Characterization Of Pirfenidone Effects In Human Cardiac Fibroblasts
by Friederike Meyer
Date of Examination:2024-04-04
Date of issue:2024-04-08
Advisor:Prof. Dr. Susanne Lutz
Referee:Dr. Ivan Prof Bogeski
Referee:Prof. Dr. Thomas Meyer
Persistent Address:
http://dx.doi.org/10.53846/goediss-10419
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Abstract
English
Background Cardiac fibrosis is a major problem in heart diseases, but it is still lacking an effective therapy. The drug pirfenidone has shown several anti-fibrotic effects in animal models of heart diseases and is already approved for the treatment of idiopathic pulmonary fibrosis. This led to the hypothesis that pirfenidone could have beneficial effects as an anti-fibrotic treatment of the human diseased heart; however, data on its effect in human cardiac fibroblasts are still missing. Methods General effects were studied in 2D cultures of human cardiac fibroblasts (hCF). Cells were treated with four different concentrations of pirfenidone (0.1, 0.3, 1.0, 3.0mg/ml). Cell number was assessed on day 0, 2 and 4 by staining with DAPI and automatic counting. The experiment was repeated with an immortalized human embryonic kidney cell line (tsA201) to assess cell specificity. Cytotoxic effects were investigated in live stained cells after 48h of pirfenidone treatment (0.1, 0.3, 1.0mg/ml) using propidium iodide and Hoechst33342. Furthermore, cells were treated with pirfenidone and afterwards incubated with 5ng/ml of TGF-β. Changes in protein phosphorylation were detected by immunoblot analysis. Engineered connective tissues (ECT) were prepared from human ventricular cardiac fibroblasts and collagen 1. Tissues were casted in moulds with flexible poles in a 48-well plate and in rigid casting moulds. Treatment of the ECT with pirfenidone (0.3, 1.0mg/ml) was carried out for 5 days. On day 5 tissue compaction and contraction were assessed by measuring the cross-sectional area and pole deflection, respectively. Viscoelastic properties were detected by destructive tensile strength measurements. Cells were re-isolated for quantifying cell number and viability by electrical current exclusion. Cell cycle was analyzed with flow cytometry. Protein expression was assessed by immunoblot analysis. Results With lower pirfenidone concentrations (0.1, 0.3mg/ml), a decrease in the proliferation rate of the hCF was observed. Concentrations above 1.0mg/ml; however, led to a substantial cell loss. The IC50 of pirfenidone on the decline in cell number was 0.4309mg/ml. In contrast, tsA201 cells were not affected by lower pirfenidone concentrations, but similar cytotoxic effects, as reflected by the IC50 of 0.7123mg/ml, were observed with concentrations above 1.0mg/ml. Treatment of confluent hCF cultures with pirfenidone demonstrated that the cytotoxic effect is dependent on cell confluency. Immunoblot analysis revealed that pirfenidone reduces the phosphorylation of ERK1/2 already under basal conditions and SMAD2 phosphorylation in the presence of TGF-β. Pirfenidone decreased the compaction and contraction of ECT and rheological analysis demonstrated that the tissues were significantly less stiff. The effects were stronger when rigid casting moulds were used instead of flexible moulds. This might be explained by the more pronounced myofibroblastic phenotype of the cells in the rigid model. The cell number and viability were only reduced in the groups with the highest treatment in both models. As a general effect, cell survival was better in tissues casted in rigid moulds compared to ones in the flexible moulds. Summary and conclusion We showed that pirfenidone displays anti-fibrotic properties in 2D and 3D cultures of human cardiac fibroblasts. Higher concentrations of the drug; however, displayed cytotoxic side effects independent of the cell type.
Keywords: Pirfenidone; Cardiac fibroblasts; Cardiac fibrosis; tissue Engineering; TGF-beta
