Celastrol inhibits laser-induced choroidal neovascularization by decreasing VEGF induced proliferation and migration

INTRODUCTION

Choroidal neovascularization (CNV) refers to the growth of abnormal new blood vessels from the choroid into the sub-retinal space accompanied by vascular leakage, retinal edema, and vision loss

. CNV is a major pathological change in ocular diseases that lead to blindness, such as age-related degeneration, pathologic myopia, angioid streaks, and trauma.Although the pathogenesis of CNV is not clearly understood,growth factors, such as vascular endothelial growth factor(VEGF) and fibroblast growth factor-induced cell proliferation and migration, play an important role in CNV development.Currently, anti-VEGF drugs are the most widely used treatment for CNV. Although these drugs are helpful in reducing the risk of visual deterioration, they only improve vision in 33% of patients

. Some patients experience worsening vision function, regardless of aggressive treatment with anti-VEGF agents

, suggesting other vascular mediators contribute to ocular angiogenesis. Furthermore, patients often exhibit CNV recurrence and require repeated treatments. A regimen of multiple intravitreal injections for months or years is associated with many complications, such as cataracts,retinal detachment, and endophthalmitis, as well as significant costs

. Studies have shown that participants in monthly dosing groups had a higher incidence of macular atrophy than those in discontinuous treatment groups, suggesting that anti-VEGF therapy induces the formation of macular atrophy in some patients

. To improve the outcomes of CNV treatment and reduce the cost to the patient, an alternative antiangiogenic treatment is needed.Celastrol, a natural products extracted from traditional Chinese herb, exhibits potent anti-inflammatory, anti-oxidant, and antiangiogenic activities

. This drug has been widely used to treat chronic inflammation, autoimmune diseases, and many types of cancer by modulating multiple pro-angiogenic and pro-inflammatory cytokines, such as hypoxia-inducible factor-1α, TNF-α, and VEGF

. These cytokines play a major role in the proliferation of endothelial cells and the progression of angiogenesis. Previous studies have reported that celastrol has anti-angiogenic effects on

and

assays

.However, few studies have evaluated the effect of celastrol on CNV. Thus, the present study investigated the effect of celastrol using a popular mouse model of laser-induced CNV.

新课标改革后，高中教育重视思维品质的培养，在高中地理学习中，无论是学业水平考试还是高考，都有较多的考查学生地理逻辑思维能力，主要包括学生对于事物的基本认知跟分析能力和地理知识的实际运用能力。在高考考核要求“认证和探讨地理问题”方面，明确要求学生“能够发现或提出科学的、具有创新意识的地理问题；能够提出必要的论据来认证和解决地理问题；能够用科学的语言、正确的逻辑关系，表达出论证和解决地理问题的过程与结果；能够运用正确的地理观念，探讨、评价现实中的地理问题。”因此，掌握正确的地理逻辑思维能力是对高中学生的一项必备的能力要求。

MATERIALS AND METHODS

总结性评价包括同行评价和教师自评。同行评价是让同行教师观摩课程，教师自评是教师课下反思，根据每节课的形成性评价和同行评价改善教学。总结性评价作用于下学期课程的设计与实施，可以在学期末和每节课后实施。

采气厂作为典型的化工企业，安全是摆在企业面前的头等大事，基于安全开展企业生产工作以及经营工作是一项基本原则，在实际业务中，采气厂由于其地理位置分散、风险系数高等特点，对安全管理有着更高的要求。

The monolayers of HUVEC and hCEC in 24-well cell plates were wounded by scratching with a pipette tip; they were then washed with PBS. ECM with 1%serum containing the vehicle, VEGF (20 ng/mL), or different concentrations of celastrol were added to the scratched monolayers. Images were taken under a microscope at 0, 6,12, 24, and 36h post-wounding. Quantification was done by measuring the number of pixels in the wound area using Image J software.

We performed an

angiogenesis assay similar to that described before

. The aorta rings from the 6-8 weeks old C57BL/6J mice were separated and cultured in ECM containing 20 ng/mL VEGF(Genzyme/Techne, Cambridge, MA) in the presence or absence of celastrol for 7d, and the sprouting of the endothelial cells was analyzed. At each time, five repeat was applied for each group and it was repeat for three times.

