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Clearance of activated stellate cells for hepatic fibrosis regression: Molecular basis and translational potential
Biomedicine & Pharmacotherapy 67 (2013) 246–250
Available online at
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Review
Clearance of activated stellate cells for hepatic fibrosis regression: Molecular basis and translational potential Desong Kong a, Feng Zhang a, Zili Zhang a, Yin Lu a,b, Shizhong Zheng a,b,* a b
Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, 282 Hanzhong Road, Nanjing, Jiangsu 210029, PR China Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2012 Accepted 22 October 2012
Hepatic fibrosis, characterized by abnormal accumulation of extracellular matrix (ECM), is a common pathological process of many chronic liver diseases. A growing number of studies have shown that the activation of hepatic stellate cells (HSCs) plays an important role in the pathogenesis of hepatic fibrosis. Inhibiting the activation of HSCs and accelerating the clearance of activated HSCs may be effective strategies for resolution of hepatic fibrosis. Therefore, understanding the underlying mechanisms of clearance of activated HSCs and the therapeutic implications is an active subject of research. Studies have shown that apoptosis, immune clearance, phenotype reversion and senescence are involved in clearance of activated HSCs. In this review, we will discuss the mechanisms of clearance of activated HSCs and their potential in resolution of hepatic fibrosis. ß 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Hepatic fibrosis Hepatic stellate cell Apoptosis Immune clearance Phenotype reversion Senescence
1. Introduction Hepatic fibrosis occurs as compensatory responses to tissue repairing process in a wide range of chronic liver injures. It is characterized by excessive deposition of extracellular matrix (ECM) in liver tissues [1]. As the pathogenesis progresses without effective management, it will lead to formation of liver fiber nodules and disruption of normal liver structure and function, and finally culminate in cirrhosis and hepatocarcinoma [1,2]. It has been well established that the central event of hepatic fibrosis is the activation and proliferation of hepatic stellate cells (HSCs) [3]. During liver fibrogenesis, the damage of liver parenchyma cells initiates the process of HSCs activation. Meanwhile, activated HSCs undergo transdifferentiation to myofibroblasts (MFBs), which have strong ability to secrete collagen and migrate to the area of necrosis and inflammation [4,5]. Consequently, the increased deposition of ECM elicits the damage of hepatocytes and impairs their metabolic functions [6]. Therefore, the activation of HSCs plays an important role in the development of hepatic fibrosis. Understanding the mechanisms of HSCs activation and aiming at activated HSCs as a target for developing effective anti-fibrotic agents are of vital significance, and have gained increasing attention in current research in hepatology. It has been proposed that inhibiting HSCs activation and reducing ECM secretion are promising strategies for the therapy of
* Corresponding author. Tel.: +86 25 86798154; fax: +86 25 86798188. E-mail address: [email protected] (S. Zheng). 0753-3322/$ – see front matter ß 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2012.10.002
hepatic fibrosis. Moreover, experimental and clinical studies have shown that the reduction in the abundance of activated HSCs is an important marker of the reversal of hepatic fibrosis [6,7]. Therefore, it would be rational to reverse hepatic fibrosis through promoting the clearance of activated HSCs. At present, a number of observations support the strategy of the clearance of activated HSCs in the treatment of hepatic fibrosis. In the following, we will update the progresses in this area, and discuss their potential implications in anti-fibrotic therapy. 2. Apoptosis Spontaneous recovery of hepatic fibrosis mainly relies on the apoptosis of activated HSCs [7,8]. This leads to decrease in the number of HSCs, and the accumulation of ECM, and to the resolution of hepatic fibrosis. Cytokines and medications, and several factors, including genetic abnormalities, autoimmunemediated damage, are involved in regulating apoptosis. Therefore, the research on the design and mechanisms of drugs to promote HSCs apoptosis may become one of promising strategies for guiding future anti-fibrotic treatment. It has been established that two main pathways are involved in apoptosis of cells, inclding HSC, i.e. the death receptor pathway and the mitochondria pathway, both of which finally activate caspases enzyme family implementing the apoptosis progress [9–11]. It is also shown that other signal transduction pathways regulate apoptosis of HSCs. Studies have shown that nerve growth factor could induce apoptosis of activated HSCs by binding to its receptor p75, which raised the activity of caspase and increased the protein
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expression of caspase-1 and caspase-3 [12]. Iwamoto et al. found a soluble integrin recognized five peptide Gly-Arg-Gly-Asp-Ser. The latter antagonized the adhesion effect of integrin, resulting in the reduction in the phosphorylation level of focal adhesion kinase and the ratio of Bcl-2/Bax, and the increase in the expression of the apoptosis regulatory protein p53. These effects collectively led to the induction of apoptosis of HSC [13]. The activation of nuclear factor-kB (NK-kB) restrains apoptosis of activated HSCs induced by tumor necrosis factor-a (TNF-a). Oakley et al. observed that inhibition of the inhibitor of kB kinase (IKK)/NF-kB pathway was sufficient to increase the rate at which activated HSCs underwent apoptosis both in vitro and in vivo. It was proposed that drugs that selectively target IKK might have potential as anti-fibrotics [14]. Moreover, it was found that C3 and Y-27632, the inhibitors of Rho, increased histone-associated DNA fragmentation and caspase-3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs, showing that Rho-associated protein kinase (ROCK) signal pathway was closely related to HSCs apoptosis [15]. Furthermore, our group demonstrated that peroxisome proliferator-activated receptor-g (PPARg) played a key role in regulating HSCs apoptosis, and that natural product curcumin could induce HSCs apoptosis dependent on PPARg activation [16]. Dickkopf-1 (Dkk-1), a Wnt coreceptor antagonist, was found to be capable of enhancing PPARg gene promoter activity and restoring HSCs quiescence in culture [17]. Induction of expression of Dkk-1 stimulated apoptosis of activated HSCs.
