Protein O-GlcNAcylation coupled to Hippo signaling drives vascular dysfunction in diabetic retinopathy.

Metabolic disorder significantly contributes to diabetic vascular complications, including diabetic retinopathy, the leading cause of blindness in the working-age population. However, the molecular mechanisms by which disturbed metabolic homeostasis causes vascular dysfunction in diabetic retinopathy remain unclear. O-GlcNAcylation modification acts as a nutrient sensor particularly sensitive to ambient glucose. Here, we observe pronounced O-GlcNAc elevation in retina endothelial cells of diabetic retinopathy patients and mouse models. Endothelial-specific depletion or pharmacological inhibition of O-GlcNAc transferase effectively mitigates vascular dysfunction. Mechanistically, we find that Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, are O-GlcNAcylated in diabetic retinopathy. We identify threonine 383 as an O-GlcNAc site on YAP, which inhibits its phosphorylation at serine 397, leading to its stabilization and activation, thereby promoting vascular dysfunction by inducing a pro-angiogenic and glucose metabolic transcriptional program. This work emphasizes the critical role of the O-GlcNAc-Hippo axis in the pathogenesis of diabetic retinopathy and suggests its potential as a therapeutic target.

Natural killer cells promote neutrophil extracellular traps and restrain macular degeneration in mice.

Neovascular age-related macular degeneration (nvAMD) is the leading cause of blindness in the elderly population. Although it is known that nvAMD is associated with focal inflammation, understanding of the precise immune components governing this process remains limited. Here, we identified natural killer (NK) cells as a prominent lymphocyte population infiltrating the perivascular space of choroidal neovascularization (CNV) lesions in patients with nvAMD and in mouse models. Olink proteomic analysis and single-cell RNA sequencing combined with knockout studies demonstrated the involvement of C-C chemokine receptor 5 (CCR5) in NK cell recruitment and extravasation at the CNV sites of mice. Depletion of NK cells or inhibition of activating receptor NK group 2, member D (NKG2D) inhibited the formation of neutrophil extracellular traps, increased vascular leakage, and exacerbated pathological angiogenesis, indicating that NK cells restrain pathogenesis in this mouse model. Age is the strongest risk factor for AMD, and we show that NK cells from aged human donors exhibited a less cytotoxic phenotype. NK cells from old mice exhibited compromised protective effects in the CNV mouse model. In addition, interleukin-2 complex-mediated expansion of NK cells improved CNV formation in mice. Collectively, our study highlights NK cells as a potential therapeutic target for patients with nvAMD.

Microglial repopulation restricts ocular inflammation and choroidal neovascularization in mice.

Introduction: Age-related macular degeneration (AMD) is a prevalent, chronic and progressive retinal degenerative disease characterized by an inflammatory response mediated by activated microglia accumulating in the retina. In this study, we demonstrate the therapeutically effects and the underlying mechanisms of microglial repopulation in the laser-induced choroidal neovascularization (CNV) model of exudative AMD. Methods: The CSF1R inhibitor PLX3397 was used to establish a treatment paradigm for microglial repopulation in the retina. Neovascular leakage and neovascular area were examined by fundus fluorescein angiography (FFA) and immunostaining of whole-mount RPE-choroid-sclera complexes in CNV mice receiving PLX3397. Altered cellular senescence was measured by beta-galactosidase (SA-ß-gal) activity and p16INK4a expression. The effect and mechanisms of repopulated microglia on leukocyte infiltration and the inflammatory response in CNV lesions were analyzed. Results: We showed that ten days of the CSF1R inhibitor PLX3397 treatment followed by 11 days of drug withdrawal was sufficient to stimulate rapid repopulation of the retina with new microglia. Microglial repopulation attenuated pathological choroid neovascularization and dampened cellular senescence in CNV lesions. Repopulating microglia exhibited lower levels of activation markers, enhanced phagocytic function and produced fewer cytokines involved in the immune response, thereby ameliorating leukocyte infiltration and attenuating the inflammatory response in CNV lesions. Discussion: The microglial repopulation described herein are therefore a promising strategy for restricting inflammation and choroidal neovascularization, which are important players in the pathophysiology of AMD.

Recalled age of myopia onset may predict risk of high adult myopia in Chinese adults.

