MicroRNA-376b-3p Suppresses Choroidal Neovascularization by Regulating Glutaminolysis in Endothelial Cells.

Purpose: Choroidal neovascularization (CNV) is a common pathological change of various ocular diseases that causes serious damage to central vision. Accumulated evidence shows that microRNAs (miRNAs) are closely related with the regulation of endothelial metabolism, which plays crucial roles in angiogenesis. Here, we investigate the molecular mechanism underlying the regulation of endothelial glutamine metabolism by miR-376b-3p in the progression of CNV. Methods: Human retinal microvascular endothelial cells (HRMECs) were transfected with control or miR-376b-3p mimics, and the expression of glutaminase 1 (GLS1), a rate-limiting enzyme in glutaminolysis, was detected by real-time PCR or Western blotting. The biological function and glutamine metabolism of transfected HRMECs were measured by related kits. Luciferase reporter assays were used to validate the CCAAT/enhancer-binding protein beta (CEBPB) was a target of miR-376b-3p. Chromatin immunoprecipitation and RNA immunoprecipitation assays were performed to verify the binding of CEBPB on the promoter region of GLS1. Fundus fluorescein angiography and immunofluorescence detected the effect of miR-376b-3p agomir on rat laser-induced CNV. Results: The expression of miR-376b-3p was decreased, whereas GLS1 expression was increased in the retinal pigment epithelial-choroidal complexes of rats with CNV. HRMECs transfected with miR-376b-3p mimic showed inhibition of CEBPB, resulting in the inactivation of GLS1 transcription and glutaminolysis. Moreover, the miR-376b-3p mimic inhibited proliferation, migration and tube formation but promoted apoptosis in HRMECs, whereas these effects counteracted by α-ketoglutarate supplementation or transfection with CEBPB overexpression plasmid. Finally, the intravitreal administration of the miR-376b-3p agomir restrained CNV formation. Conclusions: Collectively, miR-376b-3p is a suppressor of glutamine metabolism in endothelial cells that could be expected to become a therapeutic target for the treatment of CNV-related diseases.

Cytochrome P450 oxidase 2J inhibition suppresses choroidal neovascularization in mice.

INTRODUCTION: Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) leads to blindness. It has been widely reported that increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) diets reduce CNV. Of the three major pathways metabolizing ω-3 (and ω-6 LCPUFA), the cyclooxygenase and lipoxygenase pathways generally produce pro-angiogenic metabolites from ω-6 LCPUFA and anti-angiogenic ones from ω-3 LCPUFA. Howevehr, cytochrome P450 oxidase (CPY) 2C produces pro-angiogenic metabolites from both ω-6 and ω-3 LCPUFA. The effects of CYP2J2 products on ocular neovascularization are still unknown. Understanding how each metabolic pathway affects the protective effect of ω-3 LCPUFA on retinal neovascularization may lead to therapeutic interventions. OBJECTIVES: To investigate the effects of LCPUFA metabolites through CYP2J2 pathway and CYP2J2 regulation on CNV both in vivo and ex vivo. METHODS: The impact of CYP2J2 overexpression and inhibition on neovascularization in the laser-induced CNV mouse model was assessed. The plasma levels of CYP2J2 metabolites were measured by liquid chromatography and tandem mass spectroscopy. The choroidal explant sprouting assay was used to investigate the effects of CYP2J2 inhibition and specific LCPUFA CYP2J2 metabolites on angiogenesis ex vivo. RESULTS: CNV was exacerbated in Tie2-Cre CYP2J2-overexpressing mice and was associated with increased levels of plasma docosahexaenoic acids. Inhibiting CYP2J2 activity with flunarizine decreased CNV in both ω-6 and ω-3 LCPUFA-fed wild-type mice. In Tie2-Cre CYP2J2-overexpressing mice, flunarizine suppressed CNV by 33 % and 36 % in ω-6, ω-3 LCPUFA diets, respectively, and reduced plasma levels of CYP2J2 metabolites. The pro-angiogenic role of CYP2J2 was corroborated in the choroidal explant sprouting assay. Flunarizine attenuated ex vivo choroidal sprouting, and 19,20-EDP, a ω-3 LCPUFA CYP2J2 metabolite, increased sprouting. The combined inhibition of CYP2J2 with flunarizine and CYP2C8 with montelukast further enhanced CNV suppression via tumor necrosis factor-α suppression. CONCLUSIONS: CYP2J2 inhibition augmented the inhibitory effect of ω-3 LCPUFA on CNV. Flunarizine suppressed pathological choroidal angiogenesis, and co-treatment with montelukast inhibiting CYP2C8 further enhanced the effect. CYP2 inhibition might be a viable approach to suppress CNV in AMD.

