INTRODUCTION

Pathological angiogenes is in the eye often leads to serious consequences, including intractable high intraocular pressure, visual impairments, and even irreversible blindness.Choroidal neovascu larization (CNV) is a common and devastating disease that causes irreversible vision loss and has emerged as the leading cause of blindness among people aged ≥50y[1].CNV is characterized by the formation of new blood vessels in the choroid that break through the retinal pigment epithelium(RPE) and Bruch’s membrane and grow into the subretinal space, ultimately causing exudative or hemorrhagic retinal detachment[2]. The pathogenesis of CNV involves various genetic or environmental factors and is associated with angiogenesis and vasculogenesis. However, the mechanisms of neovascularization are not completely understood.

接着,Alice 和Bob协作执行一个以粒子4为受控粒子、粒子3为目标粒子的受控非门运算,于是等式(5)变为

Chemokine receptors are a superfamily of G-protein-coupled cell surface receptors. Interactions between chemokine receptors and specific chemokines induce the directional migration of cells toward a gradient of chemokines[3]. CXCR4,in parallel with its unique ligand stromal cell-derived factor-1(SDF-1), is commonly expressed in CNV and implicated in several types of ocular neovascularization associated with the mobilization, migration and adhesion of endothelial cells(ECs)[4]. However, according t o several studies, CXCR4 does not directly contribute to cell adhesion and migration but is essential for transmitting SDF-1-induced signals[5]. Therefore,other mediators may be involved in SDF-1/CXCR4 signaling to promote EC migration and adhesion.

当地创新文化、历史文化、农耕文化以及山水文化等具有渗透功能，并向农业产前产后产中蔓延与融合。农民工返乡创业集群创业在促进农村一二三产业融合发展中，具备城市打工获得人力资本优势、资金优势以及家乡的资源人脉优势等，能够利用文化渗透发展成具有特色的创意、观光、品牌等农业、乡村生态休闲、旅游观光、文化教育等产业，从而实现传统农村产业向现代产业转型与升级。

Integrins are heterodimeric receptors containing α and β subunits that function as cell adhesion molecules. Integrin α5β1 is absent or expressed at low levels in healthy ECs but is enriched during embryogenesis, tissue repair, and neovascularization[6]. In the present study, we will elaborate the roles of α5β1 in SDF-1/CXCR4-induced murine CNV and SDF-1/CXCR4-mediated EC migration and tube formation in vitro.

MATERIALS AND METHODS

In Vitro Tube Formation Assay Matrigel Basement Membrane Matrix (BD Biosciences) was thawed overnight at 4℃, diluted with medium, and used to coat the wells of 48-well plates at 37℃ for 40min. RF/6A ECs (2×105) were seeded onto the gel in 400 μL of medium and incubated at 37℃ in a 5% CO2 atmosphere for 26h. The length of the tubes was measured using IPP software, and the results are expressed as the mean fold change in tube length compared with the length of the control group. For each experiment, cells were seeded into at least three wells per condition, and each experiment was repeated three times.

RNA Interference RF/6A cells were seeded at a density of 1.0×104 cells/well in six-well plates and grown to 70%-80%con fl uence before transfection. Transfections of small interfering RNAs (siRNAs) or negative controls were performed using Lipofectamine 2000 from Invitrogen (Carlsbad, CA, USA),according to the manufacturer’s instructions. The sequences of the synthesized siRNAs were as follows: CXCR4 (CXCR4-612: sense 5’-3’GGGUGGUUGUGUUCCAGUUTT, antisense 5’-3’AACUGGAACACAACCACCCTT; CXCR4-1083:sense 5’-3’GCCUCAAGAUCCUUUCCAATT, antisense 5’-3’UUGGAAAGGAUCUUGAGGCTT; CXCR4-163:sense 5’-3’CCAUGAAGGAACCCUGUUUTT, antisense 5’-3’AAACAGGGUUCCUUCAUGGTT); and negative control siRNA (sense 5’-3’UUCUCCGAACGUGUCACGUTT,antisense 5’-3’ACGUGACACGUUCGGAGAATT). For all experiments, the cells were transfected with siRNAs 48h before further treatments.

Cell Culture Rhesus macaque choroid-retinal ECs (RF/6A)and RPE cells were obtained from the Chinese Academy of Science (Shanghai, China) and routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen,CA, USA) containing 15% fetal bovine serum (FBS; Invitrogen),100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37℃. ECs were exposed to atmosphere comprising a mixture of 5% CO2, 1% O2 and 94%N2 at 37℃ to induce hypoxia. The same final concentrations of SDF-1, AMD3100 and ATN161 used in the animal study were also used in the cell-based experiments.

