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MicroRNA-181b inhibition reduces atherosclerosis by increasing macrophage TIMP-3 expression

      Tissue inhibitor of metalloproteinases-3 (TIMP-3) characterises anti-inflammatory macrophages with potential atherosclerotic plaque stabilising properties. Human stable (n=20) and rupture-prone (n=20) atherosclerotic plaques, together with in vitro macrophage (M)-CSF or granulocyte-macrophage (GM)-CSF differentiated foam-cell macrophages (n=4), were examined for expression of TIMP-3. Fat-fed Apoe knockout and Timp-3/Apoe double knockout mice were used for in vivo analysis. Compared to M-CSF macrophages, oxLDL-induced foam-cell formation was accelerated in GM-CSF macrophages (9.6±2.8-fold increase, p<0.01) and was associated with a significant reduction in TIMP-3 protein expression (80±7 %, p<0.005), whilst mRNA expression was unaffected. Interestingly microRNA-181b can target TIMP-3 for post-transcriptional down-regulation and may underlie our observed differences in TIMP-3 protein expression. Indeed, miR-181b expression was significantly increased by 2.4-fold (p<0.005) in GM-CSF foam-cell macrophages compared to M-CSF foam-cell macrophages suggesting a regulatory role. Accordingly we observed a significant increased number of TIMP-3 positive foam-cell macrophages (62%; p<0.01) in stable plaques compared to rupture-prone lesions, and a concomitant decrease in miR-181b expression (71%; p<0.01), as assessed by in situ hybridisation. Supportingly, we observed a significant 3-fold (p<0.01) increase in brachiocephalic lesion size of Timp-3/Apoe double knockout mice compared to controls. Consequently, administration of a miR-181b inhibitor to Apoe-knockout mice with established brachiocephalic plaques significantly reduced lesion area by 37% (n=6; p<0.05) and was associated with a concurrent 42% (n=6; p<0.05) increase in intra-plaque macrophage TIMP-3 protein expression, compared to scrambled miR controls. Our results demonstrate that GM-CSF regulates foam-cell macrophage TIMP-3 expression through a miR 181b-dependent pathway, and highlight miR-181b as a therapeutic target in atherosclerosis.
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