Search Results (15)

Retinal vein occlusion (RVO) is a common vascular disease that leads to vision loss due to macular edema (ME). This study investigated the role of autotaxin (ATX), a lysophospholipase D, in the pathogenesis of RVO. In mice, RVO was induced by intravenous administration of rose bengal followed by laser irradiation of retinal veins. ATX expression in the retina was evaluated using immunohistochemistry. Intravitreal ATX was administered, and retinal changes were assessed using fluorescence angiography and optical coherence tomography (OCT). In human retinal microvascular endothelial cells (HRMECs), intercellular barrier function was evaluated using transepithelial electrical resistance (TEER). In the murine RVO model, the ATX inhibitor HA130 was administered intravitreally, and retinal thickness was measured and compared using OCT. ATX expression was increased in retinal vessels in the RVO model. Intravitreal administration of ATX induced retinal edema and serous retinal detachment (SRD). ATX significantly disrupted the barrier integrity of HRMECs and promoted the expression of vascular endothelial growth factor (VEGF), which was ameliorated by HA130. Intravitreal administration of HA130 significantly reduced retinal thickening caused by retinal edema secondary to RVO and the elevated expression of intercellular adhesion molecule (ICAM)-1 in the retina. These findings suggest that ATX plays a critical role in RVO-induced ME by disrupting endothelial barrier integrity, potentially through the upregulation of VEGF in retinal endothelial cells and subsequent ICAM-1 upregulation in the retina. Full article

Background: Diabetic retinopathy (DR), a leading cause of blindness, lacks early biomarkers and mechanism-targeted therapies. While oxidative stress drives DR pathogenesis, the role of oxeiptosis—a reactive oxygen species-induced, caspase-independent cell death pathway—remains largely unexplored. Methods: We integrated transcriptomic profiling (GSE221521: 69 DR vs. 50 controls), two-sample Mendelian randomization (MR) using blood cis-eQTLs (GTEx) as instruments and DR GWAS (FinnGen R12) as outcome, machine learning-based feature selection (SVM-RFE and Boruta algorithms), and single-cell RNA sequencing (scRNA-seq) analysis (GSE165784). Functional enrichment, immune deconvolution (CIBERSORT), and diagnostic nomogram construction were performed. We validated the key genes using human retinal microvascular endothelial cells (hRMECs) treated with high glucose (30 mM). Results: Oxeiptosis scores were elevated in DR blood samples (p < 0.001). MR analysis identified five putative causal genes: CASP2 (OR = 1.067), PLEC (OR = 1.035) and FBN2 (OR = 1.016) as risk factors, and CYP27A1 (OR = 0.960) and GPD2 (OR = 0.958) as protective factors. SVM-RFE and Boruta algorithms confirmed CASP2 and PLEC as hub genes. A nomogram incorporating both genes achieved robust DR prediction (AUC = 0.811). Functional analysis associated these genes with innate immune activation and extracellular matrix reorganization. Single-cell transcriptomics revealed PLEC was markedly overexpressed in disease-relevant cells (fibroblasts, endothelial cells), whereas CASP2 exhibited a distinct pattern, with notable enrichment in retinal CD8+ T cells. Both genes were associated with a pro-inflammatory shift in the immune landscape. Their upregulation was validated in independent datasets and high-glucose-stressed retinal cells. Conclusions: This study establishes an integrated multi-omics framework implicating oxeiptosis-related pathways in DR and nominates CASP2 and PLEC as putatively causal, biologically relevant candidate biomarkers and potential therapeutic targets. Full article

