Journal: The American Journal of Pathology

Article Title: Pulmonary Endothelial Protein Kinase C-Delta (PKCδ) Regulates Neutrophil Migration in Acute Lung Inflammation

doi: 10.1016/j.ajpath.2013.09.010

Figure Lengend Snippet: IT administration of PKCδ-TAT attenuates neutrophil migration into rat lung and attenuates ICAM-1 expression in sepsis-induced indirect lung injury. Immunohistochemical detection of MPO in representative lung tissue sections from 24 hours after surgery (n = 4 animals per group). In the sham surgery group, only a few MPO+ cells were seen in each field. In the CLP+PBS group, sepsis induced infiltration of numerous MPO+ cells throughout the lung parenchyma. Administration of the TAT-TAT control peptide had no significant effect on sepsis-induced influx of MPO+ cells into the parenchyma, compared with CLP+PBS vehicle. Administration of the inhibitory peptide led to a significant reduction of sepsis-induced MPO+ cell numbers in the lung. After primary antibody incubation, ICAM-1 was visualized using an Alexa Fluor 488–conjugated secondary antibody (green), with DAPI counterstaining (blue). Representative lung tissue sections from 24 hours after surgery are shown (n = 4 animals per group). In the sham surgery group, levels of ICAM-1 were barely detectable. In the CLP+PBS group, widespread and intense sepsis-induced ICAM-1 staining throughout the lung parenchyma was observed. In the CLP+TAT-TAT group, high levels of ICAM-1 were observed, with distribution similar to that of the CLP+PBS group. In the CLP+PKCδ-TAT group, marked reduction in sepsis-induced ICAM-1 expression was observed, with some small patches of staining seen in alveoli. Scale bars: 50 μm (left column); 100 μm (right column).

Article Snippet: The cells were incubated with 0.05 μg/mL anti-human ICAM-1 (clone 6.5B5; AbD Serotec), 0.25 μg/mL VCAM-1 (clone 51-10C9; BD Pharmingen, San Diego, CA), or 0.25 μg/mL PECAM-1 (clone P2B1; EMD Millipore) for 1 hour, washed, and incubated with goat anti-mouse IgG conjugated to HRP (1:1000) for 1 hour.

Techniques: Migration, Expressing, Immunohistochemical staining, Incubation, Staining

Journal: The American Journal of Pathology

Article Title: Pulmonary Endothelial Protein Kinase C-Delta (PKCδ) Regulates Neutrophil Migration in Acute Lung Inflammation

doi: 10.1016/j.ajpath.2013.09.010

Figure Lengend Snippet: IT administration of the PKCδ inhibitor attenuates sepsis-induced expression of the adhesion molecules ICAM-1 and VCAM-1 in the pulmonary endothelium. A: Immunohistochemical detection of ICAM-1 in representative lung tissue sections from 24 hours after surgery (minimum of n = 3 animals per group). After primary antibody incubation, ICAM-1 was visualized using HRP-conjugated secondary antibodies and the peroxidase substrate chromogen AEC, which produces a red reaction product. In the sham-surgery group, ICAM-1 staining was barely detectable, and lung architecture was normal. In the CLP+PBS group, robust ICAM-1 localization was observed throughout the distal lung tissue and in the large arteries accompanying the bronchial airways. In the CLP+PKCδ-TAT group, ICAM-1 staining was reduced to near the levels with sham surgery. B: Immunohistochemical detection of VCAM-1 in representative lung tissue sections from 24 hours after surgery (minimum of n = 3 animals per group). After primary antibody incubation, VCAM-1 was visualized using HRP-conjugated secondary antibodies and the peroxidase substrate chromogen AEC (red), which produces a red reaction product. B, top row: VCAM-1 staining was not detectable in the sham surgery group. A representative VCAM-1–positive small artery situated within the injured, inflamed parenchyma is shown for the CLP+PBS group; adhered leukocytes along the VCAM-1+ endothelium are shown at higher magnification in the inset. In the CLP+PKCδ-TAT image, VCAM-1 staining is absent in the endothelium of a similarly situated small artery in the distal parenchyma. B, bottom: Cropped views of venous and arterial localization of VCAM-1. Left to right: VCAM-1 staining in the venous endothelium (CLP+PBS); absence of VCAM-1 staining in the venous endothelium (CLP+PKCδ-TAT); a large pulmonary artery with VCAM-1+ endothelium and thickened underlying interstitium (CLP+PBS); and a large pulmonary artery with absent VCAM-1 staining in the endothelium, and with normal thickness of the underlying interstitium (CLP+PKCδ-TAT). Scale bars: 100 μm (A, top); 50 μm (A, bottom, and B, top). Original magnification, ×400 (B, bottom row). PA, pulmonary artery; PV, pulmonary vein.

Article Snippet: The cells were incubated with 0.05 μg/mL anti-human ICAM-1 (clone 6.5B5; AbD Serotec), 0.25 μg/mL VCAM-1 (clone 51-10C9; BD Pharmingen, San Diego, CA), or 0.25 μg/mL PECAM-1 (clone P2B1; EMD Millipore) for 1 hour, washed, and incubated with goat anti-mouse IgG conjugated to HRP (1:1000) for 1 hour.

