Choroidal Endothelial Cells Research Articles

Light-sensitive Ca2+ signaling in the mammalian choroid

The choroid is the thin, vasculature-filled layer of the eye situated between the sclera and the retina, where it serves the metabolic needs of the light-sensing photoreceptors in the retina. Illumination of the interior surface of the back of the eye (fundus) is a critical regulator of subretinal fluid homeostasis, which determines the overall shape of the eye, but it is also important for choroidal perfusion. Noted for having some of the highest blood flow rates in the body, the choroidal vasculature has been reported to lack intrinsic, intravascular pressure-induced (myogenic) autoregulatory mechanisms. Here, we ask how light directly regulates choroid perfusion and ocular fluid homeostasis, testing the hypothesis that light facilitates ocular fluid absorption by directly increasing choroid endothelial permeability and decreasing choroid perfusion. Utilizing ex vivo pressurized whole-choroid and whole-eye preparations from mice expressing cell-specific Ca2+ indicators, we found that the choroidal vasculature has two intrinsically light-sensitive Ca2+-signaling mechanisms: One increases Ca2+-dependent production of nitric oxide in choroidal endothelial cells; the other promotes vasoconstriction through Ca2+ elevation in vascular smooth muscle cells. In addition, we found that choroidal flow, or pressure, modulates endothelial and smooth muscle photosensitivity and trans-retinal absorption of fluid into the choroid. These results collectively suggest that the choroid vasculature exhibits an inverted form of autoregulatory control, where pressure- and light-induced mechanisms work in opposition to regulate blood flow and maintain fluid balance in response to changes in light and dark, aligning with the metabolic needs of photoreceptors.

Splice Variant of Retinal G-Protein-Coupled Receptor Deletion-Mediated Dysregulation of Autophagy Increases the Susceptibility to Age-Related Macular Degeneration-Like Defects

Introduction: The splice variant of retinal G-protein-coupled receptor deletion (RGR-d) is a persistent component of drusen and may be involved in the pathogenesis of dry age-related macular degeneration (AMD). Increasing evidence has demonstrated the critical role of autophagy in AMD. In this study, we investigated whether RGR-d disrupts autophagy in early dry AMD in vivo and in vitro. Methods: Fundus imaging and fluoroscopy were performed on RGR-d mice created by multiplex gene editing. The retina microstructure was evaluated by performing hematoxylin and eosin (H&E) staining as well as transmission electron microscopy (TEM). Retinal function was assessed by full-field electroretinography (ERG). After lentivirus transfection and stimulation, the permeability, phagocytosis, and tight junctions of ARPE-19 cells were evaluated. Western blotting of ATG5, Beclin-1, LC3II/I, and P62 was performed to detect the changes in autophagy pathways. Results: Atrophy and patchy penetrating hyperfluorescent foci, consistent with early AMD-like defects, were observed in the fundus of 12-month-old RGR-d mice. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. TEM analysis showed some diffuse granular deposits. And the morphology of choroidal microvascular endothelial cells was degraded and distorted. The morphology of the photoreceptor outer segments showed structural damage, and Bruch’s membrane was thickened. ERG indicated that the photoreceptor of RGR-d mice were dysfunctional. Changes in autophagy-related protein expression were observed in the retinal pigment epithelium and retinal neurepithelium, and autophagy regulation was decreased. Palmitic acid (PA) stimulation caused permeability, phagocytosis, and tight junction dysfunction in cells overexpressing RGR-d. Beclin-1 and LC3II/I expression levels were significantly decreased and that of P62 was elevated in RGR-d cells after PA stimulation. Conclusion: RGR-d disrupts the autophagy pathway, causing the development of an early AMD-like pathophysiology.

TLR4/TRIF/Caspase-8/Caspase-1 Pathway in Choroidal Endothelial Cells Promotes Choroidal Neovascularization