INTRODUCTION: This study aimed to investigate the relationship between age of myopia onset and high myopia and to explore if age of onset mediated the associations of high myopia with parental myopia and time spent on electronics. METHODS: This cross-sectional study enrolled 1118 myopic patients aged 18 to 40. Information was obtained via a detailed questionnaire. Multivariable logistic regression and linear regression models were utilized to assess age of onset in relation to high myopia and spherical equivalent refractive error, respectively. Structural equation models examined the mediated effect of onset age on the association between parental myopia, time spent on electronics and high myopia. RESULTS: An early age at myopia onset was negatively correlated with spherical equivalent refractive power. Subjects who developed myopia before the age of 12 were more likely to suffer from high myopia than those who developed myopia after the age of 15. Age of myopia onset was the strongest predictor of high myopia, with an area under the curve (AUC) in Receiver Operator Characteristic (ROC) analysis of 0.80. Additionally, age of myopia onset served as a mediator in the relationships between parental myopia, electronic device usage duration, and the onset of high myopia in adulthood. CONCLUSIONS: Age of myopia onset might be the single best predictor for high myopia, and age at onset appeared to mediate the associations of high myopia with parental myopia and time spent on electronics.

Lysozyme Protects Against Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Inflammation in Human Corneal Epithelial Cells.

Purpose: The purpose of this study was to investigate the effects of lysozyme, an antimicrobial enzyme found in tears that protects the eye against pathogens, on pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through corneal epithelial cells. Methods: The expression of the angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease (TMPRSS2) in human corneal epithelial cells (HCECs) was measured by RT-PCR and Western blotting. The altered expression of the pro-inflammatory molecules induced by spike protein and lysozyme was analyzed by RT-PCR. Cell toxicity was tested by CCK8 assay. The cell entry of SAR-CoV-2 in HCECs and primary rabbit corneal epithelial cells (RbCECs) was detected by luciferase assay. Results: ACE2 and TMPRSS2 were highly expressed in HCECs. The spike proteins of SARS-CoV-2 stimulated a robust inflammatory response in HCECs, characterized by increased secretion of pro-inflammatory molecules, including IL-6, TNF-α, iNOS, and MCP-1, and pretreatment with lysozyme in HCECs markedly decreased the production of proinflammatory molecules induced by spike proteins. In addition, the inflammatory cytokine TNF-α enhanced the entry of SARS-CoV-2 into HCECs, which can be mitigated by pretreatment with lysozyme. Conclusions: In this study, we analyzed the susceptibility of human corneal epithelial cells to SARS-CoV-2 infection and suggested the protective effects of lysozyme on SARS-CoV-2 infection.

Epidemiology of Sports-Related Eye Injuries Among Athletes in Tianjin, China.

Purpose: To investigate the incidence, characteristics, and risk factors of sports-related eye injuries among athletes in Tianjin, China. Methods: A cross-sectional study was carried out from March 2018 to October 2018. In this study, the athletes from Tianjin University of Sports, Tianjin Vocational College of Sports, and Tianjin provincial sports teams were selected for general investigation. In total, 1,673 athletes were invited and 1,413 participated in the study (response rate of 84.5%). Results: In total, 1,413 athletes were enrolled; 151 had suffered from sports-related eye injuries, with an incidence of 10.7% (95% CI: 9.1-12.0%). Handball (38.5%) was the sport with the highest incidence of eye injuries, followed by water polo (36.4%) and diving (26.7%). Overall, 42.4% of the athletes were injured by ball and 22.5% of injuries came from teammates. The eye injuries usually occurred during training (64.2%) and competitions (14.6%). Adnexa wound (51.7%) was the most common type of injury. About 11.9% of the athletes with eye injuries had the impaired vision; 66.7% failed to see doctors on time. The athletes <18 years of age had a higher risk of eye injuries (odds ratio [OR] =1.60, 95% CI: 1.06-2.40). The athletes with lower family income (<1,000 RMB) were at risk population for sports-related eye injuries (OR = 3.91, 95% CI: 2.24-6.82). Training >4 h a day increased the risk of eye injuries (OR = 2.21, 95% CI: 1.42-3.43). Conclusion: The incidence of sports-related eye injuries among athletes was 10.7% in Tianjin, China. Handball, water polo, and diving were the most common activities of injury. Age, family income, and training time were the risk factors for sports-related eye injuries.

Vitreous M2 Macrophage-Derived Microparticles Promote RPE Cell Proliferation and Migration in Traumatic Proliferative Vitreoretinopathy.

Purpose: To characterize vitreous microparticles (MPs) in patients with traumatic proliferative vitreoretinopathy (PVR) and investigate their role in PVR pathogenesis. Methods: Vitreous MPs were characterized in patients with traumatic PVR, patients with rhegmatogenous retinal detachment (RRD) complicated with PVR, and control subjects by flow cytometry. The presence of M2 macrophages in epiretinal membranes was measured by immunostaining. Vitreous cytokines were quantified by ELISA assay. For in vitro studies, MPs isolated from THP-1 cell differentiated M1 and M2 macrophages, termed M1-MPs and M2-MPs, were used. The effects and mechanisms of M1-MPs and M2-MPs on RPE cell proliferation, migration, and epithelial to mesenchymal transition were analyzed. Results: Vitreous MPs derived from photoreceptors, microglia, and macrophages were significantly increased in patients with traumatic PVR in comparison with control and patients with RRD (PVR), whereas no significance was identified between the two control groups. M2 macrophages were present in epiretinal membranes, and their signature cytokines were markedly elevated in the vitreous of patients with traumatic PVR. Moreover, MPs from M2 macrophages were increased in the vitreous of patients with traumatic PVR. In vitro analyses showed that M2-MPs promoted the proliferation and migration of RPE cells via activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. However, M2-MPs did not induce the expression of fibrotic proteins, including fibronectin, α-smooth muscle actin, and N-cadherin in RPE cells. Conclusions: This study demonstrated increased MP shedding in the vitreous of patients with traumatic PVR; specifically, MPs derived from M2 polarized macrophages may contribute to PVR progression by stimulating RPE cell proliferation and migration.

Association of cigarette smoking with retinal thickness and vascular structure in an elderly Chinese population.

BACKGROUND: To explore the association of cigarette smoking with retinal thickness and vascular structure in an elderly Chinese population. METHODS: This cross-sectional study enrolled employees and retirees aged over 50 years at Tianjin University of Sport from October 2020 to December 2020. Information on smoking status and lifestyle was obtained using a detailed questionnaire. All participants underwent full ophthalmic examination. OCTA image was acquired. Vascular and the thickness parameters in central fovea and peripapillary parameters were automatically calculated. Multiple linear regression analyses were utilized to assess the association of smoking with retinal thickness and vascular structure after controlling potential confounders. RESULTS: Compared with non-smoking adults, current smokers (ß=-36.78; P = 0.01) and ever smokers (ß=-35.45; P = 0.00) tended to have thinner macular fovea. Cigarettes daily, pack-years of smoking and CSI were negatively related to macular thickness (cigarettes daily: ß=-1.43; pack-years: ß=-14.73; CSI: ß=-14.70), while they were positively associated with the circumference (cigarettes daily: ß=0.03; pack-years: ß=0.30; CSI: ß=0.31) and the area of FAZ (cigarettes daily: ß=0.01; pack-years: ß=0.07). CONCLUSIONS: Cigarette smoking seems associated with decreased macular fovea thickness and elevated circumference and area of the FAZ compared to non-smokers. Our data add to evidence of smoking on retinal thickness and the microvascular system in the macular area.

Activated Blood Coagulation Factor X (FXa) Contributes to the Development of Traumatic PVR Through Promoting RPE Epithelial-Mesenchymal Transition.

Purpose: Uncontrolled coagulation reactions contribute to pathological fibroproliferation in several organs, and yet their role in proliferative vitreoretinopathy (PVR) remains to be elucidated. In this study, we evaluated the profibrotic effects of FXa in RPE cells and in a mouse model of PVR. Methods: FXa levels in the eyes of traumatic PVR patients and rabbit models of mechanical ocular trauma was measured by ELISA and immunohistochemistry. FXa-induced RPE EMT was assessed by examining cell proliferation, migration, tight junction changes, and expression of fibrotic markers. For in vivo study, FXa was injected into dispase-injured eyes, then intraocular fibrosis was evaluated by histological analysis and Western blotting. The therapeutic effect of FXa inhibitor was also examined in PVR mouse models. Results: Vitreous FXa were higher in patients with traumatic PVR compared to patients with macular hole. Moreover, expressions of FXa and PAR1 were found in the epiretinal membranes from traumatic PVR patients. Vitreous FXa were markedly increased after mechanical ocular trauma in rabbits. In vitro, FXa stimulated RPE EMT characterized as ZO-1 disruption, compromised cell polarity, and increased fibronectin expressions. Co-injection of FXa and dispase in mice induced more severely damaged retinal structures, and increased α-SMA expressions than FXa or dispase treatment alone. Oral FXa or thrombin inhibitors significantly blocked intraocular fibrosis in PVR mouse models. FXa promoted phospho-activation of p38 in ARPE19 cells, which was dependent on PAR1. Moreover, TGF-ßR inhibitor also significantly alleviated FXa-induced intraocular fibrosis in mice. Conclusions: FXa promotes intraocular fibrosis in mice via mechanisms involving RPE activation.

The Role of Intravitreal Anti-VEGF Agents in Rabbit Eye Model of Open-Globe Injury.

