VEGF signaling pathway

VEGF signaling pathway

Neuroscience 267 (2014) 232–240 CURCULIGOSIDE A INDUCES ANGIOGENESIS THROUGH VCAM-1/EGR-3/CREB/VEGF SIGNALING PATHWAY Z. KANG, a H. ZHU, b H. LUAN, a...

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Neuroscience 267 (2014) 232–240

CURCULIGOSIDE A INDUCES ANGIOGENESIS THROUGH VCAM-1/EGR-3/CREB/VEGF SIGNALING PATHWAY Z. KANG, a H. ZHU, b H. LUAN, a F. HAN a* AND W. JIANG a*

INTRODUCTION

a

School of Pharmaceutical Sciences and Institute of Material Medica, Binzhou Medical University, Yantai 264003, PR China

Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. Neurovascular responses play a central role as the damaged central nervous system transitions from initial injury into repair (Lo, 2010). Re-establishment of functional microvasculature enhances neurogenesis and functional recovery after stroke (Taguchi et al., 2004; Ohab et al., 2006; Arai et al., 2009). The signals and substrates of neurogenesis and neuroplasticity are tightly co-regulated with angiogenesis and vascular remodeling (Xiong et al., 2010). Recent findings suggest that the strategies to enhance angiogenesis provide new opportunities for stroke recovery (Beck and Plate, 2009). Vascular endothelial growth factor (VEGF) is the most important mitogen in the process of angiogenesis. The lack of a single VEGF allele shows abnormal blood vessel development and leads to embryonic lethality (Ferrara et al., 1996). The angiogenic effects of VEGF are binding to VEGF receptor 2 (VEGFR2) on the surface of endothelial cells, then activates cAMP response element-binding protein (CREB) (Lee et al., 2009a). VEGF activation of endothelial cells is mediated by early growth response 3 (Egr-3) which plays an essential role in VEGF-mediated angiogenesis (Liu et al., 2008; Suehiro et al., 2010). The dried rhizomes of Curculigo orchioides Gaertner (Amaryllidaceae) is distributed widely in Japan, China, India and Australia and used as a tonic in traditional Chinese medicine to treat a decline in physical strength. Curculigoside A, the major bioactive compound is presented in C. orchioides which stimulates the secretion of estradiol on primary cultural granulose cell and exhibits potent inhibitory activity against matrix metalloproteinase-1 in cultured human skin fibroblasts (Dong et al., 2006; Lee et al., 2009b), attenuates human umbilical vein endothelial cell injury induced by H2O2 (Wang et al., 2010), and up-regulates VEGF in MC3T3E1 Cells (Ma et al., 2011). It also showed the protective potential against cerebral ischemia injury in middle cerebral artery occlusion (MCAO) rats (Jiang et al., 2011). In the present study, we therefore investigated the hypothesis that Curculigoside A induced angiogenesis in cerebral endothelial cells, activated CREB signaling pathways, up-regulated Egr-3 and vascular cell adhesion molecule 1 (VCAM-1), and increased extracellular levels of VEGF.

b

State Key Laboratory of Long-acting and Targeting Drug Delivery Technologies (Luye Pharma Group Limited), Yantai 264003, PR China

Abstract—Curculigoside A may be a powerful way of protecting the brain against a wide variety of injury. In the present study, we sought to elucidate whether Curculigoside A contributes to induce angiogenesis and its mechanisms. To this end, we examined the role of Curculigoside A on proliferation, invasion, and tube formation in the human brain microvascular endothelial cell line (HBMEC) in vitro. For studying mechanism, the cAMP response element-binding protein (CREB) inhibitor 2-naphthol-AS-E-phosphate (KG-501), early growth response 3 (Egr-3) siRNA, vascular endothelial growth factor (VEGF) antagonist sFlt-1 and VEGF receptor 2 (VEGFR2) blocker SU-1498 were used. Human brain microvascular endothelial cell line (HMBEC) proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Scratch adhesion test was used to assess the ability of invasion. A matrigel tube formation assay was performed to test capillary tube formation ability. Vascular cell adhesion molecule 1 (VCAM-1)/Egr-3/CREB/VEGF pathway activation in HMBEC was tested by Western blot analysis. Our data suggested that Curculigoside A induced angiogenesis in vitro by enhancing the proliferation, invasion and tube formation. VEGF expression was increased by Curculigoside A and counteracted by the soluble VEGF receptor 1 (sFlt-1, VEGF antagonist) and KG-501 in HMBEC. Tube formation was enhanced by Curculigoside A and counteracted by VEGF receptor blocker-SU1498, KG-501 and Egr-3 siRNA. It may be suggested that Curculigoside A induces angiogenesis in vitro via a programed VCAM-1/Egr-3/CREB/VEGF signaling axis. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: Curculigoside A, angiogenesis, VEGF, VCAM-1, Egr-3, CREB.

