Eucalyptol Ameliorates Retinal Microvascular Defects through Modulating ER Stress and Angiopoietin-Tie Signaling in Diabetic Eyes.

Loss of the inner blood-retinal barrier (BRB) integrity is a main feature of ocular diseases such as diabetic macular edema. However, there is a lack of clarity on how inner BRB function is modulated within the diabetic retina. The current study examined whether eucalyptol inhibited inner BRB destruction and aberrant retinal angiogenesis in 33 mM glucose-exposed human retinal microvascular endothelial (RVE) cells and db/db mice. This study further examined the molecular mechanisms underlying endothelial dysfunction including retinal endoplasmic reticulum (ER) stress and angiopoietin (Ang)/Tie axis in conjunction with vascular endothelial growth factor (VEGF). Eucalyptol is a naturally occurring monoterpenoid and an achiral aromatic component of many plants including eucalyptus leaves. Nontoxic eucalyptol reduced the production of amyloid-ß (Aß) protein in glucose-loaded RVE cells and in diabetic mice. This natural compound blocked apoptosis of Aß-exposed RVE cells in diabetic mouse eyes by targeting ER stress via the inhibition of PERK-eIF2α-ATF4-CHOP signaling. Eucalyptol promoted activation of the Ang-1/Tie-2 pathway and dual inhibition of Ang-2/VEGF in Aß-exposed RVE cells and in diabetic eyes. Supply of eucalyptol reversed the induction of junction proteins in glucose/Aß-exposed RVE cells within the retina and reduced permeability. In addition, oral administration of eucalyptol reduced vascular leaks in diabetic retinal vessels. Taken together, these findings clearly show that eucalyptol inhibits glucose-induced Aß-mediated ER stress and manipulates Ang signaling in diabetic retinal vessels, which ultimately blocks abnormal angiogenesis and loss of inner BRB integrity. Therefore, eucalyptol provides new treatment strategies for diabetes-associated RVE defects through modulating diverse therapeutic targets including ER stress, Ang-1/Tie-2 signaling, and Ang-2/VEGF.

Melatonin: Unveiling the functions and implications in ocular health.

Melatonin, a versatile hormone produced by the pineal gland, has garnered considerable scientific interest due to its diverse functions. In the eye, melatonin regulates a variety of key processes like inhibiting angiogenesis by reducing vascular endothelial growth factor levels and protecting the blood-retinal barrier (BRB) integrity by enhancing tight junction proteins and pericyte coverage. Melatonin also maintains cell health by modulating autophagy via the Sirt1/mTOR pathways, reduces inflammation, promotes antioxidant enzyme activity, and regulates intraocular pressure fluctuations. Additionally, melatonin protects retinal ganglion cells by modulating aging and inflammatory pathways. Understanding melatonin's multifaceted functions in ocular health could expand the knowledge of ocular pathogenesis, and shed new light on therapeutic approaches in ocular diseases. In this review, we summarize the current evidence of ocular functions and therapeutic potential of melatonin and describe its roles in angiogenesis, BRB integrity maintenance, and modulation of various eye diseases, which leads to a conclusion that melatonin holds promising treatment potential for a wide range of ocular health conditions.

Protective Effect of Curcumin on the Tight Junction Integrity and Cellular Senescence in Human Retinal Pigment Epithelium of Early Diabetic Retinopathy.

Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood-retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo . Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.

Raddeanin A Protects the BRB Through Inhibiting Inflammation and Apoptosis in the Retina of Alzheimer's Disease.

Neuroinflammation and endothelial cell apoptosis are prominent features of blood-brain barrier (BBB) disruption, which have been described in Alzheimer's disease (AD) and can predict cognitive decline. Recent reports revealed vascular ß-amyloid (Aß) deposits, Muller cell degeneration and microglial dysfunction in the retina of AD patients. However, there has been no in-depth research on the roles of inflammation, retinal endothelial cell apoptosis, and blood-retinal barrier (BRB) damage in AD retinopathy. We found that Raddeanin A (RDA) could improve pathological and cognitive deficits in a mouse model of Alzheimer's disease by targeting ß-amyloidosis, However, the effects of RDA on AD retinal function require further study. To clarify whether RDA inhibits inflammation and apoptosis and thus improves BRB function in AD-related retinopathy. In vitro we used Aß-treated HRECs and MIO-M1 cells, and in vivo we used 3×Tg-AD mice to investigate the effect of RDA on BRB in AD-related retinopathy. We found that RDA could improve BRB function in AD-related retinopathy by inhibiting NLRP3-mediated inflammation and suppressing Wnt/ß-catenin pathway-mediated apoptosis, which is expected to improve the pathological changes in AD-related retinopathy and the quality of life of AD patients.

