Poster BB18 - Developing a model of chronic and transient senescence to interrogate the effects of the secretome on skin wound healing

Introduction: By 2050, 16% of the global population will be over the age of 65, which is a 7% increase from 2019.1 As an aging population, there has been an increased prevalence of age-related diseases, including T2D, many of which are associated with senescent cells. Senescence, the cessation of normal cell cycle and function, has a paracrine effect in which their distinct pro-inflammatory secretome promotes further senescence in healthy bystander cells, reducing the overall quality of cells. In addition to reduced cellular health, aged tissues also have poorly organized extracellular matrix, which results in overall, poor tissue quality. While chronic, prolonged senescence causes an increase in inflammation and dysfunctional wound healing, it has been shown that short-term, transient senescence plays a pivotal role in normal wound healing. Deducing the differences between chronic and transient senescence will allow researchers to determine the best methods of treating chronic wounds clinically. Additionally, there are no methods of inducing chronic senescence as there is a very fine line between cellular death and senescence induction. This research sets out to optimize an in vitro senescence model to be used to investigate skin wound healing in chronic and aged wounds. We hypothesize that transiently senescent secretome will promote skin wound healing, while chronically senescent secretome will negatively impact wound healing potential. With this study, our long-term goal is to investigate individual components in the secretome (i.e. extracellular vesicles) that are driving the paracrine effect of senescence.

Materials and

Methods: To induce transient senescence, BJ fibroblasts were incubated for 1 hour with complete media supplemented with 500 uM H2O2. Chronic senescence was induced similarly to transient, with an additional 300 uM H2O2 pulse 24 hours post-initial induction, and every other day for a total of four pulses. To our knowledge, this method has not been utilized before to induce chronically senescent fibroblasts. After senescent characterization (e.g., senescence-associated-β-galactosidase expression [SABGAL], p21 and p16 cell cycle checkpoint inhibitor gene expression, morphology changes), the complete supernatant from each group was used to investigate the effects of the total secretome using several in vitro assays associated with wound healing: vascularization – tubule formation assay; proliferation of fibroblasts; migration of fibroblasts – wound scratch assay; macrophage polarization – immune response.

Results, Conclusions, and Discussions: Following senescent induction, SABGAL expression increased 3-fold and 5.7-fold in transient and chronic senescent cells respectively, confirming our protocols' ability to induce senescence. Morphological examination of the cells' nuclear-to-cytoplasm ratio revealed a decrease of more than 50% in both transient and chronic senescent cells. To observe their potential in wound healing, cells were probed for expression of common pro-wound healing genes. The gene expression of collagen I decreased with increasing senescence severity, whereas collagen III decreased in response to both senescent groups. Following the results of cellular characterization, the effects of the total secretome of each group were investigated. In the tubule formation assay, the secretome from healthy BJFs and transient senescent cells had comparable total branching lengths, while chronic senescent secretome underperformed and displayed poor tubule formation. In response to chronic senescent secretome, there was increased IL1β gene expression in both unpolarized (M0) and pro-repair (M2) macrophages, suggesting that chronic senescence leads to macrophage polarization to a pro-inflammatory phenotype. This study suggests that chronic senescent cells play a significant role in delayed wound healing. The initial in vitro studies have shown that we have been able to induce two types of senescence, and the data is consistent with what has been previously investigated. This novel senescence induction method can be used to investigate interventions for skin wound healing. Additionally, our lab is investigating its applications for various other cell types.

Acknowledgements (Optional): T.M. acknowledges funding from the National Science Foundation Graduate Research Fellowship under grant no. 1938059.


References:
1. United Nations Department of Economic and Social Affairs, Population Division. World Population Prospects 2022: Summary of Results. UN DESA/POP/2022/TR/NO. 3. (2022).