Online Supplement for Additional Details on the Methods

Dl-3n-butylphthalide promotes angiogenesis via the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase /Akt- endothelial nitric oxide synthase signaling pathways

Xi-lin Lu1*, Dan Luo2,3*, Xiao-li Yao1, Guang-lei Wang4,5, Zhi-yong Liu1, Zhen-xing Li4,5 ,Wei Li1, Feng-jun Chang2,3, Lu Wen6, Simon Ming-yuen Lee7, Zai-jun Zhang7, Ling Li1, Jin-sheng Zeng1, Ru-xun Huang1, Zhong Pei1** and Jing-song Ou2,3 **

Online supporting information for additional details on the methods.

Materials and Methods

Preparation of reagents and cell culture

NBP was obtained from CSPC NBP Pharmaceutical Co.Ltd, China. Human umbilical vein endothelial cells (HUVEC) were obtained from Sciencell, USA. To investigate the angiogenic effects of NBP on HUVEC, NBP stock solution (100 mg/ml) was prepared in PBS containing 10% DMSO (GIBCO, USA). To investigate the angiogenic effects of NBP on zebrafish in vivo, NBP stock solution (1 mg/ml) was prepared in sterilized Milli-Q water (containing 0.5% DMSO). Vascular endothelial growth factor (VEGF) was obtained from Sigma, USA and prepared as a stock solution (100 μg/ml) in sterilized Milli-Q water.

Maintenance of zebrafish

The transgenic zebrafish line TG (fli1: EGFP), in which endothelial cells (EC) express eGFP, was kindly provided by ZFIN (Oregon) and maintained as previously described.1 Briefly, the cells were maintained in a controlled environment at a temperature of 280C on a 14 h: 10 h light/dark cycle (lights on at 08:00) as described in the zebrafish handbook. Zebrafish were stored in five gallon tanks and continuously supplied with filtered reverse osmosis H2O. The fish were fed with brine shrimp twice daily and also with general tropical fish food occasionally.

Collection of zebrafish embryos

Embryos were generated by natural pair-wise mating of fish that were 3 and 12 months old. Briefly, breeding boxes were placed in a dark place overnight until mating. In the presence of light, the fish were left undisturbed for 15-30 min. Breeding boxes were then collected and the embryos were transferred into clean Petri dishes with a fine fishing net. The embryos were maintained in Milli-Q water. Healthy, limpid, and regular embryos were collected at the 1-4 cell stage and distributed in 24-well microplates with 6-8 embryos per well.

Treatment of zebrafish embryos with NBP and fibroblast growth factor receptor 1 (FGFR-1) inhibitor, extracellular signal-regulated kinase1/2(ERK1/2) inhibitor or phosphatidylinositol 3-kinase(PI3K) inhibitor

Healthy, limpid, regular embryos were picked out at their 1-4 cell stage and distributed in 24-well microplates. Excess Milli-Q water was blotted from the microplate wells, and different concentrations (50, 100, 150 and 200 μM) of NBP solution were added to the wells. Fibroblast growth factor receptor 1 (FGFR-1) inhibitor, SU5402 (5 μM, Sigma, USA), extracellular signal-regulated kinase1/2 (ERK1/2) inhibitor, U0126 (5 μM, Sigma, USA), or phosphatidylinositol 3-kinase(PI3K) inhibitor, LY294002 (10 μM, Sigma, USA) were added to the wells as indicated. Embryos receiving DMSO (0.2%) served as a vehicle control. Embryos were treated at 280C for 96 h. All experiments were repeated at least 12 times, and with 30 embryos per group.