Purpose The purpose of this study was to investigate the role and mechanism of caspase-8 in the development of choroidal neovascularization induced by age-related macular degeneration, with the aim of identifying a potential therapeutic target for neovascular age-related macular degeneration. Methods Mouse models of laser photocoagulation-induced choroidal neovascularization and hypoxic human choroidal endothelial cells were utilized to examine the involvement of caspase-8 in choroidal neovascularization development. The toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8 pathway was explored in hypoxic human choroidal endothelial cells to elucidate its contribution to pathological angiogenesis. Various experimental techniques, including inhibition assays and immunoblotting analysis, were employed to assess the effects and mechanisms of caspase-8 activation. Results Inhibition of caspase-8 demonstrated attenuated choroidal neovascularization development in mice subjected to laser photocoagulation. Activation of the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8 pathway was observed in hypoxic human choroidal endothelial cells. Upon activation by the toll-like receptor 4/TIR domain-containing adaptor molecule 1 axis, caspase-8 directly cleaved caspase-1, leading to the cleavage of interleukin-1β and interleukin-18 by caspase-1. Consequently, activation of interleukin-1β and interleukin-18 through the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8/caspase-1 pathway promoted the proliferative, migratory, and tube-forming abilities of hypoxic human choroidal endothelial cells. Conclusion The findings of this study indicate that caspase-8 plays a crucial role in promoting choroidal neovascularization by activating interleukin-1β and interleukin-18 through the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8/caspase-1 pathway in choroidal endothelial cells. Therefore, targeting caspase-8 may hold promise as a therapeutic approach for neovascular age-related macular degeneration.

Mast cells promote choroidal neovascularization in a model of age-related macular degeneration

‘Wet’ age-related macular degeneration (AMD) is characterized by pathologic choroidal neovascularization (CNV) that destroys central vision. Abundant evidence points to inflammation and immune cell dysfunction in the progression of CNV in AMD. Mast cells are resident immune cells that control the inflammatory response. Mast cells accumulate and degranulate in the choroid of patients with AMD, suggesting they play a role in CNV. Activated mast cells secrete various biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. We investigated the role of mast cells in AMD using a model of CNV. Conditioned media from activated mast cells exerts proangiogenic effects on choroidal endothelial cells and choroidal explants. Laser-induced CNV in vivo was markedly attenuated in mice genetically depleted of mast cells (KitW−sh/W−sh) and in wild-type mice treated with mast cell stabilizer, ketotifen fumarate. Tryptase was found to elicit pronounced choroidal endothelial cell sprouting, migration and tubulogenesis; while tryptase inhibition diminished CNV. Transcriptomic analysis of laser-treated RPE/choroid complex revealed collagen catabolism and extracellular matrix (ECM) reorganization as significant events correlated in clusters of mast cell activation. Consistent with these analyses, compared to wildtype mice choroids of laser-treated mast cell-deficient mice displayed less ECM remodelling evaluated using collagen hybridizing peptide tissue binding. Findings herein provide strong support for mast cells as key players in the progression of pathologic choroidal angiogenesis and as potential therapeutic targets to prevent pathological neovascularization in ‘wet’ AMD.

Bim Expression Influences Choroidal Endothelial Cell Characteristics and Their Response to Therapeutic Intervention.

In the aging population, choroidal vessels grow through the Bruch's membrane, resulting in a loss of central vision due to choroidal neovascularization (CNV). During active neovascularization, CNV is associated with inappropriate levels of apoptosis in multiple cell types, including choroidal endothelial cells (ChECs). Bim is a pro-apoptotic member of the Bcl-2 family. It is essential for cell apoptosis due to exposure to drugs such as dexamethasone or decreased pro-survival factors, including vascular endothelial growth factor (VEGF). To better elucidate the cell autonomous contribution of Bim expression in the integrity and neovascularization of the choroidal vasculature, we isolated ChECs from wild-type and Bim-deficient (Bim-/-) mice. ChECs lacking Bim expression demonstrated increased expression of VEGF, osteopontin, and the inflammatory cytokines Rantes/Ccl5 and IL6. Bim-/- ChECs were more proliferative and demonstrated an increased capacity to undergo capillary morphogenesis. Anti-VEGF had a diminished capacity to disrupt capillary morphogenesis in Bim-/- ChECs. In vivo, utilizing the mouse laser photocoagulation model, anti-VEGF treatment mitigated CNV in wild-type but not Bim-/- mice. We also tested other modalities that are thought to not require the intrinsic death pathway for their function and showed that propranolol, anti-CTGF, and the TSP1-mimetic peptide ABT898 mitigated CNV in mice lacking Bim expression to varying degrees. Thus, in ChECs, Bim expression could impact the effectiveness of treatment modalities that require the intrinsic death pathway to mitigate CNV.