Polyvinylpyrrolidone-curcumin nanoparticles with immune regulatory and metabolism regulatory effects for the treatment of experimental autoimmune uveitis.

Uveitis comprises a cluster of intraocular inflammatory disorders characterized by uncontrolled autoimmune responses and excessive oxidative stress leading to vision loss worldwide. In the present study, curcumin (CUR) was conjugated with polyvinylpyrrolidone (PVP) to form PVP-CUR nanoparticles with significantly elevated solubility and outstanding multiple radical scavenging abilities. In vitro studies revealed that PVP-CUR nanoparticles markedly mitigated oxidative stress and reduced apoptosis in a H2O2-induced human retinal pigment epithelial cell line (ARPE-19) and promoted phenotypic polarization from M1 to M2 in an LPS-induced human microglial cell line (HMC3). Further in vivo studies demonstrated the prominent therapeutic effects of PVP-CUR nanoparticles on experimental autoimmune uveitis (EAU), which relieved clinical and pathological progression, improved perfusion and tomographic manifestations of retinal vessels, and reduced blood-retinal barrier (BRB) leakage; these effects may be mediated by mitigating oxidative stress and attenuating macrophage/microglia-elicited inflammation. Notably, treatment with PVP-CUR nanoparticles was shown to regulate metabolite alterations in EAU rats, providing novel insights into the underlying mechanisms involved. Additionally, the PVP-CUR nanoparticles showed great biocompatibility in vivo. In summary, our study revealed that PVP-CUR nanoparticles may serve as effective and safe nanodrugs for treating uveitis and other oxidative stress- and inflammation-related diseases.

Natural plant medications for the treatment of retinal diseases: The blood-retinal barrier as a clue.

BACKGROUND: Retinal diseases significantly contribute to the global burden of visual impairment and blindness. The occurrence of retinal diseases is often accompanied by destruction of the blood‒retinal barrier, a vital physiological structure responsible for maintaining the stability of the retinal microenvironment. However, detailed summaries of the factors damage the blood‒retinal barrier and treatment methods involving natural plant medications are lacking. PURPOSE: To comprehensively summarize and analyze the protective effects of active substances in natural plant medications on damage to the blood-retina barrier that occurs when retinal illnesses, particularly diabetic retinopathy, and examine their medicinal value and future development prospects. METHODS: In this study, we searched for studies published in the ScienceDirect, PubMed, and Web of Science databases. The keywords used included natural plant medications, plants, natural herbs, blood retinal barrier, retinal diseases, diabetic retinopathy, age-related macular degeneration, and uveitis. Chinese herbal compound articles, non-English articles, warning journals, and duplicates were excluded from the analysis. RESULTS: The blood‒retinal barrier is susceptible to high glucose, aging, immune responses, and other factors that destroy retinal homeostasis, resulting in pathological changes such as apoptosis and increased vascular permeability. Existing studies have shown that the active compounds or extracts of many natural plants have the effect of repairing blood-retinal barrier dysfunction. Notably, berberine, puerarin, and Lycium barbarum polysaccharides exhibited remarkable therapeutic effects. Additionally, curcumin, astragaloside IV, hesperidin, resveratrol, ginsenoside Rb1, luteolin, and Panax notoginseng saponins can effectively protect the blood‒retinal barrier by interfering with distinct pathways. The active ingredients found in natural plant medications primarily repair the blood‒retinal barrier by modulating pathological factors such as oxidative stress, inflammation, pyroptosis, and autophagy, thereby alleviating retinal diseases. CONCLUSION: This review summarizes a series of plant extracts and plant active compounds that can treat retinal diseases by preventing and treating blood‒retinal barrier damage and provides reference for the research of new drugs for treating retinal diseases.

Green Light-Triggered Intraocular Drug Release for Intravenous Chemotherapy of Retinoblastoma.

Retinoblastoma is one of the most severe ocular diseases, of which current chemotherapy is limited to the repetitive intravitreal injections of chemotherapeutics. Systemic drug administration is a less invasive route; however, it is also less efficient for ocular drug delivery because of the existence of blood-retinal barrier and systemic side effects. Here, a photoresponsive drug release system is reported, which is self-assembled from photocleavable trigonal small molecules, to achieve light-triggered intraocular drug accumulation. After intravenous injection of drug-loaded nanocarriers, green light can trigger the disassembly of the nanocarriers in retinal blood vessels, which leads to intraocular drug release and accumulation to suppress retinoblastoma growth. This proof-of-concept study would advance the development of light-triggered drug release systems for the intravenous treatment of eye diseases.

