|
Wen Jie Dai and Hong Chi Jiang
Second
Department of General Surgery, the First Clinical School, Harbin
Medical University, Harbin 150001, Heilongjiang Province, China
Dr. Wen Jie Dai, graduated from seven-year system of Harbin Medical
University in 1998, and now is a resident surgeon and doctoral
student in the Second Department of General Surgery, the First
Clinical School, Harbin Medical University, Harbin, China. Hismain
research field is hepatobiliary and splenic surgery.
Correspondence to Dr. Wen Jie Dai, Second Department of
General Surgery, the First Clinical School, Harbin Medical
University, Harbin 150001, Heilongjiang Province, China
Telephone:
0086-451-3602829, Fax.
0086-451-3670428 Email. wenjiedai@etang.com
Received:
2000-09-21 Accepted:
2000-10-29
Subject
headings
liver cirrhosis/therapy; gene therapy; transforming growth factor
beta; interleukin 10; hepatocyte growth factor; telomerase; gene
expression
Dai
WJ, Jiang HC. Advances in gene therapy of liver cirrhosis: a review.
World J Gastroenterol, 2001;7(1):1-8
INTRODUCTION
Liver fibrosis or cirrhosis is a common progressively
pathological lesion of chronic liver diseases in response to various
liver-damaging factors. The main mechanisms of fibrotic or cirrhotic
initiation and progression at the level of cellular and molecular
events have been elucidated in the past two decades[1,2].
Various causes, including hepatitis virus infections, toxification,
ischemia, congestion, parasites infection, abnormal cooper or iron
load, etc, result in chronic inflammation and/or wound healing
responses, of which the main characteristics manifest in the
absolute increase of the excessive extracellular martrix (ECM)
synthesis and the relative decrease of them, leading to ECM deposit.
With the stimulation of inflammation or toxins, activated hepatic
stellate cells(Ito cells), injuried or regenerated hepatocytes,
Kupffer cells, sinusoidal cells and natural killer (NK) cells
produce certain cytokines or immunoreactive factors, which exert
various biological effects on their respective target cells or
organs in an autocrine or paracrine manner. These consist of the
cellular basis of hepatic fibrosis advances[3,4].It is
the vital molecular event in fibrosis progression that activated
hepatic stellate cell exocrine ECM components and
fibrosis-implementing factors, for example, the transforming growth
factor β (TGF-β), which is considered the key cytokine to
accelerate cirrhotic procession[5,6]. Various factors
participate in fibrosis or cirrhosis formation. They could be simply
divided into the fibrosis-implementing factors, such as TGF-β,
platelet-derived growth factor (PDGF), epithelial growth factor (EGF),
and antifibrotic factors, such as interferon-γ and
interleukin-10. These cytokines play important and unique roles in
the interactive complicated network. Excessive ECM deposit,
disappearance of sinusoidal endothelial fenestra and subsequent
capillary vascularization cause the dysfunction of hepatocytes. The
unblocked progressively pathological lesions with inevitably result
in, lobular reconstruction, pseudolobule formation and nodular
regeneration. With the elucidation of vital cellular and molecular
events, gene delivery strategies for treatment of liver fibrosis or
cirrhosis emerge on the basis of gene manipulations. Our interest
focuses on the recent advances of gene therapy for liver fibrosis or
cirrhosis.
Effective
antifibrotic treatments, including medicinal or gene therapy, should
satisfy several essential criteria. First, any therapy should have a
sound biological basis. Additionally, the pharmacologically active
drugs or compounds should reach or reside in the liver at a high
concentration and have little side effect outside the liver.
Specific targeting to hepatic stellate cells were preferentially
chosen. Finally, in the ideal case, a therapeutic agent should keep
the regional target environment for a relatively long period[7].
BLOCKADE OF TGF-β SIGNALING
It has been demonstrated that TGF-β is of great importance in
fibrogenesis, and serves as a pivot in regulation of fibrogenic and
antifibrogenic mediators. TGF-β exhibits stimulatory or
inhibitory properties on cellular proliferation and differentiation,
and is considered to represent a fundamental regulatory molecule
acting through autocrine and/or paracrine mechanism[8].
