Retinal Vascular Leakage Research Articles

Honokiol protects against diabetic retinal microvascular injury via sirtuin 3-mediated mitochondrial fusion

IntroductionMitochondrial dysfunction and oxidative stress play important roles in diabetic retinal vascular injuries. Honokiol (HKL) is a small-molecule polyphenol that exhibits antioxidant effects and has a beneficial effect in diabetes. This study aimed to explore the potential ability of HKL to ameliorate vascular injury in diabetic retinopathy (DR) and its possible mechanisms of action.MethodsThe effect of HKL was evaluated in vascular injury in an in vivo type 2 diabetic (db/db) mouse model. In vitro, retinal microvascular endothelial cells were treated with high glucose (HG) to simulate the pathological diabetic environment. Cell viability, expression of apoptosis-related proteins, cellular reactive oxygen species, mitochondrial membrane potential, and morphological changes in the mitochondria were examined.ResultsThe diabetic mice exhibited severe retinal vascular damage, including vascular leakage in vivo and capillary endothelial cell apoptosis in vitro. HKL reversed the retinal vascular leakage in the diabetic mice. In vitro, HKL improved retinal capillary endothelial cell viability, decreased apoptosis, and reversed the HG-induced increased cellular oxidative stress and mitochondrial fragmentation. The sirtuin 3 (SIRT3) inhibitor 3-TYP blocked all the in vivo and in vitro protective effects of HKL against diabetic retinal vascular leakage and capillary endothelium and eliminated the decrease in oxidative stress levels and reduction of mitochondrial fragmentation.DiscussionIn conclusion, these findings suggest that HKL inhibits vascular injury in DR, which was likely achieved through SIRT3-mediated mitochondrial fusion. This study provides a potential new strategy for the treatment of DR.

Bruton's tyrosine kinase inhibition suppresses pathological retinal angiogenesis.

Pathological retinal angiogenesis is a typical manifestation of vision-threatening ocular diseases. Many patients exhibit poor response or resistance to anti-vascular endothelial growth factor (VEGF) agents. Bruton's tyrosine kinase (BTK) controls the proliferation and function of immune cells. Therefore, we examined the anti-inflammatory and anti-angiogenic effects of BTK inhibition on retinal angiogenesis. Retinal neovascularisation and vascular leakage in oxygen-induced retinopathy in C57/BL6J mice were assessed by whole-mount retinal immunofluorescence. PLX5622 was used to deplete microglia and Rag1-knockout mice were used to test the contribution of lymphocytes to the effects of BTK inhibition. The cytokines, activation markers, inflammatory and immune-regulatory activities of retinal microglia/macrophages were detected using qRT-PCR and immunofluorescence. NLRP3 was detected by western blotting, and the effects of BTK inhibition on the co-culture of microglia and human retinal microvascular endothelial cells (HRMECs) were examined. BTK inhibition suppressed pathological angiogenesis and vascular leakage, and significantly reduced retinal inflammation, which involved microglia/macrophages but not lymphocytes. BTK inhibition increased anti-inflammatory factors and reduced pro-inflammatory cytokines that resulted from NLRP3 inflammasome activation. BTK inhibition suppressed the inflammatory activity of microglia/macrophages, and acted synergistically with anti-VEGF without retinal toxicity. Moreover, the supernatant of microglia incubated with BTK-inhibitor reduced the proliferation, tube formation and sprouting of HRMECs. BTK inhibition suppressed retinal neovascularisation and vascular leakage by modulating the inflammatory activity of microglia and macrophages. Our study suggests BTK inhibition as a novel and promising approach for alleviating pathological retinal angiogenesis.

