Cbfα1 Expression Research Articles

Liraglutide Alleviates Diabetic Atherosclerosis through Regulating Calcification of Vascular Smooth Muscle Cells.

Background Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia, which can induce the development of atherosclerosis (AS). Calcification of vascular smooth muscle cells (VSMCs) exerts an important role in the process of AS. In this study, the effects of liraglutide (LIRA) on VSMC under high-glucose condition and its mechanism were explored. Method After VSMC was treated by high glucose with or without LIRA in vitro, the alkaline phosphatase (ALP) activity was measured by the detection kit, osteogenic marker protein expression was detected by Western blotting, and calcification was determined by alizarin red staining. Subsequently, the DM rat model was established and the ALP activity, calcification, and osteogenic marker proteins were determined in vivo. Immunohistochemical (IHC) staining and hematoxylin-eosin staining were performed on the thoracic aorta of DM rats. Result The positive rate of SMα-actin expression in the DM + AS group was significantly lower than that in control rats, but LIRA administration increased the positive rate in the model. The expression of Cbfα-1 and OPN in the DM + AS group was significantly higher than that in the control group, while it was decreased after treatment of LIRA. The ALP activity and calcium content were increased in DM + AS rats, and the treatment of LIRA decreased the phenotypes in the rats, so as to delay the progression of AS in DM rats. Meanwhile, LIRA inhibited the ALP activity, upregulated SM-α expression, and downregulated expression of OPN and Cbfα-1 in VSMC under high-glucose (HG) conditions. Mechanically, HG-enhanced ALP activity, AKT, and ERK phosphorylation were inhibited by LIRA, PI3K antagonist LY294002, or ERK1/2 antagonist PD98059, in which cotreatment of LIRA with LY294002 and PD98059 could further enhance the effect of LIRA on VSMC, and GLP-1R antagonists reversed the phenotypes in the model. LIRA blocked the osteogenic transformation of VSMC through PI3K and ERK1/2 signaling pathways, which can be reversed by GLP-1R antagonists. Conclusion LIRA inhibited the abnormalities in VSMC calcification mediated by the GLP-1R, which was related to PI3K/Akt and ERK1/2 MAPK pathways. Therefore, the prospect and significance of LIRA in the treatment of DM complicated with AS were clarified.

Icariin Accelerates Fracture Healing via Activation of the WNT1/β-catenin Osteogenic Signaling Pathway.

Icariin has been shown to enhance bone formation. The present study aimed to investigate whether icariin also promotes bone fracture healing and its mechanisms. First, we isolated and cultured rat bone marrow stromal cells (rBMSCs) with icariincontaining serum at various concentrations (0%, 2.5%, 5% and 10%) and then measured alkaline phosphatase (ALP) activity and the expression of Core-binding factor, alpha 1 (Cbfα1), bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-4 (BMP-4) in the rBMSCs. Second, we established a model of fracture healing in rats and performed gavage treatment for 20 days. Then, we detected bone biochemical markers (ELISA kits) in the serum, fracture healing (digital radiography, DR), and osteocalcin expression (immunohistochemistry). Icariin treatment increased ALP activity and induced the expression of Cbfα1, BMP-2 and BMP-4 in rBMSCs in a dose-dependent manner. In addition, Icariin increased the serum levels of osteocalcin (OC), bone-specific alkaline phosphatase (BAP), N-terminal telopeptides of type I collagen (NTX-1), C-terminal telopeptide of type I collagen (CTX-1) and tartrate-resistant acid phosphatase 5b (TRACP-5b); promoted osteocalcin secretion at the fracture site; and accelerated fracture healing. Icariin can promote the levels of bone-formation markers, accelerate fracture healing, and activate the WNT1/β-catenin osteogenic signaling pathway.

Inhibiting post-translational core fucosylation prevents vascular calcification in the model of uremia

Vascular calcification (VC) is an independent risk factor for cardiovascular disease and mortality in uremia. Post-translational core fucosylation is implicated in a number of pathological processes. First, we investigated the role of core fucosylation and key TGF-β1 pathway receptors in calcified arteries in vivo. To determine whether blocking core fucosylation effectively inhibited VC and TGF-β/Smad signaling pathway, we established an in vitro model of phosphate-induced calcification in rat vascular smooth muscle cells (VSMCs) to assess the role of core fucosylation in VC. Core fucose could be detected at markedly higher levels in calcified VSMCs than control cells. Fut8 (α-1,6 fucosyltransferase), the only enzyme responsible for core fucosylation in humans, was significantly upregulated by high phosphate. Exposed to high phosphate media and blocking core fucosylation in VSMCs by knocking down Fut8 using a siRNA markedly reduced calcium and phosphorus deposition and Cbfα1 expression (osteoblast-specific transcription factor), and increased α-Sma expression (smooth muscle cell marker). Fut8 siRNA significantly inhibited TGF-β/Smad2/3 signaling activation in VSMCs cultured in high phosphate media. In conclusion, this study provides evidence to suggest core fucosylation plays a major role in the process of VC and appropriate blockade of core fucosylation may represent a potential therapeutic strategy for treating VC in end-stage renal disease.

Bushen-Qiangdu-Zhilv decoction inhibits osteogenic differentiation of rat fibroblasts by regulating connexin 43.

Bushen-Qiangdu-Zhilv (BQZ) decoction is a traditional Chinese medicinal compound widely used for treating ankylosing spondylitis (AS). However, the mechanisms underlying effects of BQZ remain largely unknown. Osteoblast differentiation of fibroblasts plays an important role in heterotopic ossification (HO) of AS, and connexin 43 (Cx43) is crucially involved in the osteoblast differentiation of fibroblasts. The aim of the present study was to evaluate the effects of BQZ on the osteogenic differentiation of fibroblasts by regulating Cx43. Rat fibroblasts were treated with freeze-dried powder of BQZ, in the presence or absence of recombinant human bone morphogenetic protein-2 (rhBMP-2). MTS assays were performed to examine the inhibitory effects of BQZ on fibroblast proliferation. Western blot assays were conducted to detect the protein expression of core-binding factor alpha 1 (Cbfα1), Cx43 and phosphorylated Cx43 (pCx43). BQZ appeared to inhibit fibroblast proliferation in a dose-dependent manner. Furthermore, the expression of Cbfα1 and Cx43/pCx43 was significantly suppressed by BQZ, with or without rhBMP-2 stimulation. Therefore, the present results indicate that BQZ may exert an anti-AS effect by suppressing the osteogenic differentiation of fibroblasts via Cx43 regulation.

Magnesium prevents β-glycerophosphate-induced calcification in rat aortic vascular smooth muscle cells.

Vascular calcification (VC), in which high serum phosphate plays a critical role, is one major problem in patients with chronic kidney disease. Clinical studies report that magnesium has a protective effect on VC. However, the studies regarding the impact of high serum magnesium on VC at a cellular level are few and require further investigation. Therefore, the present study explored the effect of magnesium on calcification induced by β-glycerophosphate (BGP) in rat aortic vascular smooth muscle cells (RAVSMCs). In the present study, the addition of magnesium decreased calcium deposition, which was increased by BGP. Higher magