Transcript Impact of recipient age on graft 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
• 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”
Aging and the Liver
• What is known of Aging and the Liver?
• Hans Popper: Aging and the Liver; In
Progress in Liver Disease, 1986
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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:
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slight decrease in weight
accumulation of brown pigment (lipofuscin)
marker of age…significance unclear
decreased blood flow
Aging and the Liver
• Microscopic changes:
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•
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•
•
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
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
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
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
Liver Transplantation and Aging
• Livers from Older donors (>65)
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•
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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
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
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intracytoplasmic inclusions/granules in
perivenular hepatocytes
Begins in second decade and progresses
Clearance by activated macrophages in the
presence of necroinflammatory activity
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
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:
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•
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0
1
2
3
4
none
few immediate perivenular hepatocytes
all immediate perivenular hepatocytes
50-75% of zone 3 hepatocytes
> 75% of zone 3 hepatocytes
Changes in Lipofuscin over Time
Table 1
Group
Y-Y (n=9)
O-Y (n=6)
O-O (n=9)
Donor(y)
8.2
59.3
60.8
Recipient(y)
4.7
10.3
63.9
Table 2
Group Donor
Y-Y
0
O-Y
3.6(3-4)
O-O
3.3(2-4)
7d
4m
1y
0
2.5(2-4)
3.0(2-4)
0
2(1-3)
3.0(2-4)
0
1.4(0.3)
3.0
2y
0
1.0
3.0
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?
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
Gene Expression Assay
Cells
AAAA
IVT
Total
RNA
cDNA
Labeled transcript
L
L
L
L
(L-C)
Fragment
(heat, Mg2+)
L
Hybridize
Scan
Wash
Stain
(16 hours)
L
L
L
Labeled fragments
GeneChip Expression Analysis
Hybridization and Staining
Array
Hybridized Array
cRNA Target
Streptravidinphycoerythrin
conjugate
Gene Chip—Comparison Analysis
3-fold change
2-fold change
Control
NASH
Materials and Methods
• Group 1: Y-Y
• Group 2: O-Y
• Group 3: O-O
(n=2)
(n=2)
(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
Results of Gene Chip Analysis
A
B
G ene
Im m u n e fu n c tion /L ip id m eta b o lis m
á 3. 0
(á 2. 0)
H LA cla ss-I
á 3. 0
(á 2. 0)
H LA cla ss-II
á 4. 4
(á 2. 5)
P ro p. C oA carb o xyla se
á 3. 6
(á 2. 4)
3 -Oxo acyl C o A th iola se
A = ol d (donor) to young (rec ipie nt); B = ol d (donor) to old (re cipi ent).
A
B
Gene
DNA maintenance/ Protein metabolism
á4.6
(á2.4)
Glutathione peroxidase
á4.2
(á2.9)
Catalase
á6.4
(á2.3)
Elongation factor-TU
á7.3
(á3.1)
Transcription EF-SII
A= old (donor) to young (recipient); B= old (donor) to old (recipient).
A
B
Gene
Glucose metabolism
á4.6
(á2.0)
GLUT-2
á4.9
(á2.1)
Glycogen synthase
A= old (donor) to young (recipient); B= old (donor) to old (recipient).
A
B
Gene
Stress response
â7.8
(â3.2)
HSP-40
â14
(â3.5)
HSP-70
A= old (donor) to young (recipient); B= old (donor) to old (recipient).
Conclusions
• Changes in gene expression are seen
after transplantation
• Changes in gene expression in some
cellular processes associated with
aging
Future Studies
• Rat transplant model
• O-Y
• O-O
• Y-Y
• Analysis:
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mitochondrial DNA copy number
mitochondrial mutations
COX transcription
COX activity
telomerase activity
telomere length
• Others
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?