TGF-β
is clearly associated with hepatic fibrogenesis and cirrhosis in
experimental animal models and in human liver
diseases[9,10]. It regulates the gene expression of Ito
cells, which in the course of liver fibrogenesis, proliferate and
transform into the myofibroblast-like cells, synthesizing large
amounts of connective tissue components, and also being involved in
the ECM degradation by production of matrix metalloproteinases (MMP)
inhibitors of MMP (TIMP), etc[11-13]. Because of the key
role of TGF-β and Ito cells in the procession of liver
fibrogenesis, lots of studies focus on their interactions. In in
vitro studies, Ito cell cultures were found to be activated by
TGF-β to increase the production of collagen type
Ⅰ,
Ⅱ,
Ⅲ,
fibronectin, undulin and proteoglycans[14-16]. TGF-β
enhanced the Ito cell transformation from resting to activated cells
and inhibited Ito cell proliferation[17]. TGF-β
signaling was mediated by specific receptors located on the target
cell membrane. In non-activated Ito cells, type Ⅱ
and Ⅲ
were present, while in activated Ito cells, there were TGF-β/activin
receptor type Ⅰ,
Ⅱ
and Ⅲ[18,20].
All the above showed clearly that anti-TGF-β intervention
which could block the TGF-β signaling pathway may prevent liver
fibrosis. Such interventions have come into being up to date. Qi et al have
developed adenovirus-mediated gene transfer to rat liver for
expressing a truncated type Ⅱ
TGF-β receptor[21]. It inhibits TGF-β activity
by competing with binding of the cytokine to endogenous TGF-β
receptors. The expression vector, AdCAT β TR, was administrated
via the portal vein for ensured infection and high-level expression
in regional liver tissue. Apparently, there needed a large excess of
truncated receptor over the wild type full-length receptor for
effective inhibition of TGF-β signaling[22,23].
Three days after AdCAT β-TR administration, two mRNAs
corresponding to the truncated human receptor and the rat
full-length receptor (5.5kb and 0.9kb) were detected respectively,
and the former mRNA was about 20-fold that of the latter.
Pathological examination showed that the extent of liver fibrosis,
collagen type Ⅰ,
fibronectin, alpha smooth muscle actin (α-SMA), TGF-β 1
and monocytes/ macrophages (ED-1) decreased significantly in AdCAT-β-TR
administration group. This inhibitory effect was observed in all
areas of the liver. The content of liver hydroxyproline had no
significant difference between AdCAT β-TR group and the intact
group, while in the control group of dimethylnitrosamine (DMN)-treated
persistent fibrotic rats, the amount of hydroxyproline was 3.4 times
higher than that of the above two. It was evident that the
activation of Ito cells and Kupffer cells was inhibited, suggesting
that truncated type Ⅱ
TGF-β receptor gene transfer could prevent or abolish liver
fibrogenesis effectively. More important, liver function of rats
with AdCAT β-TR injection recovered with a marked decrease of
serum hepatocyte enzyme AST and ALT[21].
Reports from Ueno et al[24] supported Qi et
al ‘s results as well. They conducted an recombinant
adenovirus expressing the entire extracellular domain of the human
type Ⅱ
TGF-β receptor fused to the Fc portion of human immunoglobulin
G (AdTβ-ExR) to produce a circulating soluble form of TGF-β
receptor with remote vital organ targeting after intramuscular
injection of AdT β-ExR. Soluble TGF-β receptor was
detectable in circulation and its level peaked at 5-7 days after the
injection. AdT β-ExR-infected COS cells secreted fused type Ⅱ
TGF-β receptors, which binded to TGF-β and consequently
blocked the TGF-β signaling as the full-length TGF-β
receptor did[24]. Injection of AdT β-ExR into the
femoral muscle of DMN-induced fibrotic rats prevented hepatic
fibrosis, whileno side effects or complications were found either macroscopically
or microscopically in the main organs including brain, heart, lung,
liver and kidney. Histological examination in DMN-treated rats with i.m.
injection of AdT β-ExR was essentially as the same as that in
Qi et al ‘s report, which locally expressed truncated type Ⅱ
TGF-β receptor in the liver via the administration of AdCAT
β-TR into the portal vein[21,24]. This research also
disclosed that adenovirus-mediated expression of soluble TGF-β
receptors exerted the binding effect to TGF-β in a TGF-β
specific manner, that is, others, e.g., PDGF α-receptor and
fibroblast growth factor (FGF), could not interfere with TGF-β.
