Obesity-Related Tissue Damage

Transcription

1 Obesity-Related Tissue Damage Anna Mae Diehl, MD Duke University Sept 2018 No conflicts to disclose

2 Obesity-Related Tissue Damage Impacts virtually every organ Cardiovascular System (ASCVD, CHF) Endocrine (Type 2 DM) Kidneys (CRF) Brain (CVA, Dementia) GI Tract (BE, Cancers, NAFLD) Strongly correlates w/ Metabolic Syndrome (MetS) Dyslipidemia/Insulin-resistance/Hypertension Targets non-healthy subset of obese individuals Hepatic steatosis differentiates non-healthy from healthy obese Samocha-Bonet D, Obes Rev 2014 Why? (There s more to it than fat)

3 Obesity-related comorbidity* Associates with T2DM, MetS Extremely prevalent NonAlcoholic Fatty Liver Disease Afflicts at least 25% American adults Incidence rising Epidemic of childhood/adolescent obesity * Can occur without obesity (e.g., lipodystrophy)

4 NAFLD Two Main Subtypes US Adult Population (100%) NonAlcoholic Fatty Liver (a.k.a. simple steatosis ) NAFLD (~25%) NAFL (75%) NASH (25%) NonAlcoholic SteatoHepatitis (a.k.a. NASH )

5 NAFLD Pathologic Spectrum with Differential Prognosis NAFL NASH Dynamic! Hepatocyte Fat Hepatocyte Death Cirrhosis PLC

6 Outcome of Injury Dictated by Efficacy of Repair Dying Cells Signals Replacements Dead Cells Replacements Recovery Death Disease Acute Liver Failure Cirrhosis

7 NAFLD Fibrosis Severity Independent predictor for Liver-specific morbidity Ascites, varices, encephalopathy F0-1 (1) < F-2 (7.5x) < F-3 (13.8x) < F-4(47.5x) Liver-specific mortality/olt 7x increased in F-4 versus F-0 All-cause mortality 3x increased in F3-4 versus F-0 NOT predicted by NAFLD Activity Score (NAS), NAS components (steatosis, inflammation, ballooning) Ekstedt M, et al. Hepatology 2014 Angulo P, et al. Gastroenterology Gastroenterology 2015

8 Sensitivity Liver Gene Expression Differences in Mild (F0-1) vs. Severe (F3-4) NAFLD Heirarchical Clustering ROC curve (Mild vs. Severe NAFLD) Top 100 probes with 1000 training/testing splits N=40 N=32 1 0,8 0,6 0,4 0, , Specificity 64 gene signature differentiates Severe from Mild NAFLD Logistic Regression Analysis 20 genes Independently correlate with NAFLD severity Moylan, C. et al. Hepatology Feb, 2014.

9 Functional Analysis of Severe NAFLD Transcriptome Onco-fetal Reprogramming In Diseased Liver? Fetal-like Top Biological Functions Diseases and Disorders p value Genes (n) Cancer Reproductive System Disease Gastrointestinal Disease Cardiovascular Disease Genetic Disorder Molecular and Cellular Functions Cellular Movement Cellular Growth and Proliferation Cell Morphology Cellular Assembly and Organization Top Ingenuity Pathways Tissue remodeling Regeneration Wound healing Progenitor Cells Development Moylan, C. et al. Hepatology Feb, Protein Synthesis Top Canonical Pathways Ratio Fatty Acid Metabolism /184 Valine, Leucine and Isoleucine Degradation /107 Bile Acid Biosynthesis /105 Glycine, Serine and Threonine Metabolism /147 Butanoate Metabolism /128 Top Toxicology Functions Hepatotoxicity qrt-pcr Validation Hepatocellular Carcinoma Liver Cholestasis Liver Necrosis/Cell Death Liver Hepatitis Liver Proliferation

10 Cirrhotic Liver Pro-carcinogenic Cancer risk increases with duration of exposure Greater than 2% per year in cirrhosis Second primaries are the norm Field effect Fibrosis Marker of Defective Repair NAFLD-induced Cirrhosis Activated endothelia, immune cells, progenitors, myofibroblasts, fibrous matrix

11 Adult Hypothesis Transient Liver injury Regenerate Onco-fetal Reprogramming Persistent Adult Fetal Healthy Fetal Diseased ALF Cirrhosis Cancer

12 Implications for NASH? Pathways that regulate cell plasticity to accomplish organogenesis during development reactivate to regenerate the liver during NASH Dysregulation of these pathways promotes outgrowth of cell types that are necessary, but not sufficient, for effective regeneration/recovery Myofibroblasts, hepatocyte progenitors Persistent accumulation of these repair-related cell types impedes effective repair + promotes development of cirrhosis [and liver cancer]

