ABSTRACT
The gyrate atrophy of the choroid and retina (GACR) is a rare genetic disease for which no definitive cure is available. GACR is due to the deficit of ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme responsible for ornithine catabolism. The hallmark of the disease is plasmatic ornithine accumulation, which damages retinal epithelium leading to progressive vision loss and blindness within the fifth decade. Here, we characterized the biochemical properties of tetrameric and dimeric hOAT and evaluated hOAT loaded in red blood cells (RBCs) as a possible enzyme replacement therapy (ERT) for GACR. Our results show that (i) hOAT has a relatively wide specificity for amino acceptors, with pyruvate being the most suitable candidate for ornithine catabolism within RBCs; (ii) both the tetrameric and dimeric enzyme can be loaded in RBC retaining their activity; and (iii) hOAT displays reduced stability in plasma, but is partly protected from inactivation upon incubation in a mixture mimicking the intracellular erythrocyte environment. Preliminary ex vivo experiments indicate that hOAT-loaded RBCs are able to metabolize extracellular ornithine at a concentration mimicking that found in patients, both in buffer and, although with lower efficiency, in plasma. Overall, our data provide a proof of concept that an RBC-mediated ERT is feasible and can be exploited as a new therapeutic approach in GACR.
Subject(s)
Enzyme Replacement Therapy , Erythrocytes , Gyrate Atrophy , Ornithine-Oxo-Acid Transaminase , Ornithine , Humans , Ornithine-Oxo-Acid Transaminase/metabolism , Ornithine-Oxo-Acid Transaminase/genetics , Gyrate Atrophy/drug therapy , Gyrate Atrophy/metabolism , Gyrate Atrophy/therapy , Erythrocytes/metabolism , Ornithine/metabolism , Enzyme Replacement Therapy/methods , Retina/metabolism , Retina/pathology , Choroid/metabolism , Choroid/pathologyABSTRACT
Purpose: The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) stimulator of interferon gene (STING) pathway is a crucial cascade in the inflammatory response initiated by the recognition of cytosolic double-stranded DNA (dsDNA). The aim of this study was to evaluate the effect of STING inhibitor in murine choroidal neovascularization (CNV). Methods: To investigate whether the cGAS-STING pathway is activated during CNV, CNV was induced using laser photocoagulation in male C57BL/6J mice. The expression of change of cGAS and STING during CNV development was confirmed by Western-blotting. H-151, a potent STING palmitoylation antagonist, was used as a STING inhibitor. H-151 was administered intravitreally immediately after laser induction. To confirm the role of the cGAS-STING pathway in CNV formation, we evaluated CNV size and performed fundus fluorescein angiography. Results: The expression levels of cGAS and STING were significantly upregulated in the RPE-choroid complex after CNV induction, and dsDNA merged with cGAS was observed in CNV lesions. Intravitreal administration of H-151 suppressed CNV development and fluorescent leakage from neovessels. In CNV lesions, the high expression of STING and cGAS was observed in infiltrating F4/80+ macrophages. H-151 administration attenuated downstream signals of the cGAS-STING pathway, including the phosphorylation of nuclear factor-κB, and downregulated the expression of interleukin 1ß. Conclusions: These findings support that the inhibition of cGAS-STING pathway treats abnormal ocular angiogenesis.
Subject(s)
Choroidal Neovascularization , Membrane Proteins , Nucleotidyltransferases , Animals , Male , Mice , Blotting, Western , Choroid/metabolism , Choroid/pathology , Choroidal Neovascularization/drug therapy , Choroidal Neovascularization/metabolism , Disease Models, Animal , Fluorescein Angiography , Intravitreal Injections , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Membrane Proteins/metabolism , Membrane Proteins/antagonists & inhibitors , Mice, Inbred C57BL , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/antagonists & inhibitorsABSTRACT
The choroid, a vascularized tissue situated between the retina and the sclera, plays a crucial role in maintaining ocular homeostasis. Despite its significance, research on choroidal abnormalities and the establishment of effective in vitro models have been limited. In this study, we developed an in vitro choroid model through the co-culture of human induced pluripotent stem cells (hiPSC)-derived endothelial cells (ECs) and mouse choroidal fibroblasts (msCFs) with hiPSC-derived retinal pigment epithelial (RPE) cells via a permeable membrane. This model, inclusive of ECs, CFs, and RPE cells, exhibited similarities with in vivo choroidal vessels, as confirmed through immunohistochemistry of extracellular matrix markers and vascular-related markers, as well as choroid angiogenesis sprouting assay analysis. The effectiveness of our in vitro model was demonstrated in assessing vascular changes induced by drugs targeting vasoregulation. Our model offers a valuable tool for gaining insights into the pathological mechanisms underlying choroid development and the progression of choroidal vascular diseases.
