Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury

YTAAP-13439; No of Pages 9 Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

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Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury Haibo Zhu a,c, Jie He d, Liang Ye a,c, Fei Lin d, Jian Hou d, Yan Zhong d, Wanglin Jiang b,⁎ a

School of Public Health and Management, Binzhou Medical University, Yantai, PR China School of Pharmaceutical Sciences, Institute of Materia Medica, Binzhou Medical University, Yantai, PR China Institute of Toxicology, Binzhou Medical University, Yantai, PR China d State Key Laboratory of Long-acting Targeting Drug Delivery Technologies (Luye Pharma Group Ltd.), Yantai 264003, PR China b c

a r t i c l e

i n f o

Article history: Received 29 April 2015 Revised 23 July 2015 Accepted 5 August 2015 Available online xxxx Keywords: Curculigoside A Cerebral ischemia Angiogenesis Vascular endothelial growth factor Blood–brain barrier Wnt5a/β-catenin

a b s t r a c t Curculigoside A has shown protective effects against rat cortical neuron damage in vivo. However, the molecular mechanisms through which Curculigoside A affords this protection are unclear. In the present study, we sought to elucidate the mechanisms of angiogenesis in rat aortic endothelial cells (RAEC), rat aortic smooth muscle cells (RASMC) as well as a rat model of cerebral ischemia and reperfusion injury following treatment with Curculigoside A. We examined the role of Curculigoside A on RAEC and RASMC proliferation, migration, and tube formation in vitro and in a cerebral ischemia and reperfusion injury rat model. We used the recombinant Dickkopf (DKK)-1 protein, a Wnt/β-catenin inhibitor, and the recombinant WIF-1 protein, a Wnt5a antagonist to determine mechanisms. In addition, we measured leakage of the blood–brain barrier (BBB) and tested for angiogenesis associated proteins. Our data suggest that Curculigoside A induces angiogenesis in vitro by increasing proliferation, migration and tube formation in RAEC and RASMC. The increase in Curculigoside A-induced proliferation and tube formation was counteracted by DKK-1 and WIF-1. Curculigoside A increased expression of VEGF, p-VEGFR, p-CREB, Egr-3, VCAM-1, Ang1 and Tie2 while prohibiting BBB leakage in cerebral ischemia and reperfusion injured rats. However, Cyclosporine A, a CREB inhibitor, reduced the expression of p-CREB, Egr-3, VCAM-1, Ang1 and Tie2. These data suggest that Curculigoside A induces cell proliferation and angiogenesis through the Wnt5a/β-catenin and VEGF/CREB/Egr-3/VCAM-1 signaling axis and promotes maturation and stability of new blood vessels via increasing Ang1 and Tie-2 expression. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Cerebral ischemia results in a series of injuries, including cell death (necrosis and apoptosis) and cerebral edema. In contrast, recovery from stroke may be promoted by other cellular reactions, such as angiogenesis (Dirnagl et al., 1999) and the reestablishment of functional microvasculature. The re-establishment of functional microvasculature enhances neurogenesis and functional recovery after stroke (Arai et al., 2009), demonstrating that the formation of new blood vessels from preexisting ones, or angiogenesis, is a key factor in the regeneration process of ischemic tissue (Liman and Endres, 2012). The formation of new blood vessels from preexisting blood vessels of angiogenesis can enlarge existing blood vessels to form collaterals and the recruitment of vascular smooth muscle cells (VSMCs) (Duvall et al., 2008). Endothelial cells (ECs) and VSMCs are major cell types composing vascular structure. The neovasculogenic process in ischemic injury involves harmonized cellular interplay among various cell types, including ECs, ⁎ Corresponding author at: School of pharmaceutical sciences, Binzhou Medical University, Yantai 264003, PR China. E-mail address: [email protected] (W. Jiang).

VSMCs, hematopoietic cells, and other surrounding mesenchymal cells (Zhou et al., 2007). In addition, signals and substrates of neurogenesis and neuroplasticity are tightly co-regulated with angiogenesis and vascular remodeling (Xiong et al., 2010). The Wnt/β-catenin pathway is thought to promote the survival and/ or proliferation of primary endothelial cells. Microvascular endothelial cells cultured in vitro express Wnt5a, Wnt7a and Wnt10b, while vascular smooth muscle cells express Wnt5a (Wright et al., 1999). Ectopic expression of Wnt5a in human umbilical vein endothelial cells (HUVEC) promotes formation and proliferation of a capillary-like endothelial network that can be inhibited by experimental reduction of Wnt5a expression (Masckauchan et al., 2006). Vascular endothelial growth factor (VEGF) is stimulated by ischemia and is crucial for angiogenesis and protection against ischemic injury (Carmeliet, 2003). The up-regulation of VEGF not only promotes angiogenesis, but also increases microvascular permeability (Dvorak et al., 1995). The binding of VEGF to its receptors on the surface of endothelial cells activates the cAMP-response element binding protein (CREB) (Lee et al., 2009). VEGF activation of endothelial cells is mediated by the early growth response protein 3 (Egr-3) and plays an essential role in VEGFmediated angiogenesis (Suehiro et al., 2010). Vascular cell adhesion

http://dx.doi.org/10.1016/j.taap.2015.08.003 0041-008X/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003