*Corresponding authors. Tel/fax: +86-535-6912036. E-mail address: [email protected] (W. Jiang). Abbreviations: CREB, cAMP response element-binding protein; DMEM, Dulbecco’s Modified Eagle Medium; EDTA, ethylenediaminetetraacetic acid; Egr-3, early growth response 3; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; HBMEC, human brain microvascular endothelial cell line; HEPES, hydroxyethyl piperazineethanesulfonic acid; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; p-CREB, phosphor-CREB; PCNA, proliferating cell nuclear antigen; VCAM-1, vascular cell adhesion molecule 1; VEGFR, vascular endothelial growth factor receptor. http://dx.doi.org/10.1016/j.neuroscience.2014.02.050 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 232

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EXPERIMENTAL PROCEDURES Reagents Curculigoside A (purity >98.5%, CAS No.: 85643-19-2, molecular formula C22H26O11: 466.43. It was provided by State Key Laboratory of Long-acting and Targeting Drug Delivery Technologies, Yantai, PR China). A stock solution of Curculigoside A was made in saline at a concentration of 10 mM. The following pharmacologic agents were used: Egr-3 siRNA (sc-35268, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA), CREB inhibitor 2-naphthol-AS-E-phosphate (KG-501, Sigma Chemical Co., Ltd. Nanjing, PR China). The soluble VEGF receptor 1 (sFlt-1, VEGF antagonist Calbiochem), VEGF receptor blocker-SU1498 (572888-5MG Calbiochem). VEGF ELISA Kit (Yajie Biological Technology Company, Shanghai, PR China). VE-cadherin ELISA Kit (Shanghai Haoran Biological Technology Company, PR China). Cell culture A human brain microvascular endothelial cell line (HBMEC) was obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). The cells were seeded at 60–70% confluence and kept at 37 °C in 5% CO2. Culture media comprised RPMI 1640 containing 10% fetal bovine serum, 10% Nu-Serum, 2 mM L-glutamine, 1 mM pyruvate, essential amino acids, and vitamins. Proliferation assay For in vitro proliferation assays, HBMECs were seeded into 96-well (5  104 cells/well) flat bottom plates with medium alone (control) or medium containing different concentrations of Curculigoside A (1, 3, 9, 27 and 81 lM). Cell proliferation was tested by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, serum-starved cells were treated with Curculigoside A for 24 h. Following Curculigoside A treatment, 10 mL water-soluble tetrazolium reagent was added to 100 mL fresh culture medium in each well. Absorbance was determined at 490 nm (Spectramax/M5 multi-detection reader, Molecular devices, Sunnyvale, California, USA), and calculated as a ratio against untreated cells. In addition, serum-starved cells were treated with Curculigoside A for 24 h, then collected HBMECs to analyze the cell-cycle distribution by Flow cytometry, then counted the proportion of cells in S phase and G0/G1 phase. Scratch adhesion test HBMECs were seeded in 6-well plates (5  104 cells/well) until the cells were fused to more than 90%, discarded the culture liquid, then washed twice with PBS, diluted with different concentrations (1, 3, 9, 27 and 81 lM) of Curculigoside A by Dulbecco’s Modified Eagle Medium (DMEM) medium, then used 200-ll pipette tip to scratch and picture after 24 h, measured the distance on either side of the scratch track and counted in five random fields (100). Results were expressed as fold decrease over the control.

