| Osteoporosis (OP) often increases the risk of bone fracture and other complications, and is a major clinical problem. Previous studies have found that high blood pressure is associated with bone formation abnormalities, resulting in increased calcium loss. Here we investigate the effect of the antihypertensive drug benidipine on bone marrow stromal cell (BMSC) differentiation into osteoblasts, as well as bone formation under osteoporotic conditions. Moreover, we used a combination of in vitro and in vivo approaches to test the hypothesis that benidipine promotes murine BMSC differentiation into osteoblasts. Alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), P-catenin, and low-density lipoprotein receptor-related protein 5 (LRP5) protein expression was evaluated in primary femoral BMSCs from C57/BL6 mice cultured under osteogenic conditions for 2 weeks to examine the effects of benidipine. In addition, an ovariectomized (OVX) mouse model was used to investigate the effect of benidipine treatment for 3 months in vivo. We found that ALP, OCN, and RUNX2 expression was up-regulated and WNT/β-catenin signaling was enhanced in vitro and in vivo. In ovariectomized mice that were intragastrically administered benidipine, bone parameters (trabecular thickness, bone mineral density, and trabecular number) in the distal femoral metaphysis were significantly increased compared with control ovariectomized mice. Consistently, benidipine promoted BMSC differentiation into osteoblasts and protected against bone loss in ovariectomized mice. Therefore, it might be a suitable candidate for the treatment of patients with postmenopausal osteoporosis and hypertension.Background and Objection:Several studies have reported that high blood pressure is associated with bone formation abnormalities, resulting in increased calcium loss, as well as secondary activation of the parathyroid gland and increased calcium removal from bone [28-34]. Similarly, research conducted in hypertensive animal models has shown that hypercalciuria and subsequent hyperparathyroidism reduced growth and decreased total bone mineral content later in life [35,36]. Clinically, approximately 40% of women over the age of 50 will suffer a fracture related to postmenopausal osteoporosis during their lifetime [37]. Recently, it was also reported that higher blood pressure in elderly Caucasian women is associated with increased bone loss at the femoral neck, which may contribute to bone fracture[38].Benidipine (BD) (Fig. la) is a dihydropyridine-type calcium channel blocker that has been widely used in hypertension therapy. Calcium channel blockers primarily inhibit calcium influx through L-type voltage-dependent calcium channels on smooth muscle cell vessels, thereby disrupting the excitation contraction process [39]. Previous studies have demonstrated that BD positively affects bone metabolism[3, 5,19]. However, the mechanism remains unclear, and few studies have examined the effects of antihypertensive drugs on bone function in animal models of postmenopausal osteoporosis.Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells, and are able to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. During the aging process, BMSC differentiation into osteoblasts decreases whereas BMSC differentiation into adipocytes increases, thereby resulting in decreased osteogenesis and bone loss. Nevertheless, osteoblasts play a pivotal role in the regulation of bone formation. During differentiation, osteoblasts express osteocalcin (OCN), alkaline phosphatase (ALP), runt-related transcription factor 2(RUNX2), and other bone matrix proteins, and ultimately undergo mineral deposition. Therefore, BMSCs have a very important role in bone metabolism. Moreover, because BMSCs can differentiate into skeletal cell phenotypes,they are a good tool to study the metabolism of osteoblast differentiation.Osteoblast differentiation is predominantly regulated by WNT/β-catenin signaling (the canonical WNT pathway), which acts as the master regulator of osteogenesis. Canonical WNT signaling also functions in fate determination of mesenchymal stem cells . WNT/β-catenin signaling plays a critical role in bone tissue by controlling the differentiation of stem cells into mature osteoblasts, rather than chondrocytes and adipocytes . In the absence of β-catenin, these cells do not differentiate into mature OCN-expressing osteoblasts.In addition, low-density lipoprotein receptor-related protein 5 (LRP5), a downstream effector of WNT signaling, can promote bone formation in humans and mice. Thus, the WNT/β-catenin signaling pathway is central to osteogenesis and bone formation.In order to understand the