Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMP9 and BMP10 selectively activate SMAD1/5/8 signaling and induce proliferation in pulmonary artery endothelial cells but not pulmonary artery smooth muscle cells. ( A ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PAECs treated with the indicated TGF-β superfamily ligands (0.8 nM) or untreated control (UT); β-actin serves as a loading control. ( B ) PAEC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. ( C ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PASMCs treated with the indicated ligands (0.8 nM); β-actin serves as a loading control. ( D ) PASMC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. Data are shown as mean ± SD ( n = 3 replicate wells). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, BrdU Incorporation Assay

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMPR2 dosage-dependent model for BMP9/10 signaling output in pulmonary artery endothelial cells. Schematic illustrating how BMPR2 abundance constrains BMP9/10 (ALK1-dependent) canonical signaling output and downstream cellular programs in PAECs. ( A ) BMPR2-sufficient (~100%) state: BMP9/10 predominantly signal through ALK1–BMPR2 complexes, generating pSMAD1/5/8 output consistent with a threshold-like requirement for proliferation; bimagrumab (BiMab) produces no effect detected under BMPR2-replete conditions. ( B ) BMPR2-limiting (~50%) state: Reduced BMPR2 attenuates BMP9/10-induced canonical output and is associated with reduced proliferation and increased caspase-3/7 activity consistent with stress/injury. Under BMPR2-limiting conditions, residual canonical output becomes bimagrumab-sensitive, consistent with context-dependent contribution of Activin type II receptors (predominantly ACVR2A in PAECs; see for BMP10 affinity comparisons) to the remaining pSMAD1/5/8 signal. A putative non-canonical stress-signaling arm is shown as a proposed intermediate. Solid arrows denote observed relationships; dashed arrows and dashed-outline boxes denote proposed steps. Node shading and output gauges depict relative canonical signaling output.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 is expressed in the kidney as assessed by western blot performed on total kidney extracts. ( b ) Gata5 is essentially expressed in the glomeruli as assessed by qPCR on isolated glomeruli (Glo) and microdissected tubules (Tub) from Wt mice kidneys. ( n =3–5 per group). The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice (Mann–Whitney test). ( c , d ) Specific deletion of Gata5 in endothelial cells (e Gata5-null mice) virtually abolished renal and glomerular expression of Gata5 ( n =3 per group). The results are reported as mean±s.e.m. * P <0.05 versus eGata5 +/+ mice (Mann–Whitney test). ( e ) The expression of the glomerular genes Nphs1 (nephrin) and Nphs2 (podocin) as measured by qPCR ( n =6 per group) was increased in Gata5 -null mice. The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice; *** P <0.005 versus Gata5 +/+ mice ( t -test for nephrin; Mann–Whitney test for podocin). ( f – i ) Absence of Gata5 induces glomerular lesions (sections are stained with periodic acid Schiff; scale bar, 30 μm; n =5 per group) and renal inflammation as assessed by leucocytes CD45 immunostaining (scale bar, 200 μm; n =4 per group). The results are reported as mean±s.e.m. *** P <0.005 versus Gata5 +/+ mice (Mann–Whitney test). ( j – n ) Deletion of Gata5 from endothelial cells reproduces the renal phenotype of global Gata5 deletion: both Nephs1 and Nphs2 transcript levels were increased ( n =4–6 per group) as well as glomerular lesion score (scale bar, 30 μm; n =4–6 per group) and renal leucocytes infiltration (scale bar, 200 μm; n =4–5 per group) in e Gata5 -null mice in comparison with their controls. The results are reported as mean±s.e.m. * P <0.05 versus e Gata5 +/+ mice; *** P <0.005 versus e Gata5 +/+ mice (Mann–Whitney test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Western Blot, Isolation, MANN-WHITNEY, Expressing, Staining, Immunostaining, Comparison

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 is expressed in human cardiac microvascular (CM), coronary artery (CA), dermal microvascular (DM) and pulmonary microvascular (PM) endothelial cells. ( b , c ) The vasoconstrictor response of Gata5 -null mice mesenteric arteries to norepinephrine is unaltered ( n =7 per group), while the vasodilatory response to acetylcholine is decreased ( n =10–11 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (comparison of best-fit values—effector concentration for half-maximum response (EC 50 ) and Hill slope—using an F-test). ( d – f ) Deletion of Gata5 in endothelial (e Gata5 -null mice; n =4–5 per group) but not smooth muscle cells (sm Gata5 -null mice; n =5 per group) decreases mesenteric arteries sensitivity to acetylcholine and increases BP (e Gata5 -null mice n =6–10 per group; sm Gata5 -null mice n =6–9 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (two-factor ANOVA). ( g ) The vasodilatory response of Gata5 -null mice to diethylamine NONOate, an NO donor, is unaltered ( n =7 per group). The results are reported as mean±s.e.m. (comparison of best-fit values—EC 50 and Hill slope—using an F-test). ( h , i ) NOS3 and Akt phosphorylation are decreased in Gata5 -null mice mesenteric arteries. PTEN and PDK1 phosphorylation and expression are unaltered ( n =5–7 per group). Phosphorylated proteins are normalized to total proteins. Total proteins are normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice ( t -test). ( j ) Quantification of protein nitrotyrosination in mesenteric arteries of Gata5 -null mice and their controls as measured by ELISA. 3-Nitrotyrosine content is expressed as picomole of nitrotyrosine per milligram of protein ( n =5–7 per group). The results are reported as mean±s.e.m. ( t -test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Comparison, Concentration Assay, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 expression is significantly decreased in human dermal microvascular endothelial cells infected with a lentiviral vector containing an anti- GATA5 shRNA (HDMEC-GATA5-KD). Control cells were infected with a vector containing a control shRNA (targets no known mammalian gene) (HDMEC-pLKO-Ctrl, referred here as Ctrl). ( b ) Heatmap representation of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls as identified by transcriptomic analysis. Colour is function of Log2 RMA (Affymetrix microarray, n =3 per group). ( c ) Functional analysis of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls. Protein kinase A pathway is the most significantly enriched pathway. Fisher's exact test P value. ( d ) Validation by qPCR (upper panel) of genes predicted by microarray (lower panel) to be up- and downregulated in HDMEC-GATA5-KD endothelial cells. ( n =5 wells per condition). Downregulated genes: PRKACB codes for the PKA catalytic subunit β, PRKAR2B for the PKA regulator subunit 2β and PRKAA2 for the AMPK catalytic subunit α2. Upregulated genes: ICAM1 codes for the intercellular adhesion molecule 1, BMP4 for the bone morphogenetic protein 4 and IL6 for the interleukin 6. The results are reported as mean±s.e.m. ** P <0.01 versus Ctrl ( t -test). ( e ) Western blot representation of phospho-NOS3, NOS3 (Ser1177) and phospho-(Ser/Thr) PKA substrate motif in HDMEC-GATA5-KD cells and their controls. ( f ) Phosphorylation of NOS3 on Ser1177 is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-NOS3 is normalized to total NOS3. NOS3 is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( g ) Phosphorylation of (Ser/Thr) PKA substrate motif is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( h ) Western blot representation of phospho-(Ser/Thr) PKA substrate motif in mesenteric arteries of Gata5 -null mice and their controls. ( i ) In mesenteric arteries of Gata5 -null mice, there is a trend to decrease in the (Ser/Thr) PKA substrate motif phosphorylation ( n =4–5 per group). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. (Mann–Whitney test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Control, Microarray, Functional Assay, Biomarker Discovery, Western Blot, Phospho-proteomics, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) Administration of hydralazine (a smooth muscle cell relaxant) for 4 weeks decreased blood pressure similarly in both Gata5 -null mice and their controls ( n =5–7 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons). ( b ) Hydralazine had no effect on Gata5 -null mice endothelial dysfunction ( n =5–7 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (comparison of best-fit values—EC 50 and Hill slope—using an F-test). ( c ) Hydralazine decreased partially glomerular injuries in Gata5 -null mice (sections are stained with periodic acid Schiff; scale bar, 30 μm; n =5–7 per group). * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons). ( d ) Hydralazine decreased completely renal CD45+ cells infiltration in Gata5 -null mice and also their controls (scale bar, 200 μm; n =5–7 per group). * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Comparison, Staining

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: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC (A) and PASMC (B) attained confluence in low (0.2%) serum as determined by light microscopy and the absence of proliferation. Cells were then exposed to increasing concentrations of serum and cell number determined in triplicate seven days later. (n = 3 experiments; * indicates p<.05 compared with starting cell number).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and cell cycle profile (A and C) and BrdU incorporation (B and D) determined 24 hours later. (n = 3 experiments; * indicates p<.05).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Western Blot, Infection, BrdU Incorporation Assay

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