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The siRNA transfections HBMEC were seeded in a six-well plate at a density of 2  105 cells per well in 2 mL antibiotic-free M199 medium supplemented with 10% fetal bovine serum (FBS). The cells were incubated at 37 °C in a CO2 incubator for 24 h, washed once with 2 mL siRNA transfection medium. Medium was aspirated, then 0.8 ml siRNA transfection medium was added to each well containing the Egr-3 siRNA transfection reagent mixture. The cells were incubated for 7 h at 37 °C in a CO2 incubator and added 1 mL of normal growth medium containing two times the normal serum and antibiotics without removing the transfection mixture. After the cells were incubated Curculigoside A or KG-501 for an additional 24 h, the cells were collected and cleared for western blot analysis. Firstly, the silencing efficiency was evaluated in HBMECs with Egr-3 siRNA or Curculigoside A 9 lM. Secondly, the effects of Curculigoside A on VCAM-1 and CREB/Egr-3 signaling were investigated. Matrigel tube formation assay for angiogenesis The matrigel assay was used to assess the spontaneous formation of capillary like structures in vitro. HBMECs (2  104 cells/well) were seeded in 24-well plates in serum-free media previously coated with growth factorreduced matrigel matrix (BD Bioscience, San Jose, CA, USA) containing different concentrations of Curculigoside A (1, 3, 9, 27 and 81 lM), KG-501 (25 lM), Egr-3 siRNA, VEGF antagonist sFlt-1 (10 lM) or VEGF receptor blocker SU1498 (5 lM), then incubated at 37 °C for 24 h. The number of tube formation was determined in four random fields (100) from each well. Data were analyzed as tube formation versus untreated control wells. In vitro oxygen and glucose deprivation model To mimic the oxygen and glucose deprivation in vitro, HBMECs were incubated in a hypoxia solution for 6 h. The hypoxia solution contained 0.9 mM NaH2PO4, 6.0 mM NaHCO3, 1.0 mM CaCl2, 1.2 mM MgSO4, 40 mM Natrium lacticum, 20 mM HEPES, 98.5 mM NaCl, 10.0 mM KCl (pH adjusted to 6.8) and was bubbled with N2 for 30 min before application. The pO2 of the hypoxia solution was adjusted to reach a level of 64.0 kPa. Hypoxic condition was produced by placing the plates of cultured HBMECs in a hypoxic incubator (Kendro, Langenselbold, Germany) and oxygen was adjusted to 1.0% and CO2 to 5.0%. Prior to hypoxia, HBMECs were pretreated with various concentrations (1, 3, 9, 27 and 81 lM) of Curculigoside A for 18 h. Normal culture (DMEM containing 2% FBS under 20% oxygen and 5% CO2) served as a negative control, the hypoxia solution culture served as the control. Determination of cell viability, lactate dehydrogenase (LDH) leakage, caspase-3 activity and apoptosis HBMECs were maintained in a hypoxia solution for 6 h in the presence or absence of Curculigoside A, then cell

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viability was assessed by an MTT assay. LDH, an indicator of cell injury, was detected according to the description of the LDH assay kit (Zhongsheng Bioreagent, Beijing, PR China). LDH leakage rate (%) = Ae/At  100%. Ae indicated extracellular LDH (cells culture fluid), At indicated intracellular and extracellular LDH (cells lysate). Caspase-3 activity was measured following the procedure described by the caspase-3 assay kit. In brief, cells were lysed for 10 min in an ice bath and centrifuged at 15,000g for 10 min at 4 °C, the supernatant was incubated with acetyl-Asp-Glu-Val-Aspaldehyde-AFC at 37 °C for 1 h. Fluorescence intensity was measured with the fluorescence spectrophotometer (kex400 nm and kex505 nm). The value for each group was converted to the percentage of the normal. Apoptotic cells were evaluated by an Annexin-V FITC apoptosis detection kit. In brief, the cells were harvested, washed and incubated at 4 °C for 30 min in the dark with annexin-V FITC and propidium iodide, then analyzed on a FACS Vantage SE flow cytometer (Beckman Coulter, Fullerton, USA). VCAM-1, VEGF and VE-cadherin assays Serum-starved HBMECs were pretreated with a selective CREB inhibitor KG-501 (25 lM) or Egr-3 siRNA for 1 h before incubation with 9 lM Curculigoside A, collected the supernatants of HBMECs to determine VEGF and VCAM-1 after incubation with Curculigoside A for 23 h. VE-cadherin levels were determined in collected HBMECs. VEGF, VCAM-1 and VE-cadherin levels were confirmed in culture supernatant by ELISA Kit.