Subject(s)
Choroid , Coculture Techniques , Endothelial Cells , Induced Pluripotent Stem Cells , Retinal Pigment Epithelium , Choroid/blood supply , Choroid/metabolism , Animals , Humans , Mice , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Endothelial Cells/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/cytology , Fibroblasts/metabolism , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Cells, CulturedABSTRACT
Purpose: The purpose of this study was to investigate the presence of sex-steroid receptors in human choroidal tissue across different ages and sex, aiming to better understand the pronounced sex difference in central serous chorioretinopathy (CSC) occurrence. Methods: Paraffin-embedded enucleated eyes of 14 premenopausal women, 15 postmenopausal women, 10 young men (<45 years), and 10 older men (>60 years) were used. A clinically certified immunostaining was performed to detect the presence of the androgen receptor (AR), progesterone receptor (PR; isoform A and B), and estrogen receptor (ERα). The stained slides were scored in a blinded manner for positive endothelial cells and stromal cells in consecutive sections of the same choroidal region. Results: Our analysis revealed the presence of AR, PR, and ERα in endothelial cells and stromal cells of choroidal tissue. The mean proportion of AR-positive endothelial cells was higher in young men (46% ± 0.15) compared to aged-matched women (29% ± 0.12; P < 0.05, 95% confidence interval [CI]). Premenopausal women showed markedly lower mean proportion of ERα (5% ± 0.02) and PR-positive endothelial cells (2% ± 0.01) compared to postmenopausal women (15% ± 0.07 and 19% ± 0.13; both P < 0.05, 95% CI), young men (13% ± 0.04 and 21% ± 0.10; both P < 0.05, 95% CI), and older men (18% ± 0.09 and 27% ± 0.14; both P < 0.05, 95% CI). Mean PR-positive stromal cells were also less present in premenopausal women (12% ± 0.07) than in other groups. Conclusions: The number of sex-steroid receptors in the choroidal tissue differs between men and women across different ages, which aligns with the prevalence patterns of CSC in men and postmenopausal women.
Subject(s)
Central Serous Chorioretinopathy , Choroid , Receptors, Androgen , Receptors, Progesterone , Humans , Female , Male , Choroid/metabolism , Choroid/pathology , Middle Aged , Adult , Central Serous Chorioretinopathy/metabolism , Central Serous Chorioretinopathy/epidemiology , Central Serous Chorioretinopathy/diagnosis , Receptors, Progesterone/metabolism , Receptors, Androgen/metabolism , Aged , Sex Factors , Prevalence , Estrogen Receptor alpha/metabolismABSTRACT
The world-wide prevalence of myopia (nearsightedness) is increasing, but its pathogenesis is incompletely understood. Among many putative mechanisms, laboratory and clinical findings have implicated circadian biology in the etiology of myopia. Consistent with a circadian hypothesis, we recently reported a marked variability in diurnal patterns of gene expression in two crucial tissues controlling post-natal refractive development - the retina and choroid-at the onset of form-deprivation myopia in chick, a widely studied and validated model. To extend these observations, we assayed gene expression by RNA-Seq in retina and choroid during the progression of established unilateral form-deprivation myopia of chick. We assayed gene expression every 4 hours during a single day from myopic and contralateral control eyes. Retinal and choroidal gene expression in myopic vs. control eyes during myopia progression differed strikingly at discrete times during the day. Very few differentially expressed genes occurred at more than one time in either tissue during progressing myopia. Similarly, Gene Set Enrichment Analysis pathways varied markedly by time during the day. Some of the differentially expressed genes in progressing myopia coincided with candidate genes for human myopia, but only partially corresponded with genes previously identified at myopia onset. Considering other laboratory findings and human genetics and epidemiology, these results further link circadian biology to the pathogenesis of myopia; but they also point to important mechanistic differences between the onset of myopia and the progression of established myopia. Future laboratory and clinical investigations should systematically incorporate circadian mechanisms in studying the etiology of myopia and in seeking more effective treatments to normalize eye growth in children.
