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Matilda F Chan, Marie Wolf, Selene M Clay; MMP12 Inhibits Corneal Neovascularization Through Regulation of CCL2. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3511.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal neovascularization promotes the delivery of immune cells into the cornea and is associated with corneal disease, including corneal fibrosis and graft failure. We previously showed that chemically injured corneas of mice deficient in MMP12 (macrophage metalloelastase) have elevated levels of corneal neovascularization and increased expression of the angiogenic chemokine CCL2. MMP12 and CCL2 are both expressed by macrophages and MMP12 is able to process and inactivate CCL2. The objective of this study was to determine if the inhibitory effect of MMP12 on corneal neovascularization is directly mediated through the suppression of CCL2.
A neutralizing antibody to CCL2 or PBS control was injected into the subconjunctival space of littermate FVB wild-type (WT) and Mmp12-/- mice 2 hours prior to corneal chemical injury. Chemical injuries were created by topical application of 0.1N NaOH to corneas. Corneas were collected 7 days after injury and processed for whole mount immunostaining using primary antibodies against F4/80 and CD31 and secondary antibodies conjugated with Alexa Fluor 488 and Dylight 549. A confocal microscope was used to image the location of Alexa Fluor 488 and Dylight 549. For F4/80 stained images, the number of pixels was determined. For CD31 stained images, the lengths of neovascularization were calculated using NIH ImageJ software.
Following chemical injury, control PBS treated Mmp12-/- corneas showed significantly increased lengths of individual vessels compared with WT mice. The number of macrophages in the central corneas of Mmp12-/- mice was also significantly higher compared with WT mice. Treatment of WT and Mmp12-/- mice with an anti-CCL2 antibody resulted in a reduction in F4/80-positive cells in the cornea indicating successful neutralization of CCL2. Neutralization of CCL2 showed a reversal of the findings made in the PBS treated corneas with significantly decreased neovascular vessels lengths in injured Mmp12-/- corneas compared with WT corneas.
These results demonstrate that MMP12 has an inhibitory effect on corneal neovascularization and that neutralization of CCL2 reverses this effect. The ability of MMP12 to suppress corneal neovascularization is therefore directly mediated through its interaction with CCL2. This mechanistic link between MMP12 and CCL2 has important implications in corneal neovascularization as well as inflammation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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