Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Involvement of the coupled RAGE ligand/receptor in human corneal epithelial (HCE) cell wound healing. Representative images of scratch assays performed on HCE cells (left panel) treated with HMGB1 (high mobility group box 1, 100 ng/mL) ( A ) or AGEs (advanced glycation end products) (10/100/200 µg/mL) (B , C ). Percentage of residual wound area (right panel) after treatment with HMGB1 (100 ng/mL) ( A ) or AGEs (10/100/200 µg/mL) ( B , C ), compared with 0 hours and standardized to the untreated condition (100%) (n = 5 experiments, each conducted in duplicate). ( D ) Representative images of scratch assays performed on HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) or siRNA control (Scramble) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) (left panel). Percentage of the residual wound area of HCE cells transfected with siRNA against RAGE (100 nM) for 36 hours and then treated with AGEs (100 µg/mL), compared with 0 h (right panel) (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney test after a nonparametric ANOVA analysis; * P < 0.05; ** P < 0.01; *** P < 0.005; ns: not significant.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Transfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Functionality of the RAGE pathway. Characterization of the RAGE ( A ) mRNA and ( B ) protein expression in human cornea, primary human epithelial cells (mRNA only), and the HCE cell line (mRNA and protein) evaluated by ( A ) RT-PCR, ( B ) immunofluorescence, and ( B ) western blotting. For RT-PCR, negative controls (NC) were performed ( A ) without cDNA. ( B , left panel) Representative images of RAGE expression ( green ) in human corneas (top panel) and HCE cells (bottom panel). Nuclei were stained with Hoechst ( blue ); NC (left) were obtained by incubating HCE cells without primary antibody. ( B , right panel) Western blot experiments identified the RAGE protein at the described molecular weight (46 kDa). ( C ) Functionality of the NF-κB pathway (by luciferase reporter gene activity) was measured after treatment of HCE cells with AGEs (100 µg/mL) for 45 minutes (n = 5 experiments, each conducted in duplicate) (right panel). Positive controls (T+) were obtained by co-transfection with pMEKK (n = 5 experiments, each conducted in duplicate) (left panel). Each bar graph shows mean ± SEM. Mann-Whitney; * P < 0.05.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Staining, Molecular Weight, Luciferase, Activity Assay, Cotransfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Cx43 expression is induced by the AGEs/RAGE axis in HCE cells. Characterization of Cx43 protein expression ( arrow ) by immunostaining (bottom panel). Cells incubated without primary antibody served as a negative control (NC). ( B ) Relative quantification of Cx43 mRNA expression in untreated HCE cells 0, 6, and 24 hours after scratch wounding. Results were expressed as a ratio of the 0-hour condition (n = 5 experiments, each conducted in duplicate). ( C ) Quantification of Cx43 mRNA expression in HCE cells treated with AGEs (100 µg/mL) for 6 hours or 24 hours without scratch wounding (left panel) or with scratch wounding (right panel). Cells not treated with AGEs served as a control. Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate). (Left panel) Quantification of Cx43 mRNA expression in HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) for 6 hours. Results were expressed as a ratio of the scrambled siRNA condition (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney after a nonparametric ANOVA analysis; * P < 0.05; ns: not significant. ( D ) Relative quantification of Cx43 protein expression in HCE cells following treatment with AGEs (100 µg/mL) after scratch wounding. Untreated cells served as a control. Results were expressed as a ratio of the 0-hour, unwounded condition (n = 5 experiments) (top panel). Quantification of Cx43 protein expression in HCE cells treated with AGEs (100 µg/mL) for 12 and 24 hours after scratch wounding (bottom panel). Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate) (** P < 0.01). (E) Characterization by immunostaining of Cx43 protein expression according to the distance from the wound, time, and treatment with AGEs (100 µg/mL). Staining of Cx43 protein expression in HCE cells treated near the wound margins (left panel) and behind the wound (right panel). Cells not treated with AGEs (100 µg/mL) served as a control. Cells incubated without primary antibody served as a negative control (NC).

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Immunostaining, Incubation, Negative Control, Transfection, MANN-WHITNEY, Staining

Journal: Frontiers in Immunology

Article Title: DNA-Mediated Interferon Signature Induction by SLE Serum Occurs in Monocytes Through Two Pathways: A Mechanism to Inhibit Both Pathways

doi: 10.3389/fimmu.2018.02824

Figure Lengend Snippet: ISG induction by SLE serum requires either RAGE or FcRlla. (A) Primary human monocytes were incubated with serum from different SLE patients, B2, M91, B31, F72, or control sera, for 4 h. ISG expression was evaluated using qPCR. Data plotted represents fold induction for each gene relative to the sample incubated with control serum and is representative of 30 samples tested. (B) RAGE and FcRlla levels in monocytes treated with RAGE or FcRIIa siRNA, assessed by qPCR. (C) Monocytes were transfected with RAGE, FcRlla, or control siRNA, prior to incubation with SLE serum for 4 h. For each sample (control, RAGE, or FcRlla siRNA transfected cells) fold induction of ISGs was calculated as the ratio of gene expression between treated and untreated cells. (D) Primary human monocytes were treated with SLE serum, with or without sRAGE or monoclonal anti-FcRlla antibody for 4 h. ISG expression was analyzed by qPCR. Results in each case (B–D) indicate mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human recombinant sRAGE was purchased from ProSpec (East Brunswick NJ, USA).

