MOLECULAR CARCINOGENESIS 54:501–512 (2015)
Wogonin Influences Vascular Permeability Via Wnt/b-Catenin Pathway Xiuming Song, Yuxin Zhou, Mi Zhou, Yujie Huang, Zhiyu Li, Qidong You,* Na Lu,* and Qinglong Guo* State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, People’s Republic of China
Wogonin, a flavone from the root of Scutellaria baicalensis Georgi, has shown various biological activities. In our previous study, it was confirmed that wogonin could decrease the expression of hypoxia-inducible factor-1a (HIF-1a) by affecting its stability under hypoxia. However, it is still unknown whether wogonin could influence Wnt/b-catenin pathway under hypoxia. In this study, we found that wogonin disrupted Wnt/b-catenin signaling and reduced the secretion of vascular endothelial growth factor (VEGF, also known as vascular permeability factor, VPF), which increased vascular permeability in certain diseases. It was found that wogonin suppressed HUVECs hyperactivity and actin remodeling induced by hypoxia, inhibited transendothelial cell migration of the human breast carcinoma cell MDA-MB-231 and the extravasated Evans in vivo Miles vascular permeability assay. Wogonin-treated cells showed a decrease in the expression of Wnt protein and its co-receptors, as well as a parallel increase in the expression of Axin and GSK-3b in degradation complex, leading to degradation of b-catenin. In addition, wogonin promoted the binding between Axin and b-catenin, increased ubiquitination of b-catenin and promoted its degradation. Interestingly, wogonin decreased the expression of TCF-1, TCF3, and LEF-1 and inhibited nuclear accumulation of b-catenin as well as the binding of b-catenin and TCF-1, TCF-3, or LEF-1. All of the above results showed that wogonin could inhibit the expression of VEGF, which is an important factor regulated by b-catenin. Taken together, the results suggested that wogonin was a potent inhibitor of Wnt/b-catenin and influenced vascular permeability, and this might provide new therapeutics in certain diseases. © 2013 Wiley Periodicals, Inc. Key words: vascular permeability; Wnt/b-catenin; degradation; wogonin
INTRODUCTION Vascular permeability is closely linked with angiogenesis in a number of pathologic conditions, such as eye diseases [1], lung diseases [2], cardiovascular diseases and cancers [3]. In these diseases, the function of blood vessels is impaired because of abnormalities in their endothelial lining. For example, there are a large quantity of “abnormalized” vessels in tumors and they impair perfusion and oxygenation [4]. The resultant hypoxia promotes tumor malignancy. In the “abnormalized” vessels, the endothelial cells become more active, including cell connection damage, hypermotility and a disordered arrangement, resulting in an increase of blood vessels permeability. Thus, targeting vascular permeability may provide new therapeutics in treating certain diseases. In many diseases, hypoxia is a response to the increased vascular permeability. To maintain endothelial cell monolayer integrity, vascular endothelial cadherin (VE-cadherin), similar to many other members of the cadherin family, is linked through its cytoplasmic tail to the adherens junctions (AJ) proteins p120, b-catenin, and plakoglobin [5]. Under hypoxic condition, on one hand, VE-cadherin and bcatenin dissociate to destroy AJ; on the other hand, wingless type protein (Wnt)/b-catenin signaling is activated [6] and the expression of downstream target ß 2013 WILEY PERIODICALS, INC.
Abbreviations VE-cadherin, vascular endothelial cadherin; AJ, adherens junctions; Wnt, wingless type protein; HUVEC, human umbilical vein endothelial cell; FZD-4, frizzled 4; LRP5/6, low-density lipoprotein receptor-related protein 5/6; TCF, T-cell factor; HIF-1a, hypoxia-inducible factor-1a; VEGF, vascular endothelial growth factor; GSK-3b, glycogen synthase kinase-3b; DvL-2, 3, dishevelled-2, 3; LEF, lymphoid enhancer factor; CHX, cycloheximide; FITC, fluorescein-5isothiocyanate; Axin, axis inhibition protein; ELISA, enzyme linked immunosorbent assay. Xiuming Song and Yuxin Zhou contributed equally to this work. Conflict of interest: There are no conflicts of interest. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 30973556; 81001452; Grant sponsor: Program for Changjiang Scholars and Innovative Research Team in University; Grant number: IRT1193; Grant sponsor: Project Program of State Key Laboratory of Natural Medicines; Grant sponsor: China Pharmaceutical University; Grant numbers: JKGZ201101; SKLNMZZ201210; SKLNMZZCX201303; SKLNMZZJQ201302; Grant sponsor: Fundamental Research Funds for the Central Universities; Grant number: JKP2011003; Grant sponsor: The National Science & Technology Major Project; Grant number: 2012ZX09304-001; Grant sponsor: Natural Science Foundation of Jiangsu Province; Grant numbers: BK2009297; BK2010432; Grant sponsor: Colleges and Universities in Jiangsu Province Plans to Graduate Research and Innovation (2012) *Correspondence to: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People's Republic of China. Received 24 September 2013; Accepted 8 July 2013 DOI 10.1002/mc.22093 Published online 17 October 2013 in Wiley Online Library (wileyonlinelibrary.com).
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genes results in changes in cell adhesion and migration. For the above reasons, blood vascular permeability increases under the stimulus of hypoxia. b-Catenin was first identified at AJ where it linked cadherins with the cytoskeleton to regulate the response to cell adhesion. Later studies have found that b-catenin is a central mediator of the Wnt signaling pathway and controls the transcription of genes during both normal and malignant development [7]. There are two different Wnt/b-catenin signaling pathway: “canonical” Wnt/b-catenin signaling pathway and “non-canonical” Wnt/b-catenin signaling pathway [8]. Canonical Wnt signaling involves stabilization of cytosolic b-catenin, turning it into a nuclear transcriptional regulator. In human umbilical vein endothelial cells (HUVECs), numerous Wnt signaling components are expressed, including Wnt5a and frizzled 4 [9]. In the presence of frizzled 4 (FZD-4) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6), Wnt5a can activate canonical Wnt signaling downstream of b-catenin stabilization and promote T-cell factor (TCF) mediated transcription [10], causing cells more active. Wogonin is a naturally flavonoid isolated from the root of Scutellaria baicalensis Georgi, which has been widely used for its antioxidant [11], anti-inflammatory [12], and anticancer activities [13–15]. In our previous study, we have confirmed that wogonin could inhibit angiogenesis by decreasing the expression of hypoxia-inducible factor-1a (HIF-1a) under hypoxia. However, whether wogonin could affect vascular permeability under hypoxia is still unknown and the molecular mechanisms remain to be poorly understood and need a further investigation. In our study, we investigated the effect of wogonin on vascular permeability under hypoxia. Here, we elucidated the potential effects of wogonin on the response of HUVECs migration and actin cytoskeleton, MDAMB-231 transendothelial cell migration, and the extravasated Evans in vivo. Further mechanism research revealed that wogonin inhibited vascular permeability via influencing Wnt/b-catenin signaling pathway. We found that wogonin promoted degradation of b-catenin and inhibited its nuclear accumulation. Taken together, the results suggested that wogonin may serve as a candidate in the development of a Wnt/b-catenin inhibitor for disease therapy. MATERIALS AND METHODS Materials Wogonin was isolated from S. baicalensis Georgi according to the protocols reported previously with slight modifications [16]. Samples containing 99% or higher wogonin were used in all experiments unless otherwise indicated. Primary antibodies for b-catenin (E-5), vascular endothelial growth factor (VEGF) (147), glycogen synthase kinase 3b (GSK-3b) (H-76), FZD-4 (H-120), and b-actin were obtained from Santa Molecular Carcinogenesis
Cruz Biotechnology (Santa Cruz, CA). Primary antibody for Histone H3 (T11) and p-GSK-3b (S9) were from Bioworld (St. Louis Park, MN). Primary antibodies for Wnt5a/b (C27E8), LRP5/6 (C12A5), Dvl-2 (30D2), Dvl-3, axis inhibition protein (Axin) (C76H11), TCF-1 (C63D9), TCF-3 (D15G11), lymphoid enhancer factor-1 (LEF)-1 (C12A5), VE-Cadherin (D87F2), and ubiquitin were from Cell Signaling Technology (Danvers, MA). CoCl2, MG132, LiCl, and cycloheximide (CHX) were purchased from Sigma (St. Louis, MO). Bovine serum albumin (BSA), paraformaldehyde, Triton X-100, Tris, NaCl, EDTA, NP-40, PMSF, NaF, SDS, DTT, Evans blue and fluorescein-5isothiocyanate (FITC)-conjugated Phalloidin were purchased from Sigma-Aldrich (St. Louis, MO). IRDyeTM800 conjugated secondary antibodies were obtained from Rockland, Inc. (Philadelphia, PA). Cell Culture HUVECs were isolated from human umbilical cord veins by collagenase treatment as described previously [17]. The harvested cells were grown in medium 199 (M199, Gibco, Grand Island, NY) containing endothelial cell growth supplement (ECGS, 30 mg/mL; Sigma), epidermal growth factor (EGF, 10 ng/mL; Sigma), 20% fetal bovine serum (FBS, Gibco), 100 U/ mL penicillin, and 100 U/mL streptomycin, pH 7.4. After 3–5 passages, HUVECs were collected for use in all experiments. The human breast carcinoma cell MDA-MB-231 was originally obtained from the Cell Bank of Shanghai Institute of Cell Biology. MDA-MB231 cells were cultured in L-15 medium (Gibco) containing 10% FBS (Sijiqing, Hangzhou, China), 100 U/mL penicillin, and 100 U/mL streptomycin, and cells were grown in a stable environment with 5% CO2 at 378C. Endothelial Cell Migration Assay HUVECs were treated with wogonin (0, 1, 10, and 100 mM) under normoxic or hypoxic conditions for 4 h. Then the cells were trypsinized and suspended at a final concentration of 5 105 cells/mL in serum-free M199. Cell suspension was loaded into each of the upper wells. Following incubation at 378C in 5% CO2 for 4 h, nonmigratory cells on the upper surface were removed by a cotton swab. The migrated tumor cells on the lower surface were fixed with 100% methanol and stained with hematoxylin and eosin. The migrated cells were quantified by manual counting and five randomly chosen fields were analyzed for each group. Immunofluorescent HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 4 h under different conditions as specified. Immunofluorescent was performed according to the method described with modification [18]. Stained specimens were examined under a confocal laser scanning microscope (Fluoview FV1000, Olympus, Tokyo, Japan).
