Introduction
Cardiovascular disease (CVD) is a major cause of premature mortality, accounting for approximately one-third of mortalities (1,2). Each year, 17 million individuals succumb to CVD (3–5). Osteoporosis is a serious public health problem with an estimated worldwide incidence of >200 million (4,6). CVD and osteoporosis are the main causes of mortality in the elderly (7,8). Vascular calcification, as an independent risk factor for CVD, was originally considered to be a passive and unregulated process; however, this process is now known to be an active and tightly regulated phenomenon, in which a variety of osteogenic regulatory factors are involved (9,10). Clinical studies have demonstrated that the majority of patients with osteoporosis have vascular calcification (11), and that vascular calcification is alleviated following treatment with bisphosphonates (3,12–14). These studies are indicative of a correlation between arterial and bone pathologies (15).
Alendronate, a bisphosphonate, is widely used as a treatment for osteoporosis and other pathological conditions associated with bone loss (16–18). In addition, a number of studies have shown that alendronate is not only suitable for the treatment of the aforementioned diseases, but also for vascular calcification (3). Analysis of the mechanism by which alendronate inhibits vascular calcification is likely to clarify the regulatory mechanisms involved in arterial and bone pathologies.
Notch1 signaling plays a crucial role in cell fate determination and various developmental processes, and translates cell-cell interactions into specific transcriptional programs (19–23). In the transcriptional process, cell-cell interactions cause cleavage of the Notch intracellular domain (NICD), which migrates into the nucleus and associates with RBP-Jκ, further activating the transcription of target genes (22,23). Msx2, the target gene activated by the Notch1-RBP-Jκ signaling pathway, is considered to represent a key regulator of vascular calcification and has been identified as a homeodomain transcription factor responsible for osteoblast differentiation and mineralization (9,22,24,25).
However, to date, a limited number of studies have focused on the effect of alendronate on the expression of the Notch1-RBP-Jκ signaling pathway. In the present study, an in vitro rat model of vascular calcification was used to determine the effect of alendronate on expression of the Notch1-RBP-Jκ signaling pathway, and the possible mechanisms behind the inhibition of artery calcification by alendronate were explored.
Materials and methods
Cell culture
Primary vascular smooth muscle cells (VSMCs) were isolated from the thoracic aortas of Sprague-Dawley rats (4 weeks old; male; Animal Center, Tongji Medical College, Wuhan, China), maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin and incubated in 75-cm2 tissue culture flasks at a density of 1×104 cells/ml. VSMCs at passages 5 and 6 were used for subsequent experiments. The study was approved by the ethics committee of the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Osteoblastic differentiation was induced by culturing cells in osteogenic medium containing 50 mg/ml ascorbate-2-phosphate and 10 mmol/l β-glycerol phosphate. DMEM was used as a control. N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phen- yl-glycinet-butyl ester (DAPT; 10 mmol/l), a potent γ-secretase inhibitor, was used as a positive control for inhibition of the Notch1-RBP-Jκ-dependent signaling pathway (26). VSMCs were also cultured in the presence of various concentrations of alendronate (10−8, 10−6, 10−4 and 10−3 mmol/l). After 14 days, cultured VSMCs were subjected to the following experimental conditions in 7 groups: the control (VSMCs cultured with DMEM); osteogenic (OS; VSMCs cultured with osteogenic medium); and DAPT groups (VSMCs cultured with OS and DAPT); and the alendronate groups (VSMCs cultured with OS and alendronate), which were divided into four subgroups based on varying concentrations of alendronate (10−8, 10−6, 10−4 and 10−3 mmol/l). The medium was replaced with fresh medium every 2 days. For time-course experiments, the first day of culture in calcification medium was defined as day 0.
von Kossa staining
Cells in culture flasks were washed 3 times with PBS, followed by a fixation with 4% paraformaldehyde for 15 min. Next, cells were washed 3 times with distilled water, incubated with 5% silver nitrate solution and exposed to bright sunlight for 30 min, then washed with distilled water for 5 min and treated with 5% sodium thiosulfate for 2 min. Calcium particles were observed by microscope in visual fields (magnification, ×40).
Measurement of calcium content
Cultures were decalcified with 0.6 mol/l HCl for 24 h. The calcium content of the HCl supernatant was determined by the o-cresolphthalein complexone method (calcium kit; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Following decalcification, the cells were washed with PBS and solubilized with 0.1 mol/l NaOH-0.1% SDS. The cells were dissolved in HNO3 and then dried in an oven and redissolved with blank solution (27 nmol/l KCl and 27 μmol/l LaCl3 in deionized water). Calcium content was measured using an atomic absorption spectrophotometer (SpectrAA-240 FS; Agilent Technologies, Santa Clara, CA, USA) at 422.7 nm.