PURPOSE: To evaluate the effects of intravitreal anti-VEGF agents in a rabbit model of open-globe injury (OGI). METHODS: OGI was induced in the right eyes of 75 Belgian rabbits by making 5 mm circumferential incision placed 6 mm behind the limbus. The rabbits were divided into 4 groups: control (n = 5), OGI group (n = 40), and intravitreal Ranibizumab and Conbercept (n = 15 each). Ranibizumab or Conbercept was injected into the vitreous at 0.5 hours, 3 days, or 7 days. Vitreous fluid was collected, and levels of growth factors and cytokines were measured by enzyme-linked immunosorbent assay (ELISA). On day 28 after OGI, B scan examination and histological examination were performed to evaluate intravitreal proliferation and formation of epiretinal fibrosis. RESULTS: Vitreous levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-ß), and plasminogen activator inhibitor-1 (PAI-1) were significantly increased in rabbit eyes after OGI. Compared to eyes in OGI group, anti-VEGF treatments significantly reduced these growth factors and cytokines. Among the 7 eyes examined from each group for intravitreal proliferative changes, they were found in 7 of 7 (100%) in OGI group and were decreased by Ranibizumab and Conbercept to 5 of 7 (71.4%) and 4 of 7 (57.1%), respectively. Both Ranibizumab and Conbercept inhibited epiretinal scar formation at the wound site, with Conbercept showing the greatest effect (maximal length of scar (L), L OGI = 503 ± 82.44 µm, L Ranibizumab = 355 ± 43.66 µm, and L Conbercept = 250.33 ± 36.02 µm). CONCLUSION: Anti-VEGF treatments after OGI significantly attenuated the upregulation of growth factors and cytokines in the vitreous and prevented intravitreal proliferation and epiretinal scar formation and thus may protect against the development of posttraumatic complications such as proliferative vitreoretinopathy (PVR).

Exosome-mediated delivery of an anti-angiogenic peptide inhibits pathological retinal angiogenesis.

Background: Pathological angiogenesis is the hallmark of many vision-threatening diseases. Anti-VEGF is a primary treatment with substantial beneficial effects. However, such agents require frequent intravitreal injections. Our previous work established a method for effectively modifying exosomes (EXOs) for loading therapeutic peptides. Here, we used this system to load the anti-angiogenic peptide KV11, aiming to establish an EXO-based therapy strategy to suppress neovascularization in the retina. Methods: Using an anchoring peptide, CP05, we linked KV11 to endothelial cell (EC) derived EXOs, yielding EXOKV11. We tested the delivery efficiency of EXOKV11 via two commonly used ocular injection methods: retro-orbital injection and intravitreal injection. Deploying an oxygen-induced retinopathy (OIR) model and a VEGF injection model, we tested the effects of EXOKV11 on neovascular formation, EC proliferation, and vascular permeability. In vitro experiments were used to test the mechanism and to analyze the effects of EXOKV11 on EC proliferation, migration, and sprouting. Results: By using the EXO loading system, KV11 was more efficiently delivered to the blood vessels of the mouse retina via retro-orbital injection. In both OIR model and VEGF injection model, EXOKV11 was more effective than KV11 alone in inhibiting neovascularization and vessel leakage. The therapeutic effect of retro-orbital injection of EXOKV11 was comparable to the intravitreal injection of VEGF-trap. Mechanistically, KV11 alone inhibited VEGF-downstream signaling, while EXOKV11 showed a stronger effect. Conclusions: We used EXOs as a carrier for intraocular delivery of KV11. We showed that KV11 itself has an anti-angiogenic effect through retro-orbital injection, but that this effect was greatly enhanced when delivered with EXOs. Thus, this system has the potential to treat proliferative retinopathy via retro-orbital injection which is a less invasive manner compared with intravitreal injection.

Prevalence and causes of bilateral visual impairment in rural areas of Tianjin, China - The Tianjin Eye Study.

PURPOSE: To estimate the prevalence, causes and risk factors of bilateral visual impairment in rural areas of Tianjin, China. METHODS: A large population-based, cross-sectional study. A stratified random cluster sampling method was used to investigate 12 233 participants in all age groups living in rural Tianjin. Participants completed questionnaires and received professional ophthalmology examinations. RESULTS: According to World Health Organization best-corrected visual acuity (BCVA) criteria, the crude prevalence of bilateral visual impairment (BCVA < 20/63), bilateral low vision (BCVA < 20/63 to ≥20/400) and bilateral blindness (BCVA < 20/400) was 2.53%, 2.40% and 0.14% (age- and gender-standardized prevalence was 1.86%, 1.76% and 0.11%). The prevalence increased with age and was higher in women than men. The most common causes of bilateral visual impairment in the total population were cataract (48.39%), refractive error/amblyopia (17.74%), age-related macular degeneration (AMD) (10.00%), diabetic retinopathy (5.81%) and glaucoma (3.87%). For participants younger than 50 years, refractive error/amblyopia was the leading cause of low vision and blindness, while cataract was the major cause in the participants over 50. Female gender, older age and self-reported diabetes were associated with increased risks of visual impairment. CONCLUSION: The age- and gender-standardized prevalence of low vision, especially in the older group (50+), was higher in this study compared with previous studies in China. Refractive error/amblyopia was the leading cause of bilateral visual impairment in younger group, while cataract was the primary cause in the older group. These findings will provide useful information for planning comprehensive eye healthcare programmes in China.