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

INTRODUCTION

Age-related macular degeneration (AMD), the main cause of permanent blindness in people aged over 60y worldwide, is expected to impart a heavy burden on public health in the next few decades. Exudative (wet) AMD involves fibrovascular proliferation under the central retina (macula),which occurs with characteristic choroidal neovascularization(CNV)

. Clinically, anti-vascular endothelial growth factor(VEGF) agents such as bevacizumab, ranibizumab, and aflibercept, have been used to suppress neovascularization in wet AMD. However, a lack of long-term improvements in vision, secondary inflammation, or other side effects considerably limits the usefulness of these treatments

.Advanced subretinal fibrosis is considered an important reason for the resistance of wet AMD to anti-VEGF drugs

.

Endothelial-to-mesenchymal transition (EndMT) has been established in the pathogenesis and progression of many human conditions, including fibrosis of the heart

, lung

,liver

, and kidney

. Considered one of the important factors for inducing EndMT, hypoxia can lead to increased expression of mesenchymal marker proteins α smooth muscle actin (α-SMA) and fibroblast-specific protein 1 in rat microvascular endothelial cells

. Hypoxia can also lead to significantly decreased expression of the endothelial marker CD31 and significantly increased expression of mesenchymal markers α-SMA, collagen 1A1 (COL1A1), and COL3A1 in the pulmonary microvascular endothelium

. Hypoxiainducible factor-1α (HIF-1α), a regulator of hypoxia-induced transcription, has been identified as a key molecule that promotes EndMT by regulating transforming growth factor β(TGF-β)/SMAD

, c-Met/ETS-1

, and/or nuclear factor κB

signaling pathways.

The Hippo pathway is an evolutionarily conserved kinase cascade

. Yes-associated protein (YAP) can be phosphorylated by mammalian Ste20-like kinases 1/2 (Mst1/2) and large tumor suppressor 1/2, which prevent its entry into the nucleus to promote transcription of target proteins

. Many previous studies demonstrated crucial effects of YAP on epithelial-tomesenchymal transition (EMT) and tumor cell metastasis. In colorectal cancer cells, YAP was associated with expression of EMT markers and influenced tumor cell migration and invasion

. In lung cancer, WW and C2 domain-containing protein 3 inhibited EMT by activating Hippo-YAP signaling

,and YAP enhanced EMT through feedback regulation of WT1 and Rho family GTPases

. These YAP-dependent feedback loops result in switch-like changes in expression of signaling and EMT-related markers

. More recently, several studies reported that YAP overexpression in endothelial cells is responsible for EndMT and organ fibrosis, although the exact molecular mechanism still needs to be elucidated

.

Endothelial cells become dysfunctional under hypoxic conditions

, but whether YAP takes part in hypoxia-induced EndMT and subretinal fibrosis associated with wet AMD is unclear. To clarify the role of YAP in HIF-1α-induced EndMT, the present study employed an

hypoxia model involving exposure of human umbilical vein endothelial cells(HUVECs) to cobalt chloride (CoCl

). In addition, we show that silencing of YAP expression significantly prevented the fibrotic process in a laser-induced CNV mouse model.

MATERIALS AND METHODS

Previous reports showed hypoxia that can induce EndMT of endothelial cells.Here, we stimulated HUVECs with CoCl

to establish a hypoxia model

. To verify whether endothelial cells underwent changes characteristic of EndMT, we detected expression levels of HIF-1α, endothelial cell markers(CD31 and ZO-1), and mesenchymal cell markers (α-SMA,vimentin, and Snail) by Western blotting. Expression of HIF-1α and mesenchymal cell markers were upregulated by exposure to CoCl

in a dose-dependent manner (Figure 1). Conversely,endothelial cell markers were downregulated in HUVECs exposed to CoCl

. Because these changes were most pronounced at a concentration of 300 μmol/L CoCl

, we selected this concentration for subsequent experiments, as described in a previous study

. As shown in Figure 1, immunofluorescence microscopy results were consistent with trends observed by western blotting. Moreover, the proliferative ability and migration capacity of HUVECs exposed to CoCl

(300 μmol/L) was significantly increased compared with the control group, as indicated by Ki67 staining, CCK8 assay, and scratch woundhealing assay (Figure 1). In summary, we found that HUVECs undergo EndMT in response to CoCl

-induced hypoxia.