Preclinical Efficacy and Safety of VEGF-Grab, a Novel Anti-VEGF Drug, and Its Comparison to Aflibercept.

Purpose: VEGF-Grab is a novel anti-vascular endothelial growth factor (VEGF) candidate drug with higher affinity to both VEGF and placental growth factor (PlGF) compared to aflibercept. We investigated the preclinical efficacy of VEGF-Grab for ophthalmic therapy and compared it to that of aflibercept. Methods: The in vitro anti-VEGF efficacy of VEGF-Grab was determined using VEGF-induced cell proliferation/migration and tube formation assays. The in vivo antiangiogenic efficacy of intravitreal injection of either VEGF-Grab or aflibercept was evaluated using murine models of ocular angiogenesis: mouse oxygen-induced retinopathy (OIR) and rat laser-induced choroidal neovascularization (CNV). The in vivo retinal toxicity in the mouse eye resulting from the injection of either drug was evaluated with light and electron microscopy. Results: VEGF-Grab showed greater inhibition of VEGF-induced cell proliferation/migration than aflibercept, but it showed comparable inhibition of tube formation in vitro. In the in vivo OIR model, VEGF-Grab showed a comparable suppression of retinal neovascularization compared to aflibercept. Additionally, VEGF-Grab showed an efficacy similar to that of aflibercept in terms of CNV inhibition in the laser-induced CNV model. Histology and transmission electron microscopy showed no significant signs of toxicity in the mouse retina at 7 and 30 days following the intravitreal injection of VEGF-Grab or aflibercept. Conclusions: Compared to aflibercept, VEGF-Grab presented comparable in vivo antiangiogenic efficacy and superior in vitro anti-VEGF activity. The retinal safety profiles were comparable for the two drugs. Considering its known higher binding affinity to VEGF and PlGF compared to aflibercept, VEGF-Grab could be a potential candidate drug for neovascular retinal diseases and an alternative to aflibercept.

Attenuation of choroidal neovascularization by dietary intake of ω-3 long-chain polyunsaturated fatty acids and lutein in mice.

Dietary ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and lutein each protect against age-related macular degeneration (AMD). We here examined the effects of ω-3 LCPUFAs and lutein supplementation in a mouse model of AMD. Mice were assigned to four groups: (1) a control group fed an ω-3 LCPUFA-free diet, (2) a lutein group fed an ω-3 LCPUFA-free diet with oral administration of lutein, (3) an ω-3 group fed an ω-3 LCPUFA-supplemented diet, and (4) an ω-3 + lutein group fed an ω-3 LCPUFA-supplemented diet with oral administration of lutein. Mice were fed the defined diets beginning 2 weeks before, and received lutein with an oral gavage needle beginning 1 week before, induction of choroidal neovascularization (CNV) by laser photocoagulation. The area of CNV measured in choroidal flat-mount preparations was significantly reduced in mice fed ω-3 LCPUFAs or lutein compared with those in the control group, and it was reduced in an additive manner in those receiving both ω-3 LCPUFAs and lutein. The concentrations of various inflammatory mediators in the retina or choroid were reduced in mice fed ω-3 LCPUFAs or lutein, but no additive effect was apparent. The generation of reactive oxygen species (ROS) in chorioretinal lesions revealed by dihydroethidium staining as well as the expression of NADPH oxidase 4 (Nox4) in the retina revealed by immunohistofluorescence and immunoblot analyses were attenuated by ω-3 LCPUFAs and lutein in a synergistic manner. Our results thus show that dietary intake of ω-3 LCPUFAs and lutein attenuated CNV in an additive manner and in association with suppression of inflammatory mediator production, ROS generation, and Nox4 expression. Dietary supplementation with both ω-3 LCPUFAs and lutein warrants further study as a means to protect against AMD.

