Implications of Ocular Confounding Factors for Aqueous Humor Proteomic and Metabolomic Analyses in Retinal Diseases.

Purpose: To assess the impact of ocular confounding factors on aqueous humor (AH) proteomic and metabolomic analyses for retinal disease characterization. Methods: This study recruited 138 subjects (eyes): 102 with neovascular age-related macular degeneration (nAMD), 18 with diabetic macular edema (DME), and 18 with cataract (control group). AH samples underwent analysis using Olink Target 96 proteomics and Metabolon's metabolomics platform Data analysis included correlation, differential abundance, and gene-set analysis. Results: In total, 756 proteins and 408 metabolites were quantified in AH. Total AH protein concentration was notably higher in nAMD (3.2-fold) and DME (4.1-fold) compared to controls. Pseudophakic eyes showed higher total AH protein concentrations than phakic eyes (e.g., 1.6-fold in nAMD) and a specific protein signature indicative of matrix remodeling. Unexpectedly, pupil-dilating drugs containing phenylephrine/tropicamide increased several AH proteins, notably interleukin-6 (5.4-fold in nAMD). Correcting for these factors revealed functionally relevant protein correlation clusters and disease-relevant, differentially abundant proteins across the groups. Metabolomics analysis, for which the relevance of confounder adjustment was less apparent, suggested insufficiently controlled diabetes and chronic hyperglycemia in the DME group. Conclusions: AH protein concentration, pseudophakia, and pupil dilation with phenylephrine/tropicamide are important confounding factors for AH protein analyses. When these factors are considered, AH analyses can more clearly reveal disease-relevant factors. Translational Relevance: Considering AH protein concentration, lens status, and phenylephrine/tropicamide administration as confounders is crucial for accurate interpretation of AH protein data.

Metabolic Regulation of Endothelial Cells: A New Era for Treating Wet Age-Related Macular Degeneration.

Wet age-related macular degeneration (wet AMD) is a primary contributor to visual impairment and severe vision loss globally, but the prevailing treatments are often unsatisfactory. The development of conventional treatment strategies has largely been based on the understanding that the angiogenic switch of endothelial cells (ECs) is mainly dictated by angiogenic growth factors. Even though treatments targeting vascular endothelial growth factor (VEGF), like ranibizumab, are widely administered, more than half of patients still exhibit inadequate or null responses, suggesting the involvement of other pathogenic mechanisms. With advances in research in recent years, it has become well recognized that EC metabolic regulation plays an active rather than merely passive responsive role in angiogenesis. Disturbances of these metabolic pathways may lead to excessive neovascularization in angiogenic diseases such as wet AMD, therefore targeted modulation of EC metabolism represents a promising therapeutic strategy for wet AMD. In this review, we comprehensively discuss the potential applications of EC metabolic regulation in wet AMD treatment from multiple perspectives, including the involvement of ECs in wet AMD pathogenesis, the major endothelial metabolic pathways, and novel therapeutic approaches targeting metabolism for wet AMD.

Metrnl inhibits choroidal neovascularization by attenuating the choroidal inflammation via inactivating the UCHL-1/NF-κB signaling pathway.

Objective: Choroidal neovascularization (CNV) represents the predominant form of advanced wet Age-related Macular Degeneration (wAMD). Macrophages play a pivotal role in the pathological progression of CNV. Meteorin-like (Metrnl), a novel cytokine known for its anti-inflammatory properties in macrophages, is the focus of our investigation into its mechanism of action and its potential to impede CNV progression. Methods: Cell viability was evaluated through CCK-8 and EdU assays following Metrnl treatment. Expression levels of inflammatory cytokines and proteins were assessed using quantitative reverse-transcription polymerase chain reaction(qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot techniques. Protein-protein interactions were identified through protein mass spectrometry and co-immunoprecipitation (Co-IP). Additionally, in vivo and in vitro neovascularization models were employed to evaluate angiogenesis. Results: Our results revealed downregulated Metrnl levels in the choroid-sclera complex of CNV mice, the aqueous humor of wAMD patients, and activated macrophages. Metrnl overexpression demonstrated a reduction in pro-inflammatory cytokine production, influenced endothelial cell function, and suppressed angiogenesis in choroid explants and CNV models. Through protein mass spectrometry and Co-IP, we confirmed Metrnl binds to UCHL-1 to modulate the NF-κB signaling pathway. This interaction inhibited the transcription and expression of pro-inflammatory cytokines, ultimately suppressing angiogenesis. Conclusion: In summary, our findings indicate that Metrnl down-regulates macrophage pro-inflammatory cytokine secretion via the UCHL-1/NF-κB signaling pathway. This mechanism alleviates the inflammatory microenvironment and effectively inhibits choroidal neovascularization.

