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: Molecules

Article Title: Iloprost Attenuates Oxidative Stress-Dependent Activation of Collagen Synthesis Induced by Sera from Scleroderma Patients in Human Pulmonary Microvascular Endothelial Cells

doi: 10.3390/molecules26164729

Figure Lengend Snippet: Figure 1. (A,B) Effect of SSc and SSc iloprost-treated sera on human pulmonary microvascular endothelial cells (HPMECs) intracellular ROS levels. Before sera treatment, subconfluent HPMECs were loaded with 10 µM of H2-DCFDA, and then cultured in basal medium containing 10% (v/v) of sera from SSc patients (SSc), sera from iloprost-treated SSc patients (SSc + I),

Article Snippet: In this study, human pulmonary microvascular endothelial cells (HPMECs) isolated from the human lung of healthy donors were used (Innoprot, Bizkaia, Spain).

Techniques: Cell Culture

Journal: Respiratory Research

Article Title: The Tie-2 agonist PMC-403 inhibits endothelial damage induced by e-cigarette vapor or LPS

doi: 10.1186/s12931-026-03928-4

Figure Lengend Snippet: A A schematic setup is shown for stimulation with the Tie2 agonist PMC-403 and its effects on HUVECs and HPMECs, accordingly. Created in BioRender. Stahl, K. (2026) https://BioRender.com/yc7s3fj . B HUVECs were stimulated with different concentrations of the Tie2 agonist PMC-403 or 500 ng/ml Angpt-1 for 1 hour. A western blot was performed and pAKT and AKT were analyzed. C HPMECs were incubated with 1 µg/ml PMC-403 for 1 hour and then stimulated with LPS. TER was measured by ECIS over time and group differences over time were analyzed by a linear mixed-effects model accordingly. D Group differences are shown 6.5 hours after LPS stimulation. Results are shown as dot plots with mean ± SD and each group consisted of n =4. P -values < 0.05 are considered significant. Abbreviations: Angpt-1 - Angiopoietin-1, ECIS - Electric cell-substrate impedance sensing, HPMECs - human pulmonary microvascular endothelial cells, HUVECs – human umbilical vein endothelial cells, LPS - lipopolysaccharide, SD – standard deviation, TER – transendothelial electrical resistance

Article Snippet: As an additional endothelial cell type, primary human pulmonary microvascular endothelial cells (HPMECs; PromoCell, Heidelberg, Germany; Cat. No. C-12281) were used and cultured in endothelial cell growth medium MV2 (PromoCell; Cat. No. C-22022).

Techniques: Western Blot, Incubation, Electric Cell-substrate Impedance Sensing, Standard Deviation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Differential sex-specific expression on miR-30a in vitro. A and B: miR-30a-3p and miR-30a-5p expression in human pulmonary microvascular endothelial cells (HPMECs) exposed to room air (RA) (RA-5% CO2) and 72 h of hyperoxia (95% O2-5% CO2) (n = 3/group). Values are means ± SE. Significant differences between RA and hyperoxia within each sex are indicated by ***P < 0.001. Significant differences between male and female mice in normoxia or hyperoxia are indicated by #P < 0.05 and ##P < 0.01.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Expressing, In Vitro

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Delta-like ligand 4 (Dll4) expression in male and female human neonatal pulmonary microvascular endothelial cells upon exposure to hyperoxia in vitro: DLL4 mRNA (A) and protein (B) expression in male and female neonatal human pulmonary microvascular endothelial cells (HPMECs) exposed to room air (RA) (RA-5% CO2) and 72 h of hyperoxia (95% O2-5% CO2) (n = 3/group). Values are means ± SE. Significant differences between RA and hyperoxia within each sex are indicated by *P < 0.05 and **P < 0.01. Significant differences between male and female mice in normoxia or hyperoxia are indicated by ##P < 0.01. NS, not significant.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Expressing, In Vitro