Cytochrome P450 monooxygenase lipid metabolites are significant second messengers in the resolution of choroidal neovascularization.

Age-related macular degeneration (AMD) is the most common cause of blindness for individuals age 50 and above in the developed world. Abnormal growth of choroidal blood vessels, or choroidal neovascularization (CNV), is a hallmark of the neovascular (wet) form of advanced AMD and leads to significant vision loss. A growing body of evidence supports a strong link between neovascular disease and inflammation. Metabolites of long-chain polyunsaturated fatty acids derived from the cytochrome P450 (CYP) monooxygenase pathway serve as vital second messengers that regulate a number of hormones and growth factors involved in inflammation and vascular function. Using transgenic mice with altered CYP lipid biosynthetic pathways in a mouse model of laser-induced CNV, we characterized the role of these lipid metabolites in regulating neovascular disease. We discovered that the CYP-derived lipid metabolites epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs) are vital in dampening CNV severity. Specifically, overexpression of the monooxygenase CYP2C8 or genetic ablation or inhibition of the soluble epoxide hydrolase (sEH) enzyme led to increased levels of EDP and EEQ with attenuated CNV development. In contrast, when we promoted the degradation of these CYP-derived metabolites by transgenic overexpression of sEH, the protective effect against CNV was lost. We found that these molecules work in part through their ability to regulate the expression of key leukocyte adhesion molecules, on both leukocytes and endothelial cells, thereby mediating leukocyte recruitment. These results suggest that CYP lipid signaling molecules and their regulators are potential therapeutic targets in neovascular diseases.

Adiponectin Mediates Dietary Omega-3 Long-Chain Polyunsaturated Fatty Acid Protection Against Choroidal Neovascularization in Mice.

Purpose: Neovascular age-related macular degeneration (AMD) is a major cause of legal blindness in the elderly. Diets with omega3-long-chain-polyunsaturated-fatty-acid (ω3-LCPUFA) correlate with a decreased risk of AMD. Dietary ω3-LCPUFA versus ω6-LCPUFA inhibits mouse ocular neovascularization, but the underlying mechanism needs further exploration. The aim of this study was to investigate if adiponectin (APN) mediated ω3-LCPUFA suppression of neovessels in AMD. Methods: The mouse laser-induced choroidal neovascularization (CNV) model was used to mimic some of the inflammatory aspect of AMD. CNV was compared between wild-type (WT) and Apn-/- mice fed either otherwise matched diets with 2% ω3 or 2% ω6-LCPUFAs. Vldlr-/- mice were used to mimic some of the metabolic aspects of AMD. Choroid assay ex vivo and human retinal microvascular endothelial cell (HRMEC) proliferation assay in vitro was used to investigate the APN pathway in angiogenesis. Western blot for p-AMPKα/AMPKα and qPCR for Apn, Mmps, and IL-10 were used to define mechanism. Results: ω3-LCPUFA intake suppressed laser-induced CNV in WT mice; suppression was abolished with APN deficiency. ω3-LCPUFA, mediated by APN, decreased mouse Mmps expression. APN deficiency decreased AMPKα phosphorylation in vivo and exacerbated choroid-sprouting ex vivo. APN pathway activation inhibited HRMEC proliferation and decreased Mmps. In Vldlr-/- mice, ω3-LCPUFA increased retinal AdipoR1 and inhibited NV. ω3-LCPUFA decreased IL-10 but did not affect Mmps in Vldlr-/- retinas. Conclusions: APN in part mediated ω3-LCPUFA inhibition of neovascularization in two mouse models of AMD. Modulating the APN pathway in conjunction with a ω3-LCPUFA-enriched-diet may augment the beneficial effects of ω3-LCPUFA in AMD patients.

Targeted Intraceptor Nanoparticle for Neovascular Macular Degeneration: Preclinical Dose Optimization and Toxicology Assessment.

