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Kyuyeon Han, Jin-Hong Chang, Dimitri T Azar; Proangiogenic role of MMP14 on vascular endothelial cells: selective degradation of VEGFR1 during angiogenesis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3331. doi: https://doi.org/.
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Membrane-bound matrix metalloproteinase 14 (MMP14) plays an important role in extracellular matrix remodeling and corneal angiogenesis. Vascular endothelial growth factor 1 (VEGFR1) has been identified as tyrosine kinase receptor that serves as a decoy for VEGFR2 in angiogenesis, inflammation, and corneal wound healing. We hypothesize that MMP14 is a key regulator of VEGFA-induced angiogenesis through cleavage of VEGFR1, which enhances VEGFR2-mediated signaling in corneal angiogenesis.
Two methods were applied to modulate MMP14 expression on vascular endothelial cells (VECs): 1) MMP14_siRNA for a loss of effects and 2) transfection with MMP14 plasmid for a gain of effect. Expression of MMP14 on VECs was determined by western blotting against anti-MMP14 and pro-MMP2 using zymography. Membrane VEGFR1 (mVEGFR1) was detected by western blotting against anti-mVEGFR1. An enzyme-linked immunosorbent assay was used to measure cleaved VEGFR1 (cVEGFR1) in conditioned media. The effect of MMP14 expression on VEGFA-induced signaling was investigated by western blotting. VEGFA-induced VEC proliferation and migration were also compared using BrdU or Boyden chamber assays after modulation of MMP14 expression using either method.
mVEGFR1 was found to be reduced in VECs upon overexpression of MMP14. Moreover, more mVEGFR1 was observed when MMP14 expression was reduced by MMP14_siRNA. In contrast, cVEGFR1, cleaved by MMP14 in conditioned medium, showed the opposite trend. Upon investigating the requirement for the enzyme activity of MMP14 in the hydrolysis of mVEGFR1, we observed that proteolysis of mVEGFR1 was inhibited by the MMP inhibitors GM6001 and TIMP2. The amount of VEGFR2 observed remained the same independent of MMP14 expression. VEGFA-induced pERK was higher in MMP14-overexpressing VECs than in control VECs, whereas knockdown of MMP14 in VECs by siRNA showed the opposite result. As a result, VEGFA-induced proliferation and migration were found to differ based on MMP14 expression.
MMP14 regulates the level of mVEGFR1 on VECs through proteolysis of mVEGFR1 and potentially affects events related to VEGFA-induced angiogenesis, including VEC proliferation and migration. These results provide insight into the roles of MMP14 on VECs in support of new therapeutic methods for treating corneal neovascularization and promoting corneal wound healing.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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