CBX7 promotes choroidal neovascularization by activating the HIF-1α/VEGF pathway in choroidal vascular endothelial cells

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

Inactivation of adenosine receptor 2A suppresses endothelial-to-mesenchymal transition and inhibits subretinal fibrosis in mice.

Anti-vascular endothelial growth factor therapy has had a substantial impact on the treatment of choroidal neovascularization (CNV) in patients with neovascular age-related macular degeneration (nAMD), the leading cause of vision loss in older adults. Despite treatment, many patients with nAMD still develop severe and irreversible visual impairment because of the development of subretinal fibrosis. We recently reported the anti-inflammatory and antiangiogenic effects of inhibiting the gene encoding adenosine receptor 2A (Adora2a), which has been implicated in cardiovascular disease. Here, using two mouse models of subretinal fibrosis (mice with laser injury-induced CNV or mice with a deficiency in the very low-density lipoprotein receptor), we found that deletion of Adora2a either globally or specifically in endothelial cells reduced subretinal fibrosis independently of angiogenesis. We showed that Adora2a-dependent endothelial-to-mesenchymal transition contributed to the development of subretinal fibrosis in mice with laser injury-induced CNV. Deficiency of Adora2a in cultured mouse and human choroidal endothelial cells suppressed induction of the endothelial-to-mesenchymal transition. A metabolomics analysis of cultured human choroidal endothelial cells showed that ADORA2A knockdown with an siRNA reversed the increase in succinate because of decreased succinate dehydrogenase B expression under fibrotic conditions. Pharmacological inhibition of ADORA2A with a small-molecule KW6002 in both mouse models recapitulated the reduction in subretinal fibrosis observed in mice with genetic deletion of Adora2a. ADORA2A inhibition may be a therapeutic approach to treat subretinal fibrosis associated with nAMD.

Human Pluripotent Stem Cells Derived Endothelial Cells Repair Choroidal Ischemia.

Choroidal atrophy is a common fundus pathological change closely related to the development of age-related macular degeneration (AMD), retinitis pigmentosa, and pathological myopia. Studies suggest that choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first cells lost in choroidal atrophy. It is found that endothelial cells derived from human pluripotent stem cells (hPSC-ECs) through the MESP1+ mesodermal progenitor stage express CECs-specific markers and can integrate into choriocapillaris. Single-cell RNA-seq (scRNA-seq) studies show that hPSC-ECs upregulate angiogenesis and immune-modulatory and neural protective genes after interacting with ex vivo ischemic choroid. In a rat model of choroidal ischemia (CI), transplantation of hPSC-ECs into the suprachoroidal space increases choroid thickness and vasculature density. Close-up examination shows that engrafted hPSC-ECs integrate with all layers of rat choroidal vessels and last 90 days. Remarkably, EC transplantation improves the visual function of CI rats. The work demonstrates that hPSC-ECs can be used to repair choroidal ischemia in the animal model, which may lead to a new therapy to alleviate choroidal atrophy implicated in dry AMD, pathological myopia, and other ocular diseases.

IL1R2 promotes retinal angiogenesis to participate in retinopathy of prematurity by activating the HIF1α/PFKFB3 pathway

Retinopathy of prematurity (ROP) is the leading cause of blindness in children, but there is no safe and effective treatment available. Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor for IL-1 may affect ROP progression. This study aimed to investigate the role of IL1R2 in ROP. A microglial cell model was established under hypoxia conditions and co-cultured with choroidal endothelial cells, while an oxygen-induced retinopathy (OIR) model was also established. Microglial activation and IL1R2 levels in retinal tissues were analyzed using immunofluorescence assay. Endothelial cell migration was evaluated by Transwell assay and scratch test, angiogenesis was assessed using ELISA and tube formation assay, and proliferation was evaluated by EdU assay. The HIF1α/PFKFB3 pathway was analyzed by western blot. We observed that IL1R2 expression was predicted to be upregulated in ROP and was increased in hypoxia-treated BV2 cells. Additionally, IL1R2 levels were upregulated in the retinal tissues of OIR mice and correlated with microglial activation. In vitro experiments, we found that hypoxia promoted endothelial cell migration, angiogenesis, proliferation, and activated the HIF1α/PFKFB3 pathway, which were rescued by IL1R2 knockdown. Moreover, NHWD-870 (a HIF1α/PFKFB3 pathway inhibitor) suppressed endothelial cell migration, angiogenesis, and proliferation induced by IL1R2 overexpression. In conclusion, IL1R2 facilitates the migration, angiogenesis, and proliferation of choroidal endothelial cells by activating the HIF1α/PFKFB3 pathway to regulate ROP progression.

Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling.

The outer blood-retina barrier (oBRB), comprises tightly connected retinal pigment epithelium (RPE) cells, Bruch's membrane, and choroid blood vessels, and is essential for retinal health and normal visual function. Disruption of the RPE barrier and its dysfunction can lead to retinal disorders such as age-related macular degeneration (AMD). In the present study, we investigated the essential role of choroid endothelial cells (ECs) in the RPE barrier formation process and its dysfunction. We discovered that ECs promoted RPE barrier formation through angiocrine signaling. Through blocking or activating endothelial Notch signaling and conducting experiments in vitro and in vivo, we confirmed that endothelial Notch signaling regulated the expression of heparin-binding epidermal growth factor (HBEGF) and consequently impacted the expression and activity of matrix metalloproteinases (MMP)-9 in RPE cells. This modulation influenced the RPE extracellular matrix deposition, tight junctions and RPE barrier function. In in vivo experiments, the intravitreal administration of recombinant HBEGF (r-HBEGF) alleviated the RPE barrier disruption induced by subretinal injection (SI) or laser treatment and also rescued RPE barrier disruption in endothelial Notch-deficient mice. Our results showed that the endothelial Notch signaling drove HBEGF expression through angiocrine signaling and effectively improved RPE barrier function by regulating the MMP-9 expression in RPE cells. It suggests that the modulation of Notch signaling in the choroidal endothelium may offer a novel therapeutic strategy for retinal degenerative diseases.

Artesunate mitigates choroidal neovascularization and scar formation

Angiogenesis, although required during eye development, has a causative effect in many ocular diseases. Aberrant neovascularization contributes to the progression of neovascular age-related macular degeneration (nAMD), a vision-threaten disease in aging Americans. Since increased amounts of vascular endothelial growth factor (VEGF) drives neovascularization during the pathogenesis of nAMD the standard of care are anti-VEGF therapies attempt to disrupt this vicious cycle. These current anti-VEGF therapies try to maintain vascular homeostasis while abating aberrant neovascularization but regrettably don't prevent fibrosis or scar formation. In addition, some patients demonstrate an incomplete response to anti-VEGF therapy as demonstrated by progressive vision loss. Here, we show choroidal endothelial cells (ChEC) incubated with artesunate demonstrated decreased migration and inflammatory and fibrotic factor expression, which corresponded with decreased sprouting in a choroid/retinal pigment epithelium (RPE) explant sprouting angiogenesis assay. To assess the efficacy of artesunate to curtail neovascularization in vivo, we utilized laser photocoagulation-induced rupture of the Bruch's membrane to induce choroidal neovascularization (CNV). Artesunate significantly inhibited CNV and the accompanying fibrotic scar, perhaps due in part to its ability to inhibit mononuclear phagocyte (MP) recruitment. Thus, artesunate shows promise in inhibiting both CNV and fibrosis.

An In Vitro Bilayer Model of Human Primary Retinal Pigment Epithelial and Choroid Endothelial Cells for Permeability Studies.

The blood-retinal barrier (BRB) present in the posterior chamber of the eye plays a major role in maintaining the proper function and integrity of the retina. Retinal pigment epithelium and choriocapillaris form the outer blood retinal barrier (oBRB), and breakdown of this barrier leads to vision-threatening diseases like macular edema, macular degeneration, and diabetic retinopathy. A simplified cell culture model of oBRB will be of great importance in elucidating the molecular mechanism of the disease progression. This chapter describes methods for primary cell isolation from donor eyes to culture human retinal pigment epithelial cells (hRPE) and choroidal endothelial cells (hCEC) and the protocol for construction of a simplified in vitro model of oBRB on fibronectin-coated Transwell inserts. Further, we explained the permeability study using FITC-dextran conjugated tracers for validating the bilayer model. The permeability experiments ensured that the system could easily be manipulated to recapitulate the pathological condition in vitro. Thus, it would be an optimal system for studying the disease mechanisms related to retinal and choroidal pathologies, for screening small molecules, and for performing drug permeability kinetics. Moreover, fundamental understanding of paracellular and transcellular trafficking of cargo in hRPE and hCEC could also be studied using this model.

