Basic & Clinical Pharmacology & Toxicology, 107, 798–802
Doi: 10.1111/j.1742-7843.2010.00580.x
Atorvastatin Reduces Calcification in Rat Arteries and Vascular Smooth Muscle Cells Huan Li, Hui-ren Tao, Tao Hu, Yan-hong Fan, Rong-qing Zhang, Guoliang Jia and Hai-chang Wang Departments of Cardiology and Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China (Received 27 November 2009; Accepted 9 February 2010)
Abstract: To examine the in vivo effects of atorvastatin (AT) on arterial calcification in rats, arterial calcification was established by subcutaneous injection of vitamin D3 and Warfarin. Intragastric administration of AT began 4 days before establishment of arterial calcification in the AT group (n = 6). Blood samples were taken and abdominal aortas were collected and stained. After induction of calcification, plasma Ca2+ levels in the CA and AT groups were significantly higher than those before treatment and in the untreated controls. Plasma Ca2+ levels in the AT group were significantly lower than in the CA group. The relative calcification area in aortic specimens from the AT group was significantly smaller than in the CA group. Rat aortic vascular smooth muscle cells (VMSC) were isolated from abdominal aortic segments and pre-treated with AT (1, 5, or 10 lM) for 24 hr. Cells in the calcification (CA) group and the AT group were cultured with b-glycerophosphate, insulin and vitamin C for 14 days to induce cell calcification. Calcium deposition and alkaline phosphatase activity were significantly increased in the CA group compared to untreated controls (p < 0.01). This effect was ameliorated by AT (all p < 0.01). In vivo administration of AT reduced arterial calcification and plasma Ca2+ concentration. In vitro, AT reduced calcification markers in rat aortic vascular smooth muscle cells.
Coronary arterial calcification (CAC) has been found in over 80% of significant coronary lesions in 90% of patients with coronary artery disease. Several reviews have described the molecular mechanisms and clinical consequences of CAC [1–5]. Calcification may influence the course of coronary atherosclerosis by increasing the risk of myocardial infarction and decreasing survival. The calcium mineral deposits found in the arteries share commonalities with bone tissue. Calcified human atherosclerotic lesions express factors such as bone morphogenetic protein type-2, osteopontin, osteoprotegerin and matrix carboxyglutamic acid protein [1,4–8]. Furthermore, CAC has been shown to be an independent risk factor for coronary heart disease [9,10], however, the mechanisms by which CAC leads to vascular events remain poorly understood. The regulation of CAC is highly complex involving an interplay between a number of phenomena, including inflammation [11,12], neovascularisation [13] and various molecular mediators [1–5]. Research has shown that macrophages and vascular smooth muscle cells are among the primary cell types involved in these processes [4,5,10]. Statins, 3-hydroxy-3-methyl-gluratyl coenzyme A (HMGCoA) and reductase inhibitors have been widely prescribed for the treatment of hypercholesterolaemia. These enzymes have numerous pleiotrophic effects, resulting from their ability to block synthesis of isoprenoid intermediates and inhibit prenylation of Rho family GTPases [14,15]. These phenom-
Author for correspondence: Hui-ren Tao, Department of orthopaedics, Xijing Hospital, Fourth Military Medical University, 17th West Changle Road, Xi’an, Shaanxi, 710032, China (fax +86-02984771170, e-mail
[email protected]).