Relative Contribution of Different Mitochondrial Oxidative Phosphorylation Components to the Retinal Pigment Epithelium Barrier Function: Implications for RPE-Related Retinal Diseases.

Disruption of retinal pigment epithelial (RPE) barrier integrity is involved in the pathology of several blinding retinal diseases including age-related macular degeneration (AMD) and diabetic retinopathy (DR), but the underlying causes and pathophysiology are not completely well-defined. Mitochondria dysfunction has often been considered as a potential candidate implicated in such a process. In this study, we aimed to dissect the role of different mitochondrial components; specifically, those of oxidative phosphorylation (OxPhos), in maintaining the barrier functionality of RPE. Electric cell-substrate impedance sensing (ECIS) technology was used to collect multi-frequency electrical impedance data to assess in real-time the barrier formation of the RPE cells. For this purpose, the human retinal pigment epithelial cell line-ARPE-19-was used and treated with varying concentrations of specific mitochondrial inhibitors that target different steps in OxPhos: Rotenone for complex I (the largest protein complex in the electron transport chain (ETC)); oligomycin for ATP synthase; and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) for uncoupling ATP synthesis from the accompanying ETC. Furthermore, data were modeled using the ECIS-Zθ software to investigate in depth the effects of these inhibitors on three separate barrier parameters: cell-cell interactions (Rb), cell-matrix interactions (α), and the cell membrane capacitance (Cm). The viability of ARPE-19 cells was determined by lactate dehydrogenase (LDH) Cytotoxicity Assay. The ECIS program's modeling demonstrated that FCCP and thus OxPhos uncoupling disrupt the barrier function in the ARPE-19 cells across all three components of the total resistance (Rb, α, and Cm) in a dose-dependent manner. On the other hand, oligomycin and thus ATP synthase inhibition mostly affects the ARPE-19 cells' attachment to their substrate evident by a significant decrease in α resistance in a dose-dependent manner, both at the end and throughout the duration of the experiment. On the contrary, rotenone and complex I inhibition mostly affect the ARPE-19 paracellular resistance Rb in a dose-dependent manner compared to basolateral resistance α or Cm. Our results clearly demonstrate differential roles for different mitochondrial components in maintaining RPE cell functionality in which uncoupling of OxPhos is a major contributing factor to the disruption barrier function. Such differences can be used in investigating gene expression as well as for screening of selective agents that improve the OxPhos coupling efficiency to be used in the therapeutic approach for treating RPE-related retinal diseases.

Inflammatory Regulation of CNS Barriers After Traumatic Brain Injury: A Tale Directed by Interleukin-1.

Several barriers separate the central nervous system (CNS) from the rest of the body. These barriers are essential for regulating the movement of fluid, ions, molecules, and immune cells into and out of the brain parenchyma. Each CNS barrier is unique and highly dynamic. Endothelial cells, epithelial cells, pericytes, astrocytes, and other cellular constituents each have intricate functions that are essential to sustain the brain's health. Along with damaging neurons, a traumatic brain injury (TBI) also directly insults the CNS barrier-forming cells. Disruption to the barriers first occurs by physical damage to the cells, called the primary injury. Subsequently, during the secondary injury cascade, a further array of molecular and biochemical changes occurs at the barriers. These changes are focused on rebuilding and remodeling, as well as movement of immune cells and waste into and out of the brain. Secondary injury cascades further damage the CNS barriers. Inflammation is central to healthy remodeling of CNS barriers. However, inflammation, as a secondary pathology, also plays a role in the chronic disruption of the barriers' functions after TBI. The goal of this paper is to review the different barriers of the brain, including (1) the blood-brain barrier, (2) the blood-cerebrospinal fluid barrier, (3) the meningeal barrier, (4) the blood-retina barrier, and (5) the brain-lesion border. We then detail the changes at these barriers due to both primary and secondary injury following TBI and indicate areas open for future research and discoveries. Finally, we describe the unique function of the pro-inflammatory cytokine interleukin-1 as a central actor in the inflammatory regulation of CNS barrier function and dysfunction after a TBI.

N-Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties.