3. Immune clearance HSCs have emerged as an important target in the liver microenvironment for inflammatory cytokines. Macrophages and infiltrating monocytes participate in the development of fibrosis via several mechanisms, including increased secretion of cytokines and generation of oxidative stress-related products [18,19]. Many kinds of immunocytes participate in the clearance of activated HSCs and the reversal of hepatic fibrosis, including natural killer cells (NK cells), Kupffer cell (KC), T lymphocytes, natural killer T (NKT) cells, etc. Recent evidence indicated that NK cells possessed specifically protective effects during the development of fibrosis. Interestingly, Poly(I:C) or interferon-g (IFN-g) treatment increased the expression of the NK cells activating receptor NKG2D by intrahepatic NK cells and enhanced the cytotoxicity of NK cells against activated HSCs [19]. Increased apoptosis of HSCs was observed after direct adhesion of NK cells to HSCs [20]. NK cells could selectively kill early activated HSCs, however, NK cells had no such effects on fully activated HSC. Signal transducers and activators of transcription 1(STAT1) was indicated as a pivotal factor in mediating the inhibitory effects of NK cells on activated HSCs. Killing HSCs by NK cells was attenuated in STAT1 / mice, which was likely due to the reduced expression of NKG2D and TRAIL on STAT1 / NK cells [21]. Direct interactions were also found between HSCs and lymphocyte subsets, including NK cells. KCs are a type of macrophage populating in the hepatic sinusoid, acting as non-specific killer under normal circumstances. In the pathophysiology of hepatic fibrosis and its recovery stage, KCs play a double-edged sword role. To be specific, during the progression of fibrogenesis, KCs can promote HSCs activation, whereas during fibrosis recovery it can stimulate apoptosis of activated HSCs and ECM degradation [18,22,23]. Studies have showed that KCs could induce apoptosis of activated HSCs via the casepase-9 pathway [24]. Tang et al. confirmed that KCs could induce HSC apoptosis and inhibit their proliferation by activating casepase cascades via secretion of tumor necrosis factor-related apoptosis-inducing ligand in the recovery of liver fibrosis [25]. In
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addition, KCs may effectively inhibit the the expression of tissue inhibitor of metalloproteinase in HSCs, and promote ECM degradation via secretion of inflammation suppressive factors such as IFN-g and interleukin (IL)-10, leading to the reduction in the number of activated HSCs [26,27]. The possibility that T lymphocytes modulate the fibrogenic process was put forward almost 25 years ago. This observation could account for the increased rate of fibrosis in patients with HBV and HCV, in whom the CD4+/CD8+ ratio was reduced typically [28]. CD8+ T cells protect liver cells from pathogen infections while aggravating liver tissue damage [29]. CD4+ T cells under different conditions differentiate into varied types of auxiliary T cells, including Help T cell (Th), mediating different types of immune responses. Th1 cells secrete IFN-g that could restrain HSCs activation and proliferation, induce apoptosis, thereby exert anti-fibrotic effects [25]. Intriguingly, HSCs were also identified to be antigen-presenting cells, and might contribute to the liver immunotolerant properties through T-cell suppression [30]. NKT cells are a unique but heterogeneous group of T cells that express both ab TCR (T cell marker) and cell surface receptors that are characteristic of NK cells. Similar to NK cells, NKT cells also can directly kill activated HSCs, secrete IFN-g and play an anti-fibrotic role [31,32].