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Transendothelial Cell Migration Assay The experiment was performed as previously described [19]. Briefly, HUVECs were seeded in the amount of 1 104 cells on transwell polycarbonate filters (6.5 mm in diameter, 8 mm pore-size, CorningCostar, Cambridge, MA). After reaching a confluence, cells were preincubated for 4 h with wogonin (1, 10, or 100 mM) under normoxia or hypoxia. Then transendothelial cell migration assay using MDA-MB-231 cells was performed as described in “endothelial cell migration assay” part. Miles Vascular Permeability Assay The mice were equally randomized into five groups (each group contained seven mice): as indicated. Wogonin treatments were done at a frequency of i.v. each day for a total of 6 d. CoCl2 (100 mL) was injected intradermally each day into the back skin of mice at the same spot for 3 d to induce hypoxia-like response (0.9% normal saline was injected as control). Then the Miles assay was performed as described previously [20]. Apoptosis Assessment Cells were pretreated with wogonin (0, 1, 10, and 100 mM) as specified for 4 h. Then apoptosis induced by wogonin was detected by Annexin V-FITC apoptosis detection kit (BioVision, San Francisco, CA) according to the manufacturer’s protocol. Colony-Formation Assay Cells were pretreated with wogonin (0, 1, 10, and 100 mM) under normoxic or hypoxic conditions for 4 h, and then plated in 35-mm dishes at 10 000 cells/ well in 0.35% agar in M199 over a 0.5% agar layer. Plates were further incubated for 21 d until colonies were large enough to be visualized. Colonies were counted under inverted microscrope. Experiments were done in triplicate. Western Blot Analysis HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 4 h under normoxic and hypoxic conditions. Western blotting was done as previously reported [21]. Detection was performed by the Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE). Preparation of Cytosolic and Nuclear Extracts HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 4 h under different conditions as specified. Nuclear and cytosolic protein extracts were prepared according to the modified method as described previously [22,23]. Enzyme Linked Immunosorbent Assay Cells were pretreated with wogonin (0, 1, 10, and 100 mM) under normoxic or hypoxic conditions for 4 h, and then the medium was replaced by serum-free Molecular Carcinogenesis
medium. After 12 h, the concentration of VEGF was determined by a human VEGF Duo-set enzyme linked immunosorbent assay (ELISA) kit per manufacture’s instructions. Real-Time PCR Analysis HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 2 h under normoxic or hypoxic conditions. The mRNA level of VEGF was determined to the method described previously [24]. The primer sets used in the PCR amplification were as follows: VEGF (forward, 50 -GGT GGA CAT CTT CCA GAG TA-30 , reverse, 50 -GGC TTG TCA CAT CTG CAA GTA-30 ), bactin (forward, 50 -CTG TCC CTG TAT GCC TCT G-30 , reverse, 50 -ATG TCA CGC ACG ATT TCC-30 ). Luciferase Reporter Assay The hVEGF-pGL2 promoter reporter was provided by Dr. Bar-bara K. Vonderhaar (Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, USA). Cells were cotransfected with VEGF-pGL2 with Renilla luciferase reporter (as internal control) for 24 h and then treated with wogonin (0, 1, 10, and 100 mM) for 4 h. The luciferase activity of cell lysate was determined with the Dual-Luciferase Reporter kit (Beyotime, Haimen, China) according to the provided protocol. Luciferase signals were collected by DualLuciferase Assay system (Thermo Fisher Scientific, Rockford, IL). Immunoprecipitation HUVECs were treated with different concentrations of wogonin (0 and 40 mM) for 4 h under different conditions as specified. Immunoprecipitation was performed as previously described [25]. Proteins were precipitated from the supernatant by the addition of b-catenin antibody. Samples were stored at 208C for Western blotting assay. VE-cadherin, Axin, TCF-1, TCF-3, LEF-1 or ubiquitin antibodies were used and the secondary antibody and band detection were operated as described above. Statistical Analysis All data in different experimental groups were expressed as the mean SEM. These data shown in the study were obtained in at least three independent experiments. Statistical analyses were performed using an unpaired, two-tailed Student’s t-test. The comparisons were made relative to untreated controls and significance of difference is indicated as P < 0.05 and P < 0.01. RESULTS Wogonin Inhibited the Hypoxia-Induced Vascular Permeability HUVECs show hyperactivity during hypoxia, leading to an increase of vascular permeability. In our
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experiments, Transwell assay was used to evaluate the effect of wogonin on the migration of HUVECs. As shown in Figure 1A, HUVECs became more active under hypoxia compared with the normoxia control. When treated with 1, 10, and 100 mM wogonin, fewer HUVECs migrated to the lower side of the filter in the Transwell Chamber. Wogonin significantly inhibited the migration of HUVECs in a concentration-dependent manner. The endothelial barrier integrity is the result of a balance between the tethering and contractile forces acting on endothelial cells, which are critically dependent upon cytoskeletal components, including the actin-based microfilaments [26]. Therefore, the response of the cytoskeleton to wogonin under hypoxia in HUVECs was investigated, using FITCphalloidin to stain actin filaments. As shown in Figure 1B, HUVECs showed a polarized reorganization of the actin cytoskeleton under hypoxia compared with the normoxia control. When treated with 1, 10, and 100 mM wogonin, cytoplasmic stress fibers were blocked in a concentration-dependent manner. The transendothelial migration assay is frequently used to determine the ability of test compounds to endothelial cell permeability. As shown in Figure 1C, more cells migrated through the endothelial cells monolayer under hypoxia compared with the normoxia control. When treated with 1, 10, and 100 mM wogonin, this effect was blocked in a concentrationdependent manner. To investigate the effect of wogonin on vascular permeability in vivo, Miles vascular permeability assay was utilized. As shown in Figure 1D, vascular hyperpermeability of the mouse skin treated with CoCl2 was inhibited by wogonin at 20, 40, and 80 mg/ kg in a dose-dependent manner. These results demonstrated that wogonin inhibited CoCl2induced vascular leakage in a hypoxia-like response in vivo. Wogonin Neither Induced Apoptosis Nor Suppressed Proliferation in HUVECs at the Tested Concentrations AnnexinV-FITC apoptosis detection kit was used the assess the apoptosis effect of wogonin on HUVECs. As shown in Figure 2A, wogonin (0, 1, 10, and 100 mM) did not induce apoptosis in HUVECs. To investigate the proliferation of HUVECs, colonyformation assay was performed. As shown in Figure 2B, wogonin did not suppress proliferation of the cells. These results indicated that wogoninmediated decrease in vascular permeability was irrelevant to the difference in number of cells. Wogonin Inhibited Dissociation of VE-Cadherin and b-Catenin Endothelial permeability is regulated in part by the dynamic opening and closure of cell–cell AJ, which are largely composed of VE-cadherin binding to several protein partners, including b-catenin and Molecular Carcinogenesis
others [5]. As shown in Figure 3A, confocal images showed hypoxia led to a decrease of co-localization between VE-cadherin and b-catenin at the cell borders compared with that of control, and treatment with wogonin at 100 mM blocked the effect. Then the binding of VE-cadherin and b-catenin was further investigated by immunoprecipitation analysis. As shown in Figure 3B, wogonin inhibited the dissociation of VE-cadherin and b-catenin under hypoxia. These data provided evidence that wogonin could decrease vascular permeability by promoting the association between VE-cadherin and b-catenin to stabilize AJ. Downregulation of the Wnt Protein and its Co-Receptors by Wogonin Not only dissociation of VE-cadherin and b-catenin happened during hypoxia, but Wnt/b-catenin signaling was activated to make the endothelial cell motility and vascular permeability changed. To assess the functional relevance of Wnt/b-catenin signaling in the context of vascular permeability, we analyzed the effect of wogonin on the Wnt protein and its coreceptors. As shown in Figure 3C, Wnt protein (Wnt5a), its co-receptors (FZD-4, LRP5/6) and dishevelled-2, 3 (DvL-2, 3) were upregulated under hypoxia compared with normoxia control. When the cells were treated with wogonin, all the proteins were downregulated in a concentration-dependent manner. Wogonin Inhibited b-Catenin Expression Via Promoting its Degradation Through Modulation of Destruction Complex The most frequently studied and best-understood Wnt signaling pathway is mediated by the transcriptional activity of b-catenin. In the presence of Wnt protein, cytoplasmic b-catenin separates from the multiprotein complex and stabilizes in the cytoplasm. As shown in Figure 4A, b-catenin is upregulated under hypoxia and wogonin could inhibit its expression. In addition, Western blot analysis showed that the protein level of GSK-3b and Axin, the main components of destruction complex, increased when the cells were treated with wogonin. And p-GSK-3b that is the inactivity form of GSK-3b decreased, indicating that destruction complex was modulated to for proteasomal degradation of b-catenin (Figure 4B). To substantiate the involvement of Axin in wogoninmediated decrease in b-catenin levels, we used immunoprecipitation to assess the binding of Axin with b-catenin. As shown in Figure 4C, wogonin could enhance the binding of Axin and b-catenin, which may promote the degradation of b-catenin. To further confirm whether wogonin could modulate destruction complex for degradation of bcatenin, a well known GSK-3b inhibitor, LiCl was used. Western blot analysis showed that LiCl could block wogonin-mediated suppression of b-catenin expression (Figure 4D). The result indicated that
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Figure 1. Wogonin inhibited hypoxia-induced increasing of vascular permeability in vitro and in vivo. (A) Inhibition of hypoxia-stimulated migration of HUVECs by wogonin. (B) Effect of wogonin on cytoskeleton remodeling in HUVECs. (C) Inhibition transendothelial cell migration of MDA-MB-231 cells by wogonin. (D) Effect of wogonin on inhibiting vascular leakage in vivo. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
wogonin could promote b-catenin degradation by affecting GSK-3b. In addition, as shown in Figure 4E, in the absence of MG132, levels of b-catenin were significantly reduced Molecular Carcinogenesis
by wogonin. But the accumulation of b-catenin was unaffected by wogonin in the presence of MG132, suggesting that wogonin promoted proteasomal degradation of b-catenin. To further study the
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Figure 2. Wogonin did not induce apoptosis nor suppress proliferation at tested concertration. (A) Effect of wogonin on apoptosis of HUVECs. (B) Effect of wogonin on proliferation of HUVECs.