Novel therapeutic perspectives for wet age-related macular degeneration: RGD-modified liposomes loaded with 2-deoxy-D-glucose as a promising nanomedicine.

Choroidal neovascularization (CNV), characterized as a prominent feature of wet age-related macular degeneration (AMD), is a primary contributor to visual impairment and severe vision loss globally, while the prevailing treatments are often unsatisfactory. The development of conventional treatment strategies has largely been based on the understanding that the angiogenic switch of endothelial cells is dictated by angiogenic growth factors alone. Even though treatments targeting vascular endothelial growth factor (VEGF), like Ranibizumab, are widely administered, more than half of the patients still exhibit inadequate or null responses, emphasizing the imperative need for solutions to this problem. Here, aiming to explore therapeutic strategies from a novel perspective of endothelial cell metabolism, a biocompatible nanomedicine delivery system is constructed by loading RGD peptide-modified liposomes with 2-deoxy-D-glucose (RGD@LP-2-DG). RGD@LP-2-DG displayed good targeting performance towards endothelial cells and excellent in vitro and in vivo inhibitory effects on neovascularization were demonstrated. Moreover, our mechanistic studies revealed that 2-DG interfered with N-glycosylation, leading to the inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream signaling. Notably, the remarkable inhibitory effect on neovascularization and biocompatibility of RGD@LP-2-DG render it a highly promising and clinically translatable therapeutic candidate for the treatment of wet AMD and other angiogenic diseases, particularly in patients who are unresponsive to currently available treatments.

Examining the correlation of lymphangiogenesis biomarkers with clinical condition in Age-Related Macular Degeneration (AMD).

The aim of this study is to investigate the relationship between age-related macular degeneration (AMD) and lymphangiogenesis biomarkers, namely LYVE-1, Podoplanin, VEGF-C, VEGFR-2 and VEGFR-3. This prospective and interventional study includes 30 patients with AMD which may be dry or wet type and 30 controls for whom vitrectomy and phacoemulsification was indicated due to additional pathologies (epiretinal membrane, macular hole, retinal detachment, and cataract). 0.1-0,2 ml of aqueous humor and 0.5-1 ml of vitreous sample was taken during the operations. Before the operations 1 tube serum was also taken. All the lymphangiogenesis biomarkers in the study are examined by ELISA method. LYVE-1 (p = 0.001) and Podoplanin (p = 0.004) levels in the vitreous for the patient group are found to be significantly lower than the control group. Serum (p = 0.019), vitreous (p = 0.001), aqueous (p < 0.001) levels of VEGF-C for the patient group are significantly higher than the control group. VEGF-C/VEGFR-2 (p < 0.001), VEGF-C/VEGFR-3 (p < 0.001) ratios in the vitreous for the patient group are found to be significantly higher than the control group. Especially in wet AMD patients, LYVE-1 level is significantly lower in the vitreous (p = 0.002) and aqueous (p = 0.002) than the control group. In addition, Podoplanin level is observed as significantly lower in the vitreous (p = 0.014) and serum (p = 0.002) in comparison to control group. In the wet AMD group, VEGF-C level in the vitreous (p < 0.001), aqueous (p < 0.001) and serum (p = 0.001) is higher than the control group. The result of this study indicates a valid relationship between the weakening of lymphangiogenesis and the pathophysiology of AMD, especially for the wet type. It is observed that the levels of receptors that bind VEGF-C (VEGFR-2 and VEGFR-3) do not increase at the same rate as VEGF-C to compensate for the increase in VEGF-C. The absence of an increase in VEGFR-3, which is especially necessary for lymphangiogenesis, also suggests that lymphangiogenesis is weakened or decreased in AMD. In the future interventional studies with larger series, examination of lymphangiogenic biomarkers in inflammatory retinal diseases and glaucoma may reveal unexplored details.

