The roles of macrophage migration inhibitory factor in retinal diseases

Macrophage migration inhibitory factor(MIF),a multifunctional cytokine,is secreted by various cells and participates in inflammatory reactions,including innate and adaptive immunity.There are some evidences that MIF is involved in many vitreoretinal diseases.For example,MIF can exacerbate many types of uveitis;measurements of MIF levels can be used to monitor the effectiveness of uveitis treatment.MIF also alleviates trauma-induced and glaucoma-induced optic nerve damage.Furthermore,MIF is critical for retinal/choroidal neovascularization,especially complex neovascularization.MIF exacerbates retinal degeneration;thus,anti-MIF therapy may help to mitigate retinal degeneration.MIF protects uveal melanoma from attacks by natural killer cells.The mechanism underlying the effects of MIF in these diseases has been demonstrated:it binds to cluster of differentiation 74,inhibits the c-Jun N-terminal kinase pathway,and triggers mitogen-activated protein kinases,extracellular signal-regulated kinase-1/2,and the phosphoinositide-3-kinase/Akt pathway.MIF also upregulates Toll-like receptor 4 and activates the nuclear factor kappa-B signaling pathway.This review focuses on the structure and function of MIF and its receptors,including the effects of MIF on uveal inflammation,retinal degeneration,optic neuropathy,retinal/choroidal neovascularization,and uveal melanoma.

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.

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.

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.

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）.

CCR7/p-ERK1/2/VEGF signaling promotes retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIM： To investigate the role of CCR7/p-ERKI/2/VEGF signaling in the mouse model of oxygen-induced retinopathy （OIR）. METHODS： Neonatal C57BL/6J mice were evenly randomized into four groups： normoxia, OIR, OIR control （treated with scramble siRNA）, and OIR treated （treated with CCR7 siRNA）. Normoxia group was not specially handled. Postnatal day 7 （P7） mice in the OIR group were exposed to 75%±5% oxygen for 5d （P7-P12） and then maintained under normoxic conditions for 5d （P12-P17）. Mice in the OIR control and OIR treated groups were given injections of scramble or CCR7 siRNA plasmid on P12 before returning to normoxic conditions for 5d （P12-P17）. Retina samples were collected from all mice on P17, stained with adenosine diphosphatase （ADPase）, and retinal neovascularization （RNV） was assessed. Retinas were also stained with hematoxylin and eosin （H＆E） for RNV quantitation. The distribution and expression of CCR7, p-ERKI/2 and vas- cular endothelial growth factor （VEGF） were assessed via immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction （qRT-PCR）. RESULTS： High oxygen promoted retinal neovascularization （P〈0.05） and increased the number of endothelial nuclei in new vessels extending from the retina to the vitreous body; CCR7 promoted this process （P〈0.05）. CCR7 and VEGF mRNA were expressed at higher levels in the OIR and OIR control groups than in the normoxia and OIR treated groups. CCR7, p-ERK1/2, and VEGF protein were expressed in the retinas of mice in the OIR and OIR control groups. Intravitreal injection of CCR7 siRNA significantly reduced CCR7, p-ERKI/2, and VEGF expression in the OIR mouse model （all P〈0.05）. CCR7 significantly enhancedthe neovascularization and non-perfusion areas in the OIR group （P〈0,05）, CCR7 siRNA significantly reduced levels of p-ERK1/2 and VEGF as compared to OIR controls （P〈0.05）. CONCLUSION： These results suggest that CCR7/p-ERK I/2NEGF signaling plays an important role in OIR, CCR7 may be a potential target for the prevention and treatment of retinopathy of prematurity.

