Background/Purpose

In vitro, miR-145 exerts anti-fibrotic effects in systemic sclerosis (SSc) by downregulating TGF-β signaling. In turn, ectopic TGF-β downregulates miR-145 thereby optimizing TGF-β signaling pathways. In this study, we aimed to investigate whether therapeutic application of miR-145 could prevent fibrosis via regulation of TGF-β in vivo.

Methods

We used miR-145^-/- (n=7-8), and mouse models of dermal fibrosis induced by either bleomycin (n = 6) or by adenoviral overexpression of constitutively active TGF-β receptor type I (n = 3). MiR-145^-/- mice and wild type controls were also treated with intradermal bleomycin, while a subset of bleomycin-induced dermal fibrosis mice were simultaneously treated with intradermal injections of a synthetic miR-145 designed for in vivo transfection. Dermal thickness, myofibroblast count (α-SMA staining on paraffin-embedded skin sections), and collagen content (hydroxyproline assay), were analyzed as outcomes of skin fibrosis. The expression of miR-145 and of miR-145 targets, TGFBR2 and SMAD3, was analyzed in mice treated with the synthetic miR-145 and in the mouse model of skin fibrosis induced by the constitutive activation of TGF-β receptor type I by real-time PCR. 

Results

We found that miR-145^-/- mice were more sensitive to the effects of bleomycin than wild type controls, as shown by a stronger increase of dermal thickness (2.04 versus 1.7 fold), α-SMA count (4.4 versus 3), and collagen content (1.82 versus 1.34; all p < 0.01). According to the anti-fibrotic effects shown by miR-145 in vitro, we expected to counteract bleomycin effects in C57BL/6 mice by the simultaneous administration of synthetic miR-145. Indeed, all explored outcomes improved in these mice, as compared to controls injected with a synthetic miR-scrambled: dermal thickness reduced from 1.7 to 1.27 fold, α-SMA count from 3.7 to 1.9, collagen content from 3.6 to 2.9; all p < 0.01. MiR-145 expression in mice injected with the synthetic miR-145 increased by 37 fold (p < 0.01) confirming the efficiency of the in vivo transfection. Accordingly, the downregulation of the previously identified direct miR-145 targets, TGFBR2 and SMAD3, by 0.21 and 0.10 fold (p < 0.05) supports the hypothesis that the anti-fibrotic effects of miR-145 are mediated by the downregulation of these TGF-β signaling components. Finally, in mice overexpressing constitutively active TGF-β receptor type I, miR-145 was strongly down-regulated by 0.55 fold in fibrotic skin as compared to mock-transfected controls, confirming the existence of a regulatory feedback loop between miR-145 and TGF-β in vivo and further proofing the relevance of the miR-145/TGF-β interaction in vivo.

Conclusion

Here we show for the first time that the therapeutic application of miR-145 protects against skin fibrosis in vivo, thus opening the road to new therapeutic targeted approaches to SSc and other fibrotic disorders. We also confirm on the in vivo level, that the anti-fibrotic effects of miR-145 are mediated by the downregulation of the TGF-β signaling components TGFBR2 and SMAD3, and that the abnormal miR-145 expression that is observed in SSc is dependent, at least in part, on activation of TGF-β.

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« Back to 2014 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/mir-145-protects-against-skin-fibrosis-in-vivo-by-targeting-tgf-%ce%b2-signaling/