4. Phenotype reversion Following fibrogenic stimuli, HSCs undergo a complex transformation known as activation, in which HSCs acquire the properties of activated myofibroblast-like cells associated with an increase in the expression of a-smooth muscle actin and the loss of the vitamin A storage [33,34]. In the development of advanced fibrosis, as the disse microenvironment changes, the surface structure of HSCs is also constantly changed, such as decreased cell connection, and increased ability to move and migrate [33]. Recent report showed that MFBs could revert to an inactive phenotype during regression of hepatic fibrosis [35]. If the transformation of fibrogenic phenotypes is reversible, the antifibrotic therapy is achievable by reducing the production of ECM in HSC and restraining the development of fibrosis. The metabolism of retinols and lipids is closely associated with hepatic fibrogenesis. The mechanistic studies on activation of HSCs and differentiation of preadipocytes have revealed that HSCs and adipocytes share something in common in term of expression of key transcription factors, such as PPARg, liver X receptor-a, CCAAT/enhancer binding protein and retinoid X receptors which play fundamental roles in lipid droplet metabolism [36–38]. Treatment of HSCs with the fat cell differentiation matrix (methyl isobutyl xanthine, dexamethasone and insulin) and ectopic expression of PPARg or Sterol regulatory element-binding proteins-1c in HSCs could stimulate the continuous expression of transcription factors related to lipid generation and reverse the activated phenotype of HSCs [38]. Therefore, inducing expression of genes associated with lipid generation in activated HSCs may be a good strategy for the reversal of the profibrogenic phenotype of HSCs. Mesenchymalto epithelial transition (MET) and Epithelial to mesenchymal transition (EMT) are two fundamental developmental processes [39]. In recent years, the role of EMT in hepatic fibrosis has become an active research direction. In EMT of fibrogenesis, HSCs and hepatic epithelial progenitors obtain the fibroblast phenotype and express the stromal cell markers and eventually become MFBs, promoting the development of hepatic fibrosis [40,41]. However, there has recently been increasing evidence that supports the reverse transition, i.e. MET. This has been implicated in several pathological conditions. For example, it
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was recently discovered that renal fibroblasts could undergo MET to form epithelial aggregates that facilitated the repair of tubular injure in murine fibrotic kidney [42]. Moreover, the Wnt signalling pathway is implicated during the dynamic and reversible EMT and MET that underscore colorectal cancer progression, and it offers a new hope for anti-cancer therapy [43]. Some of key regulators including cadherins, microRNAs, transcription factors (Pax, paraxis, Fox) and growth factors (Wnts, FGFs, Ephrins) might be involved in the developmental MET [44,45]. Therefore, it is anticipated that stimulation of MET of activated HSCs would be an effective strategy to promote the clearance of activated HSCs and reversal of hepatic fibrosis. In the process of hepatic fibrosis, in addition to the overexpressed profibrogenic growth factors, another key change is the gradual replacement of the basement membrane of matrix with the internal matrix [46]. It was observed that HSCs cultured in the EHS glue (similar to the matrix in Disse gap in normal liver) could maintain their quiescence for a long time period. However, HSCs cultured in type 1 collagen (main component of ECM when hepatic fibrosis develops) were apparently activated [47]. Although activated HSCs contribute to the secretion of ECM, the latter can also retroact to HSCs, affecting HSCs behaviors and functions [5,48]. Integrin specifically regulates the connection of ECM components with cellular structures and thus is also a primary factor for maintaining cell stability [49]. Furthermore, ECM can be ligand signal molecules binding to integrin receptors on the membrane of HSCs, affecting the amount and activity of transformation growth factor-b receptors through a series of signaling transductions to keep the collagen physiological metabolism [50]. Based on these discoveries, it has been proposed
that intervention of integrin signaling could beneficially control the molecular linkage between HSCs and ECM, which probably holds promise to reduce the activated phenotype of HSCs [51,52].
5. Senescence Since Hayflick and Moorhead observed the phenomenon of senescence in human firoblasts for the first time, senescence in all kinds of cells has been observed in further studies [53]. However, the relationship between senescence of activated HSCs and its proliferation attracts increasingly attention worldwide. Sekoguchi et al. found that the turnover of cell cycle was the main reason for telomere shortening, which could drive the cells into senescent state and reduce the development of hepatic fibrosis [54]. Moreover, Jun et al. found that in the liver and skin damage, MFBs initially proliferated and secreted ECM, but they finally underwent senescence that inhibited their further proliferation [55]. Furthermore, Krizhanovsky et al. found that senescent cells accumulated in murine livers treated to produce fibrosis and the senescent cells were derived primarily from activated HSCs. Hepatic fibrosis in p53 knockout mice was severe due to the inhibited HSCs senescence [56]. A recent study found that the antifibrotic effect of IL-22 was likely to be mediated via the induction of HSCs senescence in addition to the previously discovered hepatoprotective functions [57]. Since HSCs are the main cells participating in the pathogenesis of hepatic fibrosis, it could be presumed that induction of senescence could block HSCs proliferation and activation, which may be of great significance preventing and treating hepatic fibrogenesis.
[(Fig._1)TD$IG]
Fig. 1. This diagram represents the mode for HSC-mediated liver fibrogenesis and different ways of clearance of activated HSCs. Normally, HSCs are quiescent and nonproliferating cells in space of Disse. Upon injury, they are activated and undergo transdifferentiation to myofibroblasts responsible for the oversecretion of collagen-rich ECM. Clearance of activated HSCs can reduce the number of activated HSCs and ECM generation, achieving the purpose of anti-fibrogenesis. Several ways involved in this progress include apoptosis, immune clearance, phenotype reversion and senescence. The beneficial effects of clearance of activated HSCs may provide potential treatment options for liver fibrosis.