mechanism of action of BD during bone formation, it is necessary to further characterize the effect of BD on BMSC function. To examine BMSCs, we used ovariectomized (OVX) C57/BL6 mice as an animal model that mimics postmenopausal osteoporosis in humans to investigate whether BD affects bone density and OCN and RUNX2 expression in vivo.In the present study, we report that BD increased ALP activity in long-term cultures of BMSCs, and augmented OCN and RUNX2 accumulation. In BD-treated OVX mice, trabecular thickness (Tb.Th), bone mineral density (BMD), and trabecular number (Tb.N) were significantly increased and WNT/β-catenin signaling was up-regulated.Methods and materialsBenidipine (BD) preparationA solution of BD (molecular weight,505.5622; Sigma) was prepared by dissolving solid BD in dimethylsulfoxide (DMSO, Sigma) solvent. The stock solution was stored at-20 C. Animals and drug treatmentFemale C57/BL6 mice (n=30), aged 8 weeks and weighing 18-20 g, were purchased from Inner Mongolia Agricultural University (Huhhot, China). Mice were randomly divided into control (CON), CON+BD, sham, OVX, and OVX+BD groups. Mice in the OVX+BD group were intragastrically administered BD (15 mg/kg) per day for 5 days before ovariectomy and were maintained for 3 months after surgery. The CON and CON+BD groups differed from the sham and CON+BD groups. Mice in the control group were treated with vehicle whereas those in the sham groups had some fat tissue around the ovaries removed. Cell CultureBMSCs were isolated from C57BL/6 mice (aged 4 weeks). Briefly, mice femurs were dissected free of surrounding soft tissue. The bone marrow was flushed with a-MEM (Invitrogen, Carlsbad, CA). The marrow content from 4 to 6 bones was plated in culture flasks containing BMSC growth media [a-MEM containing 10% fetal bovine serum (FBS),100 U/mL penicillin,100 mg/mL streptomycin sulfate (Gibco, New Zealand). Non-adherent cells were removed, and adherent BMSCs were cultured and expanded for further experiments. Primary cells were used in the experiments prior to the fourth passage. Cell culture media were replaced every 3 days. Cell proliferation assaysPrimary BMSCs were seeded in 96-well plates at a density of 1×104 cells/well. After 2 days in culture, cells were treated with BD at concentrations of 0.1 μM,1 μM, 10 μM,100 μM, and 1000 μM for 48 h. Cell proliferation assays were performed using Caspase-8 Colorimetric Assay Kits (CCK8; KeyGEN Biotech, China) according to the manufacturer’s instructions. Absorbance was measured at 450 nm. In vitro Q-RT-PCR analysesTotal RNA samples were isolated from BMSCs using Trizol reagent according to the manufacturer’s instruction after 2 weeks osteogenic induction. Then, the total RNA products were immediately transcribed into cDNA using a PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). PCR amplification was performed in a Chromo4 Four-Color Real-Time PCR Detection System (Bio-Rad) using the SYBRR Premix Ex TaqTM II (Tli RNaseH Plus) kit (TaKaRa). Primer sequences for each gene used in this study were shown in the Table1, synthesized by the Life Technologies Company. In vitro osteoblastic differentiationWe induced osteoblastic differentiation using differentiation media (α-MEM supplemented with 10% FBS,50 μM ascorbic acid,0.1 μM dexamethasone, and 10 mM β-glycerol phosphate) after seeded in 6-well plates at a density of1×104 cells/well. Then, we added BD (0.1 μM,1 μM,10 μM,100 μM) to the differentiation media. Media were changed every 3 days and cellular differentiation was assayed 14 days after induction using BCIP/NBT Alkaline Phosphatase Substrate Solution (Sigma; USA). Immunofluorescence analysesCell slides and paraffin-embedded sections were incubated overnight with the rabbit polyclonal OCN antibody (1:50; Santa Cruz). Slides and sections were then incubated with the goat anti-rabbit fluorescein isothiocyanate-conjugated IgG secondary antibody (1:100; Santa Cruz). Controls included substitution of primary antibody with rabbit IgG. Cells and femur histology were imaged using laser-scanning confocal microscopy (FV1000; Olympus). OCN-positive cells were evaluated using Image-Pro Plus software (Media Cybernetics; USA) to quantify cellular fluorescence intensity. Cells with fluorescence intensity≥150% of background were considered positive. Immunostaining analysesCell slides and paraffin-embedded sections were prepared for immunostaining as follows. Slides were individually incubated with RUNX2 (1:100; CST), CD105 (1:50; CST) or CD 106 (1:50; CST) for 1 h at room temperature. Secondary antibody staining was performed for 1 h at room temperature using a biotin-labeled goat anti-rabbit antibody (1:100; Santa