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H. Zhu et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

molecule 1 (VCAM-1) is a mediator of angiogenesis that induces chemotaxis in endothelial cells in vitro and angiogenesis in vivo (Koch et al., 1995). Angiopoietin 1 (Ang1) with its tie receptor (Tie2) cooperates with the VEGF system to establish dynamic blood vessel structures. Curculigoside A, a major bioactive compound present in Curculigo orchioides, attenuates H2O2-induced human umbilical vein endothelial cell injury (Wang et al., 2010) and up-regulates VEGF in MC3T3-E1 Cells (Ma et al., 2011). Recently, Curculigoside A was shown to have potentially protective properties against cerebral ischemia injury in middle cerebral artery occluded (MCAO) rats (Jiang et al., 2011). Additionally, our laboratory demonstrated that Curculigoside A induces angiogenesis through the VCAM-1/Egr-3/CREB/VEGF signaling pathway in a human brain microvascular endothelial cell line (Kang et al., 2014). In the present study, we investigated the hypothesis that Curculigoside A induces angiogenesis in a cerebral ischemia and reperfusion injury rat model, activates CREB signaling pathways, upregulates VCAM-1, and increases extracellular levels of VEGF through the Wnt5a/β-catenin and VCAM-1/Egr-3/CREB/VEGF signaling pathway.

2. Materials and methods 2.1. Chemicals and materials Curculigoside A (purity N 98.5%, CAS NO.: 85643-19-2, with molecular formula C22H26O11 and molecular weight 466.4), was obtained from the State Key Laboratory of Long-acting Extended-Release and Targeting Drug Delivery System, Yantai, PR China. Bovine Serum Albumin (BSA), streptomycin, penicillin, Amphotericin B, mitomycin C, Cyclosporine A and Type II collagenase were purchased from Sigma-Aldrich Co. LLC. Evans blue was purchased from Urchem Limited (Shanghai, P.R.China). The platelet endothelial cell adhesion molecule-1 (CD31) antibody, VEGF antibody and ABC Staining System Kits were purchased from Santa Cruz Biotechnology, Inc. The VEGF receptor 2 (p-VEGFR2), CREB, p-CREB and Egr-3 antibodies were purchased from Cell Signaling Technology Inc. The alpha-smooth muscle actin (α-SMA), VCAM-1, Wint5a, Cyclin D1 and β-Catenin antibodies were purchased from Abcam Inc. We purchased the rat Dkk-1 (4010DK-010) and WIF-1 recombinant proteins (1341-WF-050) from R&D System. The 4–0 filaments were purchased from Beijing Shadong Biology Company, P.R. China. The Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies Corporation. The matrigel matrix was purchased from BD Bioscience.

2.2. Rat aortic endothelial cell (RAEC) culture RAEC cultures were obtained by enzymatic dissociation of the aortas obtained from Sprague–Dawley rats. We conducted the isolation and culture procedure as previously described (Aley et al., 2005). Cell characterization was performed based on both cell morphology and indirect immunohistochemistry staining of CD31. Tightly confluent monolayers of RAEC from the 2nd to the 10th passage were used in all the experiments.

2.4. Cell proliferation assay RAEC or RASMC with RAEC in a 1.5:1 ratio (Molnar and Siemann, 2012) were seeded at a density of 8 × 103 cells/well in PLL-coated 96 well plates. Cells were starved for 6 h in DMEM containing 1% FBS. In one plate, the culture medium was changed to FBS-free DMEM containing Curculigoside A (1, 3, 9 or 27 μM). In another plate, cells were treated with 500 ng/mL recombinant Dickkopf (DKK)-1 protein and 3 mg/mL WIF-1 recombinant protein, followed by addition of 9 μM Curculigoside A. After incubation for 24 h, cell proliferation was determined by using the sulforhodamine B (SRB) assay (Papazisis et al., 1997). 2.5. Migration assay RAEC or RASMC with RAEC in a 1.5:1 ratio were grown in Poly-Llysine (PLL) coated 6-well plates at a density of 2 × 105 cells/well for 6 h. Then, monolayers were scratched horizontally with a yellow pipette tip to create spaces free of cells within the monolayer culture. Fresh DMEM medium containing 6 μg/mL mitomycin C and 9 μM Curculigoside A was then added and the cells were incubated for an additional 24 h. Three randomly selected fields along the scraped line were photographed for each well using an OLYMPUS inverted microscope CKX41 connected to a digital camera DP25 at 40× magnification. Migration was estimated by calculating the migration distance of cell interfaces by software Image-ProPlus 6.0 (IPP: Media Cybernetics, USA) (Du et al., 2013). 2.6. siRNA transfections RAEC or RASMC with RAEC in a 1.5:1 ratio were seeded in a 6 well plate at a density of 2 × 105 cells per well. The cells were incubated for 24 h and then washed once with 2 mL siRNA transfection medium. The medium was aspirated and 0.8 mL fresh siRNA transfection medium containing the Egr-3/VCAM-1 siRNA transfection reagent mixture was added to each well. The cells were incubated for 7 h and then 1 mL of normal growth medium added. After the cells were incubated for 24 h, Curculigoside A was added to each well (Kang et al., 2014). The cells were collected and cleared for Western blot analysis following an additional 24 h incubation period. 2.7. Matrigel capillary-like tube formation assay A standard matrigel assay was used to assess the spontaneous formation of capillary like structures in vitro (Du et al., 2013). RAEC or RASMC with RAEC in a 1.5:1 ratio were seeded in 24-well plates (2 × 104 cells/well) previously coated with a growth factor-reduced matrigel matrix containing Curculigoside A (9 μM), recombinant Dickkopf (DKK)-1 protein (500 ng/mL), WIF-1 recombinant protein (3 mg/mL), Egr-3 SiRNA and VCAM-1 siRNA in serum-free media, then incubated at 37 °C for 24 h. The number of tube formations was determined in four random fields (× 100) from each well. Data were analyzed as tube formations formed in treated wells versus untreated control wells. 2.8. Animals and MCAO model