potential mechanism, HUVECs were also treated with wogonin in the absence and presence of CHX for inhibiting de novo protein synthesis. As shown in Figure 4F, wogonin reduced the stability of b-catenin. Besides, the more extensively ubiquitinated b-catenin
protein was observed in wogonin treated cells than untreated controls under hypoxia (Figure 4G). All the findings suggested that wogonin might inhibit bcatenin protein stability by modulating the destruction complex.
Figure 3. Effects of wogonin on the binding of VE-cadherin and b-catenin, Wnt protein and its co-receptors. (A) Effect of wogonin on the co-localization of VE-cadherin (green) and b-catenin (red) in HUVECs. (B) Effect of wogonin on the binding of VE-cadherin and b-catenin in HUVECs. (C) Effect of wogonin on the Wnt protein and its coreceptors: Wnt5a, FZD-4, LRP5/6, DvL-2, DvL-3 proteins in HUVECs. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
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Figure 4. Wogonin inhibited b-catenin protein accumulation by influencing its stability. (A) Effect of wogonin on the expression of bcatenin in HUVECs. (B) Effect of wogonin on the expression of proteins in the destruction complex proteins. (C) Effect of wogonin on the binding of Axin and b-catenin. (D) Effect of LiCl on the level of b-catenin protein with or without wogonin. (E) Effect of MG132 on degradation of b-catenin induced by wogonin. (F) Effect of CHX on the level of
b-catenin protein with or without wogonin. CHX was used for inhibiting de novo protein synthesis. (G) Effect of wogonin on the ubiquitination of b-catenin. The comparisons were made relative to controls under hypoxic condition (or 100 mM wogonin-treated group under hypoxia) and significance of difference is indicated as P < 0.05 and P < 0.01 (or #P < 0.05) as specified.
Wogonin Inhibited Nuclear Accumulation of b-Catenin
examine the nuclear b-catenin level. As shown in Figure 5A, in wogonin-treated cells, the nuclear bcatenin protein levels decreased with a corresponding decrease in cytoplasmic b-catenin in a concentrationdependent manner. To sum up, wogonin could decrease b-catenin nuclear accumulation.
The nucleus localization of b-catenin is essential for the transcriptional activity. In the nucleus, b-catenin interacts with TCF/LEF transcription factors and activates cell transcription [27]. To study the effect of wogonin on the intracellular localization of bcatenin, immunofluorescence was used. As shown in Figure 3A, in the presence of wogonin, the b-catenin signal became weaker and the localization was redistributed in both nucleus and cytoplasm. To confirm the results above, Western blot was used to Molecular Carcinogenesis
Wogonin Decreased the Level of TCF/LEF Family and Influenced the Binding of TCF/LEF With b-Catenin As b-catenin transactivates gene expression in a complex with TCF/LEF family, we examined the effect of wogonin on different proteins of the TCF/LEF
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Figure 5. Wogonin decreased b-catenin nuclear accumulation (also shown in Figure 3A), inhibited the expression of TCF/LEF and the combination of b-catenin with TCF/LEF. (A) Effect of wogonin on b-catenin nuclear accumulation. (B) Effect of wogonin on the level of TCF-1, TCF-3 and LEF-1. (C) Effect of wogonin on the binding of b-catenin and TCF-1, TCF-3, or LEF-1. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
family. Wogonin caused a repression of these proteins including TCF-1, TCF-3, and LEF-1 (Figure 5B). An immunoprecipitation assay with b-catenin further identified TCF/LEF family as the key regulator of bcatenin-mediated transcription in wogonin-treated HUVECs. As shown in Figure 5C, the combination of b-catenin and TCF-1, TCF-3 or LEF-1 was significantly limited in HUVECs treated with 100 mM wogonin. Effects of Wogonin on Wnt/b-Catenin Signaling Target Gene VEGF Vijay Easwaran reported that b-catenin regulated VEGF expression [28], which was also known as VPF and closely related to blood vascular permeability [29]. Under the effect of VEGF, the endothelial cells become active, leading to hyperpermeability in several diseases. In the present study, we investigated the effect of wogonin on the secretion and expression of VEGF. ELISA was used to determine the secretion of VEGF from diluted cell-free supernatants of HUVECs cultured under normoxic condition and compared with cells cultured in a hypoxia chamber as described in the methods. As shown in Figure 6A, hypoxia induced secretion of VEGF, and wogonin significantly reduced the secretion of VEGF from the cells in a concertration-dependent manner. Western blot was used to investigate the effect of wogonin on VEGF protein in cell lysates. The result indicated that Molecular Carcinogenesis
wogonin could inhibit the expression of VEGF, which was consistent with ELISA studies. To further study the mechanism that wogonin downregulated VEGF, Real time-PCR assay was utilized. As shown in Figure 6C, mRNA expression of VEGF exhibited marked decrease after treatment of wogonin. To determine whether wogonin directly influenced the VEGF promoter, VEGF-pGL2 reporter was transfected into HUVECs. The level of repression of VEGF promoter activity was quantitatively similar to that of mRNA accumulation (Figure 6D), suggesting that the down-regulation of VEGF expression by wogonin is regulated primarily at the level of gene transcription. DISCUSSION Vascular hyperpermeability often coincides with the early stage of angiogenesis and is also found in areas of diseased tissue in diabetic retinopathy, solid tumors, myocardial infarction, wounds, and chronic inflammation [30]. Endothelial response to hypoxia mediates key processes that regulate angiogenesis progression, including endothelial cells proliferation, migration, adherence, and vascular permeability. During hypoxia, endothelial cells become more active to cause a significant increase in vascular permeability, which may form gaps or be absent in other
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Figure 6. Effects of wogonin on Wnt/b-catenin target gene VEGF. (A) Effect of wogonin on the secretion of VEGF in HUVECs. (B) Effect of wogonin on the expression of VEGF in cell lysates. (C) Effect of wogonin on VEGF mRNA level in HUVECs. (D) Effect of wogonin on VEGF transcription activation in HUVECs. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
denuded areas. So improving blood vessel function by decreasing vascular permeability may lead to new strategies for therapies. Wnt/b-catenin signaling has been separated into a “canonical” pathway and several “noncanonical” pathways, which may influence the expression of its target genes including VEGF [28], changing vascular permeability. In “canonical” pathway, b-catenin combines with several cytosolic proteins to form a large complex consisting of Axin, APC and GSK-3b, and gets phosphorylated for ubiquitin-mediated degradation. When “canonical” Wnt signaling is activated, it leads to inhibition of this turnover complex resulting in increased cytosolic b-catenin, which then transits to the nucleus, where it forms a ternary complex with transcription factors TCF/LEF to activate genes involved VEGF mentioned above. In recent years, many potential natural products have gradually gained considerable attention as a new source of drugs. Our previous study has reported that wogonin could inhibit tumor angiogenesis. In this study, we investigated that wogonin inhibited vascular hyperpermeability in vitro and in vivo. Mechanistic studies using HUVECs showed that wogonin inhibited dissociation of VE-cadherin and b-catenin, accelerated the hypoxia-induced b-catenin protein degradation and inhibited b-catenin nuclear accumulation for gene transcription. In the present study, hypoxia was shown to be associated with a significant decrease in both VEcadherin and b-catenin localized at inter-endothelial Molecular Carcinogenesis
junctions. But when HUVECs were pretreated with wogonin, hypoxia-induced dissociation of VEcadherin from b-catenin was inhibited, which was observed by confocal imaging and immunoprecipitation studies. This indicated wogonin could strengthen adherent junction and inhibit the formation of endothelial gaps. Thus, endothelial cells treated with wogonin were tightly linked and not easy to loose, leading to a decrease in vascular permeability. Wnt/b-catenin signaling plays a significant role in VEGF expression, which is closely related to endothelial cells behavior linking to vascular permeability as indicated above. In our study, Wnt/b-catenin signaling is activated during hypoxia, including the upregulated expression of Wnt proteins and its coreceptor on cell membrane, b-catenin separating from the destruction complex and stabilizing in the cytoplasm, b-catenin translocating to nucleus, interacting with TCF/LEF transcription factors and activating cell transcription in the nucleus. Wogonin treatment resulted in downregulation of Wnt5a, FZD4, LRP5/6, DvL-2 and DvL-3. Early reports suggested that in the presence of FZD4 and LRP5/6, Wnt5a could activate b-catenin/TCF signaling [10]. We conjectured that wogonin might influence Wnt/b-catenin “canonical” pathway. Then it was found wogonin could inhibit b-catenin expression. In addition, wogonin treatment led to upregulation of Axin and dephosphorylating activation of GSK-3b, which indicated that wogonin might influence the level of b-catenin protein by promoting its degradation.
