Experimental eye research最新文献

Retinal neurodegenerative diseases, including hypertensive retinopathy, involve progressive damage to retinal neurons, leading to visual impairment. In this study, we investigated the pathological mechanisms underlying retinal neurodegeneration in spontaneously hypertensive rats (SHR), using Wistar Kyoto (WKY) rats as normotensive controls. We observed that SHR exhibited significantly higher blood pressure and decreased retinal thickness, indicating retinal neurodegeneration. Molecular tests including quantitative real-time polymerase chain reaction, immunoblot, and immunofluorescent staining showed elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α, apoptotic markers (Fas, FasL, caspase-8, active caspase-3, and cleaved poly (ADP-ribose) polymerase), and necroptotic markers (receptor-interacting protein kinase-1 and -3) in SHR retinas. Additionally, we found elevated transforming growth factor-β (TGF-β) levels in the retinal pigment epithelium (RPE) of SHR, with a decrease in lecithin retinol acyltransferase (LRAT), which regulates retinoid metabolism and photoreceptor health. In human RPE cells (ARPE-19), TGF-β administration suppressed mRNA and protein levels of LRAT; and vactosertib, a selective inhibitor of TGF-β receptor kinase type 1, reversed the effect of TGF-β. These findings suggest that hypertension-induced retinal neurodegeneration involves inflammation, apoptosis, necroptosis, and disrupted retinoid metabolism, providing potential therapeutic targets for hypertensive retinopathy.

The main symptom of acute glaucoma is acute ocular hypertension (AOH), which leads to the death of retinal ganglion cells (RGCs) and permanent loss of vision. However, effective treatments for these conditions are lacking. This study aimed to identify major regulators and overall protein changes involved in AOH-induced RGC death. Proteomic patterns of the retinal protein extracts from the AOH and sham groups were analyzed using mass spectrometry (MS), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.

Proteomic analysis revealed 92 proteins in the AOH group compared to the control group; 58 proteins were upregulated and 34 were downregulated. Alterations in fatty acid-binding protein 7 (FABP7) and caveolin-1 (Cav-1), which are related to fatty acid metabolism and ocular inflammatory signaling, were detected using western blotting and biochemical assays. Variations in the expression of galectin-1 (Gal-1), S100 calcium-binding protein A6 (S100a6), and visinin-like protein-1 (VILIP) have been associated with neuronal ischemia. Our investigation demonstrates that neuroinflammation and fatty acid metabolism are involved in retinal impairment following AOH, suggesting a possible treatment approach for acute glaucoma.

Ceramides with varying acyl-chain lengths can have unique biological actions and hence, cellular responses to ceramides may depend not on their overall concentration but on that of individual ceramide species. The purpose of this study was to determine individual ceramide species impacting retinal ganglion cell (RGC) loss under the ocular hypertensive condition.

Induced pluripotent stem cell (iPSC)-derived RGCs and primary cultures of human astrocytes were used to determine the effect of individual ceramide species on both RGC viability and astrocyte secretion of inflammatory cytokines in vitro. In in vivo experiments with wild-type (WT) and ceramide synthase 5 (CerS5) knockout mice, intraocular pressure was unilaterally elevated with microbead injection. Retinal function and morphology were evaluated using pattern electroretinography (pERG) and immunofluorescence, respectively. Ceramide levels were determined by LC-MS/MS analysis.

Exposure to C16:0-, C18:0-, C18:1-, C20:0- and C24:0-ceramides significantly reduces RGC viability in vitro, with the very long chain C24:0-ceramide being the most neurotoxic; treatment with C18:0-, C18:1- and C24:0-ceramides stimulates an increase of TNF-α secretion by astrocytes. The retinas of CerS5 KO mice have significantly reduced levels of C16:0- and C18:1-ceramides compared to WT; ocular hypertensive eyes of these mice maintain higher pERG amplitudes and RGC numbers compared to WT.

Individual ceramides with different chain lengths have different effects on RGCs and astrocytes. Our results demonstrate that suppressing C16:0- and C18:1-ceramide species effectively protects RGCs against ocular hypertensive injury. These results provide a basis for targeting specific ceramide species in the treatment of glaucoma.

