Antibody-targeted myofibroblast apoptosis reduces fibrosis during sustained liver injury

https://doi.org/10.1016/j.jhep.2008.01.032Get rights and content

Background/Aims

Myofibroblast apoptosis promotes the resolution of liver fibrosis. However, retaining macrophages may enhance reversal. The effects of specifically stimulating myofibroblast apoptosis in vivo were assessed.

Methods

A single chain antibody (C1-3) to an extracellular domain of a myofibroblast membrane protein was injected as a fluorescent- or gliotoxin conjugate into mice with liver fibrosis.

Results

C1-3 specifically targeted α-smooth muscle actin positive liver myofibroblasts within scar regions of the liver in vivo and did not co-localise with liver monocytes/macrophages. Injection of free gliotoxin stimulated a 2-fold increase in non-parenchymal cell apoptosis and depleted liver myofibroblasts by 30% and monocytes/macrophages by 50% but had no effect on fibrosis severity in the sustained injury model employed. In contrast, C1-3-targeted gliotoxin stimulated a 5-fold increase in non-parenchymal cell apoptosis, depleted liver myofibroblasts by 60%, did not affect the number of monocytes/macrophages and significantly reduced fibrosis severity. Fibrosis reduction was associated with increased metalloproteinase-13 levels.

Conclusions

These data demonstrate that specific targeting of liver myofibroblast apoptosis is the most effective anti-fibrogenic therapy, supporting a role for liver monocytes and/or macrophages in the promotion of liver fibrosis reduction.

Introduction

Liver fibrosis is a common outcome in patients with chronic liver damage of any cause [1]. It is associated with the activity of liver-resident fibroblasts, derived from the trans-differentiation of hepatic stellate cells (HSCs) to myofibroblast-like cells or the proliferation of portal fibroblasts [1]. The fibrogenic response is a component of the inflammation that occurs in response to hepatocyte necrosis, with liver myofibroblasts-like functioning to re-model the extracellular matrix prior to tissue regeneration [1].

Most clinical liver disease is associated with chronic liver injuries and excessive inflammation [1]. Liver myofibroblasts generate scarring within the tissue that ultimately leads to cirrhosis. Under these conditions, the liver is unable to regenerate and the only treatment available at present is liver transplantation [1]. This laboratory [2], [3] and others [4], [5], [6], [7], [8] have shown that a stimulation of pro-fibrogenic liver myofibroblast apoptosis reduces scarring in recovery animal models of liver fibrosis. These experiments suggest that reductions in the numbers of liver myofibroblasts in a fibrotic liver may be an effective therapeutic approach to chronic liver disease. However, it is now clear that a complex relationship exists between various liver cell types in the diseased liver. The initial switch that triggers a fibrogenic response in liver myofibroblasts has not been unequivocally identified but there is good evidence to suspect that liver-resident macrophages (Kupffer cells) are its (major) source [1]. Further, many of the pro-inflammatory cytokines produced by Kupffer cells promote liver myofibroblast fibrogenic activity, most notably interleukin 6, TNF-α and TGF-β [9], [10], [11], [12], [13]. The prominent role of Kupffer cells in fibrogenesis was elegantly demonstrated by Duffield et al. in which fibrosis was significantly reduced in a mouse model of disease when Kupffer cells were depleted from the liver [14]. Surprisingly however, Kupffer depletion also inhibited the normal reversal of fibrosis that can occur prior to cirrhosis when liver damage was halted [14]. Therefore, an effective anti-fibrotic therapy reliant on the stimulation of apoptosis may likely need to be specifically targeted to liver myofibroblasts for optimum efficacy.

Synaptophysin is a membrane protein present in a restricted number of cell types such as neural cells. It is thought to function in the release and/or uptake of neurotransmitters [15]. In the liver, synaptophysin expression is reported to be restricted to HSC-derived myofibroblasts (myofibroblasts express a number of genes associated with neural tissue) [14], [15], [16], [17], [18], [19]. Synaptophysin’s external cellular location and cycling to intracellular location(s) make it a potential site for targeting liver myofibroblasts with therapeutics. Recombinant human monoclonal single chain antibodies (scAbs) were therefore generated to a conserved peptide sequence present in an extracellular domain of synaptophysin [20].

In this study we show that the C1-3 scAb does not cross the blood brain barrier and targets liver myofibroblasts in vivo in a mouse model of liver fibrosis. In addition, gliotoxin – a compound shown to stimulate apoptotic-resistant fibrogenic human HSC apoptosis in vitro [21], when chemically conjugated to C1-3, significantly reduced the numbers of liver myofibroblasts present in the fibrotic disease model without affecting Kupffer cell numbers. The effects of the C1-3–gliotoxin conjugate were compared to the effects of free gliotoxin and a control scAb–gliotoxin conjugate (CSBD9, a scAb raised to microcystin [22], which should not bind to the surface of liver cells).

Section snippets

Recombinant scAb expression and conjugation

The polyhistidine tagged recombinant scAbs – C1-3 (to synaptophysin) and CSBD9 (to microcystin-LR [22] and used as a control in these studies) were expressed in Escherichia coli and purified by nickel chromatography and gel filtration essentially as previously described [20], [22]. Endotoxin was removed from preparations using Q Maxi H columns (Sartorius Vivascience, Epsom, UK) and all preparations were tested (<0.2 EU/ml) by Lonza Biologics (Slough, UK) prior to use in vivo. Purified scAbs were

ScAb conjugation does not significantly affect antigen affinity and conjugate activity in vitro

C1-3 and CSBD9 ScAbs were conjugated with FITC or gliotoxin. Fig. 1A demonstrates that the mean molecular weight of the C1-3 scAb after conjugation with gliotoxin increased by an average of 1.5 kDa indicating that on average 3.5 molecules of gliotoxin had been conjugated to the C1-3 scAb protein (see supplementary Fig. 1). Similar results were observed with the CSDB9 scAb (data not shown). FITC conjugation generally resulted in the conjugation of 4–5 molecules of fluorescein per molecule of

Discussion

There are currently no treatments indicated for liver fibrosis [30]. In animal models of reversible fibrosis where the liver damaging agent is withdrawn (i.e. recovery), fibrosis resolution is accompanied by the apoptosis of liver myofibroblasts [31]. Previous work demonstrated – with free gliotoxin – that fibrosis resolution in rats was accelerated by stimulating the apoptosis of myofibroblasts [2], [4]. In these studies, there was evidence in liver sections for an increase in the apoptosis of

Acknowledgements

A.D. and K.W. are supported by studentships from the BBSRC.

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