Anti-Apoptotic Effects of Cellular Therapy Jason Lapetoda 1, Lee K Landeen, PhD 1, George K Michalopoulos, MD 2, Patricia W Bedard, PhD 1 1 Vital Therapies, Inc., San Diego, CA, USA 2 University of Pittsburgh Medical Center, Pittsburgh, PA, USA
Alcoholic Hepatitis: Pathogenesis, Anti-Apoptotic Component Louvet A, Mathurin P. Nat Rev Gastro & Hepat 12, 231 242 (2015) 17th International Symposium on Albumin Dialysis 2
Studies Currently Targeting AH and Alcoholic Cirrhosis Anti-Apoptosis, Anti-Oxidation and Pro-Regeneration are all strategies currently being targeted in the clinic Louvet A, Mathurin P. Nat Rev Gastro & Hepat 12, 231 242 (2015) 17th International Symposium on Albumin Dialysis 3
The Two Major Routes to Apoptosis Fas Intrinsic Mitochondrial Pathway Triggered by cellular stress Extrinsic Death Receptor Pathway Initiated by ligands binding to death receptors Hepatocyte apoptosis involves both death receptormediated and mitochondrial-mediated pathways 2 2) Malhi. Physiol Rev. 2010 17th International Symposium on Albumin Dialysis 4
Current Model of ELAD Inhibition of Hepatocyte Apoptosis 1 sfasr in ELAD CM ELAD CM Fas-Agonist 2 Amphiregulin in ELAD CM ELAD CM AR 3 Antioxidants in ELAD CM FasR EGFR ELAD CM GSH X DEATH SURVIVAL X Oxidative Stress 17th International Symposium on Albumin Dialysis 5
Primary Human Hepatocyte (PHH) Apoptosis Model Western Blotting Caspase Assay Annexin V Thaw, Count, Plate 4-hrs ELAD CM 4-hrs Fas Agonist Antibody 4-hrs 0 4 8 12 14 Time (hrs) Assess Apoptosis Modified from Berasain. J Biol Chem. 2005 17th International Symposium on Albumin Dialysis 6
ELAD Conditioned Media (CM) Reduced Apoptosis in PHH Caspase activity was reduced in both untreated and Fas agonisttreated PHH cultures in the presence of ELAD CM Result repeated in different lots of PHH and different lots of ELAD cartridges Error is SD of n=8 wells in 96-well format. (*** p<0.001 for ELAD-treated vs. untreated or Fas agonist-treated. ANOVA with Tukey post-hoc test) 17th International Symposium on Albumin Dialysis 7
ELAD CM Reduced Apoptotic Phenotypic Shift in PHH Untreated Fas-Agonist Fas-Agonist + ELAD CM Untreated controls show cobblestone morphology and minimal fluorescence Fas-agonist-treated cells are condensed and show greater annexin V fluorescent staining Fas-agonist + ELAD CM-treated PHH retain cobblestone morphology and minimal fluorescence 17th International Symposium on Albumin Dialysis 8
ELAD CM Reduced Apoptotic Cleavage Products of Caspase 8 and poly ADP ribose polymerase (PARP) Fas agonist-treated PHH lysates showed both increased caspase 8 (p-18) and PARP cleavage products Lysates from PHH treated with Fas agonist in the presence of CM showed a reduction of both cleavage products compared to controls EGFR inhibition reversed the protective effect 17th International Symposium on Albumin Dialysis 9
Caspase Activity (Luminescent Units) Amphiregulin Reduced Caspase Activity (ANOVA, Tukey s post test) Amphiregulin reduced PHH apoptosis, as measured by caspase activity in Fas agonist-treated PHH lysates Addition of the EGFR-inhibitor, canertinib, blocked the effects of amphiregulin 17th International Symposium on Albumin Dialysis 10
ELAD CM Induced Epidermal Growth Factor Receptor (EGFR) Signaling in PHH EGFR, MEK1/2, ERK1/2 and STAT3 were phosphorylated in lysates from ELAD CM-treated PHH EGFR-inhibitor, canertinib, reduced this signal, however, not completely 17th International Symposium on Albumin Dialysis 11
Caspase Activity (Luminescent Units) sfas Reduced Caspase Activity (ANOVA, Tukey s post test) sfas reduced apoptosis, as measured by caspase activity in Fas agonist-treated PHH lysates 17th International Symposium on Albumin Dialysis 12
The Anti-Oxidant Role of Glutathione (GSH) in AH GSH protects cells against ROS Ethanol exposure: Depletes GSH Increases ROS (reactive oxygen species) ROS cause: Oxidative stress and endoplasmic reticulum (ER) stress Steatosis Inflammation and apoptosis Figure adapted from: Odile S. 2012.DOI: 10.5772/1440 17th International Symposium on Albumin Dialysis 13
ELAD-Treatment Downregulates TCA Cycle Intermediates Treated Untreated Metabolomics data- Metabolon, MA, USA Dark green shading highlights TCA cycle intermediates significantly down regulated in ELAD-treated subjects Consistent with increased glutathione synthesis GSH is not measurable in plasma 17th International Symposium on Albumin Dialysis 14