2.3. Rat aortic smooth muscle cell (RASMC) culture RASMC cultures were obtained by enzymatic dissociation of the aortas obtained from Sprague–Dawley rats. We conducted the isolation and culture procedure as previously described (Bochaton-Piallat et al., 1992). Cell characterization was performed based on both cell morphology and indirect immunohistochemistry staining of α-SMA. Tightly confluent monolayers of RASMC from the 2nd to the 10th passage were used in all the experiments.

Male Sprague–Dawley rats (220–250 g) were obtained from the Experimental Animal Center of Shandong Luye Pharmaceutical Ltd. (SPF grade, Certificate NO. SYXK 20090013) and were allowed free access to food and water. All procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996. The middle cerebral artery occlusion (MCAO) operation was carried out as previously described (Jiang et al., 2011).

Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003

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2.9. Experimental protocol After performing MCAO operation on 144 rats, the rats were randomly divided into 5 groups (24 rats in sham group and 30 rats in each of the treatment groups). The rats in the sham group (Group A) and vehicle-treated (Model) group (Group B) received 0.9% saline intravenously once per day. Rats in the Curculigoside A (CuA) treatment group (Group C) were given a bolus injection of Curculigoside A to the tail vein at a dose of 10 mg/kg. Rats in the Cyclosporine A (CyA) group (Group D) were given Cyclosporine A intraperitoneally at a dose of 20 mg/kg. Rats in the Curculigoside A + Cyclosporine A (CuA + CyA) group (Group E) were given Curculigoside A at a dose of 10 mg/kg and Cyclosporine A at a dose of 20 mg/kg. All the drugs were administered to the rats 9 h after reperfusion and then once per day. The modified Neurological Severity Score (mNSS) was determined for each rat at 3, 7 and 14 days after ischemia and reperfusion. We performed the Evans blue extravasation experiment on six rats in each group on days 6 and 13. The other rats in each group were killed with intramuscular ketamine (100 mg/kg) and xylazine (10 mg/kg) on day 7 (6 rats in each group) and day 14 (6, 7, 9, 6, and 7 rats were killed in groups A, B, C, D and E, respectively) after ischemia. After performing transcardial perfusion with phosphate-buffered saline (PBS), the brains corresponding to the peri-infarct cortex were collected and cut into two pieces. One piece was fixed with 4% paraformaldehyde in PBS overnight at 4 °C for subsequent immunohistochemical staining to identify VEGF, VEGFR2, CREB, p-CREB, Egr-3 and VCAM-1 proteins. The other piece was utilized for Western blot analysis. 2.10. Evaluation of blood–brain barrier (BBB) leakage using Evans blue extravasation A quantitative assay using Evans blue was performed to determine blood–brain barrier (BBB) leakage based on a method described previously (Vakili et al., 2007). The BBB leakage was detected on days 6 and 13. 2.11. Immunohistochemical staining Immunohistochemical staining was conducted based on a previously described method (Zhu et al., 2014). Briefly, the sliced sections were incubated with an antibody against CD31 (rabbit, 1:100) and VCAM-1 (rabbit, 1:100) overnight at 4 °C. Protein positive cells were stained brown in the cytoplasm. Sections were then mounted and 3 vision test areas were randomly selected for examination with a high-power microscope (200 ×). The positive expression was analyzed by the Image-Pro Plus 6.0 analysis system. The positive area of the protein expression was defined as follows: the positive area (g%) = the positive area⁄the total area. 2.12. Neurological functional tests Neurological function tests were performed before MCAO and at 3, 7 and 14 days after MCAO by an investigator who was blinded to the experimental groups. The mNSS was evaluated using the flowing method (Chen et al., 2001). Briefly, neurological function was graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18). The mNSS is a composite of sensory, reflex, and balance tests. In the severity scores of injury, 1 score point is awarded for the inability to perform the test or for the lack of a tested reflex. Thus, a higher score corresponds to a more severe injury. 2.13. Western blot analysis For the Ang1,Tie2,VEGF, p-VEGFR2, Egr-3, p-CREB, Wint5a, βCatenin and Cyclin D1 protein assay, 50 μg of protein was resolved on a 15% SDS polyacrylamide gel, then transferred onto nitrocellulose

