Retinal Vascular Inflammation Research Articles

MDSCs promote pathological angiogenesis in ocular neovascular disease

BackgroundOcular neovascular diseases, which contribute significantly to vision loss, lack effective preventive treatments. Recent studies have highlighted the significant involvement of immune cells in neovascular retinopathy. Myeloid-derived suppressor cells (MDSCs) promote the development of neovascularization, but it is unknown whether they participate in pathological neovascularization and whether they are expected to be a therapeutic target. MethodWe investigated the role of MDSCs in promoting pathological angiogenesis using an oxygen-induced retinopathy (OIR) model, employing flow cytometry, immunofluorescence, and smart-seq analysis. Then, we evaluated the proportion of MDSCs in patient blood samples using flow cytometry. Additionally, we assessed the effect of MDSC depletion using an anti-Gr-1 monoclonal antibody on retinal vasculopathy and alterations in retinal microglia. ResultsIn the OIR model, an elevated ratio of MDSCs was observed in both blood and retinal tissue during phase II (Neovascularization). The depletion of MDSCs resulted in reduced retinal neovascularization and vaso-obliteration, along with a decrease in microglia within the neovascularization area. Furthermore, analysis of gene transcripts associated with MDSCs indicated activation of vascular endothelial growth factor (VEGF) regulation and inflammation. Importantly, infants with ROP exhibited a higher proportion of MDSCs in their blood samples. ConclusionOur results suggested that excessive MDSCs represent an unrecognized feature of ocular neovascular diseases and be responsible for the retinal vascular inflammation and angiogenesis, providing opportunities for new therapeutic approaches to ocular neovascular disease.

Defining Retinal Vasculitis

PurposeTo assess the validity of retinal vasculitis as the preferred diagnostic term for multiple conditions. DesignPerspective MethodsExpert opinion and review of literature focused on the current nosology and pathology of retinal vasculitis. Interpretation of the subset of intraocular inflammation named retinal vasculitis based on fundamental knowledge of the blood-retinal barrier, the neurovascular unit and pathological and functional responses to a variety of stimuli. Correlation with multimodal imaging and known mechanisms of immunologically mediated disease. ResultsA search of Medline in early 2024 for the phrase “retinal vasculitis” resulted in 2041 citations encompassing immunologic, genetic, neoplastic, infectious, drug- and ischemia-related disorders. Classification schemes and angiographic grading systems are descriptive and do not address pathologic mechanisms adequately, in part due to lack of histologic confirmation. Although OCT angiography holds promise for better imaging of retinal vascular changes, it does not reveal the key feature of leakage and only partially improves understanding of pathophysiology. Diagnosing catastrophic retinal vascular occlusion after intravitreal injections as a retinal vasculitis is the most recent example of speculative application of the term to complex and rare disorders. ConclusionsRetinal vasculitis is a diagnostic term that is over-used and imprecise. Revised nosology should limit the term to primary inflammation of the retinal vasculature itself that results in opening of the blood-retinal barrier with or without retinal vascular occlusions. Pending new histologic or mechanistic evidence, the provisional term of retinal vascular inflammation or retinal vasculopathy should be used for leakage or occlusion occurring in the context of intraocular inflammation.

1809-LB: Hyperglycemia Activated the STING Pathway to Induce Retinal Vascular Endothelial Inflammation and Breakdown of Retinal Tight Junctions

1809-LB: Hyperglycemia Activated the STING Pathway to Induce Retinal Vascular Endothelial Inflammation and Breakdown of Retinal Tight Junctions

Intravitreal faricimab as an adjuvant in the management of tubercular uveitis.

To report the use of intravitreal faricimab used as an adjunct in the management of intraocular tuberculosis (IOTB) in a patient with occlusive retinal vasculitis (ORV) and choroiditis. A case report. A 27-year-old Asian male presented with bilateral choroiditis and ORV complicated by retinal neovascularization and vitreous hemorrhage in one eye. The patient had a positive tuberculin skin test, indeterminate interferon-gamma release assay, and Ghon's focus on chest radiography. With a diagnosis of IOTB, the patient was treated with anti-tubercular therapy. Due to the significant vitreous hemorrhage, he underwent pars plana vitrectomy in one eye. The patient received bilateral faricimab. Within a week of the injection, he had a near-complete resolution of the extensive choroidal and retinal vascular inflammation. Intravitreal faricimab, given its angiopoietin-2 (ang2) inhibition and anti-vascular endothelial growth factor action, maybe a helpful adjunct in IOTB. Whether ang2 inhibition provides any additional benefit needs further evaluation in extensive studies.

