Chronic liver injury results in hepatic fibrosis, in which quiescent hepatic stellate cells (qHSCs) activate into myofibroblasts, depositing extensive extracellular matrix (ECM) proteins. Cessation of fibrogenic stimuli often results in regression of liver fibrosis and is associated with the disappearance of activated HSCs (aHSCs)/myofibroblasts. We have recently demonstrated that some aHSCs apoptose, while other aHSCs inactivate (iHSCs) into a quiescent-like phenotype. The overall goals of this Project are to identify the molecular factors that may prevent HSC activation into myofibroblasts, or that revert aHSC into an inactivated state. Our central hypothesis is that genome wide epigenetic changes regulate HSC phenotype by activation (or suppression) of transcriptional activity in HSCs. We also hypothesize that activation of PPAR?-target genes regulates quiescent and inactivated HSC phenotypes.
AIM1 : We will assess the genome wide methylation and acetylation sites using ChIP-Seq in qHSCs, aHSCs and iHSCs in order to identify motifs and transcription factors critical for HSC inactivation. Using in vitro systems, we will determine f siRNA knock-down or overexpression of these factors blocks HSC activation, or triggers HSC inactivation.
AIM2 : We have demonstrated that PPAR? is re-expressed in HSCs during inactivation. To gain a greater insight into the mechanisms of HSC inactivation, we will conduct a broad investigation of the epigenetic changes that regulate PPAR?-target genes in distinct HSC phenotypes (qHSCs, aHSCs and iHSCs). We will test if functional inhibition of these genes affects the qHSC and iHSC phenotypes.
AIM3 : We will assess the role of PPAR? and PPAR? target genes in HSC biology in vivo using knockout mice, in which PPAR? is constitutively or inducibly deleted specifically in HSCs. A specific role of PPAR? in the maintenance of quiescent HSC phenotype and inactivation of aHSCs will be assessed in PPAR?-deficient HSCs versus wild type HSCs. We anticipate that the collective results obtained in AIMs 1-3 will identify specific factors that can revert aHSCs into an inactivated quiescent-like state.
AIM4 : Our findings in mice must be translated to patients with fibrotic liver disease. To examine how human HSCs inactivate, quiescent human HSCs will be engrafted into livers of Rag2-/-?c-/- mice, and their activation will be induced in vivo by CCl4 followed by recovery. Human qHSCs, aHSCs and iHSCs will be isolated at each time point and analyzed by RT-PCR, Human genome microarray and flow cytometry. Next, human iHSCs will be subjected to ChIP-Seq analysis, and inactivation-specific targets will be identified (and compared to that in mouse iHSCs). The ability of these targets to inactivate human aHSCs into a quiescent-like state will be tested in vitro and in vivo using siRNA knock-down or intrahepatic transplantation of human HSCs into Rag2-/-?c-/- mice. The results of our studies will give new insight into mechanisms underlying HSC inactivation in mice and patients, identifying potential therapeutic targets that can induce inactivation of activated HSCs/myofibroblasts in fibrotic liver.

Public Health Relevance

Hepatic fibrosis and its end stage, cirrhosis, represent an enormous health care burden worldwide. The goal of this study is to determine the mechanis of activation of Hepatic Stellate cells/myofibroblasts, and identify the pathways of their inactivatio into queiscent-like state. Understanding of the mechanisms of myofibroblast inactivation is critical for the treatment of hepatic fibrosis, and will advance the field by providing novel targes for the anti-fibrotic therapy.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01DK099205-01A1
Application #
8694267
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Doo, Edward
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Surgery
Type
Schools of Medicine
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093