Neovascular age-related macular degeneration (AMD) is treated with anti-VEGF intravitreal injections, which can cause geographic atrophy, infection, and retinal fibrosis. To minimize these toxicities, we developed a nanoparticle delivery system for recombinant Flt23k intraceptor plasmid (RGD.Flt23k.NP) to suppress VEGF intracellularly within choroidal neovascular (CNV) lesions in a laser-induced CNV mouse model through intravenous administration. In the current study, we examined the efficacy and safety of RGD.Flt23k.NP in mice. The effect of various doses was determined using fluorescein angiography and optical coherence tomography to evaluate CNV leakage and volume. Efficacy was determined by the rate of inhibition of CNV volume at 2 weeks post-treatment. RGD.Flt23k.NP had peak efficacy at a dose range of 30-60 µg pFlt23k/mouse. Using the lower dose (30 µg pFlt23k/mouse), RGD.Flt23k.NP safety was determined both in single-dose groups and in repeat-dose (three times) groups by measuring body weight, organ weight, hemoglobin levels, complement C3 levels, and histological changes in vital organs. Neither toxicity nor inflammation from RGD.Flt23k.NP was detected. No side effect was detected on visual function. Thus, systemic RGD.Flt23k.NP may be an alternative to standard intravitreal anti-VEGF therapy for the treatment of neovascular AMD.

Fenofibrate Inhibits Cytochrome P450 Epoxygenase 2C Activity to Suppress Pathological Ocular Angiogenesis.

Neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related-macular-degeneration are major causes of blindness. Fenofibrate treatment in type 2 diabetes patients reduces progression of diabetic retinopathy independent of its peroxisome proliferator-activated receptor (PPAR)α agonist lipid lowering effect. The mechanism is unknown. Fenofibrate binds to and inhibits cytochrome P450 epoxygenase (CYP)2C with higher affinity than to PPARα. CYP2C metabolizes ω-3 long-chain polyunsaturated fatty acids (LCPUFAs). While ω-3 LCPUFA products from other metabolizing pathways decrease retinal and choroidal neovascularization, CYP2C products of both ω-3 and ω-6 LCPUFAs promote angiogenesis. We hypothesized that fenofibrate inhibits retinopathy by reducing CYP2C ω-3 LCPUFA (and ω-6 LCPUFA) pro-angiogenic metabolites. Fenofibrate reduced retinal and choroidal neovascularization in PPARα-/-mice and augmented ω-3 LCPUFA protection via CYP2C inhibition. Fenofibrate suppressed retinal and choroidal neovascularization in mice overexpressing human CYP2C8 in endothelial cells and reduced plasma levels of the pro-angiogenic ω-3 LCPUFA CYP2C8 product, 19,20-epoxydocosapentaenoic acid. 19,20-epoxydocosapentaenoic acid reversed fenofibrate-induced suppression of angiogenesis ex vivo and suppression of endothelial cell functions in vitro. In summary fenofibrate suppressed retinal and choroidal neovascularization via CYP2C inhibition as well as by acting as an agonist of PPARα. Fenofibrate augmented the overall protective effects of ω-3 LCPUFAs on neovascular eye diseases.

Anti-angiogenic effect of ALS-L1023, an extract of Melissa officinalis L., on experimental choroidal neovascularization in mice.

BACKGROUND: The effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) leaves, on experimental choroidal neovascularization (CNV) in mice was evaluated. METHODS: C57BL/6 mice were given either vehicle or ALS-L1023 daily via oral gavage for 3 weeks (days 0-21). CNV was induced by rupturing Bruch's membrane using laser photocoagulation (day 7). Two weeks after laser injury (day 21), the CNV lesions were evaluated by an examination of choroidal flat mounts using fluorescein-labelled dextran, immunofluorescence staining with isolectin B4 and fluorescence angiography. The effects of ALS-L1023 on endothelial cell tube formation and the expression of phosphorylated extracellular signal-regulated kinase 1/2 were evaluated using human umbilical vein endothelial cells. RESULTS: The extent of CNV was reduced by ALS-L1023. Mice treated with 100 and 200 mg/kg/day of the material exhibited 44.3 and 68.1% reductions in the extent of CNV lesions, respectively, compared to the vehicle group (P < 0.001). The size of the isolectin B4-labelled area was also significantly decreased in the ALS-L1023-treated groups (P < 0.001). On fluorescein angiography, ALS-L1023-treated mice exhibited significantly less leakage of fluorescent material than did vehicle-treated mice. ALS-L1023 decreased vascular endothelial growth factor-induced human umbilical vein endothelial cell tube formation in a dose-dependent manner. The expression of phosphorylated extracellular signal-regulated kinase 1/2 was suppressed by ALS-L1023. CONCLUSIONS: The laser-induced CNV in mice can be inhibited by ALS-L1023. Therefore, it may have therapeutic potential for the treatment of diseases involving CNV.

Cinidium officinale and its Bioactive Compound, Butylidenephthalide, Inhibit Laser-Induced Choroidal Neovascularization in a Rat Model.

Choroidal neovascularization (CNV) is a common pathology in age-related macular degeneration. In this study, we evaluated in a rat model the effect of an extract of Cinidium officinale Makino and its bioactive compound, butylidenephthalide, on laser-induced CNV. Experimental CNV was induced in Long-Evans rats by laser photocoagulation. C. officinale extract (COE) and butylidenephthalide was intraperitoneally injected once per day for ten days after laser photocoagulation. Choroidal flat mounts were prepared to measure CNV areas and macrophage infiltration. We used a protein array to evaluate the expression levels of angiogenic factors. The CNV area and macrophage infiltration in COE-treated rats were significantly lower than in vehicle-treated rats. COE decreased the expression levels of IGFBP-1, MCP-1, PAI-1, and VEGF. Additionally, butylidenephthalide also inhibited the laser-induced CNV formation and macrophage infiltration and down-regulated the expression of IGFBP-1, MCP-1 and VEGF. These results suggest that COE exerts anti-angiogenic effects on laser-induced CNV by inhibiting the expression of IGFBP-1, MCP-1, and VEGF, indicating that anti-angiogenic activities of COE may be in part due to its bioactive compound, butylidenephthalide.

Anti-VEGF treatment for myopic choroid neovascularization: from molecular characterization to update on clinical application.

Choroidal neovascularization (CNV) secondary to pathologic myopia has a very high incidence in global, especially in Asian, populations. It is a common cause of irreversible central vision loss, and severely affects the quality of life in the patients with pathologic myopia. The traditional therapeutic modalities for CNV secondary to pathologic myopia include thermal laser photocoagulation, surgical management, transpupillary thermotherapy, and photodynamic therapy with verteporfin. However, the long-term outcomes of these modalities are disappointing. Recently, intravitreal administration of anti-VEGF biological agents, including bevacizumab, ranibizumab, pegaptanib, aflibercept, and conbercept, has demonstrated promising outcomes for this ocular disease. The anti-VEGF regimens are more effective on improving visual acuity, reducing central fundus thickness and central retina thickness than the traditional modalities. These anti-VEGF agents thus hold the potential to become the first-line medicine for treatment of CNV secondary to pathologic myopia. This review follows the trend of "from bench to bedside", initially discussing the pathogenesis of myopic CNV, delineating the molecular structures and mechanisms of action of the currently available anti-VEGF drugs, and then systematically comparing the up to date clinical applications as well as the efficacy and safety of the anti-VEGF drugs to the CNV secondary to pathologic myopia.

Melissa officinalis extract inhibits laser-induced choroidal neovascularization in a rat model.

