Liver regeneration- principles, measurement and status

Perceptor Prof. S K Acharya

Introduction• Liver possesses an

ability to regenerate following partial resection until it attains its original size

• Clinically important- this can be an alternative to liver transplantation

Introduction• Regeneration means the reconstitution of a

structure that has been excised. Eg. complete re-growth of the limb of a newt or tail of lizard

• In c/o liver it is actually compensatory hyperplasia rather than true regeneration

• Total liver mass rather than the lobulated anatomic configuration is restored

Introduction• In normal liver fewer than 1 in 10,000 hepatocytes

undergo mitosis at any given time, they remain in Go

• Normal hepatocytes respond poorly to mitogens like TGF, EGF & HGF

• Liver possesses a unique capacity to replace tissue mass after injury or loss of liver mass

• Regenerative capacity is insufficient - CVH or alcoholism- cirrhosis

Experimental models

• Model to study liver regeneration is removal of 2/3 of the liver of rats/mice a technique known as 2/3 PHx • Liver cells proliferate - original liver mass is restored within 5-7 days• No massive inflammation, fibrosis or scarring

Bucher NL. Regeneration of Mammalian Liver.Int. Rev. Cytol.1963;15: 245–300

The regenerating liver

Rodent model

• Hepatocytes are the first to enter the cell cycle• Undergo 2 rounds of cell division within 2–3

days• Proliferation of hepatic stellate cells, Kupffer

cells and biliary epithelial cells• Angiogenesis (endothelial cells) occur to re-

establish the liver vasculature

Bucher NL. Regeneration of Mammalian Liver.Int. Rev. Cytol.1963;15: 245–300

Rodent model

• The advent of gene knock out mice - various specific molecules and dissection of pathways

• No single GM mouse model demonstrates 100% mortality and a complete blockage of both DNA replication and cell proliferation

• No single gene can be considered “essential” for liver regeneration

Severity of liver injury and regeneration

Networks during liver regeneration

Check points in cell division• Hepatocytes in the

normal liver are quiescent (Go phase) and respond minimally to in vivo mitogens such as TGF EGF and HGF

• These mitogens can induce replication following infusion of TNF-a

Timing of Regeneration

• In rat, DNA synthesis peaks at 24 hours after PH, when approximately 35% of hepatocytes are in cell cycle

• Observed- circadian clock controls the G2/M transition

• G2-M transition always occur at the same time of day

Matsuo T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, Okamura H. Control mechanism of the circadian clock for timing of cell division in vivo. Science 2003;302:255-25

Timing of Regeneration

• Mammals- Circadian timing system controlled by pacemaker in the suprachiasmatic nucleus of the brain

• Synchronizes subsidiary circadian oscillators in peripheral cell types including hepatocytes

Matsuo T. Control mechanism of the circadian clock for timing of cell division in vivo. Science 2003;302:255-25

Timing of Regeneration

Genes and cyclins in regeneration

Immediate Early Genes

• Activated almost immediately (within minutes) after partial hepatectomy in mice

• 70 immediate early genes and more– Proto-oncogenes c-fos, c-jun, c-myc, and c-ets– Transcription factors- NFκB , STAT3, IGF

binding protein-1 etc

NFΚB AND IMMEDIATE EARLY GENES

• Quiescent liver NFκB in the cytosol bound to IkB

• Binding of TNF-a to TNF-R1- IkB kinase- Phosphrln of IκB

• Free NFkB Translocates to nucleus

• Transcription of IL-6• Activation of STAT3

Delayed Early Genes

• Transcribed after the immediate early gene response – Expression occurs during later phases – Depends on protein synthesis; Tr Factors– HRS/SRp40 (splicing factor and modulator of alternative

splicing of RNA transcripts) and the anti apoptotic gene bcl-x

• Pro-apoptotic genes BAK, BAD, BAX - down-regulated

Cyclins and CDKs• Family of proteins that

control the progression of cells through the cell cycle by activating Cdk

• CDk- ser/threo kinases inactive

• Cyclin attachment activation• All CDKs perform

phosphorylation of target proteins

Cyclin concentration wrt cell cycle

Cyclin_Expression.svg

Cyclins and CDKs• G1 phase; CDKs catalyze the phosphorylation of

the pRb and cause its dissociation from the E2F family of proteins

• This eliminates the repression of gene expression by pRB

• Cyclin D1 also may sequester the cell cycle inhibitor p27 with advancement in cell cycle

Integration of Cytokines and Growth Factors in Liver Regeneration

• Priming– 0-5 hrs– Reversible phase in liver regeneration, G0 - G1

phase– Cytokines, eg. TNF-α and IL-6– NFκB and STAT3 are activated– Sensitize hepatocytes to growth factors

Integration of Cytokines and Growth Factors in Liver Regeneration

• Progression– Requires HGF (mesenchymal cells) and TGF-α (hepatocyte) &

cyclins– During the progression phase, the cells move past the

restriction point in G1 to S and beyond– GH, PTH, T3 are permissive for liver regeneration – Insulin and norepinephrine are adjuvant factors

