Dopamine D4 receptors inhibit proliferation and migration of vascular smooth muscle cells induced by insulin via down-regulation of insulin receptor expression

Vascular smooth muscle cells (VSMCs) proliferation and migration, which are central in the development of vascular diseases, are regulated by numerous hormones and humoral factors. Activation of the insulin receptor stimulates VSMCs proliferation while dopamine receptors, via D1 and D3 receptors, inhibit the stimulatory effects of norepinephrine on VSMCs proliferation. We hypothesize that activation of the D4 dopamine receptor may also inhibit the proliferation and migration of VSMCs, therefore, inhibit atherosclerosis. Our current study found that insulin increased the proliferation and migration of A10 cells, an effect that was reduced in the presence of a D4 receptor agonist, PD168077. The negative effect of the D4 receptor on insulin’s action may be via decreasing insulin receptor expression, because activation of the D4 receptor inhibited insulin receptor protein and mRNA expressions, indicating that the regulation occured at the transcriptional or post-transcriptional levels. To determine whether or not the inhibition of D4 receptor on insulin-mediated proliferation and migration of VSMCs has physiological significance, hyper-insulinemic Sprague–Dawley rats with balloon-injured carotid artery were treated with a D4 agonist, PD168077, (6 mg/kg/d) for 14 days. We found that PD168077 significantly inhibited neointimal formation by inhibition of VSMC proliferation. This study suggests that activation of the D4 receptor suppresses the proliferation and migration of VSMCs, therefore, inhibit atherosclerosis. The D4 receptor may be a potential therapeutic target to reduce the effects of insulin on artery remodeling.


Introduction
The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play a crucial role in neointimal formation and vascular remodeling during atherosclerosis and restenosis [1][2][3]. It is currently accepted that proliferation and migration of medial VSMCs are involved in neointimal formation after injury, which could be induced by cytokines and growth factors, including insulin. Insulin initiates many biological effects by activating the insulin signaling pathways, such as mitogen activated protein kinase, triggering hypertrophy, proliferation, and migration of VSMCs [4][5][6]. Therefore, inhibition of insulinmediated VSMCs proliferation and migration would be helpful in preventing the development of atherosclerosis and restenosis after angioplasty.
Dopamine receptors exert beneficial effects by regulating epithelial sodium transport and vascular smooth muscle tone in hypertension [7][8][9][10][11][12]. Dopamine receptors are classified into D 1 -like and D 2 -like subtypes based on their structure and pharmacology. D 1 -like receptors are composed of D 1 and D 5 receptors while D 2 -like receptors are composed of D 2 , D 3 and D 4 receptors [6][7][8][9][10][11]. We have previously shown that activation of D 1 -like or D 3 receptor inhibits the insulin-mediated VSMCs proliferation [13,14]. Activation of D 1 -like and D 3 receptors has an additive inhibitory effect on norepinephrine-mediated proliferation of VSMCs [15]. We hypothesized that D 4 receptors may also have inhibitory effect on insulin-mediated VSMCs proliferation and migration, which may inhibit the formation of atherosclerosis. To test this hypothesis, we used cultured rat aortic smooth muscle cells line (A10) to examine the potential effect of D 4 receptors on insulin-mediated proliferation and migration, and investigated the role of D 4 receptors on neointimal hyperplasia in Sprague-Dawley (SD) rats with hyperinsulinemia.
Cell culture A10 cell [13], 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% CO 2 atmosphere. After reaching subconfluence, 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 [ 3 H]-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 × 10 4 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 [ 3 H]-thymidine incorporation assay, the cells were treated with the same reagents then labeled with [ 3 H]thymidine (1 μCi/ml) 6 h and assessed for [ 3 H] incorporation into newly synthesized DNA as previously described [13]. 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 × 10 4 /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][14][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 D 4 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-D 4 receptor antibody (1:100) and rabbit polyclonal anti-α-SM-actin antibody (1:100). The colocalization of D 4 receptors and α-SM-actin was performed as described previously [18].
Immunoblotting A10 cells were treated with vehicle (ddH 2 O), D 4 receptor agonist (PD168077) and/or D 4 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 D 4 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).

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 (μm 2 ) and intima-tomedia 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.

Statistical analysis
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.

D 4 receptor in artery
D 4 receptor expression in rat thoracic aorta was determined by double-immunostaining, immunoblotting, and RT-PCR analysis. The D 4 receptor is expressed in the vascular smooth muscle, proved by its colocalization with α-SM-actin ( Figure 1A). Immunoblotting revealed that a 47 kDa band, expected molecular size for the D 4 receptor protein, disappeared when the antibody was pre-adsorbed with the corresponding immunizing peptide (1/10 w/w) ( Figure 1B). RT-PCR for the D 4 receptor in rat thoracic aorta and A10 cells showed the expected D 4 receptor size of 367 bp ( Figure 1C).