Matrix metalloproteinase-9 and vascular endothelial growth factor expression change in experimental retinal neovascularization

AIM： To investigate the signal transduction mechanism of matrix metalloproteinase-9 （MMP-9） mediatedvascular endothelial growth factor （VEGF） expression and retinal neovascularization （RNV） in oxygen-induced retinopathy （OIR） model. METHODS： C57BL/6J mice were divided into four groups： control group, OIR group, OIR control group （phosphatebuffered saline by intravitreal injection） and treated group [tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） by intravitreal injection]. OIR model was established in C57BIJ6J mice exposed to 75% ＋2% oxygen for 5d. mRNA level and protein expression of MMP-9, TIMP-1 and VEGF were measured by real-time polymerase chain reaction and Western blotting, and located by immunohistochemistry. RESULTS： Levels of MMP-9 and VEGF in retina were significantly increased in animals with OIR and OIR control group. Levels of TIMP -1 in retina was significantly reduced in animals with OIR and OIR control group. Furthermore, a significant correlation was found between MMP-9 and VEGF. Intravitreal injection of TIMP- 1 significantly reduced MMP-9 and VEGF expression of the OIR mouse model （all P〈0.05）. CONCLUSION： These results demonstrate that MMP- 9-mediated up-regulation of VEGF promotes RNV in retinopathy of prematurity （ROP）. TIMP-1 may be a potential target for the prevention and treatment of ROP.

Ultrasound-targeted cationic microbubble-mediated gene transfection and inhibition of retinal neovascularization

AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in mice.METHODS:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy.Experiments assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after treatment.RESULTS:The gene expression of endostatin was the highest in the group of the CMBs.Besides,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro.In vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups.CONCLUSION:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.

Effect of cytokeratin 17 on retinal pigment epithelium degeneration and choroidal neovascularization

AIM： To study the effects of cytokeratin 17 （CK17） on sodium iodate （NalOs） induced rat retinal pigment epithelium （RPE） degeneration, laser induced rat choroidal neovascularization （CNV）, and oxidative stress of human retinal pigment epithelium cells （ARPE-19） and human umbilical vein endothelial cell （HUVEC）. METHODS： Thirty 8-week-old male Brown Norway rats were randomly divided into 3 groups, 10 rats in control group treated with solvent alone; 10 rats in NalOs group treated with solvent and 35 mg/kg NalO3 injection through hypoglossal vein and 10 rats in CK17 ＋NaIOs group treated with 1% CK17 eye drop 3 times a day for lwk before and 4wk after NalOs injection. RPE function was measured with c-wave of electroretinogram （ERG）. Another 20 rats were randomly divided into 2 groups. Of them 10 rats in CK17 group were anesthetized to receive Nd：YAG laser and given 1% CK17 eye drop before same as above; 10 rats in control were received Nd：YAG and treated with solvent. The development of choroidal neovascularization （CNV） was determined by fundus fluorescein angiography （FFA） performed on 4wk after laser. Methylthiazoly tetrazolium （MTT） assay was used to study effect of CK17 on various oxidants induced injury in ARPE-19 and HUVEC /n vitro RESULTS： Four weeks after NalOs injection, the c- wave amplitude of ERG was 0.393±0.02 V in the control group, 0.184±0.018 V in NalOs group and 0.3±0.01 V in CK17＋NalOs group. There was a significant reversal of the c-wave by CK17 as compared to NalOs group （P〈0.01）. Four weeks after laser, the size of the CNV lesion was 2.57±0.27 mm2 in control group and 1.64 ±0.08 mm2 in CK17 group. The lesion size significantly diminished in CK17 group （P〈0.01）. The inn vitro results showed CK17 also reversed the various oxidants induced injuries in ARPE-19 at the dose of 100 μg/mL and enhanced the injury in HUVECs at different concentrations. CONCLUSION： CK17 can significantly protect RPE from NalOs induced degeneration in vivo and /n vito and also could reverse the various oxidants induced injuries in vitro. It inhibits the development of CNV in rat model, interfered with vascular endothelial cell proliferation in ivtro.

