Methods of Inducing Cellular Senescence in Lung Fibroblasts
Abstract/Description/Artist Statement
Methods of Inducing Cellular Senescence in Lung Fibroblasts
Sara Palega1, Jennifer Q Zhou2, Shan Zhu2, Yan Y Sanders2
1VHS Biomedical Sciences PhD program, 2Department of Biomedical and Translational Sciences, Macon & Joan Brock Virginia Health Sciences at Old Dominion University (ODU)
Introduction: Idiopathic Pulmonary Fibrosis (IPF) is the most prevalent and severe of interstitial lung diseases. This chronic and progressive condition lacks treatment options and is ultimately fatal without a lung transplant. IPF arises from aberrant wound healing that leads to excessive collagen deposition, inflammation, and cystic alveolar growths which makes breathing increasingly difficult as the disease progresses. The accumulation of senescent cells with abnormal secretory phenotypes exacerbates this condition by worsening inflammation and impairing tissue regeneration and repair. Because of this, investigating ways to eliminate senescent cells has emerged as a potential therapeutic option. A key limitation to studying senescence is its heterogeneity; various stimuli are known to cause cellular senescence, and senescent cells may exhibit different secretory phenotypes. Because of this, the various methods to induce senescence in lung fibroblast cultures have not been well established. Furthermore, it is not clear whether cells would present the same markers under senescence induced by different stimuli. In this project, various methods of inducing senescence in IMR90 human lung fibroblast cells, including chemical and genotoxic stress, will be characterized by evaluating the RNA and protein expression of known senescent markers p16 and p21 and inflammatory markers such as IL-1B, IL-6, and IL-8. Additionally, senolytics will be tested for their ability to clear these induced senescent fibroblast cells.
Methods: Methods of inducing cellular senescence were tested by exposing human lung IMR90 cells to various genotoxic and chemical stressors. After treatment, the cells were collected for senescence-associated β-gal staining, RNA harvesting, and protein harvesting to examine their phenotype. Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the senescent markers p21, p16, IL-1 β, IL-6, and IL-8. The p21 and p16 protein expression were also assessed with western blots.
Results: So far, it has been shown that both acute X-ray radiation (2Gy and 8Gy) as well as repeated, low dose X-ray radiation increases the expression of senescence markers and inflammatory cytokines in IMR90 human lung fibroblasts. UV treatment (10mJ/cm^2 and 15mJ/cm^2) also increased p21 protein expression. This project is ongoing, but it is expected that chemical treatment with doxorubicin as well as oxidative stress via hydrogen peroxide treatment will also result in an upregulation of senescence-related and inflammatory biomarkers; although we are not sure if all, or only some, will be up-regulated. It is expected that these effects will be mediated by treatment with senolytic compounds, which eliminate senescent cells.
Conclusion:
The various methods to induce senescence in IMR90 cells were tested by evaluating the expression of senescence markers including p21, p16, and senescence-associated β-gal staining and by quantifying changes in inflammatory cytokines such as IL-1β, IL-6, and IL-8. After establishing these models, the effectiveness of senolytic compounds can be assessed. Overall, this project will lead to a better understanding of how environmental stress leads to cellular senescence, so better models for aging lung injury repair can be created, leading to stronger medical interventions for patients with IPF.
Faculty Advisor/Mentor
Dr. Yan Y Sanders
Faculty Advisor/Mentor Email
Sandery@odu.edu
Faculty Advisor/Mentor Department
Department of Biomedical and Translational Sciences, Macon & Joan Brock Virginia Health Sciences at Old Dominion University (ODU)
College/School Affiliation
The Graduate School
Student Level Group
Graduate/Professional
Presentation Type
Poster
Methods of Inducing Cellular Senescence in Lung Fibroblasts
Methods of Inducing Cellular Senescence in Lung Fibroblasts
Sara Palega1, Jennifer Q Zhou2, Shan Zhu2, Yan Y Sanders2
1VHS Biomedical Sciences PhD program, 2Department of Biomedical and Translational Sciences, Macon & Joan Brock Virginia Health Sciences at Old Dominion University (ODU)
Introduction: Idiopathic Pulmonary Fibrosis (IPF) is the most prevalent and severe of interstitial lung diseases. This chronic and progressive condition lacks treatment options and is ultimately fatal without a lung transplant. IPF arises from aberrant wound healing that leads to excessive collagen deposition, inflammation, and cystic alveolar growths which makes breathing increasingly difficult as the disease progresses. The accumulation of senescent cells with abnormal secretory phenotypes exacerbates this condition by worsening inflammation and impairing tissue regeneration and repair. Because of this, investigating ways to eliminate senescent cells has emerged as a potential therapeutic option. A key limitation to studying senescence is its heterogeneity; various stimuli are known to cause cellular senescence, and senescent cells may exhibit different secretory phenotypes. Because of this, the various methods to induce senescence in lung fibroblast cultures have not been well established. Furthermore, it is not clear whether cells would present the same markers under senescence induced by different stimuli. In this project, various methods of inducing senescence in IMR90 human lung fibroblast cells, including chemical and genotoxic stress, will be characterized by evaluating the RNA and protein expression of known senescent markers p16 and p21 and inflammatory markers such as IL-1B, IL-6, and IL-8. Additionally, senolytics will be tested for their ability to clear these induced senescent fibroblast cells.
Methods: Methods of inducing cellular senescence were tested by exposing human lung IMR90 cells to various genotoxic and chemical stressors. After treatment, the cells were collected for senescence-associated β-gal staining, RNA harvesting, and protein harvesting to examine their phenotype. Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the senescent markers p21, p16, IL-1 β, IL-6, and IL-8. The p21 and p16 protein expression were also assessed with western blots.
Results: So far, it has been shown that both acute X-ray radiation (2Gy and 8Gy) as well as repeated, low dose X-ray radiation increases the expression of senescence markers and inflammatory cytokines in IMR90 human lung fibroblasts. UV treatment (10mJ/cm^2 and 15mJ/cm^2) also increased p21 protein expression. This project is ongoing, but it is expected that chemical treatment with doxorubicin as well as oxidative stress via hydrogen peroxide treatment will also result in an upregulation of senescence-related and inflammatory biomarkers; although we are not sure if all, or only some, will be up-regulated. It is expected that these effects will be mediated by treatment with senolytic compounds, which eliminate senescent cells.
Conclusion:
The various methods to induce senescence in IMR90 cells were tested by evaluating the expression of senescence markers including p21, p16, and senescence-associated β-gal staining and by quantifying changes in inflammatory cytokines such as IL-1β, IL-6, and IL-8. After establishing these models, the effectiveness of senolytic compounds can be assessed. Overall, this project will lead to a better understanding of how environmental stress leads to cellular senescence, so better models for aging lung injury repair can be created, leading to stronger medical interventions for patients with IPF.