Marfan syndrome aortic smooth muscle cells express high levels of microRNA miR-29b

Objective: Marfan syndrome (MFS) is an inherited connective tissue disorder leading to excessive TGF-b signaling, vascular remodeling and aortic root aneurysm development, although the mechanism(s) remains unknown. MicroRNAs are known to regulate post-transcriptional gene expression and play key roles in cardiovascular disease. Smooth muscle cells (SMC) part of the aortic wall is discussed to be involved in the pathogenesis of aneurysm development in MFS. This study examines the in vitro effects of TGF-b1 on miR-29b expression and matrix metalloproteinases (MMP) activity in aortic smooth muscles cells derived from aortas of Fbn1 ^C1039G/+ mice or littermate controls (WT).

Methods: Aortic smooth muscle cells were derived from either 4week old Fbn1 ^C1039G/+ mice or WT, isolated from five pooled murine aortas and cultured using an established protocol. Before actual treatment cells were starved for 24h before treated with 10ng/ml TGF-b1 in the media for 24h and 48h. miR-29b expression was measured by Taqman PCR and MMPs activity was quantified by zymography.

Results: NfKb (p65) a known repressor of miR-29b, showed in MFS SMCs treated with TGF-β1 reduced activation by immunofluorescence immunohistochemistry as well as a significant 2.1-fold±027 increase in miR-29b expression over WT SMC. 48h TGF-b1 treatment showed a further significant increase of miR-29b expression of 4.5-fold±0.29 over WT. In addition showed treated MFS SMCs vs. untreated MFS SMCs at 48h a significant overexpression of 2.7-fold±0.67 in miR-29b. MMPs activity of MMP-2 and -9 direct targets of miR-29b showed at baseline in MFS SMCs a significant higher activity over WT for both MMPs. MMP-2 activity significantly further increased over 24h to reach a significant 7.09-fold±0.045 higher activity at 48h over WT. The same significant effect is seen in MMP-9 activity reaching at 48h a significant 6.27-fold±0.045 increase.

Conclusion: Based on our previous results, showing a significant impact of miR-29b on aortic root aneurysm development in MFS. We here present for the first time in vitro effects of TGF-β1 on murine derived MFS SMCs on miR-29b expression and MMPs activity. Our results strongly suggest the involvement of SMCs expressing miR-29b and high MMP-2 and -9 activity and therefore playing an important role in early aneurysmal development in MFS by influencing elastin fragmentation.