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membranes as described previously (Wang et al., 2007). Optical densities of the bands were scanned and quantified using a Gel Doc 2000 (Bio-Rad). Data were normalized to the corresponding β-actin levels. Results are expressed for each group as a percentage increase over the MCAO or control group.

2.14. Statistical analysis Data are reported as mean ± S.D., with the exception of the survival rate, which was analyzed by chi-square analysis. A Levene's test was performed to test for variance homogeneity. When the result showed no significance (P N 0.05), a one-way analysis of variance (ANOVA) was performed. One-way analysis of variance (ANOVA) followed by Dunnett's test was performed to compare the differences of other parameters among groups. A value of P b 0.05 was considered statistically significant.

3. Results 3.1. Curculigoside A improves functional recovery after MCAO To test whether Curculigoside A affected functional outcomes after stroke, we performed a neurological function test. Rats treated with Curculigoside A (group C) showed significantly better functional recovery (based on mNSS testing) compared with rats in the vehicle-treated group (P b 0.01 or P b 0.05) on days 3, 7, and 14. However, rats in groups D and E treated with Cyclosporine A had significantly lower functional recovery than the Curculigoside A treated rats (group B) (P b 0.01 or P b 0.05) (Table 1).

3.2. Curculigoside A inhibits BBB leakage after MCAO Vascular permeability was quantitatively evaluated by measuring extravasated Evans blue. Treatment with 10 mg/kg of Curculigoside following ischemia resulted in less BBB leakage in Curculigoside A treated group compared to vehicle-treated group on days 7 and 14 (P b 0.01). However, the BBB leakage values in the groups treated with the CREB inhibitor Cyclosporine A in groups D and E were significantly greater than that of the Curculigoside A treated group rats (P b 0.01) (Table 1).

3.3. Curculigoside A augments proliferation, migration and capillary-like tube formation on RASMC and RAEC The proliferation, migration and tube formation assays were used as markers of angiogenesis in vitro. RAEC and a co-culture of RVSMC and RAEC displayed a basal migration in the absence of Curculigoside A after 24 h incubation. However, the cell cultures displayed a faster migration and induced proliferation in a concentration-dependent manner following treatment with 3 to 27 μM Curculigoside A (Fig. 1a). Along with increased proliferation, Curculigoside A also enhanced migration as quantified with a scratch adhesion test (Fig. 1b). Matrigel assays showed that Curculigoside A induced tube formation in a concentration-dependent manner (Fig. 1c). RVSMC co-cultured with RAEC displayed a faster migration, proliferation and tube formation effect than RVECs alone. The effects of Curculigoside A treatment appeared to peak around a dosage of 9 μM. In order to identify the roles of Egr-3 and VCAM-1 in tube formation, we used Egr-3 SiRNA and VCAM-1 SiRNA to detect tube formation in a co-culture of RVSMCs and RAEC following treatment with 9 μM of Curculigoside A. Egr-3 SiRNA and VCAM-1 SiRNA significantly inhibited the tube formation effect of Curculigoside A (P b 0.05), suggesting that Egr-3 and Vcam-1 contribute to the tube formation response (Fig. 1d).

Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003

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Table 1 Effects of Curculigoside A on BBB leakage with the Evans blue and neurological scores in ischemia and reperfusion injury rats (x ± SD).

Evans blue (μg/g) Neurological scores

7th 14th 3rd 7th 14th

Sham

Model

CuA 10 mg/kg

CyA

CuA + CyA

1.01 ± 0.13 0.98 ± 0.12 / / /

2.87 ± 0.73 2.25 ± 0.55 8.52 ± 1.61 7.64 ± 1.31 6.53 ± 0.81

1.68 ± 0.42⁎⁎ 1.39 ± 0.38⁎⁎ 5.63 ± 1.50⁎ 4.69 ± 0.97⁎ 4.10 ± 0.86⁎

3.11 ± 0.52 2.25 ± 0.41 8.75 ± 1.85 7.88 ± 2.02 6.54 ± 1.63

2.81 ± 0.62 1.87 ± 0.41 8.05 ± 1.71 7.31 ± 1.83 5.89 ± 1.21

⁎ p b 0.05 ⁎⁎ p b 0.01 vs. Model group

3.4. Effect of Curculigoside A on Wint/β-Catenin pathway, CyclinD1, VEGF and Ang1/Tie2 expression in a co-culture of RVSMC and RAEC The Wnt/β-catenin pathway is thought to promote the survival and/ or proliferation of primary endothelial cells. Co-cultures exposed to

