A 3D in vitro model for assessing the influence of intraocular lens: Posterior lens capsule interactions on lens epithelial cell responses.

Posterior Capsule Opacification (PCO), the most frequent complication of cataract surgery, is caused by the infiltration and proliferation of lens epithelial cells (LECs) at the interface between the intraocular lens (IOL) and posterior lens capsule (PLC). According to the "no space, no cells, no PCO" theory, high affinity (or adhesion force) between the IOL and PLC would decrease the IOL: PLC interface space, hinder LEC migration, and thus reduce PCO formation. To test this hypothesis, an in vitro hemisphere-shaped simulated PLC (sPLC) was made to mimic the human IOL: PLC physical interactions and to assess their influence on LEC responses. Three commercially available IOLs with different affinities/adhesion forces toward the sPLC, including Acrylic foldable IOL, Silicone IOL, and PMMA IOL, were used in this investigation. Using the system, the physical interactions between IOLs and sPLC were quantified by measuring the adhesion force and interface space using an adhesion force apparatus and Optical Coherence Tomography, respectively. Our data shows that high adhesion force and tight binding between IOL and sPLC contribute to a small interface space (or "no space"). By introducing LECs into the in vitro system, we found that, with small interface space, among all IOLs, acrylic foldable IOLs permitted the least extent of LEC infiltration, proliferation, and differentiation (or "no cells"). Further statistical analyses using clinical data revealed that weak LEC responses are associated with low clinical PCO incidence rates (or "no PCO"). The findings support that the in vitro system could simulate IOL: PLC interplays and predict IOLs' PCO potential in support of the "no space, no cells, no PCO" hypothesis.

ELOVL4 Mutations That Cause Spinocerebellar Ataxia-34 Differentially Alter Very Long Chain Fatty Acid Biosynthesis.

The FA Elongase-4 (ELOVL4) enzyme mediates biosynthesis of both very long chain (VLC)-PUFAs and VLC-saturated FA (VLC-SFAs). VLC-PUFAs play critical roles in retina and sperm function, whereas VLC-SFAs are predominantly associated with brain function and maintenance of the skin permeability barrier. While some ELOVL4 mutations cause Autosomal Dominant Stargardt-like Macular Dystrophy (STGD3), other ELOVL4 point mutations, such as L168F and W246G, affect the brain and/or skin, leading to Spinocerebellar Ataxia-34 (SCA34) and Erythrokeratodermia variabilis. The mechanisms by which these ELOVL4 mutations alter VLC-PUFA and VLC-SFA biosynthesis to cause the different tissue-specific pathologies are not well understood. To understand how these mutations alter VLC-PUFA and VLC-SFA biosynthesis, we expressed WT-ELOVL4, L168F, and W246G ELOVL4 variants in cell culture and supplemented the cultures with VLC-PUFA or VLC-SFA precursors. Total lipids were extracted, converted to FA methyl esters, and quantified by gas chromatography. We showed that L168F and W246G mutants were capable of VLC-PUFA biosynthesis. W246G synthesized and accumulated 32:6n3, while L168F exhibited gain of function in VLC-PUFA biosynthesis as it made 38:5n3, which we did not detect in WT-ELOVL4 or W246G-expressing cells. However, compared with WT-ELOVL4, both L168F and W246G mutants were deficient in VLC-SFA biosynthesis, especially the W246G protein, which showed negligible VLC-SFA biosynthesis. These results suggest VLC-PUFA biosynthetic capabilities of L168F and W246G in the retina, which may explain the lack of retinal phenotype in SCA34. Defects in VLC-SFA biosynthesis by these variants may be a contributing factor to the pathogenic mechanism of SCA34 and Erythrokeratodermia variabilis.

Novel Regulators of Retina Neovascularization: A Proteomics Approach.

The purpose of this study was to identify proteins that regulate vascular remodeling in an ROP mouse model. Pups were subjected to fluctuating oxygen levels and retinas sampled during vessel regression (PN12) or neovascularization (PN17) for comparative SWATH-MS proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed a human retinal endothelial cell (HREC) ROP correlate to validate the expression of retina neovascular-specific markers. A total of 5191 proteins were identified in OIR retinas with 498 significantly regulated in elevated oxygen and 345 after a return to normoxia. A total of 122 proteins were uniquely regulated during vessel regression and 69 during neovascularization (FC ≥ 1.5; p ≤ 0.05), with several validated by western blot analyses. Expressions of 56/69 neovascular-specific proteins were confirmed in hypoxic HRECs with 23 regulated in the same direction as OIR neovascular retinas. These proteins control angiogenesis-related processes including matrix remodeling, cell migration, adhesion, and proliferation. RNAi and transfection overexpression studies confirmed that VASP and ECH1, showing the highest levels in hypoxic HRECs, promoted human umbilical vein (HUVEC) and HREC cell proliferation, while SNX1 and CD109, showing the lowest levels, inhibited their proliferation. These proteins are potential biomarkers and exploitable intervention tools for vascular-related disorders. The proteomics data set generated has been deposited to the ProteomeXchange/iProX Consortium with the Identifier:PXD029208.

Proteomic Analysis of Plasma sEVs Reveals That TNFAIP8 Is a New Biomarker of Cell Proliferation in Diabetic Retinopathy.