13 Hedgehog Pathway Regulates fate decisions in stem/progenitor cells Oncofetal pathway Active in development Down-regulated at birth Re-activates in many adult cancers Activated in multiple liver diseases before cancer develops Promotes myofibroblast accumulation Drives fibrosis progression in NASH animal models Promotes outgrowth of immature hepatocytes Adenomas in obese women with NAFLD characterized by chromosomal translocation that activates HH signaling Machado & Diehl, J Hepatol, Oct 2017 Nault, Gastroenterology 2017

14 NASH Lipotoxity ER Stress Ubiquitinated cytoskeletal proteins accumulate Stop making Cytoskeletal proteins K8/18 (brown)/ub (red) pretreatment VitE responder (patient X ) (400x) Hepatocytes swell (balloon) Damaged hepatocytes generate DAMS (Shh) Rangwala F, et al. J Pathol 2011 Guy CD, et al. Hepatology 2015 Shh (brown) pretreatment VitE responder (patient X ) (400x)

15 Ballooned hepatocytes surrounded by Hh-responsive myofibroblasts Guy CD, et al. Hepatology; Jan 2015 IHC: gli-2 (red, Hh-responsive) α-sma (brown, myofibroblast)

16 Reducing hepatocyte lipotoxicity (ballooning) reduces hepatic production of Hedgehog ligands Vit E Shh (brown) pre-treatment VitE responder (patient X ) (400x) Shh post-treatment VitE responder (patient X ) (400x) Guy CD, et al. Hepatology; Jan 2015

17 Reducing Hedgehog ligands reduces accumulation of Hh-responsive myofibroblasts Vit E Pre-treatment VitE responder (patient X ) (400x) Post-treatment VitE responder (patient X ) (400x) Guy CD, et al. Hepatology; Jan 2015 IHC: gli-2 (red, Hh-responsive) α-sma (brown, myofibroblasts)

18 Hepatocyte derived Hh ligands drive fibrosis in NASH Wang X, et al. Cell Metab 2016

19 Autocrine-Paracrine Hedgehog Signaling Orchestrates Wound Healing Response SIGNALS Promote outgrowth of liver progenitors Stimulate vascular remodeling Recruit immune cells Regulate immune cell polarization Regulate matrix remodeling Control size of MF populations

20 Wound Healing Response Double Edged Sword SIGNALS Promote outgrowth of liver progenitors CANCER Stimulate vascular remodeling PORTAL HTN Recruit immune cells Regulate immune cell polarization INFLAMMATION Regulate matrix remodeling Control size of MF populations FIBROSIS

21 Hedgehog Interacts with Other Onco-Fetal Pathways Shh promotes activation of Yap1 (Hippo kinase target) in neural progenitors Hedgehog signaling promotes Yap1 activation in hepatic stellate cells Yap1 activity is necessary to stellate cells to be myofibroblastic

22 mrna Fold Change mrna Fold Change mrna Fold Change Hedgehog MF-HSC Q-HSC Smo Gli1 asma Collagen 1a1 PPARg * LRAT * * * * * Yap mrna Yap target genes Yap activity YAP CTGF AREG AdGFP AdCre * * * pyap S127 YAP plats1 T1079 Lats1 β-actin 1,5 1 0,5 0 YAP Lamin B NT * * * * c NT YAPΔ1 YAPΔ2 * Yap MF-HSC Hh signaling Swiderska-Syn et al. Hepatology 2017

23 Hippo Kinase Pathway Regulates fate decisions in stem/progenitor cells Hippo kinases INactivate pluripotent transcription co-factors (YAP1, TAZ) Phospho-YAP1 degraded, not retained in nucleus target genes off LOSS of YAP1 formation of epithelia Oncofetal regulation INactive in development UP-regulated at birth SILENCED in many adult cancers Silenced during adult liver regeneration (YAP1/TAZ accumulate) YAP1 (ductal cells)/taz (hepatocytes) MF accumulation NASH fibrosis

24 YAP Accumulation of Hh-responsive MF and Immature Ductal Cells Correlate with Fibrosis Stage in Human NAFLD Not-NASH Fibrosing NASH 2 P= ,5 Shh 1 0,5 0 F0-F1 F2-F4 asma (MF) R=0.642 P<0.001 R=0.562 P<0.001 K19 Yap1 YAP = brown K19 = green Immature Ductal Cells Machado et al. J Hepatol 2015

25 Fibrosis Severity Parallels YAP1 Accumulation in Ductular Cells In Mouse Models of NASH Machado MV, et al. J Hepatol; Oct 2015