Fluorescein angiography (FFA) examinations were conducted on the mice to examine the laser-induced CNV. The animals were anesthetized, and their pupils were dilated as described for the induction of CNV. They were then positioned on the stage of the microscope, and hypromellose coupling fluid was applied to the eye. The camera and eye position were adjusted to ensure correct alignment and to focus on the optic nerve head plane. Standard color fundus photography was used before adjusting to the appropriate filter set for FFA; 0.1 mL/kg of 5%fluorescein sodium was then administered

intraperitoneal injection. Images were captured using the Micron IV (Phoenix Research Laboratories, Pleasanton, CA, USA). Images were taken at 3, 7, and 14d after laser treatment. FFA images with fluorescein administration by intraperitoneal injection were taken five minutes after fluorescein sodium was injected. Mice were sacrificed by intravenous injection of air after anesthesia 3, 7, or 14d after laser photocoagulation, and the eyecups were removed and incubated with 4% paraformaldehyde at 4℃ overnight. The choroid/retinal pigment epithelium (RPE)/sclera was set in 24-well culture plates, and 0.5% bovine serum albumin (BSA) with 0.1% triton was added for 2h

at room temperature for blocking. After being washed with PBS,fluorescein-isothiocyanate-conjugated isolectin B4 (Vector,Burlingame, CA, USA, 1:500) was added and the sample were incubated at 4℃ overnight. The fluorescence-labeling tissue was flat mounted on glass slides (ThermoFisher Scientific)and covered with a cover slip. CNV was visualized using a fluorescerin microscope (FV1000; Olympus, Tokyo, Japan).For each group at least five mice were used for result analysis.

此次研究结果指出了,CTn T阳性组病变率大于CTn T阴性组,P<0.05;CK-MB阳性组病变率比照CK-MB阴性组,P>0.05,符合任焕民等[6]研究结果。

The laser-induced CNV mouse model is commonly used to evaluate the effects of treatments for CNV. To analyze the effects of celastrol

, we calculated the area of CNV and its leakage 3, 7, and 14d after induction in mice treated with the vehicle, 0.1 mg/kg celastrol, and 0.5 mg/kg celastrol. CNV area was reduced significantly in the celastrol treatment groups(

<0.001; Figure 6). To our surprise, as the dosage increased,so did the inhibition of celastrol on CNV (

<0.001), which is opposite to the results of the

experiments. On day 14,celastrol attenuated the area of CNV by 49.15% in the 0.1 mg/kg celastrol-treated group and 80.26% in the 0.5 mg/kg celastrol treated group as compared to the vehicle-treated group. In the 0.5 mg/kg celastrol group, CNV was barely seen two weeks after photocoagulation. Celastrol also decreased the leakage of CNV in a dosage-dependent manner (Figure 7). By the end of our observation, the leakage area decreased to 59.99% in the 0.1 mg/kg celastrol-treated group and 41.77% in the 0.5 mg/kg celastrol-treated group compared to the vehicle-treated group.

The aortic ring assay is a more physiologically relevant

model for angiogenesis, as it develops blood vessels from aortic explants using the surrounding endothelial cells,which is akin to angiogenesis

. This study found that 20 ng/mL VEGF increased both the number and length of vascular sprouting by 60%. Similar to the tube formation assay, 0.1 µmol/L celastrol attenuated VEGF-induced vascular sprouting to the level of the control group. Although 0.5 µmol/L celastrol decreased vascular sprouting by 30%, a statistical analysis showed no difference (Figure 2). Since 0.5 µmol/L celastrol did not significantly decrease vascular sprouting, a higher concentration of celastrol (1 µmol/L) was not applied to the aortic ring assay.

This study used 6-8 weeks old C57BL/6J mice. CNV was induced in mice by laser photocoagulation. Briefly, the procedure was performed on anesthetized [10% ketamin(100 mg/kg) and 1% xylazine (10 mg/kg) intraperitoneally]animals with dilated pupils using a laser photocoagulator(Micron IV, Phoenix Research Laboratories, Pleasanton,CA, USA) and the following parameters: spot size, 50 μm;duration, 100ms; and power, 450 mW. Four spots were applied to each eye between the major retina vessels and around the optic disc at a distance of 1-1.5 optic disc diameters from the optic disc head. Burns that generated bubbles were included in the evaluation. Power analysis determined that 5 mice per group would provide 80% or more power to detect statistically significant differences in outcomes.

The HUVEC and hCEC were cultured at a density of 5000 cells/well for VEGF-induced proliferation or 10 000 cells/well for toxicity testing in 96-well plates. Cell counting kit-8 (CCK-8; Dojindo, Shanghai,China) assays were performed following the manufacturer’s instructions. The OD ratio was read using BioTek’s Gen5TM microplate reader (Biotek, Winooski, VT, USA) 24h after seeding.

