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Fufang Xueshuantong protects retinal vascular endothelial cells from high glucose by targeting YAP
Biomedicine & Pharmacotherapy 120 (2019) 109470
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Fufang Xueshuantong protects retinal vascular endothelial cells from high glucose by targeting YAP
T
Wei Xinga,b, Yongli Songa, Hongli Lia, Zhenglin Wanga, Yan Wuc, Chun Lid, Yong Wanga, ⁎⁎ ⁎⁎⁎ ⁎ Yonggang Liue, , Wei Wanga, , Jing Hanc, a
College of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China Tsing biomedical research center, Lanzhou University Second Hospital, Lanzhou 730030, China c Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China d Modern research center of traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China e School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing 100029, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Traditional Chinese medicine Diabetic retinopathy Endothelial cells YAP
Fufang Xueshuantong (FXST), a Chinese patent medicine, is composed of Panax notoginseng, Salviae miltiorrhizae, Astragali Radix and Radix Scrophulariae and has been found to prevent diabetic retinopathy. Yes-associated protein (YAP) participates in the pathophysiology of retinal disease and promotes endothelial cell proliferation and angiogenesis. Although it is known that YAP activity is altered by FXST, the role of YAP in mediating the effect of FXST remains unclear. In high glucose-treated retinal vascular endothelial cells (RVECs), FXST significantly reduced cell viability, the number of migrating cells and tube length in the present study. Moreover, FXST decreased the levels of YAP mRNA and protein and inhibited the expression of vascular endothelial growth factor (VEGF). Transfection of sh-YAP into the cells decreased the ability of FXST to modulate cell migration and tube formation. The effect of FXST on VEGF expression was also decreased. Similar results were obtained when the cells were stimulated with a YAP inhibitor in combination with FXST. Thus, FXST is shown to protect high glucose-injured RVECs via YAP-mediated effects.
1. Introduction Diabetes mellitus has become a significant public health issue that leads to the occurrence of diabetic retinopathy (DR) [1]. DR-induced blindness represents a disastrous complication in terms of quality of life [2]. DR is characterized by changes in retinal microvessel function and integrity, leading to progressive retinal ischaemia. At the late stage of DR, new blood vessels grow along the interface of the retina. Angiogenesis is a complex process that involves the migration, proliferation
and formation of tubes of endothelial cells [3]. Despite on-going research in the field of angiogenesis, the underlying molecular mechanisms remain unclear. Yes-associated protein (YAP) is a transcriptional coactivator and a major effector of the Hippo signalling pathway [4,5]. Based on accumulating evidence, YAP plays a prominent role in the formation and pathology of retinal vessels. YAP is ubiquitously distributed in developing mouse retinal vessels [6]. The vascular density and number of branch points are reduced in mouse retinas injected with YAP siRNA.
Abbreviations: CCK-8, cell counting kit-8; DR, diabetic retinopathy; ECs, endothelial cells; FXST, FufangXueshuantong; HPLC-MS, High-performance liquid chromatography-mass spectrometry; NG, normal glucose; HG, high glucose; ICAM, intercellular cell adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase; OD, optical density; PEDF, pigment epithelium derived factor; RVECs, retinal vascular endothelial cells; NC, negative control; TEAD, TEA domain family member; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VP, verteporfin; YAP, yes-associated protein ⁎ Corresponding author at: Institute of Chinese Medicine, Beijing University of Chinese medicine, No.11 Bei San Huan Dong Lu, Chao Yang district, Beijing 100029, China. ⁎⁎ Corresponding author at: School of Chinese Material Medica, Beijing University of Chinese Medicine, No.11 Bei San Huan Dong Lu, Chao Yang district, Beijing 100029, China. ⁎⁎⁎ Corresponding author at: College of Traditional Chinese Medicine, Beijing University of Chinese Medicine, No.11 Bei San Huan Dong Lu, Chao Yang district, Beijing 100029, China. E-mail addresses: [email protected] (W. Xing), [email protected] (Y. Song), [email protected] (H. Li), [email protected] (Z. Wang), [email protected] (Y. Wu), [email protected] (C. Li), [email protected] (Y. Wang), [email protected] (Y. Liu), [email protected] (W. Wang), [email protected] (J. Han). https://doi.org/10.1016/j.biopha.2019.109470 Received 16 April 2019; Received in revised form 14 September 2019; Accepted 16 September 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Furthermore, YAP is considered a new and attractive drug target [7]. Verteporfin disrupts YAP-TEAD (TEA domain family member) signalling and is a potent inhibitor of the proliferation of retinoblastoma cells [8] and hepatic tumours [9]. The discovery of YAP has provided new opportunities to develop novel treatments for DR; however, new therapies have not made substantial progress towards clinical use. The Fufang Xueshuantong (FXST) formula is composed of Panax notoginseng (San-Qi), Salviae miltiorrhizae (Dan-Shen), Astragali Radix (Huang-Qi) and Radix Scrophulariae (Xuan-Shen). FXST is a traditional Chinese patent medicine used for the effective treatment of vascular diseases of the fundus, including central retinal vein, central retinal artery and branch artery occlusions [10]. FXST exerts a beneficial effect on DR by improving retinal haemodynamics and visual acuity. Additionally, FXST prevents haemorrhaging and microaneurysm of the fundus in patients with DR [11,12]. In the previous animal experiments, we found that the blood flow velocity in the central retinal artery of diabetic rats decreased, the number of acellular vessels increased, and the proportion of endothelial cells increased. Meanwhile the expression of YAP protein in the retinal tissue changed. Moreover, the blood flow velocity of the central retinal artery increased in FXST group, the number of acellular vessels decreased, the proportion of endothelial cells decreased, and the expression of YAP protein was restored in the retinal tissue. Therefore, we speculated that YAP might be involved in the pathogenesis of DR and played a key role in the efficacy of FXST [13]. We designed these experiments to assess whether YAP is the drug target of FXST. First, the effect of FXST on retinal vascular endothelial cells (RVECs) was evaluated. Second, tubular network formation and cell migration were investigated after YAP knockdown with an sh-RNA plasmid. Third, the effect of FXST on the expression of YAP target genes was determined. The results show that the efficacy of FXST depends on YAP to a significant extent; i.e., YAP is the target of FXST. The findings presented in this work will assist in the identification of small molecule modulators of YAP present in FXST and may lead to exciting new approaches for DR therapy.
Table 1 Flow chart of gradient elution. Time (min)
A%
B%
0 1 8 9 12
98 85 65 40 2
2 15 35 60 98
scanning. 2.3. Cell lines and culture RF/6A cells are monkey choroid-retina (endothelial) cells that were purchased from Shanghai Institutes for Biological Sciences, CAS. RF/6A cells were cultured in RPMI 1640 medium (5.5 mM glucose, Gibco) supplemented with 10% foetal bovine serum and 1% penicillin–streptomycin in a 37 °C incubator with a 5% CO2 atmosphere. The medium was replaced every 48 h. 2.4. Cell viability The toxicity and efficacy of FXST were assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). RF/ 6A cells were seeded into 96-well plates at a density of 4000 cells per well. After starvation with fresh serum-free medium for 24 h, cells were exposed to various media for 72 h. The stimuli were 5.5 mM glucose, 25 mM glucose, or 25 mM glucose plus various concentrations of FXST. Then, the cells were cultured in serum-free medium containing 10% CCK-8. Subsequently, the plate was incubated in the dark at 37 °C for 2 h. Finally, the optical density (OD) was measured at 450 nm using a SpectraMax M4 multiplate reader (Molecular Devices, USA). 2.5. Transwell assay
2. Materials and methods The transwell assay was performed using a 24-well plate and transwell chambers with porous membranes (Corning, N.Y., USA). The cells (4 × 105 cells/well) were seeded into the upper chamber. The inserts contained various media. Cells were incubated at 37 °C for 16 h. Then, the cells in the upper chamber were removed using cotton swabs. The cells that migrated to the bottom of the inserts were fixed with paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI). The migrated cells were imaged with an inverted fluorescence microscope and counted using Image-Pro Plus software.
2.1. Plant material and preparation of FXST extracts P. notoginseng, S. miltiorrhizae, Astragali Radix and Radix Scrophulariae were obtained from Tongrentang (Beijing, China). One of the authors (Y.G. Liu) authenticated the processed Chinese medicinal materials. The plant specimens (Nos. FXST1801, 1802, 1803, and 1804) were preserved in the YIFU Research Building of Beijing University of Chinese Medicine. According to the Pharmacopoeia, P. notoginseng, S. miltiorrhizae, Astragali Radix and Radix Scrophulariae were extracted for 2 h under reflux with water. The plants were extracted twice, and the extracts were pooled and concentrated under a vacuum at 60 °C.
2.6. Tube formation Cells (2 × 105 cells/well) were seeded into a 96-well plate coated with Matrigel (Corning, NY, USA). Various media were added to the plate and incubated with the cells at 37 °C for 12 h. Then, the tubes were imaged using an inverted microscope. The length of the tube network was calculated using ImageJ software.
