Medicinal preparation of Yangzheng Xiaoji (Yiling Pharmaceutical, Hebei) was subject to extraction using DMSO, balanced salt solution and ethanol, on rotating wheel for 24 h at 4°C as we recently reported (17). Insolubles were removed after centrifugation at 15,000 × g. DMSO preparation was found to be more consistent, reproducible and with better yield compared with the other two solvents. DMSO extract was hence used in the subsequent experiments. The extract was standardised by quantifying the optical density of the preparation using a spectrophotometer at a wavelength of 405 nM. A master preparation of the extract which gave 0.25 OD was stocked as the master stock and so named as DME25 for the experiments.

This was based on a previous published method (18). HECV cells were seeded into 96 well plates at a density of 3,000 cells/well. Triplicate plates were set up for incubation periods of overnight, 3 days and 5 days. Following sufficient incubation, the plates were removed from the incubator, fixed in 4% formaldehyde (v/v) and stained with 0.5% (w/v) crystal violet. The crystal violet stain was subsequently extracted using a 10% acetic acid (v/v) allowing the detection of cell density through spectrophotmeric analysis of the resulting solutions absorbance using a Bio-Tek ELx800 multi-plate reader (Bio-Tek Instruments Inc., VT, USA).

ECIS-Zθ instrument (Applied Biophysics Inc., NJ, USA) were used for cell adhesion and motility (wounding assay) assays in the study (19,20). Cell modelling was carried out using the ECIS RbA modelling software, supplied by the manufacturer. The 96W1E ECIS arrays were used in the present study. ECIS measures the interaction between cells and the substrate to which they were attached via gold-film electrodes placed on the surface of culture dishes. Following treating the array surface with a cysteine solution, the arrays were incubated with complete medium for 1 h. The same number of the respective cells was added to each well. In the cell adhesion assay, the adhesion was tracked immediately after adding the cells into the arrays. For cell migration assay, the arrays with cells were allowed to reach confluence after 3 h. The monolayer of the cells was electrically wounded at 2,000 mA for 20 sec. Impedance and resistance of the cell layer were immediately recorded for a period of up to 20 h. For signalling transduction inhibitor assays, the respective inhibitors was included in the wells. Adhesion and migration were modelled using the ECIS RbA cell modelling software as we recently reported (21,22).

In vitro microvessel tubule formation was assessed using a Matrigel endothelial cell tubule formation assay. This was modified from a previously reported method (23,24). Briefly, 250 μg of Matrigel was seeded, in serum-free medium, into a 96-well plate and placed in an incubator for a minimum of 40 min to set. Following this, 35,000 HECV were seeded onto the Matrigel layer and incubated for 4–5 h, in the presence or absence of DME25, FAK inhibitor or their combination. Tubule formation that occurred over the incubation period was visualised under low magnification and images captured. Total tubule perimeter per field was quantified using ImageJ software.

Cell culture plate (96-well) was first coated with 2 μg Matrigel and allowed to air dry. After rehydration for 1 h, the wells were gently washed with DMEM medium, HECV cells (20,000 per well) were added together with the respective treatments. Wells were gently washed with BSS 5 times, before cells were fixed with 4% formalin and stained with crystal violet. Adherent cells were counted and shown as number of adherent cell per high power field of an upright microscope.

HECV cells were seeded at a density of 20,000 cells per well in a 16-well chamber slide (LAB-TEK Fisher Scientific UK, Longhborough, UK), together with the respective treatment for 2 h (25,26). Medium was carefully aspirated from the wells and the cells were fixed in 4% formalin for 20 min. Following fixation, the cells were permeabilised for 5 min in a 0.1% Triton X-100 BSS solution. A blocking solution of (Tris buffer 25 mM Tris, pH 7.4) with 10% semi-skimmed milk was used to block non-specific binding for 40 min. Cells were subsequently washed twice with wash buffer before probing for specific antibodies to FAK and phopho-FAK (SC-1688 and SC-11766, respectively, from Santa-Cruz Biotechnologies, Inc., Santa Cruz, CA, USA). Primary antibodies were made up in the Tris buffer with 3% milk at a 1:100 concentration for 1 h. The primary antibody was then completely removed by washing the cells 5 times in the same buffer. FITC conjugated anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich) was subsequently added to the cells and the slides were incubated on a shaker platform in the dark for 1 h. The slides were finally washed 3 times to remove unbound secondary antibody, mounted with Fluor-save (Calbiochem-Novabiochem Ltd., Nottingham, UK) and visualised under an Olympus BX51 fluorescent microscope at ×100 objective magnification.

