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.

Comparative study between swept-source and spectraldomain OCTA for imaging of choroidal neovascularization in age-related macular degeneration

AIM:To compare the dif ferences of choroidal neovascularization(CNV)measurements between sweptsource and spectral-domain optical coherence tomography angiography(SS-OCTA and SD-OCTA)in neovascular agerelated macular degeneration(nAMD)and the imaging reliability of the two devices.METHODS:Prospective comparative study.SS-OCTA and SD-OCTA were used to scan the same eye with the modes of 3×3 and 6×6 mm2 centered on the neovascularization.Only qualified images were chosen and the border of CNV was manually delineated by two graders independently.The area of CNV(ACNV),vascular perfusion density(PD),and vessel length density(VLD)within the delineation were calculated using Image J.The differences of CNV measurements between the two OCTA devices were compared using Bland-Altman analysis.The agreement between the two graders on the measurements of each device was compared using the intraclass correlation coefficient(ICC).RESULTS:A total of 18 patients(22 eyes)with nAMD were included.The measurements of ACNV,PD,and VLD were 7.247±4.586 and 4.901±3.741 mm^(2),43.202±9.636 and 34.904±10.489,6.339±1.228 and 5.908±1.741 mm^(-1) for SS-OCTA and SD-OCTA,respectively.The differences between the two devices were 2.346±3.030 mm^(2)(Z=-3.782,P<0.0001),8.298±14.160(Z=-2.419,P=0.016),and 0.431±2.114 mm^(-1)(Z=-0.828,P=0.408)for ACNV,PD and VLD,respectively.The ICC between two graders were 0.893(P<0.001),0.902(P<0.001),0.885(P<0.001)for ACNV,PD,VLD in SS-OCTA,and 0.971(P<0.001),0.976(P<0.001),0.973(P<0.001)in SD-OCTA,respectively.CONCLUSION:Both OCTA devices have high imaging reliability.Compared with SD-OCTA,SS-OCTA has a larger ACNV measurements,but doesn’t show better resolution of internal vessels of CNV and well signal strength.

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.

Subconjunctival conbercept for the treatment of corneal neovascularization

·AIM:To investigate the effectiveness and safety of subconjunctival injection of conbercept in the treatment of corneal neovascularization(CNV).·METHODS:The data on 10 consecutively recruited patients with CNV who received a subconjunctival conbercept(1 mg)once,and measured the area,length,and diameter of neovascularization before and after(1d,1,2wk,and 1mo)treatment as well as the occurrence of systemic and ocular complications after treatment were analyzed.·RESULTS:There was a statistically significant reduction in the area of CNV one day after treatment(mean±SD:38.46±11.36 mm^(2)),compared with before treatment(42.46±12.80 mm^(2),P<0.01).There was also a statistically significant reduction in the length(3.86±1.80 mm vs 4.64±1.77 mm,P<0.01)and diameter(0.044±0.022 vs 0.060±0.026,P<0.05)of CNV,one week after treatment comparing to before treatment.The reduction in all three parameters was maximized at two weeks after treatment(area:29.49±8.83 mm^(2),P<0.001;length:3.50±1.88 mm,P<0.001;and diameter:0.038±0.017 mm,P<0.01).No severe systemic or ocular complication was observed during the study.·CONCLUSION:During the observation period of onemonth,subconjunctival injection of conbercept is an effective and safe method for the reduction of CNV.It may be effective as a preoperative drug for neovascular corneal transplantation.

Efficacy and safety of anti-vascular endothelial growth factor agents on corneal neovascularization: A meta-analysis

