Raw data was downloaded from the Gene Expression Omnibus

y and AFSC rescue is further supported by the recruitment of CCL2 responsive immune cell populations into the BAL with and without AFSC treatment. Furthermore, these data show the direct impact of recombinant CCL2 alone or BAL containing varying levels of CCL2, on collagen synthesis by fibroblasts in vitro. 8 AFSC Treatment Inhibits Pulmonary Fibrosis AFSC Modulate CCL2 through MMP-2 Mediated Proteolytic 21560248 Cleavage To investigate a potential mechanism of CCL2 regulation in BAL following AFSC treatment, we performed Western blot analysis of CCL2 in AECII cellular fractions 3 days postbleomycin injury, using a gradient gel with high resolving capacity. Since we previously demonstrated that AFSC treatment most significantly attenuates CCL2 secreted into the BAL following bleomycin injury, Western blots of cell fractions were not used to measure changes in levels of CCL2 secretion, but instead as an indicator of the presence and type of CCL2. In controls, as well as following bleomycin injury, mouse-specific CCL2 was present as a band of the expected molecular weight, 25 kDa. However, following murine AFSC treatment, a subtle shift downward in the CCL2 band was visualized, indicating the cleavage of a 0.4 kDa peptide. This shortened, cleaved form of CCL2 has been previously reported to function as a CCR2 receptor antagonist, thereby rendering the cleaved form of CCL2 found here a putative CCR2 receptor antagonist in BAL. CCL2 cleavage has previously been shown to occur in the presence of MMP-2. To confirm the hypothesis that the truncated form of CCL2 could be due to proteolytic cleavage, Western blots of BAL fluid, with the cellular fraction removed, demonstrated increased MMP-2 levels in bleomycin injured+AFSC treated cohorts. To determine the levels of endogenous active MMP-2 in BAL samples, we performed an MMP-2 activity assay and measured at 6 and 22 hours of incubation. Three days post bleomycin injury; MMP-2 was significantly increased when compared to controls. Also at the 3-day time point, AFSC treated cohorts exhibited marked increases in MMP-2 activity when compared to bleomycin-injured cohorts. MMP-2 activity significantly decreased at 28 days post bleomycin injury and in day 0 and day 14 AFSC treated cohorts measured 28 days post bleomycin injury. Finally, gelatin zymography of BAL harvested 3 days post-bleomycin injury plus murine AFSC day 0 treatment showed a significant increase in MMP-2 activity. This effect was transient, as seen in the MMP-2 activity assay, as elevated levels of MMP-2 did not persist to 28 days and this was true whether AFSC were given at 0 or 14 days post-bleomycin injury.. These data are significant, as CCL2 is a known target of MMP-2 proteolytic cleavage, with the CCL2 cleavage product forming the aforementioned putative receptor antagonist for CCR2. These data suggest a potential mechanism for CCL2 modulation: the proteolytic cleavage of CCL2, forming a receptor antagonist, which maintains its binding affinity for CCR2, yet does not induce a response upon binding. We hypothesize that the transient increased MMP-2 secretion into BAL, 7906496 detected after AFSC treatment, contributes to proteolytic cleavage of CCL2, thereby regulating its activity. and did not observe co-localization of a-SMA expression with AFSC. It has been previously characterized that in both human IPF and murine bleomycin-induced lung injury increased CCL2 expression is noted within activated Neuromedin N chemical information epithelium in fibrotic areas. To determine if th