MicroRNA signature and function in retinal neovascularization

Ischemic retinopathies are clinically well-defined chronic microvascular complications characterized by gradually progressive alterations in the retinal microvasculature and a compensatory aberrant neovascularization of the eye. The subsequent metabolic deficiencies result in structural and functional alterations in the retina which is highly susceptible to injurious stimuli such as diabe-tes, trauma, hyperoxia, inflammation, aging and dys-plipidemia. Emerging evidence indicates that an effec-tive therapy may require targeting multiple components of the angiogenic pathway. Conceptually, mircoRNA(miRNA)-based therapy provides the rationale basis for an effective antiangiogenic treatment. miRNAs are an evolutionarily conserved family of short RNAs, each regulating the expression of multiple protein-coding genes. The activity of specific miRNAs is important for vascular cell signaling and blood vessel formation and function. Recently, important progress has been made in mapping the miRNA-gene target network andmiRNA-mediated gene expression control. Here wehighlight the latest findings on angiogenic and antian-giogenic miRNAs and their targets as well as potentiaimplications in ocular neovascular diseases. Emphasis isplaced on how specific vascular-enriched miRNAs regu-late cell responses to various cues by targeting severafactors, receptors and/or signaling molecules in orderto maintain either vascular function or dysfunction. Fur-ther improvement of our knowledge in not only miRNAspecificity, turnover, and transport but also how miRNAsequences and functions can be altered will enhancethe therapeutic utility of such molecules.

Erythropoietin receptor antibody inhibits oxidative stress induced retinal neovascularization in mice

AIM: To observe the effect of erythropoietin receptor antibody (EpoRA) on oxygen-induced retinal neovascularization. METHODS: C57BL / 63 mice, newly born 7 days, were exposed in high oxygen for 5 days and then placed in normal air for another 5 days, thus the animal models of retinal neovascularization were made. Experimental animals were allocated into 3 groups: normal, experimental and therapeutic. The normal group was fed in the normal environment. Into the vitreous cavity of mice in the therapeutic group were injected 2 mu L of EpoRA for 5 successive days. And the experimental group was injected the same amount of normal saline. Mice were sacrificed 17 days after birth and their eyeballs were removed for detection of malonaldehyde (MDA) content in the retina and by HE staining endothelial cells were counted the breaking through internal limiting membrane. RESULTS: In the experimental group, MDA content in the retina was 25.11 +/- 3.46 mu mol/g, which was obviously less than those in the normal group (5.34 +/- 1.79 mu mol/g, P<0.01) and those in the therapeutic group (12.04 +/- 1.91 mu mol/g). Pathological sections showed the nuclear number of the endothelial cells breaking through internal limiting membrane was 0.7 +/- 0.2 in normal group, and 46.2 +/- 6.5 in high oxygen induced experimental group. In the therapeutic group injected with EpoRA, it was lowered to 24.0 +/- 5.0(P < 0.01). CONCLUSION: EpoRA can effectively inhibit oxygen-induced neovascularization in retina of mouse by reducing oxidative damage.

Bevacizumab treatment reduces retinal neovascularization in a mouse model of retinopathy of prematurity

·AIM: To evaluate the effect of different bevacizumab concentrations on retinal neovascularization in a retinopathy of prematurity(ROP) mouse model.·METHODS: A total of 60 of C57BL/6 J mice were exposed to 75% ±2% oxygen from postnatal d7 to postnatal d12. Fifteen nonexposed mice served as negative controls(group A). On d12, 30 mice(group C)were injected with 2.5 μg intravitreal bevacizumab(IVB),30 mice(group D) were injected with 1.25 μg IVB in one eye. The contralateral eyes were injected with balanced salt solution(BSS)(control group =group B). The adenosine diphosphatase(ADPase) histochemical technique was used for retinal flat mount to assess the oxygen-induced changes of retinal vessels.Neovascularization was quantified by counting the endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina. Histological changes were examined by light microscopy. The mRNA levels of vascular endothelial growth factor(VEGF) were quantified by Real-time PCR. Western-blotting analysis was performed to examine the expression of P-VEGFR.· RESULTS: Comparing with the control group B,regular distributions and reduced tortuosity of vessels were observed in our retinal flat mounts in groups C and D. The endothelial cell count per histological section was lower in groups C(P 【0.0001) and D(P 【0.0001) compared with the control group B. Histological evaluation showed no retinal toxicity in any group. In all oxygen treated groups VEGF mRNA expression was significantly increased as compared to age-matched controls. No significant change in VEGF mRNA expression could be achieved in either of the treatments or the oxygen controls. The results of the Western blot were consistent with that of the Real-time PCR analysis.·CONCLUSION: An intravitreal injection of bevacizumab is able to reduce angioproliferative retinopathy in a mouse model for oxygen-induced retinopathy.

Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model:an in-vivo study

AIM:To explore whether the retinal neovascularization(NV)in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa(RP)mouse,which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic.METHODS:The Vldlr^(-/-)mice,the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene,with the rd1 mice,the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred.Intercrossing of the above two mice led to the birth of the F1 hybrids,further inbreeding of which gave birth to the F2 offspring.The ocular genotypes and phenotypes of the mice from all generations were examined,with the F2 offspring grouped according to the genotypes.RESULTS:The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function.The Vldlr^(-/-)mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography.The F1 hydrides,with the heterozygote genotype,exhibited no phenotypes of RP or retinal NV.The F2 offspring with homozygous genotypes were grouped into four subgroups.They were the F2-Ⅰmice with the wild-type Pde6b and Vldlr genes(Pde6b~(+/+)-Vldlr~(+/+)),which had normal ocular phenotypes;the F2-Ⅱmice with homozygous mutant Vldlr gene(Pde6b~(+/+)-Vldlr^(-/-)),which exhibited the retinal NV phenotype;the F2-Ⅲmice with homozygous mutant Pde6b gene(Pde6b^(-/-)-Vldlr~(+/+)),which exhibited the RP phenotype.Specifically,the F2-Ⅳmice with homozygous mutant Vldlr and Pde6b gene(Pde6b^(-/-)-Vldlr^(-/-))showed only the RP phenotype,without the signs of retinal NV.CONCLUSION:The retinal NV can be inhibited by the RP phenotype,which implies the role of a hyperoxic state in treating retinal NV diseases.

Characterization and validation of a chronic retinal neovascularization rabbit model by evaluating the efficacy of anti-angiogenic and anti-inflammatory drugs

AIM:To establish a rabbit model with chronic condition of retinal neovascularization(RNV)induced by intravitreal(IVT)injection of DL-2-aminoadipic acid(DL-AAA),a retinal glial(Mül er)cell toxin,extensive characterization of DL-AAA induced angiographic features and the suitability of the model to evaluate anti-angiogenic and anti-inflammatory therapies for ocular vascular diseases.METHODS:DL-AAA(80 mmol/L)was administered IVT into both eyes of Dutch Belted rabbit.Post DL-AAA delivery,clinical ophthalmic examinations were performed weekly following modified Mc Donald-Shadduck Scoring System.Color fundus photography,fluorescein angiography(FA),and optical coherence tomography(OCT)procedures were performed every 2 or 4 wk until stable retinal vascular leakage was observed.Once stable retinal leakage(12 wk post DL-AAA administration)was established,anti-vascular endothelial growth factor(VEGF)(bevacizumab,ranibizumab and aflibercept)and anti-inflammatory(triamcinolone,TAA)drugs were tested for their efficacy after IVT administration.Fluorescein angiograms were scored before and after treatment following a novel grading system,developed for the DL-AAA rabbit model.RESULTS:Post DL-AAA administration,eyes were presented with moderate to severe retinal/choroidal inflammation which was accompanied by intense vitreous flare and presence of inflammatory cells in the vitreous humor.Retinal hemorrhage was restricted to the tips of neo-retinal vessels.FA revealed maximum retinal vascular leakage at 2 wk after DL-AAA injection and then persisted as evidenced by stable mean FA scores in weeks 8 and 12.Retinal vascular angiographic and tomographic features were stable and consistent up to 36 mo among two different staggers induced for RNV at two different occasions.Day 7,mean FA scores showed that 1μg/eye of bevacizumab,ranibizumab,aflibercept and 2μg/eye of TAA suppress 65%,90%,100%and 50%retinal vascular leakage,respectively.Day 30,bevacizumab and TAA continued to show 66%and 44%suppression while ranibizumab effect was becoming less effective(68%).In contrast,aflibercept was still able to fully(100%)suppress vascular leakage on day 30.On day 60,bevacizumab,ranibizumab and TAA showed suppression of 7%,12%,and 9%retinal vascular leakage,respectively,however,aflibercept continued to be more effective showing 50%suppression of vascular leakage.CONCLUSION:The DL-AAA rabbit model mimics RNV angiographic features like RNV and chronic retinal leakage.Based on these features the DL-AAA rabbit model provides an invaluable tool that could be used to test the therapeutic efficacy and duration of action of novel anti-angiogenic formulations,alone or in combination with anti-inflammatory compounds.