Celastrol (34157-83-0, purity ≥98%)was purchased from Desite Biology, Chengdu, China and dissolved in phosphate-buffered saline (PBS) containing 1% dimethyl sulfoxide and 10% ethanol. After laser photocoagulation, the mice were randomly divided into three groups (15 mice/group) and treated by intraperitoneal injection at a daily dose of 0.1 mg or 0.5 mg celastrol per kilogram (kg)body weight or with PBS containing 1% dimethyl sulfoxide and 10% ethanol as the vehicle control. Random numbers were generated using a computer based random order generator.

Data are expressed as mean±standard error of mean (SEM). All images were analyzed using Image J software (NIH). One-way analysis of variance (ANOVA; for comparisons of three or more groups)followed by Tukey’s post hoc tests were used for statistical analyses with SPSS software version 17.0 (IBM SPSS software). Statistical significance was identified as

<0.05.

RESULTS

Reducing Vascular Endothelial Growth Factor-induced Neovascularization

To evaluate whether celastrol prevented VEGF-induced neovascularization, two

models of neovascularization—a tube-formation assay and an aorta-ring culture assay—were performed. As 2 µmol/L celastrol is toxic to HUVEC

and both low (0.1 µmol/L)

and high (1 µmol/L)

inhibited migration of HUVEC, three concentration of celastrol, 0.1, 0.5, and 1 µmol/L, were applied for this study. As shown in Figure 1, 20 ng/mL VEGF stimulated the tube formation of HUVEC and hCEC, while 0.1 and 0.5 µmol/L celastrol significantly decreased VEGFinduced tube formation. A higher concentration of celastrol(1 µmol/L) significantly diminished VEGF-induced HUVEC tube formation, but it had less effect on hCEC.

一次跌倒过程的持续时间一般为10 s左右,为保证时间窗截取到完整的跌倒过程,同时结合两个数据集各自的样本时长,我们对MobiAct采用时长为8 s的时间窗,对SisFall采用时长为12 s的时间窗。

Twenty-four well plates were precoated with 200 µL matrigel. HUVEC and hCEC (50 000 cells/well)were seeded into each well. The cells were treated with the vehicle (1% dimethyl sulfoxide and 10% ethanol), VEGF(20 ng/mL) or different concentrations of celastrol with VEGF. Each experiment was performed in triplicate. Images of tube formation were photographed six hours post-assay under a light microscope. The data were imported as TIFF files into Image J software to calculate the total length of all tubing within each field using an angiogenesis analysis module.

The impact of celastrol on the viability and proliferation of hCEC and HUVEC was evaluated using CCK-8 assays. No significant difference was observed between the control and celastrol-treatment groups after 24h incubation,indicating that celastrol does not have a toxic effect at these doses (Figure 3A and 3B). Although dye formation in HUVEC treated with 0.1 µmol/L celastrol was less than in the control group, a statistical analysis found no difference among the groups. The result indicating that celastrol have no influence on viability of hCEC and HUVEC. The proliferation rates of HUVEC and hCEC were significantly enhanced by 20 ng/mL VEGF, while 0.1 and 0.5 µmol/L celastrol obstructed the growth of cells induced by VEGF (Figure 3C and 3D). Consistent with tube formation, 1 µmol/L celastrol attenuated VEGF-induced cell proliferation in HUVEC but not hCEC. Among the three doses of celastrol, 0.1 µmol/L celastrol was shown to have the greatest ability to inhibit the function of VEGF.

All animal experiments were conducted in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center. Eye cup of human beings was collected from the Eye Bank of Guangdong Province with the approval of the Sun Yat-sen University Medical Ethics Committee after getting the consent from the donor in accordance with the Declaration of Helsinki.

To evaluate the effect of celastrol on migration, a wound healing assay was performed. No significant difference in cell invasion between the control and treatment groups was found during the first 12h. VEGF was shown to have a promoting effect on HUVEC and hCEC at 24h, which is similar to results reported by others

. At 24 and 36h, the cell-covered area in the 0.1 µmol/L celastrol group was less than in the VEGF group,indicating that 0.1 µmol/L celastrol weakened the VEGF-induced migration of HUVEC (

<0.05; Figure 4A and 4B). At higher concentrations of celastrol (0.5 and 1 µmol/L), no inhibitory effects were seen. Even 1 µmol/L celastrol accelerate HUVEC migration at 24h. The increase in hCEC migration with VEGF treatment translated into a significant reduction at 36h with celastrol treatment (Figure 3C and 3D). Both 0.1 and 0.5 µmol/L of celastrol restrained hCEC migration at 36h (

<0.001).As with HUVEC, the hCEC-covered area in the 1 µmol/L celastrol group was comparable to that of the VEGF group.