RESULTS Curculigoside A augments proliferation, migration and tube formation HBMECs were incubated with different concentrations of Curculigoside A (1–27 lM) 24 h. The proliferation, migration and tube formation assays were examined as the markers of angiogenesis in vitro. HBMEC displayed a basal migration in absence of Curculigoside A after 24-h incubation, while HBMEC treated with Curculigoside A 3–27 lM, the HBMEC displayed a faster migration and induced HBMEC proliferation in a concentration-dependent manner, as shown in Fig. 1A. Along with increased proliferation, Curculigoside A also enhanced endothelial cell migration as quantified with a scratch adhesion test (Fig. 1A). Flow cytometry analysis confirmed that Curculigoside A induced a significant increase in the proliferative S phase while decreasing the resting G0/G1 phase of the cell cycle, as shown in Fig. 1B. Matrigel assays showed that Curculigoside A induced tube formation in a concentration-dependent manner, as shown in Fig. 2. In addition, we compared the effects of Curculigoside A at 9, 27 and 81 lM. the results showed that Curculigoside A at 27 lM is slightly better than 9 lM in value, but almost has no difference, 81 lM is slightly inferior to 9 lM, so we selected Curculigoside A at 9 lM to investigate its effects on inducing angiogenesis and mechanisms.

Western blotting analysis Cells were cultured for 24 h, then washed twice with ice cold PBS on ice and lysed in NP40 lysis buffer (Biosource, Camarillo, CA, USA) (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% NP-40 and 0.02% NaN3) supplemented with 1 mM phenylmethanesulfonylfluoride (PMSF) and 1 protease inhibitor cocktail (Sigma, Saint Louis, MO, USA). Equal amounts of cell protein (50 lg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by western blot using specific antibodies to VEGFR2, phosphorCREB (p-CREB), VCAM-1, Egr-3 and Proliferating cell nuclear antigen (PCNA, as a loading control). Optical densities of the bands were scanned and quantified with a Gel Doc 2000 (Bio-Rad Laboratories Ltd, Shanghai, PR China). Data were normalized against those of the corresponding PCNA bands. Results were expressed as fold increase over the control. Statistical analysis Quantitative data from experiments were expressed as mean ± S.D., significance was determined by a oneway analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 was considered statistically significant.

Fig. 1. Effects of Curculigoside A on proliferation and scratch adhesion. Curculigoside A promoted proliferation and invasion. HBMEC (5  104) were incubated for 24 h in medium alone or with Curculigoside A. Fig. 1A Effects of Curculigoside A on proliferation and scratch adhesion; Fig. 1B Flow cytometry analyzed cell-cycle distribution of S phase and G0/G1 phase. Data from experiments were expressed as mean ± SD, n = 5. ⁄P < 0.05, ⁄⁄P < 0.01 vs. Control group. Significance was determined by a one-way analysis of ANOVA followed by Dunnett’s test.

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Fig. 2. Effects of Curculigoside A on capillary tube formation. Curculigoside A promoted matrigel angiogenesis. HBMEC (2  104) were plated on Matrigel-coated, 24-well plates and were incubated for 24 h in medium alone or with Curculigoside A. Photomicrographs represent the matrigel tube formation after 24 h incubation. It was detected by phase-contrast microscopy (magnification, 100). Data from experiments were expressed as mean ± SD, n = 5. ⁄P < 0.05, ⁄⁄P < 0.01 vs. Control group. Significance was determined by a one-way analysis of ANOVA followed by Dunnett’s test.

Effects of Curculigoside A on cultured HBMECs against hypoxia-induced cytotoxicity, caspase-3 activity and apoptosis As estimated by the MTT assay, cell viability was markedly decreased after hypoxia for 6 h (Table 1). However, cells were incubated with Curculigoside A, cell viability was significantly increased in a concentrationdependent manner, as shown in Table 1. To further investigate the protective effect of Curculigoside A, LDH

leakage rate was estimated, a significant increase of LDH leakage rate in HBMECs was observed after hypoxia 6 h. Incubation with various concentrations of Curculigoside A significantly inhibited hypoxia-induced LDH release in a concentration-dependent manner. Capase-3 activity in hypoxia group was increased to 357% of the normal group. Incubation with various concentrations of Curculigoside A significantly inhibited oxygen-glucose deprivation-induced caspase-3

