial cells (ECs), H 2 S reverses HIF-1α accumulation and subsequent VEGF production that might counteract hypoxia-induced neovascularization and tube formation. In addition, hypoxia differentially regulates the expression of cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MPST), and cystathionine β-synthase (CBS). Hypoxia induces 3-MPST, decreases CSE, and has no effect on CBS levels. In vascular smooth muscle cells (VSMCs), hypoxia down-regulates Specificity protein 1 (Sp1), the master regulator of CSE in VSMCs, thereby lowering overall CSE expression, leading to increased cell death. In addition, hypoxia induces the translocation of CSE to the mitochondria.

Article Snippet: In immortalized human vascular endothelial cells (EA.hy926), HIF-1α accumulation, in response to hypoxia (1% O2) and hypoxia-mimetics, is reversed by NaHS administration via enhancing eIF2α phosphorylation.

Techniques: Translocation Assay, Expressing