Disclosures. Outline. Extracellular Vesicles in Fatty Liver Disease. From Contributors to disease pathogenesis to novel biomarkers

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1 Extracellular Vesicles in Fatty Liver Disease From Contributors to disease pathogenesis to novel biomarkers Ariel Feldstein, M.D. Professor of Pediatrics Chief, Division of Pediatric Gastroenterology Hepatology and Nutrition University of California San Diego Disclosures Co inventor on pending and issued patents filed by the Cleveland Clinic and UCSD that refer to the use of biomarkers in fatty liver disorders. And Scientific Advisory Board: Gilead, Takeda, Mitsubishi Tanabe, Raptor, Conatus And My presentation does not include discussion of off label or investigational use Outline NAFLD: Clinical Spectrum Pathogenic Pathways Lipotoxicity Hepatocyte Derived Extracellular Vesicles Mediators of Angiogenesis and Fibrosis in NASH Circulating Biomarkers Summary and Conclusions 1

2 Non Alcoholic Fatty Liver Disease (NAFLD) Spectrum of disease NAFL: fatty liver (steatosis) NASH: steatosis + inflammation + liver injury Fibrotic NASH: steatosis + inflammation + liver injury + fibrosis Angulo P et al. N Engl J Med ;36:11 31 Angulo P et al. Gastroenterology. 15 Aug;19(): NAFLD Pathogenesis: complex interaction and crosstalk between environmental factors, host genetics and gut microflora hepatocyte lypotoxicity cell death Kuppfer cell IL1-β TNF-α TGF-β Collagen MMPs sterile inflammation hepatic stellate cell gut derived PAMPs and DAMPs FFAs adiopokines adipose tissue gut Lipid Partitioning and Disease Progression Lipid Types Compartmentation Adaptation Damage Non-progressive Progressive Modified from Feldstein. Semin Liver Dis. 1 Nov;3():391-1.

3 Extracellular Vesicles () in Fibrotic NASH Lipotoxicity Induce Release by Hepatocytes HepG Number of Calcein + / ml media 6 HepG- Control Palmitic Acid Number of Calcein + pmhep- / ml media 3 1 Primary mouse hepatocytes (pmhep) Control Palmitic acid pmhep- Povero et al. Science. Signal. 6, Oct (13) Hepatocyte-derived under Lipotoxic Stress are Pro-angiogenic In vitro tube formation assay Control VEGF EV EV-free sup In vivo tube formation and migration assay CTR EV-free sup. VEGF Endothelial Cells Stained in Plug ECs infiltrated in the plug (number / 1x field) 1 6 HUVECs-total tube length (px) (x1 3 ) Control VEGF EV-free sup. Control VEGF EV-free sup Number of Cells Migrated into Plug 3

4 Circulating Isolated from NASH Models Induce Angiogenesis ex vivo Total tube length (px) / Control VEGF EV-free EV EV EV supernatant chow HF/HCarb MCD Migrated HUVECs (number / 1x field) Control VEGF EV-free EV EV EV supernatant chow HF/HCarb MCD are Internalized in Endothelial Cell-forming Tubes The Angiogenic Effects of Involved Fusion with Endothelial Cells Hep- Endothelial Cells (F-actin) Nuclei (DAPI) Control EV-Vanin1 interacts w/ lipid raft on EC plasma membrane

5 Vanin-1 Neutralizing Antibody Blocks Fusion and Reduces EV-induced Angiogenesis Events HUVECs-total tube length (px) (x1 3 ) Internalization + VNN1 Ab Intensity Tube formation CTRL Ab Control VEGF EV-free sup. + VNN1 nab Number of Calcein + ECs (x1 3 ) ECs migrated into the filter (number / 1x field) EV+VNN1 nab Chemotaxis (Boyden s chamber assay) Control VEGF EV-free sup. + VNN1 nab Located in Plasma & Liver Tissue of NASH Mouse Models EV in Liver Tissue EV in Plasma H: Hepatocyte; Ld: Lipid Droplet; m: microtubule; DS: Disse space Povero D. PLoS One. 1 Dec 3;9(1):e as New NASH Biomarkers Liquid Liver Biopsy Extracellular Vesicles (EV) (hepatocyte derived) EV Derived Proteins (panel & specific) EV Derived RNA (ncrna) (panel & specific) 5

