RPE-choroid Complex Research Articles

H-151, a Selective STING Inhibitor, Has Potential as a Treatment for Neovascular Age-Related Macular Degeneration.

The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) stimulator of interferon gene (STING) pathway is a crucial cascade in the inflammatory response initiated by the recognition of cytosolic double-stranded DNA (dsDNA). The aim of this study was to evaluate the effect of STING inhibitor in murine choroidal neovascularization (CNV). To investigate whether the cGAS-STING pathway is activated during CNV, CNV was induced using laser photocoagulation in male C57BL/6J mice. The expression of change of cGAS and STING during CNV development was confirmed by Western-blotting. H-151, a potent STING palmitoylation antagonist, was used as a STING inhibitor. H-151 was administered intravitreally immediately after laser induction. To confirm the role of the cGAS-STING pathway in CNV formation, we evaluated CNV size and performed fundus fluorescein angiography. The expression levels of cGAS and STING were significantly upregulated in the RPE-choroid complex after CNV induction, and dsDNA merged with cGAS was observed in CNV lesions. Intravitreal administration of H-151 suppressed CNV development and fluorescent leakage from neovessels. In CNV lesions, the high expression of STING and cGAS was observed in infiltrating F4/80+ macrophages. H-151 administration attenuated downstream signals of the cGAS-STING pathway, including the phosphorylation of nuclear factor-κB, and downregulated the expression of interleukin 1β. These findings support that the inhibition of cGAS-STING pathway treats abnormal ocular angiogenesis.

RO4929097, a Selective γ-Secretase Inhibitor, Inhibits Subretinal Fibrosis Via Suppressing Notch and ERK1/2 Signaling in Laser-Induced Mouse Model.

PurposeThis study aimed to explore whether RO4929097 (RO), a specific γ-secretase inhibitor, could inhibit the subretinal fibrosis in laser-induced mouse model and the relevant molecular mechanisms.MethodsMale C57BL/6J mice were used to produce choroidal neovascularization (CNV) and subretinal fibrosis by laser photocoagulation, and RO was administered intravitreally 1 day after laser induction. The sizes of CNV and subretinal fibrosis were measured and quantified in both 2D and 3D constructions. The ARPE-19 cell line and primary human RPE (phRPE) cells were treated with TGFβ1, in combination with or without RO, to examine Notch related molecules, epithelial mesenchymal transition (EMT), cell viability, migration, and contractile function, as well as the crosstalk between Notch and other EMT relevant signaling pathways.ResultsIntravitreal injection of RO reduced the sizes of both CNV and subretinal fibrosis in laser-induced young and old mice at day 7 and day 14 after laser induction. Moreover, EMT and Notch activation in RPE-choroid complexes from laser-induced mice were significantly attenuated by RO. In vitro, TGFβ1 activated Notch signaling and induced EMT in ARPE-19 cells, accompanied by enhanced EMT-related function, which were inhibited by RO. The inhibition of RO on EMT was further confirmed in TGFβ1-treated phRPE cells. Blockage of Notch signaling by RO could inhibit ERK1/2 signaling; whereas ERK1/2 inhibition had no effect on Notch. The action of RO was independent of Smad2/3 or p38, and co-inhibition of Notch and Smad2/3 showed synergistic effect on EMT inhibition.ConclusionsRO exerts its antifibrotic effect by directly inhibiting Notch signaling and indirectly suppressing ERK1/2 signaling. Targeting Notch signaling might provide a therapeutic strategy in prevention and treatment of subretinal fibrosis in neovascular age-related macular degeneration (nAMD).