26 Summary #1 Pathways that are high in fetal livers/low in healthy adult livers are reactivated in NASH Hedgehog ON Activate downstream targets (Smo/Glis) fibrosis, progenitors Pathways that are low in fetal liver/high in healthy adult livers are silenced in NASH Hippo kinases OFF Activate Yap/Taz fibrosis, progenitors Fetal Reprogramming occurs in NASH

27 Clinical Implications What signals push HSC to become fibrogenic myofibroblasts? Hedgehog, Yap (Notch, Wnt, Leptin, Others??) Can HSC resist or recover from myofibroblastic (MF) reprogramming? Yes? (recover) Is there inter-individual variability in HSC susceptibility to MF programming? Impact of environmental exposures (diet, microbiome), age, genetic/epigenetic forces? Basis for heterogeneous NAFLD outcomes?? Metabolic syndrome??

28 Hypothesis Liver injury Regenerate Hh OFF Adult Fetal Healthy Transient Onco-fetal Reprogramming Hh ON Persistent Hh OFF Adult Fetal Diseased NASH Cirrhosis Cancer Hh ON

29 Hypothesis Liver injury Regenerate Hh OFF Adult Fetal Healthy Transient Insufficient Onco-fetal Reprogramming Hh ON Hh OFF Adult Fetal Hh ON Diseased Degeneration Atrophy

30 Hedgehog Controls White adipose development Vasculogenesis/angiogenesis Pancreatic islet cell mass Regulates and is regulated by lipids Ligands: lipid-modified, carried in lipoproteins Signaling activity directly regulated by sterols Modulates lipid/glucose metabolism Inhibits lipogenesis Promotes FA b-oxidation Promotes glycolysis Inhibits gluconeogenesis Liver correlate of the MetS NAFLD Pospisilik & Esterhauser, Cell 2012 & 2014; Eaton, Development 2009 & PLoS Biology 2013; Diehl, Gastroenterology 2014; Matz-Soja, Elife 2017; Myers, PNAS 2017; Huang Cell, 2018

31 Changes in Hedgehog activity linked to Obesity-Metabolic Syndrome in Humans Mutation in Hedgehog inhibitor (DYRK1B) visceral adiposity, T2DM, premature CAD in 3 kindreds Mutation potentiates DRYK1B Hedgehog inhibitory activity Hedgehog inhibition known to cause adiposity (flies and mice) reduced viability of pancreatic beta cells (mice) decreased angiogensis (mice) Keramati et al. New Engl J Med 2014; 370:

32 Hedgehog Suppression Promotes Hepatic Steatosis Humans with Smo mutations that inhibit Hedgehog signaling hepatic steatosis Mice with impaired Hedgehog signaling (haploinsufficiency of Gli2) obesity, fatty liver but protected from diet-induced NASH/fibrosis Guillen-Sacoto, J Hepatol 2017

33 Summary #2 Inability to reactivate pathways that are high in fetal livers/low in healthy adult livers promotes obesity, MetS, fatty liver (NAFL) Hedgehog OFF Can t activate downstream targets (Smo/Glis) degenerative diseases? Depletion of stem/progenitor cell populations?? Defective Fetal Reprogramming in NAFLD

34 Hypothesis Tissue injury Regenerate Adult Transient Onco-fetal Reprogramming Excessive Fetal Healthy Insufficient Adult Adult Fetal Fetal Fatty liver Degeneration Atrophy Diseased NASH Cirrhosis Cancer

35 Obesity-Related Tissue Damage Summary Characterized by dysregulation of pathways that control fetal-adult switching Both over- and under-activation of the fetal program tissue damage Over-activation may promote fibrosis, cancer Under-activation/pre-mature termination may deplete stem cell pools and promote degenerative diseases More research is needed to delineate factors that regulate fetal-adult/adult-fetal programming Genetic Epigenetic Environmental

36 Diehl Lab Jason Sicklick* Steve Choi Alessia Omenetti* Youngmi Jung* Raf Witek* Greg Michelotti* Vanessa Teaberry* Fatima Rangwala* George Philips* Isaac Chan* Guanhua Xie* Yuping Chen* Gamze Karaca* Marzena Swiderska-Syn* Wing Kin Syn* Mariana Machado* Jeongeun Hyun She Hoon Oh Richard Premont Mark Jewell George Dalton Kuo Dui * Former lab members Thanks! Duke Collaborators Cindy Guy Jack Keene Matt Friedersdorf Outside Collaborators Undayan Apte (Yap flox mice) Funding Sources NIAAA/NIDDK Duke Endowment