Small extracellular vesicles (sEVs) derived from the plasma have been increasingly recognized as important vehicles of intercellular communication and potential sources of new biomarkers for multiple diseases. In this study, proteomic profiles of plasma sEVs from normal subjects and diabetic patients with or without diabetic retinopathy (DR) were systematically compared using iTRAQ-based quantitative proteomics. Among a total of 901 identified proteins in plasma sEVs (false discovery rate (FDR) < 1%), 90 proteins were found to have significantly changed levels in DR. Based on the findings from the proteomic analysis, the role of tumor necrosis factor-α-induced protein 8 (TNFAIP8) in promoting human retinal microvascular endothelial cell (HRMEC) proliferation was investigated. The enzyme-linked immunosorbent assay (ELISA) showed that TNFAIP8 levels in plasma sEVs and vitreous are elevated in DR, whereas not statistically different in large EVs (lEVs) and plasma. In addition, in vitro experiments demonstrated that 4-hydroxynonenal (4-HNE) increased the expression of TNFAIP8 in HRMECs. TNFAIP8 significantly increased HRMECs cell viability and promote cell migration and tube formation, and the depletion of TNFAIP8 impaired HRMEC proliferation. We demonstrated that TNFAIP8 in plasma sEVs could be used as a potential biomarker of DR. Functional studies suggested that TNFAIP8 might be an important mediator of angiogenesis in DR.

Proteomics Profiling of Plasma Exosomes in VKH Patients.

OBJECTIVES: Vogt-Koyanagi-Harada syndrome is common autoimmune uveitis that can cause blindness. Recent studies have shown that plasma exosomes carry disease-related proteins that may serve as biomarkers. Here, we aimed to find candidate biomarkers of Vogt-Koyanagi-Harada disease using proteomic analysis of plasma exosomes. METHODS: Exosomes were isolated from the plasma of normal controls and Vogt- Koyanagi-Harada patients in the following groups: a) initial inflammatory attack (active stage), b) remission after one month of treatment (unstable stage), and c) stationary phase after three months of treatment (stable stage). Groups were analyzed by mass spectrometry using isobaric tags for relative and absolute quantitation. After functional analysis, proteins of interest were verified by ELISA. RESULTS: 463 proteins were identified in the exosomes. Forty-three were upregulated at the active inflammation stage, including inflammation-associated proteins. Thirty-one were downregulated. Gene ontology and pathway analyses revealed differential proteins related to cell adhesion, cell phagocytosis, cytoskeleton movement, leukocyte migration across endothelial cells, and platelet activation. By ELISA, Carbonic anhydrase 2 and Ras-related protein Rap-1b were verified as more plentiful at the active stage compared to the normal control and stationary phase in exosomes, but not, however, in microvesicles or plasma. CONCLUSION: Plasma exosomes of Vogt-Koyanagi-Harada patients contain many proteins related to the degree of inflammation. The levels of Carbonic anhydrase 2 and Ras-related protein Rap-1b in exosomes can be used as biomarkers for active inflammation in Vogt-Koyanagi-Harada disease. Further investigation could help study the pathogenesis of Vogt-Koyanagi-Harada disease and identify therapeutic targets.

Blue light-triggered optogenetic system for treating uveal melanoma.

Uveal melanoma is the most common intraocular primary malignancy in adults and has been considered a fatal disease for decades. Optogenetics is an emerging technique that can control the activation of signaling components via irradiation with visible light. The clinical translation of optogenetics has been limited because of the need for surgical implantation of electrodes and relatively shallow tissue penetration. As visible light easily penetrates the eyes, we hypothesized that an optogenetics approach can be an effective treatment of uveal melanoma without surgery. In this study, we evaluated the feasibility of this strategy by using a genetically encoded optogenetic system based on reversible blue light-induced binding pairs between Fas-CIB1-EGFP and CRY2-mCherry-FADD. Subretinal injection of B16 cells was performed to create a uveal melanoma model. Plasmids pairs were co-transfected into B16 cells. We found that blue light irradiation dynamically controlled the translocation of FADD to Fas on the plasma membrane and induced the apoptosis of B16 cells transfected with the optogenetic nanosystem in vitro. Moreover, the blue light-controlled optogenetic nanosystem suppressed the growth of uveal melanoma in vivo by inducing apoptosis. These results suggest that light-controlled optogenetic therapy can be used as a potential novel therapeutic strategy for uveal melanoma.

Recent advances of exosomes in immune-mediated eye diseases.

Exosomes, nanosized extracellular vesicles of 30-150 nm, are shed by almost all cell types. Bearing proteins, lipids, RNAs, and DNAs, exosomes have emerged as vital biological mediators in cell-to-cell communication, affecting a plethora of physiological and pathological processes. Particularly, mounting evidence indicates that immunologically active exosomes can regulate both innate and adaptive immune responses. Herein, we review recent advances in the research of exosomes in several immune-mediated eye diseases, including Sjögren's syndrome (SS) dry eye, corneal allograft rejection, autoimmune uveitis, and age-related macular degeneration (AMD). Additionally, we discuss the potential of exosomes as novel biomarkers and drug delivery vesicles for the diagnosis and treatment of eye diseases.