Fat-storing cells and liver fibrosis

Abstract

Key Words: Adipocytes liver cirrhosis, Cytokines, Extracellular matrix

INTRODUCTION

In normal liver tissue, fat-storing cells (FSCs, also known as Ito cells) lie between endothelial cells and hepatocytes in the space of Disse. One of the most characteristic features of these cells is the presence of cytoplasmic lipid droplets. Desmin has been the best specific and reliable marker for detection of FSCs up to now. Recently, however, more attention has been paid to the relationships between FSCs and liver fibrosis[1,2].

A prominent role of FSCs in human and experimental fibrogenesis has long been suspected based on the identification of transitional cells with regions of active liver injury that have features of both FSCs and fibroblasts. Transitional cells have also been termed myofibroblasts, the latter defined morphologically by the presence of prominent microfilaments, ruffled nuclear membrane, and contractile activity. Liver transitional cells/myofibroblasts additionally have hypertrophied rough endoplasmic reticulum and are associated with increased pericellular collagen. They are proliferative in situ and retain expression of desmin. The theory of FSC origin of myofibroblasts was supported by the expression of α-smooth muscle actin (α-SMA) upon activation both in culture and in vivo; α-SMA is typical of myofibroblasts[2].

In summary, FSCs activation can be defined as the acquisition of a myofibroblastic phenotype with enhanced fibrogenic and proliferative activity.

BIOLOGICAL FUNCTIONS OF FSCS

Extracellular matrix (ECM) synthesis

A combination of immunoelectron microscopy, in situ hybridization and Northern blot analysis of purified liver cell populations has shown that FSCs are major sources of matrix components in both normal and fibrotic livers[3]. When set up in primary culture, FSCs remain quiescent for 4-5 d, then undergo dramatic phenotypic changes towards a myofibroblastic appearance. They proliferate and express both basement membrane and interstitial matrix proteins at high levels. The results from immunoelectron microscopy also suggest that FSCs can synthesize ECM components, such as collagens, fibronectin, laminin, undulin, tenascin, perlecan, etc. Sinusoidal cells contain gene transcripts for α1 (I), α2 (I), α1 (III) and α1 (IV) procollagens and laminin B1 chain. Unfortunately, the current in situ hybridized technique can not provide sufficient resolution to establish firmly which sinusoidal cell populations contain the signals[4]. However, non-parachymal cells containing these transcripts in experimental models of fibrosis have morphological and phenotypic features of FSCs. Undulin, found in recent years, is a large ECM glycoprotein that is associated with dense interstitial connective tissue. With double-staining for cell type-specific antigens, Milani et al[5] showed undulin expression in mesenchymal cells, which were identified by immunostaining for vimentin. A number of cells expressing a small amount of undulin RNA located on the fibrotic septa and cirrhotic nodules also stained for α-SMA. Several investigators[6]have used Northern blot hybridization to demonstrate ECM mRNAs in freshly isolated cell fractions to avoid the error caused by phenotypic changes of FSCs. Geerts have shown that isolated FSCs contain strong transcripts for α1 (III) procollagen and type IV collagen. Maher et al demonstrated that FSCs isolated from animals with experimental liver injury showed a dramatic increase in procollagen [WTBZ] α1 (III) and α1 (I) transcripts. Although other sinusoidal cells and hepatocytes could produce some ECM components, the FSCs are quantitatively more important.

Collagenases and collagenase inhibitors synthesis

The collagenases, which are matrix metalloproteinases, have been grouped into interstitial collagenase, type [WTBZ] IV collagenase, and stromelysin. In the liver, FSCs are the major source of interstitial collagenase that degrades type I and III collagens. There are two types of IV collagenase, one produced by fibroblasts (72000) and the other derived from macrophages (92000). The activated FSCs secrete the 72000 type IV collagenase to degrade basement membrane collagen and denatured collagen (gelatin)[7]. Metalloproteinase activity is regulated in part by the tissue inhibitors of metalloproteinases (TIMPs), TIMP-1 and TIMP-2, and by general proteinase scavengers such as α-2 macroglobulin. FSCs in culture synthesize TIMP-1 and express α-2 macroglobulin, which by inhibiting collagenase activity and hence collagen degeneration may result in increased collagen accumulation. Release of metalloproteinase inhibitors by activated FSCs may contribute to progressive fibrosis by preventing degradation of interstitial collagen[2,7].