2.2. Mass spectrometry conditions Chromatographic conditions: A Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm ×1.7 μm) [column temperature: 25 °C; sample chamber temperature: 4 °C; injection volume: 2 μL; flow rate: 0.3 mL/min; A (0.1% formic acid water): mobile phase B (acetonitrile)] was used for gradient elution, and the flow chart is shown in Table 1. The HESI ion source was set to the negative ion detection mode. The sheath gas flow rate was 40 units and auxiliary gas flow rate was 20 units. The following parameters were also used: ionization source voltage: -4 KV; ion source temperature: 350 °C; capillary voltage: −35 V; lens voltage: −110 V; Fourier transform high-resolution full scan (FT); mass scan range m/z 150–1500; detection resolution: 30,000; and second- and third-order mass spectrometry using data-dependent scanning. Three peaks with the greatest abundance in the previous stage were selected for collision-induced dissociation (CID) fragment
2.7. Scratch test Cells were seeded evenly in 6-well plates at a density of 2 × 105 cells/well. After starvation for 24 h, the cells were scratched with a 10 μL tip, washed with PBS and stimulated with various media at 37 °C for 72 h. Images of the scratches were captured at 0 h and 72 h to record cell confluence. The average widths of the scratch wounds were analysed using ImageJ software. The migration distance was calculated as the width of the scratch at 72 h minus the width of the scratch at 0 h [14]. 2
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Fig. 1. The MS analysis of FXST. Fig. 2. The effect of FXST on the viability of RF/6A cells. (A) FXST (0.9-0.3 mg/mL) was not toxic to RF/ 6A cells. * p < 0.05 and ** p < 0.01 compared with the NG group. (B) FXST inhibited the viability of RF/ 6A cells compared with the HG group. * p < 0.05 and *** p < 0.001 compared with the HG group. All data are presented as means ± SEM (n = 5–6).
were detected using a gel imager (Bio-Rad, USA).
2.8. Transient transfection The plasmid used to express sh-YAP was a gift from Dr. Zengqiang Yuan (State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, China). RF/6A cells (2.5 × 105 cells/well) were plated in 6-well plates and allowed to grow for 24 h to reach 90% confluence. Before transfection, cells were incubated with Opti-MEM medium (Life Technologies, USA) for 30 min. The sh-YAP plasmid was mixed with Opti-MEM medium and Lipofectamine 3000 reagent (Life Technologies, USA). Then, the cells were transfected with 1 μg of plasmid for 4–6 h at 37 °C. It was 48 h after transfection, the cells were used for subsequent experiments. The transfected cells were used to evaluate efficacy.
2.10. Real-Time PCR RF/6A cells were seeded in 6-well plates and incubated with various media for 72 h. After two washes with PBS, the total RNA was extracted from the cells using Trizol reagent (Invitrogen, USA). The RNA concentration was determined using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA). Then, the RNA was reverse transcribed into cDNAs using a reverse transcription kit (Roche, Switzerland). The specific oligonucleotide primers were YAP, 5′-TGG AACATGGCAGAAAGACTGA-3′ and 5′-AGCACTGAATAT TGCC ACCCA3′; the primers for β-actin, the housekeeping gene, were 5′-TCATTCC AAATATGAGATGCGTTGT-3′ and 5′-TAGAGAGAAGTGGGGTGGCT-3′. The cDNA templates (1 μL) were mixed with 5 μL of Fast Start Universal SYBR Green master mix, 3.4 μL of deionized H2O and 0.6 μL of primers. The fold changes in gene expression were calculated using the 2−ΔΔCt method based on the cycle threshold.
2.9. Western blot RF/6A cells were seeded into 6-well plates and incubated with various media for 72 h before being lysed with a lysis buffer to extract proteins. Thirty micrograms of protein was loaded onto NuPAGE gels (Life Technologies, USA) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% non-fat milk for 1 h at 37 °C and incubated with anti-YAP (1:1000, 13584-1-AP, Proteintech, China), anti-vascular endothelial growth factor (VEGF, 1:1000, 19003-1-AP, Proteintech, China) or anti-vascular endothelial growth factor receptor (VEGFR, 1:1000, ab39256, Abcam, UK) and anti-β-actin (1:2000, sc-47778, Santa Cruz, USA) primary antibodies overnight at 4 °C. Membranes were washed with TBST and incubated with secondary antibodies (1:20000, ab16284, Abcam, UK) for 1 h at room temperature. After washes with TBST, the immunoreactive bands
2.11. Statistical analysis Statistical analyses were performed using one-way analysis of variance ANOVA (SPSS software, version 20.0, SPSS Inc., Chicago, IL), and the results are reported as means ± standard deviations. P < 0.05 was considered statistically significant.
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Fig. 3. FXST inhibited cell migration and tube formation. (A and D) The effect of FXST (0.9-0.6 mg/mL) on the migration of RF/6A cells was assessed using the transwell assay. Scale bar, 200 μm. (B and E) The effect of FXST (0.9-0.6 mg/mL) on tube formation by RF/6A cells was assayed. Scale bar, 200 μm. (C and F) The effect of FXST (0.9-0.6 mg/mL) on the distance travelled by RF/6A cells was measured using the scratch assay. Scale bar, 100 μm. ** p < 0.01 and *** p < 0.001 compared with the HG group. Data are presented as means ± SEM (n = 3).
3. Results
were derived from P. notoginseng, S. miltiorrhizae and Astragali Radix.
3.1. High-performance liquid chromatography-mass spectrometry (HPLCMS) analysis of FXST
3.2. FXST inhibited cell viability
The total ion chromatogram is shown in Fig. 1. A comparison of the excimer ions with the reference spectra identified the following compounds: tanshinone IIB or isotanshinone IIB, methyl salicylate, notoginsenoside A, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rg1, salvianolic acid B, and astragaloside, among others. These compounds
Initially, the toxicity of FXST was evaluated. Cells were stimulated with NG or NG plus various concentrations of FXST for 72 h. FXST did not affect cell viability at concentrations less than or equal to 0.9 mg/ mL (Fig. 2A) compared with that in the NG group. Next, the effect of FXST on the RF/6A cells was investigated. Cells were cultured with NG, HG or HG plus FXST for 72 h. Consistent with results from a previous 4
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Fig. 4. FXST reduced the levels of the YAP mRNA and protein, and VEGF and VEGFR proteins in RF/6A cells. Cells were incubated with NG, HG, or HG plus FXST for 72 h. Levels of the YAP (A and B), VEGF (D and E) and VEGFR (F and G) proteins were evaluated using Western blot, and the expression of the YAP mRNA was evaluated using qRT-PCR (C). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the HG group. Data are presented as means ± SEM (n = 3–4).
3.4. FXST inhibited tube formation
study [13], HG increased the cell viability (p < 0.05) compared with that in the NG group. Additionally, FXST (0.9 mg/mL) decreased the cell viability compared with that in the HG treatment group (p < 0.001, Fig. 2B).
Tube formation plays an important role in the formation of new blood vessels. The effect of FXST on the length of the endothelial tube was examined under high glucose conditions. The length of the tubules in the HG group was increased compared with that in the NG group (p < 0.001). The length of the tubules in the FXST group was significantly decreased compared with that in the HG group (p < 0.01 or p < 0.001, Fig. 3B and E). Thus, FXST controls tube formation by endothelial cells.
3.3. FXST suppressed cell migration Cell migration is one of the signs of neovascularization [15]. The chamber assay and scratch test were used to assess the effect of FXST on cell migration. According to the results of the chamber assay, the relative numbers of migrated cells in the HG group increased compared with those in the NG group (p < 0.001). FXST decreased the relative number of migrated cells (p < 0.01 or p < 0.001) in a dose-dependent manner (Fig. 3A and D) compared with those in the HG group. The maximum percent inhibition was 128.89%. In the HG group, the distance travelled and motility were increased compared with that in the NG group in the scratch test (p < 0.001). Furthermore, the FXST treatment reduced cell motility and decreased the distance travelled by 89.67% (p < 0.001, Fig. 3C and F). The data from the chamber assay and the scratch test confirmed that FXST attenuates the migration of RF/6A cells.
3.5. FXST reduced the expression of the YAP mRNA and protein After evaluating the pharmacological activity, the molecular mechanism of FXST was further explored. According to the PCR and WB results, HG increased the expression of YAP mRNA and protein (p < 0.05 and p < 0.001, respectively). As expected, FXST decreased the levels of YAP (p < 0.05 and p < 0.001, respectively; Fig. 4A–C).
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Fig. 5. YAP expression was significantly decreased in transfected cells. (A) The sh-YAP plasmid was transfected into RF/6A cells. GFP fluorescence was observed in the cells under a microscope; scale bar, 100 μm. After plasmid transfection, the levels of the YAP mRNA (B) and protein (C and D) were decreased. * p < 0.05 and ** p < 0.01 compared with the NG group. Levels of the VEGF (E and F) and VEGFR (G and H) proteins were decreased. * p < 0.05 compared with the NG + sh-NC group. The data are presented as means ± SEM (n = 3–4).
(p < 0.05, Fig. 5A–D). Then, the levels of VEGF and VEGFR were examined. In sh-YAPtransfected cells, levels of the VEGF protein were significantly decreased (p < 0.05) and the VEGFR levels were slightly reduced compared with those in the sh-NC group (Fig. 5E–H). The effect of YAP on cell migration was also determined. After transfection, cells were stimulated with NG or HG for 24 h. Cell migration was assessed using the transwell assay. In the presence of NG and HG, the sh-YAP group displayed a slight decrease in cell migration compared with that in the sh-NC group (Fig. 6A–D). A tube formation assay was also performed. In the NG, sh-YAP decreased the tube length by 44.74% compared with that in the sh-NC group (p < 0.05, Fig. 7A and B). In the HG, the tube length was decreased by 20.43% in the sh-YAP group (p < 0.01, Fig. 7C and D). Thus, YAP silencing decreased cell migration, the tube length and VEGF expression. Based on these data, YAP induces the migration and tube formation of RVECs through a process mediated by VEGF.
