Proteopathy Linked to Exon-Skipping Isoform of RGR-Opsin Contributes to the Pathogenesis of Age-Related Macular Degeneration.

Purpose: Proteopathy is believed to contribute to age-related macular degeneration (AMD). Much research indicates that AMD begins in the retinal pigment epithelium (RPE), which is associated with formation of extracellular drusen, a clinical hallmark of AMD. Human RPE produces a drusen-associated abnormal protein, the exon Ⅵ-skipping splice isoform of retinal G protein-coupled receptor (RGR-d). In this study, we investigate the detrimental effects of RGR-d on cultured cells and mouse retina. Methods: ARPE-19 cells were stably infected by lentivirus overexpressing RGR or RGR-d and were treated with MG132, sometimes combined with or without endoplasmic reticulum (ER) stress inducer, tunicamycin. RGR and RGR-d protein expression, degeneration pathway, and potential cytotoxicity were explored. Homozygous RGR-d mice aged 8 or 14 months were fed with a high-fat diet for 3 months and then subjected to ocular examination and histopathology experiments. Results: We confirm that RGR-d is proteotoxic under various conditions. In ARPE-19 cells, RGR-d is misfolded and almost completely degraded via the ubiquitin-proteasome system. Unlike normal RGR, RGR-d increases ER stress, triggers the unfolded protein response, and exerts potent cytotoxicity. Aged RGR-d mice manifest disrupted RPE cell integrity, apoptotic photoreceptors, choroidal deposition of complement C3, and CD86+CD32+ proinflammatory cell infiltration into retina and RPE-choroid. Furthermore, the AMD-like phenotype of RGR-d mice can be aggravated by a high-fat diet. Conclusions: Our study confirmed the pathogenicity of the RGR splice isoform and corroborated a significant role of proteopathy in AMD. These findings may contribute to greater comprehension of the multifactorial causes of AMD.

The continuous treatment of anterior segment open globe injury: an eye injury vitrectomy study.

Background: The prognostic significance of debridement has long been demonstrated for trauma in tissues other than ocular. Unfortunately, the impact of wound healing in the anterior segment (AS) was not paid as much attention as in the posterior segment (PS). This study aims to evaluate whether a better prognosis can be obtained from continuous surgical treatment (CST) before fibrosis or scar formation in an open AS injury. Methods: In this prospective comparative cohort study, 19 eyes of 19 patients with an experience of AS open globe injury (OGI) were selected from the database of the eye injury vitrectomy study (EIVS) from January 1, 2020 to July 31, 2021. Of 19 patients, 9 who received CST were assigned to group 1, and 10 patients without CST after the initial wound repair were included in group 2. Comparison between the two groups was conducted in the final best corrected visual acuity (BCVA). Significant AS complications after injury were evaluated with χ2 test. The corneal leucoma area ratio, astigmatism, and the score of AS abnormalities were analyzed using the Student's t-test. Results: The differences of baseline clinical factors between the two groups were not statistically significant. The final BCVA was better in group 1 than in group 2 (P=0.011). The complications directly caused by AS injury, namely adhesive corneal leucoma, uneven anterior chamber, block of light passing through the pupil, and fibrosis or scarring, were more frequent in group 2 than in group 1 (P=0.011, 0.022, 0.037, and 0.040, respectively). Secondary glaucoma (3 cases) and severe AS structure destruction (2 cases) occurred only in group 2 (P=0.037 and 0.474, respectively). The area ratio of leucoma (0.79±0.44, 0.82±0.50, respectively) and corneal astigmatism (3.69±1.90, 4.50±4.80, respectively) revealed no statistical significance between the two groups. On the other hand, the score of AS abnormalities, mean values being 93.33±11.18 for group 1 and 67.00±29.46 for group 2, was statistically different (P=0.022). Conclusions: Initiating CST before fibrosis or scar formation might improve the prognosis of open AS injury, which was preferable to natural wound healing after wound repair.

Distinct Role of Lycium barbarum L. Polysaccharides in Oxidative Stress-Related Ocular Diseases.

