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

Journal: The Journal of Biological Chemistry

Article Title: An Immunogenic Peptide in the A-box of HMGB1 Protein Reverses Apoptosis-induced Tolerance through RAGE Receptor * ^♦

doi: 10.1074/jbc.M113.541474

Figure Lengend Snippet: A defined region in HMGB1 A-box binds to RAGE extracellular domains to reverse the tolerogenic program of DCs fed apoptotic cells. A, HMGB1 dependent reversal of tolerance does not require TLRs-3, -4, -6, and -9. The assay was carried out as in Fig. 2B using DCs from WT, Tlr3−/−, Tlr4−/−, Tlr6−/−, or Tlr9−/− mice. B, the HMGB1 A-box signals on DCs through RAGE to reverse tolerance. The assay was conducted as in Fig. 2B using DCs from Rage+/+ or Rage−/− mice. C, RAGE is composed of a short cytosolic tail (CT) involved in signal transduction, a transmembrane domain (TM), which anchors the protein to the cell membrane, two constant domains (C1 and C2), and a variable domain (V). sRAGE lacks the CT and TM domains. D, top, caspase-1-generated HMGB1 A-box fragment amino acid sequence and overlapping synthetic peptides used to map binding sites to RAGE. Bottom, representative SPR for recombinant A-box fragment (0–100 nm; 2-fold dilution series) and A-box synthetic peptides (0–10 μm; 2-fold dilution series) titrated over amine-coupled RAGE surfaces (∼250 RU each) in HBS-EP at 50 μl/min (1-min association + 3-min dissociation). Apparent equilibrium dissociation constants (KD) are indicated. N/A = no significant binding. E, A-box activity on reversal of tolerance correlates with a RAGE-binding region between amino acids 23–50. Overlapping synthetic A-box peptides (1 μg/ml) were assayed for activity in reversing tolerance as in Fig. 2B; recombinant HMGB1 was used as a positive control.

Article Snippet: Carrier-free, soluble RAGE was from Prospec (HEK sRAGE; PRO-601), Pierce Gentle Elution was from Thermo Scientific (21027), and detergents were from Anatrace (Tween 20 APT020 and Empigen D350); all other chemicals were reagent-grade quality.

Techniques: Transduction, Generated, Sequencing, Binding Assay, Recombinant, Activity Assay, Positive Control