VEGF plays a key role in the formation and growth of vascular in both physiological and pathological conditions that involve complex signaling.Expression of three major downstream proteins of VEGF signaling—FAK, Src, and Akt—and their phosphorylation were quantified by Western blotting to explore the mechanism of celastrol’s inhibitory effect on neovascular formation.Western blotting demonstrated that VEGF has less effect on the expression and phosphorylation of Src and Akt in HUVEC (Figure 5A and 5B). However, an increasing trend was observed in FAK phosphorylation, while the expression of FAK was maintained in the VEGF-treated group in a timedependent manner. In contrast, 0.5 µmol/L celastrol diminished the FAK phosphorylation induced by VEGF after 1h of incubation. For hCEC, VEGF increased Src expression and 0.5 µmol/L celastrol decreased it (Figure 5C and 5D).

Primary human choroidal endothelial cells (hCEC) were retrieved from donors with methods described before

. Briefly, an eye was collected from the Eye Bank of Guangdong Province within 6h from death and soaked in 0.25% povidone iodine at room temperature for 30min. After the anterior segment was removed, the choroid was isolated from the sclera with forceps, and 0.2% pronase was used for the detachment of endothelial cells. After digestion at 37℃ for 4-6h, cells were collected by centrifugation. The cell pellet was resuspended in endothelial cell medium (ECM; ScienCell, USA) without serum, and CD31 beads were used to separate hCEC from the other cells. Primary cultures of human umbilical vein endothelial cells (HUVEC) were obtained and cultured as previously described

. The hCEC and HUVEC were cultured in completed ECM. All cells were cultured at 37°C in a humidified 5% CO

atmosphere. After treatment with VEGF or different concentrations of celastrol, the whole proteins of the hCEC and HUVEC were collected. Western blotting was performed by probing with anti-tyrosine-protein kinase (Src,36D10, Cell Signaling Technology, USA), anti-phosphorate-Src (D49G4, Cell Signaling Technology, USA), anti-focal adhesion kinase (FAK; D507U, Cell Signaling Technology,USA), anti-phosphorate-FAK (D20B1, Cell Signaling Technology, USA), anti-protein kinase B (PKB/Akt; 11E7, Cell Signaling Technology, USA), and anti-phosphorate-Akt (D9E,Cell Signaling Technology, USA). Primary antibodies were detected using goat anti-rabbit IgG conjugated with horseradish peroxidase and visualized using chemiluminescence detection reagents. At least three times batch of protein was collected and run for the western blot analysis. For cell related

assay, five repeat was applied for each group each time and it was repeat for three times.

DISCUSSION

In this study, we demonstrated that celastrol inhibited neovascular formation by blocking VEGF signaling and reducing VEGF-induced proliferation, migration, and tube formation of vascular endothelial cells. Our results also indicate that celastrol is a potent, natural anti-angiogenic compound for suppressing CNV development in mice. On day 3, 7, and 14 after inducing CNV, intraperitoneal administration of celastrol significantly reduced the vascular budding area and CNV leakage as seen on flat mounts of the RPE-choroid complex.

Although it is now accepted that VEGF plays a vital role in initiating and sustaining pathologic angiogenesis in the eye,animal and human studies have demonstrated that the factors involved in inflammation also contribute to these processes

.For example, the role of M2 macrophages has been reported as dominant, and they may also play an important part in the development of CNV

, as treatment targeting M2 polarization has been found to be effective

. Multiple clinical trials have proven that celastrol is an effective and well-tolerated drug in the treatment of inflammatory diseases

. Moreover, a recent

study reported on the potent anti-angiogenic effect of celastrol in the inhibition of corneal neovascularization in rats

. Given its promising results as an anti-angiogenic and anti-inflammation drug, celastrol may have efficacy in treating CNV. Our results support this conclusion, as celastrol diminished neovascularization in two

models: a tube formation assay and an aortic ring formation assay. Moreover,mean CNV area was reduced by 49.15% in 0.1 mg/kg celastroltreated eyes and 80.26% in 0.5 mg/kg celastrol-treated eyes compared to vehicle-treated eyes, which are similar results to those of previous studies involving other drugs, such as bevacizumab, an FDA-approved anti-VEGF drug that was reported to reduce CNV area by 80% compared to vehicle treatment in laser-induced CNV in mice

.