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood-brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p < 0.05). Given this in vivo evidence of brain bioavailability and neural mitohormesis induction, N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

Inhibition of Retinal Ganglion Cell Loss By a Novel ROCK Inhibitor (E212) in Ischemic Optic Nerve Injury Via Antioxidative and Anti-Inflammatory Actions.

Purpose: This study investigated the neuroprotective effects of administration of ROCK inhibitor E212 on ischemic optic neuropathy. Methods: Rats received an intravitreal injection of either E212 or PBS immediately after optic nerve infarct. The oxidative stress in the retina was detected by performing superoxide dismutase activity and CellROX assays. The integrity of retinal pigment epithelium was determined by staining of zona occludens 1. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined by using flash visual-evoked potential analysis, retrograde FluoroGold labeling, and TdT-dUTP nick end-labeling assay. Macrophage infiltration was detected by staining for ED1. The protein levels of TNF-α, p-CRMP, p-AKT1, p-STAT3, and CD206 were evaluated using Western blotting. Results: Administration of E212 resulted in a 1.23-fold increase in the superoxide dismutase activity of the retina and 2.28-fold decrease in RGC-produced reactive oxygen species as compared to the levels observed upon treatment with PBS (P < 0.05). Moreover, E212 prevented the disruption of the blood-retinal barrier (BRB) in contrast to PBS. The P1-N2 amplitude and RGC density in the E212-treated group were 1.75- and 2.05-fold higher, respectively, than those in the PBS-treated group (P < 0.05). The numbers of apoptotic RGCs and macrophages were reduced by 2.93- and 2.54-fold, respectively, in the E212-treated group compared with those in the PBS-treated group (P < 0.05). The levels of p-AKT1, p-STAT3, and CD206 were increased, whereas those of p-PTEN, p-CRMP2, and TNF-α were decreased after treatment with E212 (P < 0.05). Conclusions: Treatment with E212 suppresses oxidative stress, BRB disruption, and neuroinflammation to protect the visual function in ischemic optic neuropathy.

TNF-α released from retinal Müller cells aggravates retinal pigment epithelium cell apoptosis by upregulating mitophagy during diabetic retinopathy.

Retinal pigment epithelium (RPE) cell damage, including mitophagy-associated cell apoptosis, accelerates the pathogenesis of diabetic retinopathy (DR), a common complication of diabetes that causes blindness. Müller cells interact with RPE cells via pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α). Herein, we investigated the role of the RPE cell epidermal growth factor receptor (EGFR)/p38 mitogen-activated protein kinase (p38)/nuclear factor kappa B (NF-κB) pathway in Müller cell-derived TNF-α-induced mitophagy-associated apoptosis during DR. Our results showed that TNF-α released from Müller cells activated the EGFR/p38/NF-κB/p62 pathway to increase mitophagy and apoptosis in RPE cells under high glucose (HG) conditions. Additionally, blockade of the TNF-α/EGFR axis alleviates blood-retina barrier breakdown in diabetic mice. Our data further illustrate the effects of the Müller cell inflammatory response on RPE cell survival, implying potential molecular targets for DR treatment.

Melatonin prevents blood-retinal barrier breakdown and mitochondrial dysfunction in high glucose and hypoxia-induced in vitro diabetic macular edema model.

Diabetic macular edema (DME) is a leading cause of blindness in diabetic retinopathy. Prolonged hyperglycemia plus hypoxia contributes to DME pathogenesis. Retinal pigmented epithelial cells comprise the outer blood-retinal barrier and are essential for maintaining physiological functioning of the retina. Melatonin acts as an antioxidant and regulator of mitochondrial bioenergetics and has a protective effect against ocular diseases. However, the role of mitochondrial dysfunction and the therapeutic potential of melatonin in DME remain largely unexplored. Here, we used an in vitro model of DME to investigate blood-retinal barrier integrity and permeability, angiogenesis, mitochondrial dynamics, and apoptosis signaling to evaluate the potential protective efficacy of melatonin in DME. We found that melatonin prevents cell hyper-permeability and outer barrier breakdown by reducing HIF-1α, HIF-1ß and VEGF and VEGF receptor gene expression. In addition, melatonin reduced the expression of genes involved in mitochondrial fission (DRP1, hFis1, MIEF2, MFF), mitophagy (PINK, BNip3, NIX), and increased the expression of genes involved in mitochondrial biogenesis (PGC-1α, NRF2, PPAR-γ) to maintain mitochondrial homeostasis. Moreover, melatonin prevented apoptosis of retinal pigmented epithelial cells. Our results suggest that mitochondrial dysfunction may be involved in DME pathology, and melatonin may have therapeutic value in DME, by targeting signaling in mitochondria.