Tocilizumab for cystoid macular edema secondary to retinitis pigmentosa

PurposeTo describe the effect of tocilizumab (TCZ) on cystoid macular edema (CME) and retinal vascular leakage (RVL) in retinitis pigmentosa (RP).MethodsRetrospective case series.ResultsWe present 2 cases of RP with marked inflammatory features in the form of CME and RVL. There was initial diagnostic uncertainty with posterior uveitis. Both patients were treated with corticosteroids, conventional disease-modifying antirheumatic drugs (cDMARDs), and biological DMARDs (bDMARDs) for the inflammatory features with partial and inconsistent treatment response. When treatment was switched to intravenous (IV) TCZ, dramatic reduction in CME and RVL were observed in both patients. Diagnosis of RP was eventually made based on findings of ancillary tests (macular spectral-domain optical coherence tomography, visual fields, full-field electroretinogram). Genetic testing led to a molecular diagnosis of EYS-related autosomal recessive RP in patient 1, while patient 2 had negative gene panel results.ConclusionsIV TCZ can be an effective treatment option in RP-related CME and RVL. Whether this treatment strategy has an effect on prognosis remains to be established, but it is possible considering chronic CME-related retinal damage is a major driver of central vision loss in RP.

Therapeutic potential of histamine H4 receptor antagonist as a preventive treatment for diabetic retinopathy in mice

Diabetic retinopathy (DR) is a prevalent complication of diabetes, often resulting in vision loss and blindness. Existing treatments primarily aim to control blood sugar levels and inhibit angiogenesis. However, current therapies for DR, such as anti-VEGF and laser photocoagulation, are frequently invasive, and can cause adverse side effects. Consequently, there is a critical need for new preventive therapeutics to address DR more effectively. This study aimed to examine the therapeutic potential of a histamine H4 receptor (HRH4) antagonist as a preventive treatment for DR in mice. A mouse model of DR was established by intraperitoneally injecting 200 mg/kg of streptozotocin (STZ). Immune cell infiltration into the retina of mice with STZ-induced diabetes was measured using fluorescence-activated cell sorting (FACS) 12 weeks after STZ injection. The preventive effects of the HRH4 antagonist on inflammation and pathological retinal vessel leakage were determined in a mouse model of DR. Infiltration of HRH4-expressing macrophages increased in the retina of mice with STZ-induced DR. The HRH4 antagonist prevented macrophage infiltration and retinal vascular leakage to prevent STZ-induced DR in mice without causing any retinal toxicity. The infiltration of macrophages increased in the retina of mice with STZ-induced diabetes through HRH4, indicating that HRH4 is potentially a novel preventative therapeutic target in DR. These findings suggest that targeting HRH4 is a promising strategy for the prevention and treatment of DR.

VEGF-B prevents chronic hyperglycemia-induced retinal vascular leakage by regulating the CDC42-ZO1/VE-cadherin pathway.

Non-proliferative diabetic retinopathy (NPDR) is the early stage of diabetic retinopathy (DR) and is a chronic oxidative stress-related ocular disease. Few treatments are approved for early DR. This study aimed to investigate the pathogenic mechanisms underlying the retinal micro-vasculopathy induced by diabetes and to explore an early potential for treating early DR in a mouse model. The mouse model of type 1 diabetes was established by intraperitoneal injection of streptozotocin (STZ, 180 mg/kg), which was used as the early DR model. The body weight and blood glucose mice were measured regularly; The retinal vascular leakage in the early DR mice was determined by whole-mount staining; Label-free quantitative proteomic analysis and bioinformatics were used to explore the target proteins and signaling pathways associated with the retinal tissues of early DR mice; To detect the effects of target protein on endothelial cell proliferation, migration, and tube formation, knockdown and overexpression of VEGF-B were performed in human retinal vascular endothelial cells (HRECs); Western blotting was used to detect the expression of target proteins invitro and invivo; Meanwhile, the therapeutic effect of VEGF-B on vascular leakage has also been evaluated invitro and invivo. The protein expressions of vascular endothelial growth factor (VEGF)-B and the Rho GTPases family member CDC42 were reduced in the retinal tissues of early DR. VEGF-B upregulated the expression of CDC42/ZO1/VE-cadherin and prevented hyperglycemia-induced vascular leakage in HRECs. Standard intravitreal VEGF-B injections improved the retinal vascular leakage and neurovascular response in early DR mice. Our findings demonstrated, for the first time, that in diabetes, the retinal vessels are damaged due to decreased VEGF-B expression through downregulation of CDC42/ZO1/VE-cadherin expression. Therefore, VEGF-B could be used as a novel therapy for early DR.

