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The Impact of Age on Liver Allograft Gene Expression

The Impact of Age on Liver Allograft Gene Expression. Michael B. Ishitani, MD William J. von Liebig Transplant Center Mayo Eugenio Litta Children’s Hospital Mayo Medical School, Foundation and Clinic Rochester, Minnesota. Aging: What really happens. The Aging Process.

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The Impact of Age on Liver Allograft Gene Expression

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  1. The Impact of Age on Liver Allograft Gene Expression Michael B. Ishitani, MD William J. von Liebig Transplant Center Mayo Eugenio Litta Children’s Hospital Mayo Medical School, Foundation and Clinic Rochester, Minnesota

  2. Aging: What really happens...

  3. The Aging Process • What occurs as we Age? • Anatomic/histologic • Physiologic changes • Alterations in cellular organelles/mitochondria/DNA • Alterations in gene expression • Individual variation • What factors can be slowed or perhaps reversed? • “Fountain of Youth”

  4. Aging and the Liver • What is known of Aging and the Liver? • Hans Popper: Aging and the Liver; In Progress in Liver Disease, 1986 • Hepatocyte life span is long (years) • Liver mitochondria life span short (days) • Large functional reserve of hepatocyte mass (15-30%) • High regenerative capability • Gross changes: • slight decrease in weight • accumulation of brown pigment (lipofuscin) • marker of age…significance unclear • decreased blood flow

  5. Aging and the Liver • Microscopic changes: • polyploidy • variation in mitochondrial size • decrease in ER size • increased fibrosis • accumulation of lipofuscin • Physiology: • Alterations in enzyme activity (variable) • Normal protein synthesis (variable) • Reduction in respiratory cycle function [cytochrome C oxidase (COX)] • Slower/lower regeneration after injury

  6. Aging and the Liver • Drug metabolism • Decreased due to mass/blood flow • Altered enzyme function (cytochrome P450) • Others: • Increase in mitochondrial DNA mutations • Decrease in mitochondrial DNA repair systems • Altered telomerase function leading to cell senescence

  7. Age-related Changes vary by Organ/Tissue • Barrazoni, Short, and Nair: Jnl Bio Chem, 2000 Rat model of aging: • Mitochondrial DNA copy number • COX transcription level • COX enzyme activity • Compare: • Skeletal muscle • Heart • Liver

  8. Age-related Changes vary by Organ/Tissue • Mitochondrial DNA • Decreased in liver and skeletal muscle • Heart unchanged • COX transcription levels • Decreased in skeletal muscle • Liver, heart unchanged • COX enzyme levels • Decreased in skeletal muscle • Liver, heart unchanged

  9. Liver Transplantation and Aging • Livers from Older donors (>65) • Primary non-function rate • Delayed graft function • Ischemia-reperfusion injury • Retransplantation rate increased • Increase in use despite these problems • Does not appear livers from an older donor “wear out” • Follow-up short-term

  10. What happens to Older donor livers over time? • Innocenti, et. al. Aging-related changes reverse in Pediatric Liver transplant recipients of Old donor livers, AST 2001 • Light microscopy • Accumulation of lipofuscin as intracytoplasmic inclusions/granules in perivenular hepatocytes • Begins in second decade and progresses • Clearance by activated macrophages in the presence of necroinflammatory activity

  11. Measurement of Lipofuscin in Different Age Groups after Liver Transplant • Group 1: Young liver to Young recipient (Y-Y) • Donor < 18 yo • Recipient < 18 yo • Group 2: Old liver to Young recipient (O-Y) • Donor > 50 yo • Recipient < 18 yo • Group 3: Old liver to Old recipient (O-O) • Donor > 50 • Recipient > 65 yo

  12. Materials and Methods • Exclusion criteria: • Survival < 6 months • No follow-up biopsies at 1 year • ACR, CMV • Routine protocol biopsies: • Transplant, 7d, 4 m, then yearly • Standard H&E • Histologic review: blinded • Grading of lipofuscin: • 0 none • 1 few immediate perivenular hepatocytes • 2 all immediate perivenular hepatocytes • 3 50-75% of zone 3 hepatocytes • 4 > 75% of zone 3 hepatocytes

  13. Changes in Lipofuscin over Time Table 1 Table 2

  14. Observation/Discussion • The amount of lipofuscin decreases over time after an old donor liver is transplanted into a young recipient • Mechanism is unclear • Increased clearance (recipient) • Decreased production (donor) • Both • More efficient macrophage response/function? • Unknown factors in the young recipient environment? • Old donor hepatocytes become “young”, resulting in decreased production or increased clearance?

  15. Gene Expression • GeneChip Expression Probe Arrays • Allow screening of multiple human genes • Identifies specific gene expression and relative mRNA production • Commercially available • Gained acceptance as a means to screen for changes in gene expression at the cellular level

  16. Gene Expression Assay Cells AAAA Labeled transcript IVT (L-C) L L L L Total RNA cDNA Fragment (heat, Mg2+) L L Hybridize (16 hours) L Wash Stain L Scan Labeled fragments

  17. GeneChip Expression AnalysisHybridization and Staining Array Hybridized Array cRNA Target Streptravidin-phycoerythrin conjugate

  18. Gene Chip—Comparison Analysis 3-fold change 2-fold change Control NASH

  19. Materials and Methods • Group 1: Y-Y (n=2) • Group 2: O-Y (n=2) • Group 3: O-O (n=2) • High density oligonucleotide microarray • Hu6800 GeneChip, Affymetrix; Santa Clara, CA • Snap biopsies at reperfusion and 1 year (0.5g) • Comparisons made between 1h and 1y in each group

  20. Conclusions • Changes in gene expression are seen after transplantation • Changes in gene expression in some cellular processes associated with aging

  21. Future Studies • Rat transplant model • O-Y • O-O • Y-Y • Analysis: • mitochondrial DNA copy number • mitochondrial mutations • COX transcription • COX activity • telomerase activity • telomere length • Others

  22. Discussion • Transplant model: • Unique insights into Gene expression • Aging: • What are the mechanisms of aging? • What processes can be reversed? • If these changes are reflected in altered gene expression, than what are the mechanisms? • Are the changes due to the recipient environment, changes within the donor liver, or some combination of both?

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