Co-inhibition of PGF and VEGF blocks their expression in mononuclear phagocytes and limits neovascularization and leakage in the murine retina

Abstract

Background: Age-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. The neovascular (wet) form of AMD can be treated with intravitreal injections of different anti-vascular endothelial growth factor (VEGF) agents. Placental growth factor (PGF) is another member of the VEGF family of cytokines with pro-angiogenic and pro-inflammatory effects. Here, we aimed to compare single and combined inhibition of VEGF-A and PGF in the laser-induced mouse model of choroidal neovascularization (CNV) with a focus on the effects on retinal mononuclear phagocytes.

Methods: CNV was induced in C57BL/6J mice using a YAG-Laser. Immediately after laser damage antibodies against VEGF-A (aVEGF), anti-PGF (aPGF), aVEGF combined with aPGF, aflibercept, or IgG control were injected intravitreally in both eyes. Three and 7 days after laser damage, the vascular leakage was determined by fluorescence angiography. Lectin staining of retinal and RPE/choroidal flat mounts was used to monitor CNV. In situ mRNA co-expression of Iba1, VEGF and PGF were quantified using in situ hybridization. Retinal and RPE/choroidal protein levels of VEGF and PGF as well as the pro-inflammatory cytokines IL-6, IL1-beta, and TNF were determined by ELISA.

Results: Early (day 3) and intermediate (day 7) vascular leakage and CNV were significantly inhibited by PGF and VEGF-A co-inhibition, most effectively with the trap molecule aflibercept. While VEGF-A blockage alone had no effects, trapping PGF especially with aflibercept prevented the accumulation of reactive microglia and macrophages in laser lesions. The lesion-related mRNA expression and secretion of VEGF-A and PGF by mononuclear phagocytes were potently suppressed by PGF and partially by VEGF-A inhibition. Protein levels of IL-6 and IL1-beta were strongly reduced in all treatment groups.

Conclusions: Retinal inhibition of PGF in combination with VEGF-A prevents vascular leakage and CNV possibly via modulating their own expression in mononuclear phagocytes. PGF-related, optimized strategies to target inflammation-mediated angiogenesis may help to increase efficacy and reduce non-responders in the treatment of wet AMD patients.

Keywords: Aflibercept; Age-related macular degeneration; Choroidal neovascularization; Laser CNV; Microglia; Neovascular AMD; Placental growth factor (PGF); Retinal degeneration; Vascular endothelial growth factor (VEGF).

Fig. 1

Effects of PGF and VEGF inhibition on vascular leakage in fundus fluorescein angiography. a Representative fundus fluorescein angiography images of animals of the indicated treatment groups 3 and 7 days after laser-induced damage. b Quantification of vascular leakage by analyzing pixel intensities 3 days after laser-induced damage (n = 13 eyes for IgG, n = 14 eyes for aVEGF, n = 13 eyes for aPGF, n = 20 eyes for aVEGF/aPGF, n = 13 eyes for aflibercept). c Quantification of vascular leakage by analyzing pixel intensities 7 days after laser-induced damage (n = 26 eyes for IgG, n = 14 eyes for aVEGF, n = 14 eyes for aPGF, n = 16 eyes for aVEGF/aPGF, n = 27 eyes for aflibercept). d Quantification of vascular leakage area 3 days after laser-induced damage (n = 13 eyes for IgG, n = 14 eyes for aVEGF, n = 13 eyes for aPGF, n = 20 eyes for aVEGF/aPGF, n = 13 eyes for aflibercept). e Quantification of vascular leakage area 7 days after laser-induced damage (n = 26 eyes for IgG, n = 14 eyes for aVEGF, n = 14 eyes for aPGF, n = 16 eyes for aVEGF/aPGF, n = 27 eyes for aflibercept). Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Effects of PGF and VEGF inhibition on choroidal neovascularization. a Representative images of lectin-stained RPE/choroidal flat mounts of animals of all treatment groups 3 and 7 days after laser damage. Dashed lines indicate CNV areas, and the asterisks mark the optic nerve head. Scale bare: 100 μm. b Quantification of lectin-stained CNV areas in RPE/choroidal flat mounts 3 days after laser coagulation with ZEN software (n = 10 eyes for IgG, n = 10 eyes for aVEGF, n = 11 eyes for aPGF, n = 11 eyes for aVEGF/aPGF, n = 10 eyes for aflibercept). c Quantification of lectin-stained CNV areas in RPE/choroidal flat mounts 7 days after laser coagulation with ZEN software (n = 14 eyes for IgG, n = 13 eyes for aVEGF, n = 14 eyes for aPGF, n = 16 eyes for aVEGF/aPGF, n = 28 eyes for aflibercept). Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 3

