Novel Therapeutic Combinations for the Treatment of Hepatic Fibrosis Principal Investigator: Joseph Gosnell Faculty Advisors: Dr. Mark Blais Dr. Ben Kalu Dr. Kim Mitchell

Patient G1 50 year old male Diagnosed in 2011 with NASH syndrome (Non-Alcoholic Steatohepatitis) Has been hospitalized more than 22 times since 2011 Has been on the transplant list for more than 3 years Must have invasive procedures Muscle wasting, jaundice, extreme anorexia/weight loss, fatigue, and encephalopathy Has developed liver cancer in the past year. Experienced a heart attack, liver failure, kidney failure, hepatic encephalopathy, loss of 30 lbs. (currently 151 lbs.), and chronic vomiting/nausea all in the past 6 months

Epidemiology of Hepatic Fibrosis Hepatic fibrosis occurs from both acute and chronic conditions Acetaminophen toxicity Chronic alcoholism Non-alcoholic Steatohepatitis (NASH syndrome) Carcinogenic compounds (carbon tetrachloride) Leads to liver failure, blood clotting abnormalities, accumulation of fluid in the body tissues (especially the abdomen), and many other complications Hepatocellular carcinoma (HCC), the 5th most common cancer, has an increased occurrence from hepatic fibrosis2 33,642 people die per year from chronic liver disease and cirrhosis (latest available numbers from CDC)2

Current Hepatic Fibrosis Targets The main physiological component being examined is the Hepatic Stellate Cell (HSC); this star shaped cell is activated in response to liver injury and begins excreting extracellular matrix (ECM) of collagen type I and III fibers, forming scar tissue.3 The scar tissue made by HSCs replaces functional liver tissue, causing liver failure Research is ongoing for anti-fibrotic compounds that will keep HSCs inactive during liver repair, as well as ways to break down scar tissue after it is formed (MMP activators, TIMP inhibitors)3

Current Therapeutic Targets for Hepatic Fibrosis3

Experimental Design Isolation of Hepatic Stellate Cells (HSCs) and Kupffer Cells (KCs) from Balb/cJ mice Density gradient centrifugation to isolate the cells from liver samples4 Co-culturing of HSCs and KCs to achieve in vivo gene expression levels5 Induction of hepatic fibrosis in Balb/cJ mice with carbon tetrachloride (CCl4)6 and treating with therapeutic combinations monitoring for fibrosis progression Octreotide inhibits hepatic heme oxygenase 1 expression Losartan suppresses TGF-β, a fibrogenic cytokine Pioglitazone reduces TIMP-1 and TIMP-2 mRNA levels, while sparing MMP-13 (breaks down ECM) Halofuginone lowers TIMP-1, α-SMA, TGF-β1, and ALT/AST levels Track fibrosis progression using LICOR Odyssey imager using infrared antibodies

Communication with International Experts in Hepatic Fibrosis Dr. Florian Winau of Harvard University helped to outline the intricacies of separating the various hepatic cells once removed Dr. Alena Jiroutova from Charles University (Prague) assisted in experimental design Dr. Michael Huber from RWTH Aachen University (Germany) assisted in connecting me with leaders in the field

Research Grants Tentative approval from the Department of Biology/Chemistry Faculty Research Grant through LU has been submitted via Dr. Blais of the Biology/Chemistry Department Discussing outside funding sources with the Icahn School of Medicine, the American Gastroenterological Association, and the American Liver Foundation

Acknowledgements Dr. Blais, Dr. Kalu, and Dr. Mitchell of the LU Biology/Chemistry Department for their active participation in this project Dr. Price and Dr. Isaacs for their insight into LU funding Dr. Brewer and Dr. Swanson of LUCOM for their willingness to allow me to use LUCOM research facilities Dr. Winau of Harvard Medical School for his knowledge of density gradient centrifugation techniques Dr. Jiroutova of Charles University for her assistance in experimental design Dr. Huber of RWTH Aachen University for his directing me to experts in the field

References Dr. Kenneth Steibel1 Centers for Disease Control and Prevention. (2015)2 Fallowfield, J. A. (2010). Therapeutic targets in liver fibrosis. American Journal of Physiology: Gastrointestinal and Liver Physiology, 300. G709-7153 Winau, F., Maschmeyer, P., and Flach, M. (2011). Seven steps to hepatic stellate cells. Journal of Visualized Experiments, 51. 4 DeMinicis, S., Seki, E., Uchinami H., Kluwe, J., Zhang, Y., Brenner, D., and Schwabe, R. (2007). Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology, 132.5 Fujii, T., Fuchs, B., Yamada, S., Lauwers, G., Kulu, Y., Goodwin, J., Lanuti, M., and Tanabe, K. (2010). Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression of CD133 and epidermal growth factor. BMC Gastroenterology, 10 (79).6

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