Objectives: To measure the effects of intact and MMP-degraded bovine fetuin on mineralization in a cell-free assay.
Methods: HA crystals were grown in vitro in a calcium/phosphate medium (pH=7.4). HA content was quantified by spectrophotometry at 540 nm using Alizarin Red S stain and measured in the presence of intact bovine fetuin or fetuin digested with MMP-7 or MMP-3 (MMP:fetuin=1:60). Degradation of fetuin was confirmed by SDS-PAGE. Electron microscopy was used to characterize HA crystals. Controls included assays without essential ions and replacement of fetuin with bovine serum albumin. Experiments were repeated a minimum of 3 times with at least 8 values each. Means, standard errors of means and p values were computed.
Results: HA mineralization was slightly increased at low fetuin concentrations (0-1 µM) but was strongly inhibited by concentrations above 1 µM. There was a 2-fold reduction of mineralization (p<0.0001) with 1.4 µM fetuin and virtually no detectable mineralization at ≥2 µM fetuin. In time-courses under constant conditions, mineralization peaked at 3.5 hours incubation and remained constant thereafter. Both MMP-7 and MMP-3 degraded fetuin after 24 hours of incubation. MMP-7-cleaved fetuin did not block mineralization while MMP-3-cleaved fetuin inhibited mineralization.
Conclusions: The regulation of mineralization by fetuin is disrupted by cleavage of fetuin by MMP-7 but not by MMP-3. The increased expression of MMP-7 in periodontitis could increase the risk of calcified atheroma formation, in part rationalizing the epidemiological links between these diseases.
Keywords: Cardiovascular disease, Epidemiology, Fetuin, Inflammatory mediators and Periodontal disease
See more of: Periodontal Research - Pathogenesis