Although a large excess of soluble TGF-β receptor seems
essential for blocking of TGF-β signaling, there was still
apparent difference between specific soluble TGF-β receptor and
non-receptor-specific binding proteins, e.g., decorin (would prevent
TGF-β induced renal fibrosis), for blockade of TGF-β
signaling[25]. Because the latter was lack of receptor
specificity and selectivity when it binded to TGF-β, it may
bind to other biological molecules, probably resulting in unexpected
problems. More exact mechanism was also explored[26-29].
There was evidence that truncated type Ⅱ
TGF-β receptor may form a complex with other type TGF-β
receptor and inhibit both the anti-proliferative effect of TGF-β
and/or the transcriptional activation by TGF-β[30-31].
Each type of three TGF-β receptor isoforms may have its own
distinct signaling pathway, each mediating a separate action setting
of TGF-β[31]. Complete inhibition of TGF-β
signaling was achieved via local expression of a kind of soluble
TGF-β receptor in a remote area from the target organ. The fact
suggested that such strategy should be applicable theoretically and
therapeutically for other factors belonging to growth factor
superfamily.
As a matter of
fact, signaling pathways introduced by PDGF, FGF, epidermal growth
factor (EGF), vascular endothelial growth factor (VEGF), insulin,
etc, could be abolished specifically by their corresponding
kinase-defective mutated form of
receptors[23,32-36]. This kinase-defective or
kinase-truncated receptors for signal transduction blockade of their
growth factor may be a useful strategy for analysis of the in
vivo role of these growth factors, because signaling pathway was
blocked at the receptor level, not at the cytokine or gene level,
without affecting the normal physiological status of these growth
factors or signal transfer of other receptors[21,22,37].
Although no side effects were found during the study period, special
attention should be paid to the surveillance of the possible
hazards, since Shull et al had demonstrated that there would
be multifocal inflammation and tissue necrosis in TGF-β gene
disrupted mice[38]. Adenovirus displayed the high degree
of hepatotrophic activity. It could also mediate the targeted
expression even if it was administrated in a remote site[21,24].
Immune responses would be elicited after repeated injection of
adenovirus particles, therefore may lower the effectiveness of
target gene expression.
STIMULATION OF HEPATOCYTE REGENERATION
In the course of liver fibrosis, the absolute and relative volume of
hepatocytes was both reduced under the direct or indirect effect of
liver-damaging factors. Stimulation of hepatocyte regeneration is
the essential strategy in choice of treatment. Hepatocyte growth
activators include an expanding list of complete mitogen and
comitogen. Complete mitogen are defined as those that are capable of
stimulating DNA synthesis and mitosis of cultured hepatocytes in
serum-free media. Hepatocyte growth factor (HGF), EGF, TGF-α,
and keratinocyte growth factor are in this category. Comitogens
enhance the stimulatory action of complete mitogens and decrease the
inhibitory effect of other inhibitors, while they have no direct
proliferation- enhancing effect on cultured hepatocytes, including
glucagon, insulin, insulin-like growth factors, adrenal cortical
hormones, vasopressin, angiotensin, thyroid and parathyroid
hormones, norepinephrine, as well as calcium and vitamin D[39].
HGF was regarded as the strongest hepatocyteproliferative agent up
to date. It was a pleiotropic factor with potent morphogenic,
mitogenic and motogenic effects and has anti-apoptotic action on
hepatocytes and other several cells in culture, exhibiting a
plethora of effects in many systems and organs[40-49].
The antifibrotic effect of HGF on hepatic fibrosis was first
characterized by Yasuda et al[50]. In their
experiment, the deletion variant of hepatocyte growth factor (dHGF)
which was more mitogenic than full-length HGF, was used to examine
its role in preventing fibrosis induced with dimethylnitrosamine in
rats. Northern hybridization and immunohistochemical staining
elucidated clearly the antifibrotic effect of dHGF on DMN-treated
rats. The main mechanism appeared to be suppression of Ito cell
activation, as demonstrated by the reduction of mRNA of procollagen
α2(Ⅰ),
α1(Ⅲ),
α1(Ⅳ),
TGF-β, desmin and α -SMA. Collagen content, as measured by
hydroxyproline, was also decreased. Futhermore, dHGF exerted the
mitogenic and antifibrotic activities even after the hepatic
fibrosis had been established with DMN[50]. Their study
showed that the antifibrotic effect of dHGF is not derived from the
possible effect of dHGF on cytochrome P450-dependent metabolic
degradation of DMN, but from the ability of dHGF to reduce or
inhibit the TGF-β mRNA level[50,51], proliferation
and activation of lipocytes and to stimulate liver regeneration[50].