Oxidative stress is an imbalance between the increased production of reactive species and reduced antioxidant activity, which can cause a variety of disturbances including ocular diseases. Lycium barbarum polysaccharides (LBPs) are complex polysaccharides isolated from the fruit of L. barbarum, showing distinct roles in antioxidants. Moreover, it is relatively safe and non-toxic. In recent years, the antioxidant activities of LBPs have attracted remarkable attention. In order to illustrate its significance and underlying therapeutic value for vision, we comprehensively review the recent progress on the antioxidant mechanisms of LBP and its potential applications in ocular diseases, including diabetic retinopathy, hypertensive neuroretinopathy, age-related macular degeneration, retinitis pigmentosa, retinal ischemia/reperfusion injury, glaucoma, dry eye syndrome, and diabetic cataract.

The essential role of N6-methyladenosine RNA methylation in complex eye diseases.

There are many complex eye diseases which are the leading causes of blindness, however, the pathogenesis of the complex eye diseases is not fully understood, especially the underlying molecular mechanisms of N6-methyladenosine (m6A) RNA methylation in the eye diseases have not been extensive clarified. Our review summarizes the latest advances in the studies of m6A modification in the pathogenesis of the complex eye diseases, including cornea disease, cataract, diabetic retinopathy, age-related macular degeneration, proliferative vitreoretinopathy, Graves' disease, uveal melanoma, retinoblastoma, and traumatic optic neuropathy. We further discuss the possibility of developing m6A modification signatures as biomarkers for the diagnosis of the eye diseases, as well as potential therapeutic approaches.

Potential Impact of N6-Methyladenosine RNA Methylation on Vision Function and the Pathological Processes of Ocular Diseases: New Discoveries and Future Perspectives.

N6-methyladenosine (m6A) methylation/modification plays a critical role in various biological processes through post-transcriptional ribonucleic acid (RNA) modification, which involves RNA processing, nuclear export, translation and decay. Functionally, m6A modification may be involved in ocular cell growth and differentiation, stem cell identity, development, haemostasis and innate versus adaptive immunity. Aberrations in m6A methylation may mediate numerous pathological conditions in the eye, including microorganism infection, inflammation, autoimmune disease, senescence, degeneration, epithelial-mesenchymal transition, fibrosis, angiogenesis, tumorigenesis and complex eye diseases. In this review, we have discussed the relevance of m6A modification to precision medicine, stem cell directional differentiation, biomarkers of eye diseases and m6A methylation activators and inhibitors. In addition, we summarised the challenges and future research directions in the field related to visual function and eye diseases.

Endoplasmic reticulum stress as a novel target to inhibit transdifferentiation of human retinal pigment epithelial cells.

BACKGROUND: The epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical event in the pathogenesis of proliferative vitreoretinopathy and neovascular age-related macular degeneration, which are the leading causes of severe vision loss. Endoplasmic reticulum (ER) stress has been implicated in the EMT of many cell types and various ocular diseases. However, the relationship between ER stress and EMT in RPE cells remains unknown. Therefore, in the study, we explored the impact of ER stress on EMT in RPE cells. METHODS: Different concentrations of tunicamycin (TM) and thapsigargin (TG) were used to induce ER stress in human RPE cells. The expression of epithelial marker, mesenchymal markers and some of genes/proteins involved in TGF-ß/Smad signaling were analized by qPCR, western blot or immunostaining at the condition with or without stimulation of TGF-ß2 (10 ng/mL). Boyden chamber and scratch assay were used to evaluate the migration of RPE cells, while cell viability and apoptosis of RPE cells were measured by MTT and TUNEL assay, respectively. RESULTS: Treatment of RPE cells with TM and TG (24 h) reduced the expression of α -SMA and FN, and increased the expression of Occludin in a dose dependent manner at protein level, which was highly associated with the expression of GRP78. Treatment with TGF-ß2 significantly increased the expression of α-SMA and FN, and decreased the expression of Occludin both in protein and mRNA levels, which was significantly inhibited by a 4h pre-treatment with TM. In addition, the expression of TGF-ßRII and Smad2/3, and mRNAs of TGF-ßRII and Smad3 were also decreased by the TM treatment. TM-induced ER stress inhibited RPE cell migration, and high concentrations of TM and TG reduced cell viability and induced apoptosis of RPE cells. CONCLUSIONS: Chemical induction of ER stress inhibited EMT and migration in RPE cells, possibly by inactivation of TGF-ß signaling, suggesting that regulation of ER stress in RPE cells may be a new approach to prevent the development of intraocular fibrosis.