Antibodies We used the following antibodies against αv/β3/β5/α5/β1: rabbit monoclonal antibodies (used for Western blot double labeling) against αv (Abcam, Cambridge,UK, 179475), β3 (Abcam, 119992), β5 (Cell Signaling Technology, MA, USA, D24A5), α5 (Abcam, 150361), and β1 (Abcam, 179471). We also used a goat polyclonal antibody against CXCR4 (Abcam, 1670). The following rat monoclonal antibodies were used for double immuno fluorescence staining:α5 (Abcam, 150361), CXCR4 (Abcam, 1670), and CD31(Abcam, 28364). An α5 antibody (Abcam, 150361) was used for flow cytometry. Antibodies against β-actin were obtained from Santa Cruz Biotechnology (CA, USA). Recombinant human secondary antibodies, including goat anti-rabbit conjugated to Alexa Fluor 594/CY3 or Alexa Fluor 488/FITC,were purchased from Beijing ComWin (Beijing, China).

Hematoxylin Eosin Staining The histopathological analysis of CNV severity in animals exposed to various treatments was performed using previously reported methods (3 eyes in each group, 4-6 spots per eye)[7]. Anesthetized mice were transcardially perfused with 0.9% saline followed by 4%paraformaldehyde. Paraffin-embedded tissues were serially sectioned at 3 mm and stained with hematoxylin eosin (HE).Serial sections of each CNV sample were examined and digital images were captured under a light microscope (BX51,Olympus Corporation, Tokyo, Japan). The CNV thickness was determined in vertical sections by measuring the distance from the adjacent RPE layer to the top of the CNV. The CNV length was identified as the maximum horizontal distance of the CNV lesion using Image-Pro Plus (IPP) 6.0 software.

Optical Coherence Tomography Optical coherence tomography[8] (OCT) “TruTrack TM Active Eye Tracking” and“Automatic Real Time (ART)” technologies were used, and follow-up images were derived from the same lesion, which eliminated the subjective placement of the scan by the operator.The CNV thickness was quantified using software provided by OptoProbe Research Ltd. (Burnaby, BC, Canada). Four to six spots from lesions in each eye were selected for followup and analysis. A CNV lesion was defined by the presence of spindle-shaped, subretinal, hyperreflective material above the RPE layer. The B-scan passing through the “geometric” lesion center was chosen for analysis to evaluate the thickness of each tracked CNV lesion.

鸭苗放养。鸭苗放养应分批次，时间间隔为一个月左右，第一批鸭苗放养时间可选择子啊鱼种投放10天后。在第一批鸭苗完成饲养出栏后，就可以直接进行第二批鸭苗投放。在鸭苗30日龄时，结合鸭生长实际，有意识的进行鸭群条件反射训练，一般在2—3周后，训练即可完成，此时鸭群成功形成条件反射，有效提升了自身的捕食能力和自我防护能力，然后就可以进行自然生态饲养。

Fundus Fluorescein Angiography Fundus fluorescein angiography (FFA) images were captured under a confocal scanning laser ophthalmoscope (OptoProbe Research Ltd.,Burnaby, BC, Canada), immediately followed by OCT.Briefly, the three groups of mice were evaluated on the 7th day after CNV induction; the mice were anesthetized and intraperitoneally injected with 0.1 mL of 2.5% sodium fluorescein(Wuzhou Pharmaceutical, Guangxi, China). FFA images were recorded using a digital imaging system (Heidelberg Engineering). A blinded assessment of fluorescein angiogram images was conducted by two retinal specialists. Fluorescein leakage intensity was graded as follows: 0, no leakage; 1,slight leakage; 2, moderate leakage; 3, prominent leakage.

Choroidal Flatmount Seven days after CNV induction, mice were anesthetized with sodium pentobarbital and perfused with PBS through the left cardiac ventricle, followed by perfusion with 4% paraformaldehyde. After sacrifice, the eyes of each animal were enucleated and fixed with chilled 4%paraformaldehyde in 0.1 mol/L phosphate buffer (PB) for 2h.Next, the anterior segment and vitreous were resected; the remaining RPE-choroid-sclera complex was flatmounted with six or more radial cuts and permeabilized with 0.2% Triton X-100 for 24h. Afterwards, the flatmounts were transferred and cultured in rhodamine-conjugated Ricinus communis agglutinin(1:1000; Vector Laboratories, Burlingame, CA, USA) for 24h. The flatmounts were then washed with 0.01 mol/L Tris buffered saline-Tween (TBST) for 24h, incubated with anti-α5 or CXCR4 antibodies at 4℃ for 18 to 20h, washed with PBS,stained with lectin (1:100) overnight at 4℃ overnight, and washed with PBS three times after mounting. Images of the flatmounts were captured using a confocal laser scanning microscope (FV1000, Olympus Corporation, Tokyo, Japan),and the CNV area was calculated using IPP 6.0 software.

Immunofluorescence Staining Seven days after treatment,mice were anesthetized with sodium pentobarbital and sequentially perfused with PBS and 4% paraformaldehyde through the left cardiac ventricle. After sacrifice, the eyes of each animal were enucleated and fixed with chilled 4%paraformaldehyde in 0.1 mol/L PB for 2h. Then, the anterior segment and vitreous were resected, and the posterior eyecups were cryoprotected in graded sucrose solutions (20% and 30%in PB). The eyecups were embedded, sectioned vertically at an 8-μm thickness, permeabilized for 10min, blocked for 30min,and incubated with primary antibodies overnight at 4℃. Then,the sections were incubated with combinations of secondary antibodies for 1h and stained with diamino-2-phenyl-indol(DAPI). Immuno fluorescence staining was visualized using a confocal microscope.

