Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces GSK-3beta activation in dose- and time-dependent manners in HPMECs. Expression of P-GSK-3beta and GSK-3beta was detected after incubation with different concentrations of LPS for 1 h (A) . The expression of P-GSK-3beta was represented as a histogram according to band intensities (B) . Expression of P-GSK-3beta and GSK-3beta was examined at indicated time points after stimulation with LPS (0.1 μg/ml) in HPMECs (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Expressing, Incubation, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: Involvement of GSK-3beta in LPS-induced GEF-H1/ROCK signaling activation. HPMECs were incubated with LPS (0.1 μg/ml) at different indicated times, and the GEF-H1 and myosin-associated phosphatase type 1 (P-MYPT 1: the substrate of ROCK) were detected by Western blot assay (A) . The expression of GEF-H1 and P-MYPT 1 were represented as a histogram according to band intensities (B) . * < 0.05 vs. LPS un-treatment group. Inhibition effect of GSK-3beta activity in HPMECs was analyzed by Western blot (C,D) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group. HPMECs were pretreated with SB-216763 (20 μM) for 1 h and then were exposed to LPS (0.1 μg/ml) for 1 h. The expression of GEF-H1 and P-MYPT 1 were determined by Western blot (E) . The Western blotting results are presented as a histogram showing the band intensity values (F) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Inhibition, Activity Assay, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta signaling is involved in LPS-induced HPMECs barrier disruption. The HPMECs were plated on the gold microelectrodes. When HPMECs formed monolayers and reached stable TER values, the SB-216763 (20 μM) was added. After 1 h, the medium or LPS (0.1 μg/ml) was added for another 6 h. The HPMEC monolayers permeability was determined by real-time TER measurement (A) . The results of the 3 h LPS stimulation were represented as a histogram in (B) according to the TER curves. * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Permeability, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces degradation of beta-catenin and ZO-1 in HPMECs monolayer. LPS (0.1 μg/ml) induced down-regulation of ZO-1 expression and increase of phosphorylated degradation of beta-catenin in a time-dependent manner (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK signaling is required for LPS-induced degradation of beta-catenin and ZO-1. After transfection with GEF-H1 siRNA and Control siRNA for 48 h, HPMECs were treated with SB-216763 (20 μM) and/or Y-27632 (10 μM) for another 1 h prior to LPS stimulation (0.1 μg/ml) for 3 h. The expression of ZO-1 was determined by immunoblotting, and GAPDH protein was used as loading control (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . The expression of P-beta-catenin was determined by immunoblotting, and GSK-3beta and GAPDH proteins were used as control (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group. NS, no significance.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Transfection, Control, Expressing, Western Blot, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK pathway is involved in LPS-induced HPMECs barrier disruption by beta-catenin and ZO-1. HPMECs monolayer was pretreated with SB-216763 (20 μM) (A) , GEF-H1 siRNA (B) , or Y-27632 (10 μM) (C) , for indicated times and then was exposed to LPS (0.1 μg/ml) for 3 h before fixation and staining with anti-beta-catenin and anti-ZO-1 antibody as described in Materials and Methods. Beta-catenin (green) and ZO-1 (green) were visualized by immunofluorescence microscopy. Red arrows not only represent the expression of beta-catenin and ZO-1 in the membrane of HPMECs but also represent the cell-cell gaps formation in the ECs monolayer.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Staining, Immunofluorescence, Microscopy, Expressing, Membrane

Journal: Blood Advances

Article Title: Transfusion of target antigens to preimmunized recipients: a new mechanism in transfusion-related acute lung injury

doi: 10.1182/bloodadvances.2020003843

Figure Lengend Snippet: Antibodies against sCD177/PR3 have a functional impact on endothelial cells. (A) To evaluate the production of ROS, TNF-α–pretreated HUVECs or HPMECs were incubated with rCD177 (open bars), rPR3 (gray bars), or sCD177/PR3 (black bars), followed by antibodies or controls as indicated. Phorbol myristate (PMA) was used as assay control. ROS was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate as a fluorochrome. Values are presented as mean fluorescence intensity (MFI) ± standard deviation (n = 5). (B) Experiments were performed as in panel A using F(ab′)2 fragments instead of complete antibodies (upper panel) or pretreated with deglycosylated anti-FCGR antibodies (I, II, and IIIb) before antibody binding (lower panel). (C) To evaluate the induction of apoptosis, endothelial cells were incubated with rCD177 (open bars), rPR3 (gray bars), or sCD177/PR3 (black bars), followed by antibodies or controls as indicated. HPMECs (dotted bars) were incubated with sCD177/PR3 followed by antibodies or controls as indicated. RGD (40 mg/mL) was used as assay control. Caspase-3/7 activity was then measured. Values are presented as mean ± standard deviation (n = 5). (D) To evaluate endothelial permeability, HUVECs (upper panel) or HPMECs (lower panel) were cultured on fibronectin-coated polycarbonate filter chambers for 48 hours and treated with rCD177 (open bars), rPR3 (gray bars), or sCD177/PR3 (black bars), followed by incubation with mAbs or controls as indicated. Thrombin (0.2 U/mL) was used as an assay control. The passage of FITC-albumin through the monolayer of cells was measured at different time points (5–60 minutes). Values are presented as mean ± standard deviation (n = 5). (E) Experiments were performed as in panel D in the presence of mAb PECAM1.2 F(ab′)2 fragments. d, deglycosylated; * P < .05, ** P < . 01, ***P < .001, n.s. not significant.

