Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.

BackgroundTo identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls.MethodsAfter uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups).ResultsIncreased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen.ConclusionsDifferential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.Electronic supplementary materialThe online version of this article (10.1186/s12886-018-0941-9) contains supplementary material, which is available to authorized users.

Citation: Park KH, Choi AJ, Yoon J, et al. Wnt modulators in the aqueous humor are associated with outer retinal damage severity in patients with neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci. 2014;55:5522-5530. DOI: 10.1167/iovs.14-14566 PURPOSE. To investigate the associations of the Wnt modulators Wnt inhibitory factor 1 (WIF-1) and Dickkopf 3 (DKK-3) in the aqueous humor with neovascular age-related macular degeneration (nAMD) and to determine their clinical implications.METHODS. Seventy-four nAMD patients initially treated with an intravitreal injection of ranibizumab (IVR) and 74 age-and sex-matched controls were studied. Aqueous humor WIF-1 and DKK-3 levels were measured by Western blotting and an ELISA before and 1 month after two consecutive IVRs (pre-and post-IVR). Visual acuity assessments and spectral domain optical coherence tomography were performed pre-and post-IVR. RESULTS.Western blotting showed increased WIF-1 and DKK-3 in 12 nAMD patients compared with 12 controls. The ELISA analysis demonstrated elevated WIF-1 (pre) and DKK-3 (pre) in 62 patients compared with 62 controls (54.7 vs. 23.0 and 114.3 vs. 93.0 ng/mL, respectively). In multivariate analyses, high WIF-1 (pre) levels were associated with increased disruption in the photoreceptor junction's inner and outer segments (IS/OS) (pre and post) and high WIF-1 (post) levels. Interestingly, WIF-1 (pre) levels were significantly higher in type 3 neovascularization (NV) patients than in type 1 or 2 NV (90.5 6 36.7 vs. 48.3 6 22.5 and 41.3 6 28.8 ng/mL, respectively). However, choroidal thickness was not correlated with WIF-1 levels.CONCLUSIONS. We report, for the first time, the possibility of phenotypic, anatomic, and ocular proteomic correlations, demonstrating correlated WIF-1 and DKK-3 upregulation in nAMD patients' aqueous humor. Secreted WIF-1, reflecting the degree of retinal structure damage, may be a new biomarker for the retina's healthy and disease states.

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DKK-3 expression was elevated in the AH of DME patients and in high glucose-treated human Müller cells. The observation of increased DKK-3 expression levels in the AH of DME patients with prominent edema in the INL suggests that the area of INL thickening might correlate with the area of reactive responses by Müller cells in these patients.