BACKGROUND Corneal neovascularization(CoNV)is the second major cause of blindness.Vascular endothelial growth factor(VEGF)inhibitors,e.g.,bevacizumab,have been used to prevent CoNV.AIM We conducted an updated systematic review and meta-analysis of clinical trials to examine the efficacy and safety of anti-VEGF in CoNV.METHODS A literature search was conducted using three electronic databases.Mean difference(MD),standard mean difference(SMD),and relative risk(RR)are used to estimate the effect size.RESULTS Nine randomized controlled and three non-randomized trials were obtained.The pooled results demonstrated a significant reduction of CoNV area/Length(SMD=-1.17,95%CI:-1.58 to-0.75),best corrected visual acuity(MD=-0.54,95%CI:-0.91 to-0.17),and graft rejection(RR=0.44,95%CI:0.24 to 0.8)and failure(RR=0.39,95%CI:0.19 to 0.78)rates in the anti-VEGF group than the placebo group.A non-significant reduction of the epithelial defect was also observed in the bevacizumab group compared with the placebo(RR=0.56,95%CI:0.30 to 1.06).Compared with a placebo,the unsynthesizable trials also support that bevacizumab improves visual acuity,CoNV,graft rejection,and failure rates.Trials reporting other comparisons revealed the superiority of combined remedy with bevacizumab compared to other treatments in reducing CoNV.CONCLUSION Anti-VEGF agents,mainly bevacizumab,are an effective and safe treatment for CoNV of all causes and prevent corneal graft rejection and failure in corneal transplantation.

A review on vasohibin and ocular neovascularization

Ischemic and neovascular disease is one of the most difficult ocular diseases to deal with nowadays.Redundancy,poor visual acuity and decreased life quality are bothering patients and ophthalmologists for decades.After vascular endothelial growth factor(VEGF)was found to be a primary factor in promoting retinal angiogenesis,intravitreal injection of anti-VEGF drugs has been the firstline treatment.Whereas,some patients are refractory to this therapy and problems of economic burden,local complications and adverse effects promote researches into other possible targets.The vasohibin(VASH)family is a newly-investigated factor in modulating ocular angiogenesis.The family includes VASH1 and VASH2,which show opposite effects of inhibiting and accelerating angiogenesis respectively.Positive results have been reported in cellular and animal experiments.With further researches,it can be a promising future target of treating ocular neovascular diseases.

Assessment of neovascularization within carotid plaques in patients with ischemic stroke

AIM:To assess neovascularization within human ca-rotid atherosclerotic soft plaques in patients with isch-emic stroke.METHODS:Eighty-one patients with ischemic stroke and 95 patients without stroke who had soft athero-sclerotic plaques in the internal carotid artery were studied.The thickest soft plaque in each patient was examined using contrast-enhanced ultrasound.Time-intensity curves were collected from 5 s to 3 min after contrast injection.The neovascularization within the plaques in the internal carotid artery was evaluated using the ACQ software built into the scanner by 2 of the experienced investigators who were blinded to the clinical history of the patients.RESULTS:Ischemic stroke was present in 7 of 33 patients(21%) with grade Ⅰ plaque,in 14 of 51 pa-tients(28%) with grade Ⅱ plaque,in 26 of 43 patients(61%) with grade Ⅲ plaque,and in 34 of 49 patients(69%) with grade Ⅳ plaque(P < 0.001 comparing grade Ⅳ plaque with grade I plaque and with grade Ⅱ plaque and P = 0.001 comparing grade Ⅲ plaque with grade Ⅰ plaque and with grade Ⅱ plaque).Analysis of the time intensity curves revealed that patients with ischemic stroke had a significantly higher intensity of enhancement(IE) than those without ischemic stroke(P < 0.01).The wash-in time(WT) of plaque was signifi-cantly shorter in stroke patients(P < 0.05).The sensi-tivity and specificity for IE in the plaque were 82% and 80%,respectively,and for WT were 68% and 74%,respectively.There was no significant difference in the peak intensity or time to peak between the 2 groups.CONCLUSION:This study shows that the higher the grade of plaque enhancement,the higher the risk of ischemic stroke.The data suggest that the presence of neovascularization is a marker for unstable plaque.