To further clarify whether YAP influenced EndMT,HUVECs were exposed to CoCl

alone or in combination with CA3, a small molecule inhibitor of YAP. CA3 has a potent inhibitory effect on YAP transcriptional activity

. Western blotting and qPCR results showed that CA3 pretreatment could reverse CoCl

-induced upregulated expression of YAP,CTGF, α-SMA, vimentin, and Snail, as well as downregulated expression of CD31 and ZO-1. Immunofluorescence staining revealed the same trend for YAP expression. Ki67 staining and scratch wound-healing assay results showed that the proliferative and migratory abilities of HUVECs exposed to CoCl

combined with CA3 was lower than cells exposed to CoCl

alone (Figure 3).

CoCl

was purchased from Sigma-Aldrich (St. Louis, MO, USA). Small molecule inhibitors YC-1 (Lificiguat, a specific inhibitor of HIF-1α), CA3 (CIL56,an inhibitor of YAP), and XMU-MP-1 (an inhibitor of Hippo kinase MST1/2, which activates YAP) were purchased from Selleck Chemicals (Houston, TX, USA). Antibodies for YAP1,HIF-1α, α-SMA, CD31, and Snail were obtained from Huaan Biotechnology Company (Hangzhou, China). Antibodies for phosphorylated YAP, vimentin, and zonula occludens 1 (ZO-1)were purchased from Cell Signaling Technology (Beverly,MA, USA), while the antibody for β-actin was from Beyotime Institute of Biotechnology (Shanghai, China). Complete endothelial cell medium (ECM) containing 5% fetal bovine serum (FBS) and endothelial cell growth supplement was purchased from ScienCell Research Laboratory (Carlsbad, CA,USA).

Primary HUVECs were obtained from ScienCell and cultured in ECM at 37℃ and 5% CO

in a humidified incubator. Cells were used in experiments between passages 3 and 7.

HUVECs were plated in sixwell plates and cultured in an incubator at 37℃ and 5%CO

with normal oxygen until they reached confluency 70%. Subsequently, the medium was exchanged for medium containing 2% FBS and varying concentrations (0-500 μmol/L) of CoCl

. After 24h of culture, HUVECs were used for additional measurements.

To further verify our hypothesis, mice were intravitreally injected with siRNA YAP on the second day after injury, and the injection was repeated at 2wk after laser photocoagulation.On day 35 after CNV model establishment, we extracted the retina and choroid tissue complex, and performed paraffin sectioning of the eyeball. Western blot results showed that intravitreal injection of siRNA YAP could reverse CoCl

-induced upregulation of YAP and mesenchymal cell markers α-SMA and vimentin. Immunofluorescence staining results were consistent with previous studies (Figure 5). Taken together, these findings indicate that silencing of YAP inhibits EndMT in subretinal fibrosis of a laser-induced CNV mouse model.

The CNV mouse model was induced by laser photocoagulation according to a previously published protocol

. Briefly, C57BL/6J mice were anesthetized with 2% sodium pentobarbital (50 mg/kg, Sigma-Aldrich), and the pupils of both eyes were dilated with 1% topiramate (Alcon Laboratories, Fort Worth, TX, USA). Laser photocoagulation was performed on the fundus of mice (532-nm wavelength,100-mW power, 50-μm spot size, 100-ms duration). Four to six laser spots were made around the optic nerve to cause damage. Microscopy revealed the appearance of bubbles in all laser burns. Spots containing bleeding or an absence of bubbles at the laser site were excluded from subsequent experiments.According to established protocols, cholesterol-modified small interfering RNA (siRNA) targeting YAP or a scramble sequence (Ribobio, Guangzhou, China) were intravitreally injected at 0.1 nmol in one eye of adult mice on day 1 (d1) and d14 after photocoagulation

. Mice were sacrificed at d7.

