Beta-alanine promotes angiogenesis in laser-induced choroidal neovascularization mice models

AIM:To investigate the effect ofβ-alanine(BA)on laserinduced choroidal neovascularization(CNV)mice models.METHODS:Laser-induced CNV mice models were established,and BA was administrated for one week and two weeks in advance,separately.Furthermore,retinal pigment epithelium(RPE)-choroid flat mounts were separated,and immunohistochemical staining was performed.The laser-induced CNV lesion areas were measured and compared.In addition,liver and kidney morphologies were observed to identify potential hepatorenal toxicity.RESULTS:Enlarged CNV lesion areas were observed in the BA treated group.No significant differences were observed in the liver and kidney sections between groups.CONCLUSION:BA treatment increase CNV lesion areas,suggesting the detrimental effects of BA as a nutritional supplement in age-related macular degeneration(AMD)population.

Effects of endogenous dopamine induced by low concentration atropine eye drops on choroidal neovascularization in high myopia mice

AIM:To evaluate effects of endogenous dopamine induced by low concentration atropine eye drops on choroidal neovascularization(CNV)in high myopia mice.METHODS:The C57BL/6J mice were deprived of the right eye for 4wk,and the high myopia was diagnosed by optometry,the diopter was less than-6.00 D,and CNV was induced by 532 nm laser.The changes of dopamine D1 receptor(DRD1),dopamine D2 receptor(DRD2),and vascular endothelial growth factor A(VEGFA)were detected by Western blot technology at 0.5,1,2h,and 7d after 0.01%,0.05%,and 0.1%atropine eye drops,respectively,the area of CNV was measured.RESULTS:Significant increases were observed on the expression of DRD2 in mouse high myopia model at 0.5,1,2h,7d with 0.05%and 0.1%atropine eye drops(P<0.05).Significant decreases were observed on the expression of DRD1 and VEGFA in mouse high myopia model at 0.5,1,2h,7d with 0.05%and 0.1%atropine eye drops(P<0.05).The area of CNV induced by laser in the drug-treated group was significantly smaller than that in the control group,and the higher the concentration,the more significant the inhibitory effect(P<0.05).CONCLUSION:The 0.01%,0.05%,0.1%atropine eye drops can decrease the level of VEGFA and inhibit high myopia CNV indirectly by up-regulating the level of DRD2 and down-regulating the level of DRD1,and the effect of 0.05%and 0.1%atropine eye drops is more significant.

Different approaches for treating myopic choroidal neovascularization:a network Meta-analysis

AIM:To evaluate the efficacy of intravitreal injection of anti-vascular endothelial growth factor(anti-VEGF),photodynamic therapy(PDT),and laser treatment(LT)for anatomical and functional improvement in myopic choroidal neovascularization(mCNV)patients.METHODS:Two researchers independently searched PubMed,Cochrane Library,Web of Science,and other databases to screen studies comparing best-corrected vision acuity(BCVA)and foveal center thickness(FCT)changes after mCNV treatment.Post-treatment chorioretinal atrophy(CRA)is a secondary outcome indicator.The retrieval time limit is from the database construction to January 30,2023.RESULTS:A total of 1072 eyes in 16 articles were included.In the RCTs,intravitreal bevacizumab(IVB)and intravitreal ranibizumab(IVR)were superior to PDT(MD=0.18,95%CI:0.02,0.40,MD=0.18,95%CI:0.01,0.42)in improving BCVA of mCNV patients(P<0.05).The relative effectiveness in improving BCVA,from high to low,appeared to be IVR,intravitreal aflibercept(IVA),IVB,LT,PDT,and sham first followed by IVA(Sham/IVA).While improving the FCT from high to low was IVA,IVR,IVB,PDT.In retrospective studies,the results of BCVA after long-term treatment showed that all the therapeutic effects from high to low was IVA,intravitreal conbercept(IVC),IVR,IVB,IVB/IVR,PDT with IVB/IVR,PDT.The effect of improving FCT was IVA,IVR,IVC,PDT,and IVB from high to low.And in the effects of improving CRA,the IVB appeared to be higher than IVR,while the PDT was the smallest,but none of the differences in the results were statistically significant.CONCLUSION:Anti-VEGF has the best effect on longterm vision improvement in mCNV patients,using IVB or IVR alone to treat mCNV may be better than IVB or IVR combined with PDT.There is no significant difference in the improvement of visual acuity,macular edema,and CRA in mCNV patients treated with any different anti-VEGF drugs.

