The Impact of Age on Liver The Impact of Age on Liver Allograft Gene ExpressionAllograft Gene Expression

Michael B. Ishitani, MDMichael B. Ishitani, MDWilliam J. von Liebig Transplant CenterWilliam J. von Liebig Transplant Center

Mayo Eugenio Litta Children’s HospitalMayo Eugenio Litta Children’s Hospital

Mayo Medical School, Foundation and ClinicMayo Medical School, Foundation and Clinic

Rochester, MinnesotaRochester, Minnesota

Aging: What really happens...Aging: What really happens...

The Aging ProcessThe 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 LiverAging 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

Aging and the LiverAging 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

Aging and the LiverAging 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 Age-related Changes vary by Organ/TissueOrgan/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 Age-related Changes vary by Organ/TissueOrgan/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)• 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

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 Measurement of Lipofuscin in Different Age Groups after Liver Transplant Age Groups after Liver Transplant

• Group 1: Young liver to Young recipient (Y-Y)Group 1: Young liver to Young recipient (Y-Y)• Donor < 18 yoDonor < 18 yo• Recipient < 18 yoRecipient < 18 yo

• Group 2: Old liver to Young recipient (O-Y)Group 2: Old liver to Young recipient (O-Y)• Donor > 50 yoDonor > 50 yo• Recipient < 18 yoRecipient < 18 yo

• Group 3: Old liver to Old recipient (O-O)Group 3: Old liver to Old recipient (O-O)• Donor > 50Donor > 50• Recipient > 65 yoRecipient > 65 yo

Materials and MethodsMaterials and Methods

• Exclusion criteria:Exclusion criteria:• Survival < 6 monthsSurvival < 6 months• No follow-up biopsies at 1 yearNo follow-up biopsies at 1 year• ACR, CMVACR, CMV

• Routine protocol biopsies: Routine protocol biopsies: • Transplant, 7d, 4 m, then yearlyTransplant, 7d, 4 m, then yearly• Standard H&EStandard H&E• Histologic review: blindedHistologic review: blinded

• Grading of lipofuscin:Grading of lipofuscin:• 00 nonenone• 11 few immediate perivenular hepatocytesfew immediate perivenular hepatocytes• 2 2 all immediate perivenular hepatocytesall immediate perivenular hepatocytes• 33 50-75% of zone 3 hepatocytes50-75% of zone 3 hepatocytes• 44 > 75% of zone 3 hepatocytes> 75% of zone 3 hepatocytes

Changes in Lipofuscin over Time

Group Donor(y) Recipient(y)Y-Y (n=9) 8.2 4.7O-Y (n=6) 59.3 10.3O-O (n=9) 60.8 63.9

Group Donor 7d 4m 1y 2yY-Y 0 0 0 0 0O-Y 3.6(3-4) 2.5(2-4) 2(1-3) 1.4(0.3) 1.0O-O 3.3(2-4) 3.0(2-4) 3.0(2-4) 3.0 3.0

Table 1

Table 2

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 ExpressionGene 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 AssayGene Expression Assay

cDNAcDNAFragmentFragment(heat, Mg(heat, Mg2+2+))

LL LL LL LL

Wash Wash StainStainScanScan

HybridizeHybridize

(16 hours)(16 hours)

Labeled transcriptLabeled transcript

TotalTotalRNARNA

IVTIVT

((LL-C)-C)

Labeled fragmentsLabeled fragments

LL LL

LL

LL

CellsCellsAAAAAAAA

GeneChip Expression AnalysisGeneChip Expression AnalysisHybridization and StainingHybridization and Staining

cRNA TargetcRNA Target

ArrayArray

Streptravidin-Streptravidin-phycoerythrinphycoerythrinconjugateconjugate

Hybridized ArrayHybridized Array

Gene Chip—Comparison AnalysisGene Chip—Comparison Analysis

ControlControl

NASHNASH

3-fold change3-fold change

2-fold change2-fold change

Materials and MethodsMaterials and Methods

• Group 1: Y-YGroup 1: Y-Y (n=2)(n=2)

• Group 2: O-YGroup 2: O-Y (n=2)(n=2)

• Group 3: O-OGroup 3: O-O (n=2)(n=2)

• High density oligonucleotide microarrayHigh density oligonucleotide microarray• Hu6800 GeneChip, Affymetrix; Santa Clara, CAHu6800 GeneChip, Affymetrix; Santa Clara, CA

• Snap biopsies at reperfusion and 1 year (0.5g)Snap biopsies at reperfusion and 1 year (0.5g)

• Comparisons made between 1h and 1y in each Comparisons made between 1h and 1y in each groupgroup

Results of Gene Chip AnalysisResults of Gene Chip Analysis

A B G ene

Im m une function/Lipid m etabolismá 3.0 (á 2.0) H LA class-I

á 3.0 (á 2.0) H LA class-II

á 4.4 (á 2.5) Prop. C oA carboxylase

á 3.6 (á 2.4) 3-Oxoacyl C oA th iolase

A = old (donor) to young (rec ipie nt); B= old (donor) to old (re cipient).

A B Gene

DNA maintenance/ Protein metabolismá 4.6 (á 2.4) Glutathione peroxidase

á 4.2 (á 2.9) Catalase

á 6.4 (á 2.3) Elongation factor-TU

á 7.3 (á 3.1) Transcription EF-SII

A= old (donor) to young (recipient); B= old (donor) to old (recipient).

A B Gene

Glucose metabolismGlucose metabolismá 4.6 (á 2.0) GLUT-2

á 4.9 (á 2.1) Glycogen synthase

A= old (donor) to young (recipient); B= old (donor) to old (recipient).

A B Gene

Stress responseâ 7.8 (â 3.2) HSP-40

â 14 (â 3.5) HSP-70

A= old (donor) to young (recipient); B= old (donor) to old (recipient).

ConclusionsConclusions

• Changes in gene expression are seen after transplantation

• Changes in gene expression in some cellular processes associated with aging

Future StudiesFuture 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

DiscussionDiscussion

• 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?