PURPOSE: This study investigated the effect of Melissa officinalis extract on laser-induced choroidal neovascularization (CNV) in a rat model. The mechanism by which M. officinalis extract acted was also investigated. METHODS: Experimental CNV was induced by laser photocoagulation in Brown Norway rats. An active fraction of the Melissa leaf extract was orally administered (50 or 100 mg/kg/day) beginning 3 days before laser photocoagulation and ending 14 days after laser photocoagulation. Optical coherence tomography and fluorescein angiography were performed in vivo to evaluate the thickness and leakage of CNV. Choroidal flat mount and histological analysis were conducted to observe the CNV in vitro. Vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9 expression were measured in retinal and choroidal-scleral lysates 7 days after laser injury. Moreover, the effect of M. officinalis extract on tertiary-butylhydroperoxide (t-BH)-induced VEGF secretion and mRNA levels of VEGF, MMP-2, and MMP-9 were evaluated in human retinal epithelial cells (ARPE-19) as well as in human umbilical vein endothelial cells (HUVECs). RESULTS: The CNV thickness in M. officinalis-treated rats was significantly lower than in vehicle-treated rats by histological analysis. The CNV thickness was 33.93±7.64 µm in the high-dose group (P<0.001), 44.09±12.01 µm in the low-dose group (P = 0.016), and 51.00±12.37 µm in the control group. The proportion of CNV lesions with clinically significant fluorescein leakage was 9.2% in rats treated with high-dose M. officinalis, which was significantly lower than in control rats (53.4%, P<0.001). The levels of VEGF, MMP-2, and MMP-9 were significantly lower in the high-dose group than in the control group. Meanwhile, M. officinalis extract suppressed t-BH-induced transcription of VEGF and MMP-9 in ARPE-19 cells and HUVECs. CONCLUSIONS: Systemic administration of M. officinalis extract suppressed laser-induced CNV formation in rats. Inhibition of VEGF and MMP-9 via anti-oxidative activity may underlie this effect.

Inhibition of APE1/Ref-1 redox activity rescues human retinal pigment epithelial cells from oxidative stress and reduces choroidal neovascularization.

The effectiveness of current treatment for age related macular degeneration (AMD) by targeting one molecule is limited due to its multifactorial nature and heterogeneous pathologies. Treatment strategy to target multiple signaling pathways or pathological components in AMD pathogenesis is under investigation for better clinical outcome. Inhibition of the redox function of apurinic endonuclease 1/redox factor-1 (APE1) was found to suppress endothelial angiogenesis and promote neuronal cell recovery, thereby may serve as a potential treatment for AMD. In the current study, we for the first time have found that a specific inhibitor of APE1 redox function by a small molecule compound E3330 regulates retinal pigment epithelium (RPEs) cell response to oxidative stress. E3330 significantly blocked sub-lethal doses of oxidized low density lipoprotein (oxLDL) induced proliferation decline and senescence advancement of RPEs. At the same time, E3330 remarkably decreased the accumulation of intracellular reactive oxygen species (ROS) and down-regulated the productions of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF), as well as attenuated the level of nuclear factor-κB (NF-κB) p65 in RPEs. A panel of stress and toxicity responsive transcription factors that were significantly upregulated by oxLDL was restored by E3330, including Nrf2/Nrf1, p53, NF-κB, HIF1, CBF/NF-Y/YY1, and MTF-1. Further, a single intravitreal injection of E3330 effectively reduced the progression of laser-induced choroidal neovascularization (CNV) in mouse eyes. These data revealed that E3330 effectively rescued RPEs from oxidative stress induced senescence and dysfunctions in multiple aspects in vitro, and attenuated laser-induced damages to RPE-Bruch׳s membrane complex in vivo. Together with its previously established anti-angiogenic and neuroprotection benefits, E3330 is implicated for potential use for AMD treatment.

AMD--the retinal disease with an unprecised etiopathogenesis: in search of effective therapeutics.

AMD (age-related macular degeneration) is a progressive vision-threatening ocular disease, affecting central region of the retina--the macula--and manifesting in the elderly. AMD is a degenerative disease, and the degeneration affects primarily the retinal pigment epithelial (RPE) cells and secondarily the photoreceptors, leading consequently to disturbances or partial loss of central vision and legal blindness. Clinically, the disease is classified as: atrophic--dry AMD (in majority of cases), and neovascular--wet AMD (with choroidal neovascularization--CNV: 10-15% of all AMD cases). Pathogenesis of AMD is complex, multifactorial and only poorly recognized. Main risk factors include: advanced age, genetic predispositions, environmental determinants, history of exposure to intensive light and smoking. At least four molecular processes contribute to the development of AMD pathology: lipofuscinogenesis, drusogenesis, inflammation and choroidal neovascularization (in wet AMD). Since vascular endothelial growth factor (VEGF) is a predominant proangiogenic factor in CNV. the wet AMD can be treated with intravitreous application of "anti-VEGF" agents (Avastin, Lucentis, Eylea). Till now, there is no approved therapy for dry AMD, although several agents/treatments are currently in clinical trials. This paper briefly describes major molecular and cellular events leading to AMD, and presents currently used and new experimental therapeutic strategies against AMD.