Curculigoside A had significantly higher expression levels of Wnt5a, βcatenin and Cyclin D1 compared to unexposed cells (P b 0.01 or P b 0.05) (Fig. 2). In addition, we observed that WIF-1, a Wnt5a antagonist, significantly attenuated Curculigoside A stimulated proliferation (Fig. 1e), suggesting that Wnt5a contributes to the proliferative and

Fig. 1. Effects of Curculigoside A on proliferation, migration and capillary-like tube formation on RASMC and RAEC. Fig.1a. Effects of Curculigoside A on proliferation. RAEC and co-culture of RVSMC and RAEC in a 1.5:1 ratio were seeded at a density of 8 × 103 cells/well with medium alone or with Curculigoside A. Fig.1b. Effects of Curculigoside A on migration. RAEC and coculture of RVSMC and RAEC in a 1.5:1 ratio were seeded at a density of 2 × 105 cells/well with medium alone or with Curculigoside A. The migration distance was calculated by Image-Pro Plus6.0 and was shown in pixel. Fig.1c. Effects of Curculigoside A on tube formation. RAEC or RASMC with RAEC in a 1.5:1 ratio were seeded in 24-well plates (2 × 104 cells/well) previously coated with a growth factor-reduced matrigel matrix containing Curculigoside A (0–9 μM). Fig.1d. Effects of Curculigoside A on tube formation. RASMC with RAEC in a 1.5:1 ratio were seeded in 24-well plates (2 × 104 cells/well) previously coated with a growth factor-reduced matrigel matrix containing Curculigoside A (9 μM). Fig.1e. Effects of Curculigoside A on proliferation. RASMC with RAEC in a 1.5:1 ratio were seeded at a density of 8 × 103 cells/well with medium alone or with Curculigoside A. A: Control group, B: Curculigoside A (9 μM), C: Curculigoside A (9 μM) with DKK-1 (500 ng/mL), D: Curculigoside A (9 μM) with WIF-1 recombinant protein (3 mg/mL), E: Curculigoside A (9 μM) with Egr-3 SiRNA, F: Curculigoside A (9 μM) with VCAM-1 siRNA. Data from experiments were expressed as mean ± SD, n = 5. ⁎P b 0.05, ⁎⁎P b 0.01 vs. Control group; ##P b 0.01, #P b 0.05 vs. RAEC group; ††P b 0.01, †P b 0.05 vs. Curculigoside A 9 μM group. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test.

Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003

H. Zhu et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

Fig. 2. Effect of Curculigoside A on Wint5a, β-Catenin and CyclinD1 expression. RAEC or RASMC with RAEC in a 1.5:1 ratio were seeded in 6-well plates containing Curculigoside A (9 μM), DKK-1 protein (500 ng/mL), WIF-1 recombinant protein (3 mg/mL) in serumfree media, then incubated at 37 °C for 24 h. Total protein extracts were prepared and assayed by Western blot analysis, and blots were normalized to β-actin expression. Representative images are shown from three individual experiments. Data from experiments were expressed as mean ± SD, n = 3. ⁎P b 0.05, ⁎⁎P b 0.01 vs. Control group, ##P b 0.01 vs. Curculigoside A 9 μM group. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test.

migratory response and significantly inhibits β-catenin and Cyclin D1 expression. Finally, addition of DKK-1 (an inhibitor of Wnt/β-catenin) to a Curculigoside A-treated co-culture resulted in a significant reduction in the proliferation rate along with Cyclin D1 expression (Fig. 1e). These data suggest that the Wnt/β-catenin pathway contributes to the proliferation that occurs following treatment with Curculigoside A. The tube formation results indicate that WIF-1 and DKK-1 can significantly inhibit tube formation due to treatment with Curculigoside A (Fig. 1d). In order to determine whether VEGF, Ang-1 and Tie2 regulate angiogenesis through controlling the expression of Wnt/β-catenin and Wnt5a, we examined the expression of VEGF, Ang-1 and Tie2 in coculture of RVSMC and RAEC added DKK-1 and WIF-1 protein. The result indicated that Wnt5a antagonist WIF-1 and Wnt/β-catenin inhibitor DKK-1 significantly suppressed the expression of VEGF. However, there was no significant difference for the expression of Ang1 and Tie2 (Fig. 3).

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Fig. 3. Effects of Curculigoside A on VEGF, Ang1 and Tie2 expression co-culture of RVSMC and RAEC. RAEC or RASMC with RAEC in a 1.5:1 ratio were seeded in 6-well plates containing Curculigoside A (9 μM), DKK-1 protein (500 ng/mL), WIF-1 recombinant protein (3 mg/mL) in serum-free media, then incubated at 37 °C for 24 h. Total protein extracts were prepared and assayed by Western blot analysis, and blots were normalized to βactin expression. Representative images are shown from three individual experiments. Data from experiments were expressed as mean ± SD, n = 3. ⁎P b 0.05, ⁎⁎P b 0.01 vs. Control group, #P b 0.05, ##P b 0.01 vs. Curculigoside A group. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test.