CST3 alleviates retinal vascular leakage by regulating the Rap1 signaling pathway

Retinal vascular leakage is a major event in several retinal diseases, including diabetic retinopathy (DR). In a previous study, we demonstrated that the aqueous humor concentration of Cystatin C (CST3), a physiological inhibitor of cysteine protease, is negatively correlated with the severity of diabetic macular edema. However, its function in the retina has not been clearly elucidated. In this study, we found a significant decrease in the aqueous humor concentration of CST3 with DR progression. Furthermore, we found that CST3 was expressed in retinal endothelial cells and that its expression was significantly downregulated in high glucose-treated human retinal microvascular endothelial cells (HRMECs) and the retinal vessels of oxygen-induced retinopathy (OIR) mice. Silencing CST3 expression resulted in decreased HRMEC migration and tubule formation ability. Exogenous addition of the CST3 protein significantly improved HRMEC migration and tubular formation. In-vivo experiments demonstrated that CST3 silencing induced retinal vascular leakage in WT mice, while its intravitreal injection significantly reduced retinal leakage in OIR mice. Mechanistically, CST3 promoted the expression of the downstream adhesion molecules, claudin5, VE-cadherin, and ZO-1, in retinal vascular cells by regulating the Rap1 signaling pathway. Therefore, this study revealed a novel mechanism by which CST3 improves retinal vascular function and provided evidence that it is a potential therapeutic target for retinal vascular leakage.

Hsa_circ_0004776 regulates the retina neovascularization in progression of diabetic retinopathy via hsa-miR-382-5p/BDNF axis

The aim of this work was to identify the regulatory function of hsa_circ_0004776 in the progression of diabetic retinopathy (DR). The direct interactions between hsa_circ_0004776 and hsa-miR-382-5p and between hsa-miR-382-5p and BDNF, were confirmed via dual-luciferase reporter assays. Quantitative Real-Time PCR analysis indicated that hsa_circ_0004776 was highly expressed in aqueous humour samples of DR patients and human retinal microvascular epithelial cells (hRECs) under a high-glucose environment, whereas hsa-miR-382-5p showed the opposite trend. Overexpressed hsa_circ_0004776 significantly enhanced DNA synthesis, proliferation, migration, and tube formation in hRECs in hyperglycaemia, while hsa-miR-382-5p mimics reversed these changes. Additionally, in a streptozotocin-induced Sprague-Dawley rat model of DR, vitreous microinjection of rno-miR-382-5p agomir reversed the pathologic features in the progression of DR, including retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis. Our results indicated that under hyperglycaemic conditions, hsa_circ_0004776 influences the progression of DR via hsa-miR-382-5p and thus represents a potential therapeutic target.

Pericyte loss via glutaredoxin2 downregulation aggravates diabetes-induced microvascular dysfunction

Diabetic retinopathy (DR) is the leading cause of vision loss and blindness among working-age adults. Pericyte loss is an early pathological feature of DR. Under hyperglycemic conditions, reactive oxygen species (ROS) production increases, leading to oxidative stress and subsequent mitochondrial dysfunction and apoptosis. Dysfunctional pericyte can cause retinal vascular leakage, obliteration, and neovascularization. Glutaredoxin 2 (Grx2) is a mitochondrial glutathione-dependent oxidoreductase which protects cells against oxidative insults by safeguarding mitochondrial function. Whether Grx2 plays a protective role in diabetes-induced microvascular dysfunction remains unclear. Our findings revealed that diabetes-related stress reduced Grx2 expression in pericytes, but not in endothelial cells. Grx2 knock-in ameliorated diabetes-induced microvascular dysfunction in vivo DR models. Decreased Grx2 expression led to significant pericyte apoptosis, and pericyte dysfunction, namely reduced pericyte recruitment towards endothelial cells and increased endothelial cell permeability. Conversely, upregulating Grx2 reversed these effects. Furthermore, Grx2 regulated pericyte apoptosis by modulating complex I activity, which is crucial for pericyte mitochondrial function. Overall, our study uncovered a novel mechanism whereby high glucose inhibited Grx2 expression in vivo and in vitro. Grx2 downregulation exacerbated pericyte apoptosis, pericyte dysfunction, and retinal vascular dysfunction by inactivating complex I and mediating mitochondrial dysfunction in pericytes.

