Speaker
Description
Introduction
Cellular senescence is an irreversible cell-cycle arrest program that has been associated with numerous biological processes. While emerging studies have extended its role to tissue repair, the focus has been on the effect of the senescence-associated secretory phenotype (SASP) rather than the direct role of the program beyond its paracrine effect. Using a previously established in vitro scaffold-based tissue wound healing model[1], our group recently observed how senescence differentialy affects ECM deposition and tissue tension. Differences observed at the cellular level (morphology, adhesion, cellular forces, migration) directly point to an altered mechano-sensation. We hypothesise that the also observed differences in tissue contraction are influenced by an altered mechano-sensation of senescent cells which in turn leads to distinct cellular responses. We here aim to investigate potential changes in cellular mechanotransduction in senescence due to alterations of mechanical stiffness in the cell environment and aim to link these findings to differences in key processes of tissue regeneration.
Methodology
To investigate individual aspects of the program, senescence was induced to primary human dermal fibroblasts (hdFs) through three triggers: (1) serial passaging leading to replicative senescence, (2) induction of DNA damage through Mitomycin C, and (3) inducible over-expression of p16Ink4a cyclin-dependent kinase inhibitor. To validate the senescence phenotype, cell cycle arrest was assessed through proliferation assays and further characterised by immunoblotting of cell-cycle inhibitor markers (p16Ink4a, p21Waf1). Lysosomal mass was quantified through enzymatic staining of senescence-associated beta-galactosidasde (SA-β-gal) and the phosphorylation of the histone H2AX was identified by immunoblotting.
Substrate stiffness-mediated response was assessed using tuneable stiffness-gradient [2] and uniform-stiffness polyacrylamide (PA) gels. Stiffness of these substrates was validated through nanoindentation. Stiffness-mediated morphological alterations as well as localization of mechanosensitive proteins (YAP, LaminA) were investigated using immunofluorescence staining.
Results
The senescence phenotype was validated by a reduced proliferation, elevated SA-β-gal activity and over-expression of both cell-cycle inhibitor markers and H2AX.
Adjusting the concentration of acrylamide and bis-acrylamide to 12% and 4%, respectively, lead to the fabrication of PA gels with a linear gradient ranging from 1.5kPa to 45kPa. Preliminary results across the gradient, show alterations of senescent cells in terms of aspect ratio and cell spreading. First observations when comparing uniformly soft (1.7kPa) with uniformly stiff (48.3kPa) environments, further support these stiffness-mediated morphological alterations. In addition, nuclear expression levels of YAP on stiffer substrates appear to be reduced in senescent cells, indicating potential differences in mechanotransduction induction mechanisms of these cells in response to stiffness.
Conclusion
Overall these results will contribute to a so far unknown understanding about the mechanosensitivity of senescent cells. This can help identify particular mechanical environments favoured by these cells which could be integrated into current strategies for tissue regeneration. We plan to further investigate stiffness-mediated alterations of focal adhesion distribution as well as potential stiffness-dependent differentiation (e.g. myo-fibroblast). As a next step, we will study the consequences of senescence on cell organization and tissue defect healing in the above-mentioned scaffold-based in vitro model.
References
[1]Brauer et al., Adv Sci. 6(9):1801780 (2019)
[2]Hadden et al., PNAS, 114(22):5647-5652 (2017)
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