Essential Role of Multi-Omics Approaches in the Study of Retinal Vascular Diseases.

Retinal vascular disease is a highly prevalent vision-threatening ocular disease in the global population; however, its exact mechanism remains unclear. The expansion of omics technologies has revolutionized a new medical research methodology that combines multiple omics data derived from the same patients to generate multi-dimensional and multi-evidence-supported holistic inferences, providing unprecedented opportunities to elucidate the information flow of complex multi-factorial diseases. In this review, we summarize the applications of multi-omics technology to further elucidate the pathogenesis and complex molecular mechanisms underlying retinal vascular diseases. Moreover, we proposed multi-omics-based biomarker and therapeutic strategy discovery methodologies to optimize clinical and basic medicinal research approaches to retinal vascular diseases. Finally, the opportunities, current challenges, and future prospects of multi-omics analyses in retinal vascular disease studies are discussed in detail.

Human RGR Gene and Associated Features of Age-Related Macular Degeneration in Models of Retina-Choriocapillaris Atrophy.

Age-related macular degeneration (AMD) is a progressive eye disease and the most common cause of blindness among the elderly. AMD is characterized by early atrophy of the choriocapillaris and retinal pigment epithelium (RPE). Although AMD is a multifactorial disease with many environmental and genetic risk factors, a hallmark of the disease is the origination of extracellular deposits, or drusen, between the RPE and Bruch membrane. Human retinal G-protein-coupled receptor (RGR) gene generates an exon-skipping splice variant of RGR-opsin (RGR-d; NP_001012740) that is a persistent component of small and large drusen. Herein, the findings show that abnormal RGR proteins, including RGR-d, are pathogenic in an animal retina with degeneration of the choriocapillaris, RPE, and photoreceptors. A frameshift truncating mutation resulted in severe retinal degeneration with a continuous band of basal deposits along the Bruch membrane. RGR-d produced less severe disease with choriocapillaris and RPE atrophy, including focal accumulation of abnormal RGR-d protein at the basal boundary of the RPE. Degeneration of the choriocapillaris was marked by a decrease in endothelial CD31 protein and choriocapillaris breakdown at the ultrastructural level. Fundus lesions with patchy depigmentation were characteristic of old RGR-d mice. RGR-d was mislocalized in cultured cells and caused a strong cell growth defect. These results uphold the notion of a potential hidden link between AMD and a high-frequency RGR allele.

Microwave resonances of magnetic skyrmions in thin film multilayers.

Non-collinear magnets exhibit a rich array of dynamic properties at microwave frequencies. They can host nanometre-scale topological textures known as skyrmions, whose spin resonances are expected to be highly sensitive to their local magnetic environment. Here, we report a magnetic resonance study of an [Ir/Fe/Co/Pt] multilayer hosting Néel skyrmions at room temperature. Experiments reveal two distinct resonances of the skyrmion phase during in-plane ac excitation, with frequencies between 6-12 GHz. Complementary micromagnetic simulations indicate that the net magnetic dipole moment rotates counterclockwise (CCW) during both resonances. The magnon probability distribution for the lower-frequency resonance is localised within isolated skyrmions, unlike the higher-frequency mode which principally originates from areas between skyrmions. However, the properties of both modes depend sensitively on the out-of-plane dipolar coupling, which is controlled via the ferromagnetic layer spacing in our heterostructures. The gyrations of stable isolated skyrmions reported in this room temperature study encourage the development of new material platforms and applications based on skyrmion resonances. Moreover, our material architecture enables the resonance spectra to be tuned, thus extending the functionality of such applications over a broadband frequency range.