Iron Chelator Deferiprone Restores Iron Homeostasis and Inhibits Retinal Neovascularization in Experimental Neovascular Age-Related Macular Degeneration.

Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.

IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stagestreptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

Ultrawidefield Fluorescein Angiography and OCT Findings in Children and Young Adults with Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy

PurposeAutosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is a rare genetic (CAPN5) autoimmune condition typically diagnosed in adulthood and characterized by a triad of inflammation, retinal degeneration and neovascularization. We report novel multimodal imaging findings in children and young adults with ADNIV and early treatment response to short-duration local and/or systemic corticosteroids. DesignRetrospective consecutive case series. ParticipantsTen patients under the age of 25 with ADNIV and available multimodal imaging. MethodsThe medical records of patients under the age of 25 with a diagnosis of ADNIV with ultra-widefield fluorescein angiography (UWFFA) and optical coherence tomography (OCT) data were reviewed. Main Outcome MeasuresUWFFA and OCT findings at baseline and following local corticosteroids. ResultsMedian age at presentation was 14 years (range 9-24 years). OCT on presentation demonstrated CME in 8/20 eyes and symptomatic vitreoretinal interface disease in 2/20 eyes. Initial UWFFA demonstrated retinal vascular leakage (20/20 eyes, 100%), peripheral non-perfusion (13/20 eyes, 65%), and retinal neovascularization (6/20 eyes, 30%). Retinal vascular leakage improved with local corticosteroids and neovascularization regressed with anti-VEGF therapy. ConclusionsUWFFA findings of prefibrotic ADNIV reported in adults were also present in children and young adults. Early testing for a pathogenic CAPN5 variant in at-risk children and regularly scheduled screening for uveitis and retinal vasculitis with UWFFA and OCT may prompt earlier intervention. Short duration local steroids are effective at treating retinal vascular leakage and macular edema but are not durable suggesting a potential role for steroid-sparing immunosuppressive therapy. Early treatment may alter the natural history of disease.

Novel CCR3-targeted cyclic peptides as potential therapeutic agents for age-related macular degeneration via inhibiting angiogenesis and reducing retinal photoreceptor damage

The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development.

The Early Pathogenesis of Diabetic Retinopathy and Its Attenuation by Sodium-Glucose Transporter 2 Inhibitors.

The early pathogenetic mechanism of diabetic retinopathy (DR) and its treatment remain unclear. Therefore, we investigated the early pathogenic alterations in DR using streptozotocin-induced diabetic mice and the protective effect of sodium-glucose cotransporter 2 (SGLT2) inhibitors against these alterations. Retinal vascular leakage was assessed by dextran fluorescence angiography. Retinal thickness and vascular leakage were increased 2 and 4 weeks after onset of diabetes, respectively. Immunostaining showed that morphological change of microglia (amoeboid form) was observed at 2 weeks. Subsequently, increased angiopoietin-2 expression, simultaneous loss of pericytes and endothelial cells, decreased vessel density, retinal hypoxia, and increased vascular endothelial growth factor (VEGF)-A/VEGF receptor system occurred at 4 weeks. SGLT2 inhibitors (luseogliflozin and ipragliflozin) had a significant protective effect on retinal vascular leakage and retinal thickness at a low dose that did not show glucose-lowering effects. Furthermore, both inhibitors at this dose attenuated microglia morphological changes and these early pathogenic alterations in DR. In vitro study, both inhibitors attenuated the lipopolysaccharide-induced activation of primary microglia, along with morphological changes toward an inactive form, suggesting the direct inhibitory effect of SGLT2 inhibitors on microglia. In summary, SGLT2i may directly prevent early pathogenic mechanisms, thereby potentially playing a role in preventing DR.

