Journal: International Journal of Molecular Sciences

Article Title: Disrupting the Btk Pathway Suppresses COPD-Like Lung Alterations in Atherosclerosis Prone ApoE −/− Mice Following Regular Exposure to Cigarette Smoke

doi: 10.3390/ijms19020343

Figure Lengend Snippet: Analysis of alveolar elastin breaks in lungs of ApoE −/− mice fed WD and exposed to SHS only (WD + SHS), with Btk inhibitor treatment (WD + SHS/Btk Inh.), or treated with endothelial cell targeted siRNA for MMP-9 (WD + SHS/MECA-32 siRNA MMP-9). Representative images are shown. Black arrows point out strand breaks in alveolar walls ( a ). Destruction of alveolar walls was assessed by counting numbers of breaks in elastin fibers ( b ). Groups receiving treatment scored significantly lower than the control group ( p < 0.05). Kruskal–Wallis ANOVA on ranks was used to assess statistical significance between groups ( p < 0.001) followed by post hoc testing with Dunn’s multiple comparison test for groups of interest, four to six mice per group were evaluated. * Significant difference detected.

Article Snippet: Treatment animals were injected intravenously [tail vein injection] with either Bruton’s tyrosine kinase inhibitor (BTK Inh) PCI-32765 (Selleck Chemicals, Houston, TX, USA) or siRNA specific for MMP-9 (Invitrogen, Carlsbad, CA, USA) conjugated with F(ab’) 2 fragments of anti-mouse neutrophil antibody (clone Ly-6G 1A8, Bio X Cell, West Lebanon, NH, USA) or anti-mouse endothelial cell antibody (clone MECA-32, Bio X Cell).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Disrupting the Btk Pathway Suppresses COPD-Like Lung Alterations in Atherosclerosis Prone ApoE −/− Mice Following Regular Exposure to Cigarette Smoke

doi: 10.3390/ijms19020343

Figure Lengend Snippet: ( a ) Morphometric analysis of lungs from ApoE −/− mice fed WD and exposed to SHS only (WD + SHS), with Btk inhibitor treatment (WD + SHS/Btk Inh.), or treated with endothelial cell targeted siRNA for MMP-9 (WD + SHS/MECA-32 siRNA MMP-9) showed reduced scores in treatment groups ( p < 0.05). Kruskal-Wallis ANOVA on ranks was used to assess statistical significance between groups ( p < 0.001) followed by post hoc testing with Dunn’s multiple comparison test for groups of interest, four to six animals per group were evaluated, with two groups of control animals combined. ( b ) Analysis of MLI in lungs of ApoE −/− mice fed WD and exposed to SHS or fed WD and exposed to SHS plus treatment with neutrophil targeted siRNA for MMP-9 (WD + SHS/Ly6G siRNA MMP-9) showed reduced scores in the treatment group ( p > 0.001). Student’s t -test was used to assess statistical significance, four mice per group were analyzed. * Significant difference detected.

Article Snippet: Treatment animals were injected intravenously [tail vein injection] with either Bruton’s tyrosine kinase inhibitor (BTK Inh) PCI-32765 (Selleck Chemicals, Houston, TX, USA) or siRNA specific for MMP-9 (Invitrogen, Carlsbad, CA, USA) conjugated with F(ab’) 2 fragments of anti-mouse neutrophil antibody (clone Ly-6G 1A8, Bio X Cell, West Lebanon, NH, USA) or anti-mouse endothelial cell antibody (clone MECA-32, Bio X Cell).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Disrupting the Btk Pathway Suppresses COPD-Like Lung Alterations in Atherosclerosis Prone ApoE −/− Mice Following Regular Exposure to Cigarette Smoke

doi: 10.3390/ijms19020343

Figure Lengend Snippet: Collagen deposition in lungs of ApoE −/− mice fed WD and exposed to SHS only (WD + SHS), with Btk inhibitor treatment (WD + SHS/Btk Inh.), or treated with endothelial cell targeted siRNA for MMP-9 (WD + SHS/MECA32 siRNA MMP-9). Picro-sirius red stained lung sections from ApoE −/− mice were viewed under regular light (left panels) or polarized light (right panels). Representative images are shown ( a ). Differences in hue of Picro-sirius red stained airways were evaluated using ImageJ software. Results are expressed as mean red component intensities (airway) or mean total intensity (vasculature) ( b ). Btk inhibition or EC targeted MMP-9 specific siRNA impacted collagen deposition in the airways or lung vasculature respectively. Kruskal-Wallis ANOVA on ranks was used to assess statistical significance in red intensity between groups ( p < 0.081). As Dunn’s multiple comparison test was not possible we assessed the reduction observed in the Btk inhibitor treated group using the non-parametric Mann-Whitney test with Bonferroni correction for multiple comparisons ( p = 0.019). For mean vascular intensity one way ANOVA was used to assess differences between groups ( p = 0.227) and as Fishers least significant difference test was not possible, we assessed the reduction observed in the MECA-32 siRNA MMP-9 treated group using Student’s t -test with Bonferroni correction for multiple comparison ( p = 0.097), four to six animals per group were evaluated, with two groups of control animals combined. * Significant difference detected.