Inhibition of laser induced rats choroidal neovascularization by intravitreous injection of sEphB4-HSA.

BACKGROUND: Choroidal neovascularization (CNV) is a leading cause of central vision loss complicated with age-related macular degeneration. Although intravitreal anti-VEGF therapy is widely used in wet age-related macular degeneration, optimal treatment regimens for the disease are still under investigation. EphrinB2 and EphB4 regulate angiogenesis, and interruption of EphB4/ephrinB2 has been demonstrated to inhibit angiogenesis. In the current study, we studied the effects of soluble EphB4 (sEphB4) on laser induced CNV in a rat model by intravitreous injection and the underlying mechanism. METHODS: Male rats (Brown-Norway) were used in the study. CNV was induced by laser and the sEphB4 was injected intravitreous after laser at days 3 and 7. The CNV lesions were evaluated by three methods: fluorescein angiography (FA) in vivo, CNV volume by confocal analysis of choroidal flat-mounts and H&E staining. The expression of fibronectin (FN), VEGFR-2, phospho-VEGFR-2 (pVEGFR-2), the double labeling of EphB4 with FN was analyzed by immunofluorescence. The interaction of FN with EphB4 and the effects of intraocular injection of sEphB4 on the inhibition of pVEGFR-2 were determined by western blot. RESULTS: The FA leakage and CNV volume were significantly inhibited by the injection of the sEphB4. Further, histology analysis showed that CNV lesion was significantly smaller in the rats with sEphB4 injection than rats with placebo application. The expressions of pVEGFR-2 and FN in the CNV lesions were reduced compared with controls. CONCLUSIONS: Our study suggests that the inhibition of CNV by sEphB4 may be through suppression of VEGFR-2 phosphorylation and the expression of FN. sEphB4 may be a new potential therapeutic strategy of CNV.

The critical role of m6A methylation in the pathogenesis of Graves' ophthalmopathy.

PURPOSE: To investigate the role of N6-methyladenosine (m6A) RNA modification in the pathogenesis of Graves' ophthalmopathy (GO). METHODS: Surgically excised extraocular muscles from 7 patients with GO and 5 subjects without GO were used. The global m6A levels in the specimens were determined using an m6A RNA methylation quantification kit. RNA sequencing (RNA-seq) was used to analyze the molecules involved in the regulation of m6A RNA methylation and the differential expression of mRNAs between the two groups (4 eyes, respectively). The expression of m6A RNA modification genes was evaluated by real-time PCR. The functional implications of the gene alterations between the GO and control specimens were determined by Gene Ontology analysis. RESULTS: The m6A level was significantly increased in the specimens of GO patients compared to the control specimens (P < 0.05). The expression of m6A methylation regulators, such as WT1 associated protein (WTAP), alkylation repair homolog protein 5 (ALKBH5), E74 like ETS transcription factor 3 (ELF3), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), YTHDF3 and YTH domain containing 2 (YTHDC2), was significantly upregulated (P < 0.05). Gene Ontology enrichment analysis showed that the most highly upregulated genes and biological pathways were related to the immune response and inflammatory processes such as lymphocyte activation, leukocyte differentiation, cytokine production and cytokine-mediated signaling pathways. CONCLUSIONS: Our results suggest that m6A methylation may play a critical role in the pathogenesis of GO and that targeting genes that regulate m6A methylation may provide a new therapeutic approach for GO.

An Updated Review of the Epigenetic Mechanism Underlying the Pathogenesis of Age-related Macular Degeneration.

Epigenetics has been recognized to play an important role in physiological and pathological processes of the human body. Accumulating evidence has indicated that epigenetic mechanisms contribute to the pathogenesis of age-related macular degeneration (AMD). Although the susceptibility related to genetic variants has been revealed by genome-wide association studies, those genetic variants may predict AMD risk only in certain human populations. Other mechanisms, particularly those involving epigenetic factors, may play an important role in the pathogenesis of AMD. Therefore, we briefly summarize the most recent reports related to such epigenetic mechanisms, including DNA methylation, histone modification, and non-coding RNA, and the interplay of genetic and epigenetic factors in the pathogenesis of AMD.