Total RNA was extracted using Trizol reagent (Takara Bio, Shiga, Japan) and synthesized into cDNA using a reverse transcription kit.Amplification experiments were performed using a SYBR green kit (Takara Bio) according to the manufacturer’s instructions. Primer sequences were as follows: YAP1,5’-GAACAATGACGACCAATAGCTC-3’ (sense) and 5’-TAGTCCACTGTCTGTACTCTCA-3’ (antisense); HIF-1α, 5’-AGTTCCGCAAGCCCTGAAAGC-3’ (sense) and 5’-TCAGTGGTGGCAGTGGTAGTGG-3’ (antisense);β-actin, 5’-ACCCACACTGTGCCCATCTA-3’ (sense) and 5’-GCCACAGGATTCCATACCCA-3’ (antisense). β-actin was used as an internal reference gene to calculate the relative expression of target genes using the 2

method for three independent experimental repeats.

As the main effector of cells under hypoxic conditions, HIF-1α is responsible for regulating various cellular responses

.Because of its role in stabilizing HIF-1α, CoCl

has become one of the most commonly used hypoxia mimics

. Studies have reported that hypoxia can increase expression of HIF-1α and YAP in tumor cells

, and promotes nuclear localization of YAP with reduced phosphorylation levels by inhibiting Hippo signaling

. However, reports describing the relationship between HIF-1α and YAP are inconsistent. For example, Zhang

found that YAP binds to HIF-1α and maintains its protein stability; in contrast, Dai

revealed that hypoxia induced nuclear translocation and accumulation of YAP independent of HIF-1α, as indicated by manipulations of HIF-1α abundance with CoCl

having no effect on total or phosphorylated levels of YAP. This discrepancy suggests potential bidirectional regulation between HIF-1α and YAP. In this study, the HIF-1α inhibitor YC-1 was used to clarify the regulatory effect of HIF-1α on YAP.

Cell lysates were prepared using cell lysis buffer containing phosphatase inhibitors. The resulting protein concentrations were determined using a bicinchoninic acid protein assay. After completely separating 40 μg of total protein sample per lane, proteins were transferred to a polyvinylidene fluoride membrane and blocked with 10% skimmed milk for 1h. Next, membranes were incubated with one or more of the following primary antibodies overnight at 4℃: YAP1 (1:1000),phospho-YAP (1:1000), HIF-1α (1:500), α-SMA (1:1000),CD31 (1:500), vimentin (1:1000), ZO-1 (1:1000), Snail(1:1000), and β-actin (1:1000). Subsequently, membranes were reacted with appropriate secondary antibodies for 1h, then detected and observed using an enhanced chemiluminescence substrate kit. The gray value of each target band was analyzed by Image J software (https://imagej.nih.gov/ij/) using β-actin as an internal reference. This experiment was repeated three times independently.

3．1 北京市西城区近5年新生儿疾病筛查的整体情况 北京市西城区新生儿疾病的筛查率、复诊率分别为98．75%和98．57%，略高于北京市平均水平［4］；CH发病率为1∶2 060，PKU发病率为1∶5 408，与北京市1∶3 622和1∶9 094相比均较高，通过早期的筛查和及时的诊治使这些患儿减轻疾病造成的损害，避免了残疾的发生。

After aspirating the medium and washing three times with phosphate-buffered saline (PBS,3min each wash), cells were fixed with 4% polyoxymethylene solution for 15min at room temperature and washed again.Next, cells were permeabilized with 0.5% Triton X-100 at room temperature for 30min and washed again three times(3min each) with PBS. Cells were blocked with 5% bovine serum albumin (BSA) for 30min and then immediately incubated with diluted primary antibody (YAP1, 1:50; CD31,1:20; α-SMA, 1:50) overnight at 4℃. Subsequently, cells were incubated with a fluorescent secondary antibody for 1h,washed three times (3min each) with PBS, and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15min in the dark.Images were acquired with an upright BX63 fluorescence microscope (Olympus, Tokyo, Japan). This experiment was repeated three times.

After exposing HUVECs in a 96-well plate to CoCl

for 24h, 100 μL of high-sensitivity CCK-8 reagent from a cell proliferation detection kit (Keygen Biotech, Jiangsu Province, China) was added to each well for incubation at 37℃ for 3h. Subsequently, the absorbance value of each well was measured at a 450-nm wavelength to assess cell viability.Three replicates were used for each of three independent experiments.

