Soluble Receptor for Advanced Glycation End-products regulates age-associated Cardiac Fibrosis

Figure 1. RAGE deficiency accelerates age-dependent LV dimension changes without affecting LV function. Echocardiography was performed on WT and Rage-/- mice aged of 2.5 (Young), 12 (Middle-age, MA) and 21 months (Old) to measure Left Ventricle (LV) end-diastolic (LVEDV) and end-systolic (LVESV) volumes, LV end-diastolic (LVEDD) and end-systolic (LVESD) diameters, LV ejection fraction (LVEF), LV fractional shortening (LVFS), Stroke Volume, and LV posterior wall in diastole (LVPW, d). Young WT n=15, Young Rage-/- n=15, MA WT n=18, MA Rage-/- n=21, Old WT n=14, Old Rage-/- n=15. Each dot represents a mouse; mean ± SD are shown (*, P<0.05; **, P<0.01; ***, P<0.001; 2-way ANOVA plus Bonferroni post-hoc test for multiple comparisons).

Figure 2. RAGE deficiency promotes the expression of HF marker genes and accelerates age-associated cardiac fibrosis. (A) Relative expression of BNP (Nppb) and ANKRD1 (Ankrd1) genes. Young WT n=4, Young Rage-/- n=4-5, Middle-age (MA) WT n=4, MA Rage-/- n=4, Old WT n=5, Old Rage-/- n=6. (B) (Left panel) Representative images of LV sections stained with an antibody against Collagen I. Bar, 20 µm. (Right panel) Quantification of Collagen I signal. Young WT n=5, Young Rage-/- n=5, MA WT n=4, MA Rage-/- n=4, Old WT n=4, Old Rage-/- n=5. (C) (Left panel) Representative images of LV sections stained with Masson's trichrome. Bar, 20 µm. (Right panel) Quantification of Masson's trichrome signal. Young WT n=5, Young Rage-/- n=5, MA WT n=4, MA Rage-/- n=4, Old WT n=4, Old Rage-/- n=5. (D) (Left panel) Representative images of LV sections stained with an antibody anti-α-SMA. Bar, 20 µm. (Right panel) Quantification of α-SMA signal. Young WT n=5, Young Rage-/- n=5, MA WT n=4, MA Rage-/- n=4, Old WT n=4, Old Rage-/- n=5. Each dot represents a mouse; mean ± SD are shown (*, P<0.05; **, P<0.01; ***, P<0.001; 2-way ANOVA plus Bonferroni post-hoc test for multiple comparisons).

Figure 3. RAGE deficiency induces age-associated TGF-β signaling. (A) Relative expression of Tgfb1gene. Young WT n=4, Young Rage-/- n=4-5, Middle-age (MA) WT n=4, MA Rage-/- n=4, Old WT n=5, Old Rage-/- n=6. (B) (Left panel) Representative images of LV sections stained with an anti-Smad2-3 antibody. Bar, 20 µm. (Right panel) Quantification of Smad2/3 signal. Young WT n=5, Young Rage-/- n=5, MA WT n=4, MA Rage-/- n=4, Old WT n=4, Old Rage-/- n=5. (C) (Left panel) Representative images of LV sections stained with an anti-TFGbetaR1 antibody. Bar, 20 µm. (Right panel) Quantification of TFGbetaR1 signal. Young WT n=5, Young Rage-/- n=5, MA WT n=4, MA Rage-/- n=4, Old WT n=4, Old Rage-/- n=5. Each dot represents a mouse; mean ± SD are shown (*, P<0.05; **, P<0.01; ***, P<0.001; 2-way ANOVA plus Bonferroni post-hoc test for multiple comparisons).