Table 1. Effects of Curculigoside A on the viability, LDH leakage and apoptosis in HBMECs exposed to oxygen-glucose deprivation Groups

Content (lM)

Cell viability (%)

LDH leakage (%)

Caspase-3 (%)

Apoptosis (%)

Normal Control

– – 1 3 9 27 81

100 ± 14.6 54.0 ± 5.4a 60.9 ± 5.9 67.3 ± 7.3b 70.1 ± 6.7c 76.2 ± 9.5c 71.2 ± 8.1c

2.4 ± 0.6 20.1 ± 2.5a 16.6 ± 3.5 14.9 ± 3.3b 11.4 ± 2.2c 10.7 ± 2.6c 11.1 ± 2.1c

100 357 ± 37 320 ± 35 293 ± 31 247 ± 35 241 ± 34 245 ± 38

2.1 ± 0.5 23.3 ± 3.6a 18.8 ± 3.9 16.6 ± 2.6b 13.1 ± 2.3c 12.6 ± 3.4c 12.9 ± 3.6c

Curculigoside A

a

b c c c

After 3-h oxygen-glucose deprivation (OGD) followed by 12 h incubation with Curculigoside A, cell viability, LDH leakage, caspase-3 activity and apoptosis were assessed. Normal: no OGD; Control: OGD. Values are mean ± S.D. (n = 5). Significance was determined by a one-way ANOVA followed by Dunnett’s test. a P < 0.01 versus normal group. b P < 0.05. c P < 0.01 versus Control group.

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activation in a concentration-dependent manner, as shown in Table 1. Apoptotic cells were estimated by annexin-V/PI staining and flow cytometry analysis, as shown in Table 1. The normal HBMECs apoptosis rate is only 2.1%, while increased to 23.3% after hypoxia for 6 h. Incubation with Curculigoside A (3–27 lM) for 18 h could arrest the apoptosis in a concentration-dependent manner.

Egr-3 siRNA

---

---

+

+

Curculigoside A

---

9 µM

---

9 µM

Egr-3

A PCNA

B Curculigoside A up-regulates VCAM-1, VEGF and VE-cadherin Serum-starved cells were incubated with 9 lM Curculigoside A for 23 h, enzyme-linked immunosorbent assay (ELISA) test was used to determine VCAM-1, VEGF and VE-cadherin expression. The results indicated that Curculigoside A increased VCAM-1, VEGF and VE-cadherin expression. When pretreatment with a selective CREB inhibitor KG-501 or Egr-3 siRNA for 1 h before incubation with 9 lM Curculigoside A, the increase of VCAM-1 and VE-cadherin expression was blocked by KG-501 or Egr-3 siRNA, VEGF was blocked by KG-501.while VEGF was partially blocked by Egr-3 siRNA, as shown in Table 2.

#

Fig. 3. Effects of the Egr-3 siRNA transfection reagents on the silencing efficiency in vitro. Serum-starved HBMEC in the presence or absence of Egr-3 siRNA for 1 h before incubation with 9 lM Curculigoside A, then the silencing efficiency was evaluated. Representative images are shown from three individual experiments. PCNA is a loading control. Data from experiments were expressed as mean ± SD, n = 3. ⁄P < 0.01, vs. Control group, #P < 0.01 vs. Curculigoside A 9 lM group. Significance was determined by a oneway analysis of ANOVA followed by Dunnett’s test.

Curculigoside A up-regulates p-CREB via Egr-3/VCAM-1 signaling Firstly, the silencing efficiency of Egr-3 siRNA in HBMECs was evaluated. The results showed that Egr-3 expression were reduced 79% and 80% by Egr-3 siRNA in the presence or absence of Curculigoside A, as shown in Fig. 3B. Secondly, effects of Curculigoside A on VCAM-1 and CREB/ Egr-3 signaling in vitro were investigated. HBMECs were incubated with Curculigoside A 9 lM 24 h robustly activated Egr-3/VCAM-1 signaling, leading to a rapid increase in Egr-3 and VCAM-1 expression (P < 0.01), as shown in Fig. 4A. Activation of this pathway was significantly blocked by Egr-3 siRNA (Compared with Curculigoside A 9 lM, P < 0.01), as shown in Fig. 4A, B. Since CREB pathway is related to Egr-3 signaling, we next examined the role of CREB in Curculigoside