3.6. FXST decreased the expression of VEGF and VEGFR VEGF and VEGFR play critical roles in angiogenesis [16]. Thus, the effects of FXST on VEGF and VEGFR expression were investigated. HG substantially decreased the levels of VEGF and VEGFR compared with the NG group (p < 0.05 and p < 0.001, respectively). The levels of the VEGF and VEGFR proteins were decreased in the FXST group compared with those in the HG group (p < 0.05 and p < 0.001, respectively; Fig. 4D–G). Based on these results, FXST modulates the VEGF-VEGFR signalling pathway to inhibit angiogenesis.
3.7. YAP induced cell migration and tube formation The effects of YAP on cell migration and tube formation were examined to further investigate the role of YAP in mediating the effects of FXST. RF/6A cells were transfected with the sh-YAP plasmid or negative control (NC). The levels of the YAP mRNA and protein were reduced by 50.77% and 50.43%, respectively, in the sh-YAP group 6
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Fig. 6. The effect of FXST on cell migration strongly depended on YAP. (A and B) In normal glucose medium, the number of migrating cells was reduced in the NG + sh-YAP group. (C and D) The number of migrating cells was reduced in the HG + sh-YAP group. (E and F) YAP gene silencing in transfected cells decreased the number of migrating cells in the HG +0.9 mg/mL FXST group compared with the HG +0.9 mg/mL FXST + sh-YAP group. *** p < 0.001 compared with the HG group. ### p < 0.001 compared with the HG +0.9 mg/mL FXST group. Data are presented as means ± SEM (n = 3); scale bar, 200 μm.
3.9. YAP silencing blocked the ability of FXST to reduce VEGF levels
3.8. YAP silencing disrupted the effects of FXST on cell migration and tube formation
WB experiments were performed to determine whether the FXSTinduced downregulation of VEGF and VEGFR was mediated by YAP. YAP expression was knocked down in RF/6A cells. Cells transfected with an empty vector served as the control. In cells transfected with shYAP, the ability of FXST to reduce VEGF expression was blocked (Fig. 8A–C). Together with the data presented in Fig. 5E, these results suggest that YAP is an upstream mediator of VEGF and that the FXSTdependent downregulation of VEGF is mediated by YAP.
Cells were transiently transfected with the sh-YAP plasmid or negative control vector (sh-NC) to assess the role of YAP in mediating the effects of FXST. Then, cells were treated with HG plus FXST. Transwell migration and tube formation assays were performed. In wild-type cells, FXST decreased the migration and tube length (p < 0.001). However, YAP silencing in RF/6A cells significantly suppressed the effects of FXST (p < 0.05 and p < 0.001, respectively). In the sh-YAP plasmidtransfected cells, FXST no longer decreased cell migration (Fig. 6E and F) or tube formation (Fig. 7E and F). Thus, the effect of FXST strongly depends on YAP.
3.10. YAP inhibition blocked the ability of FXST to reduce cell migration and tube formation A YAP inhibitor, verteporfin (VP), was used to determine whether 7
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Fig. 7. YAP silencing inhibited the FXST-induced decrease in tube formation. The sh-YAP or NC plasmid was transfected into RF/6A cells. (A and B) Cells were cultured in the presence of NG, and YAP silencing decreased the total tube length. * p < 0.05 compared with the NG group. (C and D) Cells were cultured in the presence of HG, and YAP silencing reduced the total tube length; * p < 0.05 compared with the HG + shNC group. (E and F) Cells were stimulated with NG, HG or HG plus FXST. In wild-type cells, FXST reduced the tube length. However, in the sh-YAP-transfected cells, the effect of FXST effect was inhibited. * p < 0.05 and *** p < 0.001 compared with the HG group. # p < 0.05 compared with the HG +0.9 mg/mL FXST group. Data are presented as means ± SEM (n = 3); scale bar, 200 μm.
significantly decreased YAP and VEGF expression. (3) YAP suppressed tube formation and VEGF expression in the presence of high glucose. (4) After YAP activity was blocked with an sh-RNA plasmid or inhibitor, FXST-mediated protection from high glucose was abolished. These data provide additional insights into the important role of YAP in retinal diseases and the therapeutic effect of FXST on DR.
reduced YAP activity affected FXST-mediated inhibition of cell migration and tube formation and further investigate the role of YAP in mediating the effects of FXST. VP significantly decreased YAP activity without inducing substantial changes in cell viability (Supplementary Fig. 1A–C). Additionally, FXST treatment impeded cell migration and tube formation; however, treatment with VP dramatically inhibited the effect of FXST (p < 0.01, Fig. 9A–D). Thus, the control of cell migration and tube formation by FXST is mediated by the suppression of YAP activity.
4.1. FXST ameliorated the function of RVECs induced by HG A number of published studies have confirmed the beneficial effects of FXST on patients and animal models of DR. Previous studies have described the effect of FXST on angiogenesis. According to Luo et al., FXST attenuates the VEGF-induced tube formation and migration of endothelial cells and retinal pigment epithelial cells [17]. Luo and colleagues also showed that FXST suppresses angiogenesis in the chick embryo chorioallantoic membrane [18]. Consistent with the literature,
4. Discussion As shown in the present study, a FXST treatment significantly ameliorates high glucose-induced injury in RVECs by targeting YAP. The key findings are listed below. (1) FXST reduced migration and the tube length by 128.89% and 55.64%, respectively. (2) FXST 8
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Fig. 8. YAP silencing suppressed the FXSTinduced reduction in VEGF expression. The effect of FXST on the levels of VEGF (A and C) and VEGFR (B and C) in the sh-YAP or sh-NC transfected cells. The inhibitory effect of FXST on VEGF expression was attenuated when the YAP gene was silenced. However, significant differences in the levels of the VEGFR protein were not observed between the groups. Data are presented as means ± SEM (n = 3).
Fig. 9. VP attenuated the FXST-induced inhibition of cell migration and tube formation. (A and B) FXST reduced the number of migrating cells; however, VP inhibited the effect of FXST. Scale bar, 200 μm. (C and D) FXST decreased the tube length; however, VP inhibited the effect of FXST. *** p < 0.001 compared with the HG group. ## p < 0.01 compared with the HG +0.9 mg/mL FXST + VP group; scale bar 200 μm. Data are presented as means ± SEM (n = 3).
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5. Conclusions
FXST decreased the number of migrating cells, migration distance and tube length in the presence of high glucose in our study. Thus, the effect of FXST on DR is mediated by inhibiting angiogenesis.
FXST suppressed HG-induced cell migration and tube formation. Moreover, the effect of FXST was blocked by sh-YAP or a YAP inhibitor. Thus, the protective effect of FXST on high glucose-induced damage appears to be mediated by YAP. These findings provide deeper insights into the mechanisms underlying the effects of FXST and lay the foundation for its further development.
4.2. The bioactive components of FXST include saponins and phenanthraquinone Additionally, the study was designed to determine the bioactive components of FXST. Our HPLC-MS results identified a series of representative components of FXST, including ginsenosides Rb1, tanshinone IIA and astragaloside IV. In recent years, the chemical composition of FXST has been analysed. In 2016, Jiang reported 14 types of notoginsenosides in FXST, of which notoginsenosides R1 and ginsenosides Rb1 were identified in the rat eye [19]. Moreover, Rb1 inhibits the VEGF-induced proliferation of retinal pigment epithelial cells [20]. Tanshinone IIA markedly suppresses the high glucose-induced proliferation, migration and vascularization of human retinal endothelial cells [21]. Astragaloside IV exerts a significant inhibitory effect on the proliferation and migration of vascular smooth muscle cells [22]. As shown in the study by Jian, a combination of the main constituents of FXST, including saponins, harpagoside, cryptotanshinone, and tanshinone I, decreases the number of acellular vessels and pericyte loss in diabetic retinas [23]. In general, researchers have postulated that saponins and phenanthraquinone are the bioactive components of FXST.
Funding This study was financially supported by grants from the National Natural Science Foundation of China [grant numbers 81673705 and 81873165]. Authors’ contributions HJ, WW and LYG conceived and designed the experiments. XW, SYL, LHL and WZL performed the experiments. WY and LC analysed the data. XW, HJ and LYG contributed to drafting the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors have no competing interests to declare.