Burr et al also demonstrated the vital role of HGF in liver
regeneration[52]. After treatment with anti-HGF
monoclonal antibody, the serum level of immunodetectable HGF was
inhibited, as well as the parenchymal proliferative response to
acute CCl4-induced liver injury. Ueki et al
developed a novel new gene therapy approach for rat liver cirrhosis
by muscle-mediated gene transfer of HGF cDNA[53]. Plasmid
containing HGF cDNA was embedded in the liposome, fused to the
envelope protein of hemagglutinating virus of Japan (HVJ). This
HVJ-liposome complex was intramuscularly injected repeatedly to
achieve therapeutically detectable expression level of HGF.
Repetitive administration resulted in a sound level of human HGF and
increased level of endogenous rat HGF. The number of proliferative
cellular nuclear antigen (PCNA) positive cells in HVJ-liposome-HGF
injection group was markedly higher than that of control group.
Apoptotic hepatocytes decreased significantly as well. The c-met,
HGF-specific receptor located on the surface of hepatocytes,
increased considerably in HVJ-liposome-HGF treated rats and stayed
at this high level even after the repeated DMN administration,
meanwhile strong tyrosine phosphorylation of c-met was observed.
Immunohistochemistry revealed that with the administration of
HVJ-HGF-liposome, staining of TGF-β1, expressed notably in
persistent cirrhotic liver, decreased, as well as desmin and α-SMA,
which were indicators of activated Ito cells. It suggested that
expressed HGF could inhibit activation of Ito cells, decrease the
synthesis of collagen type Ⅰ
and TGF-β, and prevent apoptosis of hepatocytes induced by DMN
administration. After several injections of HVJ-HGF-liposome,
complete remission was achieved, demonstrated by histology[53].
It can be inferred that HGF not only stimulated hepatocyte
regeneration, but also remodeled the disorganized cirrhotic tissue.
That is the pivotal potential for HGF gene therapy of liver
cirrhosis[54]. It is of particular interest that
HVJ-liposome was the ideal vehicle for gene transfer, because of the
high efficiency, simplicity and lack of toxicity[55].
Another aspect of HGF on promoting neovascularization in certain
ischemic diseases was also focused for possible application in the
treatment of liver cirrhosis. Aoki et al delivered HGF cDNA
into the myocardial infarction region in rats[56]. It
promoted the neovascularization strongly in infarcted myocardium,
including capillary, small- to medium-sized vessels. As an
endothelium-specific growth factor with potent motogenic activity to
endothelial cells, HGF’s angiogenestic activity was also confirmed
by the activation and upregulation of a transcription factor-ets,
which is essential for angiogenesis[56].
Immunohistochemical staining revealed positive expression of ets 1
in endothelial cells and vascular smooth muscle cells (VSMC) around
the neovascularized vessels. The ets family participated in
angiogenesis regulation by activating the transcription of several
genes, including collagenase Ⅰ,
stromelysine 1 and urokinase plasminogen activator, which are
proteases involved in ECM degradation, as well as migration of
endothelial cells[57]. Overexpression of exogenous HGF
activated ets family, initiated auto-loop up regulation mechanism of
HGF resulting in up-regulation of endogenous HGF expression[53,56],
since HGF gene promoter region comprised several putative regulatory
elements, such as B cell- and macrophage-specific transcription
factor binding sites (PU1/ETS), besides an interleukin-6 response
element (IL-6RE), a TGF-β inhibitory element (TIE), and a cAMP
response element (CRE)[56,58].
Another candidate for stimulation of hepatocyte proliferation
is hepatic stimulatory substance (HSS), which was first described by
LaBrecque et al in 1975. As a heat-stable stimulator for
regenerating liver with partial hepatectomy, it is considered to be
the unique liver-specific stimulatory factor and is universally
located in mammal embryonic liver, weanling liver and regenerating
liver[59]. In 1994, Hagiya et al obtained a gene
product in rats, called augmenter of liver regeneration (ALR) with a
full-length of 1.2kb and open reading frame of 378bp[60].
