After densitometric analysis corrected for GAPDH expression, the expression of MMP-12 showed no significant changes across all groups (data not shown). Given the tightly regulated activity of MMPs,9, 10 it was important to detect whether active MMP-12 was present. One of the main factors in determining MMP activity is the ratio with their tissue inhibitors, especially TIMP-1. TIMP-1 mRNA and protein (Fig. 3B) were increased after 8 and 12 weeks injury. In order to establish the degree of inhibition of MMP-12
by TIMP-1 in our model system, we coimmunoprecipitated the two proteins and analyzed the samples by zymography. After immunoprecipitation of MMP-12, casein zymography (Fig. 3C) showed a similar pattern to the samples used for western blot, indicating even efficiency of precipitation. Additionally, when we immunoprecipitated TIMP-1 and performed casein zymography (Fig. 3C) the signal increased through 4 to 8 and 12 weeks (Fig. 3C), indicating that there is increasing amounts of TIMP-1 bound to MMP-12 in increasingly fibrotic liver. Thus, MMP-12
is present in the liver but held in check by noncovalent binding to TIMP-1 with increasing duration of liver injury. Taken together, these data strongly suggest that the elastin content in scars is regulated by MMP-12-mediated degradation, with active MMP-12 being inhibited by increased interaction with TIMP-1 with worsening fibrosis in vivo. Previous work by Yoshiji et al.10, 28 using a TIMP-1 overexpression, however, suggests that the Timp-1 inhibition may not be maximal and MMP-mediated enough degradation still occurs
in remodeling during progressive fibrosis. MMP-12 has been reported to be expressed by macrophages.29 We confirmed this by immunocytochemistry on human Quizartinib order monocyte-derived macrophages stained for both MMP-12 and the macrophage marker CD-68 (Fig. 4A1-2); 100% of the cells were positive for both proteins. To define which cells express MMP-12 in vivo, we stained serial sections of rat tissue for MMP-12 and CD-68 (Fig. 4A3-4). We found that the cells positive for MMP-12 were macrophages but that only a proportion of the CD-68-positive macrophages were also positive for MMP-12. To confirm the macrophage origin of MMP-12, we used the transgenic mouse CD11b-DTR in which macrophages can be selectively depleted as described.22 These mice show a 50% decrease in macrophage populations and increased accumulation of elastin compared with WT mice after CCl4 administration. Staining of liver following macrophage depletion showed a significant decrease in MMP-12-positive cells (Fig. 4B1-3). qPCR analysis of these tissues (Fig. 4B4) showed no significant changes in the expression of either tropoelastin or neutrophil elastase, whereas MMP-12 expression was significantly decreased. To further confirm that macrophages are the major hepatic source of MMP-12, we costained mouse liver after CCl4 injury for MMP-12 and key liver cell markers.