Risk factors associated with retinal neovascularization of diabetic retinopathy in type 2 diabetes mellitus

AIM: To evaluate the risk factors associated with retinal neovascularization of diabetic retinopathy in northern Chinese Han patients with type 2 diabetes mellitus (T2DM). METHODS: The clinical characteristics of 200 patients with proliferative diabetic retinopathy (PDR) and 100 age-matched healthy individuals were compared. The univariate and multivariate logistic regression analysis were performed in the patients with PDR. RESULTS: Fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), blood urea nitrogen (BUN), uric acid (UA), white blood cell count (WBC), absolute neutrophil count, hematocrit (HCT) and mean platelet volume (MPV) and mean platelet volume (MPV) were all significantly higher in patients with PDR than in the control group (P<0.05). The univariate and multivariate logistic regression analysis showed that risk factors independently associated with retinal neovascularization of DR were duration of diabetes mellitus (OR=1.112; P=0.000), BUN (OR=1.277; P=0.000), smoking (OR=3.967; P=0.000) and MPV (OR=2.472; P=0.000). On the other hand, panretinal photocoagulation was associated with reduced risk of retinal neovascularization (OR=0.983; P=0.000). CONCLUSION: Preventing and controlling T2DM in terms of risk factors, including duration of diabetes, BUN, smoking and MPV, might offer novel approaches to prevent or delay the onset of retinal neovascularization in patients with PDR.

Comparison of subconjunctivally injected bevacizumab, ranibizumab, and pegaptanib for inhibition of corneal neovascularization in a rat model

AIM: To compare the efficacies of subconjunctival bevacizumab, ranibizumab, and pegaptanib sodium injections for the inhibition of corneal neovascularization in an experimental rat model. METHODS: Sixteen corneas of 16 rats were chemically cauterized and randomized into four groups: bevacizumab group that treated with 0.05mL/1.25mg bevacizumab, ranibizumab group that treated with 0.05mL/0.5mg ranibizumab, pegaptanib group that treated with 0.05mL/0.15mg pegaptanib sodium, and control group that treated with 0.05mL saline solution. Digital photographs of the corneas were taken and analyzed using an image analysis software program. All corneas were excised and examined histologically on the 15 th day. RESULTS: Each treatment group had significantly less neovascularized corneal areas and fewer blood vessels than the control group (all P 【0.05). In addition, bevacizumab group had significantly less neovascu-larized corneal areas and fewer blood vessels than ranibizumab and pegaptanib groups (both P 【0.05). However, there was no significant difference between the ranibizumab and pegaptanib groups regarding percentage of neovascularized corneal areas and number of blood vessels (both P 】0.05). CONCLUSION: Subconjunctival bevacizumab, ranibiz-umab, and pegaptanib sodium were effective with no corneal epitheliopathy for inhibiting corneal neovascularization after corneal burn in rats .Bevacizumab was more effective than ranibizumab and pegaptanib sodium.

Impact of Lycium Barbarum Polysaccharide and Danshensu on vascular endothelial growth factor in the process of retinal neovascularization of rabbit

AIM:To discuss the impact of Lycium Barbarum Polysaccharide (LBP) and Danshensu purified from Traditional Chinese Medicine (TCM) on vascular endothelial growth factor (VEGF) of rabbits with retinal neovascularization. METHODS:Forty rabbits were divided into normal control group, model control group, LBP group and Danshensu group. Animals in the normal control group were fed in the normal oxygen environment. Animals in the other three groups were put into the environment with 70% oxygen for 5 days in order to build the model of oxygen-induced vascular proliferation retinopathy. And then different TCM extract was injected into the abdominal cavities of these annimals. After 7 days, the VEGF content of in the serum of rabbit was measured by double antibody sandwich method. RESULTS:Data analysis indicated that VEGF content was as follows:Danshensu group was lower than model control group (12.92 ±3.84ng/L vs 19.32 ±4.15ng/L, P 【 0.05); LBP group and normal control group were lower than model control group (12.92±3.84ng/L, 9.26±1.61ng/L vs 19.32±4.15ng/L, P【0.01); total blood viscosity, plasma viscosity, cholesterol content, fibrinogen content and triacylglycerol content after peritoneal injection of LBP and Danshensu were obviously lower than before injection. CONCLUSION:TCM extract-LBP and Danshensu can prominently reduce the content of VEGF in the process of vascular proliferative retinopathy of rabbit; can prevent the occurrence of retinal microvascular disease by improving partial oxygen -deficient environment or affecting all kinds of new growth factor.

Growth factor- and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization

Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes specific growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endotheli- al progenitor cells migrate and home to specific sites following ischemic stroke via growth factor/ cytokine gradients. Some growth factors are less stable under acidic conditions of tissue isch- emia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovascularization following ischemic stroke.