Zebrafish embryos microinjected with VEGF served as positive controls

VEGF was injected either into the yolk ball or into the perivitelline space between the yolk and periderm. These two sites were chosen for injection because proteins in the yolk are often taken up by the embryo, and the perivitelline space is in the path of venous return, ensuring that proteins end up in the circulation of the embryo after injection. Embryos were collected as described above. The embryos were then sorted into holding ramps made of 1% agarose in Milli-Q water, and oriented with the yolk ball projecting upwards. The injection was performed as follows: 100 μg/ml solution of VEGF (Sigma, USA) was back-filled into a pulled-glass micropipette; the micropipette was then attached to a micromanipulator and a picospritzer (Applied Scientific Instrumentation MPPI-3). The latter was attached to a nitrogen tank. With the help of the micromanipulator, the tip of the micropipette was inserted into the embryo and a small volume of protein solution was expelled from the tip using positive pressure. While the volume expelled was variable, we injected approximately 10-15 nl of solution (approximately 1.0-1.5 ng VEGF), based on the size of the droplet expelled from the pipette tip at the onset of the injection.2

Visual screening of zebrafish embryos using fluorescence microscopy

At 96 hpf, the embryos were anesthetized using 0.05% 2-phenoxyethanol in embryo water. One to three embryos were placed in each well of a 96-well plate and angiogenesis was evaluated by looking for ectopic vessels in the subintestinal vessel plexus (SIV). The following criteria were used to determine induction of angiogenesis: (a) presence of vessels spiking out of the basket structure and/or (b) extension of the SIV basket into the yolk extension region with more than seven vertical branches within the basket. Although intersecting branches were frequently seen, they were not consistent enough to be included as criteria. Each experiment included 30 Zebrafish per group. Mean percentages and SEM were computed as described previously.3

Human umbilical vein endothelial cells (HUVEC) culture

The HUVEC were purchased from Sciencell(USA) and Passage 4 were cultured at 370C in M199 media (Invitrogen, USA) supplemented with 20% FBS (Lonza, USA), 0.01% Heparin (Sigma, USA), 0.05% Endothelial Mitogen (Biomedical Technologies, USA), and 1% Penstrep: Glutamine (Invitrogen, USA). Cells were incubated at 37℃ in a humidified atmosphere of 5% CO2. Before treated with NBP, HUVEC were serum-starved with 2% FBS for 24 hours.

HUVEC invasion assay

The effect of NBP on HUVEC invasion was measured using 10 mm tissue culture inserts (transwell chambers) with polycarbonate membrane (8 μm pores) and 24-well companion plates (Corning Incorprate Costar, USA). The upper side and lower side of the membrane were pre-coated with 1:30 (v/v) and 1:100 (v/v) of Matrigel (Sigma, USA), respectively. HUVEC (5 ×104 cells) were resuspended in low serum medium (300 μl) and seeded onto the culture inserts in triplicate. They were then deposited into the 24-well companion plate with 500 μl of low serum medium containing NBP (50, 100, 150, 200 μM) with or without SU5402 (5 μM), U0126 (5 μM) Akt inhibitor 1 (1L6-hydroxymethyl-chiroinositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, 25 μM, Calbiochem, USA), eNOS inhibitor (Cavtratin,10 μM, Calbiochem, USA) or LY294002 (10 μM). The wells of the companion plate containing DMSO (0.1%) and 20 ng/ml VEGF served as a vehicle control and positive control, respectively. The inserts were removed after 8 h of incubation and washed with PBS. Non-invasive cells on the upper surface of the membrane were removed by wiping with cotton swabs. The inserts were fixed in paraformaldehyde, stained with calcein AM and mounted on microscope slides. Images of the invasive cells were captured at 50×magnification using a fluorescent inverted microscope (Axiovert 200, Carl Zeiss) and a CCD camera (AxioCam HRC, Carl Zeiss). HUVEC invasion was quantified by counting the number of cells per insert using the Metamorph Imaging Series software (Molecular Devices, Tokyo, Japan).4 The results were compared with that of the control to calculate relative ratios for all experimental groups, after setting the control ratio to 100%.