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Further studies showed a correlation between cells positioned at senescence stage and inflammatory microenvironment in organism microenvironment [58,59]. In addition, it was also found that NKG2DU receptor ligand LBP2 and adhesion molecular CD58, which participated in the interaction of natural killer cells with their target cells, were increased. The inflammatory cytokines such as IL-11, IL-8, and IL-6 in the senescent HSCs were also excessively expressed. The senescent HSCs enhanced the functions of natural killer cells and promoted accumulation of immune cells, thereby leading to accelerated clearance of senescent cells [19,56]. Taken together, senescence enables the activated HSCs to lose their proliferative capability and promotes functions of immune cell, making it an important way to eliminate activated HSCs in hepatic fibrogenesis.
6. Concluding remarks and perspectives Developing effective therapies for hepatic fibrosis is still a demanding challenge in current medical and pharmaceutical sciences [60]. Despite so, clinical therapies for this liver disorder have emerged with the increasing understanding of the pathogenesis of hepatic fibrosis [61]. In the clinical contexts, there are no apparent markers for the early phase of hepatic fibrosis, and once patients are diagnosed to be hepatic fibrosis, the syndrome has commonly been in the later phase of the pathogenesis with relatively severe impairment of hepatic functions. At this time, there have been a considerable number of activated HSCs and MFBs in the patient liver, which could cause the degree of hepatic fibrosis to worsen [3,62]. Studies have established that inhibition of HSCs activation or stimulation of their clearance could reverse hepatic fibrosis. Several mechanisms have been identified to contribute the clearance of activated HSCs. Apoptosis, immune clearance, and senescence are directly aimed at activated HSCs to reduce the abundance. On the other hand, phenotype reversion can maintain HSCs quiescence, thereby restraining the secretion of pro-fibrotic cytokines and collagens in the fibrotic liver (Fig. 1). It would be important to develop drugs that can promote the clearance of activated HSCs for therapeutic treatment of hepatic fibrosis. Pharmaceutical industry has found many candidate agents that can restrain HSCs activation and promote HSCs clearance. Especially, pharmacological induction of HSCs apoptosis has shown promise in a number of preclinical investigations. Furthermore, as mentioned above, the bidirectional role for immune cells in HSCs clearance remains to be defined, but preliminarily suggests that the potential drugs should manly limit the development of inflammation to remove activated HSCs [63]. In addition to the HSCs apoptosis-inducing agents, the emerging molecular mechanisms of other means to remove activated HSCs also give rise to high possibility to develop related pharmacological interventions, i.e. drugs that can reverse the phenotype or interfere with the EMT process. As discussed above, lots of signaling pathways and cytokines are involved in the clearance of activated HSCs, and it is also very important to strengthen exploration of potential targets for drugs. What is more, in the seek for modulators of HSCs clearance, more attentions should be paid to the selectivity of drugs. Developing drugs with high selectivity towards activated HSCs with less side effects would be a huge task for research. Researchers have made much progress, for instance, Moreno et al. used a platinum-based linker to develop a conjugate of the AT1 receptor blocker losartan and the HSC-selective drug carrier mannose-6-phosphate modified human serum albumin (losartanM6PHSA), and short-term treatment with this HSC-targeted losartan could markedly reduce advanced liver fibrosis [64]. Understanding of the mechanisms of clearance of activated HSCs
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and their potential implications in hepatic fibrosis continues to be critical in future studies.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements The financial support was from The Open Project Program of National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine (2011ZYX4-008); Doctoral Discipline Foundation of Ministry of Education of China (20103237110010); National Natural Science Foundation of China (81270514, 30873424); Jiangsu Natural Science Foundation (BK2008456); Open Program of Jiangsu Key Laboratory of Acupuncture & Medicine (KJA200801); Project for Supporting Jiangsu Provincial Talents in Six Fields (2009-B-010); A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2010) and the National Key Technologies R & D Program of China during the 11th Five-Year Plan Period (2008BAI51B02). References [1] Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008;134:1655–69. [2] Soares JB, Pimentel-Nunes P, Afonso L, et al. Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence. Innate Immun 2012. http://dx.doi.org/10.1177/1753425912436762 [Epub ahead of print]. [3] Anthony B, Allen JT, Li YS, McManus DP. Hepatic stellate cells and parasiteinduced liver fibrosis. Parasi Vectors 2010;3:60. [4] Otogawa K, Ogawa T, Shiga R, Ikeda K, Kawada N. Induction of tropomyosin during hepatic stellate cell activation and the progression of liver fibrosis. Hepatol Int 2009;3:378–83. [5] Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Semin Liver Dis 2001;21:427–36. [6] Domitrovic´ R, Jakovac H, Tomac J, Sain I. Liver fibrosis in mice induced by carbon tetrachloride and its reversion by luteolin. Toxicol Appl Pharmacol 2009;241:311–21. [7] Sua´rez-Cuenca JA, Chagoya de Sa´nchez V, Aranda-Fraustro A, et al. Partial hepatectomy-induced regeneration accelerates reversion of liver fibrosis involving participation of hepatic stellate cells. Exp Biol Med (Maywood) 2008;233:827–39. [8] Gonzalez SA, Fiel MI, Sauk J, et al. Inverse association between hepatic stellate cell apoptosis and fibrosis in chronic hepatitis C virus infection. J Viral Hepat 2009;16:141–8. [9] Kumar A, Sinha RA, Tiwari M, et al. Hyperthyroidism induces apoptosis in rat liver through activation of death receptor-mediated pathways. J Hepatol 2007;46:888–98. [10] Lin HJ, Tseng CP, Lin CF, et al. A Chinese herbal decoction, Modified Yi Guan Jian induces apoptosis in hepatic stellate cells through an ROS-mediated mitochondrial/caspase pathway. Evid Based Complement Alternat Med 2011;2011:459–531. [11] Schinoni MI, Parana R. Apoptosis and progression of hepatic fibrosis in liver diseases. Acta Gastroenterol Latinoam 2006;36:211–7. [12] Asai K, Tamakawa S, Yamamoto M, et al. Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation. Liver Int 2006;26:595–603. [13] Iwamoto H, Sakai H, Tada S, et al. Induction of apoptosis in rat hepatic stellate cells by disruption of integrin-mediated cell adhesion. J Lab Clin Med 1999;134:83–9. [14] Oakley F, Meso M, Iredale JP, et al. Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis. Gastroenterology 2005;128:108–20. [15] Ikeda H, Nagashima K, Yanase M, et al. Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2003;285:G880–6. [16] Zheng S, Chen A. Activation of PPARgamma is required for curcumin to induce apoptosis and to inhibit the expression of extracellular matrix genes in hepatic stellate cells in vitro. Biochem J 2004;384:149–57. [17] Cheng JH, She H, Han YP, et al. Wnt antagonism inhibits hepatic stellate cell activation, liver fibrosis, Am J. Physiol Gastrointest Liver Physiol 2008;294:G39–49.
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Review
Clearance of activated stellate cells for hepatic fibrosis regression: Molecular basis and translational potential Desong Kong a, Feng Zhang a, Zili Zhang a, Yin Lu a,b, Shizhong Zheng a,b,* a b
Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, 282 Hanzhong Road, Nanjing, Jiangsu 210029, PR China Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2012 Accepted 22 October 2012
Hepatic fibrosis, characterized by abnormal accumulation of extracellular matrix (ECM), is a common pathological process of many chronic liver diseases. A growing number of studies have shown that the activation of hepatic stellate cells (HSCs) plays an important role in the pathogenesis of hepatic fibrosis. Inhibiting the activation of HSCs and accelerating the clearance of activated HSCs may be effective strategies for resolution of hepatic fibrosis. Therefore, understanding the underlying mechanisms of clearance of activated HSCs and the therapeutic implications is an active subject of research. Studies have shown that apoptosis, immune clearance, phenotype reversion and senescence are involved in clearance of activated HSCs. In this review, we will discuss the mechanisms of clearance of activated HSCs and their potential in resolution of hepatic fibrosis. ß 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Hepatic fibrosis Hepatic stellate cell Apoptosis Immune clearance Phenotype reversion Senescence
1. Introduction Hepatic fibrosis occurs as compensatory responses to tissue repairing process in a wide range of chronic liver injures. It is characterized by excessive deposition of extracellular matrix (ECM) in liver tissues [1]. As the pathogenesis progresses without effective management, it will lead to formation of liver fiber nodules and disruption of normal liver structure and function, and finally culminate in cirrhosis and hepatocarcinoma [1,2]. It has been well established that the central event of hepatic fibrosis is the activation and proliferation of hepatic stellate cells (HSCs) [3]. During liver fibrogenesis, the damage of liver parenchyma cells initiates the process of HSCs activation. Meanwhile, activated HSCs undergo transdifferentiation to myofibroblasts (MFBs), which have strong ability to secrete collagen and migrate to the area of necrosis and inflammation [4,5]. Consequently, the increased deposition of ECM elicits the damage of hepatocytes and impairs their metabolic functions [6]. Therefore, the activation of HSCs plays an important role in the development of hepatic fibrosis. Understanding the mechanisms of HSCs activation and aiming at activated HSCs as a target for developing effective anti-fibrotic agents are of vital significance, and have gained increasing attention in current research in hepatology. It has been proposed that inhibiting HSCs activation and reducing ECM secretion are promising strategies for the therapy of
* Corresponding author. Tel.: +86 25 86798154; fax: +86 25 86798188. E-mail address: [email protected] (S. Zheng). 0753-3322/$ – see front matter ß 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2012.10.002
hepatic fibrosis. Moreover, experimental and clinical studies have shown that the reduction in the abundance of activated HSCs is an important marker of the reversal of hepatic fibrosis [6,7]. Therefore, it would be rational to reverse hepatic fibrosis through promoting the clearance of activated HSCs. At present, a number of observations support the strategy of the clearance of activated HSCs in the treatment of hepatic fibrosis. In the following, we will update the progresses in this area, and discuss their potential implications in anti-fibrotic therapy. 2. Apoptosis Spontaneous recovery of hepatic fibrosis mainly relies on the apoptosis of activated HSCs [7,8]. This leads to decrease in the number of HSCs, and the accumulation of ECM, and to the resolution of hepatic fibrosis. Cytokines and medications, and several factors, including genetic abnormalities, autoimmunemediated damage, are involved in regulating apoptosis. Therefore, the research on the design and mechanisms of drugs to promote HSCs apoptosis may become one of promising strategies for guiding future anti-fibrotic treatment. It has been established that two main pathways are involved in apoptosis of cells, inclding HSC, i.e. the death receptor pathway and the mitochondria pathway, both of which finally activate caspases enzyme family implementing the apoptosis progress [9–11]. It is also shown that other signal transduction pathways regulate apoptosis of HSCs. Studies have shown that nerve growth factor could induce apoptosis of activated HSCs by binding to its receptor p75, which raised the activity of caspase and increased the protein