Exposure to VEGF-A₁₆₅a over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A. In view of the clinical relevance of anti-VEGF therapy, we also studied whether blocking VEGF-A-driven signaling is sufficient to prevent barrier dysfunction induced by a combination of both growth factors. Although angiopoietin-2 stimulated proliferation of iBREC, the formed barrier was not weakened at a concentration of 3 nM: Cell index values remained high and expression or subcellular localization of claudin-1 and vascular endothelial cadherin, respectively, were not affected. Angiopoietin-2 enhanced the changes induced by VEGF-A₁₆₅a and this was more pronounced at lower concentrations of VEGF-A₁₆₅a. Specific inhibition of the VEGF receptors with tivozanib as well as interfering with binding of VEGF-A to its receptors with bevacizumab prevented the detrimental effects of the growth factors; dual binding of angiopoietin-2 and VEGF-A by faricimab was marginally more efficient. Uptake of extracellular angiopoietin-2 by iBREC can be efficiently prevented by addition of faricimab which is also internalized by the cells. Exposure of the cells to faricimab over several days stabilized their barrier, confirming that inhibition of VEGF-A signaling is not harmful to this cell type. Taken together, our results confirm the dominant role of VEGF-A₁₆₅a in processes resulting in increased permeability of retinal endothelial cells in which angiopoietin-2 might play a minor modulating role.

Oxidative stress-mediated retinal pigment epithelial (RPE) cell damage is associated with age-related macular degeneration (AMD). ST266 is the biological secretome produced by a novel population of amnion-derived multipotent progenitor cells. Herein, we investigated the effect of ST266 on RPE cell injury induced by hydroquinone (HQ), a cigarette smoke related oxidant, hydrogen peroxide (H₂O₂) and all-trans retinal (atRal), a pro-oxidant component of the retinoid cycle. We additionally investigated its effect on Müller cell injury induced by H₂O₂.

Cultured human RPE cells were pre-treated for 1 h in the presence or absence of MK-2206, a protein kinase B (Akt) inhibitor, then treated with varying concentrations of HQ, H₂O_2, or atRal for 1.5 h. Cultured human Müller cells (MIO-M1) were pre-treated for 1 h in the presence or absence of MK-2206, then treated with varying concentrations of H₂O₂ for 1.5 h. Media were then replaced with STM100 (control media into which the ST266 secretome proteins were collected) or ST266 at various times. Cell viability was determined with WST-1 reagent. Mitochondrial membrane potential (Δψm) was quantified by a fluorescence plate reader. The protein phosphorylation levels of Akt, glycogen synthase kinase 3 beta (GSK-3β), and p70 ribosomal S6 kinase (p70S6K) were measured by Western blot.

ST266 significantly improved RPE and MIO-M1 cell viability that was reduced by oxidant exposure and improved oxidant-disrupted Δψm. In both cell types, ST266 induced phosphorylation of Akt, GSK-3β, and p70S6K. MK-2206 significantly eliminated ST266-mediated protein phosphorylation of Akt, GSK-3β, and p70S6K and abolished the ST266-protective effect on cell viability. In conclusion, ST266 activates Akt, protects against oxidative stress-mediated cell injury in an Akt-dependent manner, and improves Δψm, suggesting a potential role for ST266 therapy in treating retinal diseases such as AMD.

Fuchs endothelial corneal dystrophy (FECD), a degenerative corneal condition, is characterized by the droplet-like accumulation of the extracellular matrix, known as guttae and progressive loss of corneal endothelial cells ultimately leading to visual distortion and glare. FECD can be influenced by environmental stressors and genetic conditions. However, the role of mitochondrial dysfunction for advancing FECD pathogenesis is not yet fully studied. Therefore, in the present study we sought to determine whether a combination of environmental stressors (ultraviolet-A (UVA) light and cigarette smoke condensate (CSC)) can induce mitochondrial dysfunction leading to FECD. We also investigated if MitoQ, a water-soluble antioxidant, can target mitochondrial dysfunction induced by UVA and CSC in human corneal endothelial cells mitigating FECD pathogenesis. We modeled the FECD by increasing exogenous oxidative stress with CSC (0.2%), UVA (25J/cm²) and a combination of UVA + CSC and performed a temporal analysis of their cellular and mitochondrial effects on HCEnC-21T immortalized cells in vitro before and after MitoQ (0.05 μM) treatment. Interestingly, we observed that a combination of UVA + CSC exposure increased mitochondrial ROS and fragmentation leading to a lower mitochondrial membrane potential and increased levels of cytochrome c release leading to apoptosis and cell death. MitoQ intervention successfully mitigated these effects and restored cell viability. The UVA + CSC model could be used to study stress induced mitochondrial dysfunction. Additionally, MitoQ can serve as a viable antioxidant in attenuating mitochondrial dysfunction, underscoring its potential as a molecular-focused treatment approach to combat FECD pathogenesis.