Figure 4. Circulating sRAGE decreases with age in mice. (A, B) RAGE isoforms are not detectable in LV tissue. (A) mRNA levels of mRAGE or mRAGE+mRAGE_v4 isoforms in LV or lung (positive control) of indicated WT age groups. (B) Forty µg of LV protein lysate of the indicated age groups of WT mice were probed with an antibody α-RAGE (RAGE N-term). The same amount of LV from a Young Rage-/- mouse (KO) was used as negative control, and 10 µg of lung protein lysate of a WT mouse (L) as positive control for RAGE expression. β-tubulin was used as loading control (n=3-4 per group). (C) Quantification of sRAGE in the serum of the indicated age groups of WT mice; Young n=17, MA n=17 and Old n=18. (D) (Left panel) Ten µg of lung protein lysate of the indicated age groups of WT mice were probed with an antibody against RAGE (RAGE N-term) and a long and a short exposure are shown. Ten µg of lung protein lysate from a Young Rage-/- mouse (KO) was used as negative control. α-actin was used as loading control. (Right panel) Quantification of RAGE isoforms in the lung protein lysate of the indicated age groups of WT mice (n=3-5 per group). Each dot represents a mouse; mean ± SD are shown (*, P<0.05; **,…

Figure 5. Recombinant sRAGE reduces pro-fibrotic activity and proliferation of hcFbs. (A-C) HcFbs were stimulated with vehicle (Ctrl) or the indicated concentrations of sRAGE for 24 or 48 h. TGF-β1 (10 ng/ml) was used as positive control. (A) (Left panel) Representative images of α-SMA protein expression (green). Nuclei were stained with Hoechst (blue). Bar, 20 μm. (Right panel) Quantification of α-SMA expression. (B) (Left panel) Representative images of Collagen I expression (green). Nuclei were stained with Hoechst (blue). Bar, 20 μm. (Right panel) Quantification of Collagen I expression. (C) Collagens released in hcFbs supernatant. (D) Cell cycle distribution of hcFbs stimulated for 72 h with the indicated concentration of sRAGE or TGF-β1 (10 ng/ml). (E) (Left panel) Proliferation of hcFbs treated with indicated concentrations of sRAGE and TGF-β1 (10 ng/ml) recorded every 2 h; statistical differences were calculated 84 h after treatment. (Right panel) Slope of cell proliferation from 20 to 50 h. Each dot represents a biological replicate; mean ± SD are shown (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; 1-way ANOVA plus Bonferroni post-hoc test for multiple comparisons between…

Figure 6. Recombinant sRAGE reduces TGFbetaR1 expression and inhibits Smad2/3 signaling in hcFbs. HcFbs were stimulated with nothing (Ctrl) or the indicated concentrations of sRAGE for 4, 8 or 12 h. TGF-β1 (10 ng/ml) was used as positive control when indicated. (A) TGF-β1 released in the supernatant of cells determined by ELISA; n=6. (B) TFGbetaR1 expression determined by FACS analysis; n=3-8. (C) Quantification of P-Smad2-3 expression determined by FACS analysis 4 h after stimulation; n=3. (D) (Left panel) Representative immunofluorescence images for P-Smad2-3 (green) 4 h after stimulation; nuclei were stained with Hoechst (blue). Bar, 50 μm. (Right panel) Quantification of nuclear P-Smad2-3 expression; n=3 Each dot represents a biological replicate; mean ± SD are shown (*, P<0.05; **, P<0.01; ***, P<0.001; t-test or 1-way ANOVA plus Bonferroni post-hoc test for multiple comparisons between Ctrl and sRAGE at the same time point; dotted line: t-test between Ctrl and TGF-β1).

Figure 7. Recombinant sRAGE reduces fibrosis and TGFbetaR1 expression in vivo. MA WT mice were injected daily with about 22 μg of recombinant murine sRAGE (sRAGE) for 8 days or equal volume of physiological salt solution (CTRL). (A) (Left panel) Representative images of LV sections stained with Masson's trichrome. Bar, 20 µm. (Right panel) Quantification of Masson's trichrome signal. CTRL n=4, sRAGE n=5. (B) (Left panel) Representative images of LV sections stained with an antibody anti-α-SMA. Bar, 20 µm. (Right panel) Quantification of interstitial α-SMA signal. (C) (Left panel) Representative images of LV sections stained with an anti-TFGbetaR1 antibody. Bar, 20 µm. (Right panel) Quantification of TFGbetaR1 signal. (D) (Left panel) Western blot analysis of 35 µg of LV protein lysate probed with an antibody αTGFbR1. Red Ponceau staining (Ponceau S) was used as loading control. (Right panel) Quantification of TGFbR1 expression in the LV protein lysate. CTRL n=4, sRAGE n=5. Each dot represents a mouse; mean ± SD are shown (**, P<0.01; ***, P<0.001; t test).