Pathological changes observed in CNV lesions involve various cells, cytokines and signaling cascades. Basically, after sensing complex signal inputs from other cells or cytokines,such as vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) or SDF-1, naïve or bone marrowderived cell (BMC)-derived ECs participate in CNV formation via proliferation, differentiation, recruitment or migration.Our study focused on the role and mechanism of SDF-1 in CNV, with the objective of providing evidence for potential treatments for AMD beyond targeting VEGF. VEGF is involved in the early steps of angiogenesis, but treatments designed to inhibit VEGF are successful in ＜50% of patients with neovascular AMD[13-14]. VEGF and SDF-1 were co-localized in RPE cells from surgically excised CNV lesions. VEGF and SDF-1 may also exert synergistic effects on the development of diabetic retinopathy. SDF-1 promotesVEGF production,whereas VEGF enhances the response of ECs to SDF-1[15-16]. Cai et al[15] con firmed that SDF-1 is able and sufficient to promote endothelial chemotaxis to the is chemic retina and participate in retinal neovascularization in laser-induced mice. Therefore,we postulated that the inhibition of SDF-1 signaling may also counteract the effect of VEGF on inducing angiogenesis.Zhang et al[4] reported that hypoxia-specific hypoxia-inducible factor (HIF)-1α also contributed to the induction of SDF-1 in RPE cells, which shared a similar pattern of HIF-1α on the induction of VEGF. We also detected high level of SDF-1 in RPE cells cultured under hypoxic conditions. However, further studies examining the synergistic effects of SDF-1 and VEGF are required.

Laser photocoagulation-induced murine CNV was performed as previously reported[7]. Briefly, the eyes of each animal were exposed to a 532-nm wavelength laser (Twin, Quantel,France) and burns were induced on the fundus 1.5-2 disc diameters from the optic nerve (100 μm, 125 mV and 0.1s).Any laser spots that induced the rupture of Bruch’s membrane(confirmed by a vaporization bubble without hemorrhage)were considered effective and CNV lesions available for examination in the present study. After CNV induction, all animals were assigned to the following groups for in vivo and ex vivo experiments: control, SDF-1 treatment (200 ng/mL;Sino Biological Inc., Beijing, China), SDF-1 (200 ng/mL)+agerelated macular degeneration (AMD) 3100 (200 mmol/L)treatment (Selleckchem, Houston, USA), and SDF-1 (200 ng/mL)+ATN161 (2 mg/mL; TOCRIS, Bristol, UK) treatment. All agents were dissolved in phosphate buffer solution (PBS) at the indicated doses and injected (1 μL) at a single dose into the posterior vitreous cavity; their effects were observed for 3, 7 and 14 consecutive days.

Statistical Analysis Statistical analyses were conducted using SPSS 22.0 software. Data are presented as means±SD.Comparisons between two groups were performed using Student’s t-test. Analysis of variance followed by the least significant difference (LSD) test were used for multiple comparisons. Chi-square tests were used to compare categorical data. Statistically significant differences were indicated when two-tailed P values were less than 0.05.

Quantitative Real-time Polymerase Chain Reaction Total RNA was extracted from choroidal tissue, RF/6A cells or RPE cells using TRIzol reagent (Invitrogen),according to the manufacturer’s instructions. The cDNA templates were prepared using a reverse transcription(RT) system (TaKaRa Dalian, Dalian, China). Polymerase chain reaction (PCR) was performed in triplicate using a kit(SYBR Premix EX Taq; TaKaRa) and an ABI PRISM 7500 Real-time PCR System, with β-actin serving as an internal control. The PCR primers were: SDF-1: sense 5’-3’GATTGTAGCCCGGCTGAAGA and antisense 5’-3’TTCGGGTCAATGCACACTTGT; and CXCR4-monkey-354: sense 5’-3’GCAAGGCAGUCCAUGUCAUTT and antisense 5’-3’AUGACAUGGACUGCCUUGCTT.

Flow Cytometry A fl uorescence-activated cell sorter (FACS)analysis was performed using a FACSCaliburTM flow cytometer(BD Immunocytometry Systems). Data were analyzed using FlowJo vX.0.6 software (FlowJo, LLC, Ashland, OR, USA).

Laser Photocoagulation Induction of CNV in Mice Experimental procedures performed in animals were conducted in accordance with the guidelines of the Animal Ethics Committees of the Fourth Military University and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male C57BL/6 mice (aged 6-8wk) were housed in a barrier facility with ad libitum access to food and water under a 12h light/dark cycle. Sixty male mice were used in this study.

