Integrin ανβ5 in vitro inhibition limits pro-fibrotic response in cardiac fibroblasts of spontaneously hypertensive rats

Figures

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  Fig. 1. Collagen deposition and TGF-β1 expression are higher in SHR than WKY rat cardiac tissue. a Representative images of Masson’s trichrome staining, TGF-β1 immunostaining and relative secondary antibody negative control, on normotensive (WKY) and hypertensive (SHR) rat heart tissue samples. Quantification of collagen fibres (b blue signal in Masson’s trichrome staining) and TGF-β1 posive areas (c brown signal in immunohistochemistry) in WKY (white bars) and SHR (red bars) left ventricle samples. All histological quantifications are expressed as mean ± SD, n = 5/group. Scale bars = 100 μm. d TGF-β1 expression levels in WKY and SHR heart tissue protein extracts. Western blot quantification data are expressed as mean ± SD after β-tubulin normalization, n = 5/group. Student’s t-test: *p < 0.05; ***p < 0.0001

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  Fig. 2. TGF-β1 treatment increases SMAD2/3 signaling activation and α-SMA expression levels more in SHR-CF than in WKY-CF. a Results obtained by ImageStreamX (IMX) technology, on SMAD2 nuclear translocation in WKY- and SHR-CF before and after 3 h of TGF-β1 treatment. IMX microscopy images were obtained on fixed and permeabilized CF, hybridized with anti-SMAD2-FITC antibody (green), and stained with DRAQ5 for the nucleus (red). Merged signal (yellow) indicates SMAD2 signal after nuclear translocation. b IMX fluorescence-activated cell sorting analysis on data collected from 10,000 events/sample. Bar graph indicates the similarity dilate index of WKY-CF (white bars) and SHR-CF (red bar), without and with TGF-β1 treatment. Scale bar = 10 μm. c Phospho-SMAD2/3 and total SMAD2/3 expression levels in WKY- and SHR-CF after 30 min of treatment with 5 ng/ml of recombinant TGF-β1. α-SMA protein (d) and gene (e) expression in WKY- and SHR-CF after treatment with 5 ng/ml TGF-β1. Western blot quantification data are expressed as mean ± SD after β-tubulin normalization, qRT-PCR data are expressed as fold ± SD normalized with GAPDH gene, n = 5/group. The experiments on cells were performed in…

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  Fig. 3. Integrin ανβ5 expression is higher both in SHR cardiac tissue and in SHR-CF than respective controls. a Integrin ανβ5 and ανβ3 expression levels in WKY (white bars) and SHR (red bars) heart tissue protein extracts. Western blot quantification data are expressed as mean ± SD after β-tubulin normalization, n = 5/group. Student’s t-test: *p < 0.05. b, c Integrin ανβ5 immunohistochemistry and relative secondary antibody negative control on normotensive (WKY) and hypertensive (SHR) rat heart sample (b). Integrin ανβ5 positive areas were quantified and normalized on whole sample area (c). Student’s t-test: *p < 0.05. Scale bar = 100 μm. Integrin ανβ5 protein (d) and itgav gene (e) expression in WKY- and SHR-CF after treatment with 5 ng/ml TGF-β1. Western blot quantification data are expressed as mean ± SD after β-tubulin normalization, qRT-PCR data are expressed as fold ± SD normalized with GAPDH, n = 5/group. The experiments on cells were performed in triplicate. 2-way ANOVA with Bonferroni’s post-test: *p < 0.05

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  Fig. 4. Integrin ανβ5 blockade in vitro decreases TGF-β1 release and protein production of typical MyoFB markers. a Secreted amount of active TGF-β1 in conditioned media of WKY- and SHR-CF after treatments with 0.5 μM cilengitide. ELISA data are expressed as mean ± SD, n = 5/group. The experiments on cell surnatants were performed in triplicate. Protein expression of integrin ανβ5 (b) and collagen I (c) in WKY- and SHR-CF after treatments with 5 ng/ml TGF-β1, TGF-β1 + 0.5 μM cilengitide, or cilengitide. d Representative images of immunofluorescence performed for α-SMA on WKY- and SHR-CF. Scale bar = 100 μm. e Protein expression of α-SMA in WKY- and SHR-CF after treatments with 5 ng/ml TGF-β1, TGF-β1 + 0.5 μM cilengitide, or cilengitide. All Western blot quantification data are expressed as mean ± SD after β-tubulin normalization. The experiments on cells were performed in triplicate, with a n = 5/group. 2-way ANOVA with Bonferroni’s post-test: *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 5. In vitro inhibition of integrin ανβ5 by cilengitide limits ανβ5 and collagen I expression more effectively on SHR-CF growing on a substrate with high stiffness. a Representative Western blot images of ανβ5 and collagen I protein expression in WKY- and SHR-CF cultured on substrates with two different stiffness (high and low) and contemporary treated with 5 ng/ml TGF-β1, TGF-β1 + 0.5 μM cilengitide, or cilengitide. All Western blot quantification data (b and c) are expressed as mean ± SD after β-tubulin normalization. The experiments on cells were performed in triplicate, with a n = 5/group. 2-way ANOVA with Bonferroni’s post-test: *p < 0.05, **p < 0.01, ***p < 0.001