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Delta-like ligand 4 (Dll4) is an miR-30a-5p target in neonatal human pulmonary microvascular endothelial cells. TargetScan reported miR-30a binding sites 59–66bp in the 3′UTR of DLL4 (A). Increased mir30a-5p expression after mir30a-5p mimic transfection in HPMECs (B). DLL4 mRNA (C) and protein (D) expression after mir30a-5p mimic transfection in HPMECs. DLL4 mRNA (E) and protein (F) expression after mir30a-5p inhibitor transfection in HPMECs. DLL4 protein expression (G) after mir30a-5p mimic transfection with and without target protector in HPMECs. Values are means ± SE from 3 independent experiments (n = 3/group). Significant differences between indicated groups are indicated by *P < 0.05 and **P < 0.01. HPMEC, human pulmonary microvascular endothelial cell; UTR, untranslated region; NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Binding Assay, Expressing, Transfection, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Effect of miR30a-3p overexpression and inhibition on DLL4 expression in HPMECs. Increased mir30a-3p expression after mir30a-3p mimic transfection in HPMECs (A). DLL4 mRNA (B) and protein (C) expression after mir30a-3p mimic transfection in HPMECs. DLL4 mRNA (D) and protein (E) expression after mir30a-3p inhibitor transfection in HPMECs. Values are means ± SE from three independent experiments (n = 3/group). Significant differences between indicated groups are indicated by *P < 0.05 and **P < 0.01. DLL4, delta-like ligand 4; HPMEC, human pulmonary microvascular endothelial cell. NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Over Expression, Inhibition, Expressing, Transfection, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: miR30a-5p inhibition under hyperoxic conditions decreases increases Dll4 expression in female HPMECs. DLL4 mRNA (A) and protein (B) expression after inhibition of mir30a-5p in HPMECs upon exposure to hyperoxia. Values are means ± SE from three independent experiments (n = 3/group). Significant differences between indicated groups are indicated by **P < 0.01. DLL4, delta-like ligand 4; HPMEC, human pulmonary microvascular endothelial cell. NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Inhibition, Expressing, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: miR30a-5p mimic increases sprouting angiogenesis in female HPMECs. Angiogenic potential was quantified based on the maximum length of sprouts protruding from HPMEC-coated cytodex-3 microcarrier beads suspended in a fibrin gel (n = 3/group). A: representative images from male and female HPMECs subjected to sprouting angiogenesis assay treated with miR30a-5p mimic or negative control. Scale bar represents 200 μm. B: maximum sprouting distance in male and female HPMECs. Values are means ± SE. Significant differences between treatment and control groups is indicated by *P < 0.05. HPMEC, human pulmonary microvascular endothelial cell.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Angiogenesis Assay, Negative Control, Control

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: Overexpression of FLRT3 promotes cell migration and rescues H/R-induced cell death and inhibition on cell migration. HPMECs were transduced with LV-Control, LV-FLRT3, sh-Control or sh-FLRT3 for 48 h. A Western blot confirmed successful FLRT3 overexpression and knockdown in HPMECs. B CCK-8 assay showed FLRT3 manipulation did not affect baseline cell viability. C Wound healing assay (24 h) demonstrated enhanced migration in LV-FLRT3 group and reduced migration in sh-FLRT3 group. D Transwell migration assay validated the migration changes with FLRT3 manipulation. E Western blot showed LV-FLRT3 restored H/R-induced FLRT3 reduction. F CCK-8 assay revealed FLRT3 overexpression protected against H/R-induced viability decrease. G Wound healing assay demonstrated FLRT3 overexpression preserved migration capacity under H/R conditions. H Transwell assay confirmed the protective effect of FLRT3 on H/R-impaired cell migration. Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test to compare multiple groups. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Over Expression, Migration, Inhibition, Transduction, Control, Western Blot, Knockdown, CCK-8 Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Assay

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: FLRT3 overexpression increases endothelial barrier integrity via cytoskeletal remodeling and restoration of cell–cell junctions after H/R. HPMECs were transduced with LV-Control or LV-FLRT3 for 48 h, then subjected to H/R. A TEER measurements showed FLRT3 overexpression prevented H/R-induced barrier dysfunction. B Evans blue permeability assay demonstrated FLRT3 overexpression reduced H/R-induced barrier leakage. C F-actin staining (FITC-phalloidin, green; DAPI, blue) revealed FLRT3 overexpression maintained cytoskeletal organization under H/R. Scale bar, 50 μm. D Western blot showed FLRT3 overexpression preserved junction protein levels after H/R. E VE-cadherin immunofluorescence demonstrated maintained adherens junctions with FLRT3 overexpression during H/R. Scale bar, 50 μm. F Claudin-5 immunofluorescence showed preserved tight junction organization with FLRT3 overexpression after H/R. Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Over Expression, Transduction, Control, Permeability, Staining, Western Blot, Immunofluorescence

