Pirfenidone Has Anti-fibrotic Effects in a Tissue-Engineered Model of Human Cardiac Fibrosis

Figures

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  Figure 1. A 3D co-culture of cardiac fibroblasts and iPS-derived cardiomyocytes in a GelMA hydrogel results in a functional cardiac tissue construct. (A) Schematic overview of the tissue engineering process. Primary cardiac fibroblasts and iPS-CM were suspended in a GelMA polymer solution and subsequently cross-linked by exposure to UV-light (wavelength 365 nm). The resulting cell-laden hydrogels were washed in PBS to remove uncrosslinked polymers and were then placed in culture medium in non-adhesive well plates for suspension culture. (B) Mechanical characterization of the hydrogels, showing the elastic modulus (in kPa) for different GelMA concentrations as measured by microindentation testing (n = 3 per condition). Statistical analysis was performed using a one-way ANOVA with Sidak's multiple comparison test. Data are represented as mean ± SEM. (C) Schematic overview of the outcomes of combined cell density and cell ratio optimization experiments. Synchronous contractions throughout the cardiac tissue constructs were only observed when a cell concentration of 28 or 50 million cells per milliliter was used, with a cardiomyocyte percentage of 70 or 80. In other conditions, individual…

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  Figure 2. TGF-β1 induces a fibrotic response in cardiac tissue constructs. (A) Timeline of the experiment. Cardiac tissue constructs were fabricated at day 0 and cultured until day 7. On day 7, experimental conditions were started. Two experimental conditions were created: TGF-β1 was added in the first group in a concentration of 2 ng/mL, the other group served as controls. On day 14, the constructs were harvested for analysis, consisting of RT-qPCR, immunofluorescence staining and targeted proteomics. (B) TGF-β1 stimulation results in an increased expression of fibrotic genes periostin (POSTN), alpha-smooth muscle actin (aSMA), collagen type 1 (COL1a1), and collagen type 3 (COL3), as demonstrated by RT-qPCR (n = 13). Data are represented as mean relative expression (compared to GAPDH) ± SEM. Statistical analysis was performed using paired two-tailed Student's t-test. (C,D) Immunofluorescence staining shows an increase in α-SMA (green) and periostin (red) expression in tissue-engineered cardiac constructs stimulated with TGF-β1 (n = 4). Scale bars, 50 μm. Statistical analysis was performed using paired two-tailed Student's t-test.

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  Figure 3. Targeted proteomics confirms the TGF-β1-induced fibrotic response in tissue-engineered cardiac constructs. TGF-β1 stimulation results in an increased expression of several fibrosis-related proteins. Data are represented as mean normalized protein expression (compared to internal control) per experiment (n = 12). Statistical analysis was performed using paired two-tailed Student's t-test.

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  Figure 4. Pirfenidone has distinct anti-fibrotic effects in cardiac tissue constructs. (A) Timeline of the experiment. Cardiac tissue constructs were fabricated at day 0 and cultured under regular circumstances until day 7. On day 7, experimental conditions were started. Four experimental conditions were created, in which TGF-β1 (2 ng/mL) and/or pirfenidone (1 mg/mL) were added to the culture medium and a group without TGF-β1 and pirfenidone served as controls. On day 14, the constructs were harvested for analysis, consisting of RT-qPCR, immunofluorescence staining and targeted proteomics. (B) Pirfenidone causes a decrease in periostin (POSTN), collagen type 1 (COL1a1) and collagen type 3 (COL3) expression in control conditions, but does not affect alpha-smooth muscle actin (aSMA) expression, as assessed with RT-qPCR (n = 13). Data are represented as mean relative expression (compared to GAPDH) ± SEM. Statistical analysis was performed using repeated measures two-way ANOVA. The interaction effect and the main effects are reported in the statistical box underneath the graph, the simple main effects are reported in the graph itself. (C,D) Immunofluorescence staining shows a decrease in…

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  Figure 5. Targeted proteomics reveals that pirfenidone does not undo all TGF-β1-induced pro-fibrotic effects. Pirfenidone causes a decrease in COL1a1 expression in both fibrotic and control conditions. In other fibrosis and heart failure related proteins, different effects can be seen. Data are represented as mean normalized protein expression (compared to internal control) per experiment (n = 7). Statistical analysis was performed using repeated measures two-way ANOVA. The interaction effect and the main effects are reported in the statistical box on the right side of the graph.

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  Figure 6. Proposed mechanism of action of pirfenidone. Schematic overview showing the proposed role of pirfenidone in CTC. Upregulation of pro-fibrotic gene expression through the canonical TGF-β pathway is blocked by pirfenidone, but upregulation of pro-fibrotic gene expression through the non-canonical pathway is still possible.