Table 2. Effects of Curculigoside A on VCAM-1, VEGF and VEcadherin levels in cultured HBMECs Groups

VCAM-1 (lM)

VEGF (pg/ml)

VE-cadherin (pg/ml)

Control Curculigoside A Curculigoside A + KG-501 Curculigoside A + Egr-3 siRNA

23.8 ± 4.1 40.8 ± 5.0a 30.7 ± 5.6

326 ± 55 459 ± 50a 362 ± 39b

254 ± 54 472 ± 82a 321 ± 57

28.6 ± 4.8b

393 ± 43

287 ± 53b

VCAM-1, VEGF and VE-cadherin levels in the conditioned media were confirmed by ELISA Kits. Values are mean ± S.D. (n = 5). Significance was determined by a one-way ANOVA followed by Dunnett’s test. a P < 0.01 versus Control group. b P < 0.01 versus Curculigoside A group.

A-induced angiogenesis. Incubation of HBMECs with Curculigoside A 9 lM 24 h rapidly increased p-CREB (P < 0.01), as shown in Fig. 4B. Activation of Egr-3 occurs downstream of CREB signaling since blocking the pathway with CREB inhibitor KG-501 25 lM for 1 h significantly decreased Curculigoside A-induced Egr-3 expression (compared with Curculigoside A 9 lM, P < 0.05). Consistent with the increased p-CREB, Curculigoside A amplified Egr-3 expression, as shown in Fig. 4A, B. Curculigoside A increases VEGF via CREB-dependent signaling To directly link Curculigoside A-induced signaling with angiogenesis, we assessed the well-established proangiogenic mediator VEGF. The results of Western blots showed that p-VEGFR2 expression in HBMECs was strongly up-regulated by Curculigoside A, and this effect was dependent on p-CREB (P < 0.01), as shown in Fig. 5A. Inhibition of p-CREB with KG-501 significantly attenuated the ability of Curculigoside A to up-regulate p-VEGFR2 (compared with Curculigoside A 9 lM, P < 0.01), as shown in Fig. 5B. Consistent with elevated protein levels, activation of VEGF signaling was detected in Curculigoside A-treated HBMEC. Levels of VEGF and VE-cadherin were increased by Curculigoside A, indicating that active signaling was indeed taking place, as shown in Table 2. These pathways involved autocrine signaling since blockade of the VEGF with sFlt-1 dampened the ability of Curculigoside A to activate the VEGF pathway and p-VEGFR2.

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A

A

KG-501

KG-501

Egr-3 siRNA

sFlt-1

Curculigoside A

Curculigoside A p-CREB

p-CREB CREB

p-VEGFR2

Egr-3 VCAM-1

VEGFR2

PCNA

PCNA

B

B # # #

# #

# # #

Fig. 4. Effects of Curculigoside A on VCAM-1 and CREB/ Egr-3 Signaling in vitro. VCAM-1 is regulated by Curculigoside A and downstream of CREB/ Egr-3 signaling pathway. Serum-starved HBMEC was pretreated with a selective CREB inhibitor KG-501 25 lM and Egr-3 siRNA for 1 h before incubation with 9 lM Curculigoside A. Representative images are shown from three individual experiments. Data from experiments were expressed as mean ± SD, n = 3. ⁄P < 0.05, ⁄⁄P < 0.01 vs. Control group, # P < 0.01 vs. Curculigoside A 9 lM group. Significance was determined by a one-way analysis of ANOVA followed by Dunnett’s test.

Finally, tube formation assays demonstrated that these pathways were required for Curculigoside A-induced angiogenesis in HBMECs. Blockade of any of these steps in the signaling cascade (CREB or Egr-3) significantly suppressed Curculigoside A-induced tube formation (compared with Curculigoside A 9 lM, P < 0.01), as shown in Fig. 6A, B. Curculigoside A-induced angiogenesis is dependent on VEGF Next, we investigated whether Curculigoside A-induced angiogenesis in HBMECs was indeed dependent on the control of VEGF mechanism. As expected, Curculigoside A enhanced tube formation in the matrigel assays (P < 0.01), as shown in Fig. 6A, B. Blocking VEGF signaling potently suppressed these Curculigoside A-induced angiogenesis. Cotreatments with the VEGF antagonist sFlt-1 or the VEGFR2 blocker SU1498 both significantly decreased Curculigoside A-induced tube formation (compared with Curculigoside A 9 lM, P < 0.05), as shown in Fig. 6A, B.

