Illuminating the arc of molecular events from chronic hepatic injury to fibrosis and then cirrhosis remains among the most fundamental of challenges in investigative hepatology. Today's epidemic of end-stage liver disease due to hepatic fibrosis has precipitated an urgent need for new therapies. The hepatic stellate cell (HSC), following activation during liver injury, plays a central role in the development of fibrosis. Our long-term goal is to understand how HSC activation unfolds as an integrated, homeostatic response to liver injury;this will lead to novel, targeted interventions for hepatic fibrosis. A seminal study published by others in NATURE describing loss of lipid droplets as a feature of autophagy sparked our idea that autophagy is a component of HSC activation. Indeed, the features of HSC activation are hallmarks of autophagy, which is a highly regulated cellular response that has evolved to maintain energy homeostasis during cellular stress or enhanced metabolic demand. The objective of this project, which is the next step towards our long-term goal, is to characterize the contribution of autophagy to HSC activation, thereby uncovering new regulatory pathways and predicting consequences of their inhibition. Our central hypothesis, therefore, is that autophagy is a critical and necessary component of HSC activation. We will test our central hypothesis through the following interrelated Specific Aims: 1. Define stimuli that provoke autophagy in HSCs using established culture models. This question will be addressed in models of quiescent and culture-activated HSCs in which autophagy will be documented by: Western blot to detect conversion of LC3-I protein to LC3-II, ultrastructure, and lipid &retinoid content assessed by HPLC. 2. Determine which features of HSC activation are autophagy-dependent. Following siRNA knockdown of the specific autophagic regulators, Atg7 or Atg5, we will assess the contribution of autophagy to HSC activation in culture-activated HSCs (rodent &human), and define autophagy-regulated pathways by quantitative PCR and Western for known activation markers, and exon arrays for novel targets. 3. Establish the contribution of autophagy to HSC activation in vivo and explore the therapeutic potential of blocking autophagy in HSCs. The response to TAA or bile duct ligation models of fibrosis will be assessed in: a) Mice with stellate cell-specific Atg7 deletion. Key endpoints will include histology, collagen content, fibrogenic mRNA expression, HSC activation markers (smooth m. alpha actin and 2-PDGF receptor), ultrastructure and retinoid content. Isolated HSCs from these mice will be characterized for autophagy and activation, complemented by analyzing cultured HSCs from Atg7LoxP/LoxP infected with a cre-expressing adenovirus;b) Mice treated with chloroquine, seeking specific evidence of attenuated autophagy in HSCs that cannot be explained by indirect effects of the drug on other cell types, or on the extent of injury. These studies should uncover fundamental new pathways of stellate cell activation, leading to innovative treatment approaches for patients with fibrosing liver diseases.

Public Health Relevance

Chronic liver disease leads to scarring, or fibrosis, of the liver, and is a major public health threat, affecting hundreds of millions of individuals worldwide. We propose to study a specialized pathway called autophagy that drives activation of hepatic stellate cells in liver to make scar. New insights expected to emerge from these novel studies could significantly advance our understanding of how to block scar formation in chronic liver disease, accelerate liver repair, and prevent the end-stage of liver disease called cirrhosis, thereby improving the lives of patients throughout the developed and developing world.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Doo, Edward
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Icahn School of Medicine at Mount Sinai
Internal Medicine/Medicine
Schools of Medicine
New York
United States
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