Project Grant R21AI185281

AUTOPHAGY AND HEPATIC STELLATE CELLS ACTIVATION - PROJECT SUMMARY LIVER FIBROSIS IN CHRONIC LIVER DISEASES (CLDS) SUCH AS VIRAL HEPATITIS, METABOLIC DYSFUNCTION-ASSOCIATED STEATOTIC LIVER DISEASE (MASLD), AND METABOLIC DYSFUNCTION-ASSOCIATED STEATOHEPATITIS (MASH), IS A SIGNIFICANT HEALTH PROBLEM. THE HEPATOCYTE INJURY DURING CLDS IS FOLLOWED BY REPLACEMENT OF LIVER PARENCHYMA BY FIBROTIC TISSUE WITH EXCESSIVE DEPOSITION OF COLLAGEN RICH EXTRA-CELLULAR MATRIX. THE ACTIVATED HEPATIC STELLATE CELLS (HSCS) ARE PRIMARY SOURCE OF LIVER FIBROTIC TISSUE. IN SPITE OF INTENSIVE RESEARCH, THE MECHANISMS OF FIBROSIS AND ACTIVATION OF HSCS IS NOT COMPLETELY UNDERSTOOD AND THE DEVELOPMENT OF EFFICIENT ANTI-FIBROTIC AGENTS REMAINS A PRIORITY. AUTOPHAGY, AN INTRACELLULAR DEGRADATION PATHWAY, PLAYS AN IMPORTANT ROLE IN DIVERSE CELLULAR PROCESSES IN HEALTH AND DISEASE. AUTOPHAGY HAS SEVERAL BENEFICIAL EFFECTS ON THE HEPATOCYTES INCLUDING INSULIN SENSITIVITY AND DEGRADATION OF INTRACELLULAR LIPIDS IN THE HEPATOCYTES (LIPOPHAGY). AUTOPHAGY ALSO PREVENTS HEPATOCYTE INJURIES INCLUDING THOSE CAUSED BY OXIDATIVE STRESS AND INFLAMMATORY CYTOKINES. THE ROLE OF AUTOPHAGY IN HSCS IS, HOWEVER, AMBIGUOUS. NUMEROUS STUDIES DEPICT AUTOPHAGY AS A PRO- FIBROGENIC PROCESS DUE TO ITS ASSOCIATION WITH HSC ACTIVATION AND LIVER FIBROSIS. THE MAIN MECHANISM UNDERLYING AUTOPHAGY-MEDIATED HSC ACTIVATION AND FIBROSIS IS AUTOPHAGY-MEDIATED LIPID DROPLET (LD) DEPLETION WHICH HAS BEEN SHOWN TO CONCUR WITH HSC ACTIVATION. ON THE OTHER HAND, SEVERAL STUDIES SHOW ANTI-FIBROTIC EFFECTS OF AUTOPHAGY VIA PROMOTION OF HSC DEATH, DEGRADATION OF PRO-FIBROTIC MEDIATORS INCLUDING COLLAGEN AND METALLOPROTEINASES, AND INHIBITION OF PRO-FIBROTIC SIGNAL CARRYING EXOSOMES. THESE DISCREPANCIES MAY BE ATTRIBUTABLE TO THE TRADITIONALLY INEFFICIENT AND INACCURATE AUTOPHAGY MARKERS, INSTANCES OF LACK OF CELL-TYPE SPECIFIC STUDIES, CONTEXT OF AUTOPHAGY MODULATION IN RELATION TO THE HEPATIC INJURY, AND THE ROLE OF AUTOCRINE/ PARACRINE SIGNALING IN FIBROSIS. IT IS NOTEWORTHY THAT SEVERAL ANTI-FIBROTIC AGENTS INCLUDING STATINS ARE KNOWN TO INDUCE AUTOPHAGY. WE HYPOTHESIZE THAT PRECISE MORPHOLOGICAL DETECTION OF AUTOPHAGIC PROCESS WILL CLARIFY THE ROLE OF AUTOPHAGY IN HSC ACTIVATION. WE WILL ADDRESS THIS HYPOTHESIS WITH THE FOLLOWING SPECIFIC AIMS: 1) TO MORPHOLOGICALLY DETERMINE THE STATUS OF AUTOPHAGY DURING HSC ACTIVATION USING A NOVEL HALOTAG- LC3 ASSAY; 2) TO STUDY THE INTER-RELATIONSHIP BETWEEN LIPID ACCUMULATION AND AUTOPHAGY IN THE HSC ACTIVATION; AND 3) TO DELINEATE THE EFFECT OF ROS SCAVENGING ON LIPOPHAGY AND LIPID METABOLISM IN HSC FIBROGENIC RESPONSE. BY EMPLOYING THE NOVEL HALOTAG-LC3 ASSAY WHICH CAN DETECT SPECIFIC AUTOPHAGY STRUCTURES AND HIGH PARAMETER METABOLIC FLOW CYTOMETRY (METFLOW), WE WILL DETERMINE THE STATE AND ROLE OF AUTOPHAGY IN PRIMARY HSCS IN THE CONTEXT OF HSC ACTIVATION, HSC-HEPATOCYTE INTERACTION, AND LIPID METABOLIC PROFILE. ONCE COMPLETED, THIS STUDY WILL PROVIDE A UNIQUE KNOWLEDGE ABOUT THE SPECIFIC STEPS OF AUTOPHAGY INVOLVED IN HSC ACTIVATION AND PROVIDE A FOUNDATION FOR MECHANISM-BASED THERAPEUTIC TOOLS AGAINST LIVER FIBROSIS.