45miR-24 modulates MMP-14 protein expression in macrophages suggesting a novel regulatory mechanism associated with atherosclerotic plaque progression

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Abstract

Purpose and Methods

Our recent studies have highlighted membrane type-1 MMP (MMP-14) as a selective marker for an invasive subset of macrophages related to atherosclerotic plaque progression. Moreover, colony stimulating factors (CSF) may exert divergent effects on macrophage MMP expression. We therefore investigated the mRNA and protein expression pattern of MMP-14 in macrophage (M)-CSF and granulocyte-macrophage (GM)-CSF differentiated macrophages and their subsequent invasion in vitro.

Results

Mouse macrophages differentiated with M-CSF for 7 days showed a significant up-regulation (5-fold) of MMP-14 mRNA (p≤0.005) and a significant down regulation of MMP-14 protein (80% by western blotting p≤0.005, 90% by ICC p≤0.001) compared to adherent monocytes. Contrastingly, GM-CSF differentiation resulted in a significant down-regulation of MMP-14 mRNA level (2-fold p≤0.05), whereas protein was increased (2.1-fold, p≤0.05) compared to M-CSF differentiated macrophages. Accordingly, compared to M-CSF macrophages, GM-CSF macrophages exhibited increased in vitro invasion (2.9-fold p≤0.05) which was significantly reduced by a MMP-14 neutralising antibody (2.2 fold p=0.05). Expression of the micro-RNA (miR)-24, known to target MMP-14, was significantly increased during M-CSF macrophage maturation (85%, p < 0.05). Silencing miR-24 in M-CSF macrophages significantly increased MMP-14 expression (1.4-fold, p≤0.05) and enhanced their invasive capacity (5.1-fold, p≤0.01) compared to a scrambled control. Concomitantly miR-24 expression was significantly lower in GM-CSF matured macrophages compared to M-CSF macrophages (24%, p≤0.01) suggesting that GM-CSF can modulate MMP-14 protein expression and invasion of mouse macrophages in a miR-24 dependent manner. Increased MMP-14 protein expression in foam-cell macrophages is associated with human carotid and coronary atherosclerotic plaque progression and predicts future clinical outcomes. Furthermore, miR-24 expression in atherosclerotic plaques is inversely related with MMP-14 protein expression. Moreover, stable plaques contained higher miR-24 levels than unstable plaques (p < 0.05), and co-localised with foam-cell macrophages which exhibited low MMP-14 protein expression.

Conclusions

Taken together our data suggest an essential role for MMP-14 in promoting macrophage proteolysis in a subset of macrophages and subsequent plaque progression and that the microRNA miR-24 provides a novel regulatory mechanism for MMP-14 protein levels and therefore plaque stability, highlighting its therapeutic potential.

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