Ultrawide-field fluorescein angiography features in patients with anterior uveitis

PurposeTo evaluate the utility of ultrawide-field fluorescein angiography (UWFFA) in patients with anterior uveitis by investigating the detection of retinal vascular leakage (RVL) and the subsequent implications on disease diagnosis and management.Study design/materials and methodsPatients, who were referred to the National Eye Institute (NEI) for evaluation of anterior uveitis and underwent UWFFA imaging at the initial visit, were included in this study. The electronic medical records of eligible patients were reviewed. The UWFFA images were assessed for severity of retinal vascular leakage, presence of macular leakage, and optic disc leakage by a two-grader system, and intergrader agreement was calculated using the κ-value. Patients with altered diagnoses and management attributable to UWFFA results were noted.ResultsA total of 93 eyes of 63 patients were included in the study. Of 93 eyes, 31 (33.3%) eyes had RVL on UWFFA, with 26 (28.0%) eyes and 5 (5.4%) eyes showing mild and moderate-severe RVL, respectively. Twenty-five (26.9%) eyes showed macular leakage, and 7 (7.5%) eyes showed optic disc leakage. The κ-values ranged from 0.85 – 0.87 indicating excellent intergrader agreement. Of the 31 eyes with RVL, the diagnosis was changed to anterior/intermediate uveitis for 9 (29.0%) eyes and to panuveitis for 4 (12.9%) eyes. Systemic treatment was escalated in 5 patients.ConclusionOur results suggest that UWFFA imaging is useful in detecting subclinical posterior involvement in patients with anterior uveitis. Moreover, UWFFA results in altered diagnosis and treatment approaches in a portion of patients.

Using Deep Learning to Segment Retinal Vascular Leakage and Occlusion in Retinal Vasculitis

ABSTRACT Purpose Retinal vasculitis (RV) is characterised by retinal vascular leakage, occlusion or both on fluorescein angiography (FA). There is no standard scheme available to segment RV features. We aimed to develop a deep learning model to segment both vascular leakage and occlusion in RV. Methods Four hundred and sixty-three FA images from 82 patients with retinal vasculitis were used to develop a deep learning model, in 60:20:20 ratio for training:validation:testing. Parameters, including deep learning architectures (DeeplabV3+, UNet++ and UNet), were altered to find the best binary segmentation model separately for retinal vascular leakage and occlusion, using a Dice score to determine the reliability of each model. Results Our best model for vascular leakage had a Dice score of 0.6279 (95% confidence interval (CI) 0.5584–0.6974). For occlusion, the best model achieved a Dice score of 0.6992 (95% CI 0.6109–0.7874). Conclusion Our RV segmentation models could perform reliable segmentation for retinal vascular leakage and occlusion in FAs of RV patients.

Trimethylamine N-oxide aggravates vascular permeability and endothelial cell dysfunction under diabetic condition: in vitro and in vivo study.

To provide the direct evidence for the crucial role of trimethylamine N-oxide (TMAO) in vascular permeability and endothelial cell dysfunction under diabetic condition. The role of TMAO on the in vitro biological effect of human retinal microvascular endothelial cells (HRMEC) under high glucose conditions was tested by a cell counting kit, wound healing, a transwell and a tube formation assay. The inflammation-related gene expression affected by TMAO was tested by real-time polymerase chain reaction (RT-PCR). The expression of the cell junction was measured by Western blotting (WB) and immunofluorescence staining. In addition, two groups of rat models, diabetic and non-diabetic, were fed with normal or 0.1% TMAO for 16wk, and their plasma levels of TMAO, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were tested. The vascular permeability of rat retinas was measured using FITC-Dextran, and the expression of zonula occludens (ZO)-1 and claudin-5 in rat retinas was detected by WB or immunofluorescence staining. TMAO administration significantly increased the cell proliferation, migration, and tube formation of primary HRMEC either in normal or high-glucose conditions. RT-PCR showed elevated inflammation-related gene expression of HRMEC under TMAO stimulation, while WB or immunofluorescence staining indicated decreased cell junction ZO-1 and occludin expression after high-glucose and TMAO treatment. Diabetic rats showed higher plasma levels of TMAO as well as retinal vascular leakage, which were even higher in TMAO-feeding diabetic rats. Furthermore, TMAO administration increased the rat plasma levels of VEGF, IL-6 and TNF-α while decreasing the retinal expression levels of ZO-1 and claudin-5. TMAO enhances the proliferation, migration, and tube formation of HRMEC, as well as destroys their vascular integrity and tight connection. It also regulates the expression of VEGF, IL-6, and TNF-α.