Mechanical Regulation of Retinal Vascular Inflammation and Degeneration in Diabetes.

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of collagen-crosslinking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor β-Aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular ICAM-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.

Pharmacological Modulation of β-Catenin Preserves Endothelial Barrier Integrity and Mitigates Retinal Vascular Permeability and Inflammation.

Compromised blood-retinal barrier (BRB) integrity is a significant factor in ocular diseases like uveitis and retinopathies, leading to pathological vascular permeability and retinal edema. Adherens and tight junction (AJ and TJ) dysregulation due to retinal inflammation plays a pivotal role in BRB disruption. We investigated the potential of ICG001, which inhibits β-catenin-mediated transcription, in stabilizing cell junctions and preventing BRB leakage. In vitro studies using human retinal endothelial cells (HRECs) showed that ICG001 treatment improved β-Catenin distribution within AJs post lipopolysaccharide (LPS) treatment and enhanced monolayer barrier resistance. The in vivo experiments involved a mouse model of LPS-induced ocular inflammation. LPS treatment resulted in increased albumin leakage from retinal vessels, elevated vascular endothelial growth factor (VEGF) and Plasmalemmal Vesicle-Associated Protein (PLVAP) expression, as well as microglia and macroglia activation. ICG001 treatment (i.p.) effectively mitigated albumin leakage, reduced VEGF and PLVAP expression, and reduced the number of activated microglia/macrophages. Furthermore, ICG001 treatment suppressed the surge in inflammatory cytokine synthesis induced by LPS. These findings highlight the potential of interventions targeting β-Catenin to enhance cell junction stability and improve compromised barrier integrity in various ocular inflammatory diseases, offering hope for better management and treatment options.

Autosomal dominant neovascular inflammatory vitreoretinopathy with CAPN5 c.731T > C gene mutation; clinical management of a family cohort and review of the literature

ABSTRACT Background To report a cohort of patients with clinically and genetically diagnosed autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) and showcase the spectrum of the disease utilizing multimodal imaging and genetic testing. Additionally, the utility of multimodal imaging in guiding treatment will also be illustrated. Materials/Methods Five patients from a single-family pedigree in Ohio with clinical signs of ADNIV were evaluated. Medical history, family history, and complete ocular examinations were obtained during regular clinic visits. Multimodal imaging including ocular coherence tomography, fluorescein angiography, wide-field fundus photographs, and Humphrey visual field testing was obtained for all five patients. Additionally, genetic testing for the Calpain-5 (CAPN5) gene was conducted on all patients. Results All five patients were noted to have a CAPN5 c.731T > C (p.L244P) mutation on genetic testing. Using multimodal imaging to supplement the clinical examination, pathologic changes such as retinal vascular inflammation, macular edema, and tractional retinal membranes were well illustrated and monitored over time. This allowed for earlier intervention when appropriate such as with intraocular steroid or systemic anti-inflammatory treatments. Conclusion Phenotypic presentation varied among patients in this series, but is consistent with the spectrum of pathologic changes previously described in patients with other CAPN5 gene mutations. Monitoring of patients with ADNIV utilizing multimodal imaging can help better assess progression of this disease and guide treatment decisions. Additionally, increased genetic testing in patients with inherited retinal diseases may reveal novel gene mutations that could serve as potential targets for future genetic treatment regimens.

TGR5 supresses cGAS/STING pathway by inhibiting GRP75-mediated endoplasmic reticulum-mitochondrial coupling in diabetic retinopathy

