In all animals, ~90% of all TUNEL+ cells colocalized with macrophages despite different apoptosis rates between sellectchem the groups (Fig. 4, A, B, and C). Accordingly, the severalfold elevated apoptosis at 4 wk of reversal resulted in a corresponding increase in infiltrating macrophage numbers within the portal tracts (Supplemental Fig. S4). During this active macrophage recruitment into the resolving scar tissue, no change in the numbers and distribution of lobular (resident) macrophages/Kupffer cells was observed (Supplemental Fig. S4). Fig. 4. Scar-associated macrophages increase at maximal fibrolytic remodeling and colocalize with apoptotic cholangiocytes. A: double immunohistochemistry staining for the macrophage marker CD68 (red, cytoplasmic) and apoptosis (TUNEL: brown, nuclear) on liver …

Mapping of MMP Expression and Activities Reveals a Distinct Subset of MMPs Associated With Fibrosis Reversal Both net interstitial collagenase and gelatinase activities were elevated at maximal scar remodeling (Fig. 1, D and E), indicating that induction and/or activation of the respective MMPs occurred along with diminished expression of their inhibitors, especially TIMP-1 (Fig. 2B). In situ zymography revealed that gelatinase activity was spatially associated with the connective tissue in portal tracts and septa at any time point (Supplemental Fig. S5). To assess the contribution of particular MMPs, we performed gelatin zymography, which showed that, although both gelatinases (MMP-2 and -9) were upregulated at the peak of fibrosis, they demonstrated divergent patterns of regulation during reversal.

MMP-9 was highly upregulated starting at 14 days and peaking at 4 wk of reversal, representing most of gelatinase activity, whereas MMP-2 declined during resolution and was barely detectable at day 7 of reversal (Fig. 5A). Similar results were obtained with immunodetection of MMP-9 protein in liver homogenates by Western blotting (Fig. 5B). MMP activity requires proteolytic activation, which can be achieved by several proteases including plasmin and MMPs themselves (38). We therefore determined transcript levels of MMP-2, -3, -8, -9, -12, -13, -14, and of molecules involved in the plasmin activation cascade, i.e., urokinase plasminogen activator (uPA) and uPA receptor (UPAR)-1, to test whether their expression pattern was consistent with the observed peak of MMP activity and fibrolytic remodeling.

MMP-3, -8, -9, -12, and -14 were most prominently Cilengitide upregulated at those time points that preceded or coincided with peak fibrolytic activity (Fig. 5, A�CC; Fig. 1, D and E). Whereas MMP-12 and -14 transcripts were elevated throughout reversal, MMP-13 transcripts decreased at day 7 and remained slightly elevated above sham-operated controls (Fig. 5C). MMP-8 and -9 mRNA peaked at day 14 (Fig.