The purpose of the experimental interventional study was to examine the influence of intraocularly applied amphiregulin, a member of the epidermal growth factor (EGF) family, on axial length in young non-human primates. It included three non-human primates (Macaca mulatta), aged 4–6 years. The left eyes received three intravitreal injections of amphiregulin (400ng/50 μl) in intervals of 4 weeks, while the right eyes received three intravitreal injections of phosphate buffered solution (50 μl) at the same time points. Ocular biometry was performed in weekly intervals. At baseline, the left eyes (study eyes) were shorter than the right (control) eyes (20.69 ± 0.21 mm versus 20.79 ± 0.24 mm; P < 0.001), with an inter-eye axial length (AL) difference (left minus right eye) of −0.10 ± 0.23 mm. Inter-eye AL difference increased (P < 0.001) to 0.15 ± 0.18 mm at study end, at 12 weeks after baseline. Axial elongation during the study was higher (P < 0.001) in the left eyes (20.69 ± 0.21 mm to 21.05 ± 0.29 mm or 0.36 ± 0.30 mm) than in the right eyes (20.79 ± 0.24 mm to 20.90 ± 0.31 mm or 0.11 ± 0.17 mm). In a parallel manner, inter-eye difference in vitreous cavity depth combined with lens thickness (left eye minus right eye) increased from −0.04 ± 0.17 mm at baseline to −0.02 ± 0.21 mm (P = 0.02), 0.04 ± 0.10 mm (P = 0.002), and to 0.42 ± 0.67 mm (P < 0.001) at 5, 6, and 12 weeks after baseline, respectively. The results suggest that intravitreally applied amphiregulin as EGF family member led to an increase in axial length in adolescent non-human primates. It supports the hypothesis of amphiregulin as EGF family member being involved in the process of axial elongation.

Vascular endothelial growth factor (VEGF) signaling is crucial for choroidal neovascularization (CNV), a major pathological feature of neovascular age-related macular degeneration (nAMD). Gene transcription of VEGF is mainly regulated by hypoxia-inducible factor 1-alpha (HIF-1α). The chromobox (CBX) family polycomb protein (Pc) subgroup includes CBX2, CBX4, CBX6, CBX7, and CBX8. CBX4 enhances hypoxia-induced VEGF expression and angiogenesis in hepatocellular carcinoma (HCC) cells by increasing HIF-1α′s transcriptional activity. The objective of the study was to examine the functions of members of the CBX family Pc subgroup in choroidal vascular endothelial cells (CVECs) during CNV. CBX4 and CBX7 expression was up-regulated in hypoxic human choroidal vascular endothelial cells (HCVECs). In HCVECs, CBX7 facilitated HIF-1α transcription and expression, while CBX4 did not. In HCVECs, CBX7 stimulated HIF-1α′s nuclear translocation and transcriptional activity, which in turn stimulated VEGF transcription and expression. The CBX7/HIF-1α/VEGF pathway promoted the migration, proliferation, and tube formation of HCVECs. The CBX7/HIF-1α/VEGF pathway was up-regulated in CVECs and in the mouse model with laser-induced CNV. Mouse CNV was lessened by the blockade of CBX7 through the down-regulation of HIF-1α/VEGF. In conclusion, CBX7 enhanced pro-angiogenic behaviors of hypoxic CVECs by up-regulating the HIF-1α/VEGF pathway, which contributing to the formation of mouse laser-induced CNV.

Current research using animal models to investigate retinal cell biology and model retinal degenerative diseases largely utilize small mammals that are nocturnal and lack the ability to restore lost vision. In contrast, the Mongolian gerbil (Meriones) is a diurnal rodent with good photopic vision, and the spiny mouse (Acomys) is a small desert-dwelling rodent with remarkable regenerative capabilities. The goal of this study was to identify antibodies that detect retinal cell classes in Meriones and Acomys, and to describe the retinal anatomy of these two species in comparison to outbred laboratory mice (Mus musculus). Immunohistochemistry was performed on retinal sections with antibodies for various retinal cell types. Sections were imaged by light, fluorescence, and confocal microscopy. Cell density, morphology, and placement were compared between species qualitatively and quantitatively. Our analyses revealed a classic assembly of retinal cells in Meriones and Acomys, with a few deviations compared to Mus. Meriones displayed the highest density of cones and Acomys the lowest. A higher density of bipolar cell bodies in the proximal portion of the inner nuclear layer was observed in both Acomys and Meriones compared to Mus, and both species exhibited an increase in amacrine cell density compared to Mus. Our results provide a foundation for future research into the visual system adaptations of these interesting species.