Figure 1.
Characterization and expression of RLX receptor on primary neonatal cardiac fibroblasts.
A) Representative contrast phase microscopy images of first passage neonatal murine neonatal cardiac fibroblasts. B) Representative superimposed differential interference contrast (DIC) and confocal immunofluorescence images of neonatal cardiac fibroblasts immunostained with antibodies against vimentin (green). Nuclei are counterstained in red with propidium iodide. C) Expression of Relaxin family peptide receptor 1 (RXFP1) in neonatal cardiac fibroblasts and NIH/3T3 at mRNA level determined by RT-PCR, and protein level evaluated by Western blotting analysis. The images are representative of at least three independent experiments with similar results.
Figure 2.
Relaxin attenuates TGF-β1 induced cytoskeletal assembly in NIH/3T3 and primary neonatal cardiac fibroblasts.
Representative confocal immunofluorescence images of (A–D) NIH/3T3 cells stained with TRITC-phalloidin to reveal F-actin and of (E–H) primary cardiac fibroblasts stained with TRITC-phalloidin (red) and anti-vinculin antibody (green) to detect focal adhesions, cultured for 48 h in the indicated experimental conditions. I, J) Densitometric analyses of the intensity of F-actin and vinculin fluorescence signals performed on digitized images of neonatal cardiac fibroblasts. The images are representative of at least three independent experiments with similar results. Significance of differences: *p<0.05 vs control, °p<0.05 vs TGF-β.
Figure 3.
Relaxin reduces α–sma and type I collagen expression in TGF-β1 treated NIH/3T3 and primary neonatal cardiac fibroblasts.
A,B, E,F) Representative confocal immunofluorescence images of NIH/3T3 cells (A,B) and primary cardiac fibroblasts (E, F) cultured in the indicated experimental conditions and immunostained with antibodies against α–sma (A,E; green) or type I collagen (B, F, red). Nuclei are labeled (A,E) with propidium iodide (red) or with (B,F) Syto16 (green). The histograms show the corresponding densitometric analyses of the intensity of α–sma and type I collagen fluorescence signals. C,D) Western blotting analyses of the expression of α–sma and type I collagen proteins in neonatal cardiac fibroblasts. The densitometric analysis of the bands normalized to GAPDH is reported in the histograms. Data are representative of at least three independent experiments with similar results. Significance of differences: *p<0.05 vs control, °p<0.05 vs TGF-β1.
Figure 4.
Relaxin prevents TGF-β1 induced down regulation of MMP-2 and MMP-9 expression and up-regulation of TIMP-2 in NIH/3T3 and primary neonatal cardiac fibroblasts.
A–C, F–H). Representative confocal immunofluorescence images of NIH/3T3 cells (A–C) and primary neonatal cardiac fibroblasts (F–H) cultured in the indicated experimental conditions and immunostained with antibodies against MMP-2 (A,F; cyan), MMP-9 (B,G; green) or TIMP-2 (C,H, green). In B and G the nuclei are labeled in red with propidium iodide. The histograms show the corresponding densitometric analyses of the intensity of MMP-2, MMP-9 and TIMP-2 fluorescence signals. D–E) Western blotting analyses of the expression of (D) MMP-2, and (E) MMP-9 proteins in neonatal cardiac fibroblasts. In the histograms the densitometric analyses of the bands normalized to GAPDH are reported. Data are representative of at least three independent experiments with similar results. Significance of differences: *p<0.05 vs control, °p<0.05 vs TGF-β1.
Figure 5.
Relaxin prevents the TGF-β1-induced down-regulation of Notch-1 pathway in primary neonatal cardiac fibroblasts.
A) RT-PCR of Notch-1 expression. B,C) Western blotting analysis of activated intracellular form of Notch-1 (Notch-ICD, B) ) and of Notch-1 ligand, Jagged-1(C). The densitometric analyses of the bands normalized to GAPDH are reported in the histograms. D) Confocal immunofluorescence analysis of Notch-1 (green) and Hes-1 (cyan) expression. For the analysis of Notch-1, the cells were stained with a specific antibody recognizing both the membrane Notch-1 receptor and its activated intracellular form, Notch-ICD. Densitometric analyses of Notch-ICD and Hes-1 fluorescent signals are reported in the corresponding histogram. Significance of differences: *p<0.05 vs control, °p<0.05 vs TGF-β1.
Figure 6.
RLX and Notch-1 negatively regulates TGF-β1-induced fibroblast-myofibroblast transition in cardiac fibroblasts.
Neonatal cardiac fibroblasts were cultured for 48 h and treated as indicated. A) Western blotting analysis of NICD expression in the absence (control) or presence of DAPT (5 µM) a pharmacological γ-secretase inhibitor, used to block the generation of NICD. B) Western Blotting analysis of α–sma and MMP-2 expression in the cells treated with DAPT. C) Representative confocal immunofluorescence images cardiac fibroblasts treated with DAPT, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. D) Western blotting analysis of Jagged-1 expression in control cells, cells transfected with non specific scrambled-siRNA (SCR-siRNA) or silenced for the expression of Notch-1 ligand, Jagged-1, by specific Jagged-1 siRNA (Jagged-1 siRNA). E) Western Blotting analysis of α–sma in Jagged-1 silenced cells. F) Representative confocal immunofluorescence images cardiac fibroblasts silenced for Jagged-1 expression, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. The densitometric analyses of the bands normalized to GAPDH are reported in histograms in A–E; the densitometric analyses α–sma fluorescent signal are shown in the histograms in and C, F. Significance of differences: *p<0.05 vs control, δp<0.05 vs SCR-siRNA, °p<0.05 vs TGF-β1, #p<0.05 vs TGF-β1+ RLX.
Figure 7.
RLX and Notch-1 negatively regulates TGF-β1 signaling in primary neonatal cardiac fibroblasts.
(A) Western blotting analysis of Smad3 expression and (B) phosphorylation (performed on the immunoprecipitated Smad3 proteins) in cardiac fibroblasts cultured for 48 h in the indicated experimental conditions. Data are representative of at least three independent experiments. Histograms show the densitometric analyses of the bands normalized to (A) GAPDH and (B) total Smad3. Significance of differences: *p<0.05 vs TGF-β1.