Alkaline phosphatase (ALP) assay
ALP activity of various cells was measured using a Lab Assay ALP kit (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer’s instructions.
Real-time quantitative PCR
Total RNA was isolated from the VSMCs using the TRIzol chloroform method, according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA), and reverse transcribed into cDNA with a Toyoba reverse transcription kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Real-time quantitative PCR was performed using the ABI PRISM 7900 sequence detector system (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. β-actin was used as an endogenous control. The PCR mixture contained SYBR Green/Fluorescein qPCR Master Mix (2X; Thermo Fisher Scientific Inc.), cDNA and the primers. Relative gene expression levels (the amount of target, normalized against an endogenous control gene) were calculated using the comparative Ct method formula, 2−ΔΔCt. The following primer sequences were used for real-time quantitative PCR: rat Notch1 forward, 5′-GAGGCTTGAGATGCTCCCAG-3′ and reverse, 5′-ATTCTTACATGGTGTGCTGAGG-3′; rat RBP-Jκ forward, 5′-GAGCCATTCTCAGAGCCAAC-3′ and reverse, 5′-TCCCCAAGAAACCACAAAAG-3′; rat msx2 forward, 5′-AAGGCAAAAAGACTGCAGGA-3′ and reverse, 5′-GGATGGGAAGCACAGGTCTA-3′; and β-actin forward, 5′-CACGATGGAGGGGCCGGACTCATC-3′ and reverse, 5′-TAAAGACCTCTATGCCAACACAGT-3′
Western blot analysis
Total protein was extracted from cultured VSMCs in radio immunoprecipitation assay buffer containing 50 mmol/l Tris, 150 mmol/l NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Triton X-100 in the presence of aprotinin, PMSF, okadaic acid and leupeptin. Total protein (50 μg/sample) was loaded onto 12% SDS-polyacrylamide gels and separated at 100 V, followed by transfer to PVDF membranes at 200 mA and 4°C for 70 and 110 min for Notch1 and Msx2, and RBP-Jκ signals, respectively. Membranes were blocked in 5% non-fat milk in 0.1 mol/l PBS (pH 7.4) at room temperature for 2 h and then incubated with the following primary antibodies: goat anti-Notch1 polyclonal (1:400), rabbit anti-RBP-Jκ polyclonal (1:300) or goat anti-Msx2 polyclonal (1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following washing, membranes were incubated in HRP-conjugated rabbit anti-goat or goat anti-rabbit secondary antibodies (1:40,000; Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China) for 2 h at room temperature, followed by washing and 5 min incubation with enhanced chemiluminescence reagents. The membranes were stripped and equal protein loading was determined by GAPDH expression using a mouse monoclonal antibody (1:75,000; GoodHere Biological Technology, Ltd., Hangzhou, China).
Statistical analysis
Data are presented as the mean ± SEM. Significant differences were estimated by ANOVA followed by Student-Newman-Keuls multiple comparison tests. P≤0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA).
Results
von Kossa staining for VSMC calcification
To determine the calcification of VSMCs, von Kossa staining was performed and the results showed that no calcification was detected in the control group (Fig. 1A), while a marked positive staining (black) was detected in VSMCs cultured in osteogenic medium (Fig. 1B), indicating increased calcification in VSMCs. Increased calcification was eradicated significantly by DAPT treatment (Fig. 1G). The calcification of VSMCs was markedly reduced in the presence of alendronate in a dose-dependent manner (Fig. 1C-F) compared with those in osteogenic medium only (Fig. 1B), indicating that alendronate and DAPT repressed the calcification of VSMCs induced by osteogenic media.
ALP activity and calcium content of VSMCs
To further determine the calcification of VSMCs, ALP activity and calcium content were analyzed. The results showed that ALP activity (Fig. 2A) and calcium content (Fig. 2B) were significantly increased in VSMCs treated with osteogenic media (P<0.05 vs. control). Again, the osteogenic conversion of VSMCs was repressed by DAPT and reduced in the presence of alendronate in a dose-dependent manner (P<0.01 vs. the osteogenic group). ALP activity and calcium content reached minimum levels at 10−4 mmol/l alendronate (P<0.05 vs. the osteogenic group; Fig. 2).
Alendronate downregulates the Notch1-RBP-Jκ-dependent signaling pathway
mRNA and protein levels of Notch1 (Fig. 3A and B) and RBP-Jκ (Fig. 3C and D) in VSMCs were measured by real-time RT-PCR and western blot analysis. As shown in Fig. 3, Notch1 and RBP-Jκ were significantly increased at the mRNA and protein levels in the osteogenic group compared with controls (P<0.05), indicating that the Notch1-RBP-Jκ signaling pathway was activated in the osteogenic conversion of VSMCs.