Diabetic retinopathy (DR) is a serious and relatively under-recognized complication of diabetes. Müller glial cells extend throughout the retina and play vital roles in maintaining retinal homeostasis. Previous studies have demonstrated that TGR5, a member of the bile acid-activated GPCR family, could ameliorate DR. However, the role of TGR5 in regulating Müller cell function and the underlying mechanism remains to be ascertained. To address this, high glucose (HG)-treated human Müller cells and streptozotocin-treated Sprague-Dawley rats were used in the study. The IP3R1-GRP75-VDAC1 axis and mitochondrial function were assessed after TGR5 ablation or agonism. Cytosolic mitochondrial DNA (mtDNA)-mediated cGAS-STING activation was performed. The key markers of retinal vascular leakage, apoptosis, and inflammation were examined. We found that mitochondrial Ca2+ overload and mitochondrial dysfunction were alleviated by TGR5 agonist. Mechanically, TGR5 blocked the IP3R1-GRP75-VDAC1 axis mediated Ca2+ efflux from the endoplasmic reticulum into mitochondria under diabetic condition. Mitochondrial Ca2+ overload led to the opening of the mitochondrial permeability transition pore and the release of mitochondrial DNA (mtDNA) into the cytosol. Cytoplasmic mtDNA bound to cGAS and upregulated 2’3’ cyclic GMP-AMP. Consequently, STING-mediated inflammatory responses were activated. TGR5 agonist prevented retinal injury, whereas knockdown of TGR5 exacerbated retinal damage in DR rats, which was rescued by the STING inhibitor. Based on the above results, we propose that TGR5 might be a novel therapeutic target for the treatment of DR.

Neuroprotection of SRT2104 in Murine Ischemia/Reperfusion Injury Through the Enhancement of Sirt1-Mediated Deacetylation.

Strategies for neuroprotection are the main targets of glaucoma research. The neuroprotective properties of SRT2104 administration have been proven in central nervous system degeneration diseases through the activation of nicotinamide adenine dinucleotide-dependent deacetylase-silence information regulator 1 (Sirt1). Here, we investigated whether SRT2104 could protect the retina from ischemia/reperfusion (I/R) injury and the underlying mechanisms. SRT2104 was intravitreally injected immediately after I/R induction. RNA and protein expression were detected by quantitative real-time PCR and Western blot. Protein expression and distribution were examined by immunofluorescence staining. Retinal structure and function were analyzed by hematoxylin and eosin staining, optical coherence tomography, and electroretinogram. Optic nerve axons were quantified using toluidine blue staining. Cellular apoptosis and senescence were evaluated by TUNEL assay and SA-β-gal staining. The protein expression of Sirt1 decreased dramatically after I/R injury and SRT2104 administration effectively enhanced the stability of Sirt1 protein without significantly influencing Sirt1 mRNA synthesis. SRT2104 administration alone exerted no influence on the structure and function of normal retinas. However, SRT2104 intervention significantly protected the inner retinal structure and neurons; partially restored retinal function after I/R injury. I/R-induced cellular apoptosis and senescence were effectively alleviated by SRT2104 administration. Additionally, SRT2104 intervention markedly reduced neuroinflammation, including reactive gliosis, retinal vascular inflammation, and the overexpression of pro-inflammatory cytokines after I/R injury. Mechanistically, I/R-induced acetylation of p53, NF-κB p65, and STAT3 was significantly reversed by SRT2104 intervention. We demonstrated that SRT2104 exerted potent protective effects against I/R injury by enhancing Sirt1-mediated deacetylation and suppressing apoptosis, senescence, and neuroinflammation-related pathways.

Sustained therapeutic effect of an anti-inflammatory peptide encapsulated in nanoparticles on ocular vascular leakage in diabetic retinopathy.

Pigment epithelium-derived factor (PEDF), an endogenous Wnt signaling inhibitor in the serine proteinase inhibitors (SERPIN) super family, is present in multiple organs, including the vitreous. Significantly low levels of PEDF in the vitreous are found to associate with pathological retinal vascular leakage and inflammation in diabetic retinopathy (DR). Intravitreal delivery of PEDF represents a promising therapeutic approach for DR. However, PEDF has a short half-life after intravitreal injection, which represents a major hurdle for the long-term treatment. Here we report the prolonged therapeutic effects of a 34-mer peptide of the PEDF N-terminus, encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (PEDF34-NP), on DR. PEDF34-NP inhibited hypoxia-induced expression of vascular endothelial growth factor and reduced levels of intercellular adhesion molecule 1 (ICAM-1) in cultured retinal cells. In addition, PEDF34-NP significantly ameliorated ischemia-induced retinal neovascularization in the oxygen-induced retinopathy rat model, and significantly reduced retinal vascular leakage and inflammation in streptozotocin-induced diabetic rats up to 4weeks after intravitreal injection, as compared to PLGA-NP control. Intravitreal injection of PEDF34-NP did not display any detectable toxicities to retinal structure and function. Our findings suggest that PEDF34-NP can confer sustained therapeutic effects on retinal inflammation and vascular leakage, having considerable potential to provide long-term treatment options for DR.