VEGF pathway activation triggers a series of signaling processes, stimulating vascular endothelial cell proliferation,survival, migration, and permeability, leading to angiogenesis and vascular leakage in pathological conditions. In the present study, two endothelial cells, HUVEC and hCEC, were exposed to 20 ng/mL VEGF or to different concentrations of celastrol. Consistent with previous report, VEGF promoted endothelial proliferation and migration

, while celastrol had a minimal effect on endothelial survival and an obvious effect on the proliferation and migration induced by VEGF;this indicates that celastrol may also play a role an anti-VEGF role and inhibit neovascularization by suppressing the VEGF-induced functional activity of endothelial cells. In fact, celastrol decreases VEGF expression in HUVEC under hypoxia

and the phosphorylation of VEGF receptor 2, which is the main signaling receptor whose activation promotes vascular endothelial cell mitogenesis and permeability

.Although VEGF function was restrained by celastrol in both HUVEC and hCEC, different signaling pathway activation was observed in the current study. In HUVEC, celastrol prevented FAK phosphorylation, while Src expression was decreased in hCEC, indicating that one type of endothelial cell might not represent the complexity of neovascularization

.Both FAK and Src have been reported to mediate endothelial migration and proliferation

, while Akt has been found to be more related to cell survival

. This partially explains why Akt expression did not change much in either type of endothelial cell used in our study, as celastrol has a minimal effect on cell survival.

Although data from this work demonstrated celastrol’s inhibitory effects in neovascularization, some results were contradictory, as celastrol

was found to inhibit laserinduced CNV in a dose-dependent manner, while the

experiments showed that a higher dose of celastrol (0.5 or 1 mmol/L) reduced the efficacy of celastrol to suppress VEGF-induced enthothelial proliferation, migration, and tube formation, which is consistent with previous reports that lower concentrations of celastrol have a more obvious effect on VEGF suppression and the inducing activity of endothelial cells in angiogenesis

. One explanation for this is that the concentration of celastrol

experiments may be lower than it

experiments. Although we calculated the concentration of celastrol and demonstrated that 0.5 mg/kg celastrol is equal to 1 mmol/L celastrol when we treat mice as water, since the water content of mice is 73.2% of their fat-free body weight

. The concentration of celastrol in mice eyes may not reach 1 mmol/L because of the blood-retina barrier,which could impact drug distribution. The pharmacokinetic and pharmacodynamic properties of celastrol should be considered in future studies to assure effective dosage of celastrol for antineovascularization. Another possibility for the contradictory results is that our

experiment only mimicked part of neovascularization, as the Western blot results show different signal pathway activations in the two types of endothelial cells.In summary, our study demonstrated that celastrol significantly inhibits the development of laser-induced CNV in mice.Celastrol may serve as an alternative and economical agent

for CNV treatment, either alone or in conjunction with other therapies. Further studies are needed to explore the mechanism of the inhibitory effects of celastrol on angiogenesis and the optimal celastrol dose for preventing CNV.

在上半年的2018星友颁证交流会上，当金钻明说他是从事太赫兹光谱学研究时，太赫兹这个在媒体上出现的高频词让我们对他留下印象。半年后我们有机会在上海大学物理系一间小金的工作兼实验室里作了这次启明星采访。

法国巴黎科学与生物多样性小学，其所处位置也在一个狭小的地块中，不仅包括教学空间，还有一个面向居民开放的体育馆。学校针对自己的教学特性，将体育馆置于一端对外开放，剩余部分围绕内院布置半环形的教学空间及绿带，打造了一个可循环的生态系统。这种内院与绿带既提供了学生的校园活动空间，又使学生与自然能零距离的接触，达到了需要的教学目的（图2）。

ACKNOWLEDGEMENTS

The authors wish to thank Dr. Rong Ju for field assistance.

第二，大数据时代的到来为各行各业的发展提供了更好的发展空间，同时对个行业也提出了新的和更高的要求，在大数据的作用下，数据的保存和查阅变得更加方面和快捷。在这样的背景之下，人们也开始逐渐意识到大数据时代到来的重要性，也认识到数据资料的整理所带来的益处。

Supported by National Natural Science Foundation of China (No.81570826).

Wu MX and Shang F prepared the manuscript. Li Z and Chen F performed analysis of the data.Chen F was not aware of the group allocation. Li Z and Zhou KW executed the conceptualization and design of experiments.All authors read and approved the final manuscript.

Li Z, None; Zhou KW, None; Chen F,

None; Shang F, None; Wu MX, None.

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