The molecular weight of ALR was approximately 30kDa, as a
homodimeric complex[60,61]. The recombinant ALR expressed
from ALR cDNA-transduced COS cells exerted potent and dose-dependent
proliferative activity tested in canine Eck fistula (portal caval
shunt) model[60]. Subsequent published reports have
gradually come to a common view that ALR is actually the HSS. Yang
et al cloned the human ALR gene in 1996[62,63]. Other
groups also successfully cloned the rat and human ALR cDNA
subsequently[64,65]. In comparison of the ALR encoding
sequences of human and rat, homology of nucleotide sequence is about
87%, and that of protein sequence was 84.8%. Rat, mouse and human
ALR genes and protein products were highly conserved and
preferentially expressed in the testis and the liver, probably being
involved in the synthesis and stability of the nuclear and
mitochondrial transcripts, especially in spermatogenesis in actively
regenerative tissues. Wang et al demonstrated that ALR or
hepatopoietin (HPO) exerted the biological activity via the high
affinity receptors for HPO on the surface of rat hepatocytes and
human hepatoma cells. Each of the above cells has a mean of
approximate 10000 and 55000 receptor sites respectively, as was
identified with the binding of radioisotype 125I-labelled
HPO to a 90kDa polypeptide[66]. Updated research by
Gandhi et al showed a certain new aspect of ALR funtion[67].
Hepatic levels of ALR decreased for 12 hours after 70% hepatectomy
in adult rats and rose with no corresponding increase in ALR mRNA
transcripts, while serum ALR level increased up to 12 hours. This
implied that ALR was constitutively expressed in hepatocytes in an
inactive form, and appeared to be an active form after being
released outside the hepatocytes in response to partial hepatectomy
or other regeneration-promoting circumstances[67].
Liver-derived NK cells are important regulators for liver regeneration[68]. Francavilla et al[69] connected
the regulation activity of ALR in liver regeneration with the novel
role of NK cells. Francavilla et al administrated three
hepatotrophic factors (ALR, insulin-like growth factor Ⅱ
[IGF-Ⅱ]
and HGF) with the surely inducing sound hepatotrophic activities to
assess the in vivo effects on NK cells in normal rats.
Results showed that NK cell cytotoxic activities were inhibited in
the population of mononuclear leukocytes (MNL) in the liver
(liver-resident NK cells ), but not in the MNL from the spleen or
peripleral blood. Results obtained in vitro displayed
that ALR, IGF-Ⅱ
and HGF had no effect on NK cell function in cultured MNL from the
liver, spleen or blood[69]. Results from Polimeno et
al also verified that ALR plays a pivotal role as growth factor
and as immunoregulator by controlling the mitochondrial
transcription factor A expression and lytic activity of
liver-resident NK cells through IFN-γ levels[70] and
regulates hepatocyte proliferation through enhancing cytochrome
content and oxidative phosphorylation capacity of liver-derived
mitochondia[71]. Liver injuries could also be prevented
with recombinant ALR injected intraperitoneally[72-74]. Gene transfer of human
ALR gene into cirrhotic rat may be an meaningful tool for reversing
liver cirrhosis. Interestingly, no close or stringent homology was
found between ALR and other polypeptide growth factor, while ALR has
both structural and functional homology compared with yeast scERV1
gene which is involved in biogenesis of mitochondria and regulation
of cell cycle, which may imply ALR’s
evolutional conservation and essentialness to growth and development[60,66].
INHIBITION OF INTERSTITIAL INFLAMMATION
Interstitial inflammation was characterized as the main pathological
changes in fibrogenesis. Those inhibiting inflammatory responses
would also be the target for gene transfer to block the fibrogenic
inflammation. Interleukin-10 (IL-10) is suitable for this strategy.
IL-10, originally identified as a cytokine synthesis inhibitory
factor, inhibits production of a variety of cytokines in various
cell types. It is produced under different conditions of immune
activation by the T-H 2 and TH0 subsets of helper T cells, as well
as by monocytes, macrophages, B cells, keratinocytes and stromal
cells[75]. Sequences of open reading frame, not 5’- or
3’- flanking regions of mouse and human IL-10 cDNA showed high
homology to two viruses, Epstein-Barr virus and equine herpes virus[76].
