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.
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.
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. 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. 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 (5.5 kb and 0.9 kb) 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-β1 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.
Reports from Ueno et al supported Qi et al’s results as well. They conducted an recombinant adenovirus expressing the entire extracellular domain of the human type ¢ò TGF-â 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 d 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. Injection of AdT β-ExR into the femoral muscle of DMN-induced fibrotic rats prevented hepatic fibrosis, while no 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. 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. 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. 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. 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. 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. 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. Burr et al also demonstrated the vital role of HGF in liver regeneration. 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. 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. 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. 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. 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. 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. 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. 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. In 1994, Hagiya et al obtained a gene product in rats, called augmenter of liver regeneration (ALR) with a full-length of 1.2 kb and open reading frame of 378 bp. The molecular weight of ALR was approximately 30 kDa, 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. Subsequent published reports have gradually come to a common view that ALR is actually the HSS. Yang et al[62,63] cloned the human ALR gene in 1996. 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 90 kDa polypeptide. Updated research by Gandhi et al showed a certain new aspect of ALR funtion. 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. Liver-derived NK cells are important regulators for liver regeneration. Francavilla et al 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. 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 and regulates hepatocyte proliferation through enhancing cytochrome content and oxidative phosphorylation capacity of liver-derived mitochondia. 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-H2 and TH0 subsets of helper T cells, as well as by monocytes, macrophages, B cells, keratinocytes and stromal cells. 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. 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. 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-αin 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 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. Similar results were found in alcoholic cirrhosis. 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), gelatinase (MMP-9) 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.
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 2-4 kb, 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,immortalizationandtumorigenesis. 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. 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. 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.
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. 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. 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. 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. 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.