Aqueous humour interleukin-6 and vision outcomes with anti-vascular endothelial growth factor therapy.

BACKGROUND: This analysis evaluated aqueous humour (AH) interleukin (IL)-6 concentrations and the association between AH IL-6 and visual outcomes in patients with neovascular age-related macular degeneration (nAMD) or diabetic macular oedema (DMO) receiving anti-vascular endothelial growth factor (VEGF) monotherapy. METHODS: Post hoc analysis of the multicentre, double-masked, randomised HARBOR (NCT00891735) and READ-3 (NCT01077401) trials. HARBOR enrolled treatment-naïve nAMD patients. READ-3 enrolled treatment-naïve/previously treated DMO patients. HARBOR patients received ranibizumab 0.5 or 2.0 mg monthly or as needed; AH samples were collected at month 2, after two previous intravitreal injections. READ-3 patients received ranibizumab 0.5 or 2.0 mg as needed; AH samples were collected at baseline and months 3, 6, 9, and 12. MAIN OUTCOME MEASURE: association between AH IL-6 concentrations and month 24 best-corrected visual acuity (BCVA). RESULTS: In both trials (HARBOR, N = 36; READ-3, N = 137), patients with higher AH IL-6 concentrations had worse visual outcomes. HARBOR patients with low AH IL-6 concentrations at month 2 had a mean (95% CI) BCVA change at month 24 of +2.9 (-2.6, 8.3) letters, whereas patients with high AH concentrations had a mean (95% CI) BCVA change of -9.0 (-22.7, 4.7) letters. READ-3 patients with low AH concentrations at baseline had a mean (95% CI) BCVA change at month 12 of +9.3 (7.4, 11.3) letters, whereas patients with high AH concentrations had a mean (95% CI) BCVA change of +5.6 (2.2, 9.1) letters. CONCLUSIONS: Higher IL-6 AH concentrations may predict suboptimal visual responses to anti-VEGF monotherapy in patients with nAMD/DMO.

Tear cytokine levels are reduced in patients treated with intravitreal injections.

PURPOSE: To investigate cytokine levels in the tear fluid of patients receiving serial intravitreal injections (IVI) with anti-vascular endothelial growth factor (anti-VEGF) for neovascular age-related macular degeneration (nAMD). METHODS: Concentrations of six cytokines (IFN-γ, IL-1ß, IL-6, IL-8, TNF and VEGF) in tears of patients receiving anti-VEGF in one eye were assayed using multiplex cytometric bead array. The fellow untreated eye served as control. Tear sampling was performed on a single occasion at a minimum of four weeks after IVI. Patients underwent a pre-IVI antisepsis protocol with povidone-iodine. RESULTS: Tear fluid from thirty patients with a mean age of 78.8 years (range 58-90) was assayed. Subjects received a median of 43.5 (range 22-106) IVI in one eye. The median level of IFN-γ was 0.33 (interquartile range (IQR) 0.22-0.52) pg/mg of total protein in injected eyes versus 0.41 (IQR 0.21-1.05) pg/mg in fellow eyes (p = 0.017). For TNF, a median level of 0.12 (IQR 0.08-0.18) pg/mg of total protein was found in injected eyes versus 0.14 (IQR 0.07-0.33) pg/mg of total protein in fellow eyes (p = 0.019). There were no differences between injected and fellow eyes regarding the levels of IL-1ß, IL-6, IL-8 and VEGF. CONCLUSION: Tear fluid in eyes receiving serial IVI with anti-VEGF and preoperative povidone-iodine antisepsis constitutes lower levels of the pro-inflammatory cytokines IFN-γ and TNF compared to fellow eyes. This provides biochemical support of previous findings of reduced signs of inflammation and healthier tear film parameters in patients treated with serial IVI.

Single-cell profiling transcriptomic reveals cellular heterogeneity and cellular crosstalk in choroidal neovascularization model.

Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (nAMD) and a major contributor to vision loss in nAMD cases. However, the identification of specific cell types associated with nAMD remains challenging. Herein, we performed single-cell sequencing to comprehensively explore the cellular diversity and understand the foundational components of the retinal pigment epithelium (RPE)/choroid complex. We unveiled 10 distinct cell types within the RPE/choroid complex. Notably, we observed significant heterogeneity within endothelial cells (ECs), fibroblasts, and macrophages, underscoring the intricate nature of the cellular composition in the RPE/choroid complex. Within the EC category, four distinct clusters were identified and EC cluster 0 was tightly associated with choroidal neovascularization. We identified five clusters of fibroblasts actively involved in the pathogenesis of nAMD, influencing fibrotic responses, angiogenic effects, and photoreceptor function. Additionally, three clusters of macrophages were identified, suggesting their potential roles in regulating the progression of nAMD through immunomodulation and inflammation regulation. Through CellChat analysis, we constructed a complex cell-cell communication network, revealing the role of EC clusters in interacting with fibroblasts and macrophages in the context of nAMD. These interactions were found to govern angiogenic effects, fibrotic responses, and inflammatory processes. In summary, this study reveals noteworthy cellular heterogeneity in the RPE/choroid complex and provides valuable insights into the pathogenesis of CNV. These findings will open up potential avenues for deep understanding and targeted therapeutic interventions in nAMD.

Pathologic vs. protective roles of hypoxia-inducible factor 1 in RPE and photoreceptors in wet vs. dry age-related macular degeneration.

It has previously been reported that antioxidant vitamins can help reduce the risk of vision loss associated with progression to advanced age-related macular degeneration (AMD), a leading cause of visual impairment among the elderly. Nonetheless, how oxidative stress contributes to the development of choroidal neovascularization (CNV) in some AMD patients and geographic atrophy (GA) in others is poorly understood. Here, we provide evidence demonstrating that oxidative stress cooperates with hypoxia to synergistically stimulate the accumulation of hypoxia-inducible factor (HIF)-1α in the retinal pigment epithelium (RPE), resulting in increased expression of the HIF-1-dependent angiogenic mediators that promote CNV. HIF-1 inhibition blocked the expression of these angiogenic mediators and prevented CNV development in an animal model of ocular oxidative stress, demonstrating the pathological role of HIF-1 in response to oxidative stress stimulation in neovascular AMD. While human-induced pluripotent stem cell (hiPSC)-derived RPE monolayers exposed to chemical oxidants resulted in disorganization and disruption of their normal architecture, RPE cells proved remarkably resistant to oxidative stress. Conversely, equivalent doses of chemical oxidants resulted in apoptosis of hiPSC-derived retinal photoreceptors. Pharmacologic inhibition of HIF-1 in the mouse retina enhanced-while HIF-1 augmentation reduced-photoreceptor apoptosis in two mouse models for oxidative stress, consistent with a protective role for HIF-1 in photoreceptors in patients with advanced dry AMD. Collectively, these results suggest that in patients with AMD, increased expression of HIF-1α in RPE exposed to oxidative stress promotes the development of CNV, but inadequate HIF-1α expression in photoreceptors contributes to the development of GA.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Macrophage Sult2b1 promotes pathological neovascularization in age-related macular degeneration.

Disordered immune responses and cholesterol metabolism have been implicated in age-related macular degeneration (AMD), the leading cause of blindness in elderly individuals. SULT2B1, the key enzyme of sterol sulfonation, plays important roles in inflammation and cholesterol metabolism. However, the role and underlying mechanism of SULT2B1 in AMD have not been investigated thus far. Here, we report that SULT2B1 is specifically expressed in macrophages in choroidal neovascularization lesions. Sutl2b1 deficiency significantly reduced leakage areas and inhibited pathological angiogenesis by inhibiting M2 macrophage activation in vivo and in vitro. Mechanistically, loss of Sult2b1 activated LXRs and subsequently increased ABCA1 and ABCG1 (ABCA1/G1)-mediated cholesterol efflux from M2 macrophages. LXR inhibition (GSK2033 treatment) in Sult2b1 -/- macrophages reversed M2 polarization and decreased intracellular cholesterol capacity to promote pathological angiogenesis. In contrast to SULT2B1, STS, an enzyme of sterol desulfonation, protected against choroidal neovascularization development by activating LXR-ABCA1/G1 signalling to block M2 polarization. Collectively, these data reveal a cholesterol metabolism axis related to macrophage polarization in neovascular AMD.

