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Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis
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Journal of Hepatology Dec 2006
Markus Neef, Monika Ledermann, Hans Saegesser, Vreni Schneider, Juerg Reichen
Institute of Clinical Pharmacology, University of Berne, Berne, Switzerland
"....In summary, our findings demonstrate for the first time therapeutic effectiveness of rapamycin as an antifibrotic treatment strategy in animal models of established cirrhosis. Attenuated fibrogenesis associated with improved liver function and an equally marked survival advantage was achieved with low doses of rapamycin. Given that the security profile of rapamycin is well established, our present study warrants the clinical evaluation of rapamycin as an antifibrotic drug...."
ABSTRACT
Background/Aims: Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis.
Methods: Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-α1, transforming growth factor-β1 (TGF-β1) and β2 was quantified by RT-PCR.
Results: Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-β1 mRNA was downregulated in TAA, TGF-β2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats.
Conclusions: Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.
Introduction
Chronic liver diseases of any etiology are associated with fibrosis, i.e. the accumulation of extracellular matrix (ECM) proteins [1]. The bulk of ECM in the fibrotic liver is produced by myofibroblast-like cells of different provenance. According to recent suggestions at least three different types of ECM-producing cells can be distinguished according to their histological location. These are activated hepatic stellate cells (HSCs, located in the perisinusoidal space), portal/septal myofibroblasts, and interface myofibroblasts (located at the edge of fibrous septa) [2], [3]. Among them HSCs are the best studied cell type. Following liver injury quiescent HSCs transform into proliferative, fibrogenic, and contractile myofibroblasts [1].
A central signalling step in transforming HSCs is the PI3-K/Akt dependent activation of mTOR [4], [5], [6], a serine/threonine kinase that controls cell growth and proliferation via protein synthesis. The 70kDa ribosomal s6 kinase (s6k), one of mTOR's downstream targets, is activated upon phosphorylation and promotes increased translation of mRNA [7]. mTOR/s6k signalling thus represents an attractive target for antifibrotic intervention. Rapamycin, a potent mTOR inhibitor, is clinically used as an immunosuppressive drug. We have shown previously that oral rapamycin at a dose of 2.0mg/kg per day protected rats from fibrosis when treatment was initiated concomitant with bile duct-ligation (BDL) [8]. However, in clinical practice an antifibrotic drug would have to be given to a patient with established fibrosis. It is thus imperative to confirm prophylactic studies by therapeutic models that most closely mirror advanced human fibrotic liver diseases [9]. This requires established fibrosis before and ongoing fibrogenic stimulation during treatment. We therefore evaluated long-term rapamycin treatment in two animal models of established cirrhosis, namely in biliary cirrhosis induced by bile duct-ligation (BDL) and in thioacetamide (TAA) induced cirrhosis. In contrast to our prophylactic study, rapamycin dosage was markedly reduced to 0.5mg/kg/day which lies below levels commonly used in transplant models [10]. We show reduced ECM deposition associated with attenuated liver function impairment, portal hypertension, and mortality in rapamycin treated BDL-rats, similar findings were obtained in the TAA model. Our present study is the first to demonstrate therapeutic antifibrotic effectiveness of low-dose rapamycin treatment in adequate animal models.
Discussion
Several in vitro studies have established a role for mTOR as an attractive target for antifibrotic therapy [6], [17]. Prophylactic prevention of fibrosis by rapamycin has proved the viability of this concept [8], [18]. However, in clinical practice an antifibrotic drug would have to be given to a patient with established fibrosis. Moreover, unwanted effects of rapamycin, i.e. undue immunosuppression and renal toxicity, should be held at minimum given that liver cirrhosis is inherently associated with immunologic and renal impairment. We therefore used two different models of established cirrhosis to investigate the therapeutic potential of low-dose oral rapamycin (0.5mg/kg/day). This is a subtherapeutic dose in organ transplantation models representing only a fourth of the lowest dose used in renal transplantation [10]. Moreover, oral rapamycin at 1mg/kg/day and below is not associated with immunosuppressant-induced kidney damage in rats [19], [20].
