The Stiffness of Cardiac Fibroblast Substrates Exerts a Regulatory Influence on Collagen Metabolism via α2β1 Integrin, FAK and Src Kinases

Figures

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  Figure 1. FACS profile showing the expression of the α2 integrin subunit in cardiac fibroblasts cultured on soft (A) and stiff (B) substrate (right curve) compared to isotype controls (middle curve) and cellular autofluorescence controls (left curve).

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  Figure 2. Intracellular and extracellular collagen content in cardiac fibroblasts and cardiac fibroblast media cultured on soft and stiff gels (A). Relative expression of Col1A1 (B) and Col3A1 (C) genes in cardiac fibroblast cultures on soft and stiff substrate. Each value represents mean ±SD (* p < 0.05; *** p < 0.001).

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  Figure 3. Content of MMP-1 (A), MMP-2 (B), MMP-9 (C), TIMP-1 (D), TIMP-2 (E), TIMP-3 (F) and TIMP-4 (G) within cardiac fibroblast media cultured on soft and stiff gels. Each value represents mean ±SD (* p < 0.05; ** p < 0.01).

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  Figure 4. FACS profile showing the expression of the α2 integrin subunit in cardiac fibroblasts after silencing of the α2 integrin subunit (ITGA2; left curve), in cells treated with non-targeting siRNA (NT2; right curve) and in untreated controls (CTR; middle curve).

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  Figure 5. Total collagen content in cardiac fibroblasts cultured on soft (A) and stiff (C) gels after silencing of the α2 integrin subunit (ITGA2) in cells treated with non-targeting siRNA (NT) and in untreated controls (CTR). Effects of TCI 15 (α2β1 integrin inhibitor) applied at concentrations of 10−7 mol/L(TC-I 10−7) and 10−8 mol/L (TC-I 10−8), DMSO (TC-I 15 solvent) and untreated controls (CTR) on collagen content in cardiac fibroblasts cultured on soft (B) and stiff (D) gels. Relative expression of Col1A1 (E) and Col3A1 (F) genes in cardiac fibroblasts cultured on soft gel after administration of TC-I 15 at concentrations of 10−7 mol/L (TC-I 10−7), DMSO and untreated controls (CTR). Each value represents a mean ±SD (* p < 0.05; ** p < 0.01; *** p < 0.001).

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  Figure 6. Difference in the total and phosphorylated focal adhesion kinase (FAK) levels between cardiac cells cultured on soft and stiff gels (A). Total collagen content in cardiac fibroblasts after administration of FAK inhibitor 14 (FAK kinase inhibitor) at concentrations of 10−6 mol/L(FAKi 10−6) and 10−7 mol/L (FAKi 10−7) compared with untreated controls (CTR) (B). Relative expression of Col1A1 (C) and Col3A1 (D) genes in cardiac fibroblasts after administration of FAK inhibitor 14 at a concentration of 10−7 mol/L (FAKi 10−7) compared to untreated controls (CTR). Each value represents a mean ±SD (* p < 0.05; *** p < 0.001).

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  Figure 7. Differences in the levels of total and phosphorylated Src kinase (Src) in cardiac cells cultured on soft and stiff gels (A). Total collagen content in cardiac fibroblasts after administration of AZM 475,271 (Src kinase inhibitor) at concentrations of 10−7 mol/L (Src i 10−7) and 10−8 mol/L (Src i 10−8), DMSO (AZM 475,271 solvent) and untreated controls (CTR) (B). Relative expression of Col1A1 (C) and Col3A1 (D) genes in cardiac fibroblasts after administration of AZM 475,271 at a concentration of 10−7 mol/L (Src i 10−7) compared to DMSO and untreated controls (CTR). Each value represents a mean ± SD (* p < 0.05; *** p < 0.001).

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  Figure 8. Model of the role of α2β1 integrin in the regulation of collagen deposition in cardiac fibroblast cultures. Lower collagen deposition was observed in cardiac fibroblasts settled on stiff substrate. Accumulation of total collagen and expression of the α1 chain of procollagen type I are dependent on α2β1 integrin. In addition, on the stiff gel, a higher integrin-dependent signal measured as phosphorylated FAK and Src kinases was observed. The release of profibrotic interleukin-6 (IL-6) from cardiac fibroblasts was decreased in the stiff gel cultures [5]. This effect is also dependent on α2β1 integrin.