Experimental Choroidal Neovascularization Model Research Articles

Silencing of YAP attenuates pericyte‐myofibroblast transition and subretinal fibrosis in experimental model of choroidal neovascularization

Age-related macular degeneration (AMD) is the main reason of irreversible vision loss in the elderly. The subretinal fibrosis subsequent to choroidal neovascularization (CNV) is an important feature in the late stage of wet AMD and is considered to be one reason for incomplete response to anti-VEGF drugs. Recent studies have shown that pericyte-myofibroblast transition (PMT) is an important pathological process involving fibrotic diseases of various organs. However, the specific role and mechanism of PMT in the subretinal fibrosis of CNV have not been clarified. It has been clear that the Hippo pathway along with its downstream effector Yes-associated protein (YAP) plays an important role in both epithelial and endothelial myofibroblast development. Therefore, we speculate whether YAP participates in PMT of pericytes and promotes fibrosis of CNV. In this study, experimental CNV was induced by laser photocoagulation in C57BL/6J (B6) mice, and aberrant YAP overexpression was detected in the retinal pigment epithelial/choroid/sclera tissues of the laser-injured eyes. YAP knockdown reduced the proliferation, migration, and differentiation of human retinal microvascular pericytes in vitro. It also reduced subretinal fibrosis of laser-induced CNV in vivo. Moreover, by proteomics-based analysis of pericyte conditioned medium (PC-CM) and bioinformatic analyses, we identified that the crosstalk between Hippo/YAP and MAPK/Erk was involved in expression of filamin A in hypoxic pericytes. These findings suggest that Hippo/YAP and MAPK/Erk are linked together to mediate pericyte proliferation, migration as well as differentiation, which may embody potential implications for treatment in diseases related to CNV.

Downregulation of miR-146a-5p Inhibits Choroidal Neovascularization via the NF-κB Signaling Pathway by Targeting OTUD7B

ABSTRACT Purpose Choroidal neovascularization (CNV) is the key pathological change caused by irreversible blindness resulting from neovascular AMD (nAMD). However, the pathological mechanisms underlying CNV remain largely unknown. Here, we aimed to investigate the role of miR-146a-5p in CNV formation. Materials and Methods At the cellular level, we overexpressed or downregulated miR-146a-5p in an umbilical vein endothelial cell line (EA.hy926) by transfecting cells with either a miR-146a-5p mimic or an inhibitor. CCK8, wound healing, and Matrigel assays were performed to examine the proliferation, migration, and tube formation of endothelial cells (EA.hy926). Target relationship between miR-146a-5p and OTUD7B was verified using a double luciferase reporter experiment. An experimental CNV model was established by treating fundi of male C57BL/6 J mice with 810 nm laser. Fundus fluorescein angiography (FFA) was performed to evaluate the leakage of CNV on day 7 after miR-146a-5p antagomir intravitreal injection. The CNV volume was measured using Choroidal Flatmounts in a confocal study. The expression levels of VEGF, ICAM1, and NF-κB (p50 and p65) were detected both in vitro and in vivo. Results The expression of miR-146a-5p was increased in LPS-stimulated endothelial cells and in experimental CNV RPE-choroidal complexes in mouse models. LPS-induced proliferation, migration, and tube formation were inhibited by the miR-146a-5p inhibitor. The miR-146a-5p antagomir attenuated CNV formation and fluorescent leakage in the vivo CNV model. In the LPS-stimulated endothelial cells and the CNV mouse model, the NF-κB signaling pathway was activated and the expression of VEGF and ICAM1 increased. Conversely, downregulation of miR-146a-5p inactivated the NF-κB signaling pathway and reduced the expression of VEGF and ICAM1. Conclusions Our results indicated that downregulation of miR-146a-5p inhibited experimental CNV formation via inactivation of the NF-κB signaling pathway.

Inhibition of Hypoxia-Inducible Factor-1α and Vascular Endothelial Growth Factor by Chrysin in a Rat Model of Choroidal Neovascularization.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the elderly population. Vascular endothelial growth factor (VEGF) is essential for choroidal neovascularization (CNV) development in advanced, wet AMD. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid with anti-inflammatory, anti-oxidative, and anti-angiogenic effects. We hypothesized that intravitreally injected chrysin may inhibit CNV due to its inhibitory effect on angiogenesis. To determine the effects of chrysin on an experimental CNV model, we induced CNV in Brown Norway rats with a diode laser. One week later, rats were injected intravitreally with chrysin in the right eye and vehicle in the left eye. The following week, we evaluated chrysin’s effects via the CNV grade assessed with fluorescein angiography and histologic analyses. Hypoxia-inducible factor-1 alpha (HIF-1α) and VEGF expression in the retina/choroid complex were also measured in both eyes. The mean CNV grade was significantly lower in chrysin-treated vs. control eyes (2.34 ± 1.14 vs. 2.97 ± 1.05, p < 0.001), as was the mean CNV thickness (33.90 ± 4.89 vs. 38.50 ± 5.43 μm, p < 0.001) and mean HIF-1α and VEGF levels (both p < 0.001). Compared to chrysin-treated eyes, the relative risk of control eyes developing high-leakage lesions was 2.03 (95% confidence interval: 1.46–2.83). Since chrysin inhibited laser-induced CNV and downregulated HIF-1α and VEGF expression, it is a candidate for treating wet AMD and other CNV-associated conditions.

Serine racemase deficiency attenuates choroidal neovascularization and reduces nitric oxide and VEGF levels by retinal pigment epithelial cells.

Choroidal neovascularization (CNV) is a leading cause of blindness in age-related macular degeneration. Production of vascular endothelial growth factor (VEGF) and macrophage recruitment by retinal pigment epithelial cells (RPE) significantly contributes to the process of CNV in an experimental CNV model. Serine racemase (SR) is expressed in retinal neurons and glial cells, and its product, d-serine, is an endogenous co-agonist of N-methyl-d-aspartate receptor. Activation of the receptor results in production of nitric oxide (. NO), a molecule that promotes retinal and choroidal neovascularization. These observations suggest possible roles of SR in CNV. With laser-injured CNV mice, we found that inactivation of SR-coding gene (Srrnull ) significantly reduced CNV volume, neovascular density, and invading macrophages. We exploited the underlying mechanism invivo and exvivo. RPE from wild-type (WT) mice expressed SR. To explore the possible downstream target of SR inactivation, we showed that choroid/RPE homogenates extracted from laser-injured Srrnull mice contained less inducible nitric oxide synthase and decreased phospho-VEGFR2 compared to amounts in WT mice. In vitro, inflammation-primed WT RPEs expressed more inducible NOS, produced more. NO and VEGF than did inflammation-primed Srrnull RPEs. When co-cultured with inflammation-primed Srrnull RPE, significantly fewer RF/6A-a cell line of choroidal endothelial cell, migrated to the opposite side of the insert membrane than did cells co-cultured with pre-treated WT RPE. Altogether, SR deficiency reduces RPE response to laser-induced inflammatory stimuli, resulting in decreased production of a cascade of pro-angiogenic cytokines, including. NO and VEGF, and reduced macrophage recruitment, which contribute synergistically to attenuated angiogenesis.

The Role of LOX and LOXL2 in the Pathogenesis of an Experimental Model of Choroidal Neovascularization.

We investigated whether lysyl oxidase (LOX) and lysyl oxidase-like 2 (LOXL2) play a role in an experimental model of choroidal neovascularization (CNV). The therapeutic potential of antibodies against LOX (M64) and LOXL2 (AB0023) was evaluated in a murine laser-induced CNV model. Expression of LOX and LOXL2 in the posterior eye cups (including retina, retinal pigment epithelium, choroid, and sclera) was studied by qRT-PCR and immunohistochemistry. In the murine model of CNV, both antibodies were administered intraperitoneally every other day until the day killed. On different time points after laser, treatment outcome was studied by immunohistochemical analysis of inflammation, angiogenesis and fibrosis, and by transcript analysis of different cytokines. Levels of LOX and LOXL2 in the posterior eye cups were increased after CNV-induction at different time points after laser. At day 35, their protein expression patterns appeared to correlate with retinal glial cells and endothelial cells, respectively. Both antibodies significantly inhibited fibrosis, whereas AB0023 also significantly reduced angiogenesis and inflammation. Transcript levels of α-1 type I collagen (COL1A1) in the posterior eye cups were significantly decreased in lasered mice treated with either M64 or AB0023. Vascular endothelial growth factor expression was also reduced only after AB0023 treatment, whereas activated fibroblast marker α-smooth muscle actin (αSMA) levels were not significantly changed. This study suggests that LOX and LOXL2 may play an important role in the pathogenesis of AMD. Targeting LOXL2 could have a broader efficacy than targeting LOX, by reducing angiogenesis and inflammation, as well as fibrosis.

An Experimental Study on Choroidal Neovascularization Induced by Krypton Laser in Rat Model

The purpose of this work was to study the efficacy and safety of choroidal neovascularization (CNV) formation induced by Krypton laser in Brown Norway (BN) rats, and observe the trend of the change of CNV after laser photocoagulation. Twenty-five male BN rats were involved in this study. Two eyes of one rat without any laser photocoagulation were randomly selected as the control group, and the other 48 eyes of 24 rats were selected as the experimental group. Eight eyes of four rats were randomly selected to receive the examinations of fundus fluorescein angiography (FFA), histopathology, and transmission electron microscopy 3, 7, 14, 21, 28, and 56 days after laser photocoagulation. After laser photocoagulation, the leakage appeared in burns on day 7 (59%), reached the peak on day 21 (84%), (p<0.01), and remained stable after day 21, proven by FFA and histopathology (p>0.05). The thickness of CNV increased from day 7 to day 21 (p<0.01), and remained stable after day 21 (p>0.05). The experimental model of CNV can be successfully induced by Krypton laser in rats with a stable, long-lasting, and high success rate. After laser photocoagulation, the leakages appear on day 7, reach the peak on day 21, and remain stable after day 21.

Antiangiogenic effects of tivozanib, an oral VEGF receptor tyrosine kinase inhibitor, on experimental choroidal neovascularization in mice

We investigated the effects of tivozanib, an oral vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, on experimental choroidal neovascularization (CNV) in mice. C57BL/6 mice were treated with tivozanib (1 mg/kg/day) or vehicle at the onset (day 0) of the study and experimental CNV was induced by laser photocoagulation the following day. In the other groups, tivozanib or vehicle was started 7 days after the laser photocoagulation to determine the effects of the drug on established CNV. To evaluate changes in the CNV lesions, choroidal flat mounts, fluorescein angiography, immunofluorescence staining with isolectin B4, and histological examinations were performed 14 days after CNV induction. Expression of phosphorylated ERK1/2 in choroidal tissues was measured by western blot analysis to demonstrate the inhibitory effect of tivozanib on intracellular signaling pathways involved in CNV development. Compared to vehicle-treatment, tivozanib suppressed the development of CNV lesions and led to a significant regression of established CNV, reducing the affected areas by 80.7% and 67.7%, respectively. On fluorescein angiography, tivozanib-treated mice had significantly less fluorescence leakage than vehicle-treated mice (P < 0.001). On immunofluorescence staining, the isolectin B4-labeled area was smaller in tivozanib-treated mice (P < 0.001). Phosphorylated ERK 1/2 levels increased after CNV induction by laser application and were suppressed by tivozanib treatment. Tivozanib effectively inhibited the progression of CNV in an experimental CNV model. These results suggest that tivozanib may be a therapeutic alternative for the treatment of neovascular age-related macular degeneration.

Quantitative measurement of laser-induced choroidal neovascularization with spectral-domain optical coherent tomography

Background The neovascular form of the disease usually causes severe vision loss in a number of eye diseases.Special-domain optical coherence tomography (SD-OCT) provides high-quality in retinal imaging and the possibility of the measurement in vivo.Objective This study aimed to investigate the feasibility of SD-OCT dynamically measuring choroidal neovascularization (CNV).Methods CNV was induced in 30 left eyes of 30 clean Brown Norway(BN)rats by retinal photocoagulation with the laser parameter as follows: wavelength 532 nm,exciting power 200 mW,spot diameter 100 μm and irradiating time 50 ms.Bubble or less retinal bleeding was thought as Brunch membrane breakage and CNV model establishment.Fundus fluorescein angiography(FFA) was performed to determine the establishment of CNV model and scored based on the fluorescein leakage on 3,7,14,21 days after photocoagulation.Meanwhile,CNV memberane thickness (CMT) was dynamically measured in vivo as the maxiume value from retinal inner limiting membrane through choroidal vessel layer in various time points.Histopathologic examination was used in the 14th day to evaluate and verify the result of SD-OCT.The right eyes were as controls.Results FFA examination showed that disc-like leakage of fluorescein appeared in 7 days and extended in 14 days after photocoagulation with the scores of 1.6±0.4,2.5±0.6 and 2.4±0.5 in 7,14 and 21 days,showing a significant difference among them(F=13.11,P＜0.01).The fluorescein leakage score was significantly higher in 14 and 21 days than that of 7 days(both P＜0.05).CMT measured by SD-OCT was(76.33±10.09),(102.03±14.21)and(98.03±13.76) μm in 7,14 and 21 days after photocoagulation respectively,with a significant difference among 3 time points (F=23.25,P＜0.01),and that in 14 and 21 days was significantly declined in comparison with 7 days(both P＜0.05).The results of SD-OCT showed a consistent tendency with that of FFA.Histopathological examination showed CNV formation in 14 days after photocoagulation.Conclusions Experimental CNV model was successfully induced by laser photography.SD-OCT technology allows excellent visualization of CNV in vivo. Key words: Choroidal neovascularization; Special-domain optical coherent tomography; Animal model; Biological measurement