Article Snippet: Treatment animals were injected intravenously [tail vein injection] with either Bruton’s tyrosine kinase inhibitor (BTK Inh) PCI-32765 (Selleck Chemicals, Houston, TX, USA) or siRNA specific for MMP-9 (Invitrogen, Carlsbad, CA, USA) conjugated with F(ab’) 2 fragments of anti-mouse neutrophil antibody (clone Ly-6G 1A8, Bio X Cell, West Lebanon, NH, USA) or anti-mouse endothelial cell antibody (clone MECA-32, Bio X Cell).

Techniques: Staining, Software, Inhibition, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Disrupting the Btk Pathway Suppresses COPD-Like Lung Alterations in Atherosclerosis Prone ApoE −/− Mice Following Regular Exposure to Cigarette Smoke

doi: 10.3390/ijms19020343

Figure Lengend Snippet: Levels of MMP-9 in lung homogenates ( a ). Western blot from ApoE −/− mice fed WD and exposed to SHS only (WD + SHS), with Btk inhibitor treatment (WD + SHS/Btk Inh.), or treated with endothelial cell targeted siRNA for MMP-9 (WD + SHS/MECA-32 siRNA MMP-9). Three mice per group were analyzed ( b ). Western Blot from ApoE −/− mice fed WD and exposed to SHS only (WD + SHS) or treated with neutrophil targeted siRNA (WD + SHS/Ly6G siRNA MMP-9). Four animals per group were analyzed.

Article Snippet: Treatment animals were injected intravenously [tail vein injection] with either Bruton’s tyrosine kinase inhibitor (BTK Inh) PCI-32765 (Selleck Chemicals, Houston, TX, USA) or siRNA specific for MMP-9 (Invitrogen, Carlsbad, CA, USA) conjugated with F(ab’) 2 fragments of anti-mouse neutrophil antibody (clone Ly-6G 1A8, Bio X Cell, West Lebanon, NH, USA) or anti-mouse endothelial cell antibody (clone MECA-32, Bio X Cell).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A–B) Protein levels of PV1, Cav1 and CD31 in the lung (A) and kidney (B) total membranes of Cav1−/−, Cav1+/− and WT mice were detected by immunoblotting. C) Protein levels of PV1, cavin-1 and CD31 in the lung total membranes of cavin-1−/− and WT mice were detected by immunoblotting. D) PV1 mRNA levels in the lung ( left panel ) and kidney ( right panel ) of WT, Cav1−/− and cavin-1−/− mice.

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) Protein levels of PV1 in MLEC-wt( WT ), MLEC-Cav1KO ( Cav1KO ) and MLEC-Cav1-ECRC ( ECRC ) cells detected by immunoblotting with anti-PV1 antibodies. M - Corresponds to membrane proteins, C – cytosolic proteins. Equal amount of membrane protein was loaded whereas the cytosolic proteins were normalized to membrane extract volume. The membrane and cytosolic proteins were also partially deglycosylated with PNGase F ( + ), which resulted in the drop in PV1 molecular weight. Note very low PV1 level in Cav1KO cells and increased PV1 protein level in cells reconstituted with Cav1 (Cav1-ECRC). The top and bottom panels are different exposures of the same blot. B) PV1 is predominantly associated with caveolae on the surface of lung endothelial cells. PV1 ( red ) colocalizes with Cav1-EGFP ( green ) at the plasma membrane of live MLEC, as shown by TIRFM. Insets demonstrate the extensive colocalization of the two labels ( white arrowheads ). Scale bars −20 µm.

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Western Blot, Membrane, Molecular Weight, Clinical Proteomics

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) PV1 mRNA levels in MLEC-wt ( WT ) and MLEC-Cav1KO ( Cav1KO ) cells measured by real time quantitative PCR. The data was obtained from quadruplicate samples and normalized to b-actin mRNA levels (ΔΔCt). Bars – SEM. B) Pulse 35 S metabolic labeling of MLEC-WT ( top panel ) and MLEC-Cav1KO ( bottom panel ) cells followed by PV1 immunoprecipitation at the indicated time points and 35 S fluorography. Duplicate samples are shown for each time point assessed. PV1 has four active N-glycosylation sites and therefore shows five bands, the lowest representing the non-glycosylated form and the four higher bands representing various degrees of N-glycosylation. C) Densitometric quantitation of the amount of PV1 translated after 10 min pulse with 35 S-methionine and cysteine in MLEC-WT and MLEC-Cav1KO cells. Error bars correspond to SEM (n = 3).