4.3. FXST inhibited cell migration and tube formation by targeting YAP
Appendix A. Supplementary data
Numerous studies have investigated the pharmacological mechanisms of FXST. FXST significantly decreases the levels of VEGF, intercellular adhesion molecule 1 (ICAM1), interleukin 1β (IL1β), matrix metalloproteinase 2 (MMP2) and apoptosis [24]. Duan revealed that FXST increases the expression of occludin and pigment epithelium-derived factor (PEDF) in the retinas of diabetic rats [25]. However, these studies provide only circumstantial evidence. YAP activity was blocked with a plasmid or an inhibitor in the present study to clarify the detailed mechanism of FXST. The effects FXST were found to be dependent on YAP activity. In wild-type cells, FXST reduced migration and tube formation. In contrast, in the sh-YAP-transfected cells, the effects of FXST were reversed. Moreover, when YAP activity was suppressed by an inhibitor, FXST no longer controlled RVEC function. Additionally, the FXST-induced inhibition of VEGF expression was blocked by YAP silencing. Thus, FXST inhibited cell migration and tube formation by targeting YAP. However, all of our experiments were performed in vitro, and thus, the role of YAP in mediating the effects of FXST must be confirmed in vivo in subsequent studies. This study also would contribute to understanding the function of YAP. YAP has been extensively studied in cancer. It is an oncogene that promotes cell proliferation, inhibits cell apoptosis, and promotes malignant transformation of cells [26]. When YAP was overexpressed or activated, the expression of tumor markers elevated, and the incidence of cancer increased [27–29]. On the contrary, YAP knockout or inactivation inhibited cell migration [30,31] in vitro or tumor growth in vivo [27–29]. The role of YAP in endothelial cells has also been explored. When YAP was knocked down, the cell migration, proliferation, and tube formation decreased under hypoxia conditions [32–34]. During retinal angiogenesis, a loss of YAP function led to defects in the proliferation and migration of vascular endothelial cells (ECs) in a gene dosage-dependent manner [35,36]. However, it was not clear whether YAP could regulate the function of endothelial cells under high glucose. This experiment found that HG stimulated tube formation, meanwhile YAP and VEGF expression increased in RVECs. However, YAP silencing inhibited HG-induced tube formation and VEGF levels. It suggested that YAP could actually inhibit the connection of the cells by regulating VEGF expression, and eventually it might inhibit angiogenesis under high conditions.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109470. References [1] G. Liew, V.W. Wong, I.V. Ho, Mini review: changes in the incidence of and progression to proliferative and sight-threatening diabetic retinopathy over the last 30 years, Ophthalmic Epidemiol. 24 (2) (2017) 73–80. [2] J.B. Jonas, L. Xu, J. Xu, et al., Prevalence of diabetic retinopathy and vision loss in the Beijing eye study: the potential role of the cerebrospinal fluid pressure, Curr. Diab. Rep. 15 (10) (2015) 71. [3] C. Betz, A. Lenard, H.G. Belting, et al., Cell behaviors and dynamics during angiogenesis, Development 143 (13) (2016) 2249–2260. [4] N. Li, C. Xie, N. Lu, Crosstalk between Hippo signalling and miRNAs in tumour progression, FEBS J. 284 (7) (2017) 1045–1055. [5] Y. Ma, Y. Yang, F. Wang, et al., Hippo-YAP signaling pathway: a new paradigm for cancer therapy, Int. J. Cancer 137 (10) (2015) 2275–2286. [6] H.J. Choi, H. Zhang, H. Park, et al., Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2, Nat. Commun. 6 (2015) 6943. [7] F.X. Yu, B. Zhao, K.L. Guan, Hippo pathway in organ size control, tissue homeostasis, and Cancer, Cell 163 (4) (2015) 811–828. [8] K. Brodowska, A. Al-Moujahed, A. Marmalidou, et al., The clinically used photosensitizer Verteporfin (VP) inhibits YAP-TEAD and human retinoblastoma cell growth in vitro without light activation, Exp. Eye Res. 124 (2014) 67–73. [9] Y. Liu-Chittenden, B. Huang, J.S. Shim, et al., Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP, Genes Dev. 26 (12) (2012) 1300–1305. [10] J. Hong, Intervention of compound Xueshuantong capsule on diabetic retinopathy in hemorrhage period, China Journal of Traditional Chinese Medicine & Pharmacy (2010). [11] G.M. Hao, T.T. Lv, Y. Wu, et al., The Hippo signaling pathway: a potential therapeutic target is reversed by a Chinese patent drug in rats with diabetic retinopathy, BMC Complement. Altern. Med. 17 (1) (2017) 187. [12] J. Xu, B. Mei, N. Zhang, Clinical observation on early diabetic retinopathy with Compound Xueshuantong Capsule, zhong hua zhong yi yao za zhi 27 (10) (2012) 3247–3249. [13] J. Du, X. Chai, J. Cheng, et al., The review of ophthalmic clinical use of Fufang xueshuantong, Zhong Hua Lin Chuang Yi Shi Za Zhi 6 (7) (2012) 1833–1834. [14] Y. Wu, Q. Zhang, R. Zhang, Kaempferol targets estrogen-related receptor alpha and suppresses the angiogenesis of human retinal endothelial cells under high glucose conditions, Exp. Ther. Med. 14 (6) (2017) 5576–5582. [15] Z. Jiang, X. Chen, Q. Zhou, et al., Downregulated LRRK2 gene expression inhibits proliferation and migration while promoting the apoptosis of thyroid cancer cells by inhibiting activation of the JNK signaling pathway, Int. J. Oncol. 55 (1) (2019) 21–34. [16] P.N. Bishop, The role of extracellular matrix in retinal vascular development and preretinal neovascularization, Exp. Eye Res. 133 (2015) 30–36.
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W. Xing, et al.
[27] F.D. Camargo, S. Gokhale, J.B. Johnnidis, et al., YAP1 increases organ size and expands undifferentiated progenitor cells, Curr. Biol. 17 (23) (2007) 2054–2060. [28] J. Dong, G. Feldmann, J. Huang, et al., Elucidation of a universal size-control mechanism in Drosophila and mammals, Cell 130 (6) (2007) 1120–1133. [29] M. Mohseni, J. Sun, A. Lau, et al., A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway, Nat. Cell Biol. 16 (1) (2013) 108–117. [30] T.S. Eisinger-Mathason, V. Mucaj, K.M. Biju, et al., Deregulation of the Hippo pathway in soft-tissue sarcoma promotes FOXM1 expression and tumorigenesis, Proc. Natl. Acad. Sci. 112 (26) (2015) 3402–3411. [31] X. Wang, R. Zhang, L. Gu, et al., Cell-based screening identifies the active ingredients from traditional chinese medicine formula shixiao san as the inhibitors of atherosclerotic endothelial dysfunction, PLoS One 10 (2) (2015) e0116601. [32] C. Li, J. Wang, Q. Wang, et al., Qishen granules inhibit myocardial inflammation injury through regulating arachidonic acid metabolism, Sci. Rep. 6 (2016) 36949. [33] Y.G. Guan, J.B. Liao, K.Y. Li, et al., Potential mechanisms of an antiadenomyosis chinese herbal formula shaoyao-gancao decoction in primary cell culture model, Evid. Complement. Alternat. Med. 2014 (1) (2014) 982913. [34] Y.T. Qing, S. Ji, et al., YAP knockdown in gastric cancer-derived mesenchymal stem cell inhibits the progression of gastric cancer, J. Jiangsu Univ. (Med. Ed.) 27 (5) (2017) 374–378. [35] M. Sakabe, J. Fan, Y. Odaka, et al., YAP/TAZ-CDC42 signaling regulates vascular tip cell migration, Proc. Natl. Acad. Sci. U. S. A. 114 (41) (2017) 10918. [36] M. Zhu, X. Liu, Y. Wang, et al., YAP via interacting with STAT3 regulates VEGFinduced angiogenesis in human retinal microvascular endothelial cells, Exp. Cell Res. 373 (1-2) (2018) 155–163, https://doi.org/10.1016/j.yexcr.2018.10.007 Epub 2018 Oct 17.
[17] M. Wu, H. Xiong, Y. Xu, et al., Association between VEGF-A and VEGFR-2 polymorphisms and response to treatment of neovascular AMD with anti-VEGF agents: a meta-analysis, Br. J. Ophthalmol. 101 (7) (2017) 976–984. [18] X. Luo, X. Wu, Q. Gu, et al., Study on complex Xueshuantong interfering on choroidal neovascularization concerned with RPEcells, Modern J. Int. Trad. Chinese Western Med. 19 (33) (2010) 4241–4245. [19] M.F. Jiang, Q.H. Wang, H.H. Pang, et al., [Distribution of chemical compounds of Fufang Xueshuantong capsule in eyes and kidney of beagle dogs], Zhongguo Zhong Yao Za Zhi 41 (20) (2016) 3846–3851. [20] B.S. Betts, K. Parvathaneni, B.B. Yendluri, et al., Ginsenoside-Rb1 induces ARPE-19 proliferation and reduces VEGF release, ISRN Ophthalmol. 2011 (2011) 184295. [21] K. Fan, S. Li, G. Liu, et al., Tanshinone IIA inhibits high glucoseinduced proliferation, migration and vascularization of human retinal endothelial cells, Mol. Med. Rep. 16 (6) (2017) 9023–9028. [22] Z. Chen, Y. Cai, W. Zhang, et al., Astragaloside IV inhibits platelet-derived growth factor-BB-stimulated proliferation and migration of vascular smooth muscle cells via the inhibition of p38 MAPK signaling, Exp. Ther. Med. 8 (4) (2014) 1253–1258. [23] W. Jian, S. Yu, M. Tang, et al., A combination of the main constituents of Fufang Xueshuantong Capsules shows protective effects against streptozotocin-induced retinal lesions in rats, J. Ethnopharmacol. 182 (2016) 50–56. [24] J. Du, H. Sun, Cheng J. The effect of fufang xueshuantong on diabetic retinopathy: a review, Zhong Hua Lin Chuang Yi Shi Za Zhi 6 (22) (2012) 7373–7375. [25] H. Duan, J. Huang, W. Li, et al., Protective effects of fufang xueshuantong on diabetic retinopathy in rats, Evid. Complement. Alternat. Med. 2013 (2013) 408268. [26] M. Liu, S. Zhao, Q. Lin, et al., YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues, Mol. Cell. Biol. 38 (5) (2015) 1449–1461.