• HGF and c-met – Sources of HGF in the liver are Kupffer cells and HSC– HGF binding to of c-met activates tyrosine kinase and thus

initiates a signal transduction pathway

Summary; the sequence of signals that leads to liver regeneration

Mitogenic Signals Associated With Initiation of Liver Regeneration

• Quiescent hepatocytes express a variety of GF receptors; PDGF, VEGF, FGF receptors

• Only mitogens for hepatocytes in media are HGF and ligands of the EGFR ; EGF, TGFα, AR, HBEGF, etc

• Direct mitogens- induce a strong mitogenic response in hepatocytes

• HGF, EGF, and TGFα also induce hepatocyte proliferation when injected into mice and rats

Mechanisms of regeneration in ALF• STAT3 translocates to the nucleus with induction

of transcription of target genes • Go- G1; driven by HGF and EGF receptor ligand

family • Serum levels of HGF have been found to be

markedly elevated in the serum of adults and children in ALF

Changes in serum levels of hepatocyte growth factor in patients undergoing adult-to-adult living-donor liver transplantation. Transplantation 2003;76:1769–1770

Oval cells • HPCs- generated from the biliary compartment

in response to hepatic injury • Canals of herring, or in periductular situation• Capable of generating cholangiocytes or

hepatocytes • Growth factors that stimulate oval cell

proliferation are similar to those that stimulate hepatocyte replication

Oval cells

• Murine liver +ve for – Immature markers - α-fetoprotein– Mature hepatic markers (e.g., albumin) – Biliary markers (e.g., cytokeratin-19)

• c-kit, CD34, Ov6, CK7, cgnn A + • Oval cells and hepatocytes require signaling

through TNF-R1; but not simultaneously • TGF beta - oval cell replication is less affected

Oval cells• HPCs proliferate in the

portal zone • “Streaming liver hypothesis”

– migrate toward the central vein in the liver lobules as daughter hepatocytes

• Proven with Mt DNA mutation tracking - in the human liver as well as in regenerative nodules in cirrhosis

Bone Marrow Stem Cells inLiver Disease

• Stem cells - totipotent, pluripotent, multipotent, or unipotent

• 2 endogenous populations of stem cells in liver- Hepatocytes and hepatic oval cells

• Hepatocytes ; able to self-renew limitlessly often play the principal role in liver regeneration

Hematopoetic stem cells

• Liver - location of hematopoiesis in fetus• ?Hematopoetic stem cells remain behind to

form HSC– HSCs can share cell surface markers associated

with hematopoietic stem cells such as CD34, Thy-1 and c-kit

Can Hematopoietic Cells Generate Hepatocytes?

• Data from Murine Models– BM-HSC have been

demonstrated to repopulate liver and give rise to functional hepatocytes

– BM HSC derived hepatocytes- arise from cell fusion of donor HSC and recipient hepatocytes

Can Hematopoietic Cells Generate Hepatocytes?

• Allogeneic bone marrow cells transplanted into lethally irradiated mice- generate hepatocyte-like cells in the liver at very low frequency (1/ 10,000)

Clinical Data from Sex-Mismatched BM Transplants

• Microchimerism; Presence of cells that originate from another individual and are genetically distinct from the cells of the host individual

• Frequency of BMHSC derived hepatocytes varied between <1% and 8% in sex mismatched individuals– Higher frequencies in pediatric liver allografts– Microsatellite analyses- higher percentage of

chimerism in comparison to Y chromosome evaluation by FISH

Liver regeneration in acute severe liverimpairment: a clinicopathologicalcorrelation study• N = Liver Bx samples of 74 patients with ALF/SALF• Etiology; acute HAV + drug toxicity (n 3), acute HBV (n

10), drug induced (n 21) and cryptogenic (n 40) • Mean age: 43+17 years• M/F: 28/ 46

– IHC for CK7/CK19 – HPCs activation/differentiation – Ki 67/P21 - proliferative activity/proliferation arrest – H & E - hepatocyte loss

Aezam Katoonizadeh. Liver international 2006

Liver regeneration in acute severe liverimpairment

Group Aalive (n 24)

Group Bdied (n 10)

Group Ctransplanted (n 40)

P value

>50% hepatocyte loss

8% (2/24) 80% (8/10) 95% (38/40) <0.0001

proliferating hepatocytes

14.3+9.3 2.5 +2.5 5.7+7.3 <0.0001

Number of HPC 74+55 138+52 141+58 <0.003

Intermediate hepatocytes

63% (15/24) 50% (5/10) 83% (33/40) NS

Liver regeneration in acute severe liver impairment

• Oval cell activation requires 50% loss of hepatocytes, a/w significant decrease in the proliferative activity of remaining hepatocytes

• Associated with poor prognosis

Aezam Katoonizadeh. Liver international 2006

Mesenchymal Stem Cells as a Source forLiver Regeneration

• Multipotent stromal cells that can differentiate into osteoblasts, chondrocytes , adipocytes