Mitochondria dynamics in the aged mice eye and the role in the RPE phagocytosis

Aging is a predominant risk factor for various eye diseases. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, and its etiology remains unclear. Fragmented and dysfunctional mitochondria are associated with age-related diseases. The retinal pigment epithelium (RPE), a polarized cell layer that functions in visual pigment recycling and degeneration, is suspected as the primary region site of AMD. In the present study, we investigated the relationship between mitochondrial dysfunction and RPE aging. Compared to young mice, aged pigmented mice (C57BL/6J, 12-month-old) exhibit decreased visual function without retinal thinning. Consistently, the rhodopsin expression level decreased in the outer segment of aged mice. Moreover, the cell volume of the RPE increased in aged animals. Interestingly, the expression of mitochondria dynamics-related proteins, including Drp1, was altered in the RPE-choroid complex but not in the neural retina after aging. Electron microscopy revealed that mitochondrial size decreased and cristae width increased in aged RPE. The photoreceptor outer segment (POS) treatment of ARPE-19 cells causes Drp1 activation. Furthermore, pharmacological suppression of mitochondrial fission improved the phagocytosis of the POS. These findings indicate that mitochondrial dysfunction and fission in RPE impede phagocytosis and cause retardation of the visual cycle, which can be one of the age-related defects in the retina that may contribute to the onset of AMD.

Triptolide-nanoliposome-APRPG, a novel sustained-release drug delivery system targeting vascular endothelial cells, enhances the inhibitory effects of triptolide on laser-induced choroidal neovascularization

PurposeTo investigate whether triptolide-nanoliposome-APRPG (TP-nanolip-APRPG), a novel sustained-release nano-drug delivery system that targets vascular endothelial cells, could enhance the inhibition of triptolide (TP) on laser-induced choroidal neovascularization (CNV). MethodsTP was encapsulated with or without APRPG (Ala-Pro-Arg-Pro-Gly) peptide-modified nanoliposomes. CNV was induced by laser photocoagulation in C57BL/6J mice. One microliter of 10 μg free TP monomer, TP-nanolip containing 10 μg TP, TP-nanolip-APRPG containing 10 μg TP, or an identical volume of PBS was intravitreally injected in mice immediately after laser photocoagulation. Seven days after laser photocoagulation, CNV volumes were calculated in each group. Infiltration of M2 macrophages as well as protein levels of vascular endothelial growth factor (VEGF) and inflammatory factors including ICAM-1 and MCP-1 in the RPE-choroid complex were determined. In vitro assays for cell proliferation, migration, and tube formation were also performed. ResultsTP-nanolip-APRPG was successfully synthesized and exhibited good TP delivery and enhanced the cellular uptake of TP in vitro. In vitro studies showed that TP-nanolip-APRPG was a better inhibitor of cell proliferation (31.34 ± 3.89 % vs 41.25 ± 4.67 % vs 53.55 ± 5.76 %), migration (62.60 ± 8.88 vs 104.60 ± 13.32 vs 147.00 ± 13.15), and tube formation (681.26 ± 108.15 vs 926.75 ± 54.01 vs 1189.84 ± 157.14) than TP-nanolip or free TP (all P < 0.05). Intravitreal injections of free TP (77588.10±7719.28 μm3), TP-nanolip (64628.23 ± 5857.96 μm3), and TP-nanolip-APRPG (50880.34 ± 6606.56 μm3) inhibited the development of CNV compared with the PBS control group (120338.07 ± 17428.90 μm3) (P < 0.01, n=6). TP-nanolip-APRPG and TP-nanolip significantly down-regulated the protein levels of VEGF (152.76±19.55 vs 182.24±19.98 vs 208.55±21.93 pg/mg total protein) and inflammatory factors including ICAM-1 (61.69±3.49 vs 72.04±3.49 vs 81.92±4.09 ng/mg total protein) and MCP-1 (40.14±3.50 vs 50.75±4.18 vs 60.27±5.23 pg/mg total protein) compared with the free TP monomer group (all P < 0.05, n=8), which paralleled the decreased infiltration of M2 macrophages in the CNV lesions. Moreover, no influence on retinal morphology and function was observed before or after treatment in each group (P > 0.05, n=6). ConclusionsTP-nanolip-APRPG, a novel sustained-release drug delivery system targeting endothelial cells of CNV lesions, could enhance TP inhibition of the development of CNV without toxicity in the retina, suggesting therapeutic potential for CNV-related diseases in future clinical practice.

ROS production and mitochondrial dysfunction driven by PU.1-regulated NOX4-p22phox activation in Aβ-induced retinal pigment epithelial cell injury.