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Fufang Xueshuantong protects retinal vascular endothelial cells from high glucose by targeting YAP
T
Wei Xinga,b, Yongli Songa, Hongli Lia, Zhenglin Wanga, Yan Wuc, Chun Lid, Yong Wanga, ⁎⁎ ⁎⁎⁎ ⁎ Yonggang Liue, , Wei Wanga, , Jing Hanc, a
College of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China Tsing biomedical research center, Lanzhou University Second Hospital, Lanzhou 730030, China c Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China d Modern research center of traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China e School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing 100029, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Traditional Chinese medicine Diabetic retinopathy Endothelial cells YAP
Fufang Xueshuantong (FXST), a Chinese patent medicine, is composed of Panax notoginseng, Salviae miltiorrhizae, Astragali Radix and Radix Scrophulariae and has been found to prevent diabetic retinopathy. Yes-associated protein (YAP) participates in the pathophysiology of retinal disease and promotes endothelial cell proliferation and angiogenesis. Although it is known that YAP activity is altered by FXST, the role of YAP in mediating the effect of FXST remains unclear. In high glucose-treated retinal vascular endothelial cells (RVECs), FXST significantly reduced cell viability, the number of migrating cells and tube length in the present study. Moreover, FXST decreased the levels of YAP mRNA and protein and inhibited the expression of vascular endothelial growth factor (VEGF). Transfection of sh-YAP into the cells decreased the ability of FXST to modulate cell migration and tube formation. The effect of FXST on VEGF expression was also decreased. Similar results were obtained when the cells were stimulated with a YAP inhibitor in combination with FXST. Thus, FXST is shown to protect high glucose-injured RVECs via YAP-mediated effects.
1. Introduction Diabetes mellitus has become a significant public health issue that leads to the occurrence of diabetic retinopathy (DR) [1]. DR-induced blindness represents a disastrous complication in terms of quality of life [2]. DR is characterized by changes in retinal microvessel function and integrity, leading to progressive retinal ischaemia. At the late stage of DR, new blood vessels grow along the interface of the retina. Angiogenesis is a complex process that involves the migration, proliferation
and formation of tubes of endothelial cells [3]. Despite on-going research in the field of angiogenesis, the underlying molecular mechanisms remain unclear. Yes-associated protein (YAP) is a transcriptional coactivator and a major effector of the Hippo signalling pathway [4,5]. Based on accumulating evidence, YAP plays a prominent role in the formation and pathology of retinal vessels. YAP is ubiquitously distributed in developing mouse retinal vessels [6]. The vascular density and number of branch points are reduced in mouse retinas injected with YAP siRNA.
Abbreviations: CCK-8, cell counting kit-8; DR, diabetic retinopathy; ECs, endothelial cells; FXST, FufangXueshuantong; HPLC-MS, High-performance liquid chromatography-mass spectrometry; NG, normal glucose; HG, high glucose; ICAM, intercellular cell adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase; OD, optical density; PEDF, pigment epithelium derived factor; RVECs, retinal vascular endothelial cells; NC, negative control; TEAD, TEA domain family member; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VP, verteporfin; YAP, yes-associated protein ⁎ Corresponding author at: Institute of Chinese Medicine, Beijing University of Chinese medicine, No.11 Bei San Huan Dong Lu, Chao Yang district, Beijing 100029, China. ⁎⁎ Corresponding author at: School of Chinese Material Medica, Beijing University of Chinese Medicine, No.11 Bei San Huan Dong Lu, Chao Yang district, Beijing 100029, China. ⁎⁎⁎ Corresponding author at: College of Traditional Chinese Medicine, Beijing University of Chinese Medicine, No.11 Bei San Huan Dong Lu, Chao Yang district, Beijing 100029, China. E-mail addresses: [email protected] (W. Xing), [email protected] (Y. Song), [email protected] (H. Li), [email protected] (Z. Wang), [email protected] (Y. Wu), [email protected] (C. Li), [email protected] (Y. Wang), [email protected] (Y. Liu), [email protected] (W. Wang), [email protected] (J. Han). https://doi.org/10.1016/j.biopha.2019.109470 Received 16 April 2019; Received in revised form 14 September 2019; Accepted 16 September 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Furthermore, YAP is considered a new and attractive drug target [7]. Verteporfin disrupts YAP-TEAD (TEA domain family member) signalling and is a potent inhibitor of the proliferation of retinoblastoma cells [8] and hepatic tumours [9]. The discovery of YAP has provided new opportunities to develop novel treatments for DR; however, new therapies have not made substantial progress towards clinical use. The Fufang Xueshuantong (FXST) formula is composed of Panax notoginseng (San-Qi), Salviae miltiorrhizae (Dan-Shen), Astragali Radix (Huang-Qi) and Radix Scrophulariae (Xuan-Shen). FXST is a traditional Chinese patent medicine used for the effective treatment of vascular diseases of the fundus, including central retinal vein, central retinal artery and branch artery occlusions [10]. FXST exerts a beneficial effect on DR by improving retinal haemodynamics and visual acuity. Additionally, FXST prevents haemorrhaging and microaneurysm of the fundus in patients with DR [11,12]. In the previous animal experiments, we found that the blood flow velocity in the central retinal artery of diabetic rats decreased, the number of acellular vessels increased, and the proportion of endothelial cells increased. Meanwhile the expression of YAP protein in the retinal tissue changed. Moreover, the blood flow velocity of the central retinal artery increased in FXST group, the number of acellular vessels decreased, the proportion of endothelial cells decreased, and the expression of YAP protein was restored in the retinal tissue. Therefore, we speculated that YAP might be involved in the pathogenesis of DR and played a key role in the efficacy of FXST [13]. We designed these experiments to assess whether YAP is the drug target of FXST. First, the effect of FXST on retinal vascular endothelial cells (RVECs) was evaluated. Second, tubular network formation and cell migration were investigated after YAP knockdown with an sh-RNA plasmid. Third, the effect of FXST on the expression of YAP target genes was determined. The results show that the efficacy of FXST depends on YAP to a significant extent; i.e., YAP is the target of FXST. The findings presented in this work will assist in the identification of small molecule modulators of YAP present in FXST and may lead to exciting new approaches for DR therapy.
Table 1 Flow chart of gradient elution. Time (min)
A%
B%
0 1 8 9 12
98 85 65 40 2
2 15 35 60 98
scanning. 2.3. Cell lines and culture RF/6A cells are monkey choroid-retina (endothelial) cells that were purchased from Shanghai Institutes for Biological Sciences, CAS. RF/6A cells were cultured in RPMI 1640 medium (5.5 mM glucose, Gibco) supplemented with 10% foetal bovine serum and 1% penicillin–streptomycin in a 37 °C incubator with a 5% CO2 atmosphere. The medium was replaced every 48 h. 2.4. Cell viability The toxicity and efficacy of FXST were assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). RF/ 6A cells were seeded into 96-well plates at a density of 4000 cells per well. After starvation with fresh serum-free medium for 24 h, cells were exposed to various media for 72 h. The stimuli were 5.5 mM glucose, 25 mM glucose, or 25 mM glucose plus various concentrations of FXST. Then, the cells were cultured in serum-free medium containing 10% CCK-8. Subsequently, the plate was incubated in the dark at 37 °C for 2 h. Finally, the optical density (OD) was measured at 450 nm using a SpectraMax M4 multiplate reader (Molecular Devices, USA). 2.5. Transwell assay
2. Materials and methods The transwell assay was performed using a 24-well plate and transwell chambers with porous membranes (Corning, N.Y., USA). The cells (4 × 105 cells/well) were seeded into the upper chamber. The inserts contained various media. Cells were incubated at 37 °C for 16 h. Then, the cells in the upper chamber were removed using cotton swabs. The cells that migrated to the bottom of the inserts were fixed with paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI). The migrated cells were imaged with an inverted fluorescence microscope and counted using Image-Pro Plus software.
2.1. Plant material and preparation of FXST extracts P. notoginseng, S. miltiorrhizae, Astragali Radix and Radix Scrophulariae were obtained from Tongrentang (Beijing, China). One of the authors (Y.G. Liu) authenticated the processed Chinese medicinal materials. The plant specimens (Nos. FXST1801, 1802, 1803, and 1804) were preserved in the YIFU Research Building of Beijing University of Chinese Medicine. According to the Pharmacopoeia, P. notoginseng, S. miltiorrhizae, Astragali Radix and Radix Scrophulariae were extracted for 2 h under reflux with water. The plants were extracted twice, and the extracts were pooled and concentrated under a vacuum at 60 °C.
2.6. Tube formation Cells (2 × 105 cells/well) were seeded into a 96-well plate coated with Matrigel (Corning, NY, USA). Various media were added to the plate and incubated with the cells at 37 °C for 12 h. Then, the tubes were imaged using an inverted microscope. The length of the tube network was calculated using ImageJ software.