We previously observed elevated plasma levels of citrulline and arginine in diabetic retinopathy patients compared to diabetic controls. We tested our hypothesis that citrulline plus arginine induces angiogenesis and increases permeability in retinal endothelial cells. Human retinal microvascular endothelial cells (HRMECs) were treated with citrulline, arginine, or citrulline + arginine, and angiogenesis was measured with cell proliferation, migration, and tube formation assays. Permeability was measured in HRMEC monolayers via trans-endothelial electrical resistance (TEER) and FITC-labeled dextran. We also measured arginase activity, arginase-1 and arginase-2 expression, protein expression and phosphorylation of endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) production. Citrulline + arginine induced endothelial cell proliferation (p = 0.018), migration (p = 0.011), and tube formation (p = 0.0042). Citrulline + arginine also increased FITC-dextran flow-through (p = 1.5 × 10⁻⁵) and decreased TEER (p = 0.010). Citrulline + arginine had no effect on arginase activity, but it increased eNOS (p = 6.3 × 10⁻⁴) and phosphorylated eNOS (p = 0.029), as well as NO production (p = 0.025). Inhibiting eNOS prevented the increase in NO (p = 0.0092), inhibited citrulline + arginine-induced cell migration (p = 0.0080) and tube formation (p = 0.0092), and blocked citrulline + arginine-related alterations in FITC-dextran flow-through (p = 3.6 × 10⁻⁴) and TEER (p = 3.9 × 10⁻⁴). These data suggest that citrulline + arginine treatment induces angiogenesis and increases permeability in retinal endothelial cells by activating eNOS and increasing NO production. Full article

The protease, a disintegrin and metalloproteinase with thrombospondin type 1 motif member 13 (ADAMTS13), known to cleave only the von Willebrand factor (VWF), has powerful regulatory effects on microvascular platelet adhesion, thrombosis, inflammation, and endothelial dysfunction. We study the protection against diabetes-induced retinal injury in experimental rats by supplementation with recombinant ADAMTS13. We compare human epiretinal membranes and vitreous samples from nondiabetic subjects and patients with proliferative diabetic retinopathy (PDR) and extend in vitro analyses with the use of various immunodetection and spectrofluorimetric methods on rat retina and human retinal glial and endothelial cell cultures. Functional studies include the assessment of the blood–retinal barrier (BRB), cell adhesion, and in vitro angiogenesis. In epiretinal membranes, endothelial cells and monocytes/macrophages express ADAMTS13. The levels of VWF, the platelet marker CD41, ADAMTS13, and the biomarkers of endothelial cell injury soluble VE-cadherin and soluble syndecan-1 are increased in PDR vitreous. ADAMTS13 is downregulated in diabetic rat retinas. The intravitreal administration of ADAMTS13 attenuates diabetes-induced BRB breakdown, the downregulation of VE-cadherin and β-catenin, and the upregulation of VWF, CD41, phospho-ERK1/2, HMGB1, VCAM-1, and ICAM-1. In Müller cells, ADAMTS13 attenuates MCP-1, MMP-9, and ROS upregulation induced by diabetic mimetic conditions. In HRMECs, ADAMTS13 attenuates the shedding of the soluble VE-cadherin and soluble syndecan-1 and the levels of phospho-ERK1/2, MCP-1, fractalkine, and ROS induced by diabetic mimetic conditions, the upregulation of ICAM-1 and VCAM-1 elicited by TNF-α, the adherence of monocytes induced by TNF-α, and VEGF-induced migration of human retinal microvascular endothelial cells. Our findings suggest that enhancing ADAMTS13 levels in situ ameliorates diabetes-induced retinal inflammation and vascular dysfunction. Full article