Oxidative Stress measured by GSH:GSSG Ratio 1) lyse GSH GSH GSSG reduce GSH luciferase 2) lyse GSH GSSG X GSH GSH Luciferin-NT GSH-NT ATP Luciferin Light 15 17th International Symposium on Albumin Dialysis
ELAD CM Increased the Ratio of GSH to GSSG (Oxidized Glutathione) in Cultured PHH GSH/GSSG Molar Ratio Untreated Fas-agonist ELAD CM Fas + CM ELAD CM treatment increased GSH/GSSG ratio >5-fold above untreated cells This effect was independent of Fas-induction 17th International Symposium on Albumin Dialysis 16
Current Model of ELAD Inhibition of Hepatocyte Apoptosis 1 sfasr in ELAD CM ELAD CM Fas-Agonist 2 Amphiregulin in ELAD CM ELAD CM AR 3 Antioxidants in ELAD CM FasR EGFR ELAD CM GSH X DEATH SURVIVAL X Oxidative Stress 17th International Symposium on Albumin Dialysis 17
Working Hypothesis: ELAD Secreted Factors Have Potential to Impact Multiple Cell Types in the Liver Hepatocyte Stellate ELAD C3A Cell Cartridge Goal: Identify what factors are affecting what cells and in what ways Kupffer Endothelial 17th International Symposium on Albumin Dialysis 18
Human Aortic Endothelial Cell (HAEC) Model of LPS-Induced Apoptosis HAEC + LPS 24hr. Cell Death Adapted from Bucki R. Biochemistry. 2005 Cell death measured by cytoskeleton disruption FITC-labeled phalloidin binds to F-actin Untreated 100 μg/ml LPS 17th International Symposium on Albumin Dialysis 19
ELAD CM Inhibits LPS-Induced Cell Death in HAEC Brightfield Fluorescent Untreated CM only CM + 100 μg/ml LPS 100 μg/ml LPS *Magnification: 40x 17th International Symposium on Albumin Dialysis 20
Vascular Endothelial Growth Factor (VEGF) Partially Protects HAEC Treated with LPS Brightfield Fluorescent Untreated LPS only LPS + CM LPS + VEGF VEGF at levels found in ELAD CM, reduces LPS damage, but not as well as ELAD CM, suggesting multiple mechanisms *Magnification: 40x 17th International Symposium on Albumin Dialysis 21
Human Aortic Endothelial Cell (HAEC) Model of Oxidative Stress HAEC + H 2 O 2 4hr. Increased ROS Decreased GSH Cell stress measured by GSH:GSSG Ratio 1) lyse GSH GSH GSSG reduce GSH luciferase 2) lyse GSH GSSG X GSH GSH Luciferin-NT GSH-NT ATP Luciferin Light 17th International Symposium on Albumin Dialysis 22
G S H / G S S G M o l a r R a t i o G S H / G S S G M o l a r R a t i o ELAD CM Blocked Oxidative Stress in HAEC G S H / G S S G M o l a r R a t i o 5 0 4 0 5 0 G S H / G S S G M o l a r R a t i o U n t r e a t e d C M H U2 On t2 r e( a1 tme dm ) C CM M+ H 2 O 2 ( 1 m M ) H 2 O 2 ( 1 m M ) 3 0 4 0 C M + H 2 O 2 ( 1 m M ) 3 0 2 0 2 0 1 0 01 0 H 2 O 2 reduced the GSH/GSSG ratio 6-fold below untreated 0 GSH/GSSG ratio increased over 8-fold above H 2 O 2 cells with ELAD CM treatment 17th International Symposium on Albumin Dialysis 23
Future Directions Evaluate alternate forms of hepatocyte death: Necrosis and Pyroptosis Identify other factors contributing to protective effects on endothelial cells 17th International Symposium on Albumin Dialysis
Conclusions Cellular therapy may provide significant benefit over monotherapies in treatment of AH AH is a complex multi-faceted disease ELAD treatment will deliver multiple proteins, and has the potential to impact multiple pathways and multiple cell types simultaneously and/or consecutively ELAD C3A Cell Cartridge 17th International Symposium on Albumin Dialysis 25
Acknowledgements VTL Scientific Advisory Board Cliff Steer, M.D. (University of Minnesota) Charles Dinarello, M.D. (University Colorado Denver) George Michalopoulos, M.D. (University of Pittsburgh School of Medicine) Alan Hofmann, M.D. (emeritus) (University of California, San Diego ) Fernando Camargo, Ph.D. (Harvard and Boston Children s Hospital) Nikolaos Pyrsopoulos, M.D., M.B.A. (Rutgers New Jersey Medical School) Mike Millis, M.D. (University of Chicago Medicine) 17th International Symposium on Albumin Dialysis 26
Please Visit our Poster for More Information/Discussion VTL C3A Cell-Secreted Factors Reduce in vitro Hepatocellular Injury via Multiple Mechanisms Jason Lapetoda, Lee K Landeen, PhD, George K Michalopoulos, MD, Patricia W Bedard, PhD 17th International Symposium on Albumin Dialysis 27