PD168077 (D4 receptor agonist), L745870 (D4 receptor antagonist), streptozotocin (STZ) were from Sigma Co. (Sigma, St. Louis, MO). Insulin was purchased from Roche Group (Basel, Switzerland); Rabbit polyclonal antibody against insulin receptor, cleaved caspase 3 and Histone H3 were from Cell Signaling (Beverly, MA). Antibody for proliferating cell nuclear antigen (PCNA) was from Santa Cruz. SDS-polyacrylamide gels were from Pierce (Rockford, IL). Polyvinylidene fluoride (PVDF) and protein gel apparatus were from Bio-Rad (Hercules, CA). Minimal essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were from Gibco/Invitrogen (Carlsbad, CA); fibroblast growth factor (FGF), epidermal growth factor (EGF), phosphate buffered saline (PBS), penicillin/streptomycin, and non-essential amino acids were from Sigma Co.
A10 cell , a smooth muscle cell line from rat thoracic aorta, was purchased from ATCC (A10; ATCC, Hercules, CA), and cultured in DMEM supplemented with 20% FBS and 1% penicillin/streptomycin/bFGF/EGF at 37°C in a humidified, 5% CO2 atmosphere. After reaching sub-confluence, the A10 cells were serum-starved for 24 hrs in serum-free DMEM to maintain quiescence before further treatment.
A10 cell proliferation
Cell proliferation was determined by measuring the uptake of tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) and the incorporation of [3H]-thymidine (Atomic Energy Research Establishment of China, Beijing City, China) into DNA of cells respectively. The cells were seeded into 96-well (100 μl of medium per well) culture plates at a density of 1 × 104 cells/well, made quiescent for 24 hrs, and then stimulated with the indicated reagents. Subsequently, 20 μl of MTT (5 mg/ml) were added to each well, and the incubation continued for an additional 4 hrs at 37°C. Thereafter, dimethyl sulfoxide (DMSO, 150 μl) was added to each well, and absorbance read at 490 nm on a Microplate reader (model 680, Bio-Rad). For [3H]-thymidine incorporation assay, the cells were treated with the same reagents then labeled with [3H]-thymidine (1 μCi/ml) 6 h and assessed for [3H] incorporation into newly synthesized DNA as previously described . Cell number was also used to estimate cell proliferation by plating the A10 cells in six-well plastic culture dishes at a density of 1 × 104/well in DMEM with 10% FBS. The medium was then removed and washed three times with PBS. The cells were incubated in serum-free medium, which contained the indicated reagents for the indicated times. After incubation, the number of cells was counted by a hemocytometer (trypan blue uptake, which indicates cell death, was observed in <10% of the cells). Experiments were performed independently at least three times [13–15].
A10 cell migration
Cells migration was examined using transwell and scratch-wound migration assays. The transwell migration assay was performed using 24-well tissue culture plates (BD Bioscience, Becton, NJ) with an 8-μm-pore polycarbonate membrane [16, 17]. The number of migratory A10 cells was counted in 10 randomly chosen fields of duplicate chambers at a magnification of 200× for each sample. For the scratch-wound migration assay, the A10 cells were scratched with a small tip along the ruler. After washing, the cells were cultured in serum-free DMEM for 48 hrs. The migration area (%) was analyzed in 10 randomly chosen fields under an inverted microscope (IX-70; Olympus, Tokyo, Japan), using NIH Image J software; area at 0 hr and area at 48 hrs × 100% was calculated.
Immunoflurescence image of rat thoracic aorta
Expression of D4 receptors in thoracic aorta from SD rats was examined by laser scanning confocal microscope. Briefly, the aorta was cleared of blood with ice-cold oxygenated saline and kept in Histochoice (Amresco, Solon, OH) for one to two days at 4°C, then sectioned (4 μm), embedded in paraffin, and mounted on slides. The sections were double-immunostained with mouse anti-D4 receptor antibody (1:100) and rabbit polyclonal anti-α-SM-actin antibody (1:100). The colocalization of D4 receptors and α-SM-actin was performed as described previously .