Suppression of Choroidal Neovascularization by Intravitreal Injection of Liposomal SU5416

To clarify whether use of angiogenic vessel-homing peptide, Ala-Pro-Arg-Pro-Gly (APRPG)-modified liposomes encapsulating 3-([2,4-dimethylpyrrhol-5-yl] methylidenyl)-indolin-2-one (SU5416), an angiogenesis inhibitor, can inhibit the development of experimental choroidal neovascularization (CNV) in rats. Liposomes were prepared using the thin-film hydration method. To set up the rat experimental CNV model, intense fundus laser photocoagulation at 6 spots per eye was performed on pigmented rats. After photocoagulation, the rats were divided into 4 groups (6 rats in each group): an APRPG-liposomal SU5416 treatment group and control groups treated with a balanced salt solution, APRPG liposomes, or soluble SU5416. Each rat received a single intravitreal injection immediately after the injury. One week or 2 weeks after laser injury, the extent of CNV was evaluated by perfusion with high-molecular-weight fluorescein isothiocyanate-dextran. Two weeks after injection, the CNV area was significantly (P < .05) smaller in the APRPG-liposomal SU5416-treated group compared with the CNV area in the balanced salt solution-and APRPG liposome-treated groups. Liposomes modified by APRPG and encapsulating SU5416 constitute a potential drug formulation for CNV treatment that would require only a single intravitreal injection. This liposomal delivery may enable the sustained release of small molecules and be a new treatment modality for CNV.

Expressions of COX-2,VEGF and MMP-2 in experimental choroidal neovascularization model

目的 研究实验性脉络膜新生血管(CNV)大鼠中环氧合酶-2(COX-2)、血管内皮生长因子(VEGF)和基质金属蛋白酶-2(MMP-2)的表达,并进一步探讨其在CNV中的作用机制及相互关系.方法 应用氪红激光光凝视网膜的方法诱导制作BN大鼠的CNV模型,分别选取并制作无任何干预的对照组和实验组光凝后第3、7、14、21、30天的最大CNV膜切片,采用免疫组织化学技术检测COX-2、VEGF和MMP-2在视网膜和CNV膜中的表达.应用HPIAS-1000型高清晰度彩色病理图文分析系统测定COX-2、VEGF和MMP-2阳性染色的平均灰度值.结果 COX-2、VEGF和MMP-2在正常视网膜和脉络膜中呈弱表达,视网膜光凝后,COX-2、VEGF和MMP-2在视网膜和CNV膜中的表达在7～14 d逐渐增强,21 d时三者的表达强度均达到高峰,之后略减弱.视网膜光凝后7、14、21、30 d,实验组COX-2、VEGF和MMP-2的免疫组织化学染色平均灰度值与对照组相比差异均有统计学意义(P＜0.05).COX-2的表达强度与VEGF和MMP-2的变化均呈正相关(r=0.967,P=0.007;r=0.966,P=0.007).结论 激光诱导的实验性CNV中COX-2、VEGF及MMP-2的表达随着时间的延长呈动态变化,CNV局部炎症诱导的COX-2表达可能是VEGF和MMP-2的上游调节因子。

Inhibition of choroidal neovascularization by blocking vascular endothelial growth factor receptor tyrosine kinase

To investigate the role played by receptors of vascular endothelial growth factors, Flt-1 and KDR/Flk-1, on an experimental model of choroidal neovascularization (CNV). The vascular endothelial growth factor-A (VEGF-A) receptor-specific tyrosine kinase inhibitor SU5416 was administered to a laser-induced mouse model of CNV. The formation of CNV and the degree of vascular permeability in Flt-1 tyrosine kinase domain-deficient mice were also investigated. SU5416 reduced vascularity and vascular endothelial cell proliferation, and promoted endothelial cell apoptosis within CNV. Furthermore, the formation of CNV and the degree of vascular permeability were significantly reduced in Flt-1 tyrosine kinase domain-deficient mice, and this effect was enhanced by the administration of SU5416. Both Flt-1 and KDR/Flk-1 have a significant role in CNV formation. Suppression of apoptosis may be involved in the process.