Subject(s)
Chickens , Choroid , Circadian Rhythm , Disease Progression , Myopia , Retina , Choroid/metabolism , Choroid/pathology , Retina/metabolism , Retina/pathology , Animals , Myopia/genetics , Myopia/metabolism , Circadian Rhythm/genetics , Chickens/genetics , Humans , Disease Models, Animal , Gene Expression Regulation , Gene Expression ProfilingABSTRACT
The dog retina contains a central macula-like region, and there are reports of central retinal disorders in dogs with shared genetic etiologies with humans. Defining central/peripheral gene expression profiles may provide insight into the suitability of dogs as models for human disorders. We determined central/peripheral posterior eye gene expression profiles in dogs and interrogated inherited retinal and macular disease-associated genes for differential expression between central and peripheral regions. Bulk tissue RNA sequencing was performed on 8 mm samples of the dog central and superior peripheral regions, sampling retina and retinal pigmented epithelium/choroid separately. Reads were mapped to CanFam3.1, read counts were analyzed to determine significantly differentially expressed genes (DEGs). A similar analytic pipeline was used with a published bulk-tissue RNA sequencing human dataset. Pathways and processes involved in significantly DEGs were identified (Database for Annotation, Visualization and Integrated Discovery). Dogs and humans shared the extent and direction of central retinal differential gene expression, with multiple shared biological pathways implicated in differential expression. Many genes implicated in heritable retinal disorders in dogs and humans were differentially expressed between central and periphery. Approximately half of genes associated with human age-related macular degeneration were differentially expressed in human and dog tissues. We have identified similarities and differences in central/peripheral gene expression profiles between dogs and humans which can be applied to further define the relevance of dogs as models for human retinal disorders.
Subject(s)
Retina , Dogs , Animals , Humans , Retina/metabolism , Gene Expression Regulation/physiology , Gene Expression Profiling , Disease Models, Animal , Transcriptome , Retinal Pigment Epithelium/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Retinal Diseases/genetics , Retinal Diseases/metabolism , Male , Female , Choroid/metabolismABSTRACT
Extracellular fluid (ECF) excess is common in patients with chronic kidney disease (CKD). This study (involving 284 patients with CKD) explored the association between choroidal vascularity index (CVI) and ECF excess. We categorised patients into three groups based on extracellular water/total body water: normal, mildly overhydrated, and severely overhydrated. The more severe ECF status was associated with a lower CVI after adjustment (B = - 0.902, p = 0.001). In non-diabetic patients, both vascular luminal (LA, p < 0.001) and stromal areas (SA, p = 0.003) were significantly reduced in patients with severe ECF excess compared to others, whereas diabetic patients showed no significant differences in LA (p = 0.96) and SA (p = 0.86) based on ECF excess status. These findings suggest that ECF status may influence CVI in patients with CKD, underscoring the need for further research to clarify its direct impact on choroidal changes.
Subject(s)
Choroid , Extracellular Fluid , Renal Insufficiency, Chronic , Humans , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/metabolism , Female , Male , Choroid/blood supply , Choroid/diagnostic imaging , Choroid/metabolism , Choroid/pathology , Middle Aged , Extracellular Fluid/metabolism , Aged , Tomography, Optical Coherence/methodsABSTRACT
Aging changes the responsiveness of our immune defense, and this decline in immune reactivity plays an important role in the increased susceptibility to infections that marks progressing age. Aging is also the most pronounced risk factor for development of age-related macular degeneration (AMD), a disease that is characterized by dysfunctional retinal pigment epithelial (RPE) cells and loss of central vision. We have previously shown that acute systemic viral infection has a large impact on the retina in young mice, leading to upregulation of chemokines in the RPE/choroid (RPE/c) and influx of CD8 T cells in the neuroretina. In this study, we sought to investigate the impact of systemic infection on the RPE/c in aged mice to evaluate whether infection in old age could play a role in the pathogenesis of AMD. We found that systemic infection in mice led to upregulation of genes from the crystallin family in the RPE/c from aged mice, but not in the RPE/c from young mice. Crystallin alpha A (CRYAA) was the most upregulated gene, and increased amounts of CRYAA protein were also detected in the aged RPE/c. Increased CRYAA gene and protein expression has previously been found in drusen and choroid from AMD patients, and this protein has also been linked to neovascularization. Since both drusen and neovascularization are important hallmarks of advanced AMD, it is interesting to speculate if upregulation of crystallins in response to infection in old age could be relevant for the pathogenesis of AMD.
Subject(s)
Aging , Choroid , Macular Degeneration , Mice, Inbred C57BL , Retinal Pigment Epithelium , Up-Regulation , Animals , Mice , Choroid/metabolism , Choroid/pathology , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Macular Degeneration/metabolism , Macular Degeneration/genetics , Disease Models, Animal , Blotting, Western , Eye Infections, Viral/metabolism , Eye Infections, Viral/virology , Real-Time Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Choroidal Neovascularization , NF-kappa B , Signal Transduction , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Choroidal Neovascularization/genetics , Animals , Mice , Humans , NF-kappa B/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Disease Models, Animal , Mice, Inbred C57BL , Macrophages/metabolism , Macrophages/immunology , Choroid/metabolism , Choroid/pathology , Choroid/blood supply , Male , Wet Macular Degeneration/metabolism , Wet Macular Degeneration/genetics , Wet Macular Degeneration/pathology , Inflammation/metabolism , Cytokines/metabolismABSTRACT
The annual increase in myopia prevalence poses a significant economic and health challenge. Our study investigated the effect of calcitriol role in myopia by inducing the condition in guinea pigs through form deprivation for four weeks. Untargeted metabolomics methods were used to analyze the differences in metabolites in the vitreous body, and the expression of vitamin D receptor (VDR) in the retina was detected. Following form deprivation, the guinea pigs received intraperitoneal injections of calcitriol at different concentrations. We assessed myopia progression using diopter measurements and biometric analysis after four weeks. Results indicated that form deprivation led to a pronounced shift towards myopia, characterized by reduced choroidal and scleral thickness, disorganized collagen fibers, and decreased scleral collagen fiber diameter. Notably, a reduction in calcitriol expression in vitreous body, diminished vitamin D and calcitriol levels in the blood, and decreased VDR protein expression in retinal tissues were observed in myopic guinea pigs. Calcitriol administration effectively slowed myopia progression, preserved choroidal and scleral thickness, and prevented the reduction of scleral collagen fiber diameter. Our findings highlight a significant decrease in calcitriol and VDR expressions in myopic guinea pigs and demonstrate that exogenous calcitriol supplementation can halt myopia development, enhancing choroidal and scleral thickness and scleral collagen fiber diameter.
Subject(s)
Calcitriol , Myopia , Retina , Animals , Guinea Pigs , Myopia/metabolism , Myopia/drug therapy , Myopia/pathology , Calcitriol/pharmacology , Retina/metabolism , Retina/drug effects , Retina/pathology , Receptors, Calcitriol/metabolism , Receptors, Calcitriol/genetics , Male , Disease Models, Animal , Sclera/metabolism , Sclera/drug effects , Sclera/pathology , Choroid/metabolism , Choroid/drug effects , Choroid/pathology , Vitamin D/pharmacology , Vitamin D/administration & dosage , Axial Length, Eye , Vitreous Body/metabolism , Vitreous Body/drug effects , Disease Progression , Collagen/metabolismABSTRACT
Among the environmental factors contributing to myopia, the role of correlated color temperature (CCT) of ambient light emerges as a key element warranting in-depth investigation. The choroid, a highly vascularized and dynamic structure, often undergoes thinning during the progression of myopia, though the precise mechanism remains elusive. The retinal pigment epithelium (RPE), the outermost layer of the retina, plays a pivotal role in regulating the transport of ion and fluid between the subretinal space and the choroid. A hypothesis suggests that variations in choroidal thickness (ChT) may be modulated by transepithelial fluid movement across the RPE. Our experimental results demonstrate that high CCT illumination significantly compromised the integrity of tight junctions in the RPE and disrupted chloride ion transport. This functional impairment of the RPE may lead to a reduction in fluid transfer across the RPE, consequently resulting in choroidal thinning and potentially accelerating axial elongation. Our findings provide support for the crucial role of the RPE in regulating ChT. Furthermore, we emphasize the potential hazards posed by high CCT artificial illumination on the RPE, the choroid, and refractive development, underscoring the importance of developing eye-friendly artificial light sources to aid in the prevention and control of myopia.