Molecular pathogenesis of subretinal fibrosis in neovascular AMD focusing on epithelial-mesenchymal transformation of retinal pigment epithelium.

Age-related macular degeneration (AMD) is a leading cause of vision loss among elderly people in developed countries. Neovascular AMD (nAMD) accounts for more than 90% of AMD-related vision loss. At present, intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is widely used as the first-line therapy to decrease the choroidal and retinal neovascularizations, and thus to improve or maintain the visual acuity of the patients with nAMD. However, about 1/3 patients still progress to irreversible visual impairment due to subretinal fibrosis even with adequate anti-VEGF treatment. Extensive literatures support the critical role of epithelial-mesenchymal transformation (EMT) of retinal pigment epithelium (RPE) in the pathogenesis of subretinal fibrosis in nAMD, but the underlying mechanisms still remain largely unknown. This review summarized the molecular pathogenesis of subretinal fibrosis in nAMD, especially focusing on the transforming growth factor-ß (TGF-ß)-induced EMT pathways. It was also discussed how these pathways crosstalk and respond to signals from the microenvironment to mediate EMT and contribute to the progression of nAMD-related subretinal fibrosis. Targeting EMT signaling pathways might provide a promising and effective therapeutic strategy to treat subretinal fibrosis secondary to nAMD.

Emerging Role of Adiponectin/AdipoRs Signaling in Choroidal Neovascularization, Age-Related Macular Degeneration, and Diabetic Retinopathy.

Age-related macular degeneration (AMD), a leading cause of irreversible blindness in adults, may result in poor central vision, making it difficult to see, read, and drive. AMD is generally classified in either dry or wet types. Milder cases of dry AMD may progress to geographic atrophy (GA), leading to significant visual disability; wet, or neovascular AMD, which involves choroidal neovascularization (CNV), can lead to complete loss of central vision. Adiponectin (APN) discovery in the mid-1990's and, subsequently, its two cognate receptors (AdipoRs) in the early 2000s have led to a remarkable progress in better understanding metabolic disorders, as well as metabolism-associated ocular pathology. APN/AdipoRs signaling plays a central role in a variety of molecular and cellular physiological events, including glucose and lipid metabolism, whole-body energy regulation, immune and inflammation responses, insulin sensitivity and retinal cell biological functions. This review is an amalgamation of recent information related to APN/AdipoRs in the pathophysiology of retinal diseases and furthers its association with AMD and diabetic retinopathy. Additionally, we present our original research, where we designed control peptide and CNV inhibitory peptide from the globular region of APN to see the effect of these peptides on the mouse model of laser-induced CNV. The inhibitory peptide (APN1) inhibited CNV by more than 75% while the control peptide did not inhibit CNV.

Rare CIDEC coding variants enriched in age-related macular degeneration patients with small low-luminance deficit cause lipid droplet and fat storage defects.

BACKGROUND: The basis of Age-related macular degeneration (AMD) genetic risk has been well documented; however, few studies have looked at genetic biomarkers of disease progression or treatment response within advanced AMD patients. Here we report the first genome-wide analysis of genetic determinants of low-luminance vision deficit (LLD), which is seen as predictive of visual acuity loss and anti-VEGF treatment response in neovascular AMD patients. METHODS: AMD patients were separated into small- and large-LLD groups for comparison and whole genome sequencing was performed. Genetic determinants of LLD were assessed by common and rare variant genetic analysis. Follow-up functional analysis of rare coding variants identified by the burden test was then performed in vitro. RESULTS: We identified four coding variants in the CIDEC gene. These rare variants were only present in patients with a small LLD, which has been previously shown to indicate better prognosis and better anti-VEGF treatment response. Our in vitro functional characterization of these CIDEC alleles revealed that all decrease the binding affinity between CIDEC and the lipid droplet fusion effectors PLIN1, RAB8A and AS160. The rare CIDEC alleles all cause a hypomorphic defect in lipid droplet fusion and enlargement, resulting in a decreased fat storage capability in adipocytes. CONCLUSIONS: As we did not detect CIDEC expression in the ocular tissue affected by AMD, our results suggest that the CIDEC variants do not play a direct role in the eye and influence low-luminance vision deficit via an indirect and systemic effect related to fat storage capacity.

