The current study aimed to further examine the involvement of the NMDAR of RPE cells in the HHcy-induced features of AMD. We previously reported the involvement of endothelial NMDAR in HHcy-induced BRB dysfunction. Mouse with Deletion/Inhibition of NMDAR in Retinal Pigmented Epithelia (NMDAR R −/−) The cbs +/− mice showed significant expression of NMDAR1 in the outer retina and increased activation (green) in cbs +/− RPE cells as compared to primary RPE cells isolated from wild-type mice ( Figure 3f). Finally, to further confirm our results, the protein expression of NMDAR1 was examined in the outer retina (has a good amount of RPE cells) of the mice model of HHcy ( cbs +/−) and compared to the retina of normal control mice by WB analysis ( Figure 3e), and in primary RPE cells isolated from wild-type mice and cbs +/− mice by immunofluorescence. WB and IF analyses showed that NMDAR1 is highly expressed in the ARPE-19 cell line and the expression was significantly increased by a 100 µM Hcy treatment ( Figure 3c,d) as compared to the ARPE-19 control.

The expression of NMDAR was further confirmed on the protein level by assessing the expression of NMDAR1 by WB and IF analyses in ARPE-19 cells treated with/without Hcy (20, 50, and 100 µM). And its expression was increased by Hcy treatment in a dose dependent manner ( Figure 3b). Our results showed that the NMDAR1 gene was expressed in the ARPE-19 cell line ( Figure 3a). The gene expression of NMDAR was assessed by RT-qPCR, and human neuroblastoma cells were used as a positive control. NMDAR1 expression was assessed in the human RPE (ARPE-19) cell line at both the gene and protein levels. Homocysteine Activates HIF-1α and VEGF in RPE Cells Moreover, to confirm the effect of Hcy treatment on the proliferation of choroidal endothelial cells, we examined the expression of the Ki67 proliferation factor in cultured mouse choroidal endothelial cells (MCEC), and results showed a marked increase in Ki67 expression (green) in MCEC by Hcy treatment ( Figure 1f). Retinal flat-mounts of mice that were exposed to laser induction with/without the intravitreal injection of Hcy were stained with isolectin-B4 to examine the effect of Hcy on the size and extent of laser-induced CNV this showed that Hcy significantly increased the extent of laser-induced CNV in wild-type mice ( Figure 1d,e). The results showed that Hcy-injected wild-type mice highly expressed isolectin-B4 and showed the development of blood vessels extending from the area of the choroid to the inner retina (white arrows, Figure 1c) as compared to the control wild-type mice, which revealed a normal retinal vascular pattern. Retinal section/flat-mounts were evaluated by immunofluorescence staining for vascular marker, isolectin-B4 (red).

To study the effect of Hcy on CNV induction, we examined the CNV in the retinal frozen section and retinal flat-mounts of wild-type mice (C57-BL6) injected intravitreally with/without Hcy. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features thus, NMDAR inhibition could serve as a promising therapeutic target for AMD. Knocking down RPE NMDAR improved retinal structure and CNV. Hcy-injected WT mice showed disrupted retina and CNV. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. A neovascular AMD patient’s serum showed a significant increase in Hcy and a decrease in CBS. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC) (2) in vivo using wild-type mice and mice deficient in RPE NMDAR ( NMDAR R −/−) with/without Hcy injection. Serum Hcy and cystathionine-β-synthase (CBS) were assessed by ELISA.

This study suggests that N-Methyl- d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD.

We previously reported that HHcy induces AMD-like features. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. Age-related macular degeneration (AMD) is a leading cause of vision loss.

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