Fig 1: MMP10 is expressed in ARPE-19 cells, hRPE cells under basal conditions and in the control mouse and human retina. (A) ARPE-19 cells in transwell inserts expressing MMP10 (red). (B) hRPE cells expressing MMP10 (red) in coverslip. (C) Flat mount of WT mouse retina expressing MMP10 (red) on the RGCL. (D) Flat mount of WT mouse expressing MMP10 (red) on the RPE. (E) Flat mount of a control human expressing MMP10 (red) on the RGCL. (F) Flat mount of a control human retina expressing MMP10 (red) and lectin (green) on the RPE-choroid. (G) Human eye illustration with a magnification of the retinal in a case of CNV and the main cellular components mentioned in this manuscript, the layers mentioned in figures (C–F) are marked with an asterisk. Lectin (green) is observed in endothelial cells in vessels in mice and human retinas. DAPI and Hoescht (blue) label nuclei. CNV = Choroidal neovascularisation. MMP10 = Matrix metalloproteinase 10. RGCL = retinal ganglion cell layer. RPE = retinal pigment epithelium. Scale bar: 20 µm (A,B,D,F), 50 µm (C,E).
Fig 2: MMP10 is expressed in ARPE-19 cells and overexpressed under oxidative stress conditions. (A) Control ARPE-19 cells expressing MMP10 (red) and cytokeratin 18 (CK18) (green) in coverslips. (B) ARPE-19 cells exposed to H2O2 at 800 µM during 48 h showed a prominent increase in MMP10 expression (red) in coverslips. DAPI (blue) label nuclei. (C) ARPE-19 cells in transwell inserts in saline medium. (D) ARPE-19 cells in transwell inserts exposed to H2O2 at 800 µM during 48 h showed a prominent increase in MMP10 expression (red). (E,F) WB analysis of MMP10 under basal conditions in the supernatant and cell lysate of ARPE-19 (E) and hRPE (F) cells. As can be observed, both cell lines have a marked expression of MMP10 contained intracellularly and present a measurable concentration of extracellular MMP10. No proteolytic activation of MMP10 is observed in basal conditions, neither intra- nor extracellularly. (G) WB analysis of MMP10 in supernatant of ARPE-19 cells in saline medium and exposed to H2O2 at 800 µM at different times (6, 24, 30 and 48 h). MMP10 is expressed under basal conditions over time and H2O2 exposition seems to activate the protein generating two visible bands. (H) RT-PCR shows a significative increased in MMP10 mRNA transcription over time under oxidative stress conditions. Scale bar: 20 µm. MMP10 = Matrix metalloproteinase 10. * p < 0.05.
Fig 3: MMP10-/- mice develop smaller CNV areas after laser injury. Representative laser-induced CNV area stained with CD31 by immunofluorescence (red) from WT mice (A) and for MMP10-/- mice in RPE (B). The dotted lines represent the edge of the CNV area. (C) Comparison of mean CNV area between WT and MMP10-/- mice after laser-induced CNV. AMD = age-related macular degeneration, CNV = Choroidal neovascularisation, WT = wild type, MMP10 = Matrix metalloproteinase 10. Error bars correspond to SEM. ** p < 0.01. Scale bar: 20 µm.
Fig 4: Correlation of age with the mean plasma levels of MMP10. A statistically significant negative correlation was observed in the control group (A) while no apparent correlation was found in the AMD group (B). AMD = Age-related macular degeneration, MMP10 = Matrix metalloproteinase 10. The Pearson’s r values are displayed on each graph along with the p value of statistical significance. * p < 0.05.
Fig 5: Endothelial cells (ECs) of laser-induced CNV areas are positive for MMP10 staining. Representative laser-induced CNV lesions on choroidal flat-mounts from WT mice stained with MMP10 (red), lectin or CD31 (green). (A) Whole flat-mount micrograph with three successfully induced CNV around the optic nerve, the outlined region represents the CNV shown in (B–F). (B) Magnification of dotted square in CNV area. MMP10 expression is increased in the CNV area. (C) Orthogonal view of the obtained z-stack showing that MMP10 expression is localized over the RPE Upper orthogonal projection showed a RPE nuclei disorganization and right orthogonal projection showed disruption in RPE nuclei (*). (D) Magnification of the same CNV in which the outlined region represents the magnification shown in E and F. (E) Magnification (63×) inside the CNV area showing that the endothelium (green) is positive for MMP10 (red). Some MMP10 positive cells are lectin negative (white arrow). (F) Orthogonal view of the obtained z-stack. (G) Whole flat-mount micrograph of a MM10-/- mice with laser-induced CNV. The outlined region represents the CNV shown in H and I. (H) Magnification of the CNV in a MM10-/- mice, as expected, no MMP10 expression is detected. (I) Orthogonal view of the obtained z-stack. Hoescht and 4',6-diamidino-2-phenylindole (DAPI) (blue) label nuclei. CNV = Choroidal neovascularisation. MMP10 = Matrix metalloproteinase 10. ON = optic nerve. WT = wild type. Scale bars: 10 µm (E,F), 20 µm (I), 50 µm (B–D,H), 100 µm (G) and 500 µm (A).
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