ena are independent of their effects on cholesterol. Statins play a role in reducing CAC as they can influence bone remodelling and apoptosis of macrophages by mechanisms similar to those of bisphosphonates [16,17]. Furthermore, it has been suggested that statins transform atherosclerotic plaque architecture making them less likely to rupture [3]. The objective of our study was to investigate the effects of atorvastatin (AT) on arterial calcification in an aortic vasculature. To this end, we present the first report examining both the in vitro and in vivo effects of AT administration in a rat model of arterial calcification. Materials and Methods Animal model and specimens. This investigation conforms to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals, and was approved by the ethics review board of the Fourth Military Medical University. Eighteen four-week-old male Sprague–Dawley rats (Laboratory Animal Centre of the Fourth Military Medical University) were randomly divided into three groups (n = 6 rats per group): the atorvastatin (AT) group, the calcification group (CA) and the control group (N). CAC was induced by combined treatment with warfarin and vitamin D3 (Sigma, St Louis, MO, USA) as previously described [18]. The CA and AT groups were injected subcutaneously with warfarin (15 mg ⁄ 100 g body weight) twice daily from day 1 to day 4 and vitamin D3 (30 million U ⁄ kg ⁄ day) from day 1 to day 3. Four days before establishment of arterial calcification, the AT group was fed intragastrically with AT at 10 mg ⁄ 100 g body weight ⁄ d (Pfizer Pharmaceutical Co., New York, NY, USA); and the CA and N groups were given intragastrically normal saline. These feedings were continued to day 4. From day 2 before establishment of arterial calcification to day 4, each group was injected subcutaneously with Vitamin K1 at 1.5 mg ⁄ 100 g body weight ⁄ day. Blood samples from rats in each group were drawn from orbital veins before treatment and on day 5; 4 replicate
2010 The Authors Basic & Clinical Pharmacology & Toxicology 2010 Nordic Pharmacological Society
ATORVASTATIN REDUCES ARTERIAL CALCIFICATION biochemical measurements of plasma triglyceride, LDL-CH, HDLCH and calcium were performed for each sample. All rats were killed by cervical dislocation, and abdominal aorta segments (10 mm) between the renal and iliac arteries were removed. Segments were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Staining. Paraffin sections were processed for von Kossa staining. Briefly, dehydrated and dewaxed sections were stained with 5% silver nitrate (AgNO3). Following exposure to sunlight for 20 to 30 min., sections were rinsed and stained with neutral red dye. Cross-sectional images of vasculature showing positive black-stained calcified lumen were analysed with Leica Qwin V3.2 image analysis software. Areas of aortic calcification were calculated as percentages of whole artery sections (n = 3 per sample) and average calcification areas were determined. Aortic vascular smooth muscle cell culture. The intima and adventitia of the rat thoracic aorta were aseptically removed. Tissues were cultured in DMEM (4.5 g ⁄ L glucose, 10 mM pyruvate, 2 mM glutamine; Gibco, Carlsbad, CA, USA) supplemented with antibiotics (100 U ⁄ L penicillin and streptomycin) and foetal calf serum (150 mL ⁄ L; Hangzhou Sijiqing Company, Hangzhou, China) in an incubator (5 mL ⁄ L CO2, 37C). Upon reaching 80% confluence, vascular smooth muscle cells were dislodged by trypsin (2.5 g ⁄ L) and replated. After three to eight passages, the cells were harvested and confirmed to be smooth muscle cells based on their morphology and positive immunostaining for a-SM-Actin. Cells from passages 3–8 were collected for analysis. Treatment of cultured cells. Cultured vascular smooth muscle cells were divided into five groups: untreated controls, CA and 1 lM AT (Beijing Hung Hui Pharmaceutical Ltd, Beijing, China), 5 lM AT and 10 lM AT groups. The cells were plated in 24-well plates at a density of 5 · 104 cells ⁄ well. At 1 day post plating, the AT group cells were cultured with 1, 5 or 10 lM of AT for 24 hr. The CA and AT groups were cultured with 10 mM b-glycerophosphate (Sigma, St Louis, MO, USA), 1 · 10)7 M insulin (Jiangsu Wanbang Biochemical Pharmaceutical Ltd, Xuzhou, China), and 50 lg ⁄ L of vitamin C for 14 days to induce calcification [18]. Alizarin Red S staining of calcium nodules. Calcification was assessed by Alizarin Red S staining in replicate cultures (n = 4 per group). Briefly, three cover slips were put into six wells over which cells migrated. Cover slips were washed twice in cold PBS, fixed (acetone, 0C, 20–30 min.) and stained with 1% Alizarin red S (Sigma, St Louis, MO,USA) (pH 6.3, room temp., 5–10 min.). In light microscopic images, dark red-stained calcium depositions termed as ‘calcium nodules’ were counted using Photoshop image analysis. The percentage of calcium nodules over the entire cover slip area was determined for each group. Measurement of calcium deposition. Cells were washed in PBS twice and incubated in 0.6 M hydrochloric acid at 37C for 24 hr to induce decalcification. Calcium content in the supernatant was measured colorimetrically using a calcium determination kit (Nanjing Jiancheng Institute of Biological Engineering, China). Cells were then washed in PBS (2X) and incubated with 0.1 N NaOH ⁄ 0.1% SDS (Xian Bao Bio Co. Ltd, Xi’an, China) for 20 min. Protein content in cell supernatants was measured colorimetrically in the Bradford assay. Calcium deposition was calculated as the ratio of calcium content to protein content (mmol ⁄ mg protein). Cellular alkaline phosphatase activity. Cells were washed in PBS (3X) and incubated in 1 % TritonX-100 (Hua Mei Biological Co. Ltd, Xi’an, China) ⁄ 0.9% NaCl (20 min.) and centrifuged (1000 rpm, 10 min.). Alkaline phosphatase activity in cell supernatants was measured using an alkaline phosphatase detection kit (Nanjing Jiancheng Institute of Biological Engineering. Nanjing, China). The alkaline phosphatase activity was expressed as King units ⁄ 100 mL.
