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Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl₄-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration¹

Liver Research Group and Department of Histopathology, Infection, Inflammation and Repair, South Lab and Path Block, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK; and
^* Harvard Medical School, Massachusetts General Hospital, Boston, USA

³Correspondence: Liver Research Group and Department of Histopathology, Infection, Inflammation and Repair, South Lab and Path Block, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK. E-mail: jpi{at}soton.ac.uk

SPECIFIC AIMS

We set out to investigate the hypothesis that collagen-I degradation is critical to hepatic stellate cell apoptosis and hepatocyte regeneration during recovery from liver fibrosis.

Hepatic stellate cells (HSC) are the pivotal cell type involved in the development of hepatic fibrosis. HSC are known to undergo a phenotypic change in fibrotic injury from a quiescent retinoid-storing cell to an activated myofibroblast-like phenotype. In this activated form, HSC proliferate and are the major source of the fibrillar collagens (predominantly type I collagen) that characterize liver fibrosis. HSC also express matrix-degrading metalloproteinases and the potent tissue inhibitors of metalloproteinases (TIMPs) 1 and 2. Consequently, the development and progression of fibrosis results from decreased matrix degradation in addition to enhanced matrix synthesis. Activated HSC proliferate in response to injury, so that the progression of fibrosis is associated with a net increase in HSC numbers besides the increased matrix synthesis that results from HSC activation.

We recently demonstrated that recovery from established hepatic fibrosis occurs in the CCl₄ and bile duct ligation models of liver injury and that two key elements to this process are apoptosis of activated HSC and degradation of the fibrotic matrix. Increased matrix degradation is closely correlated with a decrease of TIMP-1 and 2 expression in the liver. The close temporal correlation between HSC apoptosis and degradation of the extracellular matrix (ECM) suggests that the two events may be linked. With reference to type I collagen, tissue culture experiments have indicated that this matrix promotes or perpetuates the activated phenotype of HSC. Contact with type I collagen has been described to take epithelial cells out of the cell cycle, which they then reenter after contact with partially degraded collagen-I, an event that may be mediated by the integrin vß₃. Therefore the degradation of collagen may also influence the regenerative response in hepatocytes during recovery from fibrotic injury.

We have studied the role of collagen degradation during the process of recovery from repeated CCL₄ injury that leads to liver fibrosis using mice (Col 1a1^r/r) in which a mutation was engineered in collagen-1 that encodes amino acid substitutions around the single collagenase cleavage site in the 1(I) chains of the type I collagen triplex helix. The collagen extracted from the Col1a1^r/r mice is completely resistant to digestion by all collagenolytic MMPs tested so far. Since neither the 1(I) nor the 2(I) chains are cleaved, the mutation behaves as dominant negative. This model allows us to directly examine the influence of collagen-I on the stellate cell and hepatocyte components of the hepatic sinusoid after induction of a fibrotic injury.

PRINCIPAL FINDINGS

1. Collagen persists in the livers of Col-1a1^r/r mice during spontaneous recovery from experimental liver fibrosis
By hydroxyproline analysis, wild-type (WT) livers demonstrated a significant decrease in total liver levels during a 28 day recovery period from liver fibrosis induced by 8 wk CCl₄ treatment. In contrast, Col-1a1^r/r livers had persistently raised total liver hydroxyproline levels through the 28 day recovery period compatible with the persistence of type I collagen. Moreover, a distinct pattern of fibrosis was observed in the Col-1a1^r/r livers in which perisinusoidal fibrosis predominated and persisted.

2. Activated HSC persist in the Col-1a1^r/r livers after withdrawal of CC1₄
Protein extracts from each injured liver were subjected to analysis for SMA by Western blot. After 8 wk of CCl₄ intoxication, both genotypes demonstrated enhanced levels of SMA relative to untreated control liver (Fig. 1 ). After 28 days recovery, however, this high level of expression persisted in the Col-1a1^r/r livers), indicating persistence of activated HSC, whereas expression in the WT livers dropped to levels similar to control (Fig. 1) .

View larger version (30K): [in this window] [in a new window]   Figure 1. Activated stellate cells persist in Col 1a1r/r livers but are lost from WT livers by apoptosis during recovery from liver fibrosis. By Western analysis, SMA protein (a marker of activated HSC) is observed at comparable levels at time 0 (peak fibrosis) in WT and Col 1alr/r livers and increased relative to the low baseline expression in control (untreated) livers of each genotype. At 28 days of recovery, SMA expression remains at levels comparable to peak fibrosis (time 0) in Col 1a1r/r livers but is barely detectable in WT livers.

3. Expression of type-I collagen and SMA mRNA persists during recovery in the Col-1a1^r/r livers
To further corroborate persistence of, and determine the activity of, activated HSC in the Col-1a1^r/r livers, relative expression of collagen-I and SMA was determined by Taqman PCR. Type I collagen and SMA expression was significantly increased relative to normal (untreated mouse liver) for both genotypes at peak fibrosis. In the WT livers, collagen and SMA expression became significantly decreased throughout the 28 day recovery period, approaching the levels observed in normal untreated liver. Although some reduction in expression of type I collagen and SMA was observed in the Col-1a1^r/r livers, expression persisted and a significant reduction in expression during recovery was not observed.

4. Apoptosis of activated HSC is reduced in Col-1a1^r/r livers relative to WT livers
We earlier demonstrated that apoptosis is the major mechanism by which activated HSC are removed during recovery from fibrosis. The mean number of TUNEL positive cells in a distribution consistent with activated HSC was raised in the WT relative to the r/r livers at each time point, a difference that was significant at 4, 7, and 28 days of recovery.