MeCP2-421-mediated RPE epithelial-mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial-mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl-CpG-binding protein 2 (MeCP2), especially P-MeCP2-421 in the pathogenesis of PVR. The expressions of P-MeCP2-421, P-MeCP2-80, PPAR-γ and the double labelling of P-MeCP2-421 with α-SMA, cytokeratin, TGF-ß and PPAR-γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α-SMA, phospho-Smad2/3, collagen I, fibronectin and PPAR-γ; the expression of α-SMA stimulated by recombinant MeCP2 in ARPE-19; and the effect of TGF-ß and 5-AZA treatment on PPAR-γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF-ß. Our results showed that P-MeCP2-421 was highly expressed in PVR membranes and was double labelled with α-SMA, cytokeratin and TGF-ß, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF-ß. TGF-ß inhibited the expression of PPAR-γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5-AZA) increased the expression of PPAR-γ. α-SMA was up-regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF-ß as demonstrated by Chip assay. The results suggest that MeCP2 especially P-MeCP2-421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.

The critical role of the interplays of EphrinB2/EphB4 and VEGF in the induction of angiogenesis.

The significant role of VEGF (vascular endothelial growth factor) as an angiogenesis inducer is well recognized. Besides VEGF, EphrinB2/EphB4 also plays essential roles in vascular development and postnatal angiogenesis. Compared with classical proangiogenic factors, not only does EphrinB2/EphB4 promote sprouting of new vessels, it is also involved in the vessel maturation. Given their involvement in many physiologic and pathological conditions, EphB4 and EphrinB2 are increasingly recognized as attractive therapeutic targets for angiogenesis-related diseases through modulating their expression and function. Previous works mainly focused on the individual role of VEGF and EphrinB2/EphB4 in angiogenesis, respectively, but the correlation between EphrinB2/EphB4 and VEGF in angiogenesis has not been fully disclosed. Here, we summarize the structure and bidirectional signaling of EphrinB2/EphB4, provide an overview on the relationship between EphrinB2/EphB4 signaling and VEGF pathway in angiogenesis and highlight the associated potential usefulness in anti-angiogenetic therapy.

Autophagy and autophagy-related proteins in cancer.

Autophagy, as a type II programmed cell death, plays crucial roles with autophagy-related (ATG) proteins in cancer. Up to now, the dual role of autophagy both in cancer progression and inhibition remains controversial, in which the numerous ATG proteins and their core complexes including ULK1/2 kinase core complex, autophagy-specific class III PI3K complex, ATG9A trafficking system, ATG12 and LC3 ubiquitin-like conjugation systems, give multiple activities of autophagy pathway and are involved in autophagy initiation, nucleation, elongation, maturation, fusion and degradation. Autophagy plays a dynamic tumor-suppressive or tumor-promoting role in different contexts and stages of cancer development. In the early tumorigenesis, autophagy, as a survival pathway and quality-control mechanism, prevents tumor initiation and suppresses cancer progression. Once the tumors progress to late stage and are established and subjected to the environmental stresses, autophagy, as a dynamic degradation and recycling system, contributes to the survival and growth of the established tumors and promotes aggressiveness of the cancers by facilitating metastasis. This indicates that regulation of autophagy can be used as effective interventional strategies for cancer therapy.

Current-induced magnetization switching in all-oxide heterostructures.

The electrical switching of magnetization through spin-orbit torque (SOT)1 holds promise for application in information technologies, such as low-power, non-volatile magnetic memory. Materials with strong spin-orbit coupling, such as heavy metals2-4 and topological insulators5,6, can convert a charge current into a spin current. The spin current can then execute a transfer torque on the magnetization of a neighbouring magnetic layer, usually a ferromagnetic metal like CoFeB, and reverse its magnetization. Here, we combine a ferromagnetic transition metal oxide7 with an oxide with strong spin-orbit coupling8 to demonstrate all-oxide SOT devices. We show current-induced magnetization switching in SrIrO3/SrRuO3 bilayer structures. By controlling the magnetocrystalline anisotropy of SrRuO3 on (001)- and (110)-oriented SrTiO3 (STO) substrates, we designed two types of SOT switching schemes. For the bilayer on the STO(001) substrate, a magnetic-field-free switching was achieved, which remained undisturbed even when the external magnetic field reached 100 mT. The charge-to-spin conversion efficiency for the bilayer on the STO(110) substrate ranged from 0.58 to 0.86, depending on the directionality of the current flow with respect to the crystalline symmetry. All-oxide SOT structures may help to realize field-free switching through a magnetocrystalline anisotropy design.

