Figure 1.
Retinal morphology and function in MMP-12 KO mice.
A). Morphology of H&E-stained retinal sections from WT and MMP-12 KO mice at age 6 months. Delayed regression of the hyaloid vessel system was observed (arrow), but the overall retinal structure appeared normal in MMP-12 KO mice. B). No functional defects were detected by scotopic and photopic ERG in MMP-12 KO mice (n = 9 in the WT group and n = 8 in the MMP-12 KO group).
Figure 2.
Reduced retinal VEGF expression and vascular leakage in MMP-12 KO mice with OIR.
A). Increased MMP-12 expression measured by real-time RT-PCR in the retina from hyperoxia-exposed WT mice (OIR) compared with room-air control mice (RA) at P17. **p<0.01 and n = 4 in each group. B). Increased retinal VEGF level measured by ELISA in WT mice with OIR at P17. **p<0.01 and n = 5 in RA group and n = 4 in OIR group. C). Western blot analysis of VEGF in retina from WT and MMP-12 KO mice. Retinal VEGF expression in MMP-12 KO mice was significantly lower than in WT mice with OIR. **p<0.01, vs WT/RA; ‡p<0.01, vs WT/OIR. n = 4 in each group. D). Retinal vascular permeability was measured by the Evans blue-albumin method in MMP-12 KO and WT mice at P17. **p<0.01, WT/RA; †p<0.05, vs WT/OIR. n = 5 in WT/RA group, n = 6 in WT/OIR group, n = 4 in MMP12/RA group and n = 9 in MMP12/OIR group.
Figure 3.
Decreased levels of inflammatory factors in retina of MMP-12 KO mice with OIR.
Expression of ICAM-1(A) and TNF-α (B) in the retinas was measured by Western blot analysis in WT and MMP-12 KO mice with OIR. **p<0.01 vs WT/RA; ‡p<0.01, †p<0.05, vs WT/OIR; n = 4 in each group.
Figure 4.
Reduced retinal macrophage infiltration and impaired migration capacity of macrophages in MMP-12 KO mice.
A). Macrophage infiltration into retina was determined by western blot analysis of F4/80, a macrophage-specific membrane marker. Right panel: Semi-quantification of retinal F4/80 levels by densitometry. *p<0.05, vs WT/RA; †p<0.05, vs WT/OIR. n = 4 in each group. B-C). Migration capacity of bone-marrow derived macrophages from MMP-12 KO and WT mice. Migration was evaluated by the transwell migration assay in the presence or absence of 100 ng/ml M-CSF. B). Representative images of migrated macrophages (200x); C). Quantification of basal and M-CSF-stimulated macrophage migration. **p<0.01, vs WT/control; ‡p<0.01, vs. WT/M-CSF.
Figure 5.
Decreased retinal avascular area in MMP-12 KO mice with OIR.
A). Representative images of retinal fluorescein angiography in MMP-12 KO and WT mice with OIR at P12 and P17. B). Quantification of retinal avascular area in retinal angiographic images using Adobe Photoshop software. n = 6 in WT/P12 group, n = 4 in MMP-12 KO/P12 group, n = 5 in WT/P17 group and n = 4 in MMP-12 KO/P17 group.
Figure 6.
Reduced pathological retinal NV in OIR in MMP-12 KO mice.
A). Images of isolectin GS-IB4 staining of retinal flat mounts showing retinal NV in MMP-12 KO and WT mice with OIR at P17. B). Quantification of retinal NV using Adobe Photoshop software. *p<0.05; n = 5 in each group.
Figure 7.
Pharmaceutical inhibition of MMP-12 suppressed retinal inflammation and attenuated retinal NV in OIR mice.
At P12, OIR mouse pups were randomly chose to gavage with or without MMP408 at dose of 30 mg/kg/d for 5 days. A-C) Expression of ICAM-1 (A), TNF-α (B) and VEGF (C) were determined by western blot analysis in retinas of OIR mice at P17. D) Retinal neovascularization were displayed by isolection GS-IB4 staining in vehicle-treated (a) and MMP408-treated (b) eyes from OIR mice at P17, and quantified by Adobe Photoshop software (c). *p<0.05 or **p<0.01 vs. vehicle; n = 4 for each group. E) In vitro angiogenesis assay. Primary human retinal endothelial cells (HRECs) were pretreated with MMP408 at doses of 0 nM (a), 0.2 nM (b), 2 nM (c), and 20 nm (d) for 16 h. Cells were seeded on Matrigel for tube formation assay. After 6 h of incubation, representative pictures were randomly taken and branch numbers were counted from 3 different visual fields.
Figure 8.
Enhanced intraretinal revascularizaiton into the ischemic retina in MMP-12 KO mice.
A). Representative retinal fluorescein angiographic images demonstrating enhanced retinal re-vascularization in MMP-12 KO mice compared with WT OIR mice from P17 to P20 (3-5 mice in each group). B). Higher magnification images of retinal angiograph at P19. C). Quantification of avascular area in retinas from WT and MMP-12 KO mice with OIR at P17-P20. Results were expressed as mean ± SD, n = 4. MMP-12 KO mice had significantly smaller area of avascular retina when compared to WT mice (P<0.01).