To determine the effects of alendronate on expression of the Notch1-RBP-Jκ signaling pathway in vitro, four concentrations (10−8, 10−6, 10−4 and 10−3 mmol/l) of alendronate were added to osteogenic VSMCs and DAPT was added as a positive control for inhibition of the Notch1-RBP-Jκ signaling pathway. The results showed that alendronate reduced the expression of Notch1 and RBP-Jκ compared with the osteogenic media group in a dose-dependent manner, and the expression of Notch1 and RBP-Jκ reached minimum levels at a dose of 10−4 mmol/l (P<0.05 vs. the osteogenic and 10−4 mmol/l alendronate groups).
Alendronate downregulates the expression of Msx2
The expression of Msx2 was measured by real-time RT-PCR and western blot analysis. The mRNA and protein levels of Msx2 were significantly increased in the osteogenic group compared with the control group (P<0.05), indicating that VSMCs were transformed to osteoblast-like cells. Results in the alendronate group showed that the transformation regulated by Msx2 was inhibited in an alendronate dose-dependent manner compared with the osteogenic media group (P<0.05). The expression of Msx2 in the DAPT group, as the negative control, was inhibited, which indicated that inhibition of the Notch1-RBP-Jκ signaling pathway decreased the expression of Msx2 (P<0.05 vs. the osteogenic group; Fig. 4).
Discussion
Osteoporosis and vascular calcification share a number of common risk factors and pathogenetic mechanisms, involving proteins, hormones, elements, lipids and vitamins (27). Previous studies have indicated that bone morphogenetic proteins, the RANKL/RANK/OPG and Wnt signaling pathways, matrix Gla protein, vitamins K and D, osteopontin and the renin-angiotensin-aldosterone system are involved in this process (2,23). In addition, the Notch signaling pathway plays a key role in the development and homeostasis of the cardiovascular system and bone (19,23,29–32).
VSMC fate decisions, including cell growth, migration and apoptosis, are fundamental features in the pathogenesis of vascular disease. The signaling pathways that regulate VSMC growth, death, differentiation and migration are the focus of regulation and control targets, and recent studies have attempted to clarify the pathway for osteoblastic transformation of VSMCs, including the Wnt signaling pathway (28,33–35). The Notch signaling pathway is also important and the Notch1 signaling pathway has been reported to induce the osteogenic differentiation and mineralization of VSMCs (22). The Notch1 signaling pathway promotes the osteogenic differentiation and mineralization of VSMCs by direct activation of Msx2 gene transcription via RBP-Jκ (22,36,37). RBP-Jκ, a major mediator of Notch1 signaling, binds NICDs and forms a complex that further activates the transcription of target genes from their cognate DNA binding sequence in the nucleus (21,22,37).
Msx2, the downstream target gene of the Notch1-RBP-Jκ signaling pathway, is a key regulatory osteogenic factor of vascular calcification. Decreased levels of Msx2 imply that the osteogenic differentiation of VSMCs is inhibited (22,38). In the present study, the expression of Notch1 and RBP-Jκ was increased in osteogenic VSMCs, consistent with the increased intracellular calcium deposition (P<0.05). The osteogenic effects on VSMCs, including increased Msx2, enhanced ALP activity and the deposition of calcium in VSMCs, were eradicated by DAPT, an inhibitor of the Notch1 signaling pathway (26), indicating that the Notch1-RBP-Jκ signaling pathway contributes to the calcification of VSMCs (P<0.05).
In clinical medicine, alendronate has been successfully developed as a treatment for osteoporosis and other pathological conditions associated with bone loss (6,13,29). Clinical studies have shown that alendronate may also be used for treating vascular calcification (3,4,25). In addition, alendronate inhibits the calcification of arteries and valves without affecting serum levels of calcium or phosphate (12). Results of the present study demonstrated that alendronate represses VSMC calcification via downregulation of the Notch1-RBP-Jκ signaling pathway in vitro in a dose-dependent manner. The expression of Msx2, the downstream target of the Notch1-RBP-Jκ signaling pathway, was repressed in a dose-dependent manner. These observations indicate that osteogenic conversion of VSMCs may be repressed by alendronate through downregulation of the Notch1-RBp-Jκ signaling pathway.
In summary, results of this study indicate that VSMCs differentiate into osteoblast-like cells by upregulating the Notch1-RBP-Jκ signaling pathway, and osteogenic transformation leads to vascular calcification. Alendronate was found to inhibit osteogenic transformation regulated by this pathway to protect arteries. Alendronate, a drug that has been found to protect the arteries and bones, is likely to be important for the prevention of CVD and osteoporosis to preserve the quality of life of elderly individuals.