To evaluate YAP expression in HUVECs under hypoxia, we analyzed transcript and protein levels of YAP in HUVECs exposed to different concentrations of CoCl

. Western blot and qPCR analysis showed that CoCl

elicited a significant increase of YAP expression, consistent with changes in HIF-1α (Figure 2). To further quantify the transcriptional activity of YAP under hypoxia, we examined levels of YAP phosphorylation and expression of connective tissue growth factor (CTGF), a target of YAP, by Western blotting. When HUVECs were exposed to 0-500 μmol/L of CoCl

, the phosphorylation level of YAP-Ser127 decreased in a concentration-dependent manner. Moreover, CTGF expression was upregulated by CoCl

exposure, consistent with changes in HIF-1α. Immunofluorescence staining also showed that CoCl

promoted YAP expression and nuclear translocation in HUVECs (Figure 2). Therefore, both total YAP expression and its transcriptional activity may be of great importance in hypoxia-induced EndMT of HUVECs.

秀容川追上三人。男孩说：“你问我为什么高兴？因为我买了陀螺。”说着把手里的陀螺向他晃了晃。那大汉说：“我在杀公鸡，谁知下刀不利索，让它跑了，我正追呢。”那老头笑眯眯地说：“漂亮女人的屁股，哪个男人不喜欢呢？你不喜欢啊？”

Slices were placed at room temperature for 10min and then immersed in xylene for 15min, which was repeated twice. After deparaffinizing paraffin sections in a gradient series of ethanol, slices were incubated with citric acid antigen retrieval solution in a microwave oven for 15min under low heat to retrieve antigens.After washing and cooling, sections were washed three times with PBS. To block nonspecific binding, 5% BSA solution was added dropwise to the tissue and incubated for 30min at room temperature. After shaking off the blocking solution,prepared primary antibody solutions were dropped onto the tissue section, which was put in a humid box and incubated overnight at 4℃. The next day, sections were immersed in PBS and washed three times. After drying, fluorescent secondary antibody was added dropwise and incubated for 1h at room temperature in the dark. After placing slices in PBS and washing three times (5min each), nuclear staining was performed by incubating slice with DAPI at room temperature for 15min. Sections were again placed in PBS and washed three times. Finally, an appropriate amount of anti-quenching agent was added and sections were observed and imaged by fluorescence microscopy.

本文以南通蓝印花布的数字化纹样图像为研究对象，通过数码相机等设备为其进行数字化图像的采集。另外，针对蓝白两色的蓝印花布这一特点，对其数字化图像进行相关预处理，包括灰度化、中值滤波去噪和归一化等操作；经过大量实验后，确定采用加权值法与最大值法结合的灰度化处理来处理，其公式如下所示：

Statistical analysis was performed using SigmaPlot 14.0 (Systat Software, San Jose, CA, USA).Significant statistical differences were determined by Student’s

-test or one-way ANOVA followed by Holm-Sidak posthoc test. A

value ＜0.05 was considered significant difference.

RESULTS

Eight-week-old male C57BL/6J mice weighing 19-25 g were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The Animal Ethics Committee of Fudan University approved all experimental protocols involving animals. Animals were handled in accordance with The Association for Research in Vision and Ophthalmology statement “Use of Animals in Ophthalmology and Vision Research”. Before performing experimental operations, all animals were acclimatized for at least 7d.

After cells plated in six-well plates reached confluency, they were streaked with a sterile 200-μL pipette tip and washed three times with PBS. Next,serum-free medium (control) or serum-free medium containing CoCl

alone or in combination with inhibitors was added to the plate. Scratch widths were observed and photographed under an inverted BX63 microscope after 0, 24, 32, and 48h of culture. The distance between scratches was measured using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville,MD, USA). This experiment was repeated three times.

