MMP-19 Deficiency Is Hepatoprotective In Vivo Figure 2. MMP19KOs show faster recovery from fibrosis than WTs. Mice treated with CCl4 for 6 weeks were allowed 10 or 15 days for recovery. (A) After 15 days of recovery, significant parenchymal regeneration with less visible necrosis and fibrosis was observed in both WT and MMP19KO livers. (B) Fibrotic area (based on image analysis of Sirius red staining) was significantly lower in MMP19KOs after 15 days of recovery. (D) aSMA mRNA expression was slightly higher in WTs than in MMP19KOs throughout the course of liver damage progression but failed to drop during the recovery. Images were originally taken at 1006 (H&E) and 506 (Sirius Red) magnification, scale bars = 200 mm. *p,0.05, MMP19s vs. WTs; 5–9 animals of each strain were analyzed at each time point. doi:10.1371/journal.pone.0046271.g002 increased in MMP19KO livers (Figure 4B). When mice recovered from liver injury, pro-MMP-2 and -MMP-9 levels returned to the level of control animals and the activated form of MMP-2 was no longer observed (not shown). Expression of collagen IV, one of the proteins of normal ECM degraded also by MMP-19, correlated well with observed MMP-2 and MMP-9 turnover. We observed significantly more collagen IV in MMP19KO than in WT livers at 4 weeks of CCl4-treatment (Figure 4C) and no difference in collagen IV expression at 6 weeks (not shown). Analysis of collagen IV mRNA showed a trend towards slightly higher expression in MMP19KO than in WT livers (Figure 4D). Analysis of MMP-2 mRNA expression showed an increase with CCl4-treatment and no differences between the WTs and MMP19KOs after 4 weeks of intoxication (Figure S4). This corresponds well with the zymo- graphy data, which showed mainly differences in the regulation of MMP-2 processing. MMP-13 mRNA expression was highly elevated in CCl4-treated animals with a trend toward higher levels in WTs than in MMP19KOs (Figure 4E). During the Figure 3. Hepatocyte proliferation in liver during fibrosis resolution is higher in MMP19KOs. (A, B) Ki67 nuclear staining was higher in WTs than in MMP19 KOs after 4 weeks of CCl4 treatment. Ki67 positive cells were counted in 206 fields; n = 6 for both WT and MMP19KO. (C, D) Binucleated cells were counted in H&E stained liver sections after 4 weeks of CCl4 and in the recovery. (C) Images show representative of counted cells in livers after 10 days of recovery.. (D) During the recovery period the number of double nuclei per field of view was significantly higher in MMP19KO than in WT livers; 4–7 animals of each strain were analyzed at each time point. **p,0.01; ***p,0.001 MMP19s vs. WTs. Scale bars = 1 mm. doi:10.1371/journal.pone.0046271.g003 recovery phase, MMP13 expression decreased in both WTs and MMP19KOs. Altogether, these data show that MMP-19 deficiency appears to impact the proteolytic network of MMPs during the development and recovery of liver fibrosis, reducing their potential to proteolyze ECM, especially in the initial phase of injury when components of the basement membrane are degraded. Decreased TGF-ß and increased IGF-1-mediated signaling during fibrogenesis and recovery in MMP19KOs To reveal signaling pathways which are affected by MMP-19 deficiency, we studied the activation of TGF-ß and IGF-1 pathway components. The phosphorylation of SMAD3, a TGF-ß-signaling mediator, was significantly decreased after 6 weeks of CCl4- treatment and in the recovery period in MMP19KO livers (Figure 5A), suggesting that the TGF-ß-mediated pro-fibrotic response was lower in MMP19KO livers. Further analysis of TGF- ß1 mRNA showed a trend toward slightly higher levels of this profibrotic factor in WT livers during the recovery period, further supporting our findings (Figure 5E). Phosphorylation of IRS1 and Akt kinase was analyzed to obtain insight into IGF-mediated, anti-apoptotic signaling. Immunoblot- PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46271