Combined therapy with bevacizumab and photodynamic therapy for myopic choroidal neovascularization: A oneyear follow-up controlled study

AIMTo evaluate the efficacy and safety of a combined treatment for myopic choroidal neovascularization (CNV) using photodynamic therapy (PDT) and intravitreal bevacizumab and to compare it with intravitreal bevacizumab monotherapy.

Predictors of visual outcome in eyes with choroidal neovascularization secondary to age related macular degeneration treated with intravitreal bevacizumab monotherapy

AIM:To evaluate the predictors of visual improvement in eyes with naive choroidal neovascularization secondary to age-related macular degeneration (CNV -AMD) treated with intravitreal bevacizumab (IVB) monotherapy. METHODS:Fifty eyes with naive CNV-AMD with pretreatment best-corrected visual acuity (BCVA) better than 20/200 and treated with IVB monotherapy were evaluated. Several variables including age, sex, pre-treatment BCVA, CNV type and lesion size on fluorescein angiogram as well as SD-OCT parameters including pre-treatment central macular thickness (CMT), inner-segment/outer-segment (IS/OS) junction integrity, and external limiting membrane (ELM) integrity were analyzed to predict visual outcome.RESULTS:On univariate regression, pretreatment ELM damage was associated with less visual improvement after treatment (P =0.0145). However, ELM damage predicted only 10% of the visual outcome. On multivariate regression, pretreatment BCVA, IS/OS junction, and ELM integrity on SD-OCT were the significant predictors for the treatment effect and together predicted 37% of visual improvement. CONCLUSION:Pretreatment BCVA, ELM and IS/OS junction integrity on SD-OCT are of significant value inpredicting the visual improvement in naive wet AMD patients treated with IVB monotherapy.

Targeting Therapy of Choroidal Neovascularization by Use of Polypeptide-and PEDF-loaded Immunoliposomes under Ultrasound Exposure

Pigment epithelium derived factor (PEDF) has been proven to be an effective drug for the treatment of choroidal neovascularization (CNV).However,the lack of ideal administration route is the biggest bottleneck preventing PEDF from wider clinical use.In this study,we developed a novel PEDF-carrying system which employed immuno-nano-liposomes (INLs) under ultrasound exposure.PEDF-loaded INLs were prepared by conjugating nanoliposomes to the peptide ATWLPPR specifically targeting the receptor-2 for vascular endothelial growth factor (VEGFR-2) and reversely encapsuling PEDF.RF/6A cells were incubated with PEDF-loaded INLs.CNV models of BN rats were injected with PEDF-loaded INLs.MTT assay was used to evaluate the cytotoxicity of the INLs on RF/6A cells.Flow cytometry was conducted to detect the apoptotic rate of cells.Laser scanning confocal microscopy was employed to observe the binding and transmitting process of PEDF-loaded INLs and to calculate the area of CNV in the rat model.The results showed that the PEDF-loaded INLs could exclusively bind to CNV but not to the normal choroidal vessels.The CNV area was significantly decreased in PEDF treatment groups in comparison with control group (P【0.05).Moreover,PEDF-loaded INLs exposed under ultrasound were more efficient in reducing the CNV area (P【0.05).It was concluded that INLs in combination with ultrasonic exposure can transmit PEDF into cytoplasma with high specificity and efficiency,which strengthens the inhibitory effects of PEDF on CNV and reduces its side effects.PEDF-loaded INLs possibly represent a new treatment paradigm for patients with ocular neovascularization.

