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creTRAP for in situ Characterization of Fibrosis-Promoting Pathways in Non-alcoholic Fatty Liver Disease

Periodic Reporting for period 1 - InCisive (creTRAP for in situ Characterization of Fibrosis-Promoting Pathways in Non-alcoholic Fatty Liver Disease)

Reporting period: 2016-03-01 to 2018-02-28

Aim:
The aim of the proposed project is to develop and employ novel tools to identify and target fibrosis-promoting, molecular pathways in non-alcoholic fatty liver disease.
Importance:
Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Parallelling the global epidemic in obesity and type-2-diabetes, NAFLD affects 20-30% of the global population and is now the most common liver disease. As incidences of obesity and obesity-associated complications like NAFLD continue to steadily increase, the economical burden on public health care systems will be enormous. The estimated annual NAFLD-related expenses (accounting for societal costs) for the United States and the four European countries (Germany, France, Italy and UK) are currently US$ 292bn and 219bn, respectively. Yet, the current lack of tools for studying the initiation and progression of NAFLD at the cellular level limits our ability to improve diagnostics, risk stratification and strategies for rational, therapeutic intervention.


This current cross-disciplinary research project was aimed at addressing this challenge by setting the following objectives:
(A) Development of novel pre-clinical tools for in situ gene expression profiling and gene knock-down in select cell types in vivo.
(B) Implementation of these tools to discover mechanisms linking metabolic stress to chronic liver disease in murine models of diet-induced fibrosis.
(C) Experimental validation of hypotheses based on the bioinformatic analysis of gene expression profiles from individual cell populations. Experimental treatment of NAFLD in animal models.
"The development of hepatic fibrosis is driven by the complex interplay between multiple hepatic cell types. However, animal studies of fibrotic development have been limited by the lack of tools for cell type-specific analyses in vivo. The purpose of the project was therefor to develop a novel in vivo platform for cell type-specific analyses of the development of non-alcoholic steatohepatitis and hepatic fibrosis. The project as it has evolved can be divided into three work objectives:

A. Development and characterization of a diet-induced murine NASH models resembling human NASH.
Mice were fed a western diet (WD) supplemented with D-fructose in their drinking water for up till 52 weeks. During WD feeding mice develop massive hepatic steatosis, hepatic crown-like structures, and pericellular fibrosis. To characterize changes in the hepatic stellate cell (HSC) population, HSCs were isolated at different time points for gene expression analyses. These analyses were compared to conventional histopathological analyses.

Our data shows that WD feeding is a good NASH model resembling human disease and point to the HSC plasticity as a ""conserved"" point of convergence of chronic liver disease. These findings were combined in a recently submitted manuscript.

B. Development and characterization of a murine knock-in platform for fluorescence labelling, ribosome tagging, and cell type-specific gene targeting.
Development of our creTRAP mouse line was completed in Apr. 2016. We have now fully characterized our creTRAP mouse model with tagged hepatic stellate cells and macrophages, respectively. We find robust and reproducible expression in HSCs and macrophages allowing us to use these mice for specific analyses of the respective cell types. Changes in gene expression in situ during NASH development will now be analysed by next-generation sequencing.

C. Characterization of hepatic stellate cell activation and cellular interactions in vivo during fibrosis development.

Having the above disease model (A) and tools (B) at hand, we have conducted experiments to explore in vivo gene expression in hepatic stellate cells and interacting cell types during development of hepatic fibrosis in situ. We combine ribosome pull down and single cell sequencing providing us with unique insights into NASH development at the cellular level.

Results from these experiments will be submitted as a manuscript to an internationally recognized journal.

The project currently involves two graduate-students and has involved four undergraduate students at the Masters and Bachelors levels.
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The establishment of our creTRAP mouse models for ribosome profiling has resulted in major developments in the project. A standing collaboration with Prof. Susanne Mandrup around these models led to the funding of our national Centre of Excellence ATLAS focused on the investigation of cellular plasticity in adipose tissue and liver during development of obesity and Non-Alcoholic Fatty Liver Disease (NAFLD) in mice.

Our creTRAP technology platform is an integral part of the ATLAS design. Using advanced sequencing technologies and bioinformatics we will now explore tissue plasticity at the cell type and single cell level in obesity mice, develop integrated models and use these to analyze and understand human patient samples. The perspectives here is a cell type-resolved view of human NAFLD, which may facilitate improved diagnostics and risk stratification as wells as facilitate drug target discovery.
Hepatic Stellate Cell labeling for isolation and gene expression profiling