Apolipoprotein A1 suppresses the hypoxia-induced angiogenesis of human retinal endothelial cells by targeting PlGF

AIM:To investigate the anti-angiogenic effect of apolipoprotein A1(apoA1)on primary human retinal vascular endothelial cells(HRECs)and explore the possible mechanism.METHODS:The primary HRECs were transfected with apoA1-GFP recombinant lentiviral and were compared with cells undergoing transfection with empty lentiviral vectors.Hypoxia chambers were used to simulate the anoxic environment of cells under pathological condition.The concentrations of secreted vascular endothelial growth factor(VEGF)and placental growth factor(PlGF)were measured by enzyme-linked immunosorbent assay(ELISA).Cell migration ability was detected by wound healing assay.The sprouting of HRECs was determined by tube formation assay.The protein levels of extracellular signal regulated kinase 1/2(ERK1/2)and phosphor ylated ERK1/2(p-ERK1/2)were measured by Western blot.RESULTS:Overexpressed apoA1 in hypoxia-induced HRECs significantly suppressed PlGF(0.67±0.10 folds,P=0.007).Overexpressed apoA1 also attenuated hypoxiainduced cell migration(0.32±0.11 folds,P<0.0001),tube formation(0.66±0.01 folds,P<0.0001)and the phosphorylation levels of ERK(0.6±0.11 folds,P=0.025).Pretreatment of mitogen-activated protein kinase kinase(MEK)inhibitor(U0126)further reduced the PlGF and angiogenesis in hypoxia-induced HRECs.CONCLUSION:ApoA 1 inhibits the angiogenesis at least in part by inactivating ERK1/2 in hypoxia-induced HRECs.Moreover,apoA1 suppresses the PlGF expression,which selectively associated with pathological angiogenesis.

Surgical treatment for neovascularized retinal pigment epithelial detachment in age -related macular degeneration

Dear Sir, I am Dr. Hui Li, from the Department of Ophthalmology of Shanghai Tenth People’s Hospital Affiliated to Tongji University in Shanghai, China. I write to report a case of neovascularized pigment epithelial detachment (PED) successfully treated with vitrectomy. PED associated occult choroidal neovascular membrane, so called vascularized PED [1] , is a special subtype of neovascular age-related macular degeneration with poor

pH-Responsive polymer boosts cytosolic siRNA release for retinal neovascularization therapy

Small interfering RNA(siRNA)has a promising future in the treatment of ocular diseases due to its high efficiency,specificity,and low toxicity in inhibiting the expression of target genes and proteins.However,due to the unique anatomical structure of the eye and various barriers,delivering nucleic acids to the retina remains a significant challenge.In this study,we rationally design PACD,an A-B-C type non-viral vector copolymer composed of a hydrophilic PEG block(A),a siRNA binding block(B)and a pH-responsive block(C).PACDs can self-assemble into nanosized polymeric micelles that compact siRNAs into polyplexes through simple mixing.By evaluating its pH-responsive activity,gene silencing efficiency in retinal cells,intraocular distribution,and anti-angiogenesis therapy in a mouse model of hypoxia-induced angiogenesis,we demonstrate the efficiency and safety of PACD in delivering siRNA in the retina.We are surprised to discover that,the PACD/siRNA polyplexes exhibit remarkable intracellular endosomal escape efficiency,excellent gene silencing,and inhibit retinal angiogenesis.Our study provides design guidance for developing efficient nonviral ocular nucleic acid delivery systems.

