Non-denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) assays
Recombinant human CD147 protein (5 μg/mL, ab155636, Abcam, Cambridge, MA, USA) was added to various concentrations of SP-8356 and mixed with sample buffer lacking SDS, followed by resolution by 10% SDS-PAGE without boiling. Anti-CD147 antibody (ab108317, Abcam, Cambridge, MA, USA) was used for immunoblotting. The concentration of recombinant human CD147 protein was determined following the previous study which reported the inhibitory effect of AC-73 on CD 147 dimerization [18].
Surface plasmon resonance (SPR) assays
SPR assays were performed on an SR7500DC instrument (Reichert Inc., Buffalo, NY, USA) at 25 °C. Approximately 4000 resonance units (RU) of recombinant human CD147 (ab155636, Abcam), at a concentration of 3 μg/mL in 10 mmol/L sodium acetate (pH 4.5), were immobilized on CMDH chips containing gold (Reichert Inc., Buffalo, NY, USA), using the amine coupling kit supplied by the manufacturer. The analyte (SP-8356; 300.35 kDa) was dissolved in running buffer (1X PBS, 2% DMSO, pH 7.4) and injected over the flow cells at concentrations of 6.25, 12.5, 25, 50, 100, 200 and 400 μM, in that order, at a flow rate of 30 μL/min. As a control, 1.25–640 nM anti-CD147 antibody (ab108317, Abcam), from lowest to highest concentration, was injected over the flow cells to test its binding to immobilized CD147. The association and dissociation times were both 5 min. After each round, the surface of the sensor chip was regenerated by injection of NaOH (5–50 mM) for 1 min until the RU signal returned the baseline. An equilibrium dissociation rate constant (Kd) was calculated from the kinetic rate constants by a simple 1:1 interaction model using Scrubber 2 software (Biologic Software, Campbell, Australia).
Cell culture
The A10 vascular smooth muscle cell line (ATCC CRL 1476) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified eagle medium (DMEM; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), and 1% penicillin/streptomycin (HyClone) at 37 °C in a 5% CO2 humidified atmosphere. The contractile type of VSMC was induced by serum deprivation for 6 days [19].
Immunocytochemistry
To assess whether SP-8356 affects VSCM phenotype modulation, induced contractile VSMCs were pre-treated with SP-8356 for 30 min and subsequently treated with recombinant human protein CD147 (5 μg/mL, ab155636, Abcam, Cambridge, MA, USA). After 24 h of incubation, cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde. Cells were then permeabilized with 0.3% Triton X-100 and blocked for 30 min with 1% bovine serum albumin in PBST, followed by incubated at 4 °C with primary antibody to smooth muscle myosin heavy chain (SM-MHC) (1:200 dilution, ab683, Abcam, Cambridge, MA, USA) for overnight. Alexa Fluor 488-conjugated goat anti-mouse IgG (1:400 dilution, A28175, Invitrogen, Carlsbad, CA, USA) was used for secondary antibody and nuclei were stained with Hoechst 33,342 solution.
Gelatin zymography
MMP activities of cultured VMSCs were evaluated by gelatin zymography. A10 VSMCs were treated for 24 h with SP-8356 in the presence of recombinant human protein CD147 (5 μg/mL, ab155636, Abcam, Cambridge, MA, USA). Conditioned media were collected and centrifuged to remove cellular debris and concentrated by Microcon centrifugal filtration (Millipore, Billerica, MA, USA). These samples were mixed with a non-reducing loading buffer without heating and loaded onto 10% SDS-PAGE gels containing 1 mg/mL gelatin (JT Baker Chemical Co., Phillipsburg, NJ, USA). Proteins were separated by electrophoresis at 125 V for 90 min. A MMP-9 recombinant protein (ab168863, Abcam, Cambridge, MA, USA) was loaded as a positive control. Following electrophoresis, the gels were rinsed twice for 30 min with Novex zymography renaturing buffer (Invitrogen, Carlsbad, CA, USA), incubated overnight with Zymogram developing buffer (Invitrogen), and stained with a Simply Blue Safe Stain (Invitrogen).
Migration assay
VSMC migration assays were performed with 24-well Transwell plates and polycarbonate membranes (8-μm pore size, Costar, Corning, NY, USA) coated with Matrigel (Sigma, St Louis, MO, USA). The upper chambers were seeded with 1.5 × 105 VSMCs in 200 μL serum-free DMEM. The Transwells were placed in 24-well culture dishes in 800 μL serum-free DMEM. After incubation for 12 h with SP-8356 in the presence of recombinant human CD147 (5 μg/mL, ab155636), the membranes were fixed and stained with a Hemacolor rapid staining kit (Merck, Darmstadt, Germany). The number of cells that had migrated to the lower chambers was counted (5 random fields/membrane) using an inverted microscope (Leica DM IL, Leica Microsystems, Wetzlar, Germany).
Animals
Male Sprague–Dawley rats (7 weeks old, 200–250 g body weight) were purchased from Orient-Bio (Seongnam, Korea). All rats were housed under a 12-h light/dark cycle with ad libitum access to water and food.
Induction of neointimal hyperplasia
After the rats were acclimated for 1 week, they were subjected to partial right carotid artery ligation, as described [17, 20]. In brief, anesthesia was initiated with 3.5% isoflurane in a 2:1 N2O/O2 mixture in a vented anesthesia chamber and sustained by inhalation through a nasal cone of 2 to 2.5% isoflurane in a 2:1 N2O/O2 mixture. The right external carotid artery, occipital artery and right internal carotid artery were ligated with 6-0 silk sutures, followed by intraperitoneal injection of vitamin D3 (6 × 105 IU/kg) once daily for 2 days. Vitamin D3 is often used to stimulate VSMC proliferation and migration [21, 22]. All rats were fed with a commercially available high fat diet (D12336, Research Diets, NJ, USA) for 1 month (28 days).