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To confirm wogonin affected b-catenin through modulation of destruction complex, a well known GSK-3b inhibitor, LiCl was used. LiCl can inhibit GSK3b by acting as a competitive inhibitor of Mg2þ inducing Ser9 autophosphorylation [31]. The results revealed LiCl recovered the accumulation of bcatenin suppressed by wogonin in HUVECs. To further study the effect of wogonin on the degradation of b-catenin, MG132 and CHX were used. The results showed that wogonin promoted proteasomal degradation of b-catenin and reduced the stability of protein. In addition, the immunoprecipitation assay showed that the more extensive ubiquitination of bcatenin protein was observed in wogonin treated cells than untreated controls, suggesting that wogonin might inhibit b-catenin protein stability depending on proteasome-mediated degradation. In our study, b-catenin translocated to the nucleus to regulate target gene transcription under hypoxia. We evaluated the effects of wogonin on b-catenin nuclear accumulation. Both immunofluorescence and Western blot showed that wogonin prevented b-catenin transporting to nucleus. The transcriptional activity of b-catenin through its interaction with the TCF/LEF family is recognized as the major effector of the Wnt/b-catenin signaling pathway. We found
wogonin not only inhibited the expression of TCF/ LEF, but significantly limited the combination of bcatenin with TCF/LEF in the nucleus. As a result, wogonin inhibited the b-catenin target gene VEGF expression on the mRNA and protein expression level, which was one of the major reasons to induce vascular hyperpermeability. We have studied the effect of wogonin on HIF-1a expression during hypoxia. Wnt/b-catenin is also activated under hypoxia [6]. There are different reports on the relationship between HIF-1a and Wnt/b-catenin signaling pathway: obstructing or increasing [32,33]. This tendency may be caused in different cells and further study will be performed to investigate the effects of wogonin on the relationship between HIF-1a and Wnt/b-catenin signaling pathway in HUVECs. Wnt/b-catenin pathway signaling is highly activated in certain tumors, and it leads to inappropriate nuclear accumulation of b-catenin and gene transactivation which is an important step in cancer progression. Our previous study has revealed that wogonin could regulate Wnt/b-catenin signaling in human colorectal cancer carcinoma cells [34] and human glioma carcinoma cancer cells [35]. It needs further investigation to study whether wogonin can
Figure 7. Mechanism of wogonin on suppressing vascular hyperpermeability. Wogonin can not only inhibit the dissociation of VE-cadherin and b-catenin, but also is an inhibitor of Wnt/b-catenin signaling pathway, by promoting degradation of b-catenin through modulation of destruction complex (thereby decreasing b-catenin levels), and by decreasing b-catenin nuclear import and TCF/LEF transcription activity.
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inhibit tumors through Wnt/b-catenin signaling in other cancers. In the present study, we found that wogonin suppressed vascular permeability via Wnt/bcatenin pathway which indicated that wogonin might improve the permeability of tumor blood vessels for cancer therapy. To sum up, we firstly demonstrated that wogonin inhibited vascular permeability under hypoxia. The results extended our understanding on the molecular mechanisms of wogonin inhibiting hypoxia-induced vascular leakage (Figure 7). Wogonin may be a potent Wnt/b-catenin inhibitor and may be developed as a chemotherapeutic agent for cancer therapeutics in the future. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 30973556 and 81001452), Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGZ201101, SKLNMZZ201210, SKLNMZZCX201303 and SKLNMZZJQ201302), Fundamental Research Funds for the Central Universities (No.JKP2011003), The National Science & Technology Major Project (No. 2012ZX09304-001) and Natural Science Foundation of Jiangsu province (No. BK2009297 and No. BK2010432), Colleges and Universities in Jiangsu Province Plans to Graduate Research and Innovation (2012). REFERENCES 1. Schlingemann RO, van Hinsbergh VW. Role of vascular permeability factor/vascular endothelial growth factor in eye disease. Br J Ophthalmol 1997;81:501–512. 2. Kaplan JD, Trulock EP, Anderson D.P, Schuster DJ. Pulmonary vascular permeability in interstitial lung disease. A positron emission tomographic study. Am Rev Respir Dis 1992;145: 1495–1498. 3. Weis SM. Vascular permeability in cardiovascular disease and cancer. Curr Opin Hematol 2008;15:243–249. 4. Jain RK. Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy. Science 2005;307:58–62. 5. Dejana E, Orsenigo MG, Lampugnani F. The role of adherens junctions and VE-cadherin in the control of vascular permeability. J Cell Sci 2008;121:2115–2122. 6. Mazumdar J, O'Brien WT, Johnson RS, et al. O2 regulates stem cells through Wnt/beta-catenin signalling. Nat Cell Biol 2010;12:1007–1013. 7. Jamieson C, Sharma M, Henderson BR. Henderson Wnt signaling from membrane to nucleus: beta-catenin caught in a loop. Int J Biochem Cell Biol 2012;44:847–850. 8. Carayol NCY, Wang IK. Kalpha stabilizes cytosolic beta-catenin by inhibiting both canonical and non-canonical degradation pathways. Cell Signal 2006;18:1941–1946. 9. Goodwin AM, Sullivan PA, D'Amore KM. Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling. Dev Dyn 2006;235: 3110–3120. 10. Mikels AJR. Nusse Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol 2006;4:e115.
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11. Gao Z, Huang K, Yang H, Xu X. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochim Biophys Acta 1999; 1472:643–650. 12. You KM, Jong HP, Kim HG. Inhibition of cyclooxygenase/ lipoxygenase from human platelets by polyhydroxylated/ methoxylated flavonoids isolated from medicinal plants. Arch Pharm Res 1999;22:18–24. 13. Lee WR, Shen SC, Lin HY, et al. Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca(2þ)-dependent endonuclease. Biochem Pharmacol 2002;63:225–236. 14. Huang KF, Zhang GD, Huang Y, Diao YQ. Wogonin induces apoptosis and down-regulates survivin in human breast cancer MCF-7 cells by modulating PI3K-AKT pathway. Int Immunopharmacol 2012;12:334–341. 15. Zhang K, Guo QL, You QD, et al. Wogonin induces the granulocytic differentiation of human NB4 promyelocytic leukemia cells and up-regulates phospholipid scramblase 1 gene expression. Cancer Sci 2008;99:689–695. 16. Hui KM, Huen MS, Wang HY, et al. Anxiolytic effect of wogonin, a benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi. Biochem Pharmacol 2002;64: 1415–1424. 17. Jaffe EA, Nachman RL, Becker CR, Minick CG. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745–2756. 18. Li C, Lu N, Qi Q, et al. Gambogic acid inhibits tumor cell adhesion by suppressing integrin beta1 and membrane lipid rafts-associated integrin signaling pathway. Biochem Pharmacol 2011;82:1873–1883. 19. Eum SY, Lee YW, Hennig M, Toborek B. VEGF regulates PCB 104-mediated stimulation of permeability and transmigration of breast cancer cells in human microvascular endothelial cells. Exp Cell Res 2004;296:231–244. 20. Choi YS, Choi HJ, Min JK, et al. Interleukin-33 induces angiogenesis and vascular permeability through ST2/TRAF6mediated endothelial nitric oxide production. Blood 2009;114: 3117–3126. 21. Rong JJ, Hu R, Qi Q, et al. Gambogic acid down-regulates MDM2 oncogene and induces p21(Waf1/CIP1) expression independent of p53. Cancer Lett 2009;284:102–112. 22. Steel R, Doherty JP, Buzzard K, et al. Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1. J Biol Chem 2004;279:51490–51499. 23. Rehbein M, Kindler S, Horke D, Richter S. Two trans-acting ratbrain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs. Brain Res Mol Brain Res 2000;79:192–201. 24. Lu Z, Lu N, Li C, et al. Oroxylin A inhibits matrix metalloproteinase-2/9 expression and activation by upregulating tissue inhibitor of metalloproteinase-2 and suppressing the ERK1/2 signaling pathway. Toxicol Lett 2012;209:211–220. 25. Gu H, Wang X, Rao S, et al. Gambogic acid mediates apoptosis as a p53 inducer through down-regulation of mdm2 in wildtype p53-expressing cancer cells. Mol Cancer Ther 2008;7: 3298–3305. 26. Sakai H, Kozutsumi Y, Goto K, Chiba M, Misawa Y. Functional importance of the cytoskeletal protein in acetylcholineinduced contraction of rat bronchial smooth muscle. J Smooth Muscle Res 2009;45:97–108. 27. Dejana E. The role of wnt signaling in physiological and pathological angiogenesis. Circ Res 2010;107:943–952. 28. Easwaran V, Lee SH, Inge L, et al. beta-Catenin regulates vascular endothelial growth factor expression in colon cancer. Cancer Res 2003;63:3145–3153. 29. Dvorak HF. Discovery of vascular permeability factor (VPF). Exp Cell Res 2006;312:522–526.
