Figure 4.

Isolated collagen and Rap1a mediated AGE/RAGE-induced increase of α-SMA expression in cardiac fibroblasts wildtype for RAGE. Cardiac fibroblasts were isolated from (A) non-diabetic, (B) diabetic, (C) Rap1a KO (D) non-diabetic RKO, and (E) diabetic RKO mouse hearts. Cells were embedded into non-diabetic (black bar) or diabetic (grey bar) collagen matrices for 7 days and then used to isolate total protein. Quantification of Western blot analysis for α-SMA (42 kDa) was assessed and normalized to total protein (Brilliant Blue Coomassie Stain) in untreated, EPAC (100 µM), exogenous AGEs (0.05 mg/mL) and EPAC+exogenous AGEs (100 µM and 0.05 mg/mL, respectively) cardiac fibroblasts. (F) Representative Western blot images for each treatment and genotype (non-db = non-diabetic matrix and db matrix = diabetic matrix). Data are means ± SEM with n = 5–10. Statistical analysis consisted of a two-way ANOVA followed by a Fisher’s Least Significant Difference post hoc. Symbols above each bar indicate significance between a specific group (p < 0.05; * vs. untreated in non-db, + vs. untreated in db, # vs. EPAC in non-db, ◆ vs. EPAC in db, ▽ vs. AGE in non-db, ⊗ vs. AGE in db, and • vs. AGE+EPAC in non-db). Untreated treatment groups presented were initially presented in Figure 3B.