3.5. Curculigoside A induced capillary-like tube formation and vascular stability via Ang1/Tie2 Signaling In order to certify the role of Ang1 and Tie2 in Curculigoside Ainduced angiogenesis, we examined the expression of Ang-1 and Tie2 as well as the capability of RVSMC and/or RAEC for tube formation. Ang-1 contributes to vascular stability and is produced by smooth muscle cells and pericytes. Tie-2 is mainly expressed on endothelial cells. Ang-1 signaling through the Tie2 receptor promotes survival, quiescence and stability of blood vessels. Therefore, we examined the tube formation and Ang1 expression in RAECs and a co-culture of RVSMC and RAEC. Ang1 expression was significantly higher in co-culture of RVSMC and RAEC group than in RAEC group (Fig. 4). The number of tube formations in the co-culture of RVSMC and RAEC exceeded the number formed in RAEC alone (Fig. 1c), suggesting that Ang1 contributes to the tube formation response.

Fig. 4. Effects of Curculigoside A on Ang1 and Tie2 expression in RAEC and/or RVSMC. RAEC or RASMC with RAEC in a 1.5:1 ratio were seeded in 6-well plates containing Curculigoside A (9 μM) or medium alone in serum-free media, then incubated at 37 °C for 24 h. Total protein extracts were prepared and assayed by Western blot analysis, and blots were normalized to β-actin expression. Representative images are shown from three individual experiments. Data from experiments were expressed as mean ± SD, n = 3. #P b 0.05, ##P b 0.01 vs. Control group, ⁎P b 0.05, ⁎⁎P b 0.01 vs. RACE group. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test.

Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003

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Western blot results show that VEGF expression was significantly increased following treatment with Curculigoside A, Curculigoside A combination with Cyclosporine A compared with rats in the vehicle-treated group (P b 0.01 or P b 0.05). Similarly, Ang1 and Tie2 levels were significantly increased following treatment with Curculigoside A compared with rats in the vehicle-treated group (P b 0.01) (Fig. 5). Curculigoside A significantly inhibit the BBB leakage after the MCAO compared with vehicle-treated group (P b 0.01 or P b 0.05) in days 7 and 14 (Table 1). Therefore, we concluded that Ang1 and Tie2 enhanced the tube formation capability and improved vascular stability. 3.6. Effects of Curculigoside A on p-VEGFR2, p-CREB and Egr-3 expression To test the mechanisms underlying Curculigoside A induced angiogenesis, we measured p-VEGFR2, p-CREB and Egr-3 expression in the ischemic brain using Western blots. p-VEGFR2, p-CREB and Egr-3 expression of in Curculigoside A treated group was significantly higher than vehicle-treated group on day 7 (P b 0.01). However, the p-CREB and Egr-3 levels were significantly lower in Cyclosporine A treated in groups D and E than Curculigoside A treated group (Fig. 6). 3.7. Effects of Curculigoside A on VCAM-1 and CD31 expression Immunohistochemical analyses and Western blot analysis demonstrated that the expression of VCAM-1 in Curculigoside A treated group was significantly higher than vehicle-treated group on day 7 (P b 0.01). However, the CREB inhibitor, Cyclosporine A, significantly inhibited expression of VCAM-1 in groups D and E (Fig. 7). As shown in Fig. 7, expression of CD31 in Curculigoside A treated group is greater than vehicle-treated group on day 7 (P b 0.01). In addition, treatment with the CREB inhibitor, Cyclosporine A, significantly inhibited CD31 expression in groups D and E when compared with the vehicle-treated group (P b 0.05). 4. Discussion In a previous study, we confirmed that Curculigoside A exerts potent and long-term neuroprotective effects in a rat model for ischemia and

Fig. 5. Effects of Curculigoside A on VEGF, Ang1 and Tie2 expression in vivo. VEGF, Ang1 and Tie2 protein expression in brain was detected by Western blot analysis in day 7 after reperfusion. Total protein extracts were prepared and assayed by Western blot analysis, and blots were normalized to β-actin expression. Representative images from three individual experiments are shown. A: Sham group, B: Model group, C: Curculigoside A group, D: Cyclosporine A group, E: Curculigoside A + Cyclosporine A group. Data from experiments were expressed as mean ± SD, n = 6. ⁎P b 0.05, ⁎⁎P b 0.01 vs. vehicle-treated group, ##P b 0.01 vs. Curculigoside A 9 μM group. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test.