Asiatic acid attenuates diabetic retinopathy through TLR4/MyD88/NF-κB p65 mediated modulation of microglia polarization.

AIM: This study aimed to evaluate the effects of Asiatic acid (AA), a naturally occurring compound of pentacyclic triterpenoid, on the pathological processes of diabetic retinopathy (DR). METHODS: SD rats were induced to develop early DR by intraperitoneal injection of STZ (60 mg/kg). Four weeks after injection, the diabetic rats were orally administrated with 37.5 mg/kg or 75 mg/kg AA every day for four weeks. The integrity of blood-retinal barrier (BRB) was measured by Evans blue staining. The polarization of microglia was determined by real-time PCR, western blot, and ELISA assays. The inner BRB (iBRB) or outer BRB (oBRB) breakdown was induced in human retinal endothelial cells or APRE19 cells through co-culture with high glucose and LPS-stimulated microglia BV2 cells. The damage to the iBRB and oBRB was measured using transendothelial/transepithelial electrical resistance (TEER/TER) and FITC-conjugated dextran cell permeability assays. KEY FINDINGS: Results demonstrated that AA alleviated BRB breakdown, as evidenced by decreased protein expression of occludin, claudin-5, and ZO-1. Furthermore, AA treatment suppressed inflammation and M1 polarization, while it increased M2 polarization in the retina of DR rats. In vitro, the iBRB or oBRB breakdown was alleviated by AA. LPS-induced M1-polarization of BV2 cells under high glucose condition was also repressed through AA administration. Finally, we demonstrated that AA weakened the TLR4/MyD88/NF-κB p65 signaling pathway both in vivo and in vitro. SIGNIFICANCE: AA ameliorated early DR by regulating microglia polarization via the TLR4/MyD88/NF-κB p65 pathway. These data indicate that AA is a potential candidate for DR treatment.

Regulation of Endothelium-Reticulum-Stress-Mediated Apoptotic Cell Death by a Polymethoxylated Flavone, Nobiletin, Through the Inhibition of Nuclear Translocation of Glyceraldehyde 3-Phosphate Dehydrogenase in Retinal Müller Cells.

In the early stages of diabetic retinopathy (DR), subtle biochemical and functional alterations occur in Müller cells, which are one of the components of the blood-retinal barrier (BRB). Müller cells are the principal glia of the retina and have shown a strong involvement in the maintenance of homeostasis and the development of retinal tissue. Their functional abnormalities and eventual loss have been correlated with a decrease in the tight junctions between endothelial cells and a consequent breakdown of the BRB, leading to the development of DR. We demonstrated that the endothelium reticulum (ER) triggers Müller cell death and that nuclear accumulation of glyceraldehyde 3-phosphate dehydrogenase is closely associated with ER-induced Müller cell death. In addition, induction of ER stress in Müller cells increased vascular endothelial growth factor expression but decreased pigment-epithelium-derived factor (PEDF) expression in Müller cells. We found that nobiletin, a polymethoxylated flavone from citrus explants, exerts protective action against ER-stress-induced Müller cell death. In addition, nobiletin was found to augment PEDF expression in Müller cells, which may lead to the protection of BRB integrity. These results suggest that nobiletin can be an attractive candidate for the protection of the BRB from breakdown in DR.

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis.

BACKGROUND: The integrin VLA-4 (α4ß1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4ß1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. METHODS: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. RESULTS: There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. CONCLUSIONS: This α4ß1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4ß1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

Melatonin exerts protective effects on diabetic retinopathy via inhibition of Wnt/ß-catenin pathway as revealed by quantitative proteomics.