推荐理由:本书首次以长篇报告文学形式，从经济、政治、文化、社会、生态文明诸领域，全景展示浦东开发开放波澜壮阔的历史进程与时代画卷。全书内容生动鲜活、大气磅礴，具有很强的文学感染力和可读性，是直面当下、讴歌时代、宣传改革开放的一部重大现实题材力作。

Phosphorylation of YAP-Ser127 creates a binding consensus for 14-3-3 proteins that contributes to YAP entrapment and maintenance of YAP in an inactive state

. To clarify whether phosphorylation of YAP is involved in EndMT,HUVECs were treated with the MST1/2 inhibitor XMUMP-1 for 24h under normoxia and analyzed for changes of endothelial and mesenchymal markers using Western blotting.We found that inhibiting YAP phosphorylation in endothelial cells increased expression levels of the mesenchymal markers α-SMA and vimentin, while inhibiting endothelial markers CD31 and ZO-1. The results of immunofluorescence staining were consistent with those of Western blotting (Figure 3).Collectively, these results suggest that YAP plays a significant role in hypoxia-induced EndMT of HUVECs.

HIF-1α is proven to be essential for expression and nuclear translocation of YAP

. Therefore, we exposed HUVECs to CoCl

and YC-1, a small molecule inhibitor of HIF-1α. As shown in Figure 4, CoCl

-induced HIF-1α expression was reversed by YC-1. As predicted, expression of mesenchymal markers α-SMA, vimentin, and Snail were significantly decreased, while expression of endothelial markers CD31 and ZO-1 showed an upward trend. Moreover, expression of YAP and its target molecule CTGF was simultaneously decreased.Immunofluorescence staining of YAP showed a consistent trend. Furthermore, both the proliferative and migratory abilities of HUVECs were decreased by YC-1 (Figure 4).These results indicate that HIF-1α is a key mediator of YAP expression and transcriptional activity, as well as hypoxiainduced EndMT of HUVECs.

To clarify the role of YAP on EndMT and subsequent fibrosis in CNV, we constructed a mouse CNV model using laser-induced injury. On the 35

day after laser injury, expression levels of YAP and mesenchymal cell markers α-SMA and vimentin were detected in the mouse pigment epithelium-choroid complex. Western blot analysis revealed that expression of YAP, α-SMA, and vimentin were all upregulated in the CNV group. Co-staining of COL1A1 and CD31 reflected the occurrence of EndMT in subretinal fibrosis of the CNV model (Figure 5). Therefore, we speculate that YAP promotes the subretinal fibrosis that occurs subsequent to CNV.

Based on previous studies, the small molecule inhibitors CA3,YC-1, and XMU-MP-1 were prepared at a concentration of 1 mmol/L in dimethyl sulfoxide, and then adjusted to a final concentration of 1 μmol/L with ECM containing 2% FBS for treatment of cells

.

在我们的家庭中，90%的问题都源于不会沟通。不管是夫妻之间，还是父母和子女之间，大家要么完全沉浸在自己的世界中自说自话，要么把沟通当成宣泄情绪的一个途径，亦或者，为了避免矛盾激化保持表面的和谐，而用隐忍的方式回避沟通。

DISCUSSION

As the main cause of visual impairment in elderly individuals,wet AMD has attracted widespread attention. CNV is a feature of neovascular AMD, also known as wet AMD

. Immature blood vessels in CNV cannot supply oxygen or nutrients at the level of normal blood vessels, causing further tissue hypoxia.Moreover, repeated leakage and bleeding from immature blood vessels can damage the retina by causing subretinal fibrosis.Therefore, CNV can complicate AMD, leading to severe vision loss and blindness

. Inhibiting the occurrence of fibrosis in patients with wet AMD is vital to maintain their visual function. Therefore, further research on mechanisms that promote the occurrence of fibrosis is necessary. In this study,we clarified the importance of YAP activity in hypoxia-induced EndMT and subretinal fibrosis of a laser-induced CNV model.Using

experiments, we verified that YAP expression and activity was increased in HUVECs under hypoxic stimulation. Our work further revealed that YAP is necessary for hypoxia-induced EndMT of HUVECs. Finally, silencing of YAP inhibited EndMT associated with subretinal fibrosis in the laser-induced CNV model, indicating YAP might be a new and effective therapeutic target for CNV therapy.