Drug treatment
The day after their last vitamin D injection, the rats were subdivided into three groups. The rats were injected intraperitoneally with vehicle (0.9% normal saline), rosuvastatin (10 mg/kg in saline, used as a reference drug) or SP-8356 (50 mg/kg in saline) once daily for 4 weeks. Rosuvastatin is a hydroxymethylglutaryl coenzyme A reductase inhibitor that has been well known to reduce neointima formation in animal models [23,24,25]. Statins also reduce restenosis characterized by neointimal hyperplasia after coronary stent implantation in clinical studies [26, 27]. At doses of 1, 10, and 20 mg/kg, rosuvastatin was reported to inhibit neointimal hyperplasia [23,24,25]. At a dose of 80 mg/kg, simvastatin has been known to be toxic to VSMCs [28], and recently it was reported that 20 mg/kg of rosuvastatin is equivalent to 80 mg/kg of simavastatin [29]. Therefore, we treated rats with 10 mg/kg of rosuvastatin. The in vivo therapeutic dose of SP-8356 corresponding to in vitro dose was determined based on our previous pharmacokinetic-pharmacodynamic relationship study [30].
Pulse wave velocity (PWV) measurement
After 4 weeks of drug administration, PWV was measured using an ultrasound Doppler system (iU22, Philips Ultrasound, Bothell, WA, USA). Electrocardiography limb electrodes were placed and the Doppler probe was located parallel to the blood flow of the right carotid and left iliac arteries. The electrocardiography and Doppler signals were recorded simultaneously; the distance and time were defined as described previously [31]. Distance was measured between the site probe points over the carotid and iliac arteries, and the time was measured between the R peak waves of the electrocardiogram to the foot of the carotid or iliac wave signals. The times were averaged over three consecutive electrocardiography cycles. PWV (m/s) was calculated as:
$$ {\text{PWV }}\left( {{\text{m}}/{\text{s}}} \right) = \frac{\text{Distance from carotid to iliac artery}}{{{\text{Time }}\left( {{\text{R peak point to iliac foot}} - {\text{R peak point to carotid foot}}} \right)}} $$
Blood pressure
Blood pressure was measured using a tail-cuff method (ML125, Powerlab, AD Instruments, Castle Hill, NSW, Australia). Rats were placed in a chamber pre-heated to 35 °C for 10 min and moved to plastic restrainers. To obtain accurate and reliable blood pressure result, rats were handled gently and not forced to enter plastic restrainer. Rats remained stable and unperturbed during the measurement period. A cuff with a pneumatic sensor was applied onto the tail and blood pressure was measured, with the results averaged from three consecutive recordings.
Histopathology
All rats survived during the study period and were sacrificed by carbon dioxide inhalation after 4 weeks of drug administration. The common carotid arteries were fixed with 4% paraformaldehyde and preserved in 30% sucrose solution. The tissue was embedded in optimal cutting temperature compound (Scigen Scientific, Gardena, CA, USA). Axial sections of 4 μm thickness were cut with a cryostat microtome (Leica CM 3050 S, Leica Microsystems). Serial sections were obtained down-stream of the bifurcation of the external and internal carotid arteries. In accordance with the guidelines for experimental study of vessel by the American Heart Association [32], lesions were analyzed in a blinded manner and quantified as an average of 6 serial sections, each 100 μm apart from each other. The sections were stained with hematoxylin and eosin and evaluated using an upright light microscope (BX51, Olympus, Tokyo, Japan). The neointimal area was defined as the area between the luminal circumference and the internal elastic lamina. The media area was defined as the area between the internal and external elastic lamina. The neointima/media ratio was defined as the area of the neointima divided by the area of the media.
Immunohistochemistry
Sections were blocked for 60 min with 5% goat serum in phosphate-buffered saline containing 0.1% Triton X-100, and incubated at 4 °C with primary antibodies to α-smooth muscle-actin (α-SMA) (1:200 dilution, ab7817, Abcam, Cambridge, MA, USA), SM-MHC (1:200 dilution, ab683, Abcam, Cambridge, MA, USA), MMP-9 (1:200 dilution, AB19016, Merck Millipore, Billerica, MA, USA), and collagen type III (1:200 dilution, ab7778, Abcam, Cambridge, MA, USA). The sections were washed and incubated with the appropriate secondary antibody, Alexa Fluor 555-conjugated goat anti-mouse IgG (1:400 dilution, A21424, Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:400 dilution, A11034, Invitrogen, Carlsbad, CA, USA), followed by counterstaining of the nuclei with 4′, 6-diamidino-2-phenylindole (DAPI). All images were obtained using a confocal microscope (LSM800, Carl Zeiss, Oberkochen, Germany).
Molecular expression intensity in the neointimal area was analyzed using Image J version 1.45 s open-source software (NIH Image, Bethesda, MD, USA). A region of interest (ROI) was drawn on the neointimal area and the integrated density of pixels in the ROI was calculated. The intensity/area ratio was calculated as the integrated density divided by the area of the neointima. Unstained and secondary antibody stained control images of neointimal hyperplasia were shown in Additional file 1: Figure S1.
Statistical analysis
All data were presented as the mean ± standard deviation of at least three independent experiments. Multiple groups were compared by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test and two groups were compared by Student’s t test. All statistical analyses were performed using SPSS version 17.0 software (SPSS Inc, Chicago, IL, USA), with a p-value < 0.05 considered statistically significant.