Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Ramachandran, Rithwik

2nd Supervisor

Zhao, Liena

Co-Supervisor

Abstract

Non-alcoholic fatty liver disease (NAFLD) affects 25% of the world population underpinning the urgent need to understand molecular pathways and develop effective therapeutic interventions. Recently, yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have emerged as two important transcriptional regulators implicated in liver fibrosis and injury. Despite the growing recognition of YAP/TAZ in several liver diseases, YAP/TAZ expression has not been extensively characterized in hepatic cells, and the upstream regulators of this pathway in the setting of liver injury remain poorly understood. Here, we examined the expression pattern of YAP/TAZ in NAFLD patient samples and demonstrated that PAR receptors could be regulators of the YAP/TAZ pathway.

Archival human sections of normal liver and NAFLD including cases of simple steatosis, steatohepatitis, steatohepatitis with various stages of fibrosis and cirrhosis were analyzed using immunostaining and the activation status of YAP/TAZ and PAR receptors was monitored through assessing subcellular localization. The expression pattern of YAP in various hepatic cell types was characterized by colocalization with clinically validated cellular markers. An in vitro reporter assay was used to directly assess YAP/TAZ activation by PAR1 and PAR2 agonists.

Confocal imaging combined with standardized image analysis revealed increased nuclear YAP expression in Kupffer cells, myofibroblasts and cholangiocytes as NAFLD progressed from steatosis to fibrosis and cirrhosis. YAP/TAZ co-activators could drive inflammation in myofibroblasts and Kupffer cells and promote bile duct regeneration in cholangiocytes. PAR receptors showed different expression patterns in diseased livers indicative of receptor upregulation and activation. In complimentary in vitro studies, specific PAR1 and PAR2 agonists increased YAP/TAZ nuclear activity providing further evidence that PAR receptors can directly regulate this pathway. Immunostaining analysis and in vitro assays indicated that PARs are disease relevant regulators of YAP/TAZ in the liver and may be targeted to modulate YAP/TAZ activation. Together, our findings can help guide the development of novel therapeutic agents for treating NAFLD.

Summary for Lay Audience

Our liver, a complex organ in our body, can suffer from diseases despite its ability to heal. One such condition is called non-alcoholic fatty liver disease (NAFLD), which happens when fat builds up in the liver of people who do not drink alcohol. More than a quarter of Canadians have this disease, and the number is expected to rise. Unfortunately, there are no specific treatments for NAFLD. When liver diseases get very serious, a transplant is often the only option, but the need for organs greatly exceeds the supply.

Our research project is about finding new ways to treat NAFLD. We looked at samples of liver tissue from patients with NAFLD to understand how a certain pathway works. We learned about two special proteins, YAP and TAZ, that can make liver damage worse. There are also some proteins on the surface of liver cells, called PAR receptors, that might help sense cell damage and control YAP/TAZ.

In our study, we looked at liver samples from patients – some healthy and some with NAFLD – under a special microscope to see these proteins. We also did experiments with cells to see what happens when we activate these proteins. What we found is that as NAFLD gets worse, YAP and TAZ become more active in some liver cells. We also found that PARs on the cell surfaces are like switches that turn on YAP and TAZ and regulate the activity of YAP and TAZ.

This finding is exciting because we might be able to create new treatments for NAFLD by targeting these PAR receptor switches. In the future, we might have new ways to help people with NAFLD and stop their livers from getting sicker.

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