河南是传统的农业大省，农民是这片土地的主角儿，农业是他们赖以生存的经济基础，农耕文化是中原文化的底色。河南人对土地的眷恋，对农业的依赖，绝非北方的游牧民族和商品经济发达的沿海地区的人们所想象到的。林语堂先生在《论“土”气与思想境界之关系》一文中说：“没有到过黄河流域这些北省的人实不足与语‘土’之为何物。他们绝不明白‘土’与人生之重要，关系之密切，他们不知道我们是生于斯，长于斯，食于斯，寝于斯，呼吸于斯，思想感慨尽系焉，诚有不可与须臾离之情景。”[3]

Endothelial Cell Migration Assay All migration assays were assessed in a 24-well modified Boyden chamber (Corning,NY, USA) in which the two compartments were separated by a polycarbonate filter with a pore size of 8 mm. Briefly,200 μL of RF/6A cells in serum-free medium (1×104 cells/mL)were seeded in the upper chamber, and negative control cells suspended in the same medium were added to the bottom chamber to a final volume of 500 μL. The chambers were incubated at 37℃ in a 5% CO2 atmosphere for 24h. Migrated cells were fixed with 4% paraformaldehyde for 10min and stained with 1% crystal violet in methanol. The number of migrated cells was counted under a fluorescence microscope.Five randomly chosen fields from each insert were counted,and values were averaged. Each experiment was performed in triplicate.

Enzyme-linked Immunosorbent Assay Cells were incubated under hypoxic conditions, and media were collected at the indicated times. The level of the SDF-1 protein secreted by RPE cells into the culture medium was measured using an enzyme-linked immunosorbent assay (ELISA) kit (JingMei Biotech, Shenzhen, China) according to the manufacturer’s instructions. Representative results were obtained from three independent experiments and are expressed in pg/mL.

RESULTS

Expression of SDF-1/CXCR4 and Integrin Subunits in CNV Tissues and Cells C ult ured Under Hypoxic Conditions We established hypoxia-exposed RPE cells or ECs in vitro and in mice with laser-induced CNV to evaluate whether SDF-1/CXCR4 and integrins participated in CNV. SDF-1 expression gradually increased over time in the RPE cells cultured under hypoxic conditions and peaked after 24h of hypoxia. The expression of the SDF-1 mRNA peaked after 6h of hypoxia(Figure 1A). Additionally, we evaluated the expression of CXCR4 and integrin subunits in Rhesus macaque choroidretinal ECs (RF/6A) during hypoxia. Levels of the CXCR4 protein increased over time in ECs cultured under hypoxic conditions and peaked after 24h of hypoxia. The expression of the CXCR4 mRNA initially increased and peaked at 12h,but then decreased during the observation (Figure 1B). Next,we measured the expression of various integrin subunits after 0, 6, 12 and 24h of hypoxia. No significant differences in integrin αv and β1 expression were observed during hypoxia.Integrin β3, β5, and α5 expression initially increased and then decreased; the highest expression and greatest changes in the levels of these proteins were observed after 6h of hypoxia(Figure 1C).

We also determined the expression of integrins and CXCR4 in mice with laser-induced CNV after 3, 7 and 14d using Western blotting. No significant differences in integrin αv and β1 expression were observed in CNV mice. Integrin β3,β5 and α5 expression appeared to increase and then decrease during the progression of CNV; their expression levels were highest exhibited marked changes after 7d. CXCR4 expression increased over the course of CNV and the highest levels were observed 14d after CNV induction (Figure 2). Integrin β3, β5 and α5 and CXCR4 expression showed an increasing trend in response to hypoxia and CNV induction. In the present study,we focused on the role of integrin α5β1 in CNV.

SDF-1 Increases the Severity of CNV in Laser-induced Mice via the CXCR4 Receptor In the vertical view of cross sections, neither SDF-1 stimulation nor subsequent CXCR4 inhibition significantly affected the CNV thickness in laserinsulted animals on the indicated days, as determined by HE staining (Figure 3A-3C). The CNV length gradually increased and peaked at 7d after CNV in each group. At each time point, a significant increase in the CNV length was detected after SDF-1 treatment compared with that in the control treatment. The CNV length was substantially decreased after CXCR4 blockade using AMD3100. The changes in the laserinduced CNV in vivo were also examined using OCT at 3, 7 and 14d in each group. OCT images revealed that the CNV lesions exhibited a hyperreflective outline and hyporeflective cavity. Following laser exposure, a subretinal, spindle-shaped hyperreflective area was observed. The outline of the laserinduced lesion was less re flective in OCT images captured on day 3, reached its maximum size on day 7, and was blurred on day 14. Notably, on day 7, the maximum height of the RPE layer was observed, and the thickness of the RPE layer in the SDF-1 group was substantially increased compared with that in control animals. CXCR4 inhibition substantially reduced the SDF-1-induced increase in the thickness of the RPE layer in OCT images (Figure 3D-3E).