Honokiol Attenuates Choroidal Neovascularization by Inhibiting the Hypoxia-Inducible Factor-α/Vascular Endothelial Growth Factor Axis via Nuclear Transcription Factor-Kappa B Activation

Purpose Honokiol is a lignan isolated from Magnolia officinalis and exhibits anti-angiogenic properties. This study was conducted to investigate the role of honokiol in choroidal neovascularization. Methods C57BL/6 mice were treated with honokiol at 10–20 mg/kg by daily intraperitoneal injection from day 1 to 6 after laser photocoagulation. ARPE-19 cells were cultured under hypoxic conditions with or without the presence of honokiol. After laser photocoagulation and honokiol treatment, hematoxylin and eosin staining, immunofluorescence and fundus fluorescein angiography were used to analyze the effect of honokiol on choroidal neovascularization formation. Quantitative real-time PCR, western blot, enzyme-linked immunosorbent assay, immunofluorescence, luciferase assay, and chromatin immunoprecipitation were performed to explore the mechanism of honokiol in the pathological process of choroidal neovascularization. Finally, the role of honokiol on the human choroidal vascular endothelial cells was detected by using 5-ethynyl-20-deoxyuridine assay, Transwell and Tube formation assays. Results The results of hematoxylin and eosin staining and immunofluorescence suggested that honokiol reduced the thickness, length, and area of choroidal neovascularization lesions in laser-induced choroidal neovascularization mouse model. Fundus fluorescein angiography showed that choroidal neovascularization leakage was reduced in honokiol group and the concentration of 20 mg/kg showed better effects. Mechanism studies have shown that honokiol exerted inhibitory effects on choroidal neovascularization by inactivating hypoxia-inducible factor-1α/vascular endothelial growth factor axis through the nuclear transcription factor-kappa B signaling pathway. The same results were obtained in ARPE-19 cells under hypoxic conditions. Furthermore, the conditional medium of retinal pigmented epithelial cells promoted the proliferation, migration, and tube formation of human choroidal vascular endothelial cells, while honokiol reversed these. Conclusion We demonstrated that honokiol attenuated choroidal neovascularization formation by inactivating the hypoxia-inducible factor-1α/vascular endothelial growth factor axis through nuclear transcription factor-kappa B signaling pathway.

Neutrophil extracellular traps boost laser-induced mouse choroidal neovascularization through the activation of the choroidal endothelial cell TLR4/HIF-1α pathway.

Choroidal neovascularization (CNV) is characterized by the infiltration of immune cells, particularly neutrophils. Neutrophil extracellular trap (NET) facilitates the angiogenesis of pulmonary endothelial cells via activating Toll-like receptor 4 (TLR4). TLR4 promotes the expression of transcription factor hypoxia inducible factor-1α (HIF-1α), which promotes inflammation and angiogenesis via the up-regulation of metalloproteinase-9 (MMP-9) and interleukin-1β (IL-1β). In the present study, we aimed to identify the formation of NET and its role in CNV. Our results showed that NET levels were increased in a mouse laser-induced CNV model via oxidative stress, whereas the inhibition of NET alleviated CNV. In vitro, NET activated the TLR4/HIF-1α pathway in human choroidal endothelial cells (HCECs). Additionally, NET increased the transcription and expression of MMP-9 and IL-1β in HCECs via activating the TLR4/HIF-1α pathway. Meanwhile, NET promoted the inflammatory response accompanied by the proliferation, migration and tube formation of HCECs in a MMP-9- and IL-1β-dependent manner. In conclusion, NET was up-regulated in CNV and promoted the formation of CNV via activating the TLR4/HIF-1α pathway in choroidal endothelial cells. Our data uncovered the novel role of NET in promoting the formation of CNV. The underlying mechanism of NET could be targeted to delay the process of CNV.