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expression of caspase-1 and caspase-3 [12]. Iwamoto et al. found a soluble integrin recognized five peptide Gly-Arg-Gly-Asp-Ser. The latter antagonized the adhesion effect of integrin, resulting in the reduction in the phosphorylation level of focal adhesion kinase and the ratio of Bcl-2/Bax, and the increase in the expression of the apoptosis regulatory protein p53. These effects collectively led to the induction of apoptosis of HSC [13]. The activation of nuclear factor-kB (NK-kB) restrains apoptosis of activated HSCs induced by tumor necrosis factor-a (TNF-a). Oakley et al. observed that inhibition of the inhibitor of kB kinase (IKK)/NF-kB pathway was sufficient to increase the rate at which activated HSCs underwent apoptosis both in vitro and in vivo. It was proposed that drugs that selectively target IKK might have potential as anti-fibrotics [14]. Moreover, it was found that C3 and Y-27632, the inhibitors of Rho, increased histone-associated DNA fragmentation and caspase-3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs, showing that Rho-associated protein kinase (ROCK) signal pathway was closely related to HSCs apoptosis [15]. Furthermore, our group demonstrated that peroxisome proliferator-activated receptor-g (PPARg) played a key role in regulating HSCs apoptosis, and that natural product curcumin could induce HSCs apoptosis dependent on PPARg activation [16]. Dickkopf-1 (Dkk-1), a Wnt coreceptor antagonist, was found to be capable of enhancing PPARg gene promoter activity and restoring HSCs quiescence in culture [17]. Induction of expression of Dkk-1 stimulated apoptosis of activated HSCs.
3. Immune clearance HSCs have emerged as an important target in the liver microenvironment for inflammatory cytokines. Macrophages and infiltrating monocytes participate in the development of fibrosis via several mechanisms, including increased secretion of cytokines and generation of oxidative stress-related products [18,19]. Many kinds of immunocytes participate in the clearance of activated HSCs and the reversal of hepatic fibrosis, including natural killer cells (NK cells), Kupffer cell (KC), T lymphocytes, natural killer T (NKT) cells, etc. Recent evidence indicated that NK cells possessed specifically protective effects during the development of fibrosis. Interestingly, Poly(I:C) or interferon-g (IFN-g) treatment increased the expression of the NK cells activating receptor NKG2D by intrahepatic NK cells and enhanced the cytotoxicity of NK cells against activated HSCs [19]. Increased apoptosis of HSCs was observed after direct adhesion of NK cells to HSCs [20]. NK cells could selectively kill early activated HSCs, however, NK cells had no such effects on fully activated HSC. Signal transducers and activators of transcription 1(STAT1) was indicated as a pivotal factor in mediating the inhibitory effects of NK cells on activated HSCs. Killing HSCs by NK cells was attenuated in STAT1 / mice, which was likely due to the reduced expression of NKG2D and TRAIL on STAT1 / NK cells [21]. Direct interactions were also found between HSCs and lymphocyte subsets, including NK cells. KCs are a type of macrophage populating in the hepatic sinusoid, acting as non-specific killer under normal circumstances. In the pathophysiology of hepatic fibrosis and its recovery stage, KCs play a double-edged sword role. To be specific, during the progression of fibrogenesis, KCs can promote HSCs activation, whereas during fibrosis recovery it can stimulate apoptosis of activated HSCs and ECM degradation [18,22,23]. Studies have showed that KCs could induce apoptosis of activated HSCs via the casepase-9 pathway [24]. Tang et al. confirmed that KCs could induce HSC apoptosis and inhibit their proliferation by activating casepase cascades via secretion of tumor necrosis factor-related apoptosis-inducing ligand in the recovery of liver fibrosis [25]. In
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addition, KCs may effectively inhibit the the expression of tissue inhibitor of metalloproteinase in HSCs, and promote ECM degradation via secretion of inflammation suppressive factors such as IFN-g and interleukin (IL)-10, leading to the reduction in the number of activated HSCs [26,27]. The possibility that T lymphocytes modulate the fibrogenic process was put forward almost 25 years ago. This observation could account for the increased rate of fibrosis in patients with HBV and HCV, in whom the CD4+/CD8+ ratio was reduced typically [28]. CD8+ T cells protect liver cells from pathogen infections while aggravating liver tissue damage [29]. CD4+ T cells under different conditions differentiate into varied types of auxiliary T cells, including Help T cell (Th), mediating different types of immune responses. Th1 cells secrete IFN-g that could restrain HSCs activation and proliferation, induce apoptosis, thereby exert anti-fibrotic effects [25]. Intriguingly, HSCs were also identified to be antigen-presenting cells, and might contribute to the liver immunotolerant properties through T-cell suppression [30]. NKT cells are a unique but heterogeneous group of T cells that express both ab TCR (T cell marker) and cell surface receptors that are characteristic of NK cells. Similar to NK cells, NKT cells also can directly kill activated HSCs, secrete IFN-g and play an anti-fibrotic role [31,32].