Longitudinal characterization of retinal vasculature alterations with optical coherence tomography angiography in a mouse model of tauopathy

Tauopathies are a family of neurodegenerative diseases which predominately afflict the rapidly growing aging population suffering from various brain disorders including Alzheimer's disease, frontotemporal dementia with parkinsonism-17 and Pick disease. As the only visually accessible region of the central nervous system, in recent years, the retina has attracted extensive attention for its potential as a target for visualizing and quantifying emerging biomarkers of neurodegenerative diseases. Our previous study has found that retinal vascular inflammation and leakage occur at the very early stage of tauopathic mouse model. Here, we aimed to non-invasively visualize age-dependent alterations of retinal vasculature assessing the potential for using changes in retinal vasculature as the biomarker for the early diagnosis of tauopathy. Optical coherence tomography angiography (OCTA), a non-invasive depth-resolved high-resolution imaging technique was used to visualize and quantify tauopathy-induced alterations of retinal vasculature in P301S transgenic mice overexpressing the P301S mutant form of human tau and age-matched wild type littermate mice at 3, 6 and 10 months of age. We observed significant alterations of vascular features in the intermediate capillary plexus (ICP) and deep capillary plexus (DCP) but not in the superficial vascular complex (SVC) of P301S mice at early stages of tauopathy. With aging, alterations of vascular features in P301S mice became more prominent in all three vascular plexuses. Staining of retinal vasculature in flatmounts and trypsin digests of P301S mice at 10 months of age revealed decreased vessel density and increased acellular capillary formation, indicating that vascular degeneration also occurs during tauopathy. Overall, our results demonstrate that the changes in retinal vascular features accelerate during the progression of tauopathy. Vessels in the ICP and DCP may be more susceptible to tauopathy than vessels in the SVC. Since changes in retinal vasculature often precede tau pathology in the brain, non-invasive identification of retinal vascular alterations with OCTA may be a useful biomarker for the early diagnosis of tauopathy and monitoring its progression.

44-OR: NFAT Inhibition Reduces Retinal Vascular Leakage and Inflammation in Diabetic Retinopathy

Purpose: Macular edema resulting from retinal vascular leakage is the leading cause of vision loss in diabetic retinopathy (DR) . The nuclear factor of activated T-cells (NFAT) family of transcription factors is known to upregulate inflammatory cytokines in response to high glucose. This study explored the effect of NFAT inhibition on retinal vascular leakage and inflammation in DR in vivo and in vitro. Methods: The streptozocin-induced (STZ) model was used as the mouse model of diabetic retinopathy. Hyperglycemia was induced at 4 weeks in C57BL/6J mice and confirmed when fasting plasma glucose became persistently elevated (>300 mg/dL) . Experiments were performed at weeks of hyperglycemia. STZ mice were injected intravitreally with the NFAT signaling inhibitor INCA-6 (25 uM) or vehicle (0.1% DMSO in PBS) . Uninjected eyes of both STZ and control mice served as additional controls. Evans blue dye (EBD) was used to measure retinal vascular leakage. Primary mouse retinal microvascular endothelial cells (MRMECs) were used for in vitro experiments. MRMEC monolayers were treated with INCA-6 (2.5 uM) or with vehicle and cell permeability was induced with mouse VEGF165. The transendothelial electrical resistance (TEER) was measured with the electrical cell-substrate impedance sensing method. MRMEC IL-6 production was investigated by ELISA. Results: In the STZ model of diabetic retinopathy, EBD vascular leakage was significantly reduced in INCA-6-treated eyes (INCA-6: 0.85 ± 0.57, n=5; vehicle: 1.78 ± 0.38, n= 7; P=0.007) , and in INCA-6 treated eyes compared to untreated eyes (untreated: 2.83 ± 1.63, n=4, P=0.04) . In the VEGF-induced MRMEC monolayer permeability model, INCA-6 significantly preserved TEER compared to vehicle (n=12, P<0.05) and decreased the VEGF-induced production of IL-6 (n=5, P<0.05) . Conclusions: NFAT inhibition has therapeutic potential to prevent and treat retinal vascular leakage and inflammation in DR. Disclosure I.De la huerta: None. J.L.Nunez: None. J.M.Poloway: None. V.Reddy: None. J.Penn: None. Funding National Institutes of Health (EY032620) International Retinal Research Foundation Grant Knights Templar Foundation Career Starter Grant

Intravitreal Injection of PACAP Attenuates Acute Ocular Hypertension-Induced Retinal Injury Via Anti-Apoptosis and Anti-Inflammation in Mice.