• In vitro; Differentiate into hepatocyte like cells and hepatic epithelial cells

• Umbilical cord blood, wharton’s gelly, adipose tissue–Hypoimmunogenic and create immuno

suppressive microenvironment–1 gram of fat > no of stem cells 1 gram of

marrow

Mesenchymal Stem Cells as a Source forLiver Regeneration

• In vitro functional assays- hepatocyte characteristics – albumin production– glycogen storage– urea secretion–uptake of LDL

• Studies in humans hindered by–Safety concerns–Lack of molecular data – Immunological mismatch

Gut,vol.58,no.4

Hepatocyte transplantation

• Involves the transfer of normal hepatocytes into diseased liver

• Isolated hepatocytes injected into splenic artery or directly into the portal vein

Hepatocyte transplantation

• Host liver architecture remains intact following the integration of hepatocytes in liver cords

• Transplantation is metabolically less stressful than whole liver transplants

• Consequences of graft loss are much less severe• Does not interfere with subsequent liver

transplantation or gene therapy

Murine studies in Hepatocyte transplantation

• Murine model- u TPA over producing cells– 10 4 liver cells – replace more than 80% of the liver– donor hepatocytes capable of at least 12 rounds

of cell division

Replacement of diseased mouse liver by hepatic cell transplantation. Science.263:1149–1152

Murine studies in Hepatocyte transplantation

• Intraperitoneal injections of hepatocytes into rats with fulminant hepatic failure induced by D galactosamine– improved survival – donor liver cells did not repopulate the liver

Cellular transplantation in the treatment of experimental hepatic failure. Science. 1980. 210:901–903

Methods for Assessing Liver Regeneration

• Need to accurately document the extent of hepatic regenerative activity –Patients with liver disease –Partial hepatic resections –Receiving interventions designed to

stimulate hepatic regeneration

Methods for Assessing Liver Regeneration

• Ideally–Non invasive, in-vivo–Measuring only specific phase

of cell cycle–Sensitive and Specific

Methods for Assessing Liver Regeneration

• Tissue determination– Liver weight

– CT/MRI- estimated weight (in grams) equals 0.81 × hepatic volume in ml +372

Mitosis measurement

• Incubating hepatocytes with potassium chloride and alcohol

–Centrifugation– McNeill's stain–No of mitotic figures per 1000 cells per

hpf–No of mitotic figures available for

counting is relatively limited

DNA synthesis

• Thymidine labelling index – TK rate limiting enzyme for DNA synthesis– Phosphorylates thymidine ; subsequent

incorporation into the DNA– Liver biopsy tissues or isolated hepatocytes

are incubated with radiolabelled thymidine for 1 h prior to measuring radioactivity

– Standardized and reproducible marker of hepatic regenerative activity

DNA synthesis

• Bromodeoxyuridine incorporation – BdU- thymidine analogue , incorporated into

DNA during DNA synthesis– Incorporation detected by IHC– Labeling index per 1000 hepatocytes/HPF• 1% in resting livers• 25-36% in hepatocytes and sinusoidal cells derived

from livers 24 h and 48 h following partial hepatectomy

Immunohistochemistry

– PCNA – Ki-67 – DNA polymerase alpha – NOR proteins

KI 67 Labelling index

• KI-67 is a nuclear protein that is needed for cellular proliferation

• Inactivation - inhibition of ribosomal RNA synthesis

• Appears in early G1, peaks during S & M• Cellular marker for proliferation• Fraction of Ki-67-positive cells is correlated

with replication of cells

Proliferating Cell Nuclear Antigen

• Processivity factor for DNA polymerase delta• Processivity is the ability to catalyze

consecutive reactions without releasing substrate

• S phase of cell cycle• Antibodies against PCNA is used as a marker of

cell replication

DNA polymerase alpha

• Appears in the cell nucleus during the G1 phase & present during the S and G2 phases

• Monoclonal anti-DNA alpha antibodies have been developed

• Relatively simple to perform

NOR proteins• Nucleolar Organizer Region (NOR) associated

proteins - collection of acidic proteins with nucleolin and B-23 as components

• 3 fold increase in NOR protein levels - after partial hepatectomy in rats

• NOR proteins may be used as a marker of hepatocyte proliferation

Endogenous gene expression

• H3 histone mRNA • Transcription rates increase 10-fold at the

onset of S phase

Enzyme and protein levels in liver tissue

Thymidine kinase level Results correlate well with PCNA and

thymidine incorporation Ornithine decarboxylase and hepatic

putrescine ODC- Enzyme expressed by regenerating liver cells Synthesise polyamine putrescine

Conclusion• The pursuit of understanding liver

regeneration has yielded great progress in last few decades

• However, many aspects of this phenomenon remain to be further understood

• On learning the regulatory networks- options may open up for therapy in ALF , small for size Txn, extensive resection and Rx of cirrhosis among many other conditions

Thank you