Diabetic retinopathy (DR), the most common ocular complication resulting from diabetes in working-age adults, causes vision impairment and even blindness because of microvascular damage to the retina. Melatonin is an endogenous neurohormone possessing various biological properties, including the regulation of oxidative stress, inflammation, autophagy, and angiogenesis functions. To evaluate the effects of melatonin on DR, we first investigated the role of melatonin in retinal angiogenesis and inner blood-retina barrier (iBRB) under high glucose conditions in vitro and in vivo. Melatonin administration ameliorated high glucose-induced iBRB disruption, cell proliferation, cell migration, invasion and tube formation, and decreased the expression levels of VEGF, MMP-2, and MMP-9. Furthermore, melatonin treatment increased the level of autophagy but decreased the expression levels of inflammation-related factors under high glucose conditions. To further explore the underlying mechanism, we evaluated human retinal microvascular endothelial cells (HRMECs) via tandem mass tags (TMT)-labeled quantitative proteomics under high-glucose conditions with or without melatonin. Bioinformatics analysis results revealed that the main enrichment pathway of differentially expressed proteins (DEPs) was the Wnt pathway. We found that melatonin inhibited the activation of Wnt/ß-catenin pathway following DR. These abovementioned protective effects of melatonin under hyperglycemia were blocked by lithium chloride (LiCl; activator of the Wnt/ß-catenin signaling pathway). In summary, melatonin exerts protective effects on experimental DR via inhibiting Wnt/ß-catenin pathway by, at least partially, alleviating autophagic dysfunction and inflammatory activation.

Synthesis and in vitro study of nitro- and methoxy-2-phenylbenzofurans as human monoamine oxidase inhibitors.

A new series of 2-phenylbenzofuran derivatives were designed and synthesized to determine relevant structural features for the MAO inhibitory activity and selectivity. Methoxy substituents were introduced in the 2-phenyl ring, whereas the benzofuran moiety was not substituted or substituted at the positions 5 or 7 with a nitro group. Substitution patterns on both the phenyl ring and the benzofuran moiety determine the affinity for MAO-A or MAO-B. The 2-(3-methoxyphenyl)-5-nitrobenzofuran 9 was the most potent MAO-B inhibitor (IC50 = 0.024 µM) identified in this series, whereas 7-nitro-2-phenylbenzofuran 7 was the most potent MAO-A inhibitor (IC50 = 0.168 µM), both acting as reversible inhibitors. The number and position of the methoxyl groups on the 2-phenyl ring, have an important influence on the inhibitory activity. Molecular docking studies confirmed the experimental results and highlighted the importance of key residues in enzyme inhibition.

[Structure Activity Relationship Study of Polymethoxylated Flavones Targeted Retinal Müller Cells for Prevention of Retinal Diseases].

Diabetic retinopathy (DR) is a retinal disease representing one of the main causes of vision loss in developed countries. In the early stage of DR, disruption of blood retinal barrier (BRB) is observed, and it will lead to vascular permeability and visual impairment. Therefore, protection against the breakdown of BRB may be useful strategy for prevention of DR. Matrix metalloproteinases (MMPs) plays an important role in the degradation of extracellular matrix proteins. In DR, they attribute to increased vascular permeability by degrading the junction proteins, such as occuldin and cadherin that are important to maintain the BRB junction complex. Müller cells constitute the main glial cells of the retina and are involved in many retinal functions. They are reported to be one of the MMP-producing cells in the retina. In this symposium review, I present the molecular mechanism of MMP expression in retinal Müller cells. In addition, I would like to introduce polymethoxylated flavones, nobiletin and the derivatives isolated from natural resource as novel MMP inhibitors, which may be applicable to prevention of DR.

Melatonin maintains inner blood-retinal barrier via inhibition of p38/TXNIP/NF-κB pathway in diabetic retinopathy.

The pathophysiology of diabetic retinopathy (DR) was complex. Under hyperglycemic conditions, the release of proinflammatory cytokines and the adhesion of leukocytes to retinal capillaries contribute to endothelial damage and the subsequent increase in vascular permeability resulting in macular edema. Melatonin, produced in the retina to regulate redox reactions and dopamine metabolism, plays protective roles against inflammation and oxidative stress. Considering its anti-inflammatory and antioxidative properties, melatonin was speculated to exert beneficial effects in DR. In this study, we characterized the protective effects of melatonin on the inner blood-retinal barrier (iBRB), as well as the possible mechanisms in experimental DR. Results showed that in diabetic rat retinas, the leakage of iBRB and the expression of inflammatory factors (VEGF, TNF-α, IL-1ß, ICAM-1, and MMP9) increased dramatically, while the expression of tight junction proteins (ZO-1, occludin, JAM-A, and claudin-5) decreased significantly. The above changes were largely ameliorated by melatonin. The in vivo data were confirmed in vitro. In addition, the protein expressions of p38 MAPK, NF-κB, and TXNIP were upregulated significantly in diabetes and were downregulated following melatonin treatment. Melatonin could maintain the iBRB integrity by upregulating the expression of tight junction proteins via inhibiting p38/TXNIP/NF-κB pathway, thus decreasing the production of inflammatory factors. This study may shed light on the development of melatonin-based DR therapy.