Background
Vascular calcification is one of the common pathological changes of atherosclerosis and is an independent risk factor for cardiovascular disease. The incidence of vascular calcification increases with aging, smoking, hemodialysis, and diabetes mellitus. However, detailed molecular mechanisms of vascular calcification in diabetes mellitus remain poorly understood. In diabetes mellitus, high glucose promotes
trans-differentiation of vascular smooth muscle cells, which is thought to cause vascular calcification, and hyperinsulinemia and elevated advanced glycosylation end products may play a role in vascular calcification [
1].
Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are drugs that lower cholesterol levels by inhibiting HMG-CoA reductase. Statins are commonly used in patients with atherosclerotic diseases and diabetes mellitus. It is widely recognized that statins have pleiotropic effects unrelated to cholesterol-lowering effects, such as anti-inflammatory and anti-oxidative effects [
2]. In addition to inhibiting cholesterol synthesis, statins also block the synthesis of isoprenoid intermediates such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) [
3]. FPP and GGPP serve as important lipid attachments for the posttranslational modification of a variety of proteins, including small GTPases [
4]. Modification with FPP is necessary for proper localization of Ras family proteins, whereas GGPP is required for Rho, Rab, and Rap family proteins. In vitro studies have demonstrated that statins inhibit Rho by inhibiting GGPP, and thereby suppress its effector Rho-associated protein kinase (ROCK) [
5].
It was reported that statins inhibit in vitro calcification of vascular smooth muscle cells induced by inorganic phosphate [
6], inflammatory mediators [
7], vitamin D
3 and warfarin [
8], and transforming growth factor (TGF)-β [
9]. However, with regard to the inhibitory effects of statins on vascular calcification, conflicting results have been reported between in vitro research and clinical studies. Several clinical studies using electron beam tomography have shown that statins reduce coronary artery calcification [
10‐
12]. However, three subsequent randomized trials using electron beam tomography have shown no inhibitory effects of statins on coronary artery calcification [
13‐
15]. Furthermore, a recent analysis of eight randomized trials using intravascular ultrasonography showed that statins promoted coronary artery calcification [
16].
Bone morphogenetic protein (BMP)-binding endothelial cell precursor-derived regulator (BMPER) is a secretory protein that is known to bind to BMP-2, 4 and 6 [
17]. BMPER can work as either an activator or an inhibitor of BMP signaling, depending on its concentrations and environment [
18]. We have reported that BMPER is a regulator of the osteoblast-like
trans-differentiation of human coronary artery smooth muscle cells (HCASMCs) [
19]. Knockdown of BMPER inhibits, whereas addition of BMPER enhances the osteoblast-like
trans-differentiation of HCASMCs.
Here, we investigated the effects of a statin, ROCK inhibitors and BMPER knockdown on alkaline phosphatase (ALP) mRNA expression and activity in HCASMCs cultured in high glucose-containing media to examine the potentially critical roles of the Rho–ROCK signaling pathway and BMPER in vascular calcification in diabetes mellitus.
Methods
Mice
All animal experiments were approved by the Institutional Animal Care and Use Committee and carried out according to the Kobe University Animal Experimental Regulations (P140605). Streptozotocin (STZ)-induced type I diabetic mice were produced as described previously [
20] with slight modification. Briefly, male C57BL/6J mice (10-week-old) were intraperitoneally injected with STZ in distilled water (200 mg/kg; Wako, Osaka, Japan) or the equal volume of vehicle as a control. Blood samples were taken from mouse lateral tail vein and blood glucose was measured by OneTouch Ultra glucometer (LifeScan, Wayne, PA, USA). Fasting blood glucose was measured at 4 and 8 days after the first STZ injection, and additional STZ injections (250 mg/kg) were given when the fasting blood glucose was less than 250 mg/dl. Rosuvastatin, kindly provided by AstraZeneca, was dissolved in drinking water, which was available ad libitum, and administered with 7.2 mg/kg body weight/day for 14 days. After being anesthetized intraperitoneally with 2.5 % 2,2,2-tribromoethanol (1.6 ml/100 g), the mice were transcardially perfused with physiological saline solution and the aortas were isolated.