5. Hepatocyte regeneration is reduced in Col-1a1^r/r livers
The resolution of fibrotic injury is associated with a hepatocyte regenerative response (analogous to re-epithelialization during wound healing). In the WT livers, there was clear evidence of hepatocyte regeneration over the first 4 days of recovery after the final injection of CCl₄. In Col-1a1^r/r livers, hepatocyte regeneration was reduced, with mitotic figures being only rarely observed in these livers over the same period (Fig. 2 ). Relative to WT collagen-I, hepatocytes (Huh7 cells) plated on to r/r collagen demonstrated a 35% decrease in ³H-thymidine incorporation. Moreover, addition of the disintegrin echistatin or integrin ß₃-neutralizing antibody resulted in a fall in ³H-thymidine incorporation in hepatocytes maintained on WT collagen-I, reaching levels comparable to those observed in cells plated onto the r/r collagen-I but had no effect on hepatocytes maintained on r/r collagen.

View larger version (20K): [in this window] [in a new window]   Figure 2. Hepatocyte regeneration is impaired in the presence of persistent collagen-I as demonstrated by a significant reduction in the number of observed hepatocyte mitotic figures. The mean number of hepatocyte mitotic figures in 50 high-power fields from each specimen of liver at time 0 and 4 and 28 days of recovery for each genotype is represented graphically (n=4 for each genotype at each time point, *P<0.05 for combined data of WT (empty bars) at 0 and 4 days with Col 1al r/r (filled bars) at 0 and 4 days of recovery by Mann Whitney test).

CONCLUSIONS AND SIGNIFICANCE

Our studies with the recovery model using the Col-1a1^r/r mice demonstrate that failure to degrade type I collagen also affects hepatocyte regeneration. Our previous findings in a rat model of liver fibrosis have been reproduced in the WT mice and show that type I collagen degradation clearly occurs during recovery from liver fibrosis. This is associated with a diminution in hepatic stellate cell number, mediated by apoptosis, and a decrease in collagen-I and SMA expression. In contrast, in the presence of collagen-I resistant to MMP degradation, persistent fibrosis ensues during recovery and is associated with persistence of activated hepatic stellate cells.

By quantifying apoptotic HSC, we were able to demonstrate that HSC persistence in the Col-1a1^r/r livers is likely to result at least in part from a failure of apoptosis. It has recently been demonstrated that adenoviral-mediated hepatic overexpression of collagenase (MMP-1) is sufficient to result in the resolution of fibrosis with associated loss of the cellular elements such as HSC during experimental injury. Together with our data, this suggests that collagen-I degradation may be sufficient to reduce fibrotic injury of the liver by facilitating removal of the activated HSC (Fig. 3 ). This in turn results in the removal of the major collagen-producing cell type while simultaneously reducing the liver levels of MMP inhibitors (TIMP-1 and 2), thereby facilitating matrix degradation. HSC persistence in the Col-1a1^r/r livers may relate to the failure of effective hepatocyte regeneration. Damaged hepatocytes express survival factors such as IGF-1, which promote the survival of activated HSC. Hepatocyte regeneration is a key event in the resolution of fibrotic injury. Our observations imply that failure to degrade type I collagen inhibits the hepatocyte proliferative response and provide an in vivo model for previous studies, suggesting that matrix degradation promotes hepatocyte proliferation.

View larger version (37K): [in this window] [in a new window]   Figure 3. Model for the putative role of collagen-I degradation in regulating recovery from hepatic fibrosis. During progressive fibrosis, intact collagen-I fails to provide damaged hepatocytes with appropriate proproliferative signals. Either directly or via paracrine secretion from hepatocytes or other nonparenchymal cells, persistent collagen-I promotes activated HSC survival and HSC-derived profibrotic factors (e.g., collagen-I, TIMP-1) continue to be expressed. During resolution, degradation of collagen-I facilitates hepatocyte proliferation and restores effective hepatocellular function, probably via vß3 stimulation. The loss of survival signals for activated HSC (potentially either or both directly via cell-matrix interaction or indirectly via paracrine mechanism) results in apoptosis with consequent reduction in the expression of profibrotic genes.

Our cell culture studies suggest that the proliferative response of an immortalized hepatocyte cell line to WT collagen can be attenuated by blocking vß₃ an effect not observed in hepatocytes cultured on r/r collagen. These data suggest a role for this integrin in the hepatocyte response to collagen-I degradation. Indeed, this observation parallels other studies that have shown that cells bind to type I collagen through ₂ß₁ integrin, after ligation of which procollagenase gene transcription is induced. Collagenase-mediated cleavage of type I collagen and further unwinding of the chain ends reveals a cryptic binding site for the vß₃ integrin. Binding of the vß₃ integrin to this cryptic site promotes an adhesion-dependent survival signal necessary for cells to progress normally through the cell cycle, an observation that is clearly germane to hepatocyte regeneration after injury (Fig. 3) .

These data suggest that degradation of collagen-I may not simply promote or facilitate the hepatocyte regenerative response after injury but may be a prerequisite for effective hepatocyte regeneration. In the context of progressive fibrosis, this inhibition of hepatocyte proliferation may represent a significant mechanism preventing the restoration of effective hepatocellular function. Overall, our data suggest that degradation of collagen-I is critical to hepatocyte regeneration and loss of activated HSC during spontaneous recovery from liver fibrosis, two processes that may be intimately interlinked (Fig. 3) . These data suggest that degradation of collagen-I is an essential aspect of a strategic approach to the treatment of hepatic fibrosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0494fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0494fje

² Contributed equally to this study.

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