Cells were grow to confluence in a 25 cm³ tissue culture flask, detached and lysed in HCMF buffer containing 1% Triton X-100, 2 mM CaCl₂, 100 μg/ml phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin and, 10 mM sodium orthovanadate on a rotor wheel for 1 h before being spun at 13,000 x g to remove insolubles. The protein levels in the samples were subsequently quantified using the Bio-Rad DC Protein assay kit (Bio-Rad Laboratories, CA, USA).

Once sufficient separation had occurred the proteins were blotted onto a Hybond-C Extra nitrocellulose membrane (Amersham Biosciences UK Ltd., Bucks, UK), blocked in 10% milk and probed for the expression of specific proteins. Anti-pFAK and anti-pPaxillin were used to probe the phophorylated FAK and paxillin, respectively (27). In addition to this, GAPDH expression was also assessed using an antibody specific to this molecule (Santa Cruz Biotechnology Inc.) to assess total protein levels and uniformity throughout the test samples. Protein bands were then visualised through the Supersignal West Dura Extended Duration substrate chemiluminescent system (Perbio Science UK Ltd., Cramlington, UK) and detected using a UVIProChem camera system (UVItec Ltd., Cambridge, UK).

Using an in vitro tubule formation assay, it was shown that DME25 (shown in Fig. 1 are 1:1000 dilution) significantly reduced tubule length compared with control (p=0.046). This was seen at concentrations at which no growth inhibition was achieved at the concentrations without cytotoxicity on HECV cells (Fig. 2). DME25 over a wide concentration did not have a significant influence on the growth of endothelial cells.

DME25 demonstrated a concentration-dependent inhibitory effect on the adhesion of HECV cells, with marked inhibitory effects seen at dilutions of 1:5,000 or lower (Figs. 3A and 4). Using 3D modelling, it was seen that the inhibitory effects by DME25 were seen across the frequencies tested (Figs. 3B and 5). Using conventional cell-matrix adhesion method, DME25 had a significant inhibitory effect on the adhesion (Fig. 3C and D).

In a similar fashion to cell-matrix adhesion, cellular migration was similarly inhibited by the presence of DME25 and was further inhibited when FAK inhibitor was used together with DME25 (Fig. 6).

FAK inhibitor has a marked effect on the adhesion of HECV cells (Figs. 3C and D, 4B, 5). When administered together with DME25, the inhibitory effect appears to be synergistically strengthened as seen in these figures.

FAK inhibitor appears to have an inhibitory effect on tubule formation although this is not statistically significant (p=0.14) (Fig. 1E). However, the combination between DME25 and FAK inhibitor had a marked inhibition on tubule formation, compared with control, with FAK inhibitor along and DME25 along (p=0.006, p=0.041, p=0.011, respectively).

We further evaluated the effect of DME25 on the activation of FAK and paxillin in HECV cells, namely tyrosine phophorylation in these proteins, using phospho-tyrosine specific antibodies. As shown in Fig. 7, DME25 suppressed phosphorylation of FAK and produced more profound inhibition together with FAK inhibitor. Neither DME25 nor FAK inhibitor or their combinations had marked effect on the phosphorylation of paxillin.

Using immunofluorescence method, FAK was seen to be stained strongly in control cells at the focal adhesion sites (Fig. 8, left panel, indicated by arrows). Addition of DME25, FAK inhibitor and the combination of DMA25 and FAK inhibitor render the cells with less focal adhesion complex although the degree of staining was unchanged compared with control (Fig. 8, left panel). It is very interesting to observe the marked changes of phosphorylated FAK which was stained with phosphorylation specific anti-pFAK antibody. As shown in Fig. 8 (right panel, with extended exposure time), control cells had visible stainings of pFAK at the focal adhesion sites in control cells. Both DME25 and FAK inhibitor resulted in reduction of staining of pFAK. However, cells treated with the combination of DME25 and FAK inhibitor almost completely lost staining of pFAK (Fig. 8 right panel).