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Basel, Switzerland) and human pulmonary microvascular endothelial cells (HPMECs) were purchased from Creative Bioarray (New York, NY).

Techniques: Functional Assay, Incubation, Control, Flow Cytometry, Fluorescence, Standard Deviation, Binding Assay, Activity Assay, Permeability, Cell Culture

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β -Catenin

doi: 10.1155/2021/8978795

Figure Lengend Snippet: The downregulation of microvascular endothelial adherens junction protein VE-cadherin was accompanied by increased inflammation. (a) HE staining of lung tissues and lung injury scores of the LPS-induced ALI mouse model at different times. Magnification: ×200. (b) HE staining of lung tissues and lung injury scores of the CLP-induced ALI mouse model at different times. Magnification: ×200. (c) The protein expression and relative quantitative data of VE-cadherin in the LPS-induced ALI mouse model. (d) The protein expression and relative quantitative data of VE-cadherin in the CLP-induced ALI mouse model. (e) The serum IL-6 and TNF- α levels in the LPS-induced ALI mouse model. (f) The serum IL-6 and TNF- α levels in the CLP-induced ALI mouse model. (g) The protein expression and relative quantitative data of VE-cadherin in HPMECs after 24 h stimulation by LPS. (h) The protein expression and relative quantitative data of VE-cadherin in HPMECs after 1.0 ng/mL LPS stimulation. ∗∗ p < 0.01 compared with the CTL or sham-operated group ( n = 6). Dot presents the single data results in the bar graph.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection.

Techniques: Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β -Catenin

doi: 10.1155/2021/8978795

Figure Lengend Snippet: β -Catenin was activated during sepsis-induced ALI. (a) Immunohistochemical staining and relative quantitative data of active β -catenin in lung tissues of the LPS-induced ALI mouse model at different times. Magnification: ×400. (b) Immunohistochemical staining and relative quantitative data of active β -catenin in lung tissues of the CLP-induced ALI mouse model at different times. Magnification: ×400. (c) The protein expression and relative quantitative data of active β -catenin in HPMECs after 24 h stimulation by LPS. (d) The protein expression and relative quantitative data of active β -catenin in HPMECs after 1.0 ng/mL LPS stimulation. (e) The mRNA levels of Wnt1, Wnt2, Wnt3a, Wnt5a, Wnt11, and Wnt16 in lung tissues of the LPS-induced ALI mouse model at different times. (f) The mRNA levels of Wnt1, Wnt2, Wnt3a, Wnt5a, Wnt11, and Wnt16 in lung tissues of the CLP-induced ALI mouse model at different times. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the CTL or sham-operated group ( n = 6). Dot presents the single data results in the bar graph.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β -Catenin

doi: 10.1155/2021/8978795

Figure Lengend Snippet: The dissociation of VE-cadherin/ β -catenin complex and the activation of β -catenin contributed to microvascular endothelial adherens junction dysfunction and inflammation. (a) The protein expression and relative quantitative data of VE-cadherin in lung tissues of LPS-induced ALI mouse model at different times. (b) The protein expression and relative quantitative data of VE-cadherin in lung tissues of CLP-induced ALI mouse model at different times. (c) The luciferase activity of TCF/LEF reporter in HPMECs after 24 h stimulation by LPS or 1.0 ng/mL LPS stimulation, respectively. (d) ChIP assay results of MMP-7 in HPMECs after 24 h stimulation by LPS or 1.0 ng/mL LPS stimulation. (e) The serum IL-6 and TNF- α levels in the LPS-induced ALI mouse model after treatment with 3-TYP or ICG-001. (f) The serum IL-6 and TNF- α levels in the CLP-induced ALI mouse model after treatment with 3-TYP or ICG-001. ∗∗ p < 0.01 compared with the CTL- or sham-operated group ( n = 6). ## p < 0.01 compared with the LPS or CLP group ( n = 6). Dot presents the single data results in the bar graph.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection.

Techniques: Activation Assay, Expressing, Luciferase, Activity Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β -Catenin

doi: 10.1155/2021/8978795

Figure Lengend Snippet: The downregulation of Sirt3 was accompanied with increased lung inflammation during ALI. (a) Immunohistochemical staining and relative quantitative data of Sirt3 in lung tissues of the LPS-induced ALI mouse model at different times. Magnification: ×400. (b) Immunohistochemical staining and relative quantitative data of Sirt3 in lung tissues of the CLP-induced ALI mouse model at different times. Magnification: ×400. (c) The protein expression and relative quantitative data of Sirt3 in HPMECs after 24 h stimulation by LPS. (d) The protein expression and relative quantitative data of Sirt3 in HPMECs after 1.0 ng/mL LPS stimulation. (e) Immunofluorescence staining and relative quantitative data of Sirt3 after 24 h stimulation by LPS. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the CTL- or sham-operated group ( n = 6). Dot presents the single data results in the bar graph.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β -Catenin

doi: 10.1155/2021/8978795

Figure Lengend Snippet: Sirt3 enhanced the stability of VE-cadherin/ β -catenin complex and inhibited β -catenin transcriptional activity to maintain microvascular endothelial adherens junction integrity. (a) The protein expression and relative quantitative data of VE-cadherin and β -catenin in HPMECs after transfection with scramble or Sirt3 shRNA. (b) The protein expression and relative quantitative data of VE-cadherin and β -catenin in HPMECs after transfection with vector or Sirt3 over. (c) The protein expression and relative quantitative data of VE-cadherin in HPMECs after transfection with scramble or Sirt3 shRNA. (d) The protein expression and relative quantitative data of VE-cadherin in HPMECs after transfection with vector or Sirt3 over. (e) The luciferase activity of TCF/LEF reporter in HPMECs after transfection with Sirt3 shRNA or Sirt3 over. (f) ChIP assay results of MMP-7 in HPMECs transfected with Sirt3 shRNA or Sirt3 over after 24 h stimulation. (g) The mRNA levels of Ang-2 in HPMECs transfected with Sirt3 shRNA or Sirt3 over after 24 h stimulation. (h) The protein expression and relative quantitative data of MMP-7 and COX-2 in HPMECs after transfection with scramble or Sirt3 shRNA. (i) The protein expression and relative quantitative data of MMP-7 and COX-2 in HPMECs after transfection with vector or Sirt3 over. (j) Immunofluorescence staining and relative quantitative data of PLA signals between VE-cadherin and β -catenin in HPMECs transfected with Sirt3 shRNA or Sirt3 over after 24 h stimulation by LPS. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the CTL- or sham-operated group ( n = 6). ## p < 0.01 compared with the LPS or CLP group ( n = 6). Dot presents the single data results in the bar graph.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection.

Techniques: Activity Assay, Expressing, Transfection, shRNA, Plasmid Preparation, Luciferase, Immunofluorescence, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β -Catenin

doi: 10.1155/2021/8978795

Figure Lengend Snippet: Sirt3-mediated VE-cadherin/ β -catenin complex maintained microvascular endothelial adherens junction integrity to suppress inflammation. (a) The protein expression and relative quantitative data of VE-cadherin, β -catenin, and MMP-7 in LPS-induced ALI WT and Sirt3 −/− mouse model at 6 h. (b) The protein expression and relative quantitative data of VE-cadherin, β -catenin, and MMP-7 in CLP-induced ALI WT and Sirt3 −/− mouse model at 6 h. (c) The serum IL-6 and TNF- α levels in LPS-induced ALI WT and Sirt3 −/− mouse model at 6 h. (d) The serum IL-6 and TNF- α levels in CLP-induced ALI WT and Sirt3 −/− mouse model at 6 h. (e) HE staining, immunohistochemical staining, and relative quantitative data of COX-2 in lung tissues of LPS-induced ALI WT and Sirt3 −/− mouse model at 6 h. Magnification: ×200; HE. Magnification: ×400; COX-2. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the CTL- or sham-operated group ( n = 6). ## p < 0.01 compared with the LPS or CLP group ( n = 6). Dot presents the single data results in the bar graph. WT: wild type.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β -Catenin

doi: 10.1155/2021/8978795

Figure Lengend Snippet: Sirt3 maintained microvascular endothelial adherens junction integrity to attenuate lung inflammation by acting on the stability of VE-cadherin/ β -catenin complex. Sepsis induced VE-cadherin downregulation, β -catenin activation, and, importantly, the dissociation of VE-cadherin/ β -catenin complex in lung microvascular endothelial cells and ALI animal models. These damaged adherens junctions and triggered the β -catenin-mediated MMP-7 expression to further destroy VE-cadherin/ β -catenin complex, which eventually resulted in the breakdown of microvascular endothelial adherens junction. These events facilitated the transfer of inflammatory factor through microvascular endothelial cells into the capillary and contributed to capillary leakage and ultimately lung inflammation. Notably, we first found that Sirt3 could inhibit inflammation through maintaining microvascular endothelial adherens junction integrity by acting on the interaction of VE-cadherin and β -catenin.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs) were purchased from the China Center for Type Culture Collection.

Techniques: Activation Assay, Expressing