Techniques: Expressing, Immunohistochemical staining, Incubation, Staining

Journal: The American Journal of Pathology

Article Title: Pulmonary Endothelial Protein Kinase C-Delta (PKCδ) Regulates Neutrophil Migration in Acute Lung Inflammation

doi: 10.1016/j.ajpath.2013.09.010

Figure Lengend Snippet: Role of PKCδ in PMVEC adhesion molecule expression. The expression of VCAM-1, ICAM-1, and PECAM-1 on PMVEC monolayers was determined by a cell-surface ELISA. Human PMVEC monolayers were treated overnight with buffer or IL-1β ± PKCδ inhibitor (0.01 to 5 mol/L). A: IL-1β–stimulated VCAM-1 expression. Constitutive expression of VCAM-1 (buffer) was normalized to 1, and expression in response to IL-1β treatment ± PKCδ inhibitor was compared with constitutive VCAM-1 expression. B: IL-1β–stimulated ICAM-1 expression, relative to constitutive ICAM-1 expression (as described for A). C: IL-1β–stimulated PECAM-1 expression, relative to constitutive PECAM-1 expression (as described for A). Data are expressed as means ± SEM. n = 7 (A); n = 5 (B); n = 6 (C). ∗∗P < 0.01, buffer versus IL-1β–treated PMVECs, †P < 0.01, IL-1β–treated PMVECs versus 10 U/mL IL-1β+PKCδ inhibitor (0.1–5 μmol/L) (A). ∗P < 0.05, IL-1β–treated PMVECs versus IL-1β+PKCδ inhibitor (0.1 μmol/L); ∗∗P < 0.01, IL-1β–treated PMVECs versus IL-1β+PKCδ inhibitor (1–5 μmol/L); and ∗∗∗P < 0.001, buffer versus IL-1β–treated PMVECs (B).

Article Snippet: The cells were incubated with 0.05 μg/mL anti-human ICAM-1 (clone 6.5B5; AbD Serotec), 0.25 μg/mL VCAM-1 (clone 51-10C9; BD Pharmingen, San Diego, CA), or 0.25 μg/mL PECAM-1 (clone P2B1; EMD Millipore) for 1 hour, washed, and incubated with goat anti-mouse IgG conjugated to HRP (1:1000) for 1 hour.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting RAGE Signaling in Diabetic Atherosclerosis

doi: 10.1371/journal.pone.0147839

Figure Lengend Snippet: ( A ) Cytoplasmic and nuclear fractions were prepared after 1 h of HG. The extents of IκB phosphorylation and nuclear translocation of NF-κB were determined by Western blotting. Representative images from at least three experiments are shown. CE, cytoplasmic extracts; NE, nuclear extracts. #,* Significant difference at the P < 0.025 based on post-hoc Mann-Whitney U test following a Kruskal-Wallis test (#, Control vs. HG; *, HG vs. HG + UDCA). Error bars: SEMs. ( B ) RNAs were extracted from cells after 8 h of HG, M (osmotic control) or TG exposure. The levels of mRNAs encoding VCAM-1, ICAM-1, and MCP-1 were determined by RT-PCR. Representative images from at least three experiments are shown. #,* Significant difference at the P < 0.025 based on post-hoc Mann-Whitney U test following a Kruskal-Wallis test (#, Control vs. HG/TG; *, HG/TG vs. HG/TG + UDCA). Error bars: SEMs. ( C ) Monocyte (U937) adhesion assay. Cells were exposed to HG for 24 h after pretreatment with UDCA. U937 cells were added to ECs and adherent monocytes counted in five random optical fields in each dish (magnification, X 100; scale bars, 100 μm). #,* Significant difference at the P < 0.025 based on post-hoc Mann-Whitney U test following a Kruskal-Wallis test (#, Control vs. HG; *, HG vs. HG + UDCA). Error bars: SEMs.

Article Snippet: Anti-ICAM-1 antibody was from SouthernBiotech (Birmingham, AL).

Techniques: Translocation Assay, Western Blot, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction, Cell Adhesion Assay

Journal: PLoS ONE

Article Title: Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting RAGE Signaling in Diabetic Atherosclerosis

doi: 10.1371/journal.pone.0147839

Figure Lengend Snippet: Diabetes was induced in mice via injection of STZ and the animals were fed a chow diet with or without 0.5% (w/v) UDCA. ( A ) Quantification of atherosclerotic lesions. Mouse aortas were isolated and stained with Oil red-O (CA, Carotid artery; DTA, Descending thoracic aorta). Plaque area proportions (% values) in aortas were quantified using the Image J software. * Significant difference at the P < 0.05 based on Mann-Whitney U test (STZ vs. STZ + UDCA). Error bars: SEMs. ( B and C ) Immunofluorescence staining of the aorta. ER stress markers (p-PERK, XBP-1, and ATF6) ( B ); adhesion molecules (VCAM-1 and ICAM-1); RAGE; and a macrophage marker (CD68) ( C ), are indicated by red signals. The green color is autofluorescence from elastic tissue in vessel walls. DAPI staining of nuclei is shown in blue; merged images are also presented. Representative images from at least three experiments are shown (magnification, ×100; scale bars, 1 mm).

Article Snippet: Anti-ICAM-1 antibody was from SouthernBiotech (Birmingham, AL).

Techniques: Injection, Isolation, Staining, Software, MANN-WHITNEY, Immunofluorescence, Marker