Subject(s)
Chlorides , Choroid , Ion Transport , Retinal Pigment Epithelium , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/radiation effects , Retinal Pigment Epithelium/pathology , Choroid/metabolism , Choroid/radiation effects , Choroid/pathology , Animals , Ion Transport/radiation effects , Chlorides/metabolism , Lighting/methods , Temperature , Color , Tight Junctions/metabolism , Myopia/metabolism , Myopia/pathology , Myopia/etiologyABSTRACT
Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1ß protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1ß mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1ß.
Subject(s)
Choroidal Neovascularization , Indenes , Inflammasomes , Interleukin-1beta , Microglia , Monocytes , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Mice , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Microglia/metabolism , Monocytes/metabolism , Mice, Knockout , Sulfones/pharmacology , Mice, Inbred C57BL , Furans/pharmacology , Receptors, CCR2/metabolism , Receptors, CCR2/genetics , Macrophages/metabolism , Macrophages/immunology , Sulfonamides/pharmacology , Heterocyclic Compounds, 4 or More Rings/pharmacology , Carrier Proteins/metabolism , Carrier Proteins/genetics , Choroid/metabolism , Choroid/pathology , Disease Models, Animal , Lasers/adverse effects , Macular Degeneration/pathology , Macular Degeneration/metabolism , Macular Degeneration/geneticsABSTRACT
Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.
Subject(s)
Disease Models, Animal , Lymphocytic choriomeningitis virus , Proteomics , Retinal Degeneration , Animals , Mice , Proteomics/methods , Retinal Degeneration/immunology , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Lymphocytic choriomeningitis virus/immunology , Mice, Inbred C57BL , Lymphocytic Choriomeningitis/immunology , Lymphocytic Choriomeningitis/virology , Tandem Mass Spectrometry , Proteome , Retina/immunology , Retina/metabolism , Retina/pathology , Chromatography, Liquid , Choroid/immunology , Choroid/pathology , Choroid/metabolismABSTRACT
Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD.
Subject(s)
Choroidal Neovascularization , Extracellular Matrix , Granzymes , Inflammation , Mast Cells , Retinal Pigment Epithelium , Granzymes/metabolism , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Animals , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Humans , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Inflammation/pathology , Inflammation/metabolism , Mice , Mast Cells/metabolism , Mast Cells/pathology , Mast Cells/enzymology , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Mice, Inbred C57BL , Choroid/pathology , Choroid/metabolism , Choroid/blood supply , Macular Degeneration/pathology , Macular Degeneration/metabolism , Mice, KnockoutABSTRACT
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.