Etiological Roles of p75NTR in a Mouse Model of Wet Age-Related Macular Degeneration.

Choroidal neovascularization (CNV) is a pathological angiogenesis of the choroidal plexus of the retina and is a key feature in the wet form of age-related macular degeneration. Mononuclear phagocytic cells (MPCs) are known to accumulate in the subretinal space, generating a chronic inflammatory state that promotes the growth of the choroidal neovasculature. However, how the MPCs are recruited and activated to promote CNV pathology is not fully understood. Using genetic and pharmacological tools in a mouse model of laser-induced CNV, we demonstrate a role for the p75 neurotrophin receptor (p75NTR) in the recruitment of MPCs, in glial activation, and in vascular alterations. After laser injury, expression of p75NTR is increased in activated Muller glial cells near the CNV area in the retina and the retinal pigmented epithelium (RPE)-choroid. In p75NTR knockout mice (p75NTR KO) with CNV, there is significantly reduced recruitment of MPCs, reduced glial activation, reduced CNV area, and the retinal function is preserved, as compared to wild type mice with CNV. Notably, a single intravitreal injection of a pharmacological p75NTR antagonist in wild type mice with CNV phenocopied the results of the p75NTR KO mice. Our results demonstrate that p75NTR is etiological in the development of CNV.

Factors That Can Prolong Ocular Treatment Duration in Age-Related Macular Degeneration.

Intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents are used to treat wet age-related macular degeneration (wAMD); however, they are associated with a considerable treatment burden and poor real-world outcomes. The molecular size and charge of anti-VEGF agents influence drug pharmacokinetics in the vitreous and peak drug efficacy. This article reviews the established and novel strategies to prolong drug action, in the vitreal cavity, and thus reduce dosing frequency. Increased ocular residency can be attained by increasing drug size as with large molecules, such as KSI-301; adding polyethylene glycol to pegcetacoplan (APL-2) or avacincaptad pegol to increase molecular size; or binding to other targets that increase molecular size, such as vitreal albumin in the case of BI-X. Faricimab is a bispecific antibody in which the fragment crystallizable portion is engineered to prolong ocular residency and reduce systemic exposure. Conversely, small VEGF-binding molecules, such as brolucizumab, can be administered at higher clinical doses, with the potential for prolonged clinical activity versus larger molecules. Other important considerations include sustained drug delivery routes, such as the ranibizumab port delivery system or subconjunctival or suprachoroidal injection. More effective and longer-lasting treatments are needed for wAMD to prolong drug action and reduce dosing frequency. Several strategies are under investigation and the prevention of vision loss in patients with AMD or other retinal diseases may be attainable in the near future.

CircRNA Uxs1/miR-335-5p/PGF axis regulates choroidal neovascularization via the mTOR/p70 S6k pathway.

Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness in the elderly population. Neovascular AMD is the late stage, characterized by choroidal neovascularization (CNV). Non-coding RNAs have been implicated in CNV; however, the role of circular RNAs (circRNAs) has not yet been elucidated. Herein, we comprehensively investigated circRNA profiles in laser-induced CNV mouse models and patient specimens. A novel circRNA, circRNA Uxs1, was identified, and its function in CNV regulation was investigated in the present study. CircRNA Uxs1 was consistently upregulated in CNV patient specimens and CNV mouse models. Knockdown of circRNA Uxs1 interrupted the tube formation, migration, and proliferation of endothelial cells in vitro. Silencing circRNA Uxs1 in vivo alleviated neovascularization formation, as shown by the decreased size of laser spots. Mechanistically, circRNA Uxs1 functioned by binding to miR-335-5p, which further upregulated the expression of placental growth factor (PGF) gene and activated the mammalian target of rapamycin/p70 S6 Kinase (mTOR/p70 S6k) pathway. By subretinal injections of adeno-associated virus (AAV), we demonstrated the anti-angiogenic function of circRNA Uxs1 knockdown in vivo. In conclusion, circRNA Uxs1 promoted CNV by sponging miR-335-5p, which stimulated PGF expression and subsequently activated the mTOR/p70 S6k pathway. Therefore, circRNA Uxs1 may serve as a promising therapeutic target for CNV.