LncRNA MALAT1 knockdown inhibits the development of choroidal neovascularization

In the pathogenesis of age-related macular degeneration, long non-coding RNAs have become important regulators. This study aimed to investigate the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the progression of choroidal neovascularization (CNV) and the underlying mechanisms. The in vivo and in vitro model of CNV was established using laser-induced mouse CNV model and human choroidal vascular endothelial cells (HCVECs) exposed to hypoxia respectively. We explore the role of MALAT1 in the pathogenesis of CNV by using the small interference RNA both in vivo and in vitro. MALAT1 expression was found to be upregulated in the retinal pigment epithelial-choroidal complexes. MALAT1 knockdown inhibited CNV development and leakage in vivo and decreased HCVECs proliferation, migration, and tube formation in vitro. MALAT1 performed the task as a miR-17-5p sponge to regulate the expression of vascular endothelial growth factor A (VEGFA) and E26 transformation specific-1 (ETS1). This study provides a new perspective on the pathogenesis of CNV and suggests that the axis MALAT/miR-17-5p/VEGFA or ETS1 may be an effective therapeutic target for CNV.

Targeting the cochlin/SFRP1/CaMKII axis in the ocular posterior pole prevents the progression of nonpathologic myopia

Myopia is a major public health issue. However, interventional modalities for nonpathologic myopia are limited due to its complicated pathogenesis and the lack of precise targets. Here, we show that in guinea pig form-deprived myopia (FDM) and lens-induced myopia (LIM) models, the early initiation, phenotypic correlation, and stable maintenance of cochlin protein upregulation at the interface between retinal photoreceptors and retinal pigment epithelium (RPE) is identified by a proteomic analysis of ocular posterior pole tissues. Then, a microarray analysis reveals that cochlin upregulates the expression of the secreted frizzled-related protein 1 (SFRP1) gene in human RPE cells. Moreover, SFRP-1 elevates the intracellular Ca2+ concentration and activates Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling in a simian choroidal vascular endothelial cell line, and elicits vascular endothelial cell dysfunction. Furthermore, genetic knockdown of the cochlin gene and pharmacological blockade of SFRP1 abrogates the reduced choroidal blood perfusion and prevents myopia progression in the FDM model. Collectively, this study identifies a novel signaling axis that may involve cochlin in the retina, SFRP1 in the RPE, and CaMKII in choroidal vascular endothelial cells and contribute to the pathogenesis of nonpathologic myopia, implicating the potential of cochlin and SFRP1 as myopia interventional targets.

Molecular, Cellular, and Functional Heterogeneity of Retinal and Choroidal Endothelial Cells.

To investigate the endothelial heterogeneity across distinct vascular beds in the inner and outer blood-retinal barriers. We evaluated the molecular, cellular, and functional differences between primary human retinal endothelial cells (HRECs) and human choroidal endothelial cells (HCECs) in terms of angiogenic and vasculogenic properties, permeability, and transcytosis. Tube formation assay, cell migration assay, in vitro permeability assay, microfluidic sprouting assay, and transcriptome analysis were performed. HRECs showed higher proliferation and migration activity than did HCECs, whereas the tube formation ability was similar between HRECs and HCECs. Under angiogenic stimuli, HCECs displayed earlier sprouting angiogenesis, but the overall speed was faster and more stable in HRECs. HRECs expressed higher levels of adherens junctional proteins, whereas the tight junctional genes and transcytosis-related genes were more highly expressed in HCECs. Angiopoietin-2 was predominantly expressed in HRECs, but vascular endothelial growth factor (VEGF) receptors were more strongly expressed in HCECs. Platelet-derived growth factor subunit B (PDGFB) was more highly expressed in HRECs, which correlates to the lower degree of pericyte coverage in choroidal blood vessels. Retinal and choroidal ECs showed significant cellular and molecular heterogeneities that correlated with their functional characteristics. Retinal ECs are vasculogenic with high migratory characteristics and faster angiogenic sprouting, and they are more responsive to VEGF-induced permeability. In contrast, choroidal ECs express high levels of transcytosis genes, and they are vasculogenic, rather proliferative, adept in generating tip cells, and less responsive to VEGF-induced permeability.

Exosomal miR-184 in the aqueous humor of patients with central serous chorioretinopathy: a potential diagnostic and prognostic biomarker