2.2. Mass spectrometry conditions Chromatographic conditions: A Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm ×1.7 μm) [column temperature: 25 °C; sample chamber temperature: 4 °C; injection volume: 2 μL; flow rate: 0.3 mL/min; A (0.1% formic acid water): mobile phase B (acetonitrile)] was used for gradient elution, and the flow chart is shown in Table 1. The HESI ion source was set to the negative ion detection mode. The sheath gas flow rate was 40 units and auxiliary gas flow rate was 20 units. The following parameters were also used: ionization source voltage: -4 KV; ion source temperature: 350 °C; capillary voltage: −35 V; lens voltage: −110 V; Fourier transform high-resolution full scan (FT); mass scan range m/z 150–1500; detection resolution: 30,000; and second- and third-order mass spectrometry using data-dependent scanning. Three peaks with the greatest abundance in the previous stage were selected for collision-induced dissociation (CID) fragment
2.7. Scratch test Cells were seeded evenly in 6-well plates at a density of 2 × 105 cells/well. After starvation for 24 h, the cells were scratched with a 10 μL tip, washed with PBS and stimulated with various media at 37 °C for 72 h. Images of the scratches were captured at 0 h and 72 h to record cell confluence. The average widths of the scratch wounds were analysed using ImageJ software. The migration distance was calculated as the width of the scratch at 72 h minus the width of the scratch at 0 h [14]. 2
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Fig. 1. The MS analysis of FXST. Fig. 2. The effect of FXST on the viability of RF/6A cells. (A) FXST (0.9-0.3 mg/mL) was not toxic to RF/ 6A cells. * p < 0.05 and ** p < 0.01 compared with the NG group. (B) FXST inhibited the viability of RF/ 6A cells compared with the HG group. * p < 0.05 and *** p < 0.001 compared with the HG group. All data are presented as means ± SEM (n = 5–6).
were detected using a gel imager (Bio-Rad, USA).
2.8. Transient transfection The plasmid used to express sh-YAP was a gift from Dr. Zengqiang Yuan (State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, China). RF/6A cells (2.5 × 105 cells/well) were plated in 6-well plates and allowed to grow for 24 h to reach 90% confluence. Before transfection, cells were incubated with Opti-MEM medium (Life Technologies, USA) for 30 min. The sh-YAP plasmid was mixed with Opti-MEM medium and Lipofectamine 3000 reagent (Life Technologies, USA). Then, the cells were transfected with 1 μg of plasmid for 4–6 h at 37 °C. It was 48 h after transfection, the cells were used for subsequent experiments. The transfected cells were used to evaluate efficacy.
2.10. Real-Time PCR RF/6A cells were seeded in 6-well plates and incubated with various media for 72 h. After two washes with PBS, the total RNA was extracted from the cells using Trizol reagent (Invitrogen, USA). The RNA concentration was determined using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA). Then, the RNA was reverse transcribed into cDNAs using a reverse transcription kit (Roche, Switzerland). The specific oligonucleotide primers were YAP, 5′-TGG AACATGGCAGAAAGACTGA-3′ and 5′-AGCACTGAATAT TGCC ACCCA3′; the primers for β-actin, the housekeeping gene, were 5′-TCATTCC AAATATGAGATGCGTTGT-3′ and 5′-TAGAGAGAAGTGGGGTGGCT-3′. The cDNA templates (1 μL) were mixed with 5 μL of Fast Start Universal SYBR Green master mix, 3.4 μL of deionized H2O and 0.6 μL of primers. The fold changes in gene expression were calculated using the 2−ΔΔCt method based on the cycle threshold.
2.9. Western blot RF/6A cells were seeded into 6-well plates and incubated with various media for 72 h before being lysed with a lysis buffer to extract proteins. Thirty micrograms of protein was loaded onto NuPAGE gels (Life Technologies, USA) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% non-fat milk for 1 h at 37 °C and incubated with anti-YAP (1:1000, 13584-1-AP, Proteintech, China), anti-vascular endothelial growth factor (VEGF, 1:1000, 19003-1-AP, Proteintech, China) or anti-vascular endothelial growth factor receptor (VEGFR, 1:1000, ab39256, Abcam, UK) and anti-β-actin (1:2000, sc-47778, Santa Cruz, USA) primary antibodies overnight at 4 °C. Membranes were washed with TBST and incubated with secondary antibodies (1:20000, ab16284, Abcam, UK) for 1 h at room temperature. After washes with TBST, the immunoreactive bands
2.11. Statistical analysis Statistical analyses were performed using one-way analysis of variance ANOVA (SPSS software, version 20.0, SPSS Inc., Chicago, IL), and the results are reported as means ± standard deviations. P < 0.05 was considered statistically significant.
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Fig. 3. FXST inhibited cell migration and tube formation. (A and D) The effect of FXST (0.9-0.6 mg/mL) on the migration of RF/6A cells was assessed using the transwell assay. Scale bar, 200 μm. (B and E) The effect of FXST (0.9-0.6 mg/mL) on tube formation by RF/6A cells was assayed. Scale bar, 200 μm. (C and F) The effect of FXST (0.9-0.6 mg/mL) on the distance travelled by RF/6A cells was measured using the scratch assay. Scale bar, 100 μm. ** p < 0.01 and *** p < 0.001 compared with the HG group. Data are presented as means ± SEM (n = 3).
3. Results
were derived from P. notoginseng, S. miltiorrhizae and Astragali Radix.
3.1. High-performance liquid chromatography-mass spectrometry (HPLCMS) analysis of FXST
3.2. FXST inhibited cell viability
The total ion chromatogram is shown in Fig. 1. A comparison of the excimer ions with the reference spectra identified the following compounds: tanshinone IIB or isotanshinone IIB, methyl salicylate, notoginsenoside A, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rg1, salvianolic acid B, and astragaloside, among others. These compounds
Initially, the toxicity of FXST was evaluated. Cells were stimulated with NG or NG plus various concentrations of FXST for 72 h. FXST did not affect cell viability at concentrations less than or equal to 0.9 mg/ mL (Fig. 2A) compared with that in the NG group. Next, the effect of FXST on the RF/6A cells was investigated. Cells were cultured with NG, HG or HG plus FXST for 72 h. Consistent with results from a previous 4
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Fig. 4. FXST reduced the levels of the YAP mRNA and protein, and VEGF and VEGFR proteins in RF/6A cells. Cells were incubated with NG, HG, or HG plus FXST for 72 h. Levels of the YAP (A and B), VEGF (D and E) and VEGFR (F and G) proteins were evaluated using Western blot, and the expression of the YAP mRNA was evaluated using qRT-PCR (C). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the HG group. Data are presented as means ± SEM (n = 3–4).
3.4. FXST inhibited tube formation
study [13], HG increased the cell viability (p < 0.05) compared with that in the NG group. Additionally, FXST (0.9 mg/mL) decreased the cell viability compared with that in the HG treatment group (p < 0.001, Fig. 2B).
Tube formation plays an important role in the formation of new blood vessels. The effect of FXST on the length of the endothelial tube was examined under high glucose conditions. The length of the tubules in the HG group was increased compared with that in the NG group (p < 0.001). The length of the tubules in the FXST group was significantly decreased compared with that in the HG group (p < 0.01 or p < 0.001, Fig. 3B and E). Thus, FXST controls tube formation by endothelial cells.
3.3. FXST suppressed cell migration Cell migration is one of the signs of neovascularization [15]. The chamber assay and scratch test were used to assess the effect of FXST on cell migration. According to the results of the chamber assay, the relative numbers of migrated cells in the HG group increased compared with those in the NG group (p < 0.001). FXST decreased the relative number of migrated cells (p < 0.01 or p < 0.001) in a dose-dependent manner (Fig. 3A and D) compared with those in the HG group. The maximum percent inhibition was 128.89%. In the HG group, the distance travelled and motility were increased compared with that in the NG group in the scratch test (p < 0.001). Furthermore, the FXST treatment reduced cell motility and decreased the distance travelled by 89.67% (p < 0.001, Fig. 3C and F). The data from the chamber assay and the scratch test confirmed that FXST attenuates the migration of RF/6A cells.
3.5. FXST reduced the expression of the YAP mRNA and protein After evaluating the pharmacological activity, the molecular mechanism of FXST was further explored. According to the PCR and WB results, HG increased the expression of YAP mRNA and protein (p < 0.05 and p < 0.001, respectively). As expected, FXST decreased the levels of YAP (p < 0.05 and p < 0.001, respectively; Fig. 4A–C).
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Fig. 5. YAP expression was significantly decreased in transfected cells. (A) The sh-YAP plasmid was transfected into RF/6A cells. GFP fluorescence was observed in the cells under a microscope; scale bar, 100 μm. After plasmid transfection, the levels of the YAP mRNA (B) and protein (C and D) were decreased. * p < 0.05 and ** p < 0.01 compared with the NG group. Levels of the VEGF (E and F) and VEGFR (G and H) proteins were decreased. * p < 0.05 compared with the NG + sh-NC group. The data are presented as means ± SEM (n = 3–4).
(p < 0.05, Fig. 5A–D). Then, the levels of VEGF and VEGFR were examined. In sh-YAPtransfected cells, levels of the VEGF protein were significantly decreased (p < 0.05) and the VEGFR levels were slightly reduced compared with those in the sh-NC group (Fig. 5E–H). The effect of YAP on cell migration was also determined. After transfection, cells were stimulated with NG or HG for 24 h. Cell migration was assessed using the transwell assay. In the presence of NG and HG, the sh-YAP group displayed a slight decrease in cell migration compared with that in the sh-NC group (Fig. 6A–D). A tube formation assay was also performed. In the NG, sh-YAP decreased the tube length by 44.74% compared with that in the sh-NC group (p < 0.05, Fig. 7A and B). In the HG, the tube length was decreased by 20.43% in the sh-YAP group (p < 0.01, Fig. 7C and D). Thus, YAP silencing decreased cell migration, the tube length and VEGF expression. Based on these data, YAP induces the migration and tube formation of RVECs through a process mediated by VEGF.