Suppression of choroidal neovascularization by intramuscular polymer-based gene delivery of vasostatin

The purpose of this study was to evaluate the efficacy of gene delivery of angiogenesis inhibitor, vasostatin (VS), in suppressing experimental model of choroidal neovascularization (CNV). A mammalian expression vector carrying VS, pCMV3-VS, was constructed and evaluated for its ability to produce VS in transfected cells using western blot analysis and a cell viability assay. CNV was induced in Brown Norway rats by fundus argon laser photocoagulation and evaluated by fundus fluorescein angiography (FAG). Ten days post-laser treatment, gene delivery was achieved by intramuscular (IM) injection of poly-( N-vinyl pyrrolidone) (PVP) polymer conjugated with pCMV3-VS (PVP-VS) or a control vector (PVP-vector). Systemic VS expression was analysed by western blot analysis and enzyme linked immunosorbent assay (ELISA), and the extent of CNV was monitored by FAG analysis at different time intervals post-PVP treatment. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Besides, IM injection of PVP-VS, but not PVP-vector, led to elevated VS level in plasma for 30 days. After laser photocoagulation, rats injected with PVP-VS exhibited significantly lower incidence of CNV comparing with animals of control groups ( P<0.01) for at least 42 days. Moreover, rats treated with PVP-VS also showed a significant reduction in the CNV lesions compared with control groups ( P<0.001) for at least 42 days. Above all, no overt adverse effects were observed in rats received PVP-VS. These results demonstrate the potential of IM VS gene delivery for CNV treatment.

Suppression of experimental choroidal neovascularization utilizing KDR selective receptor tyrosine kinase inhibitor.

We investigated the role of the VEGF-VEGF receptor 2 (KDR) system in the development of choroidal neovascularization (CNV) and its possibility as a therapeutic target utilizing KDR selective receptor tyrosine kinase (RTK) inhibitor (SU5416) both in vitro and in an experimental CNV model. VEGF-induced phosphorylation of KDR and p44/p42 MAPK in cultured bovine choroidal endothelial cells (BCECs) was determined by Western blot analysis. The proliferation and in vitro tube formation were analyzed by [3H]thymidine uptake and three-dimensional collagen gel model. For experimental CNV model, intense fundus laser photocoagulation was performed on pigmented rats. The anti-angiogenic efficacy of intraperitoneally injected SU5416 on experimental CNV was evaluated by fluorescein angiography and histology. The extent of fluorescein leakage on late-phase angiograms was scored, and the thickness of CNV membrane was histologically measured under a light microscope. VEGF-induced KDR phosphorylation in cultured BCECs was inhibited by SU5416 in a dose-dependent manner (0-3 microM) with IC50 of 0.29 +/- 0.071 microM. SU5416 treatment also resulted in a dose-dependent prohibition of VEGF-induced p44/p42 MAPK phosphorylation, [3H]thymidine uptake and in vitro tube formation with corresponding concentrations that inhibited KDR phosphorylation. The leakage score on fluorescein angiography for experimental CNV was significantly lower in the SU5416-treated group than in the control group (P<0.01). Histologically, the CNV membranes in the SU5416-treated group were 31.6% thinner than those in the control group (P<0.01). These results strengthen the evidence for a critical role of the VEGF-KDR system in the development of CNV, indicating that KDR selective inhibitor might be beneficial for the treatment of intraocular angiogenic diseases, including age-related macular degeneration.

Basic fibroblast growth factor experimentally induced choroidal angiogenesis in the minipig.

Basic fibroblast growth factor (bFGF), a soluble mitogen, has been isolated and purified from various organs, including the retina. In vivo angiogenic activity of bFGF has been demonstrated with several assays. An experimental model of choroidal neovascularization was developed in the mini pig by perfusion of recombinant human bFGF through an osmotic minipump. Endogenous bFGF and bFGF receptors were localized in the normal pig retina by immunohistochemistry and autoradiography after binding. The perfusion of exogenous bFGF induced well-organized new vessels along the last 3 mm of the catheter in the suprachoroidal space. This neovascularization did not penetrate the normal Bruch's membrane. Vascular cells (identified by von Willebrand factor antibody staining) increased in number and in surface from the proximal part to the end of the intraocular catheter in all bFGF perfused eyes. In eyes perfused with phosphate buffered saline (controls), but not in the bFGF perfused eyes, an inflammatory response occurred (identified by a macrophage specific antibody). These results demonstrate that choroidal angiogenesis can be achieved without an inflammatory response by perfusing an excess of bFGF in the suprachoroidal space.