Rationale: Amyloid β (Aβ) deposition, an essential pathological process in age-related macular degeneration (AMD), causes retinal pigment epithelium (RPE) degeneration driven mostly by oxidative stress. However, despite intense investigations, the extent to which overoxidation contributes to Aβ-mediated RPE damage and its potential mechanism has not been fully elucidated.Methods: We performed tandem mass-tagged (TMT) mass spectrometry (MS) and bioinformatic analysis of the RPE-choroid complex in an Aβ1-40-induced mouse model of retinal degeneration to obtain a comprehensive proteomic profile. Key regulators in this model were confirmed by reactive oxygen species (ROS) detection, mitochondrial ROS assay, oxygen consumption rate (OCR) measurement, gene knockout experiment, chromatin immunoprecipitation (ChIP), and luciferase assay.Results: A total of 4243 proteins were identified, 1069 of which were significantly affected by Aβ1-40 and found to be enriched in oxidation-related pathways by bioinformatic analysis. Moreover, NADPH oxidases were identified as hub proteins in Aβ1-40-mediated oxidative stress, as evidenced by mitochondrial dysfunction and reactive oxygen species overproduction. By motif and binding site analyses, we found that the transcription factor PU.1/Spi1 acted as a master regulator of the activation of NADPH oxidases, especially the NOX4-p22phox complex. Also, PU.1 silencing impeded RPE oxidative stress and mitochondrial dysfunction and rescued the retinal structure and function.Conclusion: Our study suggests that PU.1 is a novel therapeutic target for AMD, and the regulation of PU.1 expression represents a potentially novel approach against excessive oxidative stress in Aβ-driven RPE injury.

Effect of acute and chronic aldosterone exposure on the retinal pigment epithelium-choroid complex in rodents

Preclinical and clinical evidences show that aldosterone and/or mineralocorticoid receptor (MR) over-activation by glucocorticoids can be deleterious to the retina and to the retinal pigment epithelium (RPE)-choroid complex. However, the exact molecular mechanisms driving these effects remain poorly understood and pathological consequences of chronic exposure of the retina and RPE/choroid to aldosterone have not been completely explored. We aimed to decipher the transcriptomic regulation in the RPE-choroid complex in rats in response to acute intraocular aldosterone injection and to explore the consequences of systemic chronic aldosterone exposure on the morphology and the gene regulation in RPE/choroid in mice. High dose of aldosterone (100 nM) was intravitreously injected in Lewis rat eyes in order to yield an aldosterone dose able to induce a molecular response at the apical side of the RPE-choroid complex. The posterior segment morphology was evaluated in vivo using optical coherence tomography (OCT) before and 24 h after aldosterone injection. Rat RPE-choroid complexes were used for RNA sequencing and analysis. Uninephrectomy/aldosterone/salt (NAS) model was created in wild-type C57BL/6 mice. After 6 weeks, histology of mouse posterior segments were observed ex vivo. Gene expression in the RPE-choroid complex was analyzed using quantitative PCR. Acute intravitreous injection of aldosterone induced posterior segment inflammation observed on OCT. RNA sequencing of rat RPE-choroid complexes revealed up-regulation of pathways involved in inflammation, oxidative stress and RNA procession, and down-regulation of genes involved in synaptic activity, muscle contraction, cytoskeleton, cell junction and transporters. Chronic aldosterone/salt exposure in NAS model induces retinal edema, choroidal vasodilation and RPE cell dysfunction and migration. Quantitative PCR showed deregulation of genes involved in inflammatory response, oxidative stress, particularly the NOX pathway, angiogenesis and cell contractility. Both rodent models share some common phenotypes and molecular regulations in the RPE-choroid complex that could contribute to pachychoroid epitheliopathy in humans. The difference in inflammatory status relies on different intraocular or systemic route of aldosterone administration and on the different doses of aldosterone exposed to the RPE-choroid complex.

Dynamic DNA Methylation Changes of Tbx21 and Rorc during Experimental Autoimmune Uveitis in Mice.

The key transcription factors of T helper cell subpopulations, including T-bet, GATA3, RORγt, and Foxp3 are involved in various autoimmune diseases. Whether methylation of these master transcription factors is associated with the development of experimental autoimmune uveitis (EAU) and the possible epigenetic regulatory mechanisms involved has however not yet been addressed. In our study, significant methylation changes in both Tbx21 and Rorc were observed in one CpG site in the retinas of EAU mice. Two CpG sites of Tbx21 and one CpG site of Rorc showed significant dynamic methylation changes in the RPE-choroid complex during EAU. The mRNA expressions of Tbx21 and Rorc in both the retinas and RPE-choroid complexes correlated with the methylation changes at the various time points during EAU development. The methylation changes were associated with the production of the Th1/Th17 cells' signature cytokines, IFN-γ and IL-17. Dynamic changes in mRNA expression of DNA methyltransferases (DNMT1) were also noted, which may be related to the observed methylation changes of these genes. The present study provides evidence that DNA methylation of Tbx21 and Rorc may be associated with the development of EAU. DNMT1 activation may have an important effect on regulating DNA methylation dynamics.

Rhythmic Regulation of Photoreceptor and RPE Genes Important for Vision and Genetically Associated With Severe Retinal Diseases.

PurposeThe aim of the present study was to identify candidate genes for mediating daily adjustment of vision.MethodsGenes important for vision and genetically associated with severe retinal diseases were tested for 24-hour rhythms in transcript levels in neuronal retina, microdissected photoreceptors, photoreceptor-related pinealocytes, and retinal pigment epithelium-choroid (RPE-choroid) complex by using quantitative PCR.ResultsPhotoreceptors of wildtype mice display circadian clock-dependent regulation of visual arrestins (Arr1, Arr4) and the visual cycle gene Rdh12, whereas cells of the RPE-choroid exhibit light-dependent regulation of the visual cycle key genes Lrat, Rpe65, and Rdh5. Clock-driven rhythmicity of Arr1, Arr4, and Rdh12 was observed also in rat pinealocytes, to persist in a mouse model of diabetic retinopathy (db/db) and, in the case of Arr1, to be abolished in retinae of mice deficient for dopamine D4 receptors. Therefore, the expression rhythms appear to be evolutionary conserved, to be unaffected in diabetic retinopathy, and, for Arr1, to require dopamine signaling via dopamine D4 receptors.ConclusionsThe data of the present study suggest that daily adjustment of retinal function combines clock-dependent regulation of genes responsible for phototransduction termination (Arr1, Arr4) and detoxification (Rdh12) in photoreceptors with light-dependent regulation of genes responsible for retinoid recycling (Lrat, Rpe65, and Rdh5) in RPE. Furthermore, they indicate circadian and light-dependent regulation of genes genetically associated with severe retinal diseases.

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis.

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

Intravitreal itraconazole inhibits laser-induced choroidal neovascularization in rats.

Choroidal neovascularization (CNV) is a major cause of severe visual loss in patients with age-related macular degeneration (AMD). Recently, itraconazole has shown potent and dose-dependent inhibition of tumor-associated angiogenesis. We evaluated the anti-angiogenic effect of itraconazole in a rat model of laser-induced CNV. After laser photocoagulation in each eye to cause CNV, right eyes were administered intravitreal injections of itraconazole; left eyes received balanced salt solution (BSS) as controls. On day 14 after laser induction, fluorescein angiography (FA) was used to assess abnormal vascular leakage. Flattened retinal pigment epithelium (RPE)-choroid tissue complex was stained with Alexa Fluor 594-conjugated isolectin B4 to measure the CNV area and volume. Vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression was determined 1, 4, 7, and 14 days after intravitreal injection by quantitative RT-PCR or Western blot. VEGF levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Intravitreal itraconazole significantly reduced leakage from CNV as assessed by FA and CNV area and volume on flat mounts compared with intravitreal BSS (p = 0.002 for CNV leakage, p<0.001 for CNV area and volume). Quantitative RT-PCR showed significantly lower expression of VEGFR2 mRNA in the RPE-choroid complexes of itraconazole-injected eyes than those of BSS-injected eyes on days 7 and 14 (p = 0.003 and p = 0.006). Western blots indicated that VEGFR2 was downregulated after itraconazole treatment. ELISA showed a significant difference in VEGF level between itraconazole-injected and BSS-injected eyes on days 7 and 14 (p = 0.04 and p = 0.001). Our study demonstrated that intravitreal itraconazole significantly inhibited the development of laser-induced CNV in rats. Itraconazole had anti-angiogenic activity along with the reduction of VEGFR2 and VEGF levels. Itraconazole may prove beneficial for treating CNV as an alternative or adjunct to other therapies.

Wet-AMD on a Chip: Modeling Outer Blood-Retinal Barrier In Vitro.

Choroidal neovascularization (CNV) in the retinal pigment epithelium (RPE)-choroid complex constituting outer blood retinal barrier (oBRB) is a critical pathological step in various ophthalmic diseases, which results in blindness, such as wet type age-related macula degeneration. Current in vitro experimental models using petri dishes or transwell are unable to study CNV morphogenesis. Here, a unique organotypic eye-on-a-chip model is described that mimics the RPE-choroid complex in vitro. This model consists of an RPE monolayer and adjacent perfusable blood vessel network, which is supporting barrier function of oBRB. The intact barrier function of the RPE-choroid complex is reconstituted while maintaining important structural features. Further, this model can successfully mimic the pathogenesis of CNV especially in terms of morphogenesis, which is penetrating angiogenic sprouts from pre-existing choroidal vessels that result in breakdown of RPE monolayer. The alleviation of the pathological angiogenesis can be modeled with bevacizumab, a clinical drug for CNV treatment. It is believed that this model can be used to aid in the development of advanced in vitro eye drug evaluation in conjunction with animal models.

Quantifying melanin concentration in retinal pigment epithelium using broadband photoacoustic microscopy.

Melanin is the dominant light absorber in retinal pigment epithelium (RPE). The loss of RPE melanin is a sign of ocular senescence and is both a risk factor and a symptom of age-related macular degeneration (AMD). Quantifying the RPE melanin concentration provides insight into the pathological role of RPE in ocular aging and the onset and progression of AMD. The main challenge in accurate quantification of RPE melanin concentration is to distinguish this ten-micrometer-thick cell monolayer from the underlying choroid, which also contains melanin but carries different pathognomonic information. In this work, we investigated a three-dimensional photoacoustic microscopic (PAM) method with high axial resolution, empowered by broad acoustic detection bandwidth, to distinguish RPE from choroid and quantify melanin concentrations in the RPE ex vivo. We first conducted numerical simulation on photoacoustic generation in the RPE, which suggested that a PAM system with at least 100-MHz detection bandwidth provided sufficient axial resolution to distinguish the melanin in RPE from that in choroid. Based on simulation results, we integrated a transparent broadband micro-ring resonator (MRR) based detector in a homebuilt PAM system. We imaged ex vivo RPE-choroid complexes (RCCs) from both porcine and human eyes and quantified the absolute melanin concentrations in the RPE and choroid, respectively. In our study, the measured melanin concentrations were 14.7 mg/mL and 17.0 mg/mL in human and porcine RPE, and 12 mg/mL and 61 mg/mL in human and porcine choroid, respectively. This study suggests that broadband PAM is capable of quantifying the RPE melanin concentration from RCCs ex vivo.

Different distributions of M1 and M2 macrophages in a mouse model of laser-induced choroidal neovascularization.

Choroidal neovascularization (CNV) is a serious complication of age-related macular degeneration. The aim of the present study was to investigate the expression and distribution of M1 and M2 macrophages in a laser-induced CNV adult mouse model. The mRNA expression levels of M1, M2 and pan macrophage markers, and macrophage-associated angiogenic cytokines, were determined by reverse transcription-quantitative polymerase chain reaction. Immunofluorescence studies were performed to determine the location of the macrophages. The expression levels of M1 macrophage markers increased to a greater extent compared with M2 markers in the retinal pigment epithelium (RPE)-choroid complexes following laser photocoagulation. By contrast, the expression levels of M2 macrophage markers increased primarily in the retinas. Immunofluorescence studies revealed that the increased number of cluster of differentiation (CD)206-positive cells were located primarily in the retina, whereas the CD80-positive cells were located around the site of CNVs in the RPE-choroid. In addition, the M1-associated cytokines increased to a greater extent in the RPE-choroid complexes, whereas the M2-associated cytokines were highly expressed in the retinas. These findings indicate that M1 and M2 macrophage numbers increased following CNV; however, the locations were different in this mouse model of laser-induced CNV. The results of the present study suggest that M1 macrophages have a more direct role in inhibiting the development of CNV.

The progress of cell-replacement therapy for age-related macular degeneration

Based on the pathogenic mechanisms of age-related macular degeneration (AMD), tremendous preclinical and clinical trials have demonstrated that cell transplantation which aim to replace impaired retinal pigment epithelium (RPE) with healthy RPE cells is a promising approach to treat AMD. So far, choices of cell sources mainly are autologous RPE, iris pigment epithelium, fetal RPE, human embryonic stem cell-derived RPE and human induced pluripotent stem cell-derived RPE, and some of them are undergoing clinical researches. Grafting manners in cell-based therapies are various including RPE sheet or RPE-choroid complex transplantation, RPE cell suspension injection, and RPE sheet transplantation with scaffolds. This review is limited to cell-based therapies for RPE that damaged first in the progress of AMD and focus on recent advances in cell sources, transplantation methods, preclinical and clinical trials, and the obstacles that must be overcome. Key words: Macular degeneration/therapy; Retinal pigment epithelium; Cell transplantation; Review

Protective Effects of Antiplacental Growth Factor Antibody Against Light-Induced Retinal Damage in Mice.

Placental growth factor (PlGF) is part of the VEGF family and is known to be involved in angiogenesis, vasopermeability, and neuroprotection. Recently, PlGF has been reported as a novel therapeutic target for wet AMD. However, there are few reports about the effect of PlGF against dry AMD. Previously, we reported that PlGF has protective effects against retinal neuronal cell damage in vitro. Therefore, we investigated the effects of PlGF against photoreceptor degeneration. In this study, mice were exposed to white light at 8000 lx for 3 hours to induce retinal damage, which was evaluated by recording the electroretinogram amplitude and measuring the outer nuclear layer (ONL) thickness. The mice were injected intravitreally with PlGF before light exposure, PlGF after light exposure, or anti-PlGF antibody before light exposure. RPE-choroid-sclera flat mounts were immunostained with anti-ZO-1 antibody to evaluate the disruption of retinal pigmented epithelium (RPE) cell-cell junctional integrity after light exposure. Furthermore, the expression of VEGF receptor in the retina and RPE-choroid complex after light exposure was measured using Western blot analysis. Contrary to the expected outcome, PlGF treatment exacerbated the light-induced retinal functional damage and ONL thinning. In contrast, anti-PlGF treatment significantly improved the light-induced retinal degeneration. The disruption of RPE cell-cell junctional integrity after light exposure was suppressed by anti-PlGF treatment. Moreover, the VEGF receptor, which is involved in blood-retinal barrier breakdown, was up-regulated after light exposure. These findings suggest that anti-PlGF antibody has protective effects against light-induced retinal degeneration in the murine retina through inhibition of RPE breakdown after light exposure. Thus, anti-PlGF antibody may be useful therapeutic agents in dry AMD.

Blue light-induced inflammatory marker expression in the retinal pigment epithelium-choroid of mice and the protective effect of a yellow intraocular lens material in vivo

Oxidative stress in the retinal pigment epithelium (RPE) is a well-accepted pathogenic change in vision-threatening diseases such as age-related macular degeneration. One source of oxidative stress is excessive light exposure, which causes excessive activation of the visual cycle. Because short wavelength light (blue light) has more energy, it is reported to be more harmful to photoreceptor cells than the other wavelengths of light. However, the biological effect of blue light in the RPE of living animals and the protective effect of a yellow intraocular lens (IOL) material that blocks blue light is still obscure. Therefore, we compared the pathogenic effect in the RPE-choroid complexes of mice exposed to light in a box made of a clear or a yellow IOL material. We measured the level of reactive oxygen species (ROS) using 2′, 7′-dichlorodihydrofluorescein diacetate, the mRNA levels of inflammatory cytokines and a macrophage marker by real-time polymerase chain reaction, and the protein level of monocyte chemotactic protein-1 (MCP-1) by ELISA. The ROS level after light exposure was suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. In parallel, all the inflammatory cytokines that we measured and a macrophage marker were also suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. Therefore, a yellow IOL material suppressed, and thus blue light exacerbated, the increase in the ROS level and inflammatory cytokine expression as well as macrophage recruitment in the RPE-choroid in vivo after light exposure.

视网膜下瘢痕形成小鼠视网膜色素上皮-脉络膜中血管内皮生长因子、促纤维化因子及炎症因子的表达

Objective To observe the expression of vascular endothelial growth factor (VEGF),fibrogenic mediators and inflammatory mediators in the retinal pigment epithelium (RPE)-choroid complex of mice with experimental subretinal fibrosis.Methods By subretinal injection of inflammatory macrophages after retinal photocoagulation,experimental subretinal fibrosis was induced in 64 adult C57BL/6(B6) female mice (7-8 weeks).Masson staining and glial fibrillary acidic protein (GFAP) staining of choroidal wholemont were performed to verify that the subretinal fibrosis at day 7 after subretinal injection.Before subretinal injection and at day 1,2,3,5 and 7 after subretinal injection,the mRNA expression level of VEGF,transforming growth factor (TGF)-β1,TGF-β2,TGF-β3,interleukin (IL)-6,IL-10 and IL-13 in RPE-choroid complex were evaluated by quantitative reverse transcription-polymerase chain reaction.Enzyme-linked immune sorbent assay was next used to detect protein expression of these factors.Results At seven days after subretinal injection of inflammatory macrophages,experimental subretinal fibrosis was detected by Masson staining and GFAP staining.The mRNA level of VEGF,TGF-β1 and TGF-β2 reached the peak at day 5 after modeling,while the mRNA expression of IL-6,IL-10,IL-13 reached the peak at day 2 after modeling.TGF-β3 mRNA was not detected either in naive mice or during the development of experimental subretinal fibrosis.At day 2,3,5 and 7 after modeling,compared with the pre-modeling,the mRNA expression of VEGF (t =2.38,3.65,4.03,2.26),TGF-β1 (t =2.58,2.30,3.89,4.15) and TGF-β2 (t=4.37,4.20,3.77,3.98) were significantly increased (P＜0.05).At day 1,2 and 3 after modeling,compared with the pre-modeling,the mRNA expression of IL-6 were significantly increased (t=2.36,4.54,4.01; P＜0.05).At day 2 and 3 after modeling,compared with the pre-modeling,the mRNA expression of IL-10 and IL-13 were significantly increased (t=3.87,4.20,2.44,2.58; P＜0.05).At day 5 after modeling,compared with the pre-modeling,the protein expression of VEGF,total TGF-β1,activeTGF-β1,total TGF-β2 and active TGF-β2 were significantly increased (t=2.57,3.37,2.45,3.83,2.74; P＜0.05).IL-6,IL-10 and IL-13 protein were not detected at pre-modeling eyeballs,but were found at day 2 after modeling.Conclusion The expression of VEGF,fibrogenic mediators and inflammatory mediators in RPE-choroid complex in mice with experimental subretinal fibrosis are increase significantly. Key words: Wet macular degeneration/etiology; Cicatrix/physiopathology; Animal experimentation

