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Terete Borras, Priyadarsini Asokan, Millena Sivakumar, Michael R. Falvo; Matrix Gla Functions as a Keeper of Physiological Intraocular Pressure (IOP) in the Mouse Eye. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5209.
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© ARVO (1962-2015); The Authors (2016-present)
Matrix Gla (MGP) is an inhibitor of calcification gene highly expressed in arteries’ smooth muscle cells, chondrocytes, trabecular meshwork (TM) & peripapillary sclera. In mice, ablation of Mgp is lethal. Here we seek to determine the role of MGP in maintaining physiological IOP by generating and assessing a TM conditional knock-out mouse (Mgp-TM.cKO).
A Mgp floxed transgenic mouse (Mgp-loxP) was generated by inserting loxP sequences flanking Mgp exons 3 & 4 using CRISPR/CAS9 technology. Primary mouse angle cells were generated from Mgp-loxP mice and age-matched WT. Viral vectors Ad-GFP-2A-iCre (Ad.Cre) and Ad-CMV-GFP (control) were grown to high titers. Mgp floxed cells and Mgp-loxP mice were infected/ injected with Ad.Cre and control viruses. Mice injections were intracameral (IC) with 1-5 X 109 ifu at 2.5 month using a 33G NanoFil needle and a Micro-2T syringe pump. IOPs were measured at pre-injection and weekly post-injection for 75 d on sedated mice using a TonoLab. Mechanical force is being determined on the TM region of 10 µm unfixed cryosections using an MFP-3D Bio AFM (Asylum).
Ad.Cre gene delivery to Mgp-floxed cells and TM of Mgp-LoxP living mice resulted in 80-90% floxed Mgp genomic DNA, ablation of Mgp protein and generation of a Mgp-TM.cKO mice. Mgp ablation did not occur on Mgp-LoxP cells & mice treated with control viruses, on WT cells & mice treated with Ad.Cre, or when left untreated. The eyes of Mgp-TM.cKO mice (n=31) exhibit significant elevated IOP while those of the three control groups (n=29, n=22 & n=10) had baseline pressures at the same time-periods. Pressures in the Mgp-TM.cKO mice began to increase at 2 wks post-injection and continued to day 75, when the experiment was terminated. At 4 wks the Mgp-TM.cKO’s IOP was 13.0±0.6 mmHg vs 8.9±0.1, 8.4±0.2 & 8.0±0.03 mmHg on the three controls (P<0.0001 all comparisons). At 10 wks (last point), the Mgp-TM.cKO’s IOP was 24.1±0.9 mmHg vs 9.3±0.1, 8.0±0.1 & 8.1±0.3 mmHg on controls (P<0.0001 all). A preliminary non-statistical AFM study assay suggests Mgp-TM.cKO TM tissue may be stiffer than that of the control.
Mgp’s ablation in the TM leads to elevated IOP in living mice. This suggests that Mgp’s role in the eye is that of maintaining physiological IOP. Because Mgp prevents arterial calcification, it is possible that Mgp is preventing hardening and stiffness of the TM.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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