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.

新課標改革后，高中教育重視思維品質的培養，在高中地理學習中，無論是學業水平考試還是高考，都有較多的考查學生地理邏輯思維能力，主要包括學生對于事物的基本認知跟分析能力和地理知識的實際運用能力。在高考考核要求“認證和探討地理問題”方面，明確要求學生“能夠發現或提出科學的、具有創新意識的地理問題；能夠提出必要的論據來認證和解決地理問題；能夠用科學的語言、正確的邏輯關系，表達出論證和解決地理問題的過程與結果；能夠運用正確的地理觀念，探討、評價現實中的地理問題。”因此，掌握正確的地理邏輯思維能力是對高中學生的一項必備的能力要求。

MATERIALS AND METHODS

總結性評價包括同行評價和教師自評。同行評價是讓同行教師觀摩課程，教師自評是教師課下反思，根據每節課的形成性評價和同行評價改善教學。總結性評價作用于下學期課程的設計與實施，可以在學期末和每節課后實施。

采氣廠作為典型的化工企業，安全是擺在企業面前的頭等大事，基于安全開展企業生產工作以及經營工作是一項基本原則，在實際業務中，采氣廠由于其地理位置分散、風險系數高等特點，對安全管理有著更高的要求。

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.

此次研究結果指出了,CTn T陽性組病變率大于CTn T陰性組,P<0.05;CK-MB陽性組病變率比照CK-MB陰性組,P>0.05,符合任煥民等[6]研究結果。

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.

一次跌倒過程的持續時間一般為10 s左右,為保證時間窗截取到完整的跌倒過程,同時結合兩個數據集各自的樣本時長,我們對MobiAct采用時長為8 s的時間窗,對SisFall采用時長為12 s的時間窗。……