Cytokines synthesis

Transforming growth factor β (TGFβ) is the most important cytokine in liver fibrosis. FSCs express TGFβ1 mRNA in the CCl4-induced rat model of liver fibrosis. FSC clones (CFSC) derived from CCl4-induced rat liver tissue are heterogeneous with regard to the expression of cytokines and various ECM components. While clone CSFC-2G expresses low basal levels of α1 (I) procollagen, TGFβ and IL-6 mRNAs, clone CFSC-5H expresses high levels of these mRNAs. Inagaki et al[6] suggested that CFSC-5H cells have been already activated and express type I collagen due to an autocrine stimulation with TGFβ. However, although CFSC-2G cells are activated and probably contain the required nuclear transcriptional factors, they need paracrine stimulation with TGFβ to produce type I collagen. FSCs are believed to be the major source of circulating insulin-like growth factor-1 (IGF-1) and FSCs express IGF binding proteins and their mRNAs[8]. Cultured human FSCs can express the genes encoding for the platelet-derived growth factor (PDGF) A and B chains. The gene expression of these two PDGF chains is associated with the secretion of bioactive PDGF. Moreover, FSCs are able to synthesize and release platelet-activating factor (PAF) and its 10-acyl analogue, a fluid-phase and cell-associated mediator of inflammation having potent effects on hepatic circulation and metabolism[9]. FSCs also express a 47 kD collagen-binding heat shock protein, which closely correlates with the transition of FSCs and development of hepatic fibrosis. Some investigators have demonstrated that FSCs can release other soluble factors, such as macrophage-colony stimulating factor and monocyte chemotactic peptide 1, heparin binding growth factor-2, etc. Several cytokines, including TGFβ, PDGF, IL-1 and IGF-1, are mitogenic for FSCs but the most important profibrogenic factor appears to be TGFβ[2]. TGFβ1 induces the synthesis of fibronectin, laminin, collagens, proteoglycans and TIMPs. In addition, TGFβ1 down-regulates the expression of stromelysin and interstitial collagenase. The key function of TGFβ1 is to activate phenotypic changes that occur in FSC primary cultures, from a 'resting' state toward a myofibroblastic-like state. Interestingly, TGFβ has been shown to increase the steady-state levels of its own mRNA in FSCs, which forms positive feedback effects[11]. DNA synthesis and proliferation of FSCs is stimulated by PDGF, EGF, TGFβ, basic FGF, IL-1 and TNFα. PDGF may play a critical role during the activation of FSCs. Both stimulatory and inhibitory effects of TNFα on collagen synthesis in FSCs have been described. More interestingly, however, TGFβ has been shown to strongly inhibit EGF or TGFβ-induced proliferation of FSCs. The interferons (IFNs), which are potent inhibitors of FSC activation and proliferation. Therefore, the functional imbalance of up- and down-regulated cytokines on FSCs may affect fibrogenesis.

Other functions

Integrins are the main receptors of ECM. Integrin β1 chain expression is found in sinusoidal cells in liver tissue under conditions of alcoholic hepatitis and cirrhosis. Unfortunately, concrete cell types can not be identified[10]. The FSCs also have functions in storing and metabolizing vitamin A under normal and fibrotic conditions.