Cruz). Immunoreactivity was detected using DAB Horseradish Peroxidase Color Development Kit (Sigma; USA) for 2-5 min. Slides were counterstained with Mayer’s hematoxylin before dehydration and mounting. RUNX2 expression was analyzed by comparing the staining intensities between samples in the presence and absence of primary antibody under the same conditions. Histological analysesFemurs were fixed in buffered aqueous formalin, embedded in paraffin, sectioned at 2 μm, and stained with hematoxylin and eosin (HE). Micro-computed tomography (CT) analysesWe obtained long bones from mice, dissected them free of soft tissue, fixed the bones overnight in 4% paraformaldehyde, and analyzed them using high-resolution micro-CT (μCT 80; Scanco Medical; Briittisellen; Zurich; Switzerland). We set the scanner at a voltage of 89 kV, a current of 112 μA, and 20 μm scan thickness. We established cross-sectional images of the proximal tibiae and femora to perform three-dimensional histomorphometric analyses of the trabecular bone. Our analyses included various bone parameters:Tb.Th, BMD, and Tb.N. Western blot analysesProteins isolated from 6-well plates were subjected to SDS-PAGE and transferred to PVDF membranes. Membranes were probed with rabbit polyclonal antibodies to RUNX2 (1:2000; CST), OCN (1:1000; Santa Cruz), CD105 (1:2000; CST), CD106 (1:2000; CST), GAPDH (1:1000; Santa Cruz), p-catenin (1:1000; CST), LRP5 (1:1000; Santa Cruz) and goat anti-rabbit second antibody (1:1000; Santa Cruz). PVDF membranes were incubated with primary antibodies for 8 hours at 4 C and washed three times with TBST (5 min per wash). The secondary antibody was incubated for 1 hour at room temperature, followed by three washes with TBST (5 min per wash). Bound antibodies were detected using Amersham ECL Plus Western blotting detection reagent according to the manufacturer’s instructions (ECL Plus Kit; GE Healthcare, UK). Statistical analysesData were analyzed using one-way analyses of variance (ANOVA). Homogeneity of variance tests were used to evaluate data homogeneity (IBM SPSS Statistics 21.0 software). If the variances were equal, least-significant difference tests were employed. If the variances were unequal, Dunnett’s tests were used. Results are presented as the mean ±SD. P<0.05 was considered statistically significant.Results:Effect ofBD on cell proliferationAs shown in Fig. lb, we measured the effect of BD on primary murine BMSC proliferation using CCK.8 assays. We found that BD at concentrations of 1-100 μM did not significantly affect cell growth after treatment for 2 days. Effect of BD on BMSC in vitro osteogenesisFirstly, we have supplemented the negative effect of BD alone on BMSC differentiation as Supplementary Fig. la. Besides, BD is not able to significantly promote changes in osteogenic markers such as OCN (Supplementary Fig. 1b) and RUNX2 (Supplementary Fig. 1c) in the absence of differentiation medium for 2 weeks. However, to investigate the consequences of adding BD in vitro further, BMSCs from C57/BL6 mice were cultured 14 days in osteogenic media. The addition of BD increased the expression of ALP (Fig. 1c-c’’’ and d-d’’’) by approximately 10-25%(Fig.1le) in cultured BMSCs. RUNX2 was also up-regulated in western blotting (Fig. 1f, h) and immunocytochemistry experiments (Fig.2b-b’’’ and d). In addition, BD increased the expression of OCN in western blotting (Fig. 1f, g) and immunofluorescence (Fig.2a-a’’’ and c) analyses. Therefore, our data suggest that BD promotes in vitro osteogenesis. Effect of BD on femur bone microstructure morphometrics and histologic appearanceTo evaluate the effect of BD on mice bone formation, we performed μCT analyses on sham (Fig.3b, c and d-f), OVX (Fig.3b’, c’and d-f), and OVX+BD (Fig.3b", c" and d-f) mice. Micro-CT results from long bones of OVX+BD (Fig.3b", c" and d-f) mice demonstrated a clear trend in which Tb.Th (Fig.3d) was increased relative to the OVX group (Fig.3d). Moreover, BMD (Fig.3e) and Tb.N (Fig.3f) in OVX+ BD bones were significantly higher compared to OVX (Fig.3e-f) mice. However, these parameters were not significantly different between the OVX and sham groups (Fig.3e-f). HE staining of femur bone sections (Fig.3a-a") indicated that the number of trabeculae in OVX (Fig.3a’) mice was significantly decreased compared to the OVX+BD (Fig.3a") group. Effect of BD on RUNX2 and OCN expressionAbove all, we found that BD did not have significant effect on endogenous stem cells as results of IHC (Supplementary Fig.2) through the characteristic markers of marrow stromal cells, such as CD105 (Supplementary Fig.2a-a" and c) and CD106 (Supplementary Fig.2b-b" and d). That means endogenous stem cells