FIGURE 4.

Inhibition of CTGF/CCN2 gene expression or protein activity reduced retinal neovascularization and inhibited MMP-2 gene expression and activity. A, injection of lnv-AS-CTGF or lnv-CTGF-ΔCT delays blood vessel development and expansion to the retinal edge. Mouse pups were intravitreously injected at P3 with either lnv-luc (panel a), lnv-AS-CTGF (panel b), or lnv-CTGF-ΔCT (panel c) vector. Retinal flat mounts were prepared at P7 and stained with rhodamine-labeled UEA-1. B–D, effects of lentivirus-mediated expression of AS-CTGF and CTGF-ΔCT on retinal neovascularization following OIR. Representative flat-mount preparations of rhodamine-UEA-1-stained retinas from OIR eyes at P17 previously injected with either lnv-luc (panel a), lnv-AS-CTGF (panel b), or lnv-CTGF-ΔCT (panel c) are shown. Areas of vaso-obliteration at P12 and pre-retinal neovascular tufts at P17 were measured by computer-assisted image analyses, and the compiled data showing percentage of avascular and neovascular areas are shown in C and D, respectively. **, p < 0.05 versus OIR/lnv-luc (n = 6–8). E and F, transcript levels of VEGF and MMP-2 as determined by qPCR in retinas from OIR mice injected with either lnv-luc, lnv-AS-CTGF, or lnv-CTGF-ΔCT. The data shown are the means ± S.E. of three determinations each performed in triplicate. RNA levels at P12 were set to 100% to facilitate comparison among experiments. *, p < 0.01 versus P12 (n = 6). **, p < 0.05 versus either P12 or P17/lnv-luc (n = 6). ***, p < 0.05 versus P17/lnv-luc (n = 6). G and H, levels of latent pro-MMP-2 and active MMP-2 in retinal homogenates from OIR eyes injected with either lnv-luc, lnv-AS-CTGF, or lnv-CTGF-ΔCT lentiviral vectors. Latent and active MMP-2 activities were determined using MMP-2-specific Biotrack assay. Amounts of MMP-2 in the samples were determined by interpolation from a standard curve. Values are means ± S.E. from three sets of eyes. *, p < 0.01 versus P12/lnv-luc. **, p < 0.05 versus P17/lnv-luc.