We aimed to investigate the role of the CD40-CD40 ligand (CD40L) pathway in inflammation-mediated angiogenesis in proliferative diabetic retinopathy (PDR). We analyzed vitreous fluids and epiretinal fibrovascular membranes from PDR and nondiabetic patients, cultures of human retinal microvascular endothelial cells (HRMECs) and Müller glial cells and rat retinas with ELISA, immunohistochemistry, flow cytometry and Western blot analysis. Functional tests included measurement of blood–retinal barrier breakdown, in vitro angiogenesis and assessment of monocyte-HRMEC adherence. CD40L and CD40 levels were significantly increased in PDR vitreous samples. We demonstrated CD40L and CD40 expression in vascular endothelial cells, leukocytes and myofibroblasts in epiretinal membranes. Intravitreal administration of soluble (s)CD40L in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, VEGF, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). sCD40L induced upregulation of VEGF, MMP-9, MCP-1 and HMGB1 in cultured Müller cells and phospo-ERK1/2, p65 subunit of NF-ĸB, VCAM-1 and VEGF in cultured HRMECS. TNF-α induced significant upregulation of CD40 in HRMECs and Müller cells and VEGF induced significant upregulation of CD40 in HRMECs. sCD40L induced proliferation and migration of HRMECs. We provide experimental evidence supporting the involvement of the CD40L-CD40 pathway and how it regulates inflammatory angiogenesis in PDR. Full article

Inflammation and fibrosis are key features of proliferative vitreoretinal disorders. We aimed to define the macrophage phenotype and investigate the role of macrophage-myofibroblast transition (MMT) in the contribution to myofibroblast populations present in epiretinal membranes. Vitreous samples from proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and nondiabetic control patients, epiretinal fibrovascular membranes from PDR patients and fibrocellular membranes from PVR patients, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by ELISA, immunohistochemistry and flow cytometry analysis. Myofibroblasts expressing α-SMA, fibroblast activation protein-α (FAP-α) and fibroblast-specific protein-1 (FSP-1) were present in all membranes. The majority of CD68⁺ monocytes/macrophages co-expressed the M2 macrophage marker CD206. In epiretinal membranes, cells undergoing MMT were identified by co-expression of the macrophage marker CD68 and myofibroblast markers α-SMA and FSP-1. Further analysis revealed that CD206⁺ M2 macrophages co-expressed α-SMA, FSP-1, FAP-α and ß-catenin. Soluble (s) CD206 and sFAP-α levels were significantly higher in vitreous samples from PDR and PVR patients than in nondiabetic control patients. The proinflammatory cytokine TNF-α and the hypoxia mimetic agent cobalt chloride induced upregulation of sFAP-α in culture media of Müller cells but not of HRMECs. The NF-ĸß inhibitor BAY11-7085 significantly attenuated TNF-α-induced upregulation of sFAP-α in Müller cells. Our findings suggest that the process of MMT might contribute to myofibroblast formation in epiretinal membranes, and this transition involved macrophages with a predominant M2 phenotype. In addition, sFAP-α as a vitreous biomarker may be derived from M2 macrophages transitioned to myofibroblasts and from Müller cells. Full article

Background: Retinal ischemia-reperfusion (I/R) injury often results in intractable visual impairments. The survival of retinal capillary endothelial cells is crucial for the treatment of retinal I/R injury. How to protect retinal endothelia from damage is a challenging work. Withaferin A, a small molecule derived from plants, has antibacterial and anti-inflammatory effects and has been used for about millennia in traditional medicine. The present study aimed to investigate the potential protective effect of withaferin A on retinal I/R injury. Methods: The drug-likeness of withaferin A was evaluated by the SwissADME web tool. The potential protective effect of withaferin A on the I/R-induced injury of human retinal microvascular endothelial cells (HRMECs) was investigated using multiple approaches. RNA sequencing was performed and associated mechanistic signaling pathways were analyzed based on the Kyoto Encyclopedia of Genes and Genomes data. The analytical results of RNA sequencing data were further validated by in vitro and in vivo experiments. Results: Withaferin A reduced the I/R injury-induced apoptotic death of HRMECs in vitro with a good drug-like property. RNA sequencing and experimental validation results indicated that withaferin A increased the production of the crucial antioxidant molecules heme oxygenase 1 (HO-1) and peroxiredoxin 1 (Prdx-1) during I/R. In addition, withaferin A activated the Akt signaling pathway and increased the expression of HO-1 and Prdx-1, thereby exerting an antioxidant effect, attenuated the retinal I/R injury, and decreased the apoptosis of HRMECs. The blockade of Akt completely abolished the effects of withaferin A. Conclusions: The study identified for the first time that withaferin A can protect against the I/R-induced apoptosis of human microvascular retinal endothelial cells via increasing the production of the antioxidants Prdx-1 and HO-1. Results suggest that withaferin A is a promising drug candidate for the treatment of retinal I/R injury. Full article