DISCUSSION It has recently shown that Curculigoside A is a potent neuroprotective agent which reduced neuronal death in experimental model of stroke (Jiang et al., 2011).

#

Fig. 5. Effects of Curculigoside A on vascular endothelial growth factor and CREB signaling in vitro. Vascular endothelial growth factor (VEGF) induced by Curculigoside A is CREB dependent. Serumstarved HBMEC was pretreated with KG-501 (25 lM) and a selective VEGF antagonist (sFlt-1, 10 lM) for 1 h before incubation with 9 lM Curculigoside A for 20 min. Representative images from three individual experiments are shown. Data from experiments were expressed as mean ± SD, n = 3. ⁄P < 0.05, ⁄⁄P < 0.01 vs. Control group, #P < 0.01 vs. Curculigoside A 9 lM group. Significance was determined by a one-way analysis of ANOVA followed by Dunnett’s test.

Among the major findings of the present study, Curculigoside A can induce the proliferation, migration and tube formation in cerebral endothelial cells at concentration higher than 3 lM in vitro. The mechanisms of this phenomenon appear to involve upstream control of VCAM-1 via Egr-3/CREB signaling, and downstream induction of VEGF. These data provide a mechanistic basis for the potential application of Curculigoside A as a potential neurovascular repair therapy for stroke and brain injury. CREB is involved in multiple signaling pathways to regulate cell proliferation, differentiation, survival and migration (Shaywitz and Greenberg, 1999; Mayr and Montminy, 2001; Sakamoto and Frank, 2009), and closely related to the occurrence and development of angiogenesis (Jiang et al., 2010). P-CREB activates downstream of Egr-3, induces the release of Egr-3, launches endothelial cell division, proliferation and migration, induces the occurrence of angiogenesis. Our results showed that Curculigoside A enhanced Egr-3 expression in HBMEC. As a mediator of angiogenesis, VCAM-1 an Ig-like adhesion molecule expressed on the activated endothelial cells induces chemotaxis in endothelial cells in vitro and angiogenesis in vivo (Koch et al., 1995).

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Fig. 6. Involvement of signaling pathway in Curculigoside A-induced capillary tube formation. Curculigoside A-induced angiogenesis is dependent on VEGF. Serum-starved brain endothelial cells were pretreated with KG-501 (25 lM), sFLT-1 (10 lM) and VEGF receptor blocker-SU1498 (10 lM) for 1 h before incubation with 9 lM Curculigoside A or Egr-3 siRNA. A1: Control; A2: 9 lM Curculigoside A; A3: 9 lM Curculigoside A + KG-501; A4: 9 lM Curculigoside A + sFlt-1; A5: 9 lM Curculigoside A + SU1498; A6: 9 lM Curculigoside A + Egr-3 siRNA. Data from experiments were expressed as mean ± SD, n = 5. ⁄P < 0.01, vs. Control group, #P < 0.05, ##P < 0.01 vs. Curculigoside A 9 lM group. Significance was determined by a one-way analysis of ANOVA followed by Dunnett’s test.

To dissect how Curculigoside A regulates Egr-3 and VCAM-1, we assessed p-CREB expression in HBMECs. Incubation of HBMECs with the CREB inhibitor KG-501 abrogated Egr-3 induced by Curculigoside A. It is interesting to note that p-CREB is required for angiogenesis, since CREB inhibitor and Egr-3 siRNA both reduced Curculigoside A-induced tube formation. Functionally, the balance between the signals may allow fine-tuning of Egr-3 and VCAM-1 expression. Collectively, our observations highlighted the importance of CREB pathway in Curculigoside A-induced Egr-3 production, and appeared to be necessary for cerebral endothelial angiogenesis. Vascular endothelial cell adhesion, proliferation and migration play a very crucial role in angiogenesis, is the premise and foundation of angiogenesis. The binding of VEGF to its receptor VEGR2 induces endothelial proliferation, adhesion and migration, and enhances angiogenesis (Chen et al., 2007). VEGF is an important mediator executes the angiogenic program downstream of CREB. Vascular VEGF activates CREB signaling through VEGFR2 (Mayo et al., 2001). In the developing brain, VEGF/VEGFR2 signaling leading to CREB