BackgroundCentral serous chorioretinopathy (CSC) is the fourth most prevalent retinal disease leading to age-related macular degeneration (AMD) and retinal atrophy. However, CSC's pathogenesis and therapeutic target need to be better understood.ResultsWe investigated exosomal microRNA in the aqueous humor of CSC patients using next-generation sequencing (NGS) to identify potential biomarkers associated with CSC pathogenesis. Bioinformatic evaluations and NGS were performed on exosomal miRNAs obtained from AH samples of 62 eyes (42 CSC and 20 controls). For subgroup analysis, patients were divided into treatment responders (CSC-R, 17 eyes) and non-responders (CSC-NR, 25 eyes). To validate the functions of miRNA in CECs, primary cultured-human choroidal endothelial cells (hCEC) of the donor eyes were utilized for in vitro assays. NGS detected 376 miRNAs. Our results showed that patients with CSC had 12 significantly upregulated and 17 downregulated miRNAs compared to controls. miR-184 was significantly upregulated in CSC-R and CSC-NR patients compared to controls and higher in CSC-NR than CSC-R. In vitro assays using primary cultured-human choroidal endothelial cells (hCEC) demonstrated that miR-184 suppressed the proliferation and migration of hCECs. STC2 was identified as a strong candidate for the posttranscriptional down-regulated target gene of miR-184.ConclusionOur findings suggest that exosomal miR-184 may serve as a biomarker reflecting the angiostatic capacity of CEC in patients with CSC.

Targeting choroidal vasculopathy via up-regulation of tRNA-derived fragment tRF-22 expression for controlling progression of myopia

BackgroundMyopia has emerged as a major public health concern globally, which is tightly associated with scleral extracellular matrix (ECM) remodeling and choroidal vasculopathy. Choroidal vasculopathy has gradually been recognized as a critical trigger of myopic pathology. However, the precise mechanism controlling choroidal vasculopathy remains unclear. Transfer RNA-derived fragments (tRFs) are known as a novel class of small non-coding RNAs that plays important roles in several biological and pathological processes. In this study, we investigated the role of tRF-22-8BWS72092 (tRF-22) in choroidal vasculopathy and myopia progression.MethodsThe tRF-22 expression pattern under myopia-related stresses was detected by qRT-PCR. MTT assays, EdU incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of tRF-22 in choroidal endothelial cell function in vitro. Isolectin B4 staining and choroidal sprouting assay ex vivo were conducted to detect the role of tRF-22 in choroidal vascular dysfunction in vivo. Immunofluorescent staining, western blot assays and ocular biometric parameters measurement were performed to examine whether altering tRF-22 expression in choroid affects scleral hypoxia and ECM remodeling and myopia progression in vivo. Bioinformatics analysis and luciferase activity assays were conducted to identify the downstream targets of tRF-22. RNA-sequencing combined with m6A-qPCR assays were used to identify the m6A modified targets of METTL3. Gain-of-function and Loss-of-function analysis were performed to reveal the mechanism of tRF-22/METTL3-mediated choroidal vascular dysfunction.ResultsThe results revealed that tRF-22 expression was significantly down-regulated in myopic choroid. tRF-22 overexpression alleviated choroidal vasculopathy and retarded the progression of myopia in vivo. tRF-22 regulated choroidal endothelial cell viability, proliferation, migration, and tube formation ability in vitro. Mechanistically, tRF-22 interacted with METTL3 and blocked m6A methylation of Axin1 and Arid1b mRNA transcripts, which led to increased expression of Axin1 and Arid1b.ConclusionsOur study reveals that the intervention of choroidal vasculopathy via tRF-22-METTL3- Axin1/Arid1b axis is a promising strategy for the treatment of patients with myopic pathology.Graphical

Beyond the Complement Cascade: Insights into Systemic Immunosenescence and Inflammaging in Age-Related Macular Degeneration and Current Barriers to Treatment.

Landmark genetic studies have revealed the effect of complement biology and its regulation on the pathogenesis of age-related macular degeneration (AMD). Limited phase 3 clinical trial data showing a benefit of complement inhibition in AMD raises the prospect of more complex mediators at play. Substantial evidence supports the role of para-inflammation in maintaining homeostasis in the retina and choroid. With increasing age, a decline in immune system regulation, known as immunosenescence, has been shown to alter the equilibrium maintained by para-inflammation. The altered equilibrium results in chronic, sterile inflammation with aging, termed 'inflammaging', including in the retina and choroid. The chronic inflammatory state in AMD is complex, with contributions from cells of the innate and adaptive branches of the immune system, sometimes with overlapping features, and the interaction of their secretory products with retinal cells such as microglia and retinal pigment epithelium (RPE), extracellular matrix and choroidal vascular endothelial cells. In this review, the chronic inflammatory state in AMD will be explored by immune cell type, with a discussion of factors that will need to be overcome in the development of curative therapies.