Subject(s)
Choroid , Choroidal Neovascularization , Disease Models, Animal , Macrophages , Retinal Pigment Epithelium , Single-Cell Analysis , Animals , Mice , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Choroidal Neovascularization/genetics , Choroid/pathology , Choroid/metabolism , Macrophages/metabolism , Macrophages/pathology , Transcriptome , Mice, Inbred C57BL , Fibroblasts/metabolism , Fibroblasts/pathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Cell Communication/physiology , Wet Macular Degeneration/genetics , Wet Macular Degeneration/metabolism , Gene Expression ProfilingABSTRACT
PURPOSE: To investigate the association between pretreatment blood flow velocity in the choroid and optic nerve head (ONH) and retinal oxygen metabolism in the acute uveitic phase and the development of 'sunset glow fundus' in Vogt-Koyanagi-Harada (VKH) disease. METHODS: Retrospective analysis of 41 patients (82 eyes). Laser speckle flowgraphy and retinal oximetry measurements were performed at the presentation. The main outcome measure was the development of 'sunset glow fundus'. RESULTS: Twenty patients (40 eyes) presented in the phase preceding anterior segment inflammation (early presentation), and 21 patients (42 eyes) presented with anterior segment inflammation (late presentation). In ONH, mean blur rate (MBR)-vessel, representing blood flow velocity in retinal vessels, was significantly lower in the late presentation group, while choroidal MBR was not significantly different. The late presentation group had significantly lower oxygen saturation in retinal venules, a higher arteriovenous oxygen saturation difference and a smaller calibre of retinal arterioles compared with the early presentation group. Eyes that subsequently developed 'sunset glow fundus' had significantly lower ONH MBR-vessels, lower oxygen saturation in retinal venules, a higher arteriovenous oxygen saturation difference and a smaller calibre of retinal arterioles compared with eyes without 'sunset glow fundus'. ONH MBR-vessel had a significant negative correlation with arteriovenous oxygen saturation difference and a significant positive correlation with calibre of retinal arterioles. CONCLUSIONS: In the acute uveitic phase of VKH disease, the development of 'sunset glow fundus' is associated with pretreatment reduced retinal blood flow velocity, calibre of retinal arterioles and oxygen saturation in retinal venules, as well as an increased arteriovenous oxygen saturation difference.
Subject(s)
Choroid , Laser-Doppler Flowmetry , Oxygen Consumption , Oxygen , Regional Blood Flow , Retinal Vessels , Uveomeningoencephalitic Syndrome , Humans , Uveomeningoencephalitic Syndrome/physiopathology , Uveomeningoencephalitic Syndrome/metabolism , Uveomeningoencephalitic Syndrome/diagnosis , Male , Retrospective Studies , Female , Adult , Regional Blood Flow/physiology , Oxygen/metabolism , Middle Aged , Acute Disease , Choroid/blood supply , Choroid/metabolism , Retinal Vessels/physiopathology , Retinal Vessels/metabolism , Blood Flow Velocity/physiology , Oxygen Consumption/physiology , Oximetry/methods , Optic Disk/blood supply , Optic Disk/metabolism , Fluorescein Angiography/methods , Oxygen Saturation/physiology , Visual Acuity , Young Adult , Follow-Up Studies , Fundus OculiABSTRACT
The prevalence of myopia (nearsightedness) is increasing to alarming levels, but its etiology remains poorly understood. Because both laboratory and clinical findings suggest an etiologic role for circadian rhythms in myopia development, we assayed gene expression by RNA-Seq in retina and choroid at the onset of unilateral experimental myopia in chick, isolating tissues every 4 h during a single 24-h period from myopic and contralateral control eyes. Occluded versus open eye gene expression differences varied considerably over the 24-h sampling period, with some occurring at multiple times of day but with others showing differences at only a single investigated timepoint. Some of the genes identified in retina or choroid of chick myopia were previously identified as candidate genes for common human myopia. Like differentially expressed genes, pathways identified by Gene Set Enrichment Analysis also varied dramatically by sampling time. Considered with other laboratory data, human genetic and epidemiology data, these findings further implicate circadian events in myopia pathogenesis. The present results emphasize a need to include time of day in mechanistic studies of myopia and to assess circadian biology directly in trying to understand better the origin of myopia and to develop more effective therapies.
Subject(s)
Myopia , Retina , Humans , Animals , Retina/metabolism , Myopia/genetics , Myopia/metabolism , Choroid/metabolism , Circadian Rhythm/genetics , Gene Expression , Biology , Chickens/geneticsABSTRACT
Age-related macular degeneration (AMD) is a leading cause of blindness featuring pathogenic neovascularization of the choroidal vasculature (CNV). Although systemic immunity plays a role in AMD, the ocular signals that recruit and activate immune cells remain poorly defined. Using single-cell RNA sequencing, we prospectively profile peripheral blood mononuclear cells from 65 individuals including AMD and controls, which we integrate with existing choroid data. We generate a network of choroid-peripheral immune interactions dysregulated in AMD, including known AMD-relevant gene vascular endothelial growth factor (VEGF) receptor 2. Additionally, we find CYR61 is upregulated in choroidal veins and may signal to circulating monocytes. In mice, we validate that CYR61 is abundant in endothelial cells within CNV lesions neighboring monocyte-derived macrophages. Mechanistically, CYR61 activates macrophage anti-angiogenic gene expression, and ocular Cyr61 knockdown increases murine CNV size, indicating CYR61 inhibits CNV. This study highlights the potential of multi-tissue human datasets to identify disease-relevant and potentially therapeutically modifiable targets.