In both animal models two weeks of rapamycin treatment markedly inhibited the accumulation of ECM-producing cells and ECM components. Interstitial MMP-2 activity, which is considered to shift the metabolic balance towards ECM accumulation [21], was markedly reduced. These effects were associated with a marked preservation of metabolic liver function. Although portal vein pressure was not measured in this study, spleen weight may serve as an indicator for portal hypertension [22], [23]. Markedly reduced spleen weights in the limited treatment experiments support the assumption that portal pressure was lowered. Subsequent extension of treatment time to four weeks yielded discrepant results. In rapamycin treated BDL-rats survival was prolonged, which in turn implicated a survival bias for the evaluation of additional readouts. We found reduced volume fractions of ECM and reduced MMP-2 activity, but no beneficial effects on liver function or numbers of ECM-producing cells. In contrast, extended rapamycin treatment was ineffective in the TAA model despite low mortality.
Attenuation of antifibrotic effectiveness in extended compared to limited treatment of BDL-rats can partly be attributed to survival bias because of the following reasons: The apparent disappearance of sinusoidal α-SMA+ cells between BDL-14 and BDL-28 could be explained by drop out of animals that died of cirrhosis with high numbers of activated HSCs. Analogous arguments apply to the apparent improvement of liver function in BDL-28. Poor liver function per se (with ABT-k <0.47h-1) has been shown to predict death of BDL-rats [13]. Indeed, retrospective analysis shows significantly poorer pre-treatment liver function in those animals that subsequently died during the experiment compared to those who survived (p=0.0007, data not shown).
The PI3K-Akt/mTOR/p70s6k pathway is central to the signal transmission of profibrotic growth factors to HSCs [4], [5], [6], [24]. In turn, expression of profibrotic TGF-β1 is regulated by mTOR/p70s6k dependent mechanisms in fibroblasts [25]. Inhibition of p70s6k phosphorylation by rapamycin inhibits cell cycle progression and posttranscriptional collagen 1 expression in isolated HSCs [6]. However, increased collagen mRNA transcription is conferred through s6k independent signalling [17]. Our results are in line with these concepts as we show inhibition of p70s6k phosphorylation associated with reduced proliferation of activated HSCs and attenuated ECM deposition without reduction of collagen mRNA expression. In contrast, mRNA expression of TGF-β1 and TGF-β2 was inhibited significantly. These findings suggest a potential mechanism of action since profibrotic effects mediated by TGF-β include increasing the expression of ECM proteins [26] and promoting MMP-2 activation [27].
Failure of extended treatment to inhibit fibrogenesis in the TAA model suggests two conclusions: firstly, effectiveness of rapamycin may vary with different etiologies of liver disease; and secondly, alternative mechanisms can compensate for the inhibited mTOR signalling and thus confer resistance to rapamycin. To identify these mechanisms is beyond the scope of this paper. One can speculate that rapamycin might be more effective in BDL-rats because of its antiproliferative effect on bile ducts. However, bile duct proliferation was not significantly changed but numbers of periductular α-SMA+ cells were reduced by rapamycin as well as mRNA expression of TGF-β2, which is predominantly expressed in cholangiocytes [28]. In contrast, TAA-induced liver damage caused only a very mild ductular reaction associated with a 10-fold lower increase in TGF-β2 expression compared to BDL.
With respect to resistance to rapamycin one should not prematurely conclude that fibrosis would be equally resistant in human disease than in the animal models used herein because BDL and TAA pose much stronger profibrotic stimuli than most etiologies of liver disease in men. Moreover, delaying progression of fibrosis would be a valuable first therapeutic step in this otherwise incurable condition.
In summary, our findings demonstrate for the first time therapeutic effectiveness of rapamycin as an antifibrotic treatment strategy in animal models of established cirrhosis. Attenuated fibrogenesis associated with improved liver function and an equally marked survival advantage was achieved with low doses of rapamycin. Given that the security profile of rapamycin is well established, our present study warrants the clinical evaluation of rapamycin as an antifibrotic drug.
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