Factors affecting visual outcome of myopic choroidal neovascularization treated with verteporfin photodynamic therapy

AIM: To evaluate the visual outcomes of choroidal neovascularization (CNV) secondary to pathological myopia and the impact of novel risk factors affecting the final visual outcome. ·METHODS: Interventional case series of 18 consecutive patients with pathological myopia treated with photodynamic therapy (PDT). Inclusion criteria were spherical equivalent -6D or worse or features of pathological myopia on retinal examination. The main outcome measure was final best -corrected visual acuity (BCVA). ·RESULTS: Of 18 eyes, 13 (72.2%) avoided moderate visual loss (≥3 lines of LogMAR BCVA) and 5 eyes (27.8%) improved by at least 1 line after 1 year. Patients with LogMAR BCVA ≤0.3 (Snellen equivalent 20/40) at one year were younger than those with BCVA 】0.3 (mean age 39.0 vs 61.6 years, P =0.001). A higher proportion of eyes with greatest linear dimension (GLD) of ≤1 000μm avoided moderate visual loss (100% vs 50%, P =0.026). Among patients who were treated within 2 weeks of visual symptoms, 88.9% avoided the loss of 3 or more lines compared to 55.6% for those who presented later. The mean improvement in LogMAR BCVA of those with GLD ≤1 000μm was +0.12 compared to a loss of 0.55 LogMAR units for those with GLD 】1 000μm (P =0.02). Visual outcomes were not associated with gender or refractive error.·CONCLUSION: Good visual outcome in myopic CNV is associated with younger age, smaller lesion size and earlier initiation of treatment. These factors are relevant for ophthalmologists considering treatment options for myopic CNV.

Approved pharmacotherapy for myopic choroidal neovascularization:a review of randomized controlled trials in ranibizumab and aflibercept

Myopic choroidal neovascularization(mCNV) can cause severe visual impairment in highly myopic patients. We review the randomized trials of two approved pharmacotherapy for treating mCNV, including intravitreal injections of ranibizumab and aflibercept. These two vascular endothelial growth factor(VEGF) antagonists show superior ability to improve vision and reduce macular thickness, comparing with sham injections or verteporfin photodynamic therapy(vPDT). There is no severe ocular or systemic adverse reaction reported in studies associated with ranibizumab and aflibercept for mCNV. Prompt treatment with these agents can lead to a better outcome.

Inhibition of choroidal neovascularization by lentivirusmediated PEDF gene transfer in rats

AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.

Focal choroidal excavation complicated with choroidal neovascularization in young and middle aged patients

AIM: To investigate the clinical and optical coherence tomography(OCT) features of focal choroidal excavation(FCE) complicated with choroidal neovascularization(CNV) in young and middle aged patients. METHODS: We performed a retrospective review of 26 patients with FCE accompanied by CNV. All patients underwent a complete ophthalmic examination. We analyzed the clinical characteristics of patients, focusing on the spectral-domain OCT features. All patients received intravitreal injection of anti-vascular endothelial growth factor(anti-VEGF) agents. And we assessed the changes of central retinal thickness and best-corrected visual acuity(BCVA) after anti-VEGF therapy. RESULTS: The mean age of 26 patients was 35.5±7.3 y(range, 21-48 y). Of the 26 FCE lesions, 11 were located subfoveal, 6 were parafoveal, and 9 were extrafoveal. The mean FCE depth was 129.8±50.3 μm, and the mean width was 901.3±306.0 μm. The FCE depth was correlated positively with the width, but not correlated with age or refractive error. CNV was located within the excavation(19 eyes) or adjacent to the excavation(7 eyes). After anti-VEGF therapy, the central retinal thickness was significantly reduced and the BCVA was significantly improved. In the absorption process of subretinal fluid, we found that the fluid in the excavations needed to be absorbed at the last. A small amount of residual fluid could still be seen in a few deep excavations even after a longterm follow-up.CONCLUSION: FCE may be an important reason to cause CNV. Especially in young patients with idiopathic CNV, we should pay attention to the use of OCT to check the presence of FCE. Anti-VEGF therapy is generally effective for CNV associated with FCE.

