Cellular senescence occurs in the liver in health and disease. Senescence relates to a status of cell cycle arrest, which becomes more prevalent with increasing age and which develops as the consequence of liver disease.1 Due to subsequent changes in cell morphology and functionality senescent cells may prompt disease progression and the development of disease-related complications.1-3 Detection of senescence and deciphering of its underlying mechanisms may help identifying novel targets to develop preventive treatment strategies to halt the development of liver disease related complications such as fibrosis, inflammation, and ultimately cirrhosis.4 Therefore, this project seeks to address the following aspects:
Aim: Cellular senescence is a well-known consequence of diseases in general but in-depth characterization of triggers (DNA damage, Telomere shortening, etc.), pathways leading to cell cycle arrest (e.g. p16 dependent pathway, p53 dependent pathway), and subsequent cell-phenotypic changes (e.g. GATA4 driven SASP, Cyp450 expression, metabolic liver zonation) are sparse. Therefore, the main aim of this project is to decipher the characteristics of hepatocytes senescence in different types and stages of liver disease. It will serve as the basis for any type of senolytic intervention, first with respect to the type of senolytic compound [broad spectrum (e.g. BCL2 inhibitor) vs. pathway specific (e.g. p53 interacting proteins)] and second, with respect to the time point of intervention. It will be based on an in-depth analysis of human and rodent tissue samples.
Workplan: Healthy livers from different ages will be included to understand to what extent aging drives cellular senescence in the liver. The following liver diseases are relevant in Europe thus being involved in this project: 1) Paracetamol-induced liver injury 2) Drug-induced liver injury 3) Alcoholic hepatitis 4) Non-alcoholic steatohepatitis 5) Cirrhosis 6) Acute-on-chronic liver failure (ACLF). Human FFPE liver samples with different disease stages from the Pathology department Charité will be analyzed by conventional immunostaining and multiplex immunofluorescence staining with respect to DNA-damage, cell cycle arrest, autophagy, and cell death. Image analysis will be attempted to be on single-cell level including descriptive neighboring analysis. In addition, whilst regenerative mechanisms generally appreciate the proliferative activity of cells and preservation of tissue integrity, restoration of metabolic activity by alteration of liver zonation to maintain organ function is another incremental part of liver regeneration although being frequently disregarded. Therefore, expression of senescence markers will be correlated with alteration of liver zonation and metabolic characterization of hepatocytes. Mechanistic information will be obtained from already performed animal models for the above mentioned diseases as well as conditional knockout mouse lines [AhCreMdm2fl/fl (inducing senescence in hepatocytes); AhCrep53fl/fl (preventing senescence in hepatocytes)] after exposure to toxins such as lipopolysaccharides or ethanol. In addition to immunostaining analyses from paraffin-embedded tissue, frozen tissue will allow generating proteomic (mass spectrometry) and transcriptomic (mRNA sequencing) data. By using laser capture microdissection to extract proteins and RNA from areas of interest spatial information can be added to this analysis. Performing Seahorse from frozen section of liver tissue will generate more data on the metabolic/mitochondrial tissue function and will be correlated with changes of metabolic liver zonation and presence of cellular senescence in different liver diseases. All experimental techniques are well established in our lab and respective animal experiments were performed previously.
Impact: Results from this project will provide highly relevant information regarding the role of cellular senescence in homeostasis and different types of liver disease, will allow generating disease related hypothesis and may serve as the basis for later interventional study (e.g. senolytic therapies) aiming at restoring “liver health” with respect to timing of intervention and choice of senolytic compounds.