Overall Objectives of this proposal are to define mechanisms linking hepatocyte injury during lipotoxicity with hepatic inflammation in nonalcoholic steatohepatitis (NASH). NASH is the most common pediatric liver disease characterized by abundant circulating saturated free fatty acids (SFAs), along with hepatocyte lipotoxicity and monocyte-derived macrophage mediated liver inflammation. Hepatocyte lipotoxicity and liver injury are, in part, induced by SFAs and their intracellular metabolite lysophosphatidyl choline (LPC). However, cellular and molecular mechanisms linking hepatocyte lipotoxicity to liver inflammation are not completely understood. Emerging data implicate extracellular vesicles (EVs) released during hepatocyte lipotoxic stress in liver inflammation. In published and preliminary experiments, we have discovered that, lipotoxic hepatocytes release a large number of proinflammatory EVs; these EVs are enriched with the adhesion molecule integrin ?1 (ITG?1) and promote monocytes adhesion to liver sinusoidal endothelial cells (LSECs) in vitro. We also demonstrated that the expression of ITG?1 ligand, vascular cell adhesion molecule (VCAM) 1, on LSECs is increased during lipotoxicity. Based on these novel observations, we have formulated the CENTRAL HYPOTHESIS that lipotoxic hepatocytes release ITG?1-enriched EVs that recruit and retain monocyte in the liver promoting inflammation. We will employ current biochemical and cell biological approaches that include microfluidic technology, Nanoscale flow cytometry, and 89Zirconium isotopically labelled EVs visualized with positron emission tomography (PET) scan to test this hypothesis. Our independent SPECIFIC AIMS will test three integrated hypotheses. First, we will demonstrate that hepatocyte lipotoxicity induces an active conformation switch of ITG?1, enhancing its endocytic trafficking and release into EVs. Second, we will define the mechanism of increased VCAM1 expression during lipotoxicity. We will also directly test the hypothesis that lipotoxic hepatocyte-derived EVs mediate monocytes adhesion to LSECs, through ITG?1-VCAM1 binding interaction, in vitro by using microfluidic technology. Third, using a mouse model of NASH, we will test the hypothesis that pharmacological inhibition of integrin ?1 or conditional deletion of endothelial VCAM1 is protective against liver inflammation. We will also demonstrate that adoptively-transferred lipotoxic hepatocyte- derived EVs home to the LSECs of recipient mice through their high affinity integrin ?1cargo. We have established the requisite cell and mouse models to study lipotoxicity, integrin signaling and EV biology. This proposal is technically and conceptually innovative, as it seeks to integrate the molecular mechanisms underlying hepatocyte injury, integrin activation and trafficking with liver inflammation, and links hepatic pathophysiology with nanomedicine. This research has the potential to identify new therapeutic strategies, namely integrin and VCAM1 inhibitors, to prevent or reverse liver injury and inflammation in human NASH.

Public Health Relevance

The proposal examines the mechanisms of liver injury and inflammation in nonalcoholic fatty liver disease by employing models related to obesity. We propose that liver cells exposed to excess circulating toxic lipids such as saturated free fatty acids and their metabolites release abundant extracellular vesicles, which induce recruitment and retention of white blood cells into the liver and promote liver inflammation. The results of the proposal have the potential to identify new mechanisms of liver inflammation in nonalcoholic fatty liver disease (the most common pediatric liver disease), and discover new anti-inflammatory therapeutic targets for this disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK122948-01
Application #
9854281
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Doo, Edward
Project Start
2020-04-15
Project End
2024-01-31
Budget Start
2020-04-15
Budget End
2021-01-31
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905