Effects of PGF and VEGF inhibition on microgliosis in retinal flat mounts. a Representative images of Iba1-stained microglia/macrophages in single laser spots of retinal flat mounts 3 and 7 days after laser coagulation. Scale bar: 100 μm. b Quantification of mononuclear phagocytes per laser spot in retinal flat mounts 3 days after laser-induced damage (n = 16 laser spots for IgG, n = 14 laser spots for aVEGF, n = 14 laser spots for aPGF, n = 15 laser spots for aVEGF/aPGF, n = 14 laser spots for aflibercept). c Quantification of mononuclear phagocytes per laser spot in retinal flat mounts 7 days after laser-induced damage (n = 14 laser spots for IgG, n = 14 laser spots for aVEGF, n = 15 laser spots for aPGF, n = 27 laser spots for aVEGF/aPGF, n = 28 laser spots for aflibercept). d Quantification of Iba1 signals 3 days after laser coagulation in retinal flat mounts by counting the mean of colored pixels per image (n = 16 laser spots for IgG, n = 14 laser spots for aVEGF, n = 14 laser spots for aPGF, n = 15 laser spots for aVEGF/aPGF, n = 14 laser spots for aflibercept). e Quantification of Iba1 signals 7 days after laser coagulation in retinal flat mounts by counting the mean of colored pixels per image (n = 14 laser spots for IgG, n = 14 laser spots for aVEGF, n = 15 laser spots for aPGF, n = 27 laser spots for aVEGF/aPGF, n = 28 laser spots for aflibercept). f Interleukin 6 (IL-6) levels in retinal flat mounts 6 h after laser damage quantified by ELISA (n = 8 flat mounts per group). Naive (not lasered) animals were used as controls. g Interleukin 1β (IL-1β) levels in retinal flat mounts 6 h after laser damage quantified by ELISA (n = 8 flat mounts per group). h Tumor necrosis factor (TNF) levels in retinal flat mounts 6 h after laser damage quantified by ELISA (n = 8 flat mounts per group). Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

Effects of PGF and VEGF inhibition on mononuclear phagocytes in RPE/choroidal flat mounts. a Representative images of Iba1-stained microglia/macrophages in single laser spots of RPE/choroidal flat mounts 3 and 7 days after laser coagulation. Scale bar: 100 μm. b Quantification of mononuclear phagocytes per laser spot in retinal flat mounts 3 days after laser-induced damage (n = 20 laser spots for IgG, n = 10 laser spots for aVEGF, n = 12 laser spots for aPGF, n = 13 laser spots for aVEGF/aPGF, n = 27 laser spots for aflibercept). c Quantification of mononuclear phagocytes per laser spot in RPE/choroidal flat mounts 7 days after laser-induced damage (n = 11 laser spots for IgG, n = 10 laser spots for aVEGF, n = 10 laser spots for aPGF, n = 15 laser spots for aVEGF/aPGF, n = 30 laser spots for aflibercept). d Quantification of Iba1 signals 3 days after laser coagulation in RPE/choroidal flat mounts by counting the mean of colored pixels per image (n = 20 laser spots for IgG, n = 10 laser spots for aVEGF, n = 12 laser spots for aPGF, n = 13 laser spots for aVEGF/aPGF, n = 27 laser spots for aflibercept). e Quantification of Iba1 signals 7 days after laser coagulation in RPE/choroidal flat mounts by counting the mean of colored pixels per image (n = 11 laser spots for IgG, n = 10 laser spots for aVEGF, n = 10 laser spots for aPGF, n = 15 laser spots for aVEGF/aPGF, n = 30 laser spots for aflibercept). f Interleukin 6 (IL-6) levels in RPE/choroidal flat mounts 6 h after laser damage quantified by ELISA (n = 8 flat mounts per group). Naive (not lasered) animals were used as controls. g Interleukin 1β (IL-1β) levels in RPE/choroidal flat mounts 6 h after laser damage quantified by ELISA (n = 8 flat mounts per group). h Tumor necrosis factor (TNF) levels in RPE/choroidal flat mounts 6 h after laser damage quantified by ELISA (n = 8 flat mounts per group). Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

Mononuclear phagocytes co-express PGF and VEGF mRNAs in laser lesions. Scale bar: 100 μm. a Representative images of in situ hybridization of single laser spots in RPE/choroidal flat mounts of IgG-treated animals at days 3 and 7. Probes targeted both Iba1 and VEGF. b Representative images of in situ hybridization of laser spots in RPE/choroidal flat mounts of IgG-treated animals at days 3 and 7. Probes targeted both Iba1 and PGF. c Representative images of in situ hybridization of laser spots in RPE/choroidal flat mounts of IgG-treated animals at days 3 and 7. Probes targeted both PGF and VEGF. The frames show higher magnification areas

Fig. 6

Effects of PGF and VEGF inhibition on their transcription and secretion in phagocytes. a Representative images of in situ hybridization (ISH) in single laser spots of RPE/choroidal flat mounts 7 days after laser damage. Probes bind specifically to mRNAs of PGF and VEGF. Scale bar: 100 μm. b Mean RNA expression of VEGF per laser spot in RPE/choroidal flat mounts 7 days after laser coagulation. Expression was determined by quantifying the mean colored pixels per area (n = 10 laser spots for IgG, n = 10 laser spots for aVEGF, n = 11 laser spots for aPGF, n = 10 laser spots for aVEGF/aPGF, n = 14 laser spots for aflibercept). c Mean RNA expression of PGF per laser spot in RPE/choroidal flat mounts 7 days after laser coagulation. Expression was determined by quantifying the mean colored pixels per area (n = 10 laser spots for IgG, n = 10 laser spots for aVEGF, n = 11 laser spots for aPGF, n = 10 laser spots for aVEGF/aPGF, n = 14 laser spots for aflibercept). d Cytokine levels of VEGF in RPE/choroidal flat mounts 7 days after laser damage measured by ELISA (n = 6 flat mounts per group). e Cytokine levels of PGF in RPE/choroidal flat mounts 7 days after laser damage measured by ELISA (n = 6 flat mounts per group). Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001)