Matrix metalloproteases (MMPs) regulate Opticin expression in the Microglia Under Hypoxic Stress

Abstract

Purpose : Retinopathy of prematurity (ROP) is a vasoproliferative eye disease, characterized by incomplete retinal vascularization in premature infants. Recently we have shown that activated microglia in the vitreous of ROP patients caused an increased expression of C3 and MMPs. Parallelly, vitreous proteomics revealed a lower expression of Opticin, an antiangiogenic protein in ROP patients. The, present study hypothesized that activated MMPs under hypoxia degrades Opticin in microglia thereby contributing to ROP pathogenesis

Methods : The levels of MMP2, MMP9 and Opticin in the vitreous samples of ROP patients (n=10) and controls (n=10) were assessed by zymography and western blotting. Human microglia cells treated with MMP inhibitors (10µg of EDTA and/or doxycycline) for 24 hours were exposed to hypoxia for studying the effect of activation of MMPs on the expression of opticin. The levels of expressions of MMP9, Opticin, VEGF, and TIMP2 in microglia cells were assessed by quantitative real time PCR and immunofluorescence. Immunohistochemistry (IHC) of retinal membranes was done to further validate the interactions of opticin and MMP in ROP eyes. The differential expression of genes was analyzed using the unpaired student’s t-test and p-value <0.05 was considered to be statistically significant.

Results : An elevated level of the MMP’s and down regulation of opticin was observed in ROP vitreous compared to the controls. In vitro analysis confirmed a significantly higher level of MMPs (p= 0.0001) in microglia cells and reduced Opticin expression (p=0.0179) under hypoxia. Further, the inhibition of MMP9 activation rescued the opticin degradation by MMP9. Further, co-localization of MMP9 and opticin in retinal microglia of ROP on IHC supported the findings of the in vitro experiments.

Conclusions : Our study confirms the regulation of opticin expression by activated MMP9 in microglial cells under hypoxia. Thus targeting MMPs could potentially check the underlying inflammation and prevent abnormal proliferation of blood vessels in the ROP eyes.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.