Abstract
Purpose :
MiR-21 is a STAT3-dependent miR which has been involved in pathological angiogenesis. We have previously shown that miR-21 is up-regulated during the neovascular stages in a mouse model of oxygen-induced retinopathy (OIR). Here we wanted to determine the effects of in vivo delivery of a miR-21 inhibitor [antagomiR21 (a.miR-21)] in affecting retinal neovascularization (RNV) and the expression of its gene target TIMP3 in a mouse model of OIR.
Methods :
OIR was induced in mouse pups by subjecting them to different oxygen tensions, following the method of Smith LE et al. A nuclease-resistant locked nucleic acid (LNA)-a.miR-21 was injected intra-orbitally (2.5 – 5.0 mg/kg) at postnatal day 12 (P12). The contralateral eye was used as not-injected control. Another set of animals was injected with a non-targeted LNA-a.miR to assess potential off-side effects. After the injection, the mice were sacrificed at P14 or P17. Retinas were excised and processed to perform morphological and molecular analyses to assess retinal vessels distribution and expression pattern (qPCR and Western analysis) of the miR-21 target gene TIMP3. Zymography was performed to assess the activity of matrix-metalloprotease-2 and -9 (MMP-2 and MMP-9, respectively) as a measure of TIMP3 function.
Results :
In vivo inhibition of miR-21 expression was confirmed by qPCR in eyes treated with LNA-a.miR-21 and not with the non-specific LNA-a.miR. RNV, visualized in flat mounted retinas stained with Isolectin B4, was significantly down-regulated in P17 mice treated with LNA-a.miR-21 and subjected to OIR and not in mice treated with the non-specific LNA-a.miR. The observed effects were associated with the rescue of TIMP3 expression, measured by qPCR and immunoblotting at both P17 and P14, in LNA-a.miR-21 injected eyes only. Finally, zymography of retinal extracts from mice subjected to the different treatments revealed that the rescue of TIMP3 in LNA-a.miR-21 injected eyes (at both P14 and P17) resulted in down-regulation of MMP-2 and MMP-9.
Conclusions :
The obtained results suggest a potential role for miR-21 in the regulation of pathological neovascularization in ischemic retinopathies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.