did not change significantly after BD treatment. In addition, CD105 (Supplementary Fig.2c) and CD 106 (Supplementary Fig.2d) positive cells got even distribution in all groups under microscope in IHC staining and had no significant difference. However, to examine the molecular and cellular changes associated with BD-mediated protection against bone loss in vivo, we examined the epiphyseal growth plate where bone formation occurs on a cartilaginous template [48,49]. RUNX2 (Fig.4a-a", c) and OCN (Fig.4b-b", d) expression were markedly reduced in OVX (Fig.4a’, b’, c and d) mice compared to OVX+BD (Fig.4a", b", c and d) mice. However, there were no statistically significant differences between the sham (Fig.4a, b, c and d) and OVX+ BD (Fig.4a", b", c and d) groups. Therefore, our in vitro and in vivo data indicate that BD-induced osteogenesis positively regulates bone formation. Effect of BD on WNT/fl-catenin signalingTo gain further insight into the function of BD in osteogenic differentiation of cultured BMSCs, we examined the expression of β-catenin and LRP5 in vitro. Our western blotting results showed that β-catenin (Fig.5a, b) and LRP5 (Fig.5a, c) expression was enhanced. In addition, we have investigated the β-catenin mRNA (Fig. 5d) level and GSK-3β mRNA expression (Fig.5e), they both increased in BMSCs after BD treatment. Collectively, these data indicate that WNT/β-catenin signaling is up-regulated in BMSCs in the presence of BD, and suggest that BD cooperates with WNT/β-catenin signaling to regulate ossification.ConclusionIn this study, we performed experiments to examine the effect of BD on anti-osteoporosis and elucidate the molecular targets through which BD exerts its effects. Cells in previous studies were derived from neonatal mouse calvarias [50] or MC3T3-E1 cell lines. However, these cells were not a pure population of osteoblasts, and thus could not fully mimic osteoblast physiological function. Because BD treatment did not decrease bone absorption in cultured neonatal mouse calvarias in vitro, the action of BD may be via augmentation of bone formation by osteoblasts.Marrow stem cells are contained within the bones. Some mesenchymal stem cells (MSCs) develop into osteoblasts and osteocytes. Studies have revealed that the relationship between osteoporosis and osteoblast differentiation in BMSCs occurs concomitantly with decreased BMSC differentiation into osteoblasts in bone marrow in age-related osteoporosis[.In our present research, we utilized the BMSCs to investigate the action of BD on bone metabolism. Cells were cultured in osteogenic conditions, which mimics human osteoporosis.First, we assessed cell viability and did not detect obvious toxicity when BD was used at doses up to 100 μM. However, at 1000 μM, BD showed toxic effects (Figure 1B).Next, in our in vitro experiments, we found that enhanced ALP activity dose dependently induced osteogenesis in BMSCs. To the best of our knowledge, this is the first report to confirm the action of BD on BMSC differentiation into osteoblasts. This mechanism focuses on BMSCs and differs from currently available agents for osteoporosis, which target mature osteoblasts, osteoclasts, or adipocytes [15,55].To explore the signaling pathways involved in BD-induced BMSC osteogenesis, we examined the expression of P-catenin and LRP5, which are key factors in WNT signaling that regulate bone formation [43]. We found that β-catenin and LRP5 expression was enhanced by BD, indicating that BD may promote BMSC osteogenesis by promoting WNT signaling.Finally, to examine the effects of BD in vivo, we designed animal experiments. Micro-CT data showed that bone loss induced by OVX could be significantly rescued by BD treatment. We also examined expression of OCN and RUNX2 in femurs, which are important cytokines during bone remodeling. RUNX2 and OCN expression were up-regulated, suggesting that BD promotes bone formation and growth. Although our findings indicate a protective effect of BD on osteoporotic bone, we did not include a positive control group, such as estrogen treatment. In summary, the present study suggests that BD promotes osteogenesis, which is dependent on increasing RUNX2 and OCN expression and at the tissue and cellular levels, thereby increasing the lineage differentiation of BMSCs toward osteoblasts. Moreover, we suggest that WNT signaling, in part, might be the specific signaling pathway mechanism in this process. Furthermore, appropriate concentrations of BD might positively affect BMSC differentiation into osteoblasts, and thus be a suitable candidate for the treatment of patients with postmenopausal osteoporosis and hypertension. |
PDF Full Text Request