PurposePituitary adenylate cyclase-activating polypeptide (PACAP) has shown potent neuroprotective effects in central nervous system and retina disorders. However, whether PACAP can attenuate retinal neurodegeneration induced by acute ocular hypertension (AOH) and the underlying mechanisms remain unknown. In this study, we aimed to investigate the effects of PACAP on the survival and function of retinal ganglion cells (RGCs), apoptosis, and inflammation in a mouse model of AOH injury.MethodsPACAP was injected into the vitreous body immediately after inducing AOH injury. Hematoxylin and eosin staining and optical coherence tomography were used to evaluate the loss of retina tissue. Pattern electroretinogram was used to evaluate the function of RGCs. TUNEL assay was used to detect apoptosis. Immunofluorescence and western blot were employed to evaluate protein expression levels.ResultsPACAP treatment significantly reduced the losses of whole retina and inner retina thicknesses, Tuj1-positive RGCs, and the amplitudes of pattern electroretinograms induced by AOH injury. Additionally, PACAP treatment remarkably reduced the number of TUNEL-positive cells and inhibited the upregulation of Bim, Bax, and cleaved caspase-3 and downregulation of Bcl-xL after AOH injury. Moreover, PACAP markedly inhibited retinal reactive gliosis and vascular inflammation, as demonstrated by the downregulation of GFAP, Iba1, CD68, and CD45 in PACAP-treated mice. Furthermore, upregulated expression of NF-κB and phosphorylated NF-κB induced by AOH injury was attenuated by PACAP treatment.ConclusionsPACAP could prevent the loss of retinal tissue and improve the survival and function of RGCs. The neuroprotective effect of PACAP is probably associated with its potent anti-apoptotic and anti-inflammatory effects.

Abstract 53: Covid-19 Spike-protein Causes Cerebrovascular Rarefaction And Deteriorates Cognitive Functions In A Mouse Model Of Humanized ACE2

COVID-19 pandemic has affected our health and economy. Clinical trials confirmed multiple neurological symptoms due to COVID-19, ranging from headaches, insomnia to stroke, and encephalopathy. More studies are required to unravel the cellular and molecular mechanisms to find a cure for these neurological symptoms. Here, we investigate the effect of COVID-19 spike protein (S-protein) on the cerebrovasculature and cognitive functions in two mouse models that express humanized ACE-2 (h ACE2), a receptor essential for cellular infection and COVID-19 internalization. We hypothesize that COVID-19 S-protein causes cognitive dysfunction via the deterioration of cerebrovascular functions. Methods: S-protein was either injected intravenously or directly into the hippocampus of K-18 (h ACE2 in epithelial cells) or global h-ACE2 knock-in (h ACE2 KI) mice or wild-type mice. Cognitive functions were assessed by Y-maze and Barnes maze. Cerebrovascular density was determined using confocal 3-D image reconstruction. Human brain microvascular endothelial cells (HBMVEC) were treated with S-protein and assessed for apoptosis and inflammatory markers using immunoblotting and RT-PCR. K-18 and h-ACE2 KI mice received intraocular injections of S-protein; retinas were evaluated for vascular cell death and inflammation. Results: S-protein injections caused significant deterioration in memory and learning function of K-18 and h-ACE2 KI mice but not in the wild-type mice (P<0.05). S-protein significantly increased inflammatory mediators, cytokine production, and apoptosis in the brains and HBMVECs (P<0.05). Significant cerebrovascular rarefaction was detected only in K-18 and h-ACE2 KI mice compared to wild-type mice (P<0.05). Retinal vascular cell death and inflammation were significantly increased after S-protein injection. (P<0.05) Conclusions: COVID-19 spike protein decreases cognitive function via increased endothelial cell inflammation, apoptosis, and cerebrovascular rarefaction. Humanized ACE2 animal models are excellent and reliable for investigating the neurological symptoms of COVID-19.