4. Phenotype reversion Following fibrogenic stimuli, HSCs undergo a complex transformation known as activation, in which HSCs acquire the properties of activated myofibroblast-like cells associated with an increase in the expression of a-smooth muscle actin and the loss of the vitamin A storage [33,34]. In the development of advanced fibrosis, as the disse microenvironment changes, the surface structure of HSCs is also constantly changed, such as decreased cell connection, and increased ability to move and migrate [33]. Recent report showed that MFBs could revert to an inactive phenotype during regression of hepatic fibrosis [35]. If the transformation of fibrogenic phenotypes is reversible, the antifibrotic therapy is achievable by reducing the production of ECM in HSC and restraining the development of fibrosis. The metabolism of retinols and lipids is closely associated with hepatic fibrogenesis. The mechanistic studies on activation of HSCs and differentiation of preadipocytes have revealed that HSCs and adipocytes share something in common in term of expression of key transcription factors, such as PPARg, liver X receptor-a, CCAAT/enhancer binding protein and retinoid X receptors which play fundamental roles in lipid droplet metabolism [36–38]. Treatment of HSCs with the fat cell differentiation matrix (methyl isobutyl xanthine, dexamethasone and insulin) and ectopic expression of PPARg or Sterol regulatory element-binding proteins-1c in HSCs could stimulate the continuous expression of transcription factors related to lipid generation and reverse the activated phenotype of HSCs [38]. Therefore, inducing expression of genes associated with lipid generation in activated HSCs may be a good strategy for the reversal of the profibrogenic phenotype of HSCs. Mesenchymalto epithelial transition (MET) and Epithelial to mesenchymal transition (EMT) are two fundamental developmental processes [39]. In recent years, the role of EMT in hepatic fibrosis has become an active research direction. In EMT of fibrogenesis, HSCs and hepatic epithelial progenitors obtain the fibroblast phenotype and express the stromal cell markers and eventually become MFBs, promoting the development of hepatic fibrosis [40,41]. However, there has recently been increasing evidence that supports the reverse transition, i.e. MET. This has been implicated in several pathological conditions. For example, it
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was recently discovered that renal fibroblasts could undergo MET to form epithelial aggregates that facilitated the repair of tubular injure in murine fibrotic kidney [42]. Moreover, the Wnt signalling pathway is implicated during the dynamic and reversible EMT and MET that underscore colorectal cancer progression, and it offers a new hope for anti-cancer therapy [43]. Some of key regulators including cadherins, microRNAs, transcription factors (Pax, paraxis, Fox) and growth factors (Wnts, FGFs, Ephrins) might be involved in the developmental MET [44,45]. Therefore, it is anticipated that stimulation of MET of activated HSCs would be an effective strategy to promote the clearance of activated HSCs and reversal of hepatic fibrosis. In the process of hepatic fibrosis, in addition to the overexpressed profibrogenic growth factors, another key change is the gradual replacement of the basement membrane of matrix with the internal matrix [46]. It was observed that HSCs cultured in the EHS glue (similar to the matrix in Disse gap in normal liver) could maintain their quiescence for a long time period. However, HSCs cultured in type 1 collagen (main component of ECM when hepatic fibrosis develops) were apparently activated [47]. Although activated HSCs contribute to the secretion of ECM, the latter can also retroact to HSCs, affecting HSCs behaviors and functions [5,48]. Integrin specifically regulates the connection of ECM components with cellular structures and thus is also a primary factor for maintaining cell stability [49]. Furthermore, ECM can be ligand signal molecules binding to integrin receptors on the membrane of HSCs, affecting the amount and activity of transformation growth factor-b receptors through a series of signaling transductions to keep the collagen physiological metabolism [50]. Based on these discoveries, it has been proposed
that intervention of integrin signaling could beneficially control the molecular linkage between HSCs and ECM, which probably holds promise to reduce the activated phenotype of HSCs [51,52].