日本监管体系的独特性十分明显，相关的监管机构需要对一些进口食品的安全性进行监管，而其他的安全监管工作需要地方性的监管机构负责，这和日本食品的产业结构存在很大联系。日本农产品的自给率非常低，食品监管成为重点，所以中国在进行食品监管时，可以将日本的工作经验作为参考。对中日两国的企业进行比对分析发现，日本企业更重视自身职责，很多企业在生产与加工中都能达到严格规范，特别是在原料加工和出厂方面，整个过程都更严格。

Figure 1 Pro files of SDF-1/CXCR4 and integrin expression in hypoxia-exposed RPE or RF/6A cells A: ELISA and qRT-PCR analysis of SDF-1 levels in hypoxia-stimulated RPE cells at the indicated time points; B: Western blot and qRT-PCR analyses of CXCR4 expression in hypoxia-exposed RF/6A cells; C: Western blot and quantitative analyses (D) of the levels of integrin subunits and CXCR4 in RF/6A cells cultured under hypoxic conditions for the indicated times. aP＜0.01; bP＜0.05; NS: P＞0.05 compared with the control group.

Figure 2 Pro files of SDF-1/CXCR4 and integrin expression in mice with laser-induced CNV Western blot and quantitative analyses of the levels of integrin subunits and CXCR4 in mice with laser-induced CNV at the indicated times. n=3, aP＜0.01; bP＜0.05; NS: P＞0.05 compared with the control group.

CNV activity evaluations were performed using FFA. The laser spots exhibited a large hyper fluorescent core surrounded by a lace-like boundary on day 7 in control mice. Compared with CNV lesions in the control group, CNV lesions in the SDF-1-treated group showed obvious fluorescence leakage with a large and diffuse area, as well as a higher ratio of grades 2 and 3 CNV leakage. However, no difference in fluorescence leakage was observed between the SDF-1 group and SDF-1+AMD3100 group (Figure 3F-3G). Flatmount examinations revealed a larger CNV area and volume in the SDF-1-stimulated group than in the control group. The addition of AMD3100 significantly suppressed the increase in CNV area and volume induced by SDF-1 (Figure 3H-3J).

万洋山序列岩体稀土元素特征为∑REE为122.27×10-6～289.34×10-6，平均值为203.19×10-6，∑Ce/∑Y比值为1.90～5.20，平均值为3.34，δEu为0.28～0.72，平均值为0.41，反映出区内岩体稀土总量较高，但为富轻稀土型。

Figure 3 SDF-1/CXCR4 signaling promotes laser-induced CNV in mice CNV was induced in animals using laser photocoagulation,followed by SDF-1 and SDF-1+AMD3100 injections on the indicated days. A-C: HE staining of CNV lesions and quantification of the CNV thickness and length in cross sections from the three groups. The margins of the CNV lesion are outlined by solid red lines. Scale bar, 50 μm; D-E:Representative OCT images and results of the analysis of CNV thickness using OCT in laser-treated mice; FFA imaging (F) and CNV grading(G) of the three groups on day 7. CNV lesions are circled by dashed black lines (the line represents the median of each group); H: Representative CNV areas in choroidal flatmounts from the indicated groups. Blood vessels in choroidal flatmounts were stained with rhodamine-conjugated agglutinin. A CNV lesion (dashed line) formed around the optic disc (solid line). Scale bar, 100 μm. Magnified image of a single CNV lesion.Scale bar, 50 μm; I-J: CNV areas and volumes were quantified in laser-induced mice. n=6; aP＜0.01, bP＜0.05.

SDF-1 Increases CXCR4 and α5 Expression in CNV Mice We confirmed that the SDF-1 group exhibited increased α5 and CXCR4 expression in the CNV region. The α5- and CXCR4-positive areas of the CNV lesions were significantly increased on day 7 after SDF-1 treatment compared with those in the control treatment, paralleling the increase in α5/CNV and CXCR4/CNV area ratios. However, CXCR4 blockade using AMD3100 exerted a significant inhibitory effect on α5/CXCR4-positive areas, as well as α5/CNV and CXCR4/CNV area ratios (Figure 4).

Additionally, we observed the effect of SDF-1 on α5 and CXCR4 expression in ECs using double-immuno fluorescence staining with a CD31 antibody. The number of α5- and CXCR4-positive ECs was counted in the laser spots of CNV cross sections. The numbers of α5- or CXCR4-positive cells and α5/CNV- or CXCR4/CNV-labeled cells in the SDF-1 group were significantly increased, but were substantially decreased after CXCR4 inhibition (Figure 5).

这世上的人有很多爱，一如这世上的人有很多恨，都似乎只是沉浸在自己的世界中，像波斯猫在明前茶的玻璃杯前照镜子一样，像我们固执地认为远方有一个让自己拿不起也放不下的人一样。

SDF-1/CXCR4 Upregulates Integrin α5 Expression in Choroidal Endothelial Cells We silenced CXCR4 using siRNAs to determine whether integrin α5 expression was controlled by SDF-1/CXCR4 signaling. We transfected RF/6A cells with a combination of three different siRNA sequences(each at a 1/3 dose) to achieve optimal silencing efficiency(Figure 6A). The expression of integrin α5 in the SDF-1 group was significantly elevatedcompared with that in the control(P＜0.05). However, pharmacological or genetic CXCR4 inhibition significantly suppressed the level of integrin α5 observed in response to SDF-1 stimulation (Figure 6B). In addition, the percentage of α5 integrin-positive cells after each treatment was consistent with the results obtained using Western blotting (Figure 6C).