Cell culture
Human coronary artery smooth muscle cells (Lonza, Basel, Switzerland) were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (Nacalai, Kyoto, Japan) (glucose; 5.5 mM) supplemented with 15 % fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 IU/ml penicillin and 100 g/ml streptomycin (Nacalai) (normal glucose-containing media). High glucose-containing media (final 25 mM) was made by addition of 19.5 mM glucose to normal glucose-containing media. In like manner, 19.5 mM mannitol (Sigma-Aldrich, St. Louis, MO, USA) was added to normal glucose-containing media as an osmolality control (mannitol-containing media). Cells between passages 8 and 14 were used for the experiments. Unless otherwise noted, cells were cultured for the indicated time periods without changing media. In some experiments, HCASMCs were cultured with rosuvastatin (10 µM), mevalonate (Sigma-Aldrich) (100 µM), FPP (Sigma-Aldrich) (10 µM), GGPP (Sigma-Aldrich) (10 µM), fasudil (Wako) (10 µM) and Y-27632 (Wako) (10 µM). Human umbilical vein endothelial cells (HUVECs) (Lonza) were cultured at 37 °C in the EGM-2 BulletKit (Lonza).
Small interfering RNA (siRNA) experiments
Knockdown of BMPER by siRNAs was performed as described previously [
19]. HCASMCs were transfected with Stealth siRNAs for BMPER (Life Technologies) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. A Stealth siRNA non-silencing negative control (Life Technologies) was used as a control. Forty-eight hours after transfection, HCASMCs were subjected to each experiment.
Real-time polymerase chain reaction (PCR)
Real-time PCR was performed as described previously [
19]. Total mRNAs were extracted from mouse aortas and HCASMCs using TRIzol Reagent (Life Technologies) and subjected to real-time PCR using a 7500 Real-Time PCR System (Life Technologies) with a SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio, Otsu, Japan). The following primers were used: mouse ALP: forward, 5-ACACCTTGACTGTGGTTACTGCTGA-3 and reverse, 5-CCTTGTAGCCAGGCCCGTTA-3; mouse BMPER: forward, 5-ATTACCTGCTGCGTCTTGCT-3 and reverse, 5-TTCTCTCACGCACTGTGTCC-3; mouse GAPDH: forward, 5-GACCCCTTCATTGACCTCAACTAC-3 and reverse, 5-TTTCTTACTCCTTGGAGGCCATGT-3; and human ALP: forward, 5-GGACCATTCCCACGTCTTCAC-3 and reverse, 5-CCTTGTAGCCAGGCCCATTG-3; human BMPER: forward, 5-AGGACAGTGCTGCCCCAAATG-3 and reverse, 5-TACTGACACGTCCCCTGAAAG-3; human glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward, 5-CTGATGCCCCCATGTTCGTC-3 and reverse, 5-CACCCTGTTGCTGTAGCCAAATTC-3. Primer pairs were purchased from Takara Bio. GAPDH was used for standardization, and the comparative threshold method was used to assess the relative abundance of the targets.
ALP staining
Alkaline phosphatase staining was performed essentially as described previously [
19]. HCASMCs cultured in 24-well plates were washed with phosphate-buffered saline, and fixed in 4 % paraformaldehyde for 2 min. The cells were incubated in substrate working solution [100 mg/ml naphthol AS-MX phosphatase (Sigma-Aldrich), 600 mg/ml fast red TR salt (Sigma-Aldrich), 0.5 % (v/v)
N,
N-dimethylformamide, 2 mM MgCl
2 and 0.1 M Tris–HCl pH 8.8] at 37 °C for 20 min. The cells were washed until the intense red color became indicative. The ratio of ALP-positive area was calculated from 10 wells, with at least 500 cells counted per well, using ImageJ software.
Western blotting
Western blotting was performed as described previously [
21]. ROCK activity was measured by myosin phosphatase target subunit 1 (MYPT1) phosphorylation as described previously [
22]. Rabbit anti-phospho-MYPT1 polyclonal antibody (#4563, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-MYPT1 polyclonal antibody (#2634, Cell Signaling Technology) and anti-rabbit immunoglobulin G, horseradish peroxidase-linked whole antibody donkey (GE Healthcare Bioscience, Pittsburgh, PA, USA) were used at 1:1,000. The signals were detected using the Amersham Imager 600 (GE Healthcare Bioscience, Little Chalfont, UK). Densitometric analysis was performed using ImageJ software.
Statistical analysis
All experiments were performed at least three times, and the results are expressed as mean ± standard error of the mean (SEM). Student’s t-test was used when comparing differences between two groups. Differences between more than three groups were analyzed by one-way analysis of variance, followed by Tukey’s or Dunnett’s multiple comparison tests, as appropriate. Values of P < 0.05 were considered significant.
Authors’ contributions
YT, SSK performed the experiments. YT, SSK, KH and YR analyzed the data. YR conceived and designed the experiments. YT, YR wrote the paper. All authors read and approved the final manuscript.
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