Techniques: Incubation, Control, Expressing, Transfection, Gene Expression

Journal: Frontiers in Immunology

Article Title: DNA-Mediated Interferon Signature Induction by SLE Serum Occurs in Monocytes Through Two Pathways: A Mechanism to Inhibit Both Pathways

doi: 10.3389/fimmu.2018.02824

Figure Lengend Snippet: (A) Biacore T200 analysis of DWEYS inhibition of HMGB1-RAGE binding. sRAGE was immobilized on a CM5 sensor chip, HMGB1 with or without varying concentrations of DWEYS were added as analytes. IC50 was 34.7 μM. (B) Inhibition curve used for IC50 evaluation as noted in the methods section. (C,D) Human monocytes were incubated with labeled HMGB1 (red) alone ( C , top panel) or HMGB1 and DWEYS ( C , middle panel) or HMGB1 and control peptide ( C , bottom panel) for 20 min. Cells were counterstained with DAPI and imaged using a confocal laser scanning microscope. (D) Indicates adjusted fluorescence intensity for three experiments. (E) Primary human monocytes were incubated with HMGB1 and with or without DWEYS or control peptide for 4 h. lSG expression was evaluated using qPCR. Results indicate mean ± SD of three independent experiments. * p < 0.05.

Article Snippet: Human recombinant sRAGE was purchased from ProSpec (East Brunswick NJ, USA).

Techniques: Inhibition, Binding Assay, Incubation, Labeling, Control, Laser-Scanning Microscopy, Fluorescence, Expressing

Journal: Cells

Article Title: Replacement of Lost Substance P Reduces Fibrosis in the Diabetic Heart by Preventing Adverse Fibroblast and Macrophage Phenotype Changes

doi: 10.3390/cells10102659

Figure Lengend Snippet: SP reduces RAGE and oxidative stress while increasing nitric oxide in isolated cardiac fibroblasts in response to high glucose. ( A ) Receptor for advanced glycation end product (RAGE) in cardiac fibroblast lysates; ( B ) soluble RAGE (sRAGE); and ( C ) hydrogen peroxide (H 2 O 2 ) in the culture media of isolated cardiac fibroblasts in response to normal glucose (control, 5 mM), high glucose (HG, 25 mM), and HG with increasing concentrations of SP (10 to 1000 nM); ( D ) superoxide dismutase levels in cardiac fibroblast lysates in response to control, HG, and HG with increasing concentrations of SP; ( E ) total nitrate/nitrite as a marker of nitric oxide (NO) production in the culture media of isolated cardiac fibroblasts in response to control, HG, and HG with increasing concentrations of SP. SP alters cytokine production by isolated cardiac fibroblasts in response to high glucose. ( F ) TNF-α, ( G ) CCL2, ( H ) IL-4, ( I ) IL-6, and ( J ) IL-10 levels in isolated cardiac fibroblast cell culture media in response to normal glucose (control, 5 mM), high glucose (HG, 25 mM), and HG with increasing concentrations of SP (10 to 1000 nM). Data are expressed as mean ± SEM and were analyzed by one-way ANOVA with Tukey post hoc test; * p < 0.05 vs. control, ** p < 0.01 vs. control, **** p < 0.0001 vs. control, † p < 0.05 vs. HG, †† p < 0.01 vs. HG, ††† p < 0.001 vs. HG. n = 4–6 for control and n = 5–6 for all other groups.

Article Snippet: Lysyl oxidase (LOX, MyBioSource, San Diego, CA, USA), bone morphogenic protein 1 (BMP-1, Novus Biologicals, Littleton, CO, USA), soluble RAGE (sRAGE, MyBioSource, San Diego, CA, USA), hydrogen peroxide (H 2 O 2, Cell BioLabs Inc, San Diego, CA, USA), superoxide dismutase (Cell BioLabs Inc, San Diego, CA, USA), and nitrate/nitrite (nitric oxide, NO, Cell BioLabs Inc, San Diego, CA, USA) were measured in fibroblast media.

Techniques: Isolation, Marker, Cell Culture