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: The FLRT3 protein is destabilized through the autophagic–lysosomal pathway after H/R. A qRT-PCR revealed unchanged FLRT3 mRNA levels in HPMECs after H/R ( n = 3/group). B qRT-PCR showed stable FLRT3 mRNA levels in lung tissue after I/R ( n = 5/group). C , D Protein stability assay using CHX (20 μg/ml) demonstrated accelerated FLRT3 degradation in H/R-treated HPMECs, with significant reduction at 2 h post-CHX ( n = 3/group). E Western blot showed proteasome inhibitor MG132 (5 μM) failed to prevent H/R-induced FLRT3 degradation ( n = 3/group). F Lysosomal inhibitors NH 4 Cl (10 µM) and CQ (10 μM) restored FLRT3 levels in H/R-treated HPMECs. Western blot quantification showed significant restoration of FLRT3 levels with both inhibitors ( n = 3/group). G Autophagy inhibitor Baf-A1 (100 nM) increased both LC3-II/LC3-I ratio and FLRT3 levels under H/R conditions ( n = 3/group). Data are presented as mean ± SD. Statistical significance was determined using unpaired Student’s t test to compare two groups and one-way ANOVA and Tukey’s multiple comparisons test to compare multiple groups. Two‐way ANOVA with Tukey’s post hoc test was used to compare multiple groups with two categorical variables. ** P < 0.01 compared with the indicated groups. Baf-A1 Bafilomycin-A1, CHX cycloheximide, CQ chloroquine, NH 4 Cl ammonium chloride

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Quantitative RT-PCR, Stability Assay, Western Blot

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: RND3 is a target of FLRT3. A HumanBase Gene Networks analysis predicted functional correlation between FLRT3 and RND3 in lung tissue. B Co-IP assay demonstrated endogenous FLRT3–RND3 protein interaction in HPMECs, with input and IgG controls confirming specificity. C GST pulldown assay validated direct FLRT3-RND3 binding. D Double immunofluorescence (FLRT3-red, RND3-green) showed protein co-localization in HPMECs. Scale bar, 50 μm. E Western blot revealed FLRT3 overexpression increased RND3 levels while FLRT3 knockdown reduced RND3 expression. F Western blot showed FLRT3 overexpression restored H/R-induced RND3 reduction ( P < 0.01). Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Functional Assay, Co-Immunoprecipitation Assay, GST Pulldown Assay, Binding Assay, Immunofluorescence, Western Blot, Over Expression, Knockdown, Expressing

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: RND3 is required for FLRT3 overexpression related cytoprotective effects after H/R in HPMEC cells. H/R-induced HPMECs were transduced with LV-FLRT3 and/or sh-RND3. A Western blot showed that overexpression of FLRT3 led to an increase in RND3 protein expression, while knockdown of RND3 in FLRT3-overexpressing HPMECs resulted in a reduction in RND3 protein expression levels. B , C Western blot showed RND3 knockdown reversed FLRT3-mediated protein expression under H/R. D TEER measurements revealed RND3 knockdown compromised FLRT3’s barrier-protective effect. E Evans blue assay validated RND3’s requirement in FLRT3-mediated barrier protection. F F-actin staining (phalloidin, green; DAPI, blue) showed RND3 knockdown prevented FLRT3’s cytoskeletal preservation. Scale bar, 50 μm. G Western blot demonstrated RND3 knockdown reversed FLRT3’s protection of junction proteins (VE-cadherin, ZO-1, claudin-5, and occludin). H , I Immunofluorescence of VE-cadherin ( H ) and claudin-5 ( I ) confirmed RND3’s role in FLRT3-mediated junction maintenance. Scale bar, 50 μm. Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Over Expression, Transduction, Western Blot, Expressing, Knockdown, Evans Blue Assay, Staining, Preserving, 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: 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