6 Increase Detected in Blood of NASH Mouse Model Steatosis NASH SSC-A Control Steatosis NASH Mouse model FSC-A/FITC+ Povero D. PLoS One. 1 Dec 3;9(1):e EV-Derived Protein Profile from Blood as a barcode to Discriminate NASH Chow: Control CSAA: Steatosis CDAA: NASH Steatosis NASH Blood Contain Liver-Specific MicroRNAs Chow: Control CS-CSAA: Steatosis CD-CDAA: NASH 6

7 Increase in Mouse NASH Model Over Time and Correlation with Liver Biopsy Chow CD w k CD w k CD w k 56 Chow CD w k CD w k CD w k Relative CD31 staining (% Area) (Log) 1.5 Spearman r=.79 p< Number / L blood (Log1) # TUNEL positive cells (Log) Spearman r=.6397 p< Number / L blood (Log1) Chow: Control CD: Experimental NASH Povero D. PLoS One. 1 Dec 3;9(1):e Depleting mirnas reduces EV-mediated HSC pro-fibrogenic responses Dicer/Drosha Scramble RNA sirna EV (PKH6) MERGE RNA (SYTO) + + Palmitic acid Hepatocyte Control Hep Hep scrna Hep Dcr/DrosiRNA Hep-EV Fluorescence intensity (% of control) Proliferation Migrated LX (number / 1x field) Migration EV-miRNAs Control Hep-EV ScRNA Dcr/Dro- Control Hep-EV scrna Dcr/Dro- Hep-EV EV 7

8 Mir-1-1/ is upregulated in liver in experimental models of 6. NASH mir-1-3p expression in liver (% of control x1 3 ).1 mir-1-3p expression in liver (% of control x1 3 ) CHOW CS wk CD wk. CHOW HFAT 1wk mir-1a expression in HepG (% control) Control PA AntagomiR1-3p Anti control mir-1-3p expression in HepG-EV (% of control ) Anti control + PA AntagomiR-1-3p + PA Dr. Davide Povero AASLD November 15, 15 Novel Molecular Targets in NASH + Palmitic acid Blocking mir-1a resulted in reduced HSC activation mediated by PPAR- mrna / BM (fold change) PPAR-γ α-sma Collagen-Iα SMA mrna / BM (fold change) Col1 1 mrna / BM (fold change). mir-1-3p control Loss of function Anti mir-1a EV HSC Migrated LX (number / 1x field) 6 mir-1-3p control Migration mir-1-3p control Fluorescence intensity in LX (% of control) 3 1 mir-1-3p control Proliferation mir-1-3p control Silencing Dicer/Drosha Anti mir-1a VNN1 mir-1a MIMIC mirnas Protein/Lipids Internalization mir-1a Protein/Lipids mirnas Protein/Lipids PPAR-γ - Proliferation - Migration - Pro-fibrogenic activation Modulation Hepatic Stellate Cell

9 Summary / Conclusion NAFLD Pathogenesis involves a complex interaction between environmental factors, host genetics and gut microflora and depends on both intrahepatic and extrahepatic events Lipid accumulation in hepatocytes may result in lipotoxicity A key consequence of lipotoxicity is the formation and release of extracellular vesicles () may act on neighboring cells and trigger angiogenic and fibrotic responses. also carry a footprint of the cell/tissue of origin and are release into the bloodstream suggesting that monitoring in circulation as a promising novel disease biomarker Collaborators Stanley Hazen (CC) Thomas McIntyre (CC) Maurizio Parola (Univ. of Torino) Laura Nagy (CC) Leon Adams (Perth, Australia) Jonathan Smith (CC) Valerio Nobili (Rome, Italy) Marco Arrese (Santiago, Chile) Craig McClain (Louisville) Chris Ramsden (NIAAA Funding Mathew Cooper (Univ. of Queensland) Rohit Kohli (Univ. of Cincinnati) Sonia Caprio (Yale University) Susan Fisher-Hoch (UT Houston) Michael Fallon (UT Houston) Santiago Horgan (UCSD) Rohit Loomba (UCSD) Ekihiro Seki (UCSD) Hal Hoffman (UCSD) Vivian Hook (UCSD) Michael Karin (UCSD) NIH: R1 DK51, U1 AA9, R1AA357, Gilead Sciences, Conatus Pharmaceutical 9