Blueberry anthocyanins extract attenuates oxidative stress and angiogenesis on an in vitro high glucose-induced retinopathy model through the miR-33/GLCCI1 axis

Background:Diabetes retinopathy(DR)is a complication of diabetes that affects patients’vision.Previous studies have found blueberry anthocyanins extract(BAE)can inhibit the progression of DR,but its mechanism is not completely clear.Methods:To study the role of BAE in diabetes retinopathy,we treated human retinal endothelial cells(HRCECs)with 30 mM high glucose to simulate the microenvironment of diabetes retinopathy and used BAE to intervene the in vitro high glucose-induced retinopathy model.HRCEC cell viability and apoptosis rates were examined by Cell Counting Kit 8(CCK-8)assay and flow cytometry assay.The binding sites between miR-33 and glucocorticoid-induced transcript 1(GLCCI1)were assessed by luciferase reporter assay.Retinal neovascularization and oxidative stress contribute to diabetic retinopathy.The tubule formation assay was applied to detect the retinal neovascularization.The oxidative stress in the HRCECs was manifested by the reactive oxygen species(ROS)level,the malondialdehyde(MDA)level,and the superoxide dismutase(SOD)activity.Results:Compared with HRCECs cells cultured under normal conditions,high glucose(HG)can induce oxidative stress in HRCRCs,specifically manifested in the increase of ROS and MDA levels,and the decrease of SOD activity.BAE relieved the tubule formation in n the HRCEC.BAE also relieved the ROS and MDA levels and increased the SOD activity.Luciferase reporter assay revealed that GLCCI1 is a target molecule downstream of miR-33.In HRCEC,BAE significantly inhibited the expression of miR-33 induced by HG.miR-33 mimic inhibited the BAE’s effects on oxidative stress and angiogenesis in an in vitro high glucose-induced retinopathy model.Conclusion:BAE alleviated the oxidative stress and microangiogenesis of HRCEC by regulating the miR-33/GLCCI1 axis.

Homocysteine mediates transcriptional changes of the inflammatory pathway signature genes in human retinal pigment epithelial cells

AIM：To test whether homocysteine（Hcy） can influence the transcriptional profile, we hypothesized that Hcy can lead to the induction of proinflammatory molecules in the retinal cells of aging people.METHODS：An unbiased in vitro inflammatory pathway focused study was designed employing retinal pigment epithelial（RPE） cell line, ARPE-19. Cells were cultured in the presence or absence of Hcy to capture target genes＇ expression profile. Three different concentrations of Hcy were added in the culture medium of confluent monolayers. c RNAs were made from the isolated total RNAs and the labeled c RNA probes were hybridized to microarrays specific for human disease pathway inflammatory cytokines, chemokines and their receptor gene micro-array panels as per manufacture＇s recommendations. Two Hcy up-regulated molecules：IL6 and CEBPB were further validated via Western blot analysis. Hcy＇s effect on ARPE-19 cellular morphology and genomic DNA integrity were also evaluated.RESULTS：Gene microarray analyses of RPE cells in response to Hcy treatment revealed alterations in the expressions of several inflammatory gene transcripts such as CCL5, CEBPB, IL13RA2, IL15 RA, IL6, IL8 and CXCL3 that were up-regulated. The transcripts for C3, CCL2, IL11 RA and IL18 genes exhibited down-regulation. The IL6 and CEBPB expressions were subsequently validated at the protein levels. Treatment of the retinal cells with increasing Hcy concentration influenced their density in culture however their morphology and DNA integrity remained unaffected. CONCLUSION：These findings suggest that Hcy can potentially mediate the expression of chemokines,cytokines and interleukins receptors in the retinal cells without having any debilitating effects on their morphology and the genomic DNA integrity.

Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro

AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL63 mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer. Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-LS-LOX-for Ad-vectorvia Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR(2) could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.