Integrin α5 Mediates SDF-1/CXCR4-induced EC Migration and Tube Formation RF/6A cells gradually formed tubular structures after treatment with SDF-1. Notably, compared with the control, SDF-1 significantly increased lumen formation at 20h (Figure 7A). A decrease in lumen formation was observed after CXCR4 inhibition or α5 integrin inhibition using ATN161, despite the presence of SDF-1. Moreover,the addition of CXCR4 functional inhibitors and specific siRNAs, as well as ATN161, inhibited retinal EC angiogenesis(Figure 7B). A markedly greater number of migrated cells was observed in the SDF-1 group than in the control group (P＜0.05).After treatment with SDF-1+AMD3100, SDF-1+si-CXCR4 and SDF-1+ATN161, a marked decrease in the number of migrated cells was observed. Furthermore, the injection of ATN161 in mice greatly reduced the CNV length and leakage areas induced by SDF-1 (Figure 7C-7D).

Figure 4 Expression of integrin α5 and CXCR4 in CNV mice CNV was induced in animals using laser photocoagulation, followed by SDF-1 and SDF-1+AMD3100 injections on day 7. Representative images of choroidal flatmounts (A, C) and quanti fi cation of the α5- and CXCR4-expressing areas and ratios in CNV lesions (B, D) after laser induction. CNV lesions (dashed line) formed around the optic disc (solid line). Scale bar, 100 μm. n=6; aP＜0.01, bP＜0.05.

Figure 5 Expression of integrin α5 and CXCR4 in ECs from CNV mice CNV was induced in animals using laser photocoagulation, followed by SDF-1 and SDF-1+AMD3100 injections on day 7. Representative confocal images of the CNV lesion areas in different groups on day 7 (A, C)and quanti fi cation of α5 and CXCR4 expression relative to CD31 (B, D). Scale bar, 100 μm. n=6; aP＜0.01, bP＜0.05.

Figure 6 Effects of SDF-1/CXCR4 signaling on integrin α5 expression on ECs A: Inhibitory effect of the CXCR4 siRNA on CXCR4 expression in RF/6A cells. aP＜0.01 compared with the si-NC group; bP＜0.01, cP＜0.05 compared with the group transfected with the three siRNA sequences. Integrin α5 expression (B) and flow cytometry analysis of the percentage of α5-positive RF/6A cells (C) after CXCR4 inhibition in the presence of SDF-1, aP＜0.05 compared with the control, bP＜0.05 compared with the SDF-1 group, cP＜0.05 compared with the SDF-1+si-NC group.

Figure 7 Effects of SDF-1/CXCR4 and integrin α5 on EC migration and tube formation A: Tube formation of RF/6A cells in response to SDF-1 induction and CXCR4 inhibition or integrin α5β1 inhibition using ATN161 (2 mg/mL), scale bar, 500 μm; B: Transwell assay of RF/6A cell migration, scale bar, 500 μm; C: HE staining of CNV lesions and quantification of the CNV length in cross sections from the two groups on day 7; D: Fundus fluorescence angiography imaging of the two groups on day 7. n=6; aP＜0.05 compared with the control, bP＜0.05 compared with the SDF-1 group.

DISCUSSION

Choroidal neovascularization represents an important pathological cause of intractable and severe vision loss in approximately 40 ocular diseases; the most common of these diseases is AMD[9]. Neovascularization is induced by several factors,such as ischemia, hypoxia or inflammation[10-11]. Newly constructed vessels are always structurally fragile and prone to hemorrhage, thus leading to edema, exudates and accompanying fibrosis[12]. Therefore, the suppression of neovascularization plays a pivotal role in the treatment of choroidal lesion-related ocular diseases. Inhibition of SDF-1/CXCR4 signaling and subsequent integrin α5β1 expression substantially suppressed CNV lesions in mice, as well as the migration and tube formation of ECs in the present study.Agents targeting this signaling pathway could become an alternative treatment option for CNV.

Western Blot Tissue or cell lysates were prepared in lysis buffer [50 nmol/L Tris-HCl, 150 nmol/L NaCl, 1% Nonidet P-40 (NP40), 0.1% sodium dodecyl sulfate (SDS), 0.5%deoxycholate, and 1 mmol/L phenyl methylsulfonyl fluoride],and protein concentrations were determined using a BCA protein assay kit (Sangon Biotech, Shanghai, China). Equal amounts (20 μg) of protein were electrophoresed on a 10%SDS polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). Primary antibodies incubated with the membranes overnight at 4℃.Membranes were incubated with secondary antibodies for 30min. After three washes with TBST, signals from the protein band were detected with an enhanced chemiluminescence system (Millipore) and standardized to β-actin levels.