Combination of targeted PDT and anti-VEGF therapy for rat CNV by RGD-modified liposomal photocyanine and sorafenib.

PURPOSE: To achieve a combination therapy of targeted PDT and anti-VEGF for choroidal neovascularization (CNV). METHODS: Arg-Gly-Asp (RGD)-modified liposomes encapsulating photocyanine and sorafenib (RGD-SSL-[P]-[S]) were prepared and characterized. Drug concentration in RGD-SSL-[P]-[S] and irradiation time were optimized on ARPE-19 and HUVEC cells in vitro. A laser-induced CNV rat model was used to assess the efficacy of this targeted combinational system. The effect of RGD-SSL-[P]-[S] on the retinal structure of BN rats was also examined. RESULTS: The particle size of RGD-SSL-[P]-[S] was approximately 100 nm, with a encapsulation efficiency more than 90% for both photocyanine and sorafenib. From RGD-SSL-[P]-[S], the release rate of photocyanine was approximately 22%, whereas that of sorafeinb was approximately 40% at 48 hours. With the optimal drug concentration and irradiation time, RGD-SSL-[P]-[S] exhibited cytotoxicity only to HUVEC without obvious damage to normal ARPE-19 cells. Rats treated with RGD-SSL-[P]-[S] showed the least CNV area and fluorescein leakage in fluorescein fundus angiography. RGD-SSL-[P]-[S] was also found safe to the rat retina. CONCLUSIONS: Combination of targeted PDT and anti-VEGF might be an effective therapy for CNV.

Hyperglycaemia exacerbates choroidal neovascularisation in mice via the oxidative stress-induced activation of STAT3 signalling in RPE cells.

Choroidal neovascularisation (CNV) that occurs as a result of age-related macular degeneration (AMD) causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG) antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and vascular endothelial growth factor (VEGF), in mice with CNV. The production of reactive oxygen species (ROS) in retinal pigment epithelial (RPE) cells that had been cultured under high glucose was quantitated using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC) rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV. Thus, our results reveal a potential strategy for the treatment and prevention of diseases involving CNV.

The Chinese medicine formula HB01 reduces choroidal neovascularization by regulating the expression of vascular endothelial growth factor.

BACKGROUND: Choroidal neovascularization (CNV) remains the leading cause of newly acquired blindness in the developed world. Currently anti-vascular endothelial growth factor (VEGF) therapies are broadly used to treat neovascular ocular disorders. Here we demonstrate the effect of a traditional Chinese medicine formula, HB01, on CNV. METHODS: A rat model of laser-induced CNV was used to investigate the effect of HB01 in vivo. The CNV lesions in the eye were evaluated using fundus fluorescein angiography and visualized/quantified using confocal microscopy. Expression of VEGF in the choroidal and retinal tissues was measured using quantitative real-time PCR and immunohistochemistry. RESULTS: We demonstrated that a traditional Chinese Medicine formula, named HB01, significantly reduced neovascularization in a rat CNV model. The effect of HB01 on CNV was comparable to the intravitreal injection of bevacizumab (Avastin). Our results also suggested that HB01 may reduce CNV partially through inhibiting the expression of VEGF. CONCLUSIONS: These data support HB01 as an alternative therapy for ocular neovascular disorders.

Inhibition of choroidal neovascularization with an anti-inflammatory carotenoid astaxanthin.

PURPOSE: Astaxanthin (AST) is a carotenoid found in marine animals and vegetables. The purpose of the present study was to investigate the effect of AST on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms. METHODS: Laser photocoagulation was used to induce CNV in C57BL/6J mice. Mice were pretreated with intraperitoneal injections of AST daily for 3 days before photocoagulation, and treatments were continued daily until the end of the study. CNV response was analyzed by volumetric measurements 1 week after laser injury. Retinal pigment epithelium-choroid levels of IkappaB-alpha, intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2 were examined by Western blotting or ELISA. AST was applied to capillary endothelial (b-End3) cells, macrophages, and RPE cells to analyze the activation of NF-kappaB and the expression of inflammatory molecules. RESULTS: The index of CNV volume was significantly suppressed by treatment with AST compared with that in vehicle-treated animals. AST treatment led to significant inhibition of macrophage infiltration into CNV and of the in vivo and in vitro expression of inflammation-related molecules, including VEGF, IL-6, ICAM-1, MCP-1, VEGFR-1, and VEGFR-2. Importantly, AST suppressed the activation of the NF-kappaB pathway, including IkappaB-alpha degradation and p65 nuclear translocation. CONCLUSIONS: AST treatment, together with inflammatory processes including NF-kappaB activation, subsequent upregulation of inflammatory molecules, and macrophage infiltration, led to significant suppression of CNV development. The present study suggests the possibility of AST supplementation as a therapeutic strategy to suppress CNV associated with AMD.

