Diabetic retinopathy (DR), with increasing incidence, is the major cause of vision loss and blindness worldwide in working-age adults. Diabetic macular edema (DME) remains the main cause of vision impairment in diabetic patients, with its pathogenesis still not completely elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in the pathogenesis of DR and DME. Currently, intravitreal injection of anti-VEGF agents remains as the first-line therapy in DME treatment due to the superior anatomic and functional outcomes. However, some patients do not respond satisfactorily to anti-VEGF injections. More than 30% patients still exist with persistent DME even after regular intravitreal injection for at least 4 injections within 24 weeks, suggesting other pathogenic factors, beyond VEGF, might contribute to the pathogenesis of DME. Recent advances showed nearly all the retinal cells are involved in DR and DME, including breakdown of blood-retinal barrier (BRB), drainage dysfunction of Müller glia and retinal pigment epithelium (RPE), involvement of inflammation, oxidative stress, and neurodegeneration, all complicating the pathogenesis of DME. The profound understanding of the changes in proteomics and metabolomics helps improve the elucidation of the pathogenesis of DR and DME and leads to the identification of novel targets, biomarkers and potential therapeutic strategies for DME treatment. The present review aimed to summarize the current understanding of DME, the involved molecular mechanisms, and the changes in proteomics and metabolomics, thus to propose the potential therapeutic recommendations for personalized treatment of DME.

Purpose. To investigate the hyperreflective foci (HRF) as an inflammatory biomarker using optical coherence tomography angiography (OCTA) in neovascular age‐related macular degeneration (AMD) patients after antivascular endothelial growth factor (anti‐VEGF) treatment and its association with the retinal microcapillary density. Methods. Twenty‐five eyes from 25 patients with neovascular AMD were included in the study. All eyes were imaged with OCTA at baseline (M0) and after 3 consecutive injections (M3; injection performed each month) of anti‐VEGF. The number of HRF in the superficial capillary plexus (SCP), deep capillary plexus (DCP), and outer retina was counted. The vascular density of the fovea, parafovea, and the whole macula, as well as the area of the foveal avascular zone (FAZ), was measured. Results. The mean interval between baseline and follow‐up with OCTA was 93.08 ± 5.00 (range, 85‐101) days. Compared with the baseline, the number of HRF significantly decreased in DCP (7.52 ± 3.06 vs. 3.76 ± 1.48, P < 0.01) and outer retina (12.04 ± 4.91 vs. 5.88 ± 3.32, P < 0.01) after treatment. There was no significant difference for HRF number in the SCP, the vascular density (containing foveal, parafoveal, and whole macular), and FAZ area before and after treatments. Conclusion. The number of HRF in DCP and outer retina might serve as an inflammatory biomarker in patients with neovascular AMD. The reduced HRF possibly represents the alleviation of inflammation after anti‐VEGF treatment in patients with AMD.

The protective effects and mechanisms of metformin against oxidative stress were evaluated bothin vivoandin vitro. ARPE-19 cells comprised the normal group, the glyoxal-treated group (0.5 mM glyoxal), and the glyoxal+metformin group (0.5 mM glyoxal and 0.1 mM metformin). In thein vitromodel, differences in cell viability, ROS production, NO products, cellular apoptosis, and the expressions of phospho-AMPK alpha, total-AMPK alpha, Sirt1, Nrf2, TXNIP, ZO-1, and Occludin were assessed. In the glyoxal-treated group, cell viability and NO production were decreased, while ROS production and cell apoptosis were increased (p<0.05), compared with the control group. These changes were prevented by metformin treatment. Protein expressions of phospho-AMPK alpha, Sirt1, TXNIP, ZO-1, and Occludin, but not Nrf2, were decreased significantly in the glyoxal-treated group compared to normal controls. Metformin treatment significantly increased the above protein expressions and slightly increased TXNIP expression. Immunofluorescence showed that metformin prevented the glyoxal-induced, disorganized tight junctions in ARPE-19 cells. To confirm metformin's protection, Sprague-Dawley rats were injected intravenously with sodium iodate (SI) to induce oxidative stress in the retinal pigment epithelium (RPE). Metformin was then delivered intraperitoneally or intravitreally. One day and three days after SI and metformin treatments, the RPE-Bruch's membrane-choriocapillaris complex was isolated and immune-stained with ZO-1 antibodies. The morphology of the RPE showed enlarged cellular bodies and disorganized ZO-1 staining in SI-treated rats. Metformin treatment prevented these changes. The results indicated that metformin maintained the barrier functions of RPE cells bothin vivoandin vitro. Metformin exerted its protection against oxidative stress possibly via activating AMPK/Sirt1 and increasing TXNIP. Metformin has been proposed as a candidate drug for age-related macular degeneration (AMD) by both preclinical and clinical studies. The cellular and animal models used in this study might be useful for the interpretation of the molecular mechanisms involved in the drug activity.

Background: Neovascular age-related macular degeneration (nAMD), accounts for up to 90% of AMD-associated vision loss, ultimately resulting in the formation of fibrotic scar in the macular region. The pathogenesis of subretinal fibrosis in nAMD involves the process of epithelial–mesenchymal transition (EMT) occurring in retinal pigment epithelium (RPE). Here, we aim to investigate the underlying mechanisms involved in the Wnt signaling during the EMT of RPE cells and in the pathological process of subretinal fibrosis secondary to nAMD. Methods: In vivo, the induction of subretinal fibrosis was performed in male C57BL/6J mice through laser photocoagulation. Either FH535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) was intravitreally administered on the same day or 14 days following laser induction. The RPE-Bruch's membrane-choriocapillaris complex (RBCC) tissues were collected and subjected to Western blot analysis and immunofluorescence to examine fibrovascular and Wnt-related markers. In vitro, transforming growth factor beta 1 (TGFβ1)-treated ARPE-19 cells were co-incubated with or without FH535, Foxy-5 (a Wnt5a-mimicking peptide), Box5, or Wnt5a shRNA, respectively. The changes in EMT- and Wnt-related signaling molecules, as well as cell functions were assessed using qRT-PCR, nuclear-cytoplasmic fractionation assay, Western blot, immunofluorescence, scratch assay or transwell migration assay. The cell viability of ARPE-19 cells was determined using Cell Counting Kit (CCK)-8. Results: The in vivo analysis demonstrated Wnt5a/ROR1, but not Wnt3a, was upregulated in the RBCCs of the laser-induced CNV mice compared to the normal control group. Intravitreal injection of FH535 effectively reduced Wnt5a protein expression. Both FH535 and Box5 effectively attenuated subretinal fibrosis and EMT, as well as the activation of β-catenin in laser-induced CNV mice, as evidenced by the significant reduction in areas positive for fibronectin, alpha-smooth muscle actin (α-SMA), collagen I, and active β-catenin labeling. In vitro, Wnt5a/ROR1, active β-catenin, and some other Wnt signaling molecules were upregulated in the TGFβ1-induced EMT cell model using ARPE-19 cells. Co-treatment with FH535, Box5, or Wnt5a shRNA markedly suppressed the activation of Wnt5a, nuclear translocation of active β-catenin, as well as the EMT in TGFβ1-treated ARPE-19 cells. Conversely, treatment with Foxy-5 independently resulted in the activation of abovementioned molecules and subsequent induction of EMT in ARPE-19 cells. Conclusions: Our study reveals a reciprocal activation between Wnt5a and β-catenin to mediate EMT as a pivotal driver of subretinal fibrosis in nAMD. This positive feedback loop provides valuable insights into potential therapeutic strategies to treat subretinal fibrosis in nAMD patients.

Fibrotic scar is a severe side effect of trabeculectomy, resulting in unsatisfactory outcomes for glaucoma surgery. Accumulating evidence showed human Tenon's fibroblasts (HTFs) play an important role in fibrosis formation. We previously reported that the aqueous level of secreted protein acidic and rich in cysteine (SPARC) was higher in the patients with primary angle closure glaucoma, which was associated with the failure of trabeculectomy. In this study, the potential effect and mechanism of SPARC in promoting fibrosis were explored by using HTFs. HTFs were employed in this study and examined under a phase-contrast microscope. Cell viability was determined by CCK-8. The expressions of SPARC-YAP/TAZ signaling and the fibrosis-related markers were examined with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and immunofluorescence, subcellular fractionation was conducted to further determined the variation of YAP and phosphorylated YAP. The differential gene expressions were analyzed with RNA sequencing (RNAseq), followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Exogenous SPARC induced HTFs-myofibroblast transformation, as evidenced by the increased expression of α-SMA, collagen I and fibronectin in both protein and mRNA levels. SPARC knockdown decreased the expressions of the above genes in TGF-β2-treated HTFs. KEGG analysis showed that the Hippo signaling pathway was mostly enriched. SPARC treatment increased the expressions of YAP, TAZ, CTGF and CYR61 as well as enhanced YAP translocation from cytoplasm to nucleus, and decreased the phosphorylation of YAP and LAST1/2, which was reversed by SPARC knockdown. Knockdown of YAP1 decreased the fibrosis-related markers, such as α-SMA, collagen I and Fibronectin, in SPARC-treated HTFs. SPARC induced HTFs-myofibroblast transformation via activating YAP/TAZ signaling. Targeting SPARC-YAP/TAZ axis in HTFs might provide a novel strategy for inhibiting fibrosis formation after trabeculectomy.