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33. Genetos DC, Toupadakis CA, Raheja LF, et al. Hypoxia decreases sclerostin expression and increases Wnt signaling in osteoblasts. J Cell Biochem 2010;110:457–467. 34. He L, Lu N, Dai Q, et al. Wogonin induced G1 cell cycle arrest by regulating Wnt/beta-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells. Toxicology 2013;312C:36–47. 35. Wang Y, Zhang Y, Qian C, et al. GSK3beta/beta-catenin signaling is correlated with the differentiation of glioma cells induced by wogonin. Toxicol Lett 2013;222: 212–223.
Wogonin Influences Vascular Permeability Via Wnt/b-Catenin Pathway Xiuming Song, Yuxin Zhou, Mi Zhou, Yujie Huang, Zhiyu Li, Qidong You,* Na Lu,* and Qinglong Guo* State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, People’s Republic of China
Wogonin, a flavone from the root of Scutellaria baicalensis Georgi, has shown various biological activities. In our previous study, it was confirmed that wogonin could decrease the expression of hypoxia-inducible factor-1a (HIF-1a) by affecting its stability under hypoxia. However, it is still unknown whether wogonin could influence Wnt/b-catenin pathway under hypoxia. In this study, we found that wogonin disrupted Wnt/b-catenin signaling and reduced the secretion of vascular endothelial growth factor (VEGF, also known as vascular permeability factor, VPF), which increased vascular permeability in certain diseases. It was found that wogonin suppressed HUVECs hyperactivity and actin remodeling induced by hypoxia, inhibited transendothelial cell migration of the human breast carcinoma cell MDA-MB-231 and the extravasated Evans in vivo Miles vascular permeability assay. Wogonin-treated cells showed a decrease in the expression of Wnt protein and its co-receptors, as well as a parallel increase in the expression of Axin and GSK-3b in degradation complex, leading to degradation of b-catenin. In addition, wogonin promoted the binding between Axin and b-catenin, increased ubiquitination of b-catenin and promoted its degradation. Interestingly, wogonin decreased the expression of TCF-1, TCF3, and LEF-1 and inhibited nuclear accumulation of b-catenin as well as the binding of b-catenin and TCF-1, TCF-3, or LEF-1. All of the above results showed that wogonin could inhibit the expression of VEGF, which is an important factor regulated by b-catenin. Taken together, the results suggested that wogonin was a potent inhibitor of Wnt/b-catenin and influenced vascular permeability, and this might provide new therapeutics in certain diseases. © 2013 Wiley Periodicals, Inc. Key words: vascular permeability; Wnt/b-catenin; degradation; wogonin
INTRODUCTION Vascular permeability is closely linked with angiogenesis in a number of pathologic conditions, such as eye diseases [1], lung diseases [2], cardiovascular diseases and cancers [3]. In these diseases, the function of blood vessels is impaired because of abnormalities in their endothelial lining. For example, there are a large quantity of “abnormalized” vessels in tumors and they impair perfusion and oxygenation [4]. The resultant hypoxia promotes tumor malignancy. In the “abnormalized” vessels, the endothelial cells become more active, including cell connection damage, hypermotility and a disordered arrangement, resulting in an increase of blood vessels permeability. Thus, targeting vascular permeability may provide new therapeutics in treating certain diseases. In many diseases, hypoxia is a response to the increased vascular permeability. To maintain endothelial cell monolayer integrity, vascular endothelial cadherin (VE-cadherin), similar to many other members of the cadherin family, is linked through its cytoplasmic tail to the adherens junctions (AJ) proteins p120, b-catenin, and plakoglobin [5]. Under hypoxic condition, on one hand, VE-cadherin and bcatenin dissociate to destroy AJ; on the other hand, wingless type protein (Wnt)/b-catenin signaling is activated [6] and the expression of downstream target ß 2013 WILEY PERIODICALS, INC.
Abbreviations VE-cadherin, vascular endothelial cadherin; AJ, adherens junctions; Wnt, wingless type protein; HUVEC, human umbilical vein endothelial cell; FZD-4, frizzled 4; LRP5/6, low-density lipoprotein receptor-related protein 5/6; TCF, T-cell factor; HIF-1a, hypoxia-inducible factor-1a; VEGF, vascular endothelial growth factor; GSK-3b, glycogen synthase kinase-3b; DvL-2, 3, dishevelled-2, 3; LEF, lymphoid enhancer factor; CHX, cycloheximide; FITC, fluorescein-5isothiocyanate; Axin, axis inhibition protein; ELISA, enzyme linked immunosorbent assay. Xiuming Song and Yuxin Zhou contributed equally to this work. Conflict of interest: There are no conflicts of interest. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 30973556; 81001452; Grant sponsor: Program for Changjiang Scholars and Innovative Research Team in University; Grant number: IRT1193; Grant sponsor: Project Program of State Key Laboratory of Natural Medicines; Grant sponsor: China Pharmaceutical University; Grant numbers: JKGZ201101; SKLNMZZ201210; SKLNMZZCX201303; SKLNMZZJQ201302; Grant sponsor: Fundamental Research Funds for the Central Universities; Grant number: JKP2011003; Grant sponsor: The National Science & Technology Major Project; Grant number: 2012ZX09304-001; Grant sponsor: Natural Science Foundation of Jiangsu Province; Grant numbers: BK2009297; BK2010432; Grant sponsor: Colleges and Universities in Jiangsu Province Plans to Graduate Research and Innovation (2012) *Correspondence to: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People's Republic of China. Received 24 September 2013; Accepted 8 July 2013 DOI 10.1002/mc.22093 Published online 17 October 2013 in Wiley Online Library (wileyonlinelibrary.com).
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genes results in changes in cell adhesion and migration. For the above reasons, blood vascular permeability increases under the stimulus of hypoxia. b-Catenin was first identified at AJ where it linked cadherins with the cytoskeleton to regulate the response to cell adhesion. Later studies have found that b-catenin is a central mediator of the Wnt signaling pathway and controls the transcription of genes during both normal and malignant development [7]. There are two different Wnt/b-catenin signaling pathway: “canonical” Wnt/b-catenin signaling pathway and “non-canonical” Wnt/b-catenin signaling pathway [8]. Canonical Wnt signaling involves stabilization of cytosolic b-catenin, turning it into a nuclear transcriptional regulator. In human umbilical vein endothelial cells (HUVECs), numerous Wnt signaling components are expressed, including Wnt5a and frizzled 4 [9]. In the presence of frizzled 4 (FZD-4) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6), Wnt5a can activate canonical Wnt signaling downstream of b-catenin stabilization and promote T-cell factor (TCF) mediated transcription [10], causing cells more active. Wogonin is a naturally flavonoid isolated from the root of Scutellaria baicalensis Georgi, which has been widely used for its antioxidant [11], anti-inflammatory [12], and anticancer activities [13–15]. In our previous study, we have confirmed that wogonin could inhibit angiogenesis by decreasing the expression of hypoxia-inducible factor-1a (HIF-1a) under hypoxia. However, whether wogonin could affect vascular permeability under hypoxia is still unknown and the molecular mechanisms remain to be poorly understood and need a further investigation. In our study, we investigated the effect of wogonin on vascular permeability under hypoxia. Here, we elucidated the potential effects of wogonin on the response of HUVECs migration and actin cytoskeleton, MDAMB-231 transendothelial cell migration, and the extravasated Evans in vivo. Further mechanism research revealed that wogonin inhibited vascular permeability via influencing Wnt/b-catenin signaling pathway. We found that wogonin promoted degradation of b-catenin and inhibited its nuclear accumulation. Taken together, the results suggested that wogonin may serve as a candidate in the development of a Wnt/b-catenin inhibitor for disease therapy. MATERIALS AND METHODS Materials Wogonin was isolated from S. baicalensis Georgi according to the protocols reported previously with slight modifications [16]. Samples containing 99% or higher wogonin were used in all experiments unless otherwise indicated. Primary antibodies for b-catenin (E-5), vascular endothelial growth factor (VEGF) (147), glycogen synthase kinase 3b (GSK-3b) (H-76), FZD-4 (H-120), and b-actin were obtained from Santa Molecular Carcinogenesis
Cruz Biotechnology (Santa Cruz, CA). Primary antibody for Histone H3 (T11) and p-GSK-3b (S9) were from Bioworld (St. Louis Park, MN). Primary antibodies for Wnt5a/b (C27E8), LRP5/6 (C12A5), Dvl-2 (30D2), Dvl-3, axis inhibition protein (Axin) (C76H11), TCF-1 (C63D9), TCF-3 (D15G11), lymphoid enhancer factor-1 (LEF)-1 (C12A5), VE-Cadherin (D87F2), and ubiquitin were from Cell Signaling Technology (Danvers, MA). CoCl2, MG132, LiCl, and cycloheximide (CHX) were purchased from Sigma (St. Louis, MO). Bovine serum albumin (BSA), paraformaldehyde, Triton X-100, Tris, NaCl, EDTA, NP-40, PMSF, NaF, SDS, DTT, Evans blue and fluorescein-5isothiocyanate (FITC)-conjugated Phalloidin were purchased from Sigma-Aldrich (St. Louis, MO). IRDyeTM800 conjugated secondary antibodies were obtained from Rockland, Inc. (Philadelphia, PA). Cell Culture HUVECs were isolated from human umbilical cord veins by collagenase treatment as described previously [17]. The harvested cells were grown in medium 199 (M199, Gibco, Grand Island, NY) containing endothelial cell growth supplement (ECGS, 30 mg/mL; Sigma), epidermal growth factor (EGF, 10 ng/mL; Sigma), 20% fetal bovine serum (FBS, Gibco), 100 U/ mL penicillin, and 100 U/mL streptomycin, pH 7.4. After 3–5 passages, HUVECs were collected for use in all experiments. The human breast carcinoma cell MDA-MB-231 was originally obtained from the Cell Bank of Shanghai Institute of Cell Biology. MDA-MB231 cells were cultured in L-15 medium (Gibco) containing 10% FBS (Sijiqing, Hangzhou, China), 100 U/mL penicillin, and 100 U/mL streptomycin, and cells were grown in a stable environment with 5% CO2 at 378C. Endothelial Cell Migration Assay HUVECs were treated with wogonin (0, 1, 10, and 100 mM) under normoxic or hypoxic conditions for 4 h. Then the cells were trypsinized and suspended at a final concentration of 5 105 cells/mL in serum-free M199. Cell suspension was loaded into each of the upper wells. Following incubation at 378C in 5% CO2 for 4 h, nonmigratory cells on the upper surface were removed by a cotton swab. The migrated tumor cells on the lower surface were fixed with 100% methanol and stained with hematoxylin and eosin. The migrated cells were quantified by manual counting and five randomly chosen fields were analyzed for each group. Immunofluorescent HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 4 h under different conditions as specified. Immunofluorescent was performed according to the method described with modification [18]. Stained specimens were examined under a confocal laser scanning microscope (Fluoview FV1000, Olympus, Tokyo, Japan).
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Transendothelial Cell Migration Assay The experiment was performed as previously described [19]. Briefly, HUVECs were seeded in the amount of 1 104 cells on transwell polycarbonate filters (6.5 mm in diameter, 8 mm pore-size, CorningCostar, Cambridge, MA). After reaching a confluence, cells were preincubated for 4 h with wogonin (1, 10, or 100 mM) under normoxia or hypoxia. Then transendothelial cell migration assay using MDA-MB-231 cells was performed as described in “endothelial cell migration assay” part. Miles Vascular Permeability Assay The mice were equally randomized into five groups (each group contained seven mice): as indicated. Wogonin treatments were done at a frequency of i.v. each day for a total of 6 d. CoCl2 (100 mL) was injected intradermally each day into the back skin of mice at the same spot for 3 d to induce hypoxia-like response (0.9% normal saline was injected as control). Then the Miles assay was performed as described previously [20]. Apoptosis Assessment Cells were pretreated with wogonin (0, 1, 10, and 100 mM) as specified for 4 h. Then apoptosis induced by wogonin was detected by Annexin V-FITC apoptosis detection kit (BioVision, San Francisco, CA) according to the manufacturer’s protocol. Colony-Formation Assay Cells were pretreated with wogonin (0, 1, 10, and 100 mM) under normoxic or hypoxic conditions for 4 h, and then plated in 35-mm dishes at 10 000 cells/ well in 0.35% agar in M199 over a 0.5% agar layer. Plates were further incubated for 21 d until colonies were large enough to be visualized. Colonies were counted under inverted microscrope. Experiments were done in triplicate. Western Blot Analysis HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 4 h under normoxic and hypoxic conditions. Western blotting was done as previously reported [21]. Detection was performed by the Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE). Preparation of Cytosolic and Nuclear Extracts HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 4 h under different conditions as specified. Nuclear and cytosolic protein extracts were prepared according to the modified method as described previously [22,23]. Enzyme Linked Immunosorbent Assay Cells were pretreated with wogonin (0, 1, 10, and 100 mM) under normoxic or hypoxic conditions for 4 h, and then the medium was replaced by serum-free Molecular Carcinogenesis
medium. After 12 h, the concentration of VEGF was determined by a human VEGF Duo-set enzyme linked immunosorbent assay (ELISA) kit per manufacture’s instructions. Real-Time PCR Analysis HUVECs were treated with wogonin (0, 1, 10, and 100 mM) for 2 h under normoxic or hypoxic conditions. The mRNA level of VEGF was determined to the method described previously [24]. The primer sets used in the PCR amplification were as follows: VEGF (forward, 50 -GGT GGA CAT CTT CCA GAG TA-30 , reverse, 50 -GGC TTG TCA CAT CTG CAA GTA-30 ), bactin (forward, 50 -CTG TCC CTG TAT GCC TCT G-30 , reverse, 50 -ATG TCA CGC ACG ATT TCC-30 ). Luciferase Reporter Assay The hVEGF-pGL2 promoter reporter was provided by Dr. Bar-bara K. Vonderhaar (Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, USA). Cells were cotransfected with VEGF-pGL2 with Renilla luciferase reporter (as internal control) for 24 h and then treated with wogonin (0, 1, 10, and 100 mM) for 4 h. The luciferase activity of cell lysate was determined with the Dual-Luciferase Reporter kit (Beyotime, Haimen, China) according to the provided protocol. Luciferase signals were collected by DualLuciferase Assay system (Thermo Fisher Scientific, Rockford, IL). Immunoprecipitation HUVECs were treated with different concentrations of wogonin (0 and 40 mM) for 4 h under different conditions as specified. Immunoprecipitation was performed as previously described [25]. Proteins were precipitated from the supernatant by the addition of b-catenin antibody. Samples were stored at 208C for Western blotting assay. VE-cadherin, Axin, TCF-1, TCF-3, LEF-1 or ubiquitin antibodies were used and the secondary antibody and band detection were operated as described above. Statistical Analysis All data in different experimental groups were expressed as the mean SEM. These data shown in the study were obtained in at least three independent experiments. Statistical analyses were performed using an unpaired, two-tailed Student’s t-test. The comparisons were made relative to untreated controls and significance of difference is indicated as P < 0.05 and P < 0.01. RESULTS Wogonin Inhibited the Hypoxia-Induced Vascular Permeability HUVECs show hyperactivity during hypoxia, leading to an increase of vascular permeability. In our
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experiments, Transwell assay was used to evaluate the effect of wogonin on the migration of HUVECs. As shown in Figure 1A, HUVECs became more active under hypoxia compared with the normoxia control. When treated with 1, 10, and 100 mM wogonin, fewer HUVECs migrated to the lower side of the filter in the Transwell Chamber. Wogonin significantly inhibited the migration of HUVECs in a concentration-dependent manner. The endothelial barrier integrity is the result of a balance between the tethering and contractile forces acting on endothelial cells, which are critically dependent upon cytoskeletal components, including the actin-based microfilaments [26]. Therefore, the response of the cytoskeleton to wogonin under hypoxia in HUVECs was investigated, using FITCphalloidin to stain actin filaments. As shown in Figure 1B, HUVECs showed a polarized reorganization of the actin cytoskeleton under hypoxia compared with the normoxia control. When treated with 1, 10, and 100 mM wogonin, cytoplasmic stress fibers were blocked in a concentration-dependent manner. The transendothelial migration assay is frequently used to determine the ability of test compounds to endothelial cell permeability. As shown in Figure 1C, more cells migrated through the endothelial cells monolayer under hypoxia compared with the normoxia control. When treated with 1, 10, and 100 mM wogonin, this effect was blocked in a concentrationdependent manner. To investigate the effect of wogonin on vascular permeability in vivo, Miles vascular permeability assay was utilized. As shown in Figure 1D, vascular hyperpermeability of the mouse skin treated with CoCl2 was inhibited by wogonin at 20, 40, and 80 mg/ kg in a dose-dependent manner. These results demonstrated that wogonin inhibited CoCl2induced vascular leakage in a hypoxia-like response in vivo. Wogonin Neither Induced Apoptosis Nor Suppressed Proliferation in HUVECs at the Tested Concentrations AnnexinV-FITC apoptosis detection kit was used the assess the apoptosis effect of wogonin on HUVECs. As shown in Figure 2A, wogonin (0, 1, 10, and 100 mM) did not induce apoptosis in HUVECs. To investigate the proliferation of HUVECs, colonyformation assay was performed. As shown in Figure 2B, wogonin did not suppress proliferation of the cells. These results indicated that wogoninmediated decrease in vascular permeability was irrelevant to the difference in number of cells. Wogonin Inhibited Dissociation of VE-Cadherin and b-Catenin Endothelial permeability is regulated in part by the dynamic opening and closure of cell–cell AJ, which are largely composed of VE-cadherin binding to several protein partners, including b-catenin and Molecular Carcinogenesis