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Real-time Polymerase Chain Reaction, Labeling, Immunoprecipitation, Glycoproteomics, Quantitation Assay

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) Schematic of the timeline ( upper right ) and the principal steps of PV1 internalization flow cytometric assay ( right ). An example of data gating and fluorescence intensity histogram is given in the lower left panels. B) Amount of PV1 on the surface of MLEC-wt ( WT ) and MLEC-Cav1KO ( Cav1 KO) at t 0 expressed as median fluorescence intensity per cell from fluorophore-labeled anti-PV1 ( PV1 ). Labeling of cells with isotype control non-immune antibodies showed the level of unspecific binding ( control ) (error bars correspond to stdev, n = 4, *p<0.01). C) Amount of internalized PV1 at different time points in MLEC-WT at 37°C ( solid line ) and 4°C ( dashed line ) expressed as median fluorescence intensity per cell from fluorophore-labeled anti-PV1 ( PV1 ) (stdev, n = 6, *p<0.01). D) PV1 internalization in MLEC-WT ( WT, top panels ) and MLEC-Cav1KO ( Cav1 KO, bottom panels) cells at different time points, as detected by confocal microscopy. Images are maximum projections of confocal stacks in green channel (PV1, lower panels ) or green merged with blue (nuclei labeled with DAPI, upper panels ). E) Internalization rate of PV1 in MLEC-WT ( solid line, solid circles ) and Cav1KO ( dashed line, open circles ) cells, expressed as a percentage from the total amount of PV1 on the cell surface. (stdev, n = 4 per time point, *p<0.01). F) Degradation curves of 35 S labeled PV1 in MLEC-Cav1KO (Cav1KO, dashed line , open circles ) and MLEC-WT (WT, solid circles ), isolated from Cav1−/− and wild type mice, respectively. Data is representative of three experiments carried out in duplicate. G, H) PV1 degradation rates were measured in MLEC-WT (WT) and MLEC-Cav1KO (Cav1KO) treated with lysosomal or proteasomal inhibitors. G) Western blots used for densitometric quantifications of PV1 protein level. H) Quantitation of protein levels of PV1.

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Flow Cytometry, Fluorescence, Labeling, Control, Binding Assay, Confocal Microscopy, Isolation, Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

doi: 10.1371/journal.pone.0032655

Figure Lengend Snippet: A) PV1 does not colocalize with clathrin-GFP on the cell surface. Confocal micrographs of MLEC-WT transfected with clathrin-GFP ( Clathrin, green ) and labeled with fluorescent anti-PV1 antibodies ( PV1, red ). The insets represent a low power field with two transfected cells. The areas in shaded in grey are magnified in lowed panels. B–G) PV1 and transferrin internalization rates in MLECs were quantified by flow cytometry. Error bars correspond to StDev. B–D) Percentage of fluorescent antibody labeled PV1 internalized from the cell surface. B) PV1 internalization at 15 and 60 min in presence and absence of the clathrin pathway inhibitor PitStop2 ( PS2 ) or the inactive PitStop2 negative control ( NC ) (n = 4, p >0.05). C,D) PV1 internalization at 15 and 60 min in presence of dynamin inhibitors Dyngo-4a (C) (n = 4, p >0.05) or Dynasore ( D ) (n = 4, p>0.05). E) Median fluorescent intensity of transferrin-Alexa647 internalized within 10 min in the presence and absence of PitStop2, Dynasore or Dyngo4a (n = 4, * p <0.01). D–G) Internalization of PV1 (F) and transferrin (G) at 15 min in untransfected MLECs (mock) and MLECs transfected with eGFP-encoding plasmid (GFP), dynamin 2-eGFP fusion (Dyn2 wt) or dominant-negative form of dynamin 2 fused to eGFP (Dyn2 K44A MLECs (n = 4, * p <0.01). H) Schematic of PV1 ( green ) trafficking in ECs. De novo formed PV1 enters the secretory pathway and arrives at the cell surface by exocytosis ( green arrow ) using secretory vesicles (Step 1). On the plasma membrane PV1 is targeted to caveolae, fenestrae or TEC (Step 2) where it forms diaphragms. PV1 is internalized via clathrin- and dynamin-independent endocytic mechanism ( Step 3 and 4 ) followed by degradation in the lysosomes ( Step 5 ).

Article Snippet: Prior to the experiment the MLEC-wt and MLEC-Cav1KO cells were serum-starved (2 h, 37°C) in serum-free endothelial basal medium 2 (EBM2) (Lonza), followed by labeling (30 min, 10°C) with fluorophore coupled rat anti-mouse PV1 MECA-32 mAb (1.5 μg/ml) in EBM2 supplemented with 2% BSA.

Techniques: Transfection, Labeling, Flow Cytometry, Negative Control, Plasmid Preparation, Dominant Negative Mutation, Clinical Proteomics, Membrane