Fig. 6. Effects of Curculigoside A on p-VEGFR2, p-CREB and Egr-3 expression in vivo. pVEGFR2, p-CREB and Egr-3 protein expression in brain was detected by Western blot analysis in day 7 after reperfusion. Total protein extracts were prepared and assayed by Western blot analysis, and blots were normalized to β-actin expression. Representative images from three individual experiments are shown. A: Sham group, B: Model group, C: Curculigoside A group, D: Cyclosporine A group, E: Curculigoside A + Cyclosporine A group. Data from experiments were expressed as mean ± SD, n = 6. ⁎P b 0.05, ⁎⁎P b 0.01 vs. vehicle-treated group, ##P b 0.01 vs. Curculigoside A 9 μM group. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test.

reperfusion injury (Jiang et al., 2011) and induces angiogenesis in HBMEC via the VCAM-1/Egr-3/CREB signaling pathway (Kang et al., 2014). In the present study, we confirmed that Curculigoside A confers long-term brain protection via an angiogenic mechanism in a rat model of ischemia and reperfusion injury. The mechanisms of this phenomenon appear to involve angiogenic effects that rely on the Wnt5a/βcatenin and VEGF/CREB/Egr-3/VCAM-1 signaling pathways (Fig. 8) and promote maturation and stability of new blood vessels via increasing Ang1 and Tie-2 expression. Vascular endothelial cell adhesion, proliferation and migration play crucial roles in angiogenesis. The canonical Wnt/β-catenin pathway has been implicated in many aspects of angiogenesis, vascular remodeling and differentiation in various species and organ systems. In addition to being a major contributor to brain angiogenesis and barrier formation, the Wnt/β-catenin pathway influences vascular sprouting, remodeling and arterio-venous specification by modulating the Notch pathway. Growing evidence also suggests that the non-canonical Wnt pathway may play a role in vascular development by regulating VEGF availability (Reis and Liebner, 2013). Microvascular endothelial cells cultured in vitro express Wnt-5a, Wnt-7a and Wnt-10b, while vascular smooth muscle cells express Wnt-5a (Zerlin et al., 2008). Cyclin D1 is synthesized early in the G1 phase and plays a key role in the initiation and progression of cell proliferation. When cells enter the S phase, cyclin D1 is rapidly degraded by ubiquitin-proteasome dependent proteolysis. Moreover, the cyclin D1 gene is one of the target genes for the Wnt/βcatenin signaling pathway, and the degradation of β-catenin is initiated by GSK-3β (Takahashi-Yanaga and Sasaguri, 2007). Therefore, we examined the expression of Wnt5a, β-catenin and cyclin D1 in a Curculigoside A-treated co-culture of RASMC and RAEC. Curculigoside A significantly increased Wnt5a and β-catenin protein expression and enhanced migration, proliferation and tube formation. However, the Wnt5a antagonist, WIF-1, significantly attenuated these effects. In addition, Curculigoside A stimulated proliferation, suggesting that Wnt5a contributes to the proliferative and migratory response and significantly inhibits β-catenin and cyclin D1 expression. Finally, when DKK-1 (an inhibitor of Wnt/β-catenin) was added to cultures treated with Curculigoside A, the proliferation rate and cyclin D1 expression

Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003

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Fig. 7. Effects of Curculigoside A on VCAM-1 and CD31 expression in vivo. VCAM-1 and CD31 protein expression in brain was detected by immunohistochemical staining at 200× magnification and Western blot analysis in day 7 after reperfusion. Total protein extracts were prepared and assayed by Western blot analysis, and blots were normalized to β-actin expression. The above pictures are immunohistochemical staining representative images of VCAM-1 and CD31. The blow figures are representative images from three individual experiments and statistics chart. A: Sham group, B: Model group, C: Curculigoside A group, D: Cyclosporine A group, E: Curculigoside A + Cyclosporine A group. Data from experiments were expressed as mean ± SD, n = 6. ⁎P b 0.05, ⁎⁎P b 0.01 vs. vehicle-treated group, ##P b 0.01 vs. Curculigoside A 9 μM group. Significance was determined by one-way analysis of ANOVA followed by Dunnett's test.

was significantly reduced, suggesting that the Wnt/β-catenin pathway contributes to the proliferative effect. Ischemic injury has been found to stimulate both angiogenesis and vascular permeability. Furthermore, VEGF expression is stimulated by ischemia and is crucial for angiogenesis and protecting against ischemic injury. The binding of VEGF to its receptor on the surface of endothelial cells activates intracellular tyrosine kinases, triggering multiple downstream signals that promote angiogenesis. P-VEGFR2 mediates the majority of the downstream angiogenic effects of VEGF, including microvascular permeability, endothelial cell proliferation, migration, migration, and survival (Hicklin and Ellis, 2005). Vascular VEGF activates

CREB, which mediates downstream signaling through VEGFR-2. VEGF/ VEGFR-2 signaling leading to CREB phosphorylation is a shared pathway in neurons and vascular endothelial cells in the developing brain (Lee et al., 2009). Down-regulating expression of the CREB-binding protein inhibits the proliferation of endothelial cells (Jiang et al., 2010). Hence, p-CREB is required for angiogenesis. CREB is involved in multiple signaling pathways to regulate cell proliferation, differentiation, survival and migration (Sakamoto and Frank, 2009) and is closely related to the occurrence and development of angiogenesis (Jiang et al., 2010). Phosphorylation of CREB induces the release of Egr-3, which launches endothelial cell division, proliferation and migration and induces the