二、三年级学生是以具体形象思维为主，他们对抽象概念本质的领悟，都必须以足够的直观材料和充分的实践经验为基础。对比上面表格及材料，我们不难发现，十年前给二年级学生上“倍的认识”时，只有在“再识倍”这个环节中放手让学生操作“摆一摆”多倍。除此以外的教学过程都是由教师“扶”着学生感知理解“倍的意义”，生生互动较少。而十年后在三年级学生学习“倍”时，整节课学生都处在“数一数、摆一摆、画一画，再画一画、做一做、分一分”的学习中，教师在重点、难点处介入学习，师生、生生互动比较多，学习方式呈现任务驱动化，让不同的学生在完成任务过程中有不同的体悟。

掺入纳米氧化硅后，在限制膨胀约束过程中，孔隙分布趋于均匀。但约束卸除后，一方面，膨润土吸水孔隙胀开，纳米氧化硅进入胀开后孔隙；另一方面，在收缩时，硅与膨润土界面交接处产生了微观收缩裂隙[14]，大孔隙数量增加，如新增孔径>1.8μm的孔隙。同时，由于颗粒膨胀挤压约束作用，相应的小孔隙数量也在增加，因此，掺入纳米氧化硅的膨润土，在限制膨胀结束、自然风干收缩稳定后，孔隙分布范围更广。

EndMT of both choroidal and retinal endothelial cells has been studied in numerous ocular angiogenic diseases, including AMD, proliferative diabetic retinopathy, and retinopathy of prematurity

. In the present study, expression of endothelial markers CD31 and ZO-1 was decreased, while that of mesenchymal markers α-SMA, vimentin, and Snail was increased in HUVECs after hypoxia, consistent with results observed for cells exposed to 1% O

. More importantly,we also investigated the impact of YAP on HIF-1α-meditated EndMT. Our results reveal that pharmacological inhibition or genetic depletion of YAP could attenuate EndMT of HUVECs

, as well as fibrosis of a laser-induced CNV mouse model

. Savorani

recently provided novel evidence that YAP acts as SMAD3 transcriptional co-factor by preventing its phosphorylation, thus protecting against TGFβinduced EndMT. Given that Snail is an important regulator of hypoxia-induced EndMT

, whether YAP influences expression or nuclear transcription of Snail needs further elucidation.

With regard to biological function, we observed that upregulation of YAP was accompanied by increases in the proliferative capacity and migratory ability of HUVECs.Endothelial cells undergoing EndMT exhibited higher proliferation and migration abilities, both directly through transformation into smooth muscle-like cells and indirectly through paracrine secretion

. In addition, the mesenchymal transition regulator Snail functions to promote cell proliferation and migration

. Thus, we speculate that promotion of endothelial cell proliferation and migration induced by YAP may be attributed to EndMT regulation.

In conclusion, the present study identified a critical role of the HIF-1α/YAP signaling axis in EndMT, which suggests that YAP may be an attractive target for treatment of AMD with the advantage of controlling both CNV and subretinal fibrosis.

We thank Liwen Bianji (Edanz) (www.liwenbianji.cn/) for editing the English text of a draft of this manuscript.

山东琦泉目前投产运行6个项目，装机容量290MW，占全国总投产项目规模的6%左右；在建4个项目，装机容量320MW。琦泉秸秆直燃发电厂主要分布在山东地区，逐步向广西拓展[26]。

Zou R, Feng YF, Xu YH, Shen MQ and Zhang X performed the research; Yuan YZ designed the research study; and Zou R and Feng YF analyzed the data and prepared the manuscript.

女人的男朋友比她大一岁，属虎。两人在一起同居时男朋友经常跟她开玩笑地说，虎吃牛。女人就咯咯地笑，她喜欢被男朋友吃，确切点说是吃她的身体。男朋友在一所农业科研所里搞农作物的繁殖，很废寝忘食的。他不时地就出国考察，也下到全国各地的农村实验田里做试点。男朋友只有回到他们住的小屋里才会有笑容。

Supported by the National Natural Science Foundation of China (No.81970817; No.81873680).

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