The Impact of Age on Liver Allograft Gene Expression

1
The Impact of Age on Liver Allograft Gene
Expression

• Michael B. Ishitani, MD
• William J. von Liebig Transplant Center
• Mayo Eugenio Litta Childrens 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/DN
  A
• 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 agesignificance 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 (gt65)
• 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 lt 18 yo
• Recipient lt 18 yo
• Group 2 Old liver to Young recipient (O-Y)
• Donor gt 50 yo
• Recipient lt 18 yo
• Group 3 Old liver to Old recipient (O-O)
• Donor gt 50
• Recipient gt 65 yo

12
Materials and Methods

• Exclusion criteria
• Survival lt 6 months
• No follow-up biopsies at 1 year
• ACR, CMV
• Routine protocol biopsies
• Transplant, 7d, 4 m, then yearly
• Standard HE
• 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 gt 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
L
IVT (L-C)
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 ChipComparison Analysis
3-fold change
2-fold change
Control
NASH
19
Materials and Methods

• Group 1 Y-Y (n2)
• Group 2 O-Y (n2)
• Group 3 O-O (n2)
• 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
(No Transcript)
21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24
Conclusions

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

25
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

26
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?