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Cellular proliferation assay. Cells were cultured in a 96-well culture plate at a density of 5 · 104 ⁄ well. On the second day, cells were exposed to serum-free DMEM for 24 hr. The AT groups were incubated with 1, 5 or 10 lM AT for 24 hr. Following this, 10 mM b-glycerophosphate (Sigma, St Louis, MO, USA), 1 · 10)7 mol ⁄ L insulin and 50 lg ⁄ L vitamin C were added to the CA and AT groups and cells were further incubated for 72 hr. Culture media was removed and cellular proliferation was assessed by MTT assay (Beijing Amoi Biological Co., Ltd, Beijing, China). Briefly cells were exposed to MTT (2.5 g ⁄ L, 20 lL per well) for 4 hr. Upon removal of the supernatant, cells were treated with DMSO (150 ll ⁄ well) and agitated for 10 min. Absorbance (A490 nm) was measured in 12 replicate wells for each group. Statistical analysis. One-way ANOVA was used to compare variance between groups and data are presented as mean€S.D. Post-hoc t-test with Bonferroni adjustments were performed for pair-wise groups separately. Statistical significance was determined at p < 0.05. Bonferroni post-hoc tests were set at significance levels of p < 0.0167 for groups (n = 3) in the first experiment and p < 0.005 for groups (n = 5) in the second experiment. All statistical analyses were performed using the SPSS 15.0 statistics software (SPSS Inc., Chicago, IL, USA).
Results Changes plasma triglyceride, LDL-CH, HDL-CH and calcium concentrations. The changes in the plasma concentrations of triglycerides, LDL-CH, HDL-CH and Ca2+ are summarised in table 1. There was no significant difference in pre-treatment plasma Ca2+ levels between the three groups for any of the four measurements. Post-treatment, plasma Ca2+ levels in the CA and AT groups were significantly higher than pre-treatment levels. Post-treatment plasma Ca2+ levels were significantly different between the three groups (p < 0.001) such that the CA and AT groups were significantly higher versus the nontreated control group, and the Ca2+ levels in the AT group were significantly lower compared to the CA group. There was no difference between post-treatment versus pre-treatment levels among any of the groups for plasma triglyceride, LDL-CH or HDL-CH concentrations. Table 1. Assessment of plasma triglyceride, HDL-CH, LDL-CH and Ca2+ levels.
Triglyceride (mmol ⁄ L) HDL-CH (mmol ⁄ L) LDL-CH (mmol ⁄ L) Ca2+ (mmol ⁄ L)
Before After Before After Before After Before After
Control (n = 6)
CA (n = 6)
1.6 (0.38) 1.63 (0.24) 1.73 (0.31) 1.66 (0.21) 2.43 (0.23) 2.27 (0.53) 1.33 (0.02) 1.24 (0.04)
1.52 (0.55) 1.57 (0.51) 1.62 (0.23) 1.45 (0.38) 2.38 (0.16) 2.33 (0.25) 1.33 (0.03) 1.73 (0.06)†§
AT (n = 6)
p-value
1.55 (0.47) 0.9571 1.62 (0.34) 0.9581 1.53 (0.65) 0.7338 1.58 (0.42) 0.5859 2.11 (0.01) 0.4776 2.2 (0.72) 0.9159 1.35 (0.01) 0.2135 1.58 (0.05)†‡§
