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