- Top of page
- Material and methods
Background: Statins are shown to have cholesterol-independent properties such as anti-inflammation and immunomodulation. Activated hepatic stellate cells (HSCs) acquire the capacity to synthesize matrix proteins in damaged liver. We tested the hypothesis that atorvastatin may be capable of inducing apoptosis in HSCs.
Methods: Primary cultures of rat HSCs were exposed to atorvastatin, mevalonic acid and U0126. Quantification of living, apoptotic and necrotic HSCs was performed by flow cytometry and laser-scan microscopy. Cell-cycle analysis was performed by flow cytometry. Pro- and anti-apoptotic factors were investigated by Western blot and electrophoresis mobility shift assay. Protease activity of caspases was calculated using a colorimetric kit.
Results: Atorvastatin leads to a G2-arrest and induces apoptosis in activated HSCs. Atorvastatin-mediated apoptosis could be blocked by co-administration of mevalonic acid and U0126. No effects of atorvastatin on gene expression of CD95, CD95L, NF-κB, p53 and p21WAF1 could be observed. Atorvastatin-induced apoptosis in activated HSCs is related to an increased protease activity of caspase-9 and -3. Gene expression of the major proteins of the bcl-system shows that truncated Bid is involved in apoptosis mediated by atorvastatin. By blocking the extracellular signal-regulated protein kinase (ERK1/2) activation by adding U0126, we could prevent the apoptosis induced by atorvastatin. By Western blot we could not detect any change in the activation of c-jun N-terminal kinase (JNK).
Conclusions: Atorvastatin induces apoptosis in activated HSCs acting through an ERK-dependent cleavage of Bid and a highly increased protease activity of caspase-9 and -3. JNK is not involved in atorvastatin-mediated apoptosis in HSCs.
Hepatic stellate cells (HSCs) are one of the mesenchymal cell populations involved in liver fibrosis and may become the main actor in certain forms of cirrhosis like haemochromatosis (1) or alcoholic cirrhosis (2). Liver fibrosis can be classified as a wound healing response to a variety of chronic stimuli that is characterized by an excessive deposition of extracellular matrix (ECM), of which type I and type III collagen predominate. At present, liver fibrogenesis is considered a dynamic process involving complex cellular and molecular mechanisms, resulting from the chronic activation of the tissue repair mechanisms, which follows recurring liver tissue injury. If the chronic damage persists, inflammation and fibrosis can progress to liver cirrhosis, ultimately leading to organ failure and death. During recovery from liver fibrosis in the rat carbon tetrachloride or in the bile duct ligation model, there is a reduction in the number of α-smooth muscle actin (SMA)-positive cells. Further findings strongly suggested that apoptosis of activated HSCs is responsible for mediating cell loss during recovery from fibrosis (3–6). Evolving antifibrotic therapies should therefore focus on treatments of activated HSCs.
3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, or statins, are widely used in the clinic for their cholesterol-lowering properties. Although the benefits of lowering cholesterol levels in the prevention and treatment of cardiovascular diseases are proven, several clinical trials have shown that statins also have cholesterol-independent properties (7–14), including improving endothelial function (15–18), enhancing plaque stability (19, 20), decreasing vascular inflammation and oxidative stress, inhibiting the thrombogenic response in the vascular wall, and immunomodulatory properties by inhibiting expression of MHC class II proteins (21). These ‘pleiotropic’ effects can be attenuated by addition of the postreductase product, mevalonate (22), and are related to inhibition of the production of other isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) (23).
Although the impact of statin therapy on each of these processes is not fully understood, ongoing studies are likely to shed further light on the potential pleiotropic benefits of statins, some of which are focusing on ECM-producing cells to evaluate a potential antifibrogenic effect. An in vitro study on rat HSCs treated with two different statins showed that the collagen synthesis by activated cells was decreased (24). To establish whether this effect could be because of a reduction of cellular viability through the induction of apoptosis, we studied the effect of one of the statins of the newest generation, atorvastatin, on the cellular life of rat HSCs.
- Top of page
- Material and methods
In this work we studied the effect of atorvastatin on cell cycle and on the fate of activated rat liver HSCs in primary culture. Atorvastatin dose dependently induces a G2 arrest and an apoptosis rate of 68% at ‘therapeutic’ concentrations in activated HSCs. The apoptotic effect is mediated by the ERK-dependent mitochondrial pathway followed by the increase of caspase-9 activity. This effect can be fully blocked by caspase-9 inhibitors, by ERK-inhibitor U0126 as well as by mevalonic acid.
The HMG-CoA reductase inhibitors are widely used in clinics for their cholesterol-lowering properties. Recent experimental and clinical evidence indicates that some of the beneficial effects of statins are because of their pleiotropic effects, such as anti-inflammation and immunomodulation, encouraging further research aimed at evaluating the potential of statins (7–21, 23).
Activated HSCs are one of the major matrix-producing cell types during liver repair and fibrosis. During recovery from liver fibrosis in the rat carbon tetrachloride and bile duct ligation model of fibrosis, there is a reduction in α-SMA-positive cell number. Further findings strongly suggested that apoptosis of activated HSCs may be responsible for mediating cell loss during recovery from fibrosis (3–6). Evolving antifibrotic therapies are therefore targeted at inhibiting activation of HSCs or in inducing their apoptosis.
In a previous in vitro study, the effects of two different HMG-CoA inhibitors (simvastatin and lovastatin) on rat HSCs were evaluated. The results show that these statins are able to reverse the activation and to inhibit the synthesis of collagen type I and reduce the synthesis of collagen types III and IV. These effects were partially reversible with PDGF and mevalonate (24). We believe that these effects are due to apoptosis induced by statins.
In this study we have shown that atorvastatin, one of the statins of the newest generation, is capable of blocking activated HSCs in the G2 phase of the cellular cycle and of inducing their apoptosis in vitro. The apoptosis induced by atorvastatin is dose dependent in activated HSCs. The fraction of cells undergoing apoptosis increased from a control value of 19% apoptosis (spontaneous apoptosis) proportionally with increasing doses of atorvastatin to 60% at the serum concentration used in clinic (10−7–10−8 mol/L) and to 92% apoptosis at a dose of 10−3 mol/L atorvastatin. In quiescent HSCs, the fraction of cells undergoing apoptosis at the serum concentration used in clinics, does not differ significantly from the control. However, at the dosage used in clinic the concentration of atorvastatin in the tissue remains unknown; nevertheless no toxic effect and no changes at the mitochondrial level could be detected in quiescent HSCs even at higher dosage (10−3 and 10−5 mol/L).
These data suggest that atorvastatin, by up-regulating the apoptosis of activated HSCs, could decrease the extent of fibrosis in chronic liver injury while having no effects on quiescent HSCs. Induction and execution of apoptosis programmes are generally believed to be mediated through a hierarchy of caspase activation. Caspases are synthesized as catalytically inactive pro-enzymes and need to be activated by proteolytic cleavage at internal aspartate residues (41, 42). Two pathways of caspase activation during apoptosis have been described. The first one is mediated by death receptors, such as CD95 or tumour necrosis factor (TNF) receptors, controlled by caspases-8/10, which in turn activate downstream effector caspases such as caspase-3 and -7. Spontaneous apoptosis occurring in activated HSCs involves an increased expression of CD95/CD95L (Fas and Fas-ligand) (3).
We evaluated the expression of those proteins in control and in atorvastatin-treated cultures. Although a strong increase in the expression of CD95 and a significant increase in the expression of CD95L were noted in activated HSCs compared with the control cells, no significant changes between the control cells and the cells treated with atorvastatin were detectable, showing that the CD95/CD95L pathway is not involved in the apoptosis induced by atorvastatin. In the second pathway, diverse apoptotic signals converge at the mitochondrial level, inducing the release of cytochrome C from the mitochondria to the cytosol (the cytochrome C is being released into the cytoplasm following the loss of the mitochondrial transmembrane potential) (43, 44). Once in the cytosol, cytochrome C binds to its cytosolic partner apoptotic protease activating factor-1 (APAF-1) and induces the oligomerization of APAF-1–cytochrome C complex in a dATP/ATP-dependent manner (45, 46). This multimeric complex, named ‘apoptosome’, is sufficient to recruit the initiator caspase, procaspase-9, to the complex and induces procaspase-9 autoactivation (47). The activated caspase-9 is released from the apoptosome and subsequently initiates a caspase cascade involving effector caspases such as caspase-3, -6 and -7 (48, 49).
Once active, these caspases cleave various cellular targets, ultimately leading to cell death. It is known that both the death receptor and the mitochondrial pathway can be interconnected via the activation of the pro-apoptotic Bcl-2 family member, Bid, through caspase-8, possibly serving as an amplification loop (50). The mitochondrial pathway of caspase activation is largely determined by the Bcl-2 family of anti- and pro-apoptotic regulators. These proteins respond to cues from various forms of intracellular stress, such as DNA damage or cytokine deprivation, and interact with opposing family members to determine whether or not the caspase proteolytic cascade should be unleashed (51). Some Bcl-2 family members that are located on the mitochondrial membrane can alter the permeability of the mitochondrial membrane and trigger the activation of caspases. Bid is distinguished from other pro-apoptotic Bcl-2 family members in that it plays a critical role in both receptor- and granule-mediated apoptosis because Bid is a substrate for both active caspase-8 and granzyme B. Active caspase-8 cleaves Bid at aspartic acid residue 60, while granzyme B preferentially cleaves Bid as aspartic acid residue 76. The cleavage product as processed by either protease, termed t-Bid, then translocates to the mitochondria through mechanisms that include selective binding to cardiolipin, a mitochondrial membrane lipid. Once at the mitochondria, t-Bid induces the oligomerization of Bax and Bak, leading to mitochondrial membrane permeability and release of the apoptosome, which activates caspase 9.
We have previously shown that the ERK-dependent activation of the Bcl-2 family can have a pro-apoptotic effect on HSCs (52).