3.6. FXST decreased the expression of VEGF and VEGFR VEGF and VEGFR play critical roles in angiogenesis [16]. Thus, the effects of FXST on VEGF and VEGFR expression were investigated. HG substantially decreased the levels of VEGF and VEGFR compared with the NG group (p < 0.05 and p < 0.001, respectively). The levels of the VEGF and VEGFR proteins were decreased in the FXST group compared with those in the HG group (p < 0.05 and p < 0.001, respectively; Fig. 4D–G). Based on these results, FXST modulates the VEGF-VEGFR signalling pathway to inhibit angiogenesis.
3.7. YAP induced cell migration and tube formation The effects of YAP on cell migration and tube formation were examined to further investigate the role of YAP in mediating the effects of FXST. RF/6A cells were transfected with the sh-YAP plasmid or negative control (NC). The levels of the YAP mRNA and protein were reduced by 50.77% and 50.43%, respectively, in the sh-YAP group 6
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Fig. 6. The effect of FXST on cell migration strongly depended on YAP. (A and B) In normal glucose medium, the number of migrating cells was reduced in the NG + sh-YAP group. (C and D) The number of migrating cells was reduced in the HG + sh-YAP group. (E and F) YAP gene silencing in transfected cells decreased the number of migrating cells in the HG +0.9 mg/mL FXST group compared with the HG +0.9 mg/mL FXST + sh-YAP group. *** p < 0.001 compared with the HG group. ### p < 0.001 compared with the HG +0.9 mg/mL FXST group. Data are presented as means ± SEM (n = 3); scale bar, 200 μm.
3.9. YAP silencing blocked the ability of FXST to reduce VEGF levels
3.8. YAP silencing disrupted the effects of FXST on cell migration and tube formation
WB experiments were performed to determine whether the FXSTinduced downregulation of VEGF and VEGFR was mediated by YAP. YAP expression was knocked down in RF/6A cells. Cells transfected with an empty vector served as the control. In cells transfected with shYAP, the ability of FXST to reduce VEGF expression was blocked (Fig. 8A–C). Together with the data presented in Fig. 5E, these results suggest that YAP is an upstream mediator of VEGF and that the FXSTdependent downregulation of VEGF is mediated by YAP.
Cells were transiently transfected with the sh-YAP plasmid or negative control vector (sh-NC) to assess the role of YAP in mediating the effects of FXST. Then, cells were treated with HG plus FXST. Transwell migration and tube formation assays were performed. In wild-type cells, FXST decreased the migration and tube length (p < 0.001). However, YAP silencing in RF/6A cells significantly suppressed the effects of FXST (p < 0.05 and p < 0.001, respectively). In the sh-YAP plasmidtransfected cells, FXST no longer decreased cell migration (Fig. 6E and F) or tube formation (Fig. 7E and F). Thus, the effect of FXST strongly depends on YAP.
3.10. YAP inhibition blocked the ability of FXST to reduce cell migration and tube formation A YAP inhibitor, verteporfin (VP), was used to determine whether 7
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Fig. 7. YAP silencing inhibited the FXST-induced decrease in tube formation. The sh-YAP or NC plasmid was transfected into RF/6A cells. (A and B) Cells were cultured in the presence of NG, and YAP silencing decreased the total tube length. * p < 0.05 compared with the NG group. (C and D) Cells were cultured in the presence of HG, and YAP silencing reduced the total tube length; * p < 0.05 compared with the HG + shNC group. (E and F) Cells were stimulated with NG, HG or HG plus FXST. In wild-type cells, FXST reduced the tube length. However, in the sh-YAP-transfected cells, the effect of FXST effect was inhibited. * p < 0.05 and *** p < 0.001 compared with the HG group. # p < 0.05 compared with the HG +0.9 mg/mL FXST group. Data are presented as means ± SEM (n = 3); scale bar, 200 μm.
significantly decreased YAP and VEGF expression. (3) YAP suppressed tube formation and VEGF expression in the presence of high glucose. (4) After YAP activity was blocked with an sh-RNA plasmid or inhibitor, FXST-mediated protection from high glucose was abolished. These data provide additional insights into the important role of YAP in retinal diseases and the therapeutic effect of FXST on DR.
reduced YAP activity affected FXST-mediated inhibition of cell migration and tube formation and further investigate the role of YAP in mediating the effects of FXST. VP significantly decreased YAP activity without inducing substantial changes in cell viability (Supplementary Fig. 1A–C). Additionally, FXST treatment impeded cell migration and tube formation; however, treatment with VP dramatically inhibited the effect of FXST (p < 0.01, Fig. 9A–D). Thus, the control of cell migration and tube formation by FXST is mediated by the suppression of YAP activity.
4.1. FXST ameliorated the function of RVECs induced by HG A number of published studies have confirmed the beneficial effects of FXST on patients and animal models of DR. Previous studies have described the effect of FXST on angiogenesis. According to Luo et al., FXST attenuates the VEGF-induced tube formation and migration of endothelial cells and retinal pigment epithelial cells [17]. Luo and colleagues also showed that FXST suppresses angiogenesis in the chick embryo chorioallantoic membrane [18]. Consistent with the literature,
4. Discussion As shown in the present study, a FXST treatment significantly ameliorates high glucose-induced injury in RVECs by targeting YAP. The key findings are listed below. (1) FXST reduced migration and the tube length by 128.89% and 55.64%, respectively. (2) FXST 8
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Fig. 8. YAP silencing suppressed the FXSTinduced reduction in VEGF expression. The effect of FXST on the levels of VEGF (A and C) and VEGFR (B and C) in the sh-YAP or sh-NC transfected cells. The inhibitory effect of FXST on VEGF expression was attenuated when the YAP gene was silenced. However, significant differences in the levels of the VEGFR protein were not observed between the groups. Data are presented as means ± SEM (n = 3).
Fig. 9. VP attenuated the FXST-induced inhibition of cell migration and tube formation. (A and B) FXST reduced the number of migrating cells; however, VP inhibited the effect of FXST. Scale bar, 200 μm. (C and D) FXST decreased the tube length; however, VP inhibited the effect of FXST. *** p < 0.001 compared with the HG group. ## p < 0.01 compared with the HG +0.9 mg/mL FXST + VP group; scale bar 200 μm. Data are presented as means ± SEM (n = 3).
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5. Conclusions
FXST decreased the number of migrating cells, migration distance and tube length in the presence of high glucose in our study. Thus, the effect of FXST on DR is mediated by inhibiting angiogenesis.
FXST suppressed HG-induced cell migration and tube formation. Moreover, the effect of FXST was blocked by sh-YAP or a YAP inhibitor. Thus, the protective effect of FXST on high glucose-induced damage appears to be mediated by YAP. These findings provide deeper insights into the mechanisms underlying the effects of FXST and lay the foundation for its further development.
4.2. The bioactive components of FXST include saponins and phenanthraquinone Additionally, the study was designed to determine the bioactive components of FXST. Our HPLC-MS results identified a series of representative components of FXST, including ginsenosides Rb1, tanshinone IIA and astragaloside IV. In recent years, the chemical composition of FXST has been analysed. In 2016, Jiang reported 14 types of notoginsenosides in FXST, of which notoginsenosides R1 and ginsenosides Rb1 were identified in the rat eye [19]. Moreover, Rb1 inhibits the VEGF-induced proliferation of retinal pigment epithelial cells [20]. Tanshinone IIA markedly suppresses the high glucose-induced proliferation, migration and vascularization of human retinal endothelial cells [21]. Astragaloside IV exerts a significant inhibitory effect on the proliferation and migration of vascular smooth muscle cells [22]. As shown in the study by Jian, a combination of the main constituents of FXST, including saponins, harpagoside, cryptotanshinone, and tanshinone I, decreases the number of acellular vessels and pericyte loss in diabetic retinas [23]. In general, researchers have postulated that saponins and phenanthraquinone are the bioactive components of FXST.
Funding This study was financially supported by grants from the National Natural Science Foundation of China [grant numbers 81673705 and 81873165]. Authors’ contributions HJ, WW and LYG conceived and designed the experiments. XW, SYL, LHL and WZL performed the experiments. WY and LC analysed the data. XW, HJ and LYG contributed to drafting the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors have no competing interests to declare.
