Metformin inhibits pathological retinal neovascularization but promotes retinal fibrosis in experimental neovascular age-related macular degeneration

Abstract

Purpose: This study aims to investigate the effects and mechanism of action of metformin on retinal neovascularization and fibrosis in a mouse model of neovascular age-related macular degeneration (nAMD).

Methods: Very low-density lipoprotein receptor knockout (Vldlr ^-/-) mice, a mouse model of nAMD, were used in this study. Vldlr ^-/- mice were administered metformin on postnatal day (P) 20 for 20 days (early stage of pathological change) or at 5.5 months of age for 45 days (late stage of pathological change). Retinal leakage was examined by fundus fluorescein angiography (FFA). Retinal neovascularization was assessed by lectin staining. Retinal fibrosis was assessed by Western blotting, immunofluorescence staining, and Masson's trichrome staining.

Results: Retinal vascular leakage and neovascularization were significantly reduced in Vldlr ^-/- mice treated with metformin compared to those treated with the vehicle at P40. The protein levels of inflammatory factors and phospho(p)-STAT3 were decreased, and P38 and ERK signaling were suppressed in the retinas of metformin-treated Vldlr ^-/- mice relative to those in the control group at P40. Fibrotic markers were upregulated in the retinas of Vldlr ^-/- mice treated with metformin compared to those treated with the vehicle at 7 months. Levels of the inflammatory factors and p-STAT3 were increased, and PI3K/AKT, P38, and ERK signaling were upregulated in the retinas of metformin-treated Vldlr ^-/- mice compared to those in the control group at 7 months.

Conclusion: Metformin inhibits pathological retinal neovascularization but promotes fibrosis in experimental nAMD. These results provide evidence and highlight important considerations for the clinical use of metformin in different stages of nAMD.

Keywords: age-related macular degeneration; metformin; neovascular AMD; retinal fibrosis; retinal neovascularization; very low-density lipoprotein receptor.

FIGURE 1

Metformin inhibits retinal vascular leakage and neovascularization in the retinas of Vldlr ^−/− mice at P40. Vldlr ^−/− mice were treated with metformin (200 mg/kg/day) or vehicle solution (control) by daily gavage from P20 to P40. The total number of retinal neovascular sprouts was quantified at P40. (A, B) Representative images of fundus fluorescein angiography (FFA) of Vldlr ^−/− mice treated with vehicle (VEH) (A) or metformin (MET) (B). (C, D) Representative images of lectin staining from Vldlr ^−/− mice treated with vehicle (VEH) or metformin (MET). (E, F) Quantification of leakage areas of FFA images (E) or neovascular spots of lectin staining images (F) from vehicle and metformin-treated Vldlr ^−/− mice. Data are shown as mean ± SEM. N = 6, ***p < 0.001. A two-tailed Student’s t-test was used.

FIGURE 2

Metformin improves oscillatory potentials in the retinas of Vldlr ^−/− mice at P40. (A) Representative images of the a-wave and b-wave from Vldlr ^−/− mice treated with vehicle (VEH) or metformin (MET). ERGs were obtained by averaging three responses to 1.0 cd·s/m² flashes. (B, C) Amplitudes of ERG a-wave (B) and b-wave (C) of the two groups were analyzed and quantified. (D) Representative images of oscillatory potentials from Vldlr ^−/− mice treated with vehicle (VEH) or metformin (MET). (E) Oscillatory potentials from the two groups were analyzed and quantified. Data are shown as mean ± SEM; n = 6–8, *p < 0.05, **p < 0.01, and ***p < 0.001. A two-tailed Student’s t-test was used.

FIGURE 3

Metformin reduces the retinal pro-inflammatory cytokines, p-P38 and p-ERK, in an AMPK-dependent manner in the eyecups of Vldlr ^−/− mice at P40. (A–C) The protein levels of p-AMPK (A, B) and AMPK (A, C) in the eyecups of Vldlr ^−/− mice treated with vehicle (VEH) or metformin (MET) were determined by Western blot analysis and quantified by densitometry. (D–H) The protein levels of VEGF (D, E), VCAM-1 (D, F), p-STAT3 (D, G), and STAT3 (D, H) in the eyecups of Vldlr ^−/− mice treated with vehicle (VEH) or metformin (MET) were determined by Western blot analysis and quantified by densitometry. (I–M) Protein levels of p-P38 (I, J), P38 (I, K), p-ERK (I, L), and ERK (I, M) in the eyecups of the two indicated groups were determined by Western blot analysis and quantified by densitometry. Data are shown as mean ± SEM; n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001. A two-tailed Student’s t-test was used.

FIGURE 4

Metformin promotes subretinal fibrosis in Vldlr ^−/− mice at 7 months of age. Vldlr ^−/− mice were fed with a diet containing metformin from the age of 5.5 months to 7 months. The mice were euthanized at 7 months of age. (A, B) Representative retinal images of H&E staining (A) and Masson’s staining (B) of collagen deposition in the retinal paraffin sections of Vldlr ^−/− mice fed with control chow (VEH) or metformin chow (MET). (C–E) Representative images of immunostaining show the expression of collagen-1 (C), α-SMA (D), and GFAP (E) in the retinal cryosections of the two indicated groups. (F–I) The protein levels of collagen-1(F, G), vimentin (F, H), and GFAP (F, I) were determined by Western blot analysis and quantified by densitometry in the two indicated groups. Data are shown as mean ± SEM; n = 6. *p < 0.05 and **p < 0.01. A two-tailed Student’s t-test was used.

FIGURE 5

Metformin increases inflammation in the eyecups of Vldlr ^−/− mice at 7 months of age. (A) Representative images of immunostaining show the expression of VCAM-1 in the cryosection of Vldlr ^−/− mice fed with vehicle (VEH) or metformin (MET). (B–E) The protein levels of VEGF (B, C), p-STAT3 (B, D), and STAT3 (B, E) in the Vldlr ^−/− mice fed with control chow (VEH) or metformin chow (MET) were determined by Western blot analysis and quantified by densitometry. Data are shown as mean ± SEM; n = 6. *p < 0.05 and **p < 0.01. A two-tailed Student’s t-test was used.

FIGURE 6

Metformin activates PI3K/AKT, p-P38, and p-ERK pathways in the eyecups of Vldlr ^−/− mice at 7 months of age. (A–C) The protein levels of p-PI3K (A, B) and PI3K (A, C) in the Vldlr ^−/− mice fed with control chow (VEH) or metformin chow (MET) were determined by Western blot analysis and quantified by densitometry. (D–F) The protein levels of p-AKT (D, E) and AKT (D, F) in the two indicated groups were determined by Western blot analysis and quantified by densitometry. (G–K) The protein levels of p-P38 (G, H), P38 (G, I), p-ERK (G, J), and ERK (G, K) in the two indicated groups were determined by Western blot analysis and quantified by densitometry. Data are shown as mean ± SEM; n = 6. *p < 0.05 and **p < 0.01. A two-tailed Student’s t-test was used.