HUVEC migration assay

Migration of HUVEC was performed by the wound healing method. 2, 5 HUVEC were seeded in 24-well plates (3 ×105 cells/well) in complete medium and incubated for 24 h at 37°C in the presence of 5% CO2. Cells were then starved for 24 h in low serum medium (0.5% FBS). The HUVEC were then scraped away horizontally in each well using a P100 pipette tip. Three randomly selected views along the scraped line were photographed on each well at 50x magnification, using a fluorescent inverted microscope and the CCD camera attached to the microscope. The medium was replaced with fresh medium containing DMSO, VEGF (20 ng/ml) or different concentrations of NBP (50, 100, 150 and 200 μM) with or without SU5402 (5 μM), U0126 (5 μM), Akt inhibitor 1 (25 μM), Cavtratin (10 μM) or LY294002 (10 μM). After 12 h of incubation, another set of images were taken as before. Migration was evaluated by image analysis using the Metamorph Imaging Series software. The average scraped area of each well under each condition was measured and subtracted from that of the pre-treatment condition. The change in area of the experimental condition was compared with that of the control. A reduction in the scraped area indicates a sign of migration as described previously. 2 The results were compared with that of the control and the relative ratios for all experimental groups was calculated, after setting the control ratio to 100%.

Endothelial cell tube formation assay in HUVEC

Endothelial tube formation was assessed in 24-well plates using MatrigelTM as described previously.6, 7 Briefly, the plates were coated with 250 ul of complete medium containing 5 mg/ml Matrigel 4°C and allowed to polymerize at 37°C for 30 min. HUVEC were seeded at a density of 5 X104 on Matrigel-coated plates. Cells were incubated with NBP (200 uM) in the absence and presence of SU5402 (5 μM), U0126 (5 μM), Akt inhibitor 1 (25 μM), Cavtratin (10 μM) or LY294002 (10 μM) in the growth medium. Cells incubated with VEGF (20 ng/ml) served as positive controls. Tube formation was examined under a phase-contrast microscope at 18 h after incubation. The tube formation was quantified by counting the number of connecting branches between two discrete endothelial cells and measuring the length of capillary structures using the software NIH Image J. Tube length was assessed by drawing a line along each tube and measuring the length of the line in pixels as reported earlier.6, 7 Five randomly selected fields of view were photographed in each well. The average of five fields was taken as the value for each sample. Three wells were assessed at each condition.

Western immunoblot analysis

HUVEC were passaged to 60mm dishes (16,700 cells/cm2) and cultured for 2 days. The medium was replaced with fresh M199 medium containing 2% FBS. Cells were treated for 24 h with NBP (200 μM). Before cellular proteins were harvested, cells were pretreated with or without U0126 (5 μM) or LY294002 (10 μM) for 30 mins. The cultures were washed three times with HBSS and total cellular proteins were harvested in 500 μl of modified RIPA buffer, as described 8-11. Aliquots (50 μL) were combined with an equal volume of Laemmeli buffer, denatured (95°C, 5 min) and stored on ice. Total protein was separated by sodium dodecyl sulfate-polyacrylamide (7%) electrophoresis (SDS–PAGE) (15 μg protein/lane). The proteins were transferred to nitrocellulose membranes and blotted at 4°C overnight with antibodies to phosphorylated ERK1/2 (P-ERK1/2, Catalog# 9102, Cell Signaling Technology, USA), total ERK1/2 (T-ERK1/2, Catalog# 4695S, Cell Signaling Technology, USA), anti-FGF-2 (Catalog# 3196S, Cell Signaling Technology, USA), anti-eNOS (Catalog# sc-654, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylation of eNOS at S1177 (P-eNOS, Catalog# 9571, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (P-Akt, Catalog# 9271, Cell Signaling Technology, Danvers, MA, USA), anti-Akt (Catalog# 9272, Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (Catalog# 2565, Biosynthesis Biotechnology, Beijing, China). Bands were visualized using horseradish peroxidase (HRP)-linked secondary antibodies and ECL reagents (Amersham-Pharmacia Biotech). 9, 12-14 Images of the bands of interest were obtained with an EPSON scanner and Adobe PhotoShop CS. Densities of the bands were quantified from the scanned images with NIH Image 1.63 software. Relative changes of the proteins in the lysates were determined as follows. The band densities for FGF-2 were divided by the band densities for the corresponding β-actin. The band densities for P-ERK1/2, P-eNOS and P-Akt were divided by the band densities for the corresponding T-ERK1/2, eNOS and Akt respectively. The resulting ratios of P-ERK1/2/ T-ERK1/2, P-eNOS/eNOS, P-Akt/Akt and FGF-2/β-actin for the controls were used to calculate relative ratios for all experimental groups, after setting the control ratio to 1.

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