5. Senescence Since Hayflick and Moorhead observed the phenomenon of senescence in human firoblasts for the first time, senescence in all kinds of cells has been observed in further studies [53]. However, the relationship between senescence of activated HSCs and its proliferation attracts increasingly attention worldwide. Sekoguchi et al. found that the turnover of cell cycle was the main reason for telomere shortening, which could drive the cells into senescent state and reduce the development of hepatic fibrosis [54]. Moreover, Jun et al. found that in the liver and skin damage, MFBs initially proliferated and secreted ECM, but they finally underwent senescence that inhibited their further proliferation [55]. Furthermore, Krizhanovsky et al. found that senescent cells accumulated in murine livers treated to produce fibrosis and the senescent cells were derived primarily from activated HSCs. Hepatic fibrosis in p53 knockout mice was severe due to the inhibited HSCs senescence [56]. A recent study found that the antifibrotic effect of IL-22 was likely to be mediated via the induction of HSCs senescence in addition to the previously discovered hepatoprotective functions [57]. Since HSCs are the main cells participating in the pathogenesis of hepatic fibrosis, it could be presumed that induction of senescence could block HSCs proliferation and activation, which may be of great significance preventing and treating hepatic fibrogenesis.
[(Fig._1)TD$IG]
Fig. 1. This diagram represents the mode for HSC-mediated liver fibrogenesis and different ways of clearance of activated HSCs. Normally, HSCs are quiescent and nonproliferating cells in space of Disse. Upon injury, they are activated and undergo transdifferentiation to myofibroblasts responsible for the oversecretion of collagen-rich ECM. Clearance of activated HSCs can reduce the number of activated HSCs and ECM generation, achieving the purpose of anti-fibrogenesis. Several ways involved in this progress include apoptosis, immune clearance, phenotype reversion and senescence. The beneficial effects of clearance of activated HSCs may provide potential treatment options for liver fibrosis.
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Further studies showed a correlation between cells positioned at senescence stage and inflammatory microenvironment in organism microenvironment [58,59]. In addition, it was also found that NKG2DU receptor ligand LBP2 and adhesion molecular CD58, which participated in the interaction of natural killer cells with their target cells, were increased. The inflammatory cytokines such as IL-11, IL-8, and IL-6 in the senescent HSCs were also excessively expressed. The senescent HSCs enhanced the functions of natural killer cells and promoted accumulation of immune cells, thereby leading to accelerated clearance of senescent cells [19,56]. Taken together, senescence enables the activated HSCs to lose their proliferative capability and promotes functions of immune cell, making it an important way to eliminate activated HSCs in hepatic fibrogenesis.
6. Concluding remarks and perspectives Developing effective therapies for hepatic fibrosis is still a demanding challenge in current medical and pharmaceutical sciences [60]. Despite so, clinical therapies for this liver disorder have emerged with the increasing understanding of the pathogenesis of hepatic fibrosis [61]. In the clinical contexts, there are no apparent markers for the early phase of hepatic fibrosis, and once patients are diagnosed to be hepatic fibrosis, the syndrome has commonly been in the later phase of the pathogenesis with relatively severe impairment of hepatic functions. At this time, there have been a considerable number of activated HSCs and MFBs in the patient liver, which could cause the degree of hepatic fibrosis to worsen [3,62]. Studies have established that inhibition of HSCs activation or stimulation of their clearance could reverse hepatic fibrosis. Several mechanisms have been identified to contribute the clearance of activated HSCs. Apoptosis, immune clearance, and senescence are directly aimed at activated HSCs to reduce the abundance. On the other hand, phenotype reversion can maintain HSCs quiescence, thereby restraining the secretion of pro-fibrotic cytokines and collagens in the fibrotic liver (Fig. 1). It would be important to develop drugs that can promote the clearance of activated HSCs for therapeutic treatment of hepatic fibrosis. Pharmaceutical industry has found many candidate agents that can restrain HSCs activation and promote HSCs clearance. Especially, pharmacological induction of HSCs apoptosis has shown promise in a number of preclinical investigations. Furthermore, as mentioned above, the bidirectional role for immune cells in HSCs clearance remains to be defined, but preliminarily suggests that the potential drugs should manly limit the development of inflammation to remove activated HSCs [63]. In addition to the HSCs apoptosis-inducing agents, the emerging molecular mechanisms of other means to remove activated HSCs also give rise to high possibility to develop related pharmacological interventions, i.e. drugs that can reverse the phenotype or interfere with the EMT process. As discussed above, lots of signaling pathways and cytokines are involved in the clearance of activated HSCs, and it is also very important to strengthen exploration of potential targets for drugs. What is more, in the seek for modulators of HSCs clearance, more attentions should be paid to the selectivity of drugs. Developing drugs with high selectivity towards activated HSCs with less side effects would be a huge task for research. Researchers have made much progress, for instance, Moreno et al. used a platinum-based linker to develop a conjugate of the AT1 receptor blocker losartan and the HSC-selective drug carrier mannose-6-phosphate modified human serum albumin (losartanM6PHSA), and short-term treatment with this HSC-targeted losartan could markedly reduce advanced liver fibrosis [64]. Understanding of the mechanisms of clearance of activated HSCs
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and their potential implications in hepatic fibrosis continues to be critical in future studies.
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