Blockade of the Sonic Hedgehog Signalling Pathway Inhibits Choroidal Neovascularization in a Laser-induced Rat Model

Sonic hedgehog (Shh) signaling has recently been shown to be involved in the pathological angiogenesis in response to tissue hypoxia and ischemic injury.Hypoxia/ischemia is considered to play an important role in the development of choroidal neovascularization (CNV).This study was aimed to examine the effect of blockade of the Shh signaling pathway on CNV and the underlying mechanism.A total of 64 male Brown-Norway (BN) rats were used in this study.One eye of each rat underwent laser photocoagulation.The other eye served as normal control.After the laser treatment, the 64 rats were divided into four groups (n=16 in each group):Blank control group, in which no intravitreal administration was given; cyclopamine group, recombinant Shh N-terminals protein (rShh) group and phosphate-buffered saline (PBS) group, in which cyclopamine (a Shh inhibitor), rShh (a Shh activator) and PBS were intravitreally injected into the laser-treated eyes respectively every other day for a total of four intravitreal injections immediately after the laser treatment.Fourteen days after the intravitreal administration, the changes of CNV-related variables, including positive CNV lesion percentage, CNV membrane area and CNV membrane thickness, were evaluated by fluorescein anqiography, indocyanine green angiography and pathological examinations.The mRNA and protein expression of PTCH1, Gli1, HIF-1α, VEGF and DLL4 in each group on 14 days of CNV model was detected by real-time quantitative PCR and western blot analysis, and the relationship between the Shh cascade and the HIF-1α-VEGF-DLL4 cascade in CNV was analyzed.The results showed that the CNV membrane area and the CNV membrane thickness were decreased by 62.5% and 41.9% in the cyclopamine group and increased by 85.7% and 64.3% in the rShh group in comparison to those in the blank control group (P【0.01 for each).There was no significant difference in the CNV membrane area and thickness between the blank control group and PBS group (P=0.102 and P=0.063, respectively).Real-time quantitative PCR revealed a 5.23-, 4.14-, 2.97-, 2.78-and 2.39-fold up-regulation of the mRNA expression of PTCH1, Gli1, HIF-1α, VEGF and DLL4 genes in the laser-treated eyes compared with the normal control eyes in the control group.In the cyclopamine group, the mRNA and protein expression of Gli1, HIF-1α, VEGF and DLL4 was significantly down-regulated (P【0.05 for each) while the expression of PTCH1 showed no significant changes at the mRNA (P=0.293) and protein level (P=0.304).The mRNA expression and protein expression (P=0.001 and P=0.021, respectively) of PTCH1, Gli1, HIF-1α, VEGF and DLL4 was significantly increased in the rShh group when compared with the control group.The expression level of these genes was related to the severity of the CNV.It was concluded that intravitreal administration of cyclopamine can effectively inhibit the formation of laser-induced experimental CNV by down-regulating the expression of the HIF-1α-VEGF-DLL4 cascade in CNV.The Shh signaling pathway as an upstream signaling pathway of HIF-1α-VEGF-DLL4 cascade is implicated in the development of experimental CNV.

Correlation of CD105 and Vascular Endothelial Growth Factor in Laser-induced Choroidal Neovascularization in Rats