We analyzed the expression of ADAMTS proteinases ADAMTS-1, -2, -4, -5 and -13; their activating enzyme MMP-15; and the degradation products of proteoglycan substrates versican and biglycan in an ocular microenvironment of proliferative diabetic retinopathy (PDR) patients. Vitreous samples from PDR and nondiabetic patients, epiretinal fibrovascular membranes from PDR patients, rat retinas, retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied. The levels of ADAMTS proteinases and MMP-15 were increased in the vitreous from PDR patients. Both full-length and cleaved activation/degradation fragments of ADAMTS proteinases were identified. The amounts of versican and biglycan cleavage products were increased in vitreous from PDR patients. ADAMTS proteinases and MMP-15 were localized in endothelial cells, monocytes/macrophages and myofibroblasts in PDR membranes, and ADAMTS-4 was expressed in the highest number of stromal cells. The angiogenic activity of PDR membranes correlated significantly with levels of ADAMTS-1 and -4 cellular expression. ADAMTS proteinases and MMP-15 were expressed in rat retinas. ADAMTS-1 and -5 and MMP-15 levels were increased in diabetic rat retinas. HRMECs and Müller cells constitutively expressed ADAMTS proteinases but not MMP-15. The inhibition of NF-κB significantly attenuated the TNF-α-and-VEGF-induced upregulation of ADAMTS-1 and -4 in a culture medium of HRMECs and Müller cells. In conclusion, ADAMTS proteinases, MMP-15 and versican and biglycan cleavage products were increased in the ocular microenvironment of patients with PDR. Full article

The pathophysiology of retinopathy of prematurity (ROP) is postulated to first involve delayed intraretinal vascularization, followed by intravitreal neovascularization (IVNV). Although intravitreal agents that reduce the bioactivity of vascular endothelial growth factor (VEGF) are used to treat IVNV, concerns exist regarding their effects on intraretinal vascularization. In an experimental ROP model, VEGF receptor 2 (VEGFR2) knockdown in retinal endothelial cells reduced IVNV and promoted intraretinal vascularization, whereas knockdown of a downstream effector, signal transducer and activator of transcription 3 (STAT3) in retinal endothelial cells only reduced IVNV. In this study, we tested the hypothesis that the different pathways involved in VEGF-triggered VEGFR2 signaling and VEGF-triggered STAT3 signaling in retinal endothelial cells would allow us to delineate signaling pathways involved in IVNV from those involved in intraretinal vascularization in ROP. To address our hypothesis, we used RNA-sequencing and pathway enrichment analysis to determine changes in the transcriptome of cultured human retinal microvascular endothelial cells (HRMECs). Of the enriched pathways, inactivation of oncostatin M signaling was predicted by either KDR or STAT3 knockdown in the presence of VEGF. Activation of kinetochore metaphase signaling was predicted by KDR knockdown, whereas inactivation was predicted by STAT3 knockdown in the presence of VEGF. Inactivation of signaling by the Rho family of GTPases was predicted by KDR knockdown, but activation was predicted by STAT3 knockdown in the presence of VEGF. Taken together, our data identified unique signaling pathway differences between VEGF-triggered VEGFR2 and VEGF-triggered STAT3 in HRMECs that might have implications in ROP. Full article

Choroidal neovascularization (CNV) is a major cause of visual impairment that results from excessive growth of blood vessels in the eye’s choroid. The limited clinical efficacy of the current therapy for this condition requires the emergence of new treatment modalities such as microRNA (miRNAs). A recent study identified microRNA-539-5p (miR-539) as an angiogenic suppressor in a CNV animal model; however, its therapeutic delivery is limited. Therefore, this study aims to formulate miR-539 in targeted nanoparticles (NPs) prepared from polylactic-co-glycolic acid (PLGA). The NPs were decorated with internalizing arginylglycylaspartic (RGD) peptide (iRGD), which specifically targets the alpha-v-beta-3 (αvβ3) integrin receptor that is overexpressed in blood vessels of ocular tissue in CNV patients. The ¹H NMR spectra results revealed successful conjugation of iRGD peptide into PLGA NPs. The miR-539-PLGA.NPs and miR-539-iRGD-PLGA.NPs were prepared and showed a particle size of 300 ± 3 and 306.40 ± 4 nm, respectively. A reduction in human retinal microvascular endothelial cell (HRMEC) viability was shown 48 and 72 h post transfection with miR-539 incorporated in PLGA NPs and iRGD-PLGA.NPs. iRGD-functionalized PLGA NPs caused further significant reduction in cell viability when compared with plain ones, revealing an enhancement in the NP uptake with iRGD-grafted NPs. The current study showed that miR-539-PLGA.NPs and miR-539-iRGD-PLGA.NPs are promising approaches that reduced the viability of HRMECs, suggesting their therapeutic potential in the treatment of CNV. Full article

Neovascular age-related macular degeneration (nAMD) featuring choroidal neovascularization (CNV) is the principal cause of irreversible blindness in elderly people in the world. Integrated stress response (ISR) is one of the intracellular signals to be adapted to various stress conditions including endoplasmic reticulum (ER) stress. ISR signaling results in the upregulation of activating transcription factor 4 (ATF4), which is a mediator of ISR. Although recent studies have suggested ISR contributes to the progression of some age-related disorders, the effects of ATF4 on the development of CNV remain unclear. Here, we performed a murine model of laser-induced CNV and found that ATF4 was highly expressed in endothelial cells of the blood vessels of the CNV lesion site. Exposure to integrated stress inhibitor (ISRIB) reduced CNV formation, vascular leakage, and the upregulation of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)-choroid-sclera complex. In human retinal microvascular endothelial cells (HRMECs), ISRIB reduced the level of ATF4 and VEGF induced by an ER stress inducer, thapsigargin, and recombinant human VEGF. Moreover, ISRIB decreased the VEGF-induced cell proliferation and migration of HRMECs. Collectively, our findings showed that pro-angiogenic effects of ATF4 in endothelial cells may be a potentially therapeutic target for patients with nAMD. Full article

The aim of the current study was to investigate the impact of long-acting fibroblast growth factor 21 (FGF21) on retinal vascular leakage utilizing machine learning and to clarify the mechanism underlying the protection. To assess the effect on retinal vascular leakage, C57BL/6J mice were pre-treated with long-acting FGF21 analog or vehicle (Phosphate Buffered Saline; PBS) intraperitoneally (i.p.) before induction of retinal vascular leakage with intravitreal injection of mouse (m) vascular endothelial growth factor 164 (VEGF164) or PBS control. Five hours after mVEGF164 injection, we retro-orbitally injected Fluorescein isothiocyanate (FITC) -dextran and quantified fluorescence intensity as a readout of vascular leakage, using the Image Analysis Module with a machine learning algorithm. In FGF21- or vehicle-treated primary human retinal microvascular endothelial cells (HRMECs), cell permeability was induced with human (h) VEGF165 and evaluated using FITC-dextran and trans-endothelial electrical resistance (TEER). Western blots for tight junction markers were performed. Retinal vascular leakage in vivo was reduced in the FGF21 versus vehicle- treated mice. In HRMECs in vitro, FGF21 versus vehicle prevented hVEGF-induced increase in cell permeability, identified with FITC-dextran. FGF21 significantly preserved TEER compared to hVEGF. Taken together, FGF21 regulates permeability through tight junctions; in particular, FGF21 increases Claudin-1 protein levels in hVEGF-induced HRMECs. Long-acting FGF21 may help reduce retinal vascular leakage in retinal disorders and machine learning assessment can help to standardize vascular leakage quantification. Full article