Acquiring a mammalian IL-10 homology and subsequent inhibitory
properties of IL-10 toward macrophages may help the virus escape
from the antiviral immune attacks. IL-10 exerted multiple biological
activities, including inhibition of T-H1 cells and
antigen-presenting cells, stimulation of mast cell proliferation,
protease expression, stimulation of B cell proliferation, antibody
secretion and major histocompatibility complex II (MHC II)
expression[75]. IL-10 takes part in the sophisticated
regulation of fibrogenesis. In IL-10 gene knockout (KO) mice treated
with tetrachloride (CCl4), there existed no difference in
hepatic toxicity of CCl4
between KO mice and wild type (WT) mice.
In CCl4-induced
acute liver injury mice, serum TNF-α and TGF-β levels were
markedly high in KO mice than that in WT mice. Administration of
recombinant IL-10 inhibited cultured Kupffer cells producing
superoxide radicals and TNF-α in vitro . In CCl4-induced
chronic liver injury model, the degree of hepatic fibrosis was
severer and the level of tissue TNF-α was higher than that of
WT mice. IL-10 knockout also seemed to enhance monocyte infiltration[77,78].
Up-regulation of IL-10 mRNA was found in both freshly isolated
quiescent and activated Ito cells. Co-transfection of Ito cells with
an IL-10 expression vector and collagen reporter genes showed a 40%
inhibition of α1 (Ⅰ)
collagen promoter activity, suggesting the in vivo role of
IL-10 in matrix remodeling and the possibility that failure for Ito
cell to sustain IL-10 expression underlied pathologic progression to
liver cirrhosis[79,80]. Kupffer cell also played a
pivotal role in production of IL-10. Reports from Rai et al[81]
revealed the subtle role of IL-10 in regulating production of TNF-α,
a hepatocyte proliferative factor initiated after 70% partial
hepatectomy. Kupffer cell depletion induced by gadolinium chloride
(GdCl) abolished induction of IL-10, then elongated half-life of
TNF-α mRNA. Overexpression of TNF-α promoted liver
regeneration potently after partial hepatectomy. Meijer et al
strengthened the common point of view that Kupffer cell depletion,
physically induced with dichloromethylene-diphosphonate, resulted in
an imbalanced hepatic cytokine expression, thereby suppressing
important growth-stimulating factors, including HGF and TNF-α[82].
Similar results were found in alcoholic cirrhosis[83].
Contraversy existed with respect to effects of IL-10 on
extracellular matrix regulation, for IL-10 was likely to have
different effects relying upon the differently detailed experimental
materials and conditions. In general, IL-10 inhibited α1 (Ⅰ)
collagen gene expression[79,84]stimulated collagenase
(MMP-1), stromelysin (MMP-3)[84], gelatinase (MMP-9)[85]
expression and elastin promoter activity.
Antifibrotic effect of IL-10 on hepatic fibrosis was also
correlated with its inhibition of TGF-β expression in fibrotic
tissue, which was verified as the key fibrotic regulator as stated
above. This mechanism was also verified in other experiments[82,86].
As an inhibitory regulator with pleiotropic nature, IL-10 may be
used as a target gene for gene transfer in the treatment of liver
cirrhosis, based on the potent capacity of suppressing production of
proinflammatory cytokines[75,77,78,82,83,87-92]. Nelson et
al showed an
exciting report that IL-10 could reduce liver fibrosis and normalize
serum ALT levels in IFN-γ unresponsive hepatitis C patients[93].
TELOMERE AND TELOMERASE WITH CIRRHOSIS
Telomere is a special cap-like structure at the end of eukaryotic
chromosomes, composed of a tandemly (TTAGGG)-rich repeat DNA
sequences and relative catalyzing proteins. Length of telomere
within a certain range is essential and vital for normal mitosis,
for it enhances the stability of end chromosome, prevents abnormal
chromosomal rearrangement and end-end fusion, and protects against
the degradation or destruction by nuclease and/or ligase. When
telomere was shortened to a checkpoint range of 2-4kb, the
stabilization of chromosomes would collapse. Maintenance of telomere
length depends on the telomerase, which consists of RNA and two
protein subsets and serves as RNA-dependent DNA polymerase to
achieve complete and entire replication of chromosome. There are
large amounts of evidence to demonstrate that telomerase takes part
in complex regulation and plays a crucial role in cell
proliferation, aging, immortalization and tumorigenesis[94].