Targeted degradation of VEGF with bispecific aptamer-based LYTACs ameliorates pathological retinal angiogenesis.

Rationale: Neovascular ocular diseases (NODs) represent the leading cause of visual impairment globally. Despite significant advances in anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF), persistent challenges remain prevalent. As a proof-of-concept study, we herein demonstrate the effectiveness of targeted degradation of VEGF with bispecific aptamer-based lysosome-targeting chimeras (referred to as VED-LYTACs). Methods: VED-LYTACs were constructed with three distinct modules: a mannose-6-phosphate receptor (M6PR)-binding motif containing an M6PR aptamer, a VEGF-binding module with an aptamer targeting VEGF, and a linker essential for bridging and stabilizing the two-aptamer structure. The degradation efficiency of VED-LYTACs via the autophagy-lysosome system was examined using an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Subsequently, the anti-angiogenic effects of VED-LYTACs were evaluated using in vitro wound healing assay, tube formation assay, three-dimensional sprouting assay, and ex vivo aortic ring sprouting assay. Finally, the potential therapeutic effects of VED-LYTACs on pathological retinal neovascularization and vascular leakage were tested by employing mouse models of NODs. Results: The engineered VED-LYTACs promote the interaction between M6PR and VEGF, consequently facilitating the translocation and degradation of VEGF through the lysosome. Our data show that treatment with VED-LYTACs significantly suppresses VEGF-induced angiogenic activities both in vitro and ex vivo. In addition, intravitreal injection of VED-LYTACs remarkably ameliorates abnormal vascular proliferation and leakage in mouse models of NODs. Conclusion: Our findings present a novel strategy for targeting VEGF degradation with an aptamer-based LYTAC system, effectively ameliorating pathological retinal angiogenesis. These results suggest that VED-LYTACs have potential as therapeutic agents for managing NODs.

SR1375: Phase 1 results of a novel, orally administered lipoprotein‐associated phospholipase A2 inhibitor targeting diabetic macular oedema

Aims/Purpose: Lipoprotein‐associated phospholipase A2 (Lp‐PLA2) activity have been linked to a higher risk of diabetic retinopathy and inhibiting Lp‐PLA2 activity showed improvement in vision and macular oedema for patients with diabetic macular oedema (DME). SR1375 is an orally available, selective inhibitor of Lp‐PLA2 that shows significant retinal vascular leakage reduction in preclinical studies, and is being developed for DME. A phase 1, randomized, double‐blind, placebo‐controlled study is conducted to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of SR1375 in healthy subjects.Methods: A total of 72 healthy subjects were enrolled, with 54 had received SR1375. 48 subjects were in the single ascending dose arms (1, 3, 10, 30, 100 and 200 mg) while 24 subjects were in the multiple ascending dose arms (5, 15 and 50 mg once daily for 14 days).Results: SR1375 was safe and well tolerated across all doses. It had excellent oral absorption with the exposure level increasing in an approximately dose proportional manner after single and multiple doses. The elimination of SR1375 was slow with a long t1/2 ranging from 28.8 to 90.0 h. The accumulation of SR1375 was not significant after multiple administrations. The plasma inhibition on Lp‐PLA2 activity by SR1375 was strong (up to 95%) and enduring, which was correlated with the concentration of SR1375 (IC50 as 1.41 ng/mL) and almost returned to basal level when the concentration of SR1375 was low enough.Conclusions: SR1375 was safe and effective in inhibiting plasma Lp‐PLA2. The safety and PK/PD profile supports the once daily therapy. A Phase 2 study for DME patients is planned later this year. It would be a 12‐week randomized, double‐blind and placebo‐controlled trial. The primary objective is to assess the effect of SR1375 on vision and oedema.