Stimulation of the D 4 receptor inhibits insulin-mediated A10 cell proliferation and migration
VSMCs are the main cellular components of neointimal lesions after vascular injury. Their migration and proliferation contribute to the formation of these lesions. Therefore, the effect of D 4 receptor on A10 cell proliferation was explored by using MTT, [ 3 H]-thymidine incorporation and direct cell counting. Insulin increased A10 cell proliferation in a concentration-dependent manner in A10 cells (Figure 2A). Although PD168077 by itself had no effect on cell proliferation ( Figure 2B), it inhibited the insulin-mediated proliferation in A10 cells in a concentration-dependent manner (10 −8 -10 −6 mol/L) ( Figure 2C and D). The inhibitory effect of D 4 receptor was specific because the D 4 receptor antagonist L745870 (10 −7 mol/L/24 hrs) blocked the cell proliferative effect of PD168077 (Figure 2E, F and G).
We also studied the effect of insulin on cell migration by transwell and scratch-wound migration tests. Insulin increased the migration of A10 cells, an effect that was inhibited by a D 4 receptor agonist, PD168077, in a concentration-dependent manner ( Figure 3A and B). Consistent with the cell proliferation study, the inhibitory effect of PD168077 was specific to the D 4 receptor because the D 4 receptor antagonist L745870 (10 −7 mol/ L/24 hrs) blocked the effect of PD168077 on cell migration ( Figure 3C and D).

Activation of D 4 receptor decreases insulin receptor expression in A10 cells
To uncover the mechanisms underlying the negative regulation of the D 4 receptor of insulin-mediated proliferation and migration in A10 cells, we quantified the expression of insulin receptor. We found that stimulation of the D 4 receptor by a D 4 receptor agonist, PD168077, decreased insulin receptor expression in a concentration (10 −10 -10 −6 mol/L) and time (4-30 hrs)-dependent manner ( Figure 4A and B). The effect of PD168077 on insulin receptor expression was via the D 4 receptor, because a D 4 receptor antagonist, L745870 (10 −7 mol/L/24 hrs), blocked the inhibitory effect of PD168077 on insulin receptor expression in A10 cells ( Figure 4C). Stimulation of the D 4 receptor PD168077 (10 −7 mol/L/24 hrs) also decreased insulin receptor mRNA expression, an effect that was blocked by the D 4 receptor antagonist L745870 ( Figure 4D), suggesting that the regulation of insulin receptor by the D 4 receptor occurs at the transcriptional or post-transcriptional levels.

Effect of D 4 receptor on neointima formation in balloon-injured carotid arteries of SD rats
To determine whether or not the D 4 receptor-mediated inhibition of insulin-induced proliferation and migration of A10 cells had physiological significance, we studied the effect of the D 4 receptor agonist PD168077 in hyperinsulinemic type 2 diabetic SD rats (Table 1). A significant increase in the body weight was observed in the rats fed with HFD compared to rats fed with normal pellet diet (NPD). Before STZ injection, HFD-fed rats had similar glucose concentrations to NPD-fed rats, but significantly higher serum insulin, triglyceride, total cholesterol, and free fatty acids concentrations, Injection of STZ (25 mg kg −1 ), The level of blood glucose significantly increased compared to HFD-fed rats and NPD-fed rats, Figure 5 Effect of PD168077 on neointimal formation after vascular injury in SD rats with hyperinsulinemia. Carotid balloon-injured rats were intraperitoneally injected with vehicle or PD168077 (6 mg/kg/day) for 14 days. Representative histologies (A-C) were shown above the bar graphs. A: sham operation control. B: balloon-injured carotid artery and vehicle injection. C: balloon-injured carotid artery and PD168077 injection. The bar graphs show the area of the intima to media ratio (I/M ratio, D) (n = 6, *P < 0.05 vs. sham group, # P < 0.05 vs. balloon-injured and vehicle group). thus suggesting producing type 2 diabetes with insulin resistance. However, the blood pressure remained unaltered among STZ + HFD-treated rats, HFD-treated rats and control NPD-fed rats. We found no significant changes in each parameter between PD168077-treated and untreated STZ + HFD rats. The carotid artery-injured rats were then intraperitoneally injected with PD168077 (6 mg/kg/d) for 14 days to investigate the role of PD168077 in neointimal formation in vivo. Consistent with previous reports [21,23], a significant increase in neointimal formation was found 2 weeks after the balloon injury of the carotid artery ( Figure 5A and B). Compared with balloon injury and vehicle-injected group, PD168077 significantly inhibited neointimal formation, associated with a decrease in the intima/media (I/M) ratio ( Figure 5C and D).