Dual angiopoietin‐2/VEGF‐A neutralisation prevents vascular leakage, cell death and subretinal macrophage infiltration in mouse models of retinal ischaemia/reperfusion injury and spontaneous CNV

AbstractPurposeExplore impact of dual angiopoietin‐2 (Ang‐2)/vascular endothelial growth factor‐A (VEGF‐A) targeting by faricimab on vessel destabilisation and retinal inflammation in 2 mouse models: retinal ischaemia/reperfusion (I/R) injury model to assess vascular permeability, and JR5558 spontaneous choroidal neovascularisation (sCNV) model to investigate inflammation, in the context of phase 2/3 clinical data on faricimab.MethodsAntibodies against Ang‐2, VEGF‐A, or both (VA2) and IgG (control) were intravitreally injected 2 days before I/R injury in the I/R model. Vascular permeability and ongoing cell death were assessed by retinal FITC‐BSA accumulation and DNA fragmentation assay, respectively, 48h after injury. JR5558 mice were treated intraperitoneally at postnatal days P45, P52 with mouse cross‐reactive tool antibodies to Ang‐2, VEGF‐A, both (VA2) and IgG. Subretinal inflammatory cell (Iba1+, CD45+, CD11b+) infiltration was evaluated by flat‐mount retinal pigment epithelium (RPE)/choroid histology at 1 (PT1), 3 (PT2), and 5 weeks (PT3) post‐treatment.ResultsPost‐I/R injury, VA2 significantly prevented retinal vascular permeability by 64%; anti–VEGF‐A by 37%; anti–Ang‐2 produced no significant change. VA2 significantly reduced ongoing cell death by 47%; VEGF‐A, Ang‐2 alone had no significant effect. In JR5558 mice, VA2, not anti–VEGF‐A or anti–Ang‐2 alone, significantly reduced Iba1+ microglia/macrophages on RPE/choroid and around lesions versus IgG at PT1 and PT2. At PT3, anti–Ang‐2, VA2, not anti–VEGF‐A, showed significant reductions in Iba1+ cells versus IgG. CD45+, CD11b+ cells on RPE/choroid and around lesions were significantly reduced only with VA2 versus IgG.ConclusionsDual Ang‐2/VEGF‐A inhibition synergistically prevented retinal vascular leakage, cell death and inflammation in retinal I/R injury and sCNV mouse models, suggesting that vascular leakage, prolonged inflammation are driven by Ang‐2, and supporting recent phase 2/3 clinical data that demonstrated sustained efficacy of faricimab versus intravitreal anti‐VEGF monotarget therapy and extended durability up to every 16 weeks.

Histopathological assessments reveal retinal vascular changes, inflammation, and gliosis in patients with lethal COVID-19

PurposeThe purpose of this study is to assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection.MethodsEyes from seven COVID-19-positive and six similar age-matched control donors with a negative test for SARS-CoV-2 were assessed. Globes were evaluated ex vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for Epon embedding and immunocytochemistry.ResultsFundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the controls. OCT-based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes; however, the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples.ConclusionsIn situ analysis of the retinal tissue suggests that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.

Interleukin-6 Trans-Signaling Mediated Regulation of Paracellular Permeability in Human Retinal Endothelial Cells

Long-term hyperglycemia-mediated oxidative stress and inflammation lead to the blood-retinal barrier (BRB) dysfunction and increased vascular permeability associated with diabetic retinopathy (DR). Interleukin-6 (IL-6) is one of the primary mediators of retinal vascular inflammation. IL-6 signaling through its membrane-bound IL-6 receptor is known as classical signaling, and through a soluble IL-6 receptor (sIL-6R) is known as trans-signaling. Increasing evidence suggests that classical signaling is primarily anti-inflammatory, whereas trans-signaling induces the pro-inflammatory effects of IL-6. The purpose of this study was to compare the effects of these two pathways on paracellular permeability and expression of genes involved in inter-endothelial junctions in human retinal endothelial cells (HRECs). IL-6 trans-signaling activation caused significant disruption to paracellular integrity, with increased paracellular permeability, and was associated with significant changes in gene expression related to adherens, tight, and gap junctions. IL-6 classical signaling did not alter paracellular resistance in HRECs and had no distinct effects on gene expression. In conclusion, IL-6 trans-signaling, but not classical signaling, is a major mediator of the increased paracellular permeability characteristic of inner BRB breakdown in diabetic retinopathy. This study also identified potential inter-endothelial junction genes involved in the IL-6 trans-signaling mediated regulation of paracellular permeability in HRECs.