Except for the lymphocytes of peripheral bood, hematopoietic stem
cells, germ cells, embryonic somatic cells and those resident in
actively proliferating tissues, for example, hair, skin and
endometrium, also have telomerase activities[95]. Other
tissues exert no telomerase activity.
In cirrhotic
liver, evidence verified no or a variety of low activity of
telomerase, while telomere length decreased
commonly[96-100]. Hytiroglou et al found a clear
cut difference in telomerase activity levels between hepatocellular
carcinoma (HCC) (positive or strongly positive) and cirrhotic liver
tissues (weakly positive or negative). They considered that
activation of telomerase was an early event in larger nodular
cirrhosis formation, and consequently exerted facilitating effect on
other factors in the progression of carcinogenesis[101].
A proportion of 86% of large noncancerous nodules exhibited similar
telomerase activity to HCCs, and part of it was derived from large
regenerative nodules, but not dysplastic nodules[101].
The importance of maintenance of telomere length has been verified
in cultured telomerase-negative human retinal pigment epithelial
cells and foreskin fibroblasts, transfected with vectors encoding
the human telomerase catalytic subunit[102]. A comparison
between human telomerase reverse transcriptase subunit (hTRT) gene
transfer group and control group showed that with hTRT gene
transfer, the telomerase-negative cells displayed normal phenotype
and karyotype, as well as exceeded their normal life-span by at
least 20 doublings. The fact was also demonstrated by Kiyono et
al[103]. Results obtained from telomerase-deficient
mice verified the crucial role of telomerase and maintained telomere
in development, which owned the nature of progressive telomere
shortening from one generation to the next, for lack of active
telomerase[104,105].
Based on the
elucidation of telomerase’s role in senescence and tumorigenesis,
Rudolph et al brought new insight into the mechanism of telomerase gene
transfer (mTR gene) in the treatment of liver injury and cirrhosis.
They adopted three systems to gauge how telomere shortening
influenced hepatocyte proliferation, survival and ultimately
predisposition to cirrhosis[106]. The first was the
albumin-directed urokinase plasminogen activator (Alb-uPA)
transgenic mouse, in which Alb-uPA expression would cause widespread
hepatocytes death and fatal liver failure in newborn mice. Results
showed a progressive decline in telomere length of peripheral blood
lymphocytes from the first generation (G1) to the third generation
(G3), with a 3.6-fold increase in apoptotic hepatocytes and a
decrease in proliferative hepatocytes. The second and third system
was partial hepatectomy and CCl4 mediated liver injury,
respectively. Flow cytometry verified that telomerase dysfunction
inhibited mitosis of mice hepatocytes. Pathology showed abnormal
mitosis and formation of anaphase bridges in G6 mTR-/-
mice. Induced apoptosis of hepatocytes delayed the volume recovery
of hepatectomized liver. Repeated administration of CCl4,
caused liver cirrhosis first in G6 mTR-/- mice, followed
by G3 mTR-/- mice and finally by mTR+/+. It
suggested that with the shortening of telomere, damage-resistant
capacity of parenchymal cells decreased and cirrhosis was prone to
be induced.
As gene transfer
vector, adenovirus revealed good hepatotrophic property (an
infection rate of 85%-100%). Telomere was shortened by about 70% in
G6 mTR-/- mice, however, after mTR gene transfer, ascites
reduced with gain of body weight, and serum levels of ALT and AST
improved with active proliferation of hepatocytes. Compared with mTR-/-
mice, mTR-transferred G6 mTR-/- mice survived
during the experimental stage with 66% decrease of mitosis index and
58% reduction of anaphase chromatin bridges. No TGF-β was
detected in mTR-transferred G6 mTR-/- mice, implying that
mTR transfer may block TGF-β signaling and/or inhibit
activation of Ito cell and TGF-β secretion. Target transfer of
telomerase gene into affected organ created a new strategy and also
a novel pathway for the treatment of aging and certain
telomerase-related chronic disease. Before actual application,
over-high activity of telomerase may induce instablility of genomic
structure and tumor formation. From a clinical point of view,
telomerase gene therapy may be an ideal choice of treatment for
patients awaiting liver transplantation, for it could actually
improve liver function and extend survival, and the eventual
surgical removal of diseased organ should at most minimize the
potential cancer risk[105]. Telomerase gene therapy may
be regarded as an effective short-term supporting procedure for
patients with end-stage liver disease before carrying out of liver
transplantation[107].
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