Macular pigment lutein is antiinflammatory in preventing choroidal neovascularization.

BACKGROUND: Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration, the most common cause of blindness in the developed countries. The aim of the current study was to investigate the effect of lutein supplementation on the development of the murine model of laser-induced CNV together with underlying molecular mechanisms. METHODS AND RESULTS: Mice were orally pretreated with lutein daily from 3 days before laser photocoagulation until the end of the study. The index of CNV volume was significantly suppressed by the treatment with lutein, compared with vehicle-treated animals. Lutein treatment led to significant inhibition of macrophage infiltration into CNV and of the in vivo and in vitro expression of inflammation-related molecules including vascular endothelial growth factor, monocyte chemotactic protein -1, and intercellular adhesion molecule-1. Importantly, lutein suppressed IkappaB-alpha degradation and nuclear translocation of nuclear factor (NF)-kappaB p65 both in vivo and in vitro. Additionally, the development of CNV was significantly suppressed by inhibiting NF-kappaB p65 nuclear translocation, to the levels seen in the lutein treatment. CONCLUSIONS: Lutein treatment led to significant suppression of CNV development together with inflammatory processes including NF-kappaB activation and subsequent upregulation of inflammatory molecules, providing molecular evidence of potential validity of lutein supplementation as a therapeutic strategy to suppress CNV.

Eicosapentaenoic acid is anti-inflammatory in preventing choroidal neovascularization in mice.

PURPOSE: To investigate the role of eicosapentaenoic acid (EPA), the major omega-3 polyunsaturated fatty acid (PUFA), in the development of choroidal neovascularization (CNV), together with underlying molecular mechanisms. METHODS: Six-week-old C57BL/6 mice were fed with laboratory chow with 5% EPA or the omega-6 PUFA linoleic acid (LA) for 4 weeks. Laser photocoagulation was performed to induce CNV, and the volume of CNV tissue was evaluated by volumetric measurements. The expression and production of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, vascular endothelial growth factor (VEGF) and interleukin (IL)-6 in the retinal pigment epithelium (RPE)-choroid in vivo, and stimulated b-End3 endothelial cells and RAW264.7 macrophages in vitro were evaluated by RT-PCR and ELISA. Fatty acid composition in the serum and the RPE-choroid was analyzed by gas chromatography and high-performance liquid chromatography, respectively. Serum levels of C-reactive protein (CRP), IL-6, VEGF, MCP-1, and soluble ICAM-1 were examined by ELISA. RESULTS: The CNV volume in EPA-fed animals was significantly suppressed compared with that in control mice, whereas the LA-rich diet did not affect CNV. The mRNA expression and protein levels of ICAM-1, MCP-1, VEGF, and IL-6 after CNV induction were significantly reduced in EPA-supplemented mice. In vitro, EPA application led to significant inhibition of mRNA and protein levels of ICAM-1 and MCP-1 in endothelial cells and VEGF and IL-6 in macrophages. EPA-fed mice exhibited significantly higher levels of EPA and lower levels of the omega-6 PUFA arachidonic acid in the serum and the RPE-choroid than control animals. EPA supplementation also led to significant reduction of serum levels of IL-6 and CRP after CNV induction. CONCLUSIONS: The present study demonstrates for the first time that an EPA-rich diet results in significant suppression of CNV and CNV-related inflammatory molecules in vivo and in vitro. These results suggest that frequent consumption of omega-3 PUFAs may prevent CNV and lower the risk of blindness due to age-related macular degeneration.