phosphorylation is the shared pathway in neurons and vascular endothelial cells (Lee et al., 2009a). Downregulation of CREB-binding protein expression inhibits the endothelial cells proliferation (Jiang et al., 2010). Hence, VEGF may be an important mechanism by Curculigoside A inducing angiogenesis. However, VEGF increases vascular permeability and inflammation during angiogenesis (Croll et al., 2004), particularly in the formative stages of angiogenic vessels are leaky which leads to BBB leakage and edema, and damages the nervous tissue (Van Bruggen et al., 1999; Schoch et al., 2002). It is interesting that CREB promotes antiinflammatory immune responses (Wen et al., 2010), so p-CREB is required for angiogenesis by VEGF to avoid the defects. Our results showed that Curculigoside A induced p-CREB and VEGF expression. It suggests that Curculigoside A can avoid VEGF-induced BBB in the formative stages of angiogenic vessel for Curculigoside A attenuate inflammatory response, this result is confirmed in experimental brain injury (Jiang et al., 2011). VE-cadherin is indispensable for proper vascular development and maintaining newly formed vessels

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(Carmeliet et al., 1999; Gory-Faure´ et al., 1999). VEGF induces VE-cadherin tyrosine phosphorylation in endothelial cells (Esser et al., 1998), so VEGF plays a key role in E-cadherin expression. Our results demonstrated that Curculigoside A induced VEGF, VCAM-1 and VE-cadherin in HBMEC. It suggested that Curculigoside A induced vascular development and maintained newly formed vessels, the results of Curculigoside A on the proliferation, scratch adhesion and tube formation also confirmed this. Pretreatment with Egr-3 siRNA before incubation with Curculigoside A, the increase of VE-cadherin expression was blocked. It suggested that VE-cadherin expression was dependent on Egr-3 expression. The importance of these mechanisms is confirmed that blockade of any of these CREB or VEGF signaling steps potently suppressed Curculigoside A-induced angiogenesis in our cerebral endothelial models in vitro. Furthermore, positive feedback loops may also be involved. VEGFR2 can also be up-regulated by VEGF stimulation, leading to enhanced VEGF signaling and angiogenesis (Yang et al., 2002; Sima˜o et al., 2012). Our data confirmed that Curculigoside A both up-regulated VEGF and p-VEGFR2, and inhibition of p-CREB dampened all components of the VEGF response and angiogenesis. Apoptosis and survival co-exist in the process of vascular endothelial cell adhesion, proliferation and migration (Cardone et al., 1998; DeBusk et al., 2004). How to make those cells survive? Reducing the apoptosis is very important. Our results showed that oxygen-glucose deprivation increased caspase-3 activity in cultured HBMEC. Moreover, treatment with Curculigoside A inhibited oxygen-glucose deprivationinduced cell apoptosis. Taken together, our findings suggest that Curculigoside A may be a novel way to induce angiogenesis in cerebral endothelial cells. But there are several important caveats to keep in mind. First, although our data provide cellular and pharmacologic proof of principle for Curculigoside A in cerebral angiogenesis, in vivo validation of these mechanisms remains to be obtained. The pro-angiogenic utility of Curculigoside A as a potential stroke recovery therapy should be explored in future experiments. In summary, the present study provides mechanistic evidence that Curculigoside A induces angiogenesis in cerebral endothelial cells via VCAM-1/Egr-3/CREB/ VEGF signaling. Further in vivo and clinical exploration of these pathways is warranted to validate these experimental findings and develop Curculigoside A as a potential neurovascular repair therapy for stroke and brain injury.

Acknowledgements—The study was supported by National Natural Science Foundation of China (Grant No.: 31170321) and the funds of Binzhou Medical University for Scientific Research (Grant No.: BY2011KYQD05), in part financially supported by Taishan Scholar Project, Natural Science Foundation of Shandong Province (Grant No.: ZR2013HQ022) and the National Basic Research Program of China (No. 2012CB724003).

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(Accepted 27 February 2014) (Available online 12 March 2014)