In parallel with SDF-1 upregulation, its unique receptor,CXCR4, was elevated in CNV lesions and hypoxia-exposed ECs. The interaction between SDF-1 and CXCR4 regulates the entire process of endothelial mobilization, targeted migration,recruitment and differentiation. SDF-1 also promotes the migration of embryonic stem cell-derived ECs and the formation of the vascular network. The directional migration of ECs shares many similarities with the homing of leukocytes to inflammation sites. As a major chemokine receptor expressed on ECs, CXCR4 induces cell chemotaxis toward a gradient of SDF-1. Furthermore, high levels of SDF-1 have been detected in the retina and choroid, and the SDF-1/CXCR4 axis may function as a “navigation system” for ECs[17-18]. SDF-1/CXCR4 signaling itself is not directly involved in cell adhesion and migration but may be involved by transducing signals to cells via other molecules[19]. Therefore, we intended to evaluate the roles of adhesion molecules in the SDF-1/CXCR4 signaling pathway.

Integrins are cell surface adhesion molecules that not expressed in mature ocular blood vessels but selectively expressed in activated ECs. Levels of integrin subunits α5, β3 and β5,but not αv and β1, were increased in both CNV animals and hypoxia-exposed ECs, and their altered expression patterns

were similar to that of CXCR4. Integrin αvβ3 has been detected in CNV membranes in patients with AMD[20]. Patients with diabetic retinopathy and retinal neovascularization present pre-retinal membrane expression of integrins αvβ3 and αvβ5[20]. According to the study by Reynolds and colleagues[21],β3 or β5 integrin knockout mice still form new blood vessels.Therefore, we focused on the roles of integrin α5 in CNV in our study. Integrin α5 was closely associated with CNV and CXCR4 expression. SDF-1 increased integrin α5 subunit expression and the number of integrin α5-positive ECs in CNV lesions, and these effects were reversed by CXCR4 inhibition. In addition, we observed an inhibitory effect of integrin α5β1 suppression on the migration and tube formation of ECs. Integrin α5 mediates the adhesion of cells to the extracellular matrix by binding to fibronectin, which may facilitate the formation of EC tubes following recruitment by SDF-1/CXCR4 signaling. Our fi ndings provide a link between a chemokine receptor and integrins in CNV pathogenesis.Similarly, the association between SDF-1/CXCR4 and adhesion molecules has been described in renal and prostate cancer cells[22]. However, the potential induction mechanism has not been elucidated and requires further research.

例如，“工业4.0遇上工业互联网”论坛吸引了超过8000名与会者参加。汉诺威工业博览会的外贸平台——“全球商业和市场”展区共吸引了220家参展商和超过5000名观众。“初创技术企业”展区是汉诺威工业博览会的创业中心，包含展览、论坛、推介、采销配对活动和研讨会。

Above all, inhibition of CXCR4 or integrin α5β1 substantially suppressed the migration and tube formation of ECs in this study, ultimately reducing lesion areas and leakage of CNV.

ACKNOWLEDGEMENTS

Foundations: Supported by the National Natural Science Foundation of China (No.81770936; No.81570856; No.81670863;No.81500748; No.81370020).

Conflicts of Interest: Lyu Y, None; Xu WQ, None; Sun LJ,None; Pan XY, None; Zhang J, None; Wang YS, None.

生命的进化并不是一条直线，更不是一个越来越高的梯子。进化并不以人类为终点和目标。各种生物不会进化成为我们，就像我们人类不会变成他们。

1.2.2 供试品溶液的制备。咖啡豆研磨成粉末(过0.45 mm筛)，准确称取咖啡生豆粉末0.5 g(焙炒咖啡粉1.0 g)，置于50 mL容量瓶中，加入50%的甲醇约25 mL，超声提取20 min，将上清液滤入50 mL容量瓶中，滤渣再加入20 mL 50%甲醇，超声提取15 min，合并2次提取液，用50%甲醇定容至50 mL，摇匀，过0.45 μm滤膜，待测。

REFERENCES

1 Gao X, Wang Y, Hou HY, Lyu Y, Wang HY, Yao LB, Zhang J, Cao F, Wan g YS. In vivo bioluminescence imaging of hyperglycemia exacerbating stem cells on choroidal neovascularization in mice. Int J Ophthalmol 2016;9(4):519-527.

2 Yu YJ, Mo B, Liu L, Yue YK, Yue CL, Liu W. Inhibition of choroidal neovascularization by lentivirus-mediated PEDF gene transfer in rats. Int J Ophthalmol 2016;9(8):1112-1120.

3 Zeng Y, Wang X, Yin B, Xia G, Shen Z, Gu W, Wu M. Role of the stro mal cell derived factor-1/CXC chemokine receptor 4 axis in the invasion and metastasis of lung cancer and mechanism. J Thorac Dis 2017;9(12): 4947-4959.

4 Zhang ZX, Wang YS, Shi YY, Hou HY, Zhang C, Cai Y, Dou GR,Yao LB, Li FY. Hypoxia specific SDF-1 expression by retinal pigment epithelium initiates bone marrow-derived cells to participate in choroidal neovascularization in a laser-induced mouse model. Curr Eye Res 2011;36(9):838-849.

5 Feng L, Ju M, Lee KYV, Mackey A, Evangelista M, Iwata D, Adamson P, Lashkari K, Foxton R, Shima D, Ng YS. Aproin flammatory function

of toll-like receptor 2 in the retinal pigment epithelium as a novel target for reducing choroidal neovascularization in age-related macular degeneration. Am J Pathol 2017;187(10):2208-2221.

6 Wang WQ, Wang FH, Qin WX, Liu HY, Lu B, Chung C, Zhu J, Gu Q,Shi W, Wen C, Wu F, Zhang K, Sun XD. Joint antiangiogenic effect of ATN-161 and anti-VEGF antibody in a rat model of early wet age-related macular degeneration. Mol Pharm 2016;13(9):2881-2890.

7 Shi YY, Wang YS, Zhang ZX, Cai Y, Zhou J, Hou HY, van Rooijen N. Monocyte/macrophages promote vasculogenesis in choroidal neovascularization in mice by stimulating SDF-1 expression in RPE cells.Graefes Arch Clin Exp Ophthalmol 2011;249(11):1667-1679.

8 Liu T, Hui L, Wang YS, Guo JQ, Li R, Su JB, Chen JK, Xin XM, Li WH. In-vivo investigation of laser-induced choroidal neovascularization in rat using spectral-domain optical coherence tomography (SD-OCT).Graefes Arch Clin Exp Ophthalmol 2013;251(5):1293-1301.

9 Gao F, Hou H, Liang H, Weinreb RN, Wang H, Wang Y. Bone marrowder ived cells in ocular neovascularization: contribution and mechanisms.Angiogenesis 2016;19(2):107-118.

10 Cabral T, Mello LGM, Lima LH, Polido J, Regatieri CV, Belfort R Jr,Mahajan VB. Retinal and choroidal angiogenesis: a review of new targets.Int J Retina Vitreous 2017;3:31.

11 Zhao M, Xie W, Tsai SH, Hein TW, Rocke BA, Kuo L, Rosa RH Jr.In travitreal stanniocalcin-1 enhances new blood vessel growth in a rat model of laser-induced choroidal neovascularization. Invest Ophthalmol Vis Sci 2018;59(2):1125-1133.

12 Xu D, Davila JP, Rahimi M, Rebhun CB, Alibhai AY, Waheed NK,Sarr af D. Long-term progression of type 1 neovascularization in agerelated macular degeneration using optical coherence tomography angiography. Am J Ophthalmol 2018;187:10-20.

13 Wang H, Fotheringham L, Wittchen ES, Hartnett ME. Rap1 gtpase inhibits tumor necrosis factor-alpha-induced choroidal endothelial migration via NADPH oxidase- and NF-kappab-dependent activation of rac1. Am J Pathol 2015;185(12):3316-3325.

14 Sato T, Takeuchi M, Karasawa Y, Enoki T, Ito M. Intraocular in fl a mmatory cytokines in patients with neovascular age-related macular degeneration before and after initiation of intravitreal injection of anti-VEGF inhibitor. Sci Rep 2018;8(1):1098.

15 Cai Y, Li X, Wang YS, Shi YY, Ye Z, Yang GD, Dou GR, Hou HY, Yang N, Cao XR, Lu ZF. Hyperglycemia promotes vasculogenesis in choroidal neovascularization in diabetic mice by stimulating VEGF and SDF-1 expression in retinal pigment epithelial cells. Exp Eye Res 2014;123:87-96.

16 Grierson R, Meyer-Rusenberg B, Kunst F, Berna MJ, Richard G, Thil l M. Endothelial progenitor cells and plasma vascular endothelial growth factor and stromal cell-derived factor-1 during ranibizumab treatment for neovascular age-related macular degeneration. J Ocul Pharmacol Ther 2013;29(6):530-538.

17 Maddirela DR, Kesanakurti D, Gujrati M, Rao JS. MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting alphavbeta3-mediated SDF-1/CXCR4 signaling. Int J Oncol 2013;42(4):1279-1288.

18 Feng YF, Guo H, Yuan F, Shen MQ. Lipopolysaccharide promotes chor oidal neovascularization by up-regulation of CXCR4 and CXCR7 expression in choroid endothelial cell. PLoS One 2015;10(8):e0136175.

19 Wang B, Wang W, Niu W, Liu E, Liu X, Wang J, Peng C, Liu S, Xu L, Wang L, Niu J. SDF-1/CXCR4 axis promotes directional migration of colorectal cancer cells through upregulation of integrin alphavbeta6.Carcinogenesis 2014;35(2):282-291.

20 Friedlander M, The esfeld CL, Sugita M, Fruttiger M, Thomas MA,Ch ang S, Cheresh DA. Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases. Proc Natl Acad Sci U S A 1996;93(18):9764-9769.

21 Reynolds LE, Wyder L, Lively JC, Taverna D, Robinson SD, Huang X, Sheppard D, Hynes RO, Hodivaladilke KM. Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.Nature Medicine 2002;8(1):27-34.

22 Jones J, Marian D, Weich E, Engl T, Wedel S, Relja B, Jonas D,Bl aheta RA. CXCR4 chemokine receptor engagement modifies integrin dependent adhesion of renal carcinoma cells. Exp Cell Res 2007;313(19):4051-4065.