others [5]. As shown in Figure 3A, confocal images showed hypoxia led to a decrease of co-localization between VE-cadherin and b-catenin at the cell borders compared with that of control, and treatment with wogonin at 100 mM blocked the effect. Then the binding of VE-cadherin and b-catenin was further investigated by immunoprecipitation analysis. As shown in Figure 3B, wogonin inhibited the dissociation of VE-cadherin and b-catenin under hypoxia. These data provided evidence that wogonin could decrease vascular permeability by promoting the association between VE-cadherin and b-catenin to stabilize AJ. Downregulation of the Wnt Protein and its Co-Receptors by Wogonin Not only dissociation of VE-cadherin and b-catenin happened during hypoxia, but Wnt/b-catenin signaling was activated to make the endothelial cell motility and vascular permeability changed. To assess the functional relevance of Wnt/b-catenin signaling in the context of vascular permeability, we analyzed the effect of wogonin on the Wnt protein and its coreceptors. As shown in Figure 3C, Wnt protein (Wnt5a), its co-receptors (FZD-4, LRP5/6) and dishevelled-2, 3 (DvL-2, 3) were upregulated under hypoxia compared with normoxia control. When the cells were treated with wogonin, all the proteins were downregulated in a concentration-dependent manner. Wogonin Inhibited b-Catenin Expression Via Promoting its Degradation Through Modulation of Destruction Complex The most frequently studied and best-understood Wnt signaling pathway is mediated by the transcriptional activity of b-catenin. In the presence of Wnt protein, cytoplasmic b-catenin separates from the multiprotein complex and stabilizes in the cytoplasm. As shown in Figure 4A, b-catenin is upregulated under hypoxia and wogonin could inhibit its expression. In addition, Western blot analysis showed that the protein level of GSK-3b and Axin, the main components of destruction complex, increased when the cells were treated with wogonin. And p-GSK-3b that is the inactivity form of GSK-3b decreased, indicating that destruction complex was modulated to for proteasomal degradation of b-catenin (Figure 4B). To substantiate the involvement of Axin in wogoninmediated decrease in b-catenin levels, we used immunoprecipitation to assess the binding of Axin with b-catenin. As shown in Figure 4C, wogonin could enhance the binding of Axin and b-catenin, which may promote the degradation of b-catenin. To further confirm whether wogonin could modulate destruction complex for degradation of bcatenin, a well known GSK-3b inhibitor, LiCl was used. Western blot analysis showed that LiCl could block wogonin-mediated suppression of b-catenin expression (Figure 4D). The result indicated that
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Figure 1. Wogonin inhibited hypoxia-induced increasing of vascular permeability in vitro and in vivo. (A) Inhibition of hypoxia-stimulated migration of HUVECs by wogonin. (B) Effect of wogonin on cytoskeleton remodeling in HUVECs. (C) Inhibition transendothelial cell migration of MDA-MB-231 cells by wogonin. (D) Effect of wogonin on inhibiting vascular leakage in vivo. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
wogonin could promote b-catenin degradation by affecting GSK-3b. In addition, as shown in Figure 4E, in the absence of MG132, levels of b-catenin were significantly reduced Molecular Carcinogenesis
by wogonin. But the accumulation of b-catenin was unaffected by wogonin in the presence of MG132, suggesting that wogonin promoted proteasomal degradation of b-catenin. To further study the
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Figure 2. Wogonin did not induce apoptosis nor suppress proliferation at tested concertration. (A) Effect of wogonin on apoptosis of HUVECs. (B) Effect of wogonin on proliferation of HUVECs.
potential mechanism, HUVECs were also treated with wogonin in the absence and presence of CHX for inhibiting de novo protein synthesis. As shown in Figure 4F, wogonin reduced the stability of b-catenin. Besides, the more extensively ubiquitinated b-catenin
protein was observed in wogonin treated cells than untreated controls under hypoxia (Figure 4G). All the findings suggested that wogonin might inhibit bcatenin protein stability by modulating the destruction complex.
Figure 3. Effects of wogonin on the binding of VE-cadherin and b-catenin, Wnt protein and its co-receptors. (A) Effect of wogonin on the co-localization of VE-cadherin (green) and b-catenin (red) in HUVECs. (B) Effect of wogonin on the binding of VE-cadherin and b-catenin in HUVECs. (C) Effect of wogonin on the Wnt protein and its coreceptors: Wnt5a, FZD-4, LRP5/6, DvL-2, DvL-3 proteins in HUVECs. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
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Figure 4. Wogonin inhibited b-catenin protein accumulation by influencing its stability. (A) Effect of wogonin on the expression of bcatenin in HUVECs. (B) Effect of wogonin on the expression of proteins in the destruction complex proteins. (C) Effect of wogonin on the binding of Axin and b-catenin. (D) Effect of LiCl on the level of b-catenin protein with or without wogonin. (E) Effect of MG132 on degradation of b-catenin induced by wogonin. (F) Effect of CHX on the level of
b-catenin protein with or without wogonin. CHX was used for inhibiting de novo protein synthesis. (G) Effect of wogonin on the ubiquitination of b-catenin. The comparisons were made relative to controls under hypoxic condition (or 100 mM wogonin-treated group under hypoxia) and significance of difference is indicated as P < 0.05 and P < 0.01 (or #P < 0.05) as specified.
Wogonin Inhibited Nuclear Accumulation of b-Catenin
examine the nuclear b-catenin level. As shown in Figure 5A, in wogonin-treated cells, the nuclear bcatenin protein levels decreased with a corresponding decrease in cytoplasmic b-catenin in a concentrationdependent manner. To sum up, wogonin could decrease b-catenin nuclear accumulation.
The nucleus localization of b-catenin is essential for the transcriptional activity. In the nucleus, b-catenin interacts with TCF/LEF transcription factors and activates cell transcription [27]. To study the effect of wogonin on the intracellular localization of bcatenin, immunofluorescence was used. As shown in Figure 3A, in the presence of wogonin, the b-catenin signal became weaker and the localization was redistributed in both nucleus and cytoplasm. To confirm the results above, Western blot was used to Molecular Carcinogenesis
Wogonin Decreased the Level of TCF/LEF Family and Influenced the Binding of TCF/LEF With b-Catenin As b-catenin transactivates gene expression in a complex with TCF/LEF family, we examined the effect of wogonin on different proteins of the TCF/LEF
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Figure 5. Wogonin decreased b-catenin nuclear accumulation (also shown in Figure 3A), inhibited the expression of TCF/LEF and the combination of b-catenin with TCF/LEF. (A) Effect of wogonin on b-catenin nuclear accumulation. (B) Effect of wogonin on the level of TCF-1, TCF-3 and LEF-1. (C) Effect of wogonin on the binding of b-catenin and TCF-1, TCF-3, or LEF-1. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
family. Wogonin caused a repression of these proteins including TCF-1, TCF-3, and LEF-1 (Figure 5B). An immunoprecipitation assay with b-catenin further identified TCF/LEF family as the key regulator of bcatenin-mediated transcription in wogonin-treated HUVECs. As shown in Figure 5C, the combination of b-catenin and TCF-1, TCF-3 or LEF-1 was significantly limited in HUVECs treated with 100 mM wogonin. Effects of Wogonin on Wnt/b-Catenin Signaling Target Gene VEGF Vijay Easwaran reported that b-catenin regulated VEGF expression [28], which was also known as VPF and closely related to blood vascular permeability [29]. Under the effect of VEGF, the endothelial cells become active, leading to hyperpermeability in several diseases. In the present study, we investigated the effect of wogonin on the secretion and expression of VEGF. ELISA was used to determine the secretion of VEGF from diluted cell-free supernatants of HUVECs cultured under normoxic condition and compared with cells cultured in a hypoxia chamber as described in the methods. As shown in Figure 6A, hypoxia induced secretion of VEGF, and wogonin significantly reduced the secretion of VEGF from the cells in a concertration-dependent manner. Western blot was used to investigate the effect of wogonin on VEGF protein in cell lysates. The result indicated that Molecular Carcinogenesis
wogonin could inhibit the expression of VEGF, which was consistent with ELISA studies. To further study the mechanism that wogonin downregulated VEGF, Real time-PCR assay was utilized. As shown in Figure 6C, mRNA expression of VEGF exhibited marked decrease after treatment of wogonin. To determine whether wogonin directly influenced the VEGF promoter, VEGF-pGL2 reporter was transfected into HUVECs. The level of repression of VEGF promoter activity was quantitatively similar to that of mRNA accumulation (Figure 6D), suggesting that the down-regulation of VEGF expression by wogonin is regulated primarily at the level of gene transcription. DISCUSSION Vascular hyperpermeability often coincides with the early stage of angiogenesis and is also found in areas of diseased tissue in diabetic retinopathy, solid tumors, myocardial infarction, wounds, and chronic inflammation [30]. Endothelial response to hypoxia mediates key processes that regulate angiogenesis progression, including endothelial cells proliferation, migration, adherence, and vascular permeability. During hypoxia, endothelial cells become more active to cause a significant increase in vascular permeability, which may form gaps or be absent in other
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Figure 6. Effects of wogonin on Wnt/b-catenin target gene VEGF. (A) Effect of wogonin on the secretion of VEGF in HUVECs. (B) Effect of wogonin on the expression of VEGF in cell lysates. (C) Effect of wogonin on VEGF mRNA level in HUVECs. (D) Effect of wogonin on VEGF transcription activation in HUVECs. The comparisons were made relative to controls under hypoxic condition and significance of difference is indicated as P < 0.05 and P < 0.01.