Nuclear factor of activated T-cells (NFAT) regulation of IL-1β-induced retinal vascular inflammation

Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1β has the reputation of being a major driver of cytokine-induced inflammation. IL-1β and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1β-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1β-induced expression of IL-1β and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1β-induced increases of IL-1β, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1β-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1β-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes.

Classification Criteria for Syphilitic Uveitis

To determine classification criteria for syphilitic uveitis. Machine learning of cases with syphilitic uveitis and 24 other uveitides. Cases of anterior, intermediate, posterior, and panuveitides were collected in an informatics-designed preliminary database, and a final database was constructed of cases achieving supermajority agreement on the diagnosis, using formal consensus techniques. Cases were analyzed by anatomic class, and each class was split into a training set and a validation set. Machine learning using multinomial logistic regression was used on the training set to determine a parsimonious set of criteria that minimized the misclassification rate among the different uveitic classes. The resulting criteria were evaluated on the validation set. Two hundred twenty-two cases of syphilitic uveitis were evaluated by machine learning, with cases evaluated against other uveitides in the relevant uveitic class. Key criteria for syphilitic uveitis included a compatible uveitic presentation (anterior uveitis; intermediate uveitis; or posterior or panuveitis with retinal, retinal pigment epithelial, or retinal vascular inflammation) and evidence of syphilis infection with a positive treponemal test. The Centers for Disease Control and Prevention reverse screening algorithm for syphilis testing is recommended. The misclassification rates for syphilitic uveitis in the training sets were as follows: anterior uveitides 0%, intermediate uveitides 6.0%, posterior uveitides 0%, panuveitides 0%, and infectious posterior/panuveitides 8.6%. The overall accuracy of the diagnosis of syphilitic uveitis in the validation set was 100% (99% confidence interval 99.5, 100)-that is, the validation set's misclassification rates were 0% for each uveitic class. The criteria for syphilitic uveitis had a low misclassification rate and seemed to perform sufficiently well for use in clinical and translational research.

Salusin-β Mediates High Glucose-Induced Inflammation and Apoptosis in Retinal Capillary Endothelial Cells via a ROS-Dependent Pathway in Diabetic Retinopathy

BackgroundDiabetic retinopathy (DR) is characterized by retinal vascular endothelial cell death and vascular inflammation, which are microvascular complications of diabetes mellitus (DM). Salusin-β, a newly identified peptide, is closely associated with hypertension, atherosclerosis and diabetic cardiomyopathy. However, the exact role of salusin-β in high glucose (HG)-induced retinal capillary endothelial cell (REC) inflammation and apoptosis remains unclear.Patients and MethodsA total of 60 patients with type 2 diabetes and 20 healthy controls were included in this study. Based on fundus fluorescein angiography findings, the diabetic patients were divided into three subgroups: diabetes without retinopathy (DWR), non-proliferative DR (NPDR) and proliferative DR (PDR). Serum salusin-β levels were measured by enzyme-linked immunosorbent assay. Human RECs (HRECs) were cultured in normal glucose (NG) and HG medium with or without salusin-β. Salusin-β expression was analysed by Western blotting and immunofluorescence staining. Expression of the pro-inflammatory cytokines MCP-1, IL-1β, TNF-α, and VCAM-1 was analysed by Western blotting. Reactive oxygen species (ROS) production was measured with 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Cell apoptosis rates were determined by flow cytometry. The levels of p38, JNK, p-p38, and p-JNK and the apoptosis-related proteins cleaved caspase-3, Bax, and cl2 were analysed by Western blotting.ResultsSerum salusin-β levels were higher in diabetic patients than in healthy controls (p = 0.0027), especially in patients with NPDR and PDR (both p<0.01). HG upregulated salusin-β expression in HRECs in a time-dependent manner. Salusin-β exacerbated inflammation and apoptosis, upregulated intracellular ROS production in HG-induced HRECs, and activated ROS-dependent JNK and p38 MAPK signalling, while knockdown of salusin-β suppressed these effects.ConclusionOur findings indicate that salusin-β can promote inflammation and apoptosis via ROS-dependent JNK and p38 MAPK signalling in HG-induced HRECs and could be a therapeutic target for DR.