Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondria are efficiently internalized by HPMEC. (A) Representative live imaging of mitochondrial transplantation. Mitochondria isolated from MSC (MSC-mt) were prestained with MitoTracker Red and co-cultured with HPMEC prestained with MitoTracker Green for 24 h. Top panels: control. Middle panels: MSC-mt. Confocal imaging of internalized MSC-mt mitochondria (Mitotracker Red) co-localized to endogenous mitochondria network of non-stimulated HPMEC (MitoTracker Green). Lower panel: LPS + MSC-mt. MSC-mt internalized and co-localized to the endogenous mitochondrial network of HPMEC stimulated with LPS. The images were taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (B) Representative images of orthogonal views (Scale bar = 20 μm) and 3D reconstruction (Scale bar = 5 μm) on the LPS + MSC-mt group. (C) Analysis of region of interest (ROI) colocalization of control, positive control, and MSC-mt group. Data presented as mean ± SD.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Imaging, Transplantation Assay, Isolation, Cell Culture, Control, Microscopy, Positive Control

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: MSC-mt transplantation effects on mtDNA copies, viability, inflammatory activation, and mitochondrial respiration of recipient cells. (A) Quantification of mtDNA copy numbers in LPS-stimulated HPMEC after mitochondrial transplantation (24 h). (B) Effects on HPMEC viability measured by levels of LDH release. ( n = 3–4). (C) Levels of interleukin (IL)-8 secretion by HPMEC measured by ELISA ( n = 3–4). (D) Levels of TNF-α secreted by THP1 macrophages measured by ELISA ( n = 3–4). (E) Representation of Seahorse Mito Stress assay curve showing OCR in HPMEC. (F–H) Values for respiratory parameters: basal respiration (F), maximal respiration (G), and ATP production (H) ( n = 4–5). Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. Differences were assessed using Kruskal–Wallis with post-hoc Dunn’s test.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondrial transplantation alleviates mitochondrial dysfunction in HPMEC. (A) Representative images of JC-1 fluorescence in HPMEC. Green areas indicate depolarized mitochondrial membranes (JC-1 monomers), and red areas indicate polarized mitochondrial membranes (JC-1 aggregates). FCCP was used to induce mitochondria depolarization. Images taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (B) Representative images of HPMEC mitochondrial superoxide production detected with MitoSOX. Mitotempo (MT) was used as positive control for ROS quenching. Images taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (C) Quantification of red to green JC-1 fluorescence intensity ratio in HPMEC analyzed by ImageJ software. (D) Quantitative MitoSOX fluorescence intensity (MFI) analyzed by ImageJ software. Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. Differences were assessed using one-way ANOVA analysis with post hoc Bonferroni’s test.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Fluorescence, Microscopy, Positive Control, Software

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondrial transplantation restores HPMEC barrier integrity in the presence of LPS or ARDS plasma. (A) Real-time impedance analysis of HPMEC exposed to LPS and treated with MSC-mt and their respective cell impedance analysis of XCelligence RTCA measurements at a 24-h timepoint. Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. (B) Representative real-time impedance analysis of HPMEC exposed to hypoinflammatory ARDS plasma and their respective cell impedance analysis of XCelligence RTCA measurements at a 24 and 48-h timepoint. (C) Representative real-time impedance analysis of HPMEC exposed to hyperinflammatory ARDS plasma and their respective cell impedance analysis of XCelligence RTCA measurements at a 24 and 48-h timepoint. Data presented as mean ± SD. Differences were assessed using Kruskal–Wallis with post-hoc Dunn’s test (A) and one-way ANOVA analysis with post hoc Bonferroni’s test (B, C).

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Clinical Proteomics

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: Scientific Reports

Article Title: FOSL1 is a novel mediator of endotoxin/lipopolysaccharide-induced pulmonary angiogenic signaling

doi: 10.1038/s41598-020-69735-z

Figure Lengend Snippet: Validation of cell line as endothelial cells. ( A ) expression of ERG, CDH5, and vWF by immunoflourescence, and uptake of AcLDL; ( B ) assessment of novel LPS genes after treatment of primary HPMECs (n = 3) and HPMEC line (n = 4) with LPS for 6 h (p values determined by one-sample, two-tailed Student’s t test); and ( C ) assessment of angiogenesis or vasculogenesis genes after treatment of primary HPMECs (n = 3) and HPMEC line (n = 5) with LPS for 24 h (p values determined by one-sample, two-tailed Student’s t test; all data were normally distributed except 24 h primary HPMEC LRG1 which was assessed with one-sample Wilcoxon signed rank test). Data presented at mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 between LPS-treated and saline control baseline. Graphs created in SPSS 26 for Mac ( https://www.ibm.com/analytics/spss-statistics-software ) and image assembled in Adobe Illustrator 24.1.0 for Mac ( https://www.adobe.com/products/illustrator.html ).

Article Snippet: We proceeded to establish functional relationships between FOSL1 and downstream targets in an in vitro model. We used 19-week gestation, male primary HPMEC (ScienCell), derived from neonatal lungs.

Techniques: Biomarker Discovery, Expressing, Two Tailed Test, Standard Deviation, Saline, Control, Software

Journal: Chinese Medical Journal

Article Title: Down-regulation of miR-155 inhibits inflammatory response in human pulmonary microvascular endothelial cells infected with influenza A virus by targeting sphingosine-1-phosphate receptor 1

doi: 10.1097/CM9.0000000000001036

Figure Lengend Snippet: MiR-155 regulates cytokine production in H1N1-infected HPMECs. Cultured HPMECs were transfected with miR-155 mimic, inhibitor, or NC. After 48 h of transfection, the cells were infected with H1N1 at an MOI of 1 for 1 h. Total RNA was isolated at indicated time points and cDNA synthesized for analysis by real-time polymerase chain reaction. The expression levels of the proinflammatory cytokines were detected using real-time polymerase chain reaction. The experiments were repeated three times, and the data are presented as mean ± standard error. Data were analyzed by analysis of variance ( ∗ P < 0.05; † P < 0.01; compared with the cytokine levels in virus-treated NC group cells). CCL: CC motif chemokine ligand; HPMECs: Human pulmonary microvascular endothelial cells; IFN-β: Interferon-β; IL: Interleukin; MOI: Multiplicity of infection; NC: Negative control; p.i.: Post-infection; TNF-α: Tumor necrosis factor-α.

Article Snippet: Primary HPMECs were obtained from ScienCell (San Diego, CA, USA) and cultured in endothelial cell medium using the recommended supplements from the supplier, and they were used in passages 3 to 5.

Techniques: Infection, Cell Culture, Transfection, Isolation, Synthesized, Real-time Polymerase Chain Reaction, Expressing, Negative Control