Fig. 8. Proposed model for the roles of Wnt5a/β-catenin signaling and VEGF/CREB/Egr-3/VCAM-1 signaling regulated by Curculigoside A in angiogenesis. Curculigoside A increased the expression of Wnt5a, Wnt5a then binds to members of the Frizzled family of receptors on the cell surface thus activating the canonical Wnt/β-catenin signaling pathway through augmentation of β-catenin in the cells. Improved interaction of nuclear β-catenin then leads to the upregulation of typical target genes such as cyclin D1. The expression of VEGF was upregulated by β-catenin of Wnt/β-catenin signaling. Curculigoside A increased the expression of VEGF, VEGF binds to its receptor VEGFR2 on the surface of endothelial cells activates intracellular tyrosine kinases. Then VEGF/VEGFR2 pathway activates CREB, leading to CREB phosphorylation. Phosphorylation of CREB induces the release of Egr-3, which can upregulated the expression of VCAM-1.

Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003

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occurrence of angiogenesis. VCAM-1 is an Ig-like adhesion molecule expressed on activated endothelial cells and is a mediator of angiogenesis. VCAM-1 also induces chemotaxis in endothelial cells in vitro and angiogenesis in vivo (Koch et al., 1995). Our results show that p-CREB, VEGF, p-VEGFR2, Egr-3 and VCAM-1 expression are increased by Curculigoside A, whereas expression of p-CREB, Egr-3 and VCAM-1 is significantly decreased by the CREB inhibitor, Cyclosporine A. We have confirmed that Curculigoside A induces angiogenesis in HBMEC via the VCAM-1/Egr-3/CREB signaling pathway (Kang et al., 2014), so we hypothesized that Curculigoside A would display long-term neuroprotective effects via angiogenesis in a rat model for ischemia and reperfusion injury through the VEGF/CREB/Egr-3/VCAM-1 pathway. VEGF expression can be upregulated by Wnt/β-catenin pathway (Easwaran et al., 2003; Skurk et al., 2005). Our results confirmed that Wnt5a antagonist WIF-1 and Wnt/β-catenin inhibitor DKK-1 significantly suppressed the expression of VEGF. Thus the VEGF/CREB/Egr-3/VCAM-1 pathway may be influenced by Wnt/β-catenin pathway. VEGF and Ang-1 play essential and complementary roles in vascular development and angiogenesis. VEGF controls the early phases of new blood vessel formation; however, VEGF also increases vascular permeability and inflammation during this process (Croll et al., 2004), particularly in the formative stages when angiogenic vessels are leaky (Schoch et al., 2003). Ang-1 is produced by nonendothelial cells and contributes to vascular stability. Ang-1 binds to the tyrosine kinase receptor Tie-2 and induces Tie2 phosphorylation, which is essential for vasculogenesis, maintaining vascular endothelial integrity as well as controlling the maturation and stability of new blood vessels (Iurlaro et al., 2003). Ang-1 also has anti-permeability and anti-inflammatory functions (Eklund and Olsen, 2006). CD31 is a cell surface protein on major cell types associated with the vascular compartment, including platelets, leukocytes and endothelial cells and has been used as a specific marker for endothelial cells (Delisser et al., 1997). Previously, we confirmed that Curculigoside A significantly enhanced Ang1 and Tie2 expression and is capable of enhancing tube formation and improving vascular stability. Expression of CD31, Ang1 and Tie2 was also increased by Curculigoside A and counteracted by the CREB inhibitor, Cyclosporine A. Treatment with Curculigoside A also improved recovery of neurologic function and inhibited BBB leakage, effects that were counteracted by Cyclosporine A in cerebral ischemia and reperfusion injured rats. In conclusion, Curculigoside A induces cell proliferation and angiogenesis through the Wnt5a/βcatenin and VEGF/CREB/Egr-3/VCAM-1 signaling axis and promotes maturation and stability of new blood vessels via increasing Ang1 and Tie-2 expression. Conflict of interest The authors declare no conflict of interest. Transparency Document The Transparency document associated with this article can be found, in the online version. Acknowledgments The study was supported by the National Natural Science Foundation of China (Grant no.:31270391), Outstanding Young Scientist Research Award Foundation of Shandong Province (No.BS2014YY008), Natural Science Foundation of Shandong Province, China (No. ZR2014HM091) and the Scientific Research Foundation of Binzhou Medical College, Shandong Province, China (No. BY2013KYQD21), in part financially supported by Taishan Scholar Project.

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Please cite this article as: Zhu, H., et al., Mechanisms of angiogenesis in a Curculigoside A-treated rat model of cerebral ischemia and reperfusion injury, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.08.003