4.3. FXST inhibited cell migration and tube formation by targeting YAP
Appendix A. Supplementary data
Numerous studies have investigated the pharmacological mechanisms of FXST. FXST significantly decreases the levels of VEGF, intercellular adhesion molecule 1 (ICAM1), interleukin 1β (IL1β), matrix metalloproteinase 2 (MMP2) and apoptosis [24]. Duan revealed that FXST increases the expression of occludin and pigment epithelium-derived factor (PEDF) in the retinas of diabetic rats [25]. However, these studies provide only circumstantial evidence. YAP activity was blocked with a plasmid or an inhibitor in the present study to clarify the detailed mechanism of FXST. The effects FXST were found to be dependent on YAP activity. In wild-type cells, FXST reduced migration and tube formation. In contrast, in the sh-YAP-transfected cells, the effects of FXST were reversed. Moreover, when YAP activity was suppressed by an inhibitor, FXST no longer controlled RVEC function. Additionally, the FXST-induced inhibition of VEGF expression was blocked by YAP silencing. Thus, FXST inhibited cell migration and tube formation by targeting YAP. However, all of our experiments were performed in vitro, and thus, the role of YAP in mediating the effects of FXST must be confirmed in vivo in subsequent studies. This study also would contribute to understanding the function of YAP. YAP has been extensively studied in cancer. It is an oncogene that promotes cell proliferation, inhibits cell apoptosis, and promotes malignant transformation of cells [26]. When YAP was overexpressed or activated, the expression of tumor markers elevated, and the incidence of cancer increased [27–29]. On the contrary, YAP knockout or inactivation inhibited cell migration [30,31] in vitro or tumor growth in vivo [27–29]. The role of YAP in endothelial cells has also been explored. When YAP was knocked down, the cell migration, proliferation, and tube formation decreased under hypoxia conditions [32–34]. During retinal angiogenesis, a loss of YAP function led to defects in the proliferation and migration of vascular endothelial cells (ECs) in a gene dosage-dependent manner [35,36]. However, it was not clear whether YAP could regulate the function of endothelial cells under high glucose. This experiment found that HG stimulated tube formation, meanwhile YAP and VEGF expression increased in RVECs. However, YAP silencing inhibited HG-induced tube formation and VEGF levels. It suggested that YAP could actually inhibit the connection of the cells by regulating VEGF expression, and eventually it might inhibit angiogenesis under high conditions.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109470. References [1] G. Liew, V.W. Wong, I.V. Ho, Mini review: changes in the incidence of and progression to proliferative and sight-threatening diabetic retinopathy over the last 30 years, Ophthalmic Epidemiol. 24 (2) (2017) 73–80. [2] J.B. Jonas, L. Xu, J. Xu, et al., Prevalence of diabetic retinopathy and vision loss in the Beijing eye study: the potential role of the cerebrospinal fluid pressure, Curr. Diab. Rep. 15 (10) (2015) 71. [3] C. Betz, A. Lenard, H.G. Belting, et al., Cell behaviors and dynamics during angiogenesis, Development 143 (13) (2016) 2249–2260. [4] N. Li, C. Xie, N. Lu, Crosstalk between Hippo signalling and miRNAs in tumour progression, FEBS J. 284 (7) (2017) 1045–1055. [5] Y. Ma, Y. Yang, F. Wang, et al., Hippo-YAP signaling pathway: a new paradigm for cancer therapy, Int. J. Cancer 137 (10) (2015) 2275–2286. [6] H.J. Choi, H. Zhang, H. Park, et al., Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2, Nat. Commun. 6 (2015) 6943. [7] F.X. Yu, B. Zhao, K.L. Guan, Hippo pathway in organ size control, tissue homeostasis, and Cancer, Cell 163 (4) (2015) 811–828. [8] K. Brodowska, A. Al-Moujahed, A. Marmalidou, et al., The clinically used photosensitizer Verteporfin (VP) inhibits YAP-TEAD and human retinoblastoma cell growth in vitro without light activation, Exp. Eye Res. 124 (2014) 67–73. [9] Y. Liu-Chittenden, B. Huang, J.S. Shim, et al., Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP, Genes Dev. 26 (12) (2012) 1300–1305. [10] J. Hong, Intervention of compound Xueshuantong capsule on diabetic retinopathy in hemorrhage period, China Journal of Traditional Chinese Medicine & Pharmacy (2010). [11] G.M. Hao, T.T. Lv, Y. Wu, et al., The Hippo signaling pathway: a potential therapeutic target is reversed by a Chinese patent drug in rats with diabetic retinopathy, BMC Complement. Altern. Med. 17 (1) (2017) 187. [12] J. Xu, B. Mei, N. Zhang, Clinical observation on early diabetic retinopathy with Compound Xueshuantong Capsule, zhong hua zhong yi yao za zhi 27 (10) (2012) 3247–3249. [13] J. Du, X. Chai, J. Cheng, et al., The review of ophthalmic clinical use of Fufang xueshuantong, Zhong Hua Lin Chuang Yi Shi Za Zhi 6 (7) (2012) 1833–1834. [14] Y. Wu, Q. Zhang, R. Zhang, Kaempferol targets estrogen-related receptor alpha and suppresses the angiogenesis of human retinal endothelial cells under high glucose conditions, Exp. Ther. Med. 14 (6) (2017) 5576–5582. [15] Z. Jiang, X. Chen, Q. Zhou, et al., Downregulated LRRK2 gene expression inhibits proliferation and migration while promoting the apoptosis of thyroid cancer cells by inhibiting activation of the JNK signaling pathway, Int. J. Oncol. 55 (1) (2019) 21–34. [16] P.N. Bishop, The role of extracellular matrix in retinal vascular development and preretinal neovascularization, Exp. Eye Res. 133 (2015) 30–36.
10
Biomedicine & Pharmacotherapy 120 (2019) 109470
W. Xing, et al.
[27] F.D. Camargo, S. Gokhale, J.B. Johnnidis, et al., YAP1 increases organ size and expands undifferentiated progenitor cells, Curr. Biol. 17 (23) (2007) 2054–2060. [28] J. Dong, G. Feldmann, J. Huang, et al., Elucidation of a universal size-control mechanism in Drosophila and mammals, Cell 130 (6) (2007) 1120–1133. [29] M. Mohseni, J. Sun, A. Lau, et al., A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway, Nat. Cell Biol. 16 (1) (2013) 108–117. [30] T.S. Eisinger-Mathason, V. Mucaj, K.M. Biju, et al., Deregulation of the Hippo pathway in soft-tissue sarcoma promotes FOXM1 expression and tumorigenesis, Proc. Natl. Acad. Sci. 112 (26) (2015) 3402–3411. [31] X. Wang, R. Zhang, L. Gu, et al., Cell-based screening identifies the active ingredients from traditional chinese medicine formula shixiao san as the inhibitors of atherosclerotic endothelial dysfunction, PLoS One 10 (2) (2015) e0116601. [32] C. Li, J. Wang, Q. Wang, et al., Qishen granules inhibit myocardial inflammation injury through regulating arachidonic acid metabolism, Sci. Rep. 6 (2016) 36949. [33] Y.G. Guan, J.B. Liao, K.Y. Li, et al., Potential mechanisms of an antiadenomyosis chinese herbal formula shaoyao-gancao decoction in primary cell culture model, Evid. Complement. Alternat. Med. 2014 (1) (2014) 982913. [34] Y.T. Qing, S. Ji, et al., YAP knockdown in gastric cancer-derived mesenchymal stem cell inhibits the progression of gastric cancer, J. Jiangsu Univ. (Med. Ed.) 27 (5) (2017) 374–378. [35] M. Sakabe, J. Fan, Y. Odaka, et al., YAP/TAZ-CDC42 signaling regulates vascular tip cell migration, Proc. Natl. Acad. Sci. U. S. A. 114 (41) (2017) 10918. [36] M. Zhu, X. Liu, Y. Wang, et al., YAP via interacting with STAT3 regulates VEGFinduced angiogenesis in human retinal microvascular endothelial cells, Exp. Cell Res. 373 (1-2) (2018) 155–163, https://doi.org/10.1016/j.yexcr.2018.10.007 Epub 2018 Oct 17.
[17] M. Wu, H. Xiong, Y. Xu, et al., Association between VEGF-A and VEGFR-2 polymorphisms and response to treatment of neovascular AMD with anti-VEGF agents: a meta-analysis, Br. J. Ophthalmol. 101 (7) (2017) 976–984. [18] X. Luo, X. Wu, Q. Gu, et al., Study on complex Xueshuantong interfering on choroidal neovascularization concerned with RPEcells, Modern J. Int. Trad. Chinese Western Med. 19 (33) (2010) 4241–4245. [19] M.F. Jiang, Q.H. Wang, H.H. Pang, et al., [Distribution of chemical compounds of Fufang Xueshuantong capsule in eyes and kidney of beagle dogs], Zhongguo Zhong Yao Za Zhi 41 (20) (2016) 3846–3851. [20] B.S. Betts, K. Parvathaneni, B.B. Yendluri, et al., Ginsenoside-Rb1 induces ARPE-19 proliferation and reduces VEGF release, ISRN Ophthalmol. 2011 (2011) 184295. [21] K. Fan, S. Li, G. Liu, et al., Tanshinone IIA inhibits high glucoseinduced proliferation, migration and vascularization of human retinal endothelial cells, Mol. Med. Rep. 16 (6) (2017) 9023–9028. [22] Z. Chen, Y. Cai, W. Zhang, et al., Astragaloside IV inhibits platelet-derived growth factor-BB-stimulated proliferation and migration of vascular smooth muscle cells via the inhibition of p38 MAPK signaling, Exp. Ther. Med. 8 (4) (2014) 1253–1258. [23] W. Jian, S. Yu, M. Tang, et al., A combination of the main constituents of Fufang Xueshuantong Capsules shows protective effects against streptozotocin-induced retinal lesions in rats, J. Ethnopharmacol. 182 (2016) 50–56. [24] J. Du, H. Sun, Cheng J. The effect of fufang xueshuantong on diabetic retinopathy: a review, Zhong Hua Lin Chuang Yi Shi Za Zhi 6 (22) (2012) 7373–7375. [25] H. Duan, J. Huang, W. Li, et al., Protective effects of fufang xueshuantong on diabetic retinopathy in rats, Evid. Complement. Alternat. Med. 2013 (2013) 408268. [26] M. Liu, S. Zhao, Q. Lin, et al., YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues, Mol. Cell. Biol. 38 (5) (2015) 1449–1461.
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