Retinal apoptosis plays a critical role in the progression of diabetic retinopathy (DR), a common diabetic complication. Currently, the tight control of blood glucose levels is the standard approach to prevent or delay the progression of DR. However, prevalence of DR among diabetic patients remains high. Focusing on natural nutrients or herbal medicines that can prevent or delay the onset of diabetic complications, we administered an ethanol extract of the aerial portion of Osteomeles schwerinae (OSSCE), a Chinese herbal medicine, over a period of 17 weeks to spontaneously diabetic Torii (SDT) rats. OSSCE was found to ameliorate retinal apoptosis through the regulation of advanced glycation end product (AGE) accumulation, oxidative stress, and mitochondrial function via the inhibition of NF-κB activity, in turn, through the downregulation of PKCδ, P47phox, and ERK1/2. We further demonstrated in 25 mM glucose-treated human retinal microvascular endothelial cells (HRMECs) that hyperoside (3-O-galactoside-quercetin), quercitrin (3-O-rhamnoside-quercetin), and 2″-O-acetylvitexin (8-C-(2″-O-acetyl-glucoside)-apigenin) were the active components of OSSCE that mediated its pharmacological action. Our results provide evidence that OSSCE is a powerful agent that may directly mediate a delay in the development or disease improvement in patients of DR. Full article

Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1β and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in connexin43 or GFAP expression in Akita but higher expression in Akimba compared to wild-type mice. On PLVAP-positive vessels, connexin43 was higher in Akimba but unchanged in Akita compared to wild-type mice. Connexin43 expression appeared higher in donor retinas with confirmed DR compared to age-matched controls, similar to the distribution seen in Akimba mice and correlating with the in vitro results. Although connexin43 expression seems reduced in diabetes, hyperglycemia and inflammation present in the pathology of DR seem to increase connexin43 expression, suggesting a causal role of connexin43 channels in the disease progression. Full article

Epigalloccatechin-3-gallate (EGCG) is the main polyphenol component of green tea (leaves of Camellia sinensis). EGCG is known for its antioxidant, anti-inflammatory, antiviral, and anti-carcinogenic properties. Here, we identify EGCG as a new inhibitor of ocular angiogenesis and its vascular permeability. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a key role in the processes of extracellular matrix (ECM) remodeling and microvascular permeability during angiogenesis. We investigated the inhibitory effects of EGCG on ocular neovascularization and vascular permeability using the retina oriented cells and animal models induced by VEGF and alkaline burn. EGCG treatment significantly decreased mRNA and protein expression levels of MMP-9 in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and tumor necrosis factor alpha (TNF-α) in human retinal pigment epithelial cells (HRPECs). EGCG also effectively protected ARPE-19 cells from cell death and attenuated mRNA expressions of key angiogenic factors (MMP-9, VEGF, VEGF Receptor-2) by inhibiting generation of reactive oxygen species (ROS). EGCG significantly inhibited proliferation, vascular permeability, and tube formation in VEGF-induced human retinal microvascular endothelial cells (HRMECs). Furthermore, EGCG significantly reduced vascular leakage and permeability by blood-retinal barrier breakdown in VEGF-induced animal models. In addition, EGCG effectively limited upregulation of MMP-9 and platelet endothelial cell adhesion molecule (PECAM/CD31) on corneal neovascularization (CNV) induced by alkaline burn. Our data suggest that MMP-9 and VEGF are key therapeutic targets of EGCG for treatment and prevention of ocular angiogenic diseases such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization. Full article