Subject(s)
Choroidal Neovascularization , Macular Degeneration , Humans , Mice , Animals , Choroidal Neovascularization/genetics , Choroidal Neovascularization/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Leukocytes, Mononuclear/metabolism , Endothelial Cells/metabolism , Macular Degeneration/genetics , Macular Degeneration/complications , Macular Degeneration/metabolism , Choroid/metabolism , Choroid/pathologyABSTRACT
Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, however, the involvement of the choroid in disease progression is not fully understood. CHM is caused by mutations in the CHM gene, encoding the ubiquitously expressed Rab escort protein 1 (REP1). REP1 plays an important role in intracellular trafficking of vesicles, including melanosomes. In this study, we examined the ultrastructure of the choroid in chmru848 fish and Chmnull/WT mouse models using transmission electron and confocal microscopy. Significant pigmentary disruptions were observed, with lack of melanosomes in the choroid of chmru848 fish from 4 days post fertilisation (4dpf), and a reduction in choroidal blood vessel diameter and interstitial pillars suggesting a defect in vasculogenesis. Total melanin and expression of melanogenesis genes tyr, tryp1a, mitf, dct and pmel were also reduced from 4dpf. In Chmnull/WT mice, choroidal melanosomes were significantly smaller at 1 month, with reduced eumelanin at 1 year. The choroid in CHM patients were also examined using spectral domain optical coherence tomography (SD-OCT) and OCT-angiography (OCT-A) and the area of preserved choriocapillaris (CC) was found to be smaller than that of overlying photoreceptors, suggesting that the choroid is degenerating at a faster rate. Histopathology of an enucleated eye from a 74-year-old CHM male patient revealed isolated areas of RPE but no associated underlying CC. Pigmentary disruptions in CHM animal models reveal an important role for REP1 in melanogenesis, and drugs that improve melanin production represent a potential novel therapeutic avenue.
Subject(s)
Choroideremia , Aged , Animals , Humans , Male , Mice , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Choroid/metabolism , Choroideremia/genetics , Choroideremia/pathology , Choroideremia/therapy , Melanins , Melanogenesis , Mice, KnockoutABSTRACT
Myopia is one of the most prevalent eye diseases that seriously threaten the eyesight of children and adolescents worldwide. However, the pathogenesis is still unclear, and effective drugs are still scarce. In the present study, the guinea pigs were randomly divided into a normal control (NC) group, a lens-induced myopia (LIM) group, a NOS inhibitor (L-NMMA) injection group, and a NOS inhibitor solvent phosphate-buffered saline (PBS) group and the animals received relevant treatments. After 2- and 4-week different treatments, we noted that the refraction and choroidal thickness in the LIM group decreased compared with the NC group, whereas the ocular axial length increased significantly, and the choroid showed a fibrotic trend. The expression of NOS1, NOS3, TGF-ß1, COLI, and α-SMA at gene and protein levels was increased significantly in the choroid (all P < 0.05). After intravitreal injection of NOS inhibitor L-NMMA, we found that compared with the LIM group, the refraction and the choroidal thickness significantly increased, whereas the axial length reduced significantly, accompanied by an increase of choroidal thickness and an improvement of choroidal fibrosis. The expression levels of choroidal NOS1, NOS3, TGF-ß, COLI, and α-SMA were significantly reduced (all P < 0.05). In conclusion, the trend of choroidal fibrosis in LIM guinea pigs is positively correlated with the increase in axial length. The NOS inhibitor L-NMMA can alleviate the process of choroidal fibrosis in myopic guinea pigs by inhibiting NO signaling pathway.