Mineralocorticoid receptor antagonism limits experimental choroidal neovascularization and structural changes associated with neovascular age-related macular degeneration

Abstract

Choroidal neovascularization (CNV) is a major cause of visual impairment in patients suffering from wet age-related macular degeneration (AMD), particularly when refractory to intraocular anti-VEGF injections. Here we report that treatment with the oral mineralocorticoid receptor (MR) antagonist spironolactone reduces signs of CNV in patients refractory to anti-VEGF treatment. In animal models of wet AMD, pharmacological inhibition of the MR pathway or endothelial-specific deletion of MR inhibits CNV through VEGF-independent mechanisms, in part through upregulation of the extracellular matrix protein decorin. Intravitreal injections of spironolactone-loaded microspheres and systemic delivery lead to similar reductions in CNV. Together, our work suggests MR inhibition as a novel therapeutic option for wet AMD patients unresponsive to anti-VEGF drugs.

Conflict of interest statement

M.Z. and F.B.-C. are listed as inventors in a patent application related to the “Methods and pharmaceutical compositions for the treatment of choroidal neovascularization” (WO2017064121A1, https://patents.google.com/patent/WO2017064121A1/en). The remaining authors declare no competing interests.

Figures

Fig. 1

Clinical results. a Relative changes in subretinal thickness (SRF, µm) measured with Spectralis OCT during the study period, month 0 (M0) to month 6 (M6). SRF thickness is significantly reduced as compared to M0, at M1 (p = 0.012), M2 (p = 0.040), M3 (p = 0.014), M4 (p = 0.024). b Relative change in central retinal thickness (CRT, µm) automatic values with Spectralis OCT during the study period, month 0 (M0) to month 6 (M6). CRT is significantly reduced as compared to M0, at M3 (p = 0.011), M4 (p = 0.007), and M5 (p = 0.028). At each time point (Month, M), the data have been summarized using box and whisker plots, the upper and lower 95% CIs (confidence interval) are marked with the whiskers, the box represents the interquartile range, and the median is represented by the bold horizontal line bisecting the box. Paired Wilcoxon signed-rank tests were used. *p < 0.05

Fig. 2

Spironolactone and eplerenone reduce CNV in a rat nAMD model. a Spironolactone (Spiro) significantly reduces the CNV angiographic grades evaluated on fluorescein angiography (FA) (p = 0.0025) and the CNV volume labeled with FITC-isolectin (green) (p = 0.002) as compared to the control group (Ctrl). Infrared images (IR) are used to localize and check the efficient laser-induced burns. The effect of spironolactone is not different from anti-VEGF in reducing the choroidal neovascular leakage on FA and inhibiting the CNV in rat choroidal flat-mounts. Combining the two treatments allows an enhanced effect in reducing vascular permeability compared to anti-VEGF alone (p = 0.0335). b Eplerenone, a more specific MR antagonist, given orally, significantly reduces CNV angiographic grades (p = 0.0012) and CNV volumes (p < 0.0001). Bars: 100 µm. 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 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

Fig. 3

Vascular endothelial MR contributes to CNV. a Systemic spironolactone significantly reduces CNV fluorescein angiographic (FA) grades (p < 0.001) as well as CNV volume (p = 0.0348) as compared to control mice. CNV were labeled with FITC-dextran (green). Infrared (IR) images show up all the laser burns in the fundus. b Cell-type-specific MR deletion from endothelial cells using the VE-Cadherin promoter (Vecadh-MR-KO) reduces CNV leakage on FA (p = 0.0041) and decreases the volume of CNV labeled with FITC-dextran (green) in mice (p = 0.0434). IR images show up all the laser burns. Bar: 100 µm (a, b). 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 mouse. n represents the number of mice. Linear mixed model was used for statistical analyses. *p < 0.05. c In a model of corneal neovascularization using Vecadh-MR-KO mice, a reduction in corneal neovessels labeled with FITC-dextran was observed compared to control mice. Quantification of the neovascular surface on mosaic images confirms a significant decrease in the neovascularization/whole corneal area ratio in Vecadh-MR-KO mice. Data are expressed as mean ± SEM. n represents the number of mice. Non-parametric Mann–Whitney U-test was used. **p < 0.01

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