Regulation of oxidative stress and inflammatory responses in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is an eye disease, which causes impaired vision that can lead to blindness. The incidence of AMD increases with age. Retinal pigment epithelial (RPE) cells maintain retinal homeostasis and support the functionality of photoreceptors. In the pathogenesis of AMD, the degeneration of the RPE cells precedes photoreceptor cell death. RPE cells are susceptible to oxidative stress, and chronic inflammation involving nucleotide-binding domain, leucine-rich repeat and pyrin domain 3 (NLRP3) inflammasome activation and impaired autophagy are challenges faced by aged RPE cells in AMD. There are two types of AMD, dry (85-90%) and wet (10-15%) disease forms. Choroidal neovascularization is typical for wet AMD, and anti-vascular endothelial growth factor (anti-VEGF) injections are used to prevent the progression of the disease but there is no curative treatment. There is no cure for the dry disease form, but antioxidants have been proposed as a potential treatment option. Ageing is the most important risk factor of AMD, and tobacco smoke is the most important environmental risk factor that can be controlled. Hydroquinone is a cytotoxic, immunotoxic, carcinogenic and pro-oxidative component of tobacco smoke. The aim of this PhD thesis was to study hydroquinone-induced oxidative stress and NLRP3 inflammasome activation in human RPE cells (ARPE-19 cells). An age-related eye disease study (AREDS) formulation (incl. omega-3 fatty acids, vitamin C and E, copper, zinc, lutein and zeaxanthin), which is clinically investigated p.o. dosing combination of dietary supplements for AMD patients, has been evaluated as a possible treatment and restraining option for AMD. Resvega (4.1.1, Table 2) is a similar kind of product to AREDS with added resveratrol, and many of the components incorporated within Resvega can be considered as belonging to the normal antioxidative defence system of the retina. Another aim was to evaluate the effects of Resvega on hydroquinone-induced oxidative stress or NLRP3 inflammasome activation induced by impaired protein clearance. The results of this study reveal that hydroquinone elevated the activity of NADPH oxidase which subsequently mediated the production of reactive oxygen species (ROS) and predisposed RPE cells to degeneration by reducing levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Hydroquinone induced an NLRP3-independent IL-18 release and NLRP3 accumulation inside the IL-1α-primed cells. Resvega treatment reduced the extent of hydroquinone-induced ROS production and NLRP3 inflammasome activation evoked by impaired protein clearance. Thus, Resvega alleviated hydroquinone- and impaired protein clearance-induced stress in human RPE cells, but more studies are needed, for example, to reveal the most optimal route of administration for targeting the cells in the retina, since both oxidative stress and NLRP3 inflammasome activation are important contributors to the development of AMD and represent significant treatment targets.

A Linkage between Angiogenesis and Inflammation in Neovascular Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the aging population with a limited understanding of its pathogenesis and the number of patients are all the time increasing. AMD is classified into two main forms: dry and neovascular AMD (nAMD). Dry AMD is the most prevalent form (80-90%) of AMD cases. Neovascular AMD (10-20% of AMD cases) is treated with monthly or more sparsely given intravitreal anti-vascular endothelial growth factor inhibitors, but unfortunately, not all patients respond to the current treatments. A clinical hallmark of nAMD is choroidal neovascularization. The progression of AMD is initially characterized by atrophic alterations in the retinal pigment epithelium, as well as the formation of lysosomal lipofuscin and extracellular drusen deposits. Cellular damage caused by chronic oxidative stress, protein aggregation and inflammatory processes may lead to advanced geographic atrophy and/or choroidal neovascularization and fibrosis. Currently, it is not fully known why different AMD phenotypes develop. In this review, we connect angiogenesis and inflammatory regulators in the development of nAMD and discuss therapy challenges and hopes.

Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration.