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Journal Club: Liver Signaling Pathways

Journal Club: Liver Signaling Pathways. Olga Filippova, Munir Nahri, Akash Patel Group 1. Liver Regeneration Signaling. More than 100 immediate early genes identified Cytokine Pathway IL6, TNF Growth Factor Pathway HGF, EGF, TGF, VEGF Termination SOCS3. Taub, 2004.

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Journal Club: Liver Signaling Pathways

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  1. Journal Club: Liver Signaling Pathways Olga Filippova, Munir Nahri, Akash Patel Group 1

  2. Liver Regeneration Signaling • More than 100 immediate early genes identified • Cytokine Pathway • IL6, TNF • Growth Factor Pathway • HGF, EGF, TGF, VEGF • Termination • SOCS3 Taub, 2004

  3. Liver Regeneration Growth Factors Mohammed end Khokha, 2005

  4. Hepatocyte Growth Factor • Synthesized by non-parenchymal cells • Stellate • Paracrine and Endocrine effects • pro-HGF precursor activated by proteases • binds to c-MET receptor • HGF/c-met signaling essential for cell cycle entry after PH • Multiple Effects • morphogenic, motogenic, mitogenic http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/PathFinder/Pathway_Maps/HGF_Pathway.html

  5. C-MET (HGF Receptor) • α, β heterodimer with tyrosine kinase activity • Dimerizes after ligand binding • Associates with multiprotein complex • downstream signal transduction http://www.icampus.ucl.ac.be/courses/SBIM2520/document/genemol/pictures/c-met/c-met.html

  6. Article Overview • Smith, M.K., Riddle, K.W., and Mooney, D.J. Delivery of Hepatotrophic Factors Fails to Enhance Longer-Term Survival of Subcutaneously Transplanted Hepatocytes. Tissue Engineering 12, 235, 2006 • Hepatocyte transplantation limited by poor cell viability • Improved vascularization with VEGF • better survival rates • Hypothesis: HGF and EGF will increase long-term cell survival VEGF PLG Scaffold hepatocytes EGF HGF Subcutaneous implant

  7. Methods and Materials

  8. Scaffold Manufacturing • 85:15 and 75:25 PLG microspheres • Standard double emulsion process • Combined 4:1 with alginate • 75:25 PLG encapsulate EGF • VEGF and HGF directly onto scaffold 5µm Smith et al, 2004 Lima and Rodrigues, 1999

  9. EGF, HGF Release Kinetics EGF Release from PLG microspheres • 125I labeled EGF • 25±2% retained in micropsheres • 55±3% incorporated into scaffold • Initial (4 day) 50% burst • 0.7% per day • Similar HGF analysis Smith et al, 2006

  10. Scaffold Implantation • Three Scaffold Types + Control • Seeded with Lewis rat hepatocytes • 62±5% efficiency • Implanted into SCID mice • 3, 7 or 14 days Experimental Scaffold Setup Control VEGF+EGF VEGF+EGF +HGF VEGF+HGF

  11. Histological Analysis • Vascularization • CD31 antigen • Vascular endothelial cells • Hematocyte Survival • Hematoxylin and eosin • Presence/lack of nucleus • E-800 light microscope Smith et al, 2006

  12. ResultsandDiscussion

  13. Vascularization Smith et al, 2006 Smith et al, 2004

  14. Hepatocyte Survival Smith et al, 2004 Smith et al, 2006

  15. Hepatocyte Survival • Similar amounts of live cells • More nucleated cells in growth factor scaffolds • Less nucleated cells in growth factor scaffolds Smith et al, 2006

  16. Hepatocyte Survival Not Improved • Saturation • VEGF  releases HGF precursor • HGF  releases VEGF • Mesentery vs subcutaneous implantation • Not enough EGF or HGF • Improper release kinetics Taub, 2004

  17. References • Mohammed, F.F., and Khokha, R. Thinking outside the cell: proteases regulate hepatocyte division. Trends in Cell Biology 15, 555, 2005. • Lima, K.M., and Rodrigues J.M.Jr. Poly-DL-lactide-co-glycolide microspheres as a controlled release antigen delivery system. Braz J Med Biol Res 32, 171, 1999. • Smith, M.K., Peters, M.C., Richardson, T.P., Garbern, J.C., and Mooney, D.J. Locally Enhanced Angiogenesis Promotes Transplanted Cell Survival. Tissue Engineering 10, 63, 2004. • Smith, M.K., Riddle, K.W., and Mooney, D.J. Delivery of Hepatotrophic Factors Fails to Enhance Longer-Term Survival of Subcutaneously Transplanted Hepatocytes. Tissue Engineering 12, 235, 2006. • Taub, R. Liver Regeneration: From Myth to Mechanism. Nature 5, 836, 2004. • Fausto, N., Campbell, J.S., and Riehle, K.J. Liver Regeneration. Hepatology 43, S45, 2006. • Huh, C.G., Factor, V.M., Sanchez, A., Uchida, K., Conner, E.A., Thorgeirsson, S.S. Hepatocyte growth facotr/c-met signaling pathway is required for effic ient liver regeneration and repair. Cell Biology 101, 4477, 2004. • Borowiak, M., Garratt, A.N., Wustefeld, T., Strehle, M., Trautwein, C., Birchmeier, C. Met provides essential signals for liver regeneration. Cell Biology 101, 10608, 2004. • Lysy, P.A., Campard, D., Smets, F., Najimi, M., and Sokal, E.M. Stem cells for liver tissue repair: Current knowledge and perspectives. World Journal of Gastroenterology 14, 864, 2008.

  18. Questions? Olga Filippova, Munir Nahri, Akash Patel Group1

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