Fig. 4

Spironolactone inhibits CNV through induction of anti-angiogenic decorin protein. a On western blot, the decorin (DCN) level decreases in the rat retinal pigment epithelium (RPE)-choroid at different time point (day 1, 3, 7, and 10) after laser induction compared to the normal rat RPE-choroid (ctrl). Densitometric quantification shows significant decrease of DCN protein in rat RPE-choroid at day 3 after laser induction. b Intravitreal injection (IVT) of recombinant mDCN protein in rat eyes inhibits choroidal vascular leakage on fluorescein angiography (FA); DCN 10 µg/ml significantly reduces the CNV angiographic grades (p = 0.02), whereas DCN at both 1 µg/ml (p = 0.0461) and 10 µg/ml (p = 0.0388) decrease the size of CNV induced by laser. c IVT of DCN siRNA in rat eyes with laser-induced CNV. Treatment with spironolactone (Spiro) in the presence of control siRNA significantly increases the DCN protein level in the rat RPE-choroid 48 h after laser induction. IVT of DCN siRNA prevents the increase in the DCN protein induced by spironolactone. d Treatment with spironolactone in the presence of control siRNA inhibits choroidal neovascular leakage on FA at day 14 after laser induction (p = 0.0194). IVT of DCN siRNA at day 0 and 3 after laser induction abrogates the effect of spironolactone on vascular leakage (p = 0.0344). Spironolactone in the presence of control siRNA reduces significantly CNV volume as compared to laser control (p < 0.0001). DCN siRNA injected at day 0 and 3 after laser induction abrogates the effect of spironolactone on CNV volume (p = 0.0003). Western blot data are expressed as mean ± SEM. Dots represent individual RPE-choroid sample. Non-parametric Kruskal–Wallis test was used. FA Data are expressed as the incidence of CNV angiographic grades of the total laser impacts in each group. CNV volumes are expressed as mean ± SEM of the average CNV size per rat. n represents the number of rats. Linear mixed model was used for statistical analyses. *p < 0.05, **p < 0.01, ***p < 0.001