MEDIATION OF HEPATIC FIBROSIS

Interferons

IFNs are potent antifibrotic agents both in vivo and in vitro. Jimenez et al showed that both α- and γ-IFNs caused a dose-dependent inhibition of collagen synthesis in quiescent human fibroblasts. In experimentally-induced fibrotic livers, γ-IFN treatment of Schistosoma-infected mice was found to affect collagen accumulation and to decrease the steady state procollagen α1 (I) and α1 (III) mRNA levels[11]. Capra et al[12] demonstrated that in some patients with chronic viral hepatitis and cirrhosis, who had been treated with recombinant γ-IFN 2a for 6 mo at least, aminotransferase activity, serum procollagen III peptide and laminin levels had significantly decreased, while the scores for inflammation and necrosis were significantly lower than those in the control group. The data suggest that γ-IFN treatment may decrease stimuli for fibrogenesis by reducing liver inflammation and necrosis, thus preventing progression to cirrhosis. Substantial data have been reported showing that α- and γ-IFNs are potent inhibitors of FSCs activation in vitro. These factors have marked effects on FSC proliferation and significantly reduce the steady state levels of mRNAs for α-SMA, collagens and fibronectin. In addition, IFNs can modulate other cytokines so as to affect the FSC activities indirectly.

Vitamin A

Retinyl palmitate is the predominant storage form of vitamin A in FSCs. It is possible to infer that a decrease in retinyl palmitate per se may be a facilitating factor in hepatic fibrosis by lipocytes. Senoo et al have demonstrated that the administration of vitamin A inhibits CCl4-induced fibrosis, and another study has shown that vitamin A treatment inhibits the rate of collagen synthesis by FSCs[13]. There are some conflicting data suggesting that chronic hypervitaminosis A results in hepatic cirrhosis. Up to now, the majority of authors have suggested vitamin A can inhibit proliferation and transition of FSCs in fibrosis. We believe that the dose of vitamin A is the key to determining up- or down-regulation on FSCs.

Polyunsaturated lecithin (PUL)

Lieber et al[14] have shown that oral supplementation of the diet with a purified soybean PUL extract can prevent the development of septal fibrosis and cirrhosis in baboons fed ethanol. Their previous study, which involved withdrawal of the PUL from the ethanol-diet in three baboons, showed progression of perivenular and/or interstitial fibrosis to septal fibrosis and then to cirrhosis in all three baboons after 18-21 mo. The mechanism for the remarkable effect of PUL in preventing liver fibrosis remains unknown. Regarding the effects of PUL on collagen synthesis, it was noted that the percentage of transformation of lipocytes to transitional cells was decreased by PUL in the livers of the alcoholic-fed baboons. In cultured rat FSCs, PUL can increase collagenase activity of the cells. PUL has also been shown to have excellent bioavailability in patients with no known side effects[7].

Chinese herbal medicines

We have investigated the therapeutic action of Cordyceps sinensis (CS) on CCl4-induced hepatic fibrosis in Wistar rats. Serum procollagen levels in rats treated with CS were much lower than those in the control group (P < 0.01). In liver tissue, degeneration and necrosis of hepatocytes, fibrosis and deposition of type I , III and IV collagens in the Disse space were all less than observed in the controls. Moreover, the number of desmin-positive cells, after treatment for 12 wk, was fewer than that in the control group (P < 0.01). Our ultrastructural data showed that CS could inhibit proliferation and transition of FSCs to myofibroblasts. It is suggested that CS can effectively prevent and cure experimental hepatic fibrosis, and that the mechanism may involve inhibition of FSCs by CS[15]. Sun et al[16] have demonstrated that tetrandrine, a purified extract from the Chinese herb Stephania tetradra S. Moore, can prevent and treat rat hepatic fibrosis, via the possible mechanism of FSC inhibition. Their later clinical study provided the same results. Our investigation on FSC primary culture has confirmed that tetrandrine exerts an inhibitory effect on proliferation and transition of FSCs (data not shown). Some other Chinese herbs have been identified and studied in experimental animals and patients, such as Salvia miltiorrhiza, peach kernel, etc., but the mechanisms need to be studied.

Others

Prednisone is a widely utilized inhibitor of TNF production, and colchicine is used frequently in various liver diseases. These agents can decrease TNF receptor density and protect against TNF toxicity in mice in vivo. Multiple agents, such as antibodies against IL-1, IL-1 receptor antagonists, and TNF, have been used to affect the activities of FSCs indirectly.