Cardiac fibroblast phenotype regulation in engineered connective tissues with a special focus on ER adaptation and stress

by Alisa DeGrave
Doctoral thesis
Date of Examination:2022-12-15
Date of issue:2023-03-23
Advisor:Prof. Dr. Susanne Lutz
Referee:Prof. Dr. Susanne Lutz
Referee:Prof. Dr. Ivan Bogeski
Referee:Prof. Dr. Michael Zeisberg
Referee:Prof. Dr. Ralf Dressel
Referee:Prof. Dr. Thomas Meyer
Referee:Prof. Dr. Tiago F. Outeiro
Persistent Address: http://dx.doi.org/10.53846/goediss-9797

 

 

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Abstract

English

Cardiovascular diseases cause adverse myocardial remodeling due to cardiomyocyte loss and modifications to the extracellular matrix (ECM), which can ultimately lead to contractile dysfunction and pathological cardiac fibrosis. Cardiac fibroblasts (CF) are the main drivers of progressive cardiac fibrosis once they transition into myofibroblasts upon injury, in which they demonstrate an enhanced ECM secretory activity. Understanding the behavior of human CF is crucial for developing urgently needed anti-fibrotic drugs, and tissue engineering models represent a good compromise between complex in vivo models and simplified 2D cultures for the necessary investigations. In this context, our group has established a dynamic dual engineered connective tissue (ECT) model with the use of normal primary CF from one male donor and molds with different mechanical constraints as a result of their geometries (non-uniform and uniform). Thus, one primary aim was to further validate and identify differences between our dual ECT model with CF from different donors, including females and patients with end-stage heart failure. The obtained results demonstrated that the uniform geometry generates stiffer and less extensible ECT than the non-uniform geometry. Intriguingly, these two parameters showed a clear inverse regulation. ECT compaction was also closely linked to cell size, whereas ECT contraction was a highly individual parameter dependent upon each donor CF. For most CF, the uniform ECT also showed a higher expression of fibrosis-associated genes. Lastly, prolonged culture gave insight into the lack of a homeostatic steady state between the cellular phenotype and ECM organization. By characterizing these models with different CF, it was also possible to identify delicate regulations of the protein folding machinery. Therefore, the second aim was to unravel the role of ER adaptive processes in the phenotypic CF switch by interfering with the unfolded protein response (UPR). Based on the detected differential expression of RNA-like endoplasmic reticulum kinase (PERK) in non-uniform and uniform ECT, the effects of the PERK inhibitor GSK-2656157 (GSK’157) were investigated. GSK’157 reduced the contraction, compaction, and stiffness of non-uniform ECT and downregulated collagen I expression in ECT and 2D-cultured cells. Furthermore, it inhibited CF proliferation. Despite these anti-fibrotic effects, a paradoxical induction of the UPR was observed. Moreover, PERK inhibition restored collagen I expression after high ER stress induction. Taken together, our dual ECT model could be further validated as an advantageous way to modulate the phenotype of 3D-cultured CF and helped demonstrate that the UPR mediator PERK is an important regulator in ER adaptation and ER stress, as well as an effective regulator of collagen I.
Keywords: Cardiac fibroblasts; Cardiac fibrosis; Tissue engineering; ER stress
Schlagwörter: Cardiac fibroblasts; Cardiac fibrosis; Tissue engineering; ER stress