denuded areas. So improving blood vessel function by decreasing vascular permeability may lead to new strategies for therapies. Wnt/b-catenin signaling has been separated into a “canonical” pathway and several “noncanonical” pathways, which may influence the expression of its target genes including VEGF [28], changing vascular permeability. In “canonical” pathway, b-catenin combines with several cytosolic proteins to form a large complex consisting of Axin, APC and GSK-3b, and gets phosphorylated for ubiquitin-mediated degradation. When “canonical” Wnt signaling is activated, it leads to inhibition of this turnover complex resulting in increased cytosolic b-catenin, which then transits to the nucleus, where it forms a ternary complex with transcription factors TCF/LEF to activate genes involved VEGF mentioned above. In recent years, many potential natural products have gradually gained considerable attention as a new source of drugs. Our previous study has reported that wogonin could inhibit tumor angiogenesis. In this study, we investigated that wogonin inhibited vascular hyperpermeability in vitro and in vivo. Mechanistic studies using HUVECs showed that wogonin inhibited dissociation of VE-cadherin and b-catenin, accelerated the hypoxia-induced b-catenin protein degradation and inhibited b-catenin nuclear accumulation for gene transcription. In the present study, hypoxia was shown to be associated with a significant decrease in both VEcadherin and b-catenin localized at inter-endothelial Molecular Carcinogenesis
junctions. But when HUVECs were pretreated with wogonin, hypoxia-induced dissociation of VEcadherin from b-catenin was inhibited, which was observed by confocal imaging and immunoprecipitation studies. This indicated wogonin could strengthen adherent junction and inhibit the formation of endothelial gaps. Thus, endothelial cells treated with wogonin were tightly linked and not easy to loose, leading to a decrease in vascular permeability. Wnt/b-catenin signaling plays a significant role in VEGF expression, which is closely related to endothelial cells behavior linking to vascular permeability as indicated above. In our study, Wnt/b-catenin signaling is activated during hypoxia, including the upregulated expression of Wnt proteins and its coreceptor on cell membrane, b-catenin separating from the destruction complex and stabilizing in the cytoplasm, b-catenin translocating to nucleus, interacting with TCF/LEF transcription factors and activating cell transcription in the nucleus. Wogonin treatment resulted in downregulation of Wnt5a, FZD4, LRP5/6, DvL-2 and DvL-3. Early reports suggested that in the presence of FZD4 and LRP5/6, Wnt5a could activate b-catenin/TCF signaling [10]. We conjectured that wogonin might influence Wnt/b-catenin “canonical” pathway. Then it was found wogonin could inhibit b-catenin expression. In addition, wogonin treatment led to upregulation of Axin and dephosphorylating activation of GSK-3b, which indicated that wogonin might influence the level of b-catenin protein by promoting its degradation.
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To confirm wogonin affected b-catenin through modulation of destruction complex, a well known GSK-3b inhibitor, LiCl was used. LiCl can inhibit GSK3b by acting as a competitive inhibitor of Mg2þ inducing Ser9 autophosphorylation [31]. The results revealed LiCl recovered the accumulation of bcatenin suppressed by wogonin in HUVECs. To further study the effect of wogonin on the degradation of b-catenin, MG132 and CHX were used. The results showed that wogonin promoted proteasomal degradation of b-catenin and reduced the stability of protein. In addition, the immunoprecipitation assay showed that the more extensive ubiquitination of bcatenin protein was observed in wogonin treated cells than untreated controls, suggesting that wogonin might inhibit b-catenin protein stability depending on proteasome-mediated degradation. In our study, b-catenin translocated to the nucleus to regulate target gene transcription under hypoxia. We evaluated the effects of wogonin on b-catenin nuclear accumulation. Both immunofluorescence and Western blot showed that wogonin prevented b-catenin transporting to nucleus. The transcriptional activity of b-catenin through its interaction with the TCF/LEF family is recognized as the major effector of the Wnt/b-catenin signaling pathway. We found
wogonin not only inhibited the expression of TCF/ LEF, but significantly limited the combination of bcatenin with TCF/LEF in the nucleus. As a result, wogonin inhibited the b-catenin target gene VEGF expression on the mRNA and protein expression level, which was one of the major reasons to induce vascular hyperpermeability. We have studied the effect of wogonin on HIF-1a expression during hypoxia. Wnt/b-catenin is also activated under hypoxia [6]. There are different reports on the relationship between HIF-1a and Wnt/b-catenin signaling pathway: obstructing or increasing [32,33]. This tendency may be caused in different cells and further study will be performed to investigate the effects of wogonin on the relationship between HIF-1a and Wnt/b-catenin signaling pathway in HUVECs. Wnt/b-catenin pathway signaling is highly activated in certain tumors, and it leads to inappropriate nuclear accumulation of b-catenin and gene transactivation which is an important step in cancer progression. Our previous study has revealed that wogonin could regulate Wnt/b-catenin signaling in human colorectal cancer carcinoma cells [34] and human glioma carcinoma cancer cells [35]. It needs further investigation to study whether wogonin can
Figure 7. Mechanism of wogonin on suppressing vascular hyperpermeability. Wogonin can not only inhibit the dissociation of VE-cadherin and b-catenin, but also is an inhibitor of Wnt/b-catenin signaling pathway, by promoting degradation of b-catenin through modulation of destruction complex (thereby decreasing b-catenin levels), and by decreasing b-catenin nuclear import and TCF/LEF transcription activity.
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inhibit tumors through Wnt/b-catenin signaling in other cancers. In the present study, we found that wogonin suppressed vascular permeability via Wnt/bcatenin pathway which indicated that wogonin might improve the permeability of tumor blood vessels for cancer therapy. To sum up, we firstly demonstrated that wogonin inhibited vascular permeability under hypoxia. The results extended our understanding on the molecular mechanisms of wogonin inhibiting hypoxia-induced vascular leakage (Figure 7). Wogonin may be a potent Wnt/b-catenin inhibitor and may be developed as a chemotherapeutic agent for cancer therapeutics in the future. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 30973556 and 81001452), Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGZ201101, SKLNMZZ201210, SKLNMZZCX201303 and SKLNMZZJQ201302), Fundamental Research Funds for the Central Universities (No.JKP2011003), The National Science & Technology Major Project (No. 2012ZX09304-001) and Natural Science Foundation of Jiangsu province (No. BK2009297 and No. BK2010432), Colleges and Universities in Jiangsu Province Plans to Graduate Research and Innovation (2012). REFERENCES 1. Schlingemann RO, van Hinsbergh VW. Role of vascular permeability factor/vascular endothelial growth factor in eye disease. Br J Ophthalmol 1997;81:501–512. 2. Kaplan JD, Trulock EP, Anderson D.P, Schuster DJ. Pulmonary vascular permeability in interstitial lung disease. A positron emission tomographic study. Am Rev Respir Dis 1992;145: 1495–1498. 3. Weis SM. Vascular permeability in cardiovascular disease and cancer. Curr Opin Hematol 2008;15:243–249. 4. Jain RK. Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy. Science 2005;307:58–62. 5. Dejana E, Orsenigo MG, Lampugnani F. The role of adherens junctions and VE-cadherin in the control of vascular permeability. J Cell Sci 2008;121:2115–2122. 6. Mazumdar J, O'Brien WT, Johnson RS, et al. O2 regulates stem cells through Wnt/beta-catenin signalling. Nat Cell Biol 2010;12:1007–1013. 7. Jamieson C, Sharma M, Henderson BR. Henderson Wnt signaling from membrane to nucleus: beta-catenin caught in a loop. Int J Biochem Cell Biol 2012;44:847–850. 8. Carayol NCY, Wang IK. Kalpha stabilizes cytosolic beta-catenin by inhibiting both canonical and non-canonical degradation pathways. Cell Signal 2006;18:1941–1946. 9. Goodwin AM, Sullivan PA, D'Amore KM. Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling. Dev Dyn 2006;235: 3110–3120. 10. Mikels AJR. Nusse Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol 2006;4:e115.
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