ALT and AST levels in WTs but not in MMP19KOs (Figure 1A). Histology revealed typical centrolobular hepatocellular necrosis in both strains (Figure 1B, S2A); however, the necrotic areas were more often bridging in WTs and, after 6 week CCl4 treatment, also significantly larger in WTs than in MMP19KOs (Figure 1C). Image analysis of Sirius red staining for fibrillar collagen showed slightly larger fibrotic areas in WTs than in MMP19KOs at both 4 and 6 week time points (Figure S2B). Liver hydroxyproline content Figure 1. 6 week CCl4 treatment results in lower liver damage in MMP19KOs compare to WTs. (A) After 4 and 6 week treatment with CCl4, serum ALT and AST levels were significantly lower in MMP19KO than in WT mice. WT mice treated with olive oil are shown as control mice. (B) H&E staining showed larger bridging necrosis areas in WTs than in MMP19KOs. Sirius red staining showed more fibrillar collagen deposition in WTs than in MMP19KOs. (C) Liver hydroxyproline (OH-proline) content and image analysis of necrosis areas in H&E stained liver sections showed significantly more extensive damage in WTs than in MMP19KOs. Images were originally taken at 1006 (H&E) and 506 (Sirius Red) magnification, scale bars = 200 mm. *p,0.05; **p,0.01; ***p,0.001 MMP19 vs. WT mice; n = 6 olive oil controls, 7 WT and 9 MMP19KO mice. MMP-19 Deficiency Is Hepatoprotective In Vivo correlated with histology, being higher in WTs than in MMP19KOs after 6 weeks of intoxication (Figure 1C). Serum levels of MCP-1 and KC cytokines were only slightly increased in WTs than in MMP19KOs at 4 or 6 weeks of treatment (Table S1), indicating a trend toward decreased inflammatory reaction in MMP19KOs. Altogether, the observed parameters are consistent with a trend toward decreased hepatocellular injury, fibrosis development, and lower inflammatory reaction in MMP19KOs in response to chronic liver damage. MMP19KOs recover faster from fibrotic changes and show higher hepatocyte proliferation during the recovery period Mice treated for 6 weeks with CCl4 were further allowed to recover for 10 or 15 days. Liver function significantly improved after 10 days of recovery, evidenced by ALT and AST levels returning to normal (Figure 1A) in all animals. Even though hepatocytes regenerated in both WTs and MMP19KOs (Figure 2A), histology showed necrotic septa still visible, predom- inantly in WT livers. Sirius red staining revealed a decrease in fibrillar collagen deposition in both strains with the MMP19KOs showing less extensive fibrotic areas during recovery (Figure 2A, B). Interestingly, there was a further decrease in the extent of fibrotic areas from 10 to 15 days of recovery in MMP19KOs but not in WTs as judged from Sirius red staining (Figure S2B). aSMA mRNA levels, which were slightly lower in MMP19KOs than in WTs during fibrosis development, did not change with fibrosis resolution in MMP19KOs but were significantly decreased in WT animals (Figure 2B). Interestingly, after 10 days of recovery, MMP19 mRNA in WTs significantly increased compared to the period of fibrosis development and then returned to normal levels after 15 days of recovery (Figure S3), thus pointing to a time window during the healing process, in which MMP-19 is engaged in matrix remodeling or removal of fibrotic matrix. As evidenced by the lower ALT and AST levels and histology, hepatocellular damage was substantially lower in MMP19KOs. This led us to investigate the difference in regenerative potency between WT and MMP19KO hepatocytes in vivo. Hepatocyte proliferation was studied using Ki67 staining after 4 weeks of CCl4 (Figure 3A, B) and by counting binucleated cells (Figure 3C, D). After 4 weeks, both Ki67-staining and binucleated cell counts indicated higher proliferation activity in WT than in MMP19KO hepatocytes, likely reflecting the lower damage in MMP19KO hepatocytes and thus lower demand for reconstitution. However, although we did not observe any Ki67-positive nuclei in either WT or MMP19KO hepatocytes in the recovery period (not shown), binucleated cell counts were significantly higher in MMP19KOs (Figure 3C, D). Activity and expression of MMPs during liver damage progression As the initial destruction of normal ECM during liver injury is controlled by proteases, especially MMPs, we studied the expression and processing of MMP-2 and MMP-9. After 4 weeks of intoxication, both pro-MMP-9 and -MMP-2 were upregulated in both WT and MMPKO mice in comparison to control, olive oil-treated mice (Figure S4). However, MMP19KOs showed significantly lower levels of both pro-MMP-2 and -MMP-9 compared to WTs (Figure 4A). MMP19KOs also exhibited slightly decreased levels of the activated form of MMP-2. In contrast, after 6 weeks of CCl4-treatment, the expression of pro- MMP-9 was slightly higher and activated MMP-2 was significantly PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46271