A10 cells were treated with vehicle (ddH2O), D4 receptor agonist (PD168077) and/or D4 receptor antagonist (L745870) at the indicated concentrations and times. After treatment, the A10 cells were washed once in PBS and lysed in lysis buffer. Immunoblotting was performed as previously reported [13, 14]. Protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as standard. Cell lysates were boiled in sample buffer (35 mmol/L Tris–HCl, pH 6.8, 4% SDS, 9.3% dithiothreitol, 0.01% bromophenol blue, 30% glycerol) for 10 min; 50 μg of cell protein were subject to immunoblotting analysis. PVDF membranes were blocked with 5% nonfat dry milk in PBS-T [0.05% Tween20 in 10 mmol/L phosphate-buffered (isotonic) saline] at 37°C for 2 hrs with constant shaking. The membranes were then probed with rabbit polyclonal antibodies specific for insulin receptor and D4 receptor at 4°C overnight, and then incubated with goat anti-rabbit polyclonal antibodies conjugated to horseradish peroxidase. Proteins were detected using enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK). The amount of protein transferred onto the membranes was normalized by immunoblotting of α-actin (monoclonal α-actin antibody, 1:400, Santa Cruz Biotechnology, Inc. Santa Cruz, CA).
Total RNA from A10 cells was isolated using a Trizol procedure (Invitrogen, Carlsbuel, CA). Two μg of total RNA were used to synthesize cDNA, which served as template for the amplification of D4 receptor, insulin receptor, and β-actin (as housekeeping gene). Primer sequences for D4 receptor were 5′-TGC CCT GTC CGC TCA TGC TAC TGC-3′ (forward) and 5′-CAC CGG CAG GAC TCT CAT CGC CTT GC-3′ (reverse). Primer sequences for insulin receptor were 5′-GGA CTGAAG GTA TGA ATG GAG-3′ (forward) and 5′-TAA CAC AAG CCA AGG AAG GG-3′ (reverse), Primer sequences for β-actin was 5′-GTGGGTATGGGTCAGAAGGA-3′(forward) and 5′-AGCGCGTAACCCTCATAGAT-3′ (reverse). The amplification was performed with the following conditions: 95°C for 1 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 45 sec. This was followed by a final extension at 72°C for 10 min. The PCR products were electrophoresed in 2% agarose gels [13, 14].
Rat hyperinsulinemic model
Healthy male SD rats weighing 210-220 g, were obtained from the Experimental Animal Center of Daping Hospital. The rats were housed on a constant 12 hrs light/12 hrs dark cycle in temperature-controlled central facility (18-22°C) and allowed free access to normal chow and tap water. Type 2 diabetes was induced using STZ, as described previously [19, 20]. Briefly, the rats were intragastrically fed high-fat diet (HFD) consisting of 15% lard, 30% sucrose, 10% Tween-80 and 10% propylene glycol in a volume of 20 ml/kg body weight once a day for 4 weeks (normal-pellet diet rats served as the control). Then the rats were injected intraperitoneally with STZ (25 mg/kg in a 0.1 mol/L citrate buffer at a single dose, pH 4.5) to induce type 2 diabetes with hyperinsulinemia and insulin resistance. After STZ injection, body mass and blood pressure were measured; blood samples were obtained from the tail vein for assay of blood glucose, serum insulin, triglycerides, cholesterol, and free fatty acids using commercially available kits. The rats with fasting blood glucose concentrations more than 11.1 mmol/L were considered to qualify for subsequent experiments.
Rat carotid balloon injury
SD rats were anesthetized using a mixture of ketamine (80 mg/kg) and xylazine (12 mg/kg), and then the left common and external carotid arteries were exposed and isolated. A 2 F Fogarty catheter (Edwards Life Sciences, Irvine, CA) was introduced into the common carotid artery through an arteriotomy in the external carotid artery and inflated to 1.0-1.5 atm. A 10-mm injury was induced by withdrawing the catheter 4 times. The external carotid artery was then untied, and blood flow was restored. After the balloon-injury, SD rats were intraperitoneally injected with PD168077 (6 mg/kg per day) or similar volume of distilled water (2 ml/kg) daily for 14 days. Before the SD rats were euthanized, body mass and blood pressure were measured, and bloods for biochemical parameter measurements were obtained. Then, both carotid arteries were dissected and the sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope (Olympus, Tokyo, Japan). Media and neointima areas of injured arteries were quantified by planimetry (μm2) and intima-to-media ratios were calculated as reported [21, 22].
Proliferation and apoptosis of rat carotid arteries treated with PD168077 after balloon injury
Cell proliferation and apoptosis are important contributors to neointimal formation after balloon injury. We performed experiments investigating the role of PD168077 in reducing the proliferation and sustaining the apoptosis of rat carotid artery at day14 after balloon injury. PCNA (1:500) and cleaved caspase 3 (1:800) expression were evaluated the proliferation or apoptosis respectively by western-blot as previously.
The data are expressed as mean ± SEM. Comparison within groups was made by repeated measures ANOVA (or paired t-test when only 2 groups were compared), and comparison among groups (or t-test when only 2 groups were compared) was made by factorial ANOVA with Holm-Sidak test. A value of P < 0.05 was considered significant.