In atherosclerosis, macrophages lodge in the intima and subintima of arteries, leading to the formation of obstructive atherosclerotic plaques that are prone to rupture, which provokes thrombosis, myocardial infarction or stroke. Recently, we identified eight differentially expressed miRs, either in monocytes from myocardial infarction patients or in atherosclerotic plaques. Since differentiation of monocytes to M0 macrophages or pro-inflammatory M1 macrophages is a prerequisite for their invasion in the atherosclerotic plaque, we now analyzed expression of those eight miRs during the process of monocyte/macrophage differentiation.

As human monocytic cell line THP-1 cells were used. For induction of macrophage differentiation to M0, cells were stimulated with PMA (phorbol-12-myristate-13-acetate, 5 ng/ml). mRNA expression of CD14, a marker of macrophage differentiation, started to rise within one day and was upregulated 31-times compared to non-stimulated cells after three days (n=4, p<0.05). At the same time, morphology of round, non-adherent THP-1 monocytes changed to a macrophage-like phenotype with enhanced granularity, flattening, formation of pseudopodia, and adherence to the culture dish. let-7f and miR92a expression decreased under PMA stimulation (0.4 and 0.5-times vs. unstimulated control, n=10, p<0.05) and the expression of miR1 and miR143 increased 350-times and 4-times, respectively (n=5, p<0.05 vs. unstimulated control) within 3 days, while four of the eight miRs under study, namely miR21, -22, -99a, and -223, showed no change in expression. Treatment of THP-1 cells with agomiR let-7f or agomiR92a prevented the decrease of let-7f or miR-92a under PMA stimulation. Even a significant upregulation of these miRs was provoked by the agomiRs. AgomiR92a treatment attenuated enhancement of CD14-mRNA expression and thus M0-differentiation under PMA stimulation. In contrast, treatment of THP-1 cells with agomiR-let-7f amplified CD14-mRNA expression under PMA. For M1 differentiation, M0 macrophages were treated with IFN-γ (20 ng/ml) and LPS (10 pg/ml). Marker genes of M1 polarization, IL-1β and TNFα, increased (36-times within 3 days, and 27-times after 1 day, respectively, n=9, p<0.05 vs. unstimulated control). Under those conditions, four miRs under study, namely let-7f, miR1, -92a, and -99a, showed no change in expression. However, miR21 increased, and miR143 and -223 decreased under INFγ/LPS stimulation (1.72-, 0.73-, 0.78-times, respectively, n=13, p<0.05 vs. unstimulated control).

In conclusion, different subsets of miRs are expressed during M0- and M1 differentiation. While miR1 and miR143 increase, and let-7f and miR92a decrease during M0 differentiation, miR21 increases, and miR143 and miR223 decrease during M1 differentiation. Preventing downregulation of miR92a by agomiR treatment reduces M0 differentiation and thus could reduce the development of monocytes into proatherogenic macrophages.