Requirement of Site-Specific Tyrosine Phosphorylation of Cortactin in Retinal Neovascularization and Vascular Leakage.

Retinal neovascularization is a major cause of vision impairment. Therefore, the purpose of this study is to investigate the mechanisms by which hypoxia triggers the development of abnormal and leaky blood vessels. A variety of cellular and molecular approaches as well as tissue-specific knockout mice were used to investigate the role of Cttn (cortactin) in retinal neovascularization and vascular leakage. We found that VEGFA (vascular endothelial growth factor A) stimulates Cttn phosphorylation at Y421, Y453, and Y470 residues in human retinal microvascular endothelial cells. In addition, we observed that while blockade of Cttn phosphorylation at Y470 inhibited VEGFA-induced human retinal microvascular endothelial cell angiogenic events, suppression of Y421 phosphorylation protected endothelial barrier integrity from disruption by VEGFA. In line with these observations, while blockade of Cttn phosphorylation at Y470 negated oxygen-induced retinopathy-induced retinal neovascularization, interference with Y421 phosphorylation prevented VEGFA/oxygen-induced retinopathy-induced vascular leakage. Mechanistically, while phosphorylation at Y470 was required for its interaction with Arp2/3 and CDC6 facilitating actin polymerization and DNA synthesis, respectively, Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, resulting in adherens junction disruption. Furthermore, whereas Cttn phosphorylation at Y470 residue was dependent on Lyn, its phosphorylation at Y421 residue required Syk activation. Accordingly, lentivirus-mediated expression of shRNA targeting Lyn or Syk levels inhibited oxygen-induced retinopathy-induced retinal neovascularization and vascular leakage, respectively. The above observations show for the first time that phosphorylation of Cttn is involved in a site-specific manner in the regulation of retinal neovascularization and vascular leakage. In view of these findings, Cttn could be a novel target for the development of therapeutics against vascular diseases such as retinal neovascularization and vascular leakage.

Knockdown of ChREBP ameliorates retinal microvascular endothelial cell injury and angiogenic responses in diabetic retinopathy

PurposeTo examine whether and how carbohydrate response element-binding protein (ChREBP) plays a role in diabetic retinopathy. MethodsWestern blotting was used to detect ChREBP expression and location following high glucose stimulation of Human Retinal Microvascular Endothelial Cells (HRMECs). Flow cytometry, TUNEL staining, and western blotting were used to evaluate apoptosis following ChREBP siRNA silencing. Cell scratch, transwell migration, and tube formation assays were used to determine cell migration and angiogenesis. Diabetic models for wild-type (WT) and ChREBP knockout (ChKO) mice were developed. Retinas of WT and ChKO animals were cultivated in vitro with vascular endothelial growth factor + high glucose to assess neovascular development. ResultsChREBP gene knockdown inhibited thioredoxin-interacting protein and NOD-like receptor family pyrin domain containing protein 3 expression in HRMECs, which was caused by high glucose stimulation, reduced apoptosis, hindered migration, and tube formation, and repressed AKT/mTOR signaling pathway activation. Compared with WT mice, ChKO mice showed suppressed high glucose-induced alterations in retinal structure, alleviated retinal vascular leakage, and reduced retinal neovascularization. ConclusionsChREBP deficiency decreased high glucose-induced apoptosis, migration, and tube formation in HRMECs as well as structural and angiogenic responses in the mouse retina; thus, it is a potential therapeutic target for diabetic retinopathy.