Assistant Professor University of Massachusetts Amherst, United States
Introduction:: In 2021, 537 million adults lived with type II diabetes (T2D), and this number is expected to rise to 783 million by 2045.1 Those suffering from diabetes have an increased incidence rate of developing chronic, non-healing diabetic foot ulcers (DFUs). 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, results in a pro-inflammatory microenvironment and dysfunctional cellular communication. Dysregulated cellular signaling often leads to the inability of DFUs to heal on their own, which leads to the need for clinical intervention, and sometimes limb amputation. In addition to pro-inflammatory factors, senescent cells have a distinct secretome that also includes extracellular matrix degradation factors, soluble proteins, and extracellular vesicles (EVs). EVs are bi-layered nanovesicles that transport cargo (e.g., proteins, cytokines, nucleic acids) used to facilitate cellular communication. While chronic 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. We hypothesize that transiently senescent extracellular vesicles can ameliorate dysregulated cellular communication in chronic wounds, thus increasing wound healing, while chronically senescent extracellular vesicles will negatively impact wound healing potential.
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 senescence in 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. Ongoing work consists of EV isolations from each group that will be assessed using the assays stated above. Since patient-derived cells are a better representation of the chronic disease states, we have employed healthy foot fibroblasts and diabetic foot ulcer fibroblasts to further explore senescent effects, with EVs from each cell type are being investigated.
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 protocol’s 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 and fibronectin decreased with increasing senescence severity. In addition, VEGF gene expression was increased in chronic senescent cells, which has been correlated with an increase in tumorigenesis in aged tissues in vivo. Following the results of cellular characterization, the effects of the total secretome of each group were investigated. In the tubule formation assay, healthy BJFs and transient senescent cells had comparable total branching lengths while chronic senescent cells underperformed and displayed poor tubule formation. Chronic SEN performance is contradictory to the increased VEGF gene expression, suggesting that non-VEGF-mediated changes are driving a decreased vascularization. The preliminary data 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. Our next steps are to investigate the EVs from each of these groups and determine the role they play in wound healing.
Acknowledgements (Optional): : This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1938059