Angle-resolved broadband ferromagnetic resonance apparatus enabled through a spring-loaded sample mounting manipulator.

Broadband ferromagnetic resonance is a useful technique to determine the magnetic anisotropy and study the magnetization dynamics of magnetic thin films. We report a spring-loaded sample loading manipulator for reliable sample mounting and rotation. The manipulator enables maximum signal, enhances system stability, and is particularly useful for fully automated in-plane-field angle-resolved measurements. This angle-resolved broadband ferromagnetic resonance apparatus provides a viable method to study anisotropic damping and weak magnetic anisotropies, both vital for fundamental research and applications.

Histone deacetylase inhibitor attenuates experimental fungal keratitis in mice.

Fungal keratitis is one of the leading causes of blindness of infected corneal diseases, but the pathogenesis of fungal keratitis is not fully understood and therefore the treatment of the disease by medication is still under investigation. In the current study, we sought to study the effect of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on experimental fungal keratitis in mice. SAHA (25 mg/kg) (n = 30) or vehicle (DMSO) (n = 30) was delivered through intraperitoneal injection (IP) 24 hours after the fungal inoculation, and the same amount of SAHA injection or DMSO was followed at day 2. The expression of histone H3 (H3), acetylated histone H3 (AC-H3), histone deacetylase 1 (HDAC)1, tumor necrosis factor-α (TNFα), and Toll-like receptor 4 (TLR4) in surgically excised specimens from the patients and mice with fungal keratitis were detected by immunohistochemistry. The expression of mRNAs for Interleukin-1ß (IL-1ß), TNFα, and TLR4 were evaluated in the corneas of the mice with fungal infection and the control corneas by real-time PCR. The quantification of IL-1ß and TNFα in the corneas of the mice with fungal infection was determined by ELISA. The inhibitory effect of SAHA on mice fungal keratitis was revealed by GMS and H&E staining. We found that the downregulation of histone acetylation and upregulation of HDAC1 expression were associated with the increased inflammation response in fungal keratitis not only in humans but also in experimental animals. SAHA was able to inhibit experimental fungal keratitis in mouse by suppressing TLR4 and inflammatory cytokines such as TNFα and IL-1ß; the inhibition of HDAC may be a potential therapeutic approach for the treatment of fungal keratitis.

Emergence of Topological Hall Effect in a SrRuO3 Single Layer.

Topological Hall effect (THE), appearing as bumps and/or dips in the Hall resistance curves, is considered as a hallmark of the skyrmion spin texture originated from the inversion symmetry breaking and spin-orbit interaction. Recently, Néel-type skyrmion is proposed based on the observed THE in 5d transition metal oxides heterostructures such as SrRuO3 /SrIrO3 bilayers, where the interfacial Dzyaloshinskii-Moriya interaction (DMI), due to the strong spin-orbit coupling (SOC) in SrIrO3 and the broken inversion symmetry at the interface, is believed to play a significant role. Here the emergence of THE in SrRuO3 single layers with thickness ranging from 3 to 6 nm is experimentally demonstrated. It is found that the oxygen octahedron rotation in SrRuO3 also has a significant effect on the observed THE. Furthermore, the THE may be continuously tuned by an applied electrical field. It is proposed that the large SOC of Ru ions together with the broken inversion symmetry, mainly from the interface, produce the DMI that is responsible for the observed THE. The emergence of the gate-tunable DMI in SrRuO3 single layer may stimulate further investigations of new spin-orbit physics in strong SOC oxides.