Genistein attenuates choroidal neovascularization

Genistein is a dietary-derived flavonoid abundantly present in soybeans and known to possess various biological effects including anti-inflammation and anti-angiogenic activity. To investigate the effects of genistein on intraocular neovascularization, we used an animal model of laser-induced choroidal neovascularization (CNV). Male C57BL/6J mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CNV was induced by laser photocoagulation. The animals were fed a mixture diet containing 0.5% genistein or a control diet ad libitum for 7 days before laser photocoagulation and the treatment was continued until the end of the study. Seven days after laser injury, the size of CNV lesions was quantified. Retinal pigment epithelium (RPE)-choroid complex was also harvested 1 or 3 days after laser injury and the level of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and matrix metalloproteinase (MMP)-9 were measured by enzyme-linked immunosorbent assay. Expression levels of Ets-1 and F4/80 were examined by real-time PCR. A significant decrease in CNV size was observed in animals treated with genistein (15441.9±1511.8 μm2) compared to control mice (21074.0±1940.7μm2, P<.05). Genistein significantly reduced the protein level of MCP-1, ICAM-1, and MMP-9 in the RPE-choroid complex (P<.05). In addition, genistein suppressed the expression levels of Ets-1 and F4/80 (P<.05). The current data indicate the anti-angiogenic property of genistein during CNV formation.

Resveratrol prevents the development of choroidal neovascularization by modulating AMP-activated protein kinase in macrophages and other cell types

The development of choroidal neovascularization (CNV) is a critical step in the pathogenesis of age-related macular degeneration (AMD), a vision-threatening disease. In this study, we used a mouse model of AMD to study the protective effects of resveratrol (RSV) supplementation against CNV as well as the underlying molecular mechanisms. Mice were orally pretreated with RSV daily for 5days. On the fifth day, the mice underwent laser photocoagulation to induce CNV. One week after laser treatment, CNV volume was significantly lower in the RSV-treated mice compared with vehicle-treated animals. In addition, RSV treatment significantly inhibited macrophage infiltration into the retinal pigment epithelium (RPE)-choroid and suppressed the expression of inflammatory and angiogenic molecules, including vascular endothelial growth factor, monocyte chemotactic protein-1 and intercellular adhesion molecule-1. Importantly, RSV prevented the CNV-induced decrease in activated AMP-activated protein kinase and increase in activated nuclear factor-κB in the RPE-choroid complex. The regulatory effects of RSV on these molecules were confirmed in RPE, microvascular endothelial and macrophage cell lines. Inhibition of macrophage infiltration by RSV was confirmed by in vitro scratch and migration assays. RSV suppressed CNV development, reducing the levels of multiple cytokines secreted from several cell types and inhibiting macrophage migration. The direct effects of RSV on each cell type were confirmed in vitro. Although further studies are needed, RSV could potentially be applied in the clinic to prevent CNV development in AMD.

Effect of retinoic acid on the tight junctions of the retinal pigment epithelium-choroid complex of guinea pigs with lens-induced myopia in vivo

Zonula occludens-1 (ZO-1) and occludin are important tight junction (TJ)-associated proteins, which are expressed in the retinal pigment epithelium (RPE)-choroid complex. Retinoic acid (RA) is a regulator of eye growth and may play an important role in forming functional TJs. The aim of this study was to detect the changes that occur in the expression of ZO-1 and occludin in the RPE-choroid complex of guinea pigs with lens-induced myopia (LIM), and to investigate the effect of RA on TJ-associated proteins in vivo. We developed an animal model of myopia by placing a −6.00 D negative lens on the right eyes of 3-week-old guinea pigs. The refractive error and axial length of the eye were measured on days 0, 3, 7 and 14. High-performance liquid chromatography (HPLC) was performed to detect the changes in endogenous RA in the RPE-choroid complex. The expression of ZO-1 and occludin was observed by immunofluorescence and assayed by western blot analysis. Additionally, 2 μl LE540 (2.5 μg/μl), an antagonist of RA receptors (RARs), was injected into the vitreous chamber of the eyes of guinea pigs with LIM and 2 μl phosphate-buffered saline (PBS) (2.5 μg/μl) were injected as a negative control. We observed no obvious change in RA, ZO-1 and occludin expression in the normal control group within 14 days. In the LIM and LIM plus PBS groups, the level of RA and the expression of ZO-1 and occludin in the RPE-choroid complex significantly increased within 14 days along with the development of myopia. However, the level of RA was inhibited and the expression of TJ-associated proteins decreased in the eyes of guinea pigs with LIM following the injection of LE540. Thus, we consider that the expression of ZO-1 and occludin is increased in the RPE-choroid complex during the development of myopia. This change in expression may be regulated by RA, a factor known to be involved in the regulation of eye growth.