The mitogen-activated protein (MAP) kinases are a family of second-messenger kinases that are essential for transferring signals from the cell surface to the nucleus.
These kinases are a family of serine–threonine protein kinases that are activated in response to a variety of extracellular stimuli. ERK, c-Jun NH1-terminal kinase (JNK) and p38 MAP kinase constitute three major subfamilies of MAP kinases that appear to mediate cellular responses, including proliferation, differentiation and apoptosis. (53)
Extracellular signal-regulated protein kinase plays a major role in cell proliferation and differentiation, as well as in survival mediated by various growth factors (54).
On the other hand, JNK and p38 MAP kinase are activated by various inflammatory cytokines and environmental stressors, and play an important role in the signal cascades leading to the induction of cytokines and chemical mediators (55).
Cellular ERK activation either inhibits or enhances apoptosis in some cells (56, 57). Even in the same cell, the role of ERK activation is likely to differ depending on the cellular insult (56).
Several studies revealed a pathway of apoptosis induced by an ERK-dependent increased activation of the downstream kinases JNK and p38 MAP kinase. On the other hand, other studies have shown that ERK-induced apoptosis may occur with or without activation of JNK (58, 59).
In the present study we have shown the effect of atorvastatin in leading to apoptosis in activated HSCs, and we gained insight into mechanisms of apoptotic response, previously undetected in HSCs.
When we evaluated the expression of some of the pro- and anti-apoptotic members of the Bcl-2 family, in quiescent and activated HSCs, in control and in atorvastatin-treated cultures, the results showed an increased expression of Bax and a decreased expression of Bcl-2 and Bcl-xl (pro-apoptotic ratio) in activated HSCs compared with the quiescent HSCs. Between the control cells and the cells treated with atorvastatin, we could detect an activation of t-Bid in activated HSCs treated over 6 h with atorvastatin, but the activity of caspase-8 remained unchanged, showing that there is no link between the death receptor and the mitochondrial pathway. We followed the MAP kinase and the caspase activation pathways; atorvastatin induces an increased phosphorylation of ERK(1/2), but not its basal expression level. By adding the ERK-inhibitor U0126 and also by adding mevalonic acid to activated HSCs, we could fully block the apoptosis induced by atorvastatin. The increased phosphorylation of ERK(1/2) is not followed by activation of JNK.
In this study we have shown that the pro-apoptotic effect of atorvastatin on HSCs does not involve death receptors such as Fas, while it seems to be mediated by the release of cytochrome C from the mitochondria, induced by an ERK-dependent activation of t-Bid, followed by an increase in the activity of caspase-9 and -3 (Fig. 8).
Figure 8. Diagram summarizing the pathway of apoptosis induced by atorvastatin in hepatic stellate cells (HSCs). Atorvastatin induces an increased phosphorylation of extracellular signal-regulated protein kinase (ERK1/2) which is followed by activation and cleavage of Bid into truncated Bid (t-Bid). t-Bid translocates to the mitochondria leading to changes in the mitochondrial membrane permeability (see Fig. 7), which initiates activation of procaspase-9 to caspase-9. The activated caspase-9 is released from the apoptosome and subsequently initiates a caspase cascade involving the effector caspases such as caspase-3. Once active, this caspase cleaves various cellular targets, ultimately leading to DNA damage and apoptosis.
Download figure to PowerPoint
In cells treated with atorvastatin, we could detect apoptotic changes at the mitochondrial level 6 h after beginning the treatment, while the detection of apoptosis with other methods (annexin V-FITC, TUNEL) was first possible after 20 h.
Apoptosis induced by atorvastatin in activated HSCs in culture is fully inhibited by caspase-9 inhibitors, ERK-inhibitor and mevalonate, showing that this effect is specific.
We also evaluated the expression of the transcription factors NF-κB and p53. Activation of NF-κB in cultured HSCs is required for expression of specific genes associated with the activated phenotype and is anti-apoptotic for rat HSCs, while the normal function of p53 is to effect cell cycle arrest at the G1 and G2 phases in response to DNA damage to allow DNA repair and, if repair is not successful, p53 initiates programmed cell death to prevent the propagation of genetic defects to successive generations of cells. Involvement of those proteins in apoptosis regulation in HSCs could be shown for the signals of transforming growth factor (TGF)-α and TNF-α (60). Both transcription factors are increasingly expressed in activated HSCs compared with quiescent cells, but no differences were detectable in atorvastatin-treated cells compared with the control, showing that the increased ERK1/2 phosphorylation and the apoptotic pathway initiated by atorvastatin are not regulated by those transcription factors.
Considering the importance of apoptosis occurring in activated HSCs, these in vitro data suggest that atorvastatin could act not only as an anti-inflammatory agent but also as an antifibrotic agent. On the other hand, other fibrogenic cells (like the resident myofibroblasts and fibroblasts) are involved in liver fibrogenesis. Preliminary observations suggest that liver myofibroblasts show a different response to atorvastatin administration with respect to apoptosis when compared with activated HSCs.
These results suggest atorvastatin as a candidate for further in vitro as well as in vivo study in antifibrotic strategies.