Effect of trapping vascular endothelial growth factor-A in a murine model of dry eye with inflammatory neovascularization

AIM： To evaluate whether trapping vascular endothelial growth factor-A（VEGF-A） would suppress angiogenesis and inflammation in dry eye corneas in a murine corneal suture model.METHODS： We established two groups of animals, one with non-dry eyes and the other with induced dry eyes.In both groups, a corneal suture model was used to induce inflammation and neovascularization. Each of two groups was again divided into three subgroups according to the treatment; subgroup I（aflibercept）,subgroup II（dexamethasone） and subgroup III（phosphate buffered saline, PBS）. Corneas were harvested and immunohistochemical staining was performed to compare the extents of neovascularization and CD11b＋ cell infiltration. Real-time polymerase chain reaction was performed to quantify the expression of inflammatory cytokines and VEGF-A in the corneas.RESULTS： Trapping VEGF-A with aflibercept resulted in significantly decreased angiogenesis and inflammation compared with the dexamethasone and PBS treatments in the dry eye corneas（all P 〈0.05）, but with no such effects in non-dry eyes. The anti-inflammatory and antiangiogenic effects of VEGF-A trapping were stronger than those of dexamethasone in both dry eye and non-dry eye corneas（all P 〈0.05）. The levels of RNA expression of VEGF-A, TNF-alpha, and IL-6 in the aflibercept subgroup were significantly decreasedcompared with those in the PBS subgroup in the dry eye group.CONCLUSION： Compared with non-dry eye corneas,dry eye corneas have greater amounts of inflammation and neovascularization and also have a more robust response to anti-inflammatory and anti-angiogenic agents after ocular surface surgery. Trapping VEGF-A is effective in decreasing both angiogenesis and inflammation in dry eye corneas after ocular surface surgery.

Effects of nuclear factor κB expression on retinal neovascularization and apoptosis in a diabetic retinopathy rat model

AIM: To investigate the expression and role of nuclear factor κB(NF-κB) in diabetic retinopathy(DR) and its relationship with neovascularization and retinal cell apoptosis. METHODS: A total of 80 male Wistar rats were randomly assigned to control(4, 8, 12 and 16 wk, n =10 in each group) and diabetes mellitus(DM) groups(4, 8, 12 and 16wk, n =10 in each group). A diabetic rat model was established by intraperitoneal injection of streptozotocin(60 mg/kg). After 4, 8, 12 and 16 wk, rats were sacrificed.Retinal layers and retinal neovascularization growth were stained with hematoxylin-eosin and examined under light microscopy. Cell apoptosis in the retina was detected by Td T-mediated d UTP nick end labeling, and NF-κB distribution and expression in the retina was determined using immunohistochemistry. RESULTS: DM model success rate up to 100%.Diabetes model at each time point after the experimental groupcompared with the control group, the blood glucose was significantly increased, decreased body weight, each time point showed significant differences compared with the control group(P 【0.01). After 12 wk other pathological changes in the retina of diabetic rats were observed; after 16 wk, neovascularization were observed. After 1mo, retinal cell apoptosis was observed.Compared with the control group, NF-κB expression in the DM group significantly increased with disease duration.CONCLUSION: With the prolonging of DM progression,the expression NF-κB increases. NF-κB may be related to retinal cell apoptosis and neovascularization.

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.

Comparison of the inhibitory effect of different doses of subconjunctival bevacizumab application in an experimental model of corneal neovascularization