Purpose: Choroidal neovascularization (CNV) plays an important role in pathogenesis of age-related macular degeneration (AMD), ocular histoplasmosis syndrome (OHS) and so on. However, mechanisms of CNV formation are not fully understood. The aim of this study is to investigate the correlation between expressions of CD105 and vascular endothelial growth factor (VEGF) in experimental laser-induced CNV in rats. Methods: CNV model was established by 532 nm laser photocoagulation in Brown-Norway rats. The expression of CD105 and VEGF in CNV was observed by immunohistochemistry at 3, 7, 14, 21, 28 and 56 days after laser photocoagulation. The image analysis was performed with the professional software of Image-Pro Plus. Results: Fluorescein angiography showed fluorescein leakage in CNV from days 7 to 56 after photocoagulation. VEGF expression was mainly observed in vascular endothelial cells, ganglion cells, inner nuclear layers and retinal pigment epithelial cells in normal retina and vascular endothelial cells in normal choroid of the rats. On day 3 after photocoagulation, VEGF began to express in laser-induced lesions. VEGF was strongly expressed in CNV after 7 days (P<0.05) and decreased after 14 days (P>0.05). CD105 was initially presented in CNV at 7 days and obviously expressed at 14 days after photocoagulation (P<0.05). Four weeks later, when angiogenesis tended toward inactive status, expression of CD105 was markedly decreased (P>0.05). There was notablely direct correlation between CD105-positive-microvessel density and positively semiquantitative scoring of VEGF in the CNV(r=0.989, P<0.01). Conclusions: There is direct correlation between the expression of CD105 and VEGF in the laser-induced CNV in rat. It suggests that CD105 and VEGF might participate in the new blood vessel formation and promote the growth of CNV.

Microarray expression profile and functional analysis of circular RNAs in choroidal neovascularization

Choroidal neovascularization(CNV) is a leading cause of visual loss in age-related macular degeneration(AMD). However, the molecular mechanism for CNV progression is still unclear. This study aimed to identify CNV-related circular RNAs(circRNAs), a novel class of non-coding RNAs with diverse functions. A total of 117 circRNAs were differentially expressed in the murine CNV model by microarrays. Gene ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis were performed to identify the functions of selected circRNAs. The host genes of these circRNAs were predicted to be targeted to neurogenesis(ontology: biological process), proteinaceous extracellular matrix(ECM)(ontology: cellular component), and binding(ontology: molecular function). Differentially expressed circRNAs-mediated regulatory networks were enriched in ECM receptor interaction. Most of the dysregulated circRNAs could potentially bind to five different mi RNAs by Target Scan and mi Randa. Specifically, circ_15752 was identified in this circRNAs pool which may facilitate vascular endothelial cell proliferation, migration, and tube formation, suggesting a critical role in endothelial angiogenesis. Our work suggests that dysregulated circRNAs may be involved in CNV pathogenesis and serve as potential biomarkers for CNV.

Suppression of laser-induced choroidal neovascularization by intravitreal injection of tristetraprolin

AIM: To examine the effect of intravitreal adenoviral vector-mediated tristetraprolin(Ad-TTP) on VEGF m RNA expression in a rat model of laser-induced choroidal neovascularization.METHODS: Ad-TTP was prepared using a commercial kit. Retinal laser-induced photocoagulation(10 spots per eye) was performed on rats in this experimental choroidal neovascularization(CNV) model. Rats were divided into four groups: control(single intravitreal injection of balanced salt solution, n =10), laser-induced CNV(photocoagulation only, n =20), laser-induced CNV plus Ad-TTP injection(photocoagulation plus a single intravitreal Ad-TTP injection, n =20) and Ad-TTP injection only(n =10). Changes in choroidal morphology were evaluated in ten rats in the laser only and the laser plus Ad-TTP groups. Two weeks after laser injury, the size of CNV was calculated by perfusion with high-molecular-weight fluorescein isothiocyanate(FITC)-dextran. VEGF m RNA expression in retina-choroid tissue from ten rats in each group was measured by reverse transcription polymerase chain reaction(RT-PCR). RESULTS: Two weeks after treatment, the area of laser-induced CNV was reduced by approximately 60% in the rats given the Ad-TTP injection compared with that in the laser-only group. There was a tendency toward decreased VEGF m RNA expression in the Ad-TTP injection groups.CONCLUSION: A single intravitreal injection of Ad-TTP significantly suppressed CNV size in this experimental laser-induced CNV model. Ad-TTP injection also decreased VEGF m RNA expression compared with that inthe laser-induced CNV group. The present study is meaningful as the first study to investigate the effect of tristetraprolin delivered via intravitreal injection.

Effect of integrin α5β1 inhibition on SDF-l/CXCR4-mediated choroidal neovascularization

AIM：To investigate the roles of integrins in choroidal neovascularization（CNV） and their associations with the stromal cell-derived factor-1（SDF-1）/CXCR4 axis.METHODS：CNV lesions were induced in mice using laser photocoagulation.After CNV induction,all animals were randomly assigned to：control,SDF-1,SDF-1＋age-related macular degeneration（AMD） 3100（CXCR4 inhibitor）,and SDF-1＋ATN161（integrin α5β1 inhibitor） groups;their effects on CNV progression were observed using hematoxylin eosin（HE） staining,fundus fluorescein angiography（FFA） grading and optical coherence tomography（OCT）,and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining.Hypoxia-exposed endothelial cells（ECs） were used to simulate CNV in vitro,they were treated with SDF-1,combined with CXCR4 siRNA/AMD3100 or ATN161,and expression of integrin α5,cell migration and tube formation were analyzed.RESULTS：Integrin subunit α5 increased at 3^ rd and 7^ th day and decreased at 14 ^th day in CNV mice,with no significant change of β1-integrin.CXCR4 expression in CNV mice had persistent increase within 14 d after induction.SDF-1 treatment significantly promoted the CNV progression during 3-14 d.The mean CNV length in AMD3100 andATN161 group at day 7 was 270.13 and 264.23 μm in HE images,significantly lower than the mean length in SDF-1（345.70 μm） group.AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1.Mean integrin α5 positive area in SDF-1 group reached 2.31×104 μm^2,significantly higher than control（1.25×104 μm^2）,which decreased to 1.78×104 μm^2 after AMD3100 treatment.About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group,which significantly decreased to 43.12% after AMD3100 treatment.In vitro,integrin α5 peaked by 6 folds after 6 h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24 h of hypoxia.Approximately 25.12% of ECs expressed integrin α5 after SDF-1 stimulation,which decreased to 7.2%-9.5% after si-CXCR4 or AMD3100 treatment.ATN161 exerted an inhibitory effect comparable to that of si-CXCR4 on EC migration and tube formation in the presence of SDF-1.CONCLUSION：SDF-1/CXCR4 signaling induces integrin α5β1 expression in ECs to promote CNV.

Bietti crystalline dystrophy and choroidal neovascularization in childhood

Dear Editor,I am Dr.Sabiha Gungor Kobat,from the Department of Ophthalmology,Elazig Health Sciences University,Elazig,Turkey.I am writing to present an exceedingly rare case of two siblings,one of whom developed Bietti crystalline dystrophy(BCD)with choroidal neovascularization at the age of 13 years,and the other has asymptomatic BCD at 8 years old.

MicroRNAs in laser-induced choroidal neovascularization in mice and rats:their expression and potential therapeutic targets

Choroidal neovascularization characterizes wet age-related macular degeneration.Choroidal neovascularization formation involves a primarily angiogenic process that is combined with both inflammation and proteolysis.A primary cause of choroidal neovascularization pathogenesis is alterations in pro-and anti-angiogenic factors derived from the retinal pigment epithelium,with vascular endothelium growth factor being mainly responsible for both clinical and experimental choroidal neovascularization.MicroRNAs(miRNAs)which are short,non-coding,endogenous RNA molecules have a major role in regulating various pathological processes,including inflammation and angiogenesis.A review of recent studies with the mouse laser-induced choroidal neovascularization model has shown alterations in miRNA expression in choroidal neovascularization tissues and could be potential therapeutic targets for wet age-related macular degeneration.Upregulation of miR-505(days 1 and 3 post-laser),miR-155(day 14)occurred in retina;miR-342-5p(days 3 and 7),miR-126-3p(day 14)in choroid;miR-23a,miR-24,miR-27a(day 7)in retina/choroid;miR-505(days 1 and 3)in retinal pigment epithelium/choroid;downregulation of miR-155(days 1 and 3),miR-29a,miR-29b,miR-29c(day 5),miR-93(day 14),miR-126(day 14)occurred in retinal pigment epithelium/choroid.Therapies using miRNA mimics or inhibitors were found to decrease choroidal neovascularization lesions.Choroidal neovascularization development was reduced by overexpression of miR-155,miR-188-5p,miR-(5,B,7),miR-126-3p,miR-342-5p,miR-93,miR-126,miR-195a-3p,miR-24,miR-21,miR-31,miR-150,and miR-184,or suppression of miR-505,miR-126-3p,miR-155,and miR-23/27.Further studies are warranted to determine miRNA expression in mouse laser-induced choroidal neovascularization models in order to validate and extend the reported findings.Important experimental variables need to be standardized;these include the strain and age of animals,gender,number and position of laser burns to the eye,laser parameters to induce choroidal neovascularization lesions including wavelength,power,spot size,and duration.

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.

Yes-associated protein promotes endothelial-tomesenchymal transition of endothelial cells in choroidal neovascularization fibrosis

AIM:To reveal whether and how Yes-associated protein(YAP)promotes the occurrence of subretinal fibrosis in agerelated macular degeneration(AMD).METHODS:Cobalt chloride(Co Cl2)was used in primary human umbilical vein endothelial cells(HUVECs)to induce hypoxia in vitro.Eight-week-old male C57 BL/6 J mice weighing 19-25 g were used for a choroidal neovascularization(CNV)model induced by laser photocoagulation in vivo.Expression levels of YAP,phosphorylated YAP,mesenchymal markers[αsmooth muscle actin(α-SMA),vimentin,and Snail],and endothelial cell markers(CD31 and zonula occludens 1)were measured by Western blotting,quantitative real-time PCR,and immunofluorescence microscopy.Small molecules YC-1(Lificiguat,a specific inhibitor of hypoxia-inducible factor 1α),CA3(CIL56,an inhibitor of YAP),and XMU-MP-1(an inhibitor of Hippo kinase MST1/2,which activates YAP)were used to explore the underlying mechanism.RESULTS:Co Cl2 increased expression of mesenchymal markers,decreased expression of endothelial cell markers,and enhanced the ability of primary HUVECs to proliferate and migrate.YC-1 suppressed hypoxia-induced endothelialto-mesenchymal transition(End MT).Moreover,hypoxia promoted total expression,inhibited phosphorylation,and enhanced the transcriptional activity of YAP.XMU-MP-1 enhanced hypoxia-induced End MT,whereas CA3 elicited the opposite effect.Expression of YAP,α-SMA,and vimentin were upregulated in the laser-induced CNV model.However,silencing of YAP by vitreous injection of small interfering RNA targeting YAP could reverse these changes.CONCLUSION:The findings reveal a critical role of the hypoxia-inducible factor-1α(HIF-1α)/YAP signaling axis in End MT and provide a new therapeutic target for treatment of subretinal fibrosis in AMD.

Heparanase-1 activities in the development of laser induced choroidal neovascularization

AIM: To investigate the role of heparanase-1 in laser-induced choroidal neovascularization (CNV). METHODS: Experimental CNV was induced by krypton laser photocoagulation in 15 male Brown Norway rats. Fundus fluorescein angiography and histopathological examination were performed in observing the CNV development. The expression and distribution of heparanase-1 protein in the laser lesions were determined by immunohistochemistry and western blotting analysis. RESULTS: The success rate of laser induced CNV was approximately 75% on 3-4 weeks after laser photo-coagulation. The protein levels of heparanase-1 increased significantly in the retina-choroidal complex of CNV models when compared to normal rat eyes (P 【 0.01). Immunostaining confirmed strong heparanase-1 expressions in all laser lesions, and it displayed to be highest at the newly formed blood vessels within the fibrovascular complex in the subretinal space. CONCLUSION: Heparanase-1 is closely involved in the development of laser induced CNV.