Effect of D 4 receptor on proliferation and apoptosis of in balloon-injured carotid arteries of SD rats
PCNA expression revealed a significant decrease in proliferation in the PD168077-injected rats compared with vehicle-injected rats ( Figure 6A). There was no difference in cleaved caspase 3 expression between two groups ( Figure 6B), it seemed likely that the mechanisms of PD168077 inhibited neointimal hyperplasia were attributed to its antiproliferative and not apoptotic effects in balloon injured carotid artery of STZ + HFD rats.

Discussion
Insulin resistance and hyperinsulinemia are important risk factors for cardiovascular disease due, in part, to the VSM proliferative and pro-atherogenic actions of insulin [3,5,24,25]. Therefore, reducing the stimulatory effect of insulin on VSMC proliferation and migration may provide new insights in the prevention of atherosclerosis.
More and more pieces of evidence indicate an interaction between insulin and dopamine receptors in various tissues and cells. The renal dopaminergic system is impaired in hyperinsulinemic rats and type 2 diabetes patients [26][27][28]. The dopamine D 1 -like receptor agonist, fenoldopam, has been shown to improve peripheral insulin sensitivity in STZ-induced type 2 diabetic rats [29]. In pancreatic beta cells, the D 2 -like receptor (D 2 R/D 3 R) agonist quinpirole inhibits glucose-stimulated insulin secretion [30]. Another D 2 -like receptors agonist, bromocriptine, has been reported to decrease insulin levels of obese women [31]. Our previous studies also indicate a negative interaction between D 1 -like and insulin receptors because a D 1 -like receptor agonist, fenoldopam, inhibits the proliferative effect of insulin in A10 cells [13]. Stimulation of the D 3 receptor also inhibits insulin receptor mRNA and protein expressions and insulin-mediated VSMC proliferation [14]. However, whether or not the D 4 receptor has inhibitory effect on insulin-mediated VSMC proliferation and migration is unknown.
Previous studies have demonstrated that D 4 receptors are expressed in the adventitia and adventitia-media border of human pulmonary and coronary arteries [32,33]. We now show, for the first time, that D 4 receptors are expressed in thoracic aorta of SD rats, determined RT-PCR and immunoblotting and in VSM of the aorta, by double-immunofluorescence staining. Moreover, the D 4 receptor in VSMCs is functional. Stimulation of the D 4 receptor reduced insulin-mediated VSMC proliferation Figure 6 Effects of PD168077 on the proliferation and apoptosis of balloon-injured carotid in SD rats with hyperinsulinemia. Carotid balloon-injured rats were intraperitoneally injected with vehicle or PD168077 (6 mg/kg/day) for 14 days. Proliferation was measured by immunoblotting of proliferating cell nuclear antigen (PCNA) (A) and cleaved caspase 3 expression (B) in PD168077-treated and control rats. Results are expressed as the ratio of PCNA to Histone H3 densities or ratio of caspase-3 to α-actin densities (n = 4, *P < 0.05 vs. control). and migration, although D 4 receptor, by itself, had no effect.
The importance of the D 4 receptor on VSMC remodeling during insulin resistance was investigated using a rat model of insulin resistance and hyperinsulinemia (STZ-treated rats on high fat diet). Treatment of these rats with PD168077, a D 4 receptor agonist, for two weeks prevented neointimal growth in balloon-injured carotid artery. Neointimal formation after vessel injury is a complex process involving the proliferation and migration of VSMCs [21]. Apoptosis is also important in this process [34,35]. In this study, PD168077 significantly reduced the balloon injury caused neointimal hyperplasia in rat carotid artery. PCNA expression was also decreased in injured artery with PD168077 treatment. However, the expression of cleaved caspase 3 was not significant change in injured carotid arteries between the two groups which seems proliferation not apoptosis likely to be the major contributor for attenuation of neointimal formation after treatment with PD168077. Based on these findings and previous studies, we speculate that the D 4 receptor may be a target to inhibit abnormal VSMC growth induced by insulin, which may lead to a novel strategy in the treatment of atherosclerosis.
In summary, we have demonstrated that activation of the D 4 receptor inhibits insulin receptor expression and insulin-mediated proliferation and migration of A10 cells in vitro, and neointimal formation in vivo. This study implicates a critical role of the D 4 receptor in the pathogenesis of vascular remodeling and this new insight may result in the development of a novel strategy in the treatment and prevention of vascular injury associated with insulin resistance.