AIM： To evaluate the inhibitory effect of subconjunctival bevacizumab as single-and multiple-dose application, and compare their effects on corneal neovascularization in a rat model. METHODS： Thirty adult Sprague-Dawley rats were used in this experimental study. The central cornea of the rats was cauterized chemically. The rats were randomly enrolled into three groups. All groups received subconjunctival injections. In Group 1（control group, n=10）, 0.05 m L 0.9% Na Cl solution was injected on the first day. In Group 2（single-dose group, n=10）, 0.05 m L bevacizumab（1.25 mg） was injected on the first day. In Group 3（multiple-dose group, n=10）, four doses of 0.05 m L bevacizumab（1.25 mg） were injected on the first, third, fifth and seventh day. Slit-lamp examination of all rats was performed at the third and ninth day. Digital images of the corneas were taken and analyzed using image analysis software to calculate corneal neovascularization area. All rats were sacrificed on the tenth day. In corneal sections, the number of blood vessels, state of inflammation and collagen formation was evaluated histopathologically. RESULTS： In Group 3, corneal edema grades were significantly lower than Group 1 and Group 2（P=0.02, and P=0.035, respectively）. The mean percentage of neovascularized corneal area in Group 3 was significantly lower than Group 2（P=0.005）. On histopathological examination, Group 2 and Group 3 showed significantly less number of blood vessels than Group 1（P=0.005, and P=0.001, respectively）. Additionally, Group 3 showed significantly less number of blood vessels compared to Group 2（P=0.019）. Inflammation and edema grades were significantly lower in Group 3 compared to Group 1（P=0.001）. CONCLUSION： Subconjunctival bevacizumab injection is effective in inhibition of newly formed corneal neovascularization. The multiple-dose bevacizumab treatment seems to be more effective compared to single-dose treatment.

Endothelial progenitor cells as factors in neovascularization and endothelial repair

Endothelial progenitor cells(EPCs)are a heterogeneous population of cells that are provided by the bone marrow and other adult tissue in both animals and humans.They express both hematopoietic and endothelial surface markers,which challenge the classic dogma that the presumed differentiation of cells into angioblasts and subsequent endothelial and vascular differentiation occurred exclusively in embryonic development.This breakthrough stimulated research to understand the mechanism(s)underlying their physiologic function to allow development of new therapeutic options.One focus has been on their ability to form new vessels in injured tissues,and another has been on their ability to repair endothelial damage and restore both monolayer integrity and endothelial function in denuded vessels.Moreover,measures of their density have been shown to be a better predictor of cardiovascular events,both in healthy and coronary artery disease populations than the classical tools used in the clinic to evaluate the risk stratification.In the present paper we review the effects of EPCs on revascularization and endothelial repair in animal models and human studies,in an attempt to better understand their function,which may lead to potential advancement in clinical management.

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.

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.

Inhibition of LY294002 in retinal neovascularization via down-regulation the PI3K/AKT-VEGF pathway in vivo and in vitro

AIM： To investigate the effects of the phosphatidylinositol 3-kinase（PI3 K） inhibitor LY294002 on retinal neovascularization（RNV） in the oxygen-induced retinopathy（OIR） mouse model and human umbilical vein endothelial cells（HUVECs）.METHODS： C57 BL/6 J mice were randomly divided into normoxia-control, OIR-control and LY294002 treatment groups. LY294002 or phosphate-buffered solution was intraperitoneally injected daily into mouse pups from P6 to P9 in LY294002 treatment group or OIR-control group. Morphological and pathological changes in RNV, as well as expression levels of PI3 K, serine-threonine kinase（AKT） and vascular endothelial growth factor（VEGF） were observed. HUVECs treating with LY294002 were exposed to hypoxia; the expression of PI3 K, AKT and VEGF were examined by Western blot and RT-PCR analyses.RESULTS： Compared with the OIR-control group, LY294002 significantly inhibit RNV. Adenosine diphosphatase（ADPase） staining and hematoxylin and eosin staining indicated that the clock hour scores of neovascularization and the nuclei of pre-retinal neovascular cells in the LY294002 treatment group were clearly less than those in the OIR-control group（1.41±0.52 vs 6.20±1.21; 10.50±1.58 vs 22.25±1.82, both P〈0.05）. Intravitreal injection of LY294002（in the LY294002 treatment group） markedly decreased PI3 K/AKT-VEGF expression compared with the OIR-control group by immunohistochemistry, Western blotting and RT-PCR（all P〈0.05）. In HUVECs treated with hypoxia, expression of PI3 K, AKT and VEGF were downregulated in the hypoxia-LY294002 group（all P〈0.05）.CONCLUSION： The PI3 K inhibitor LY294002 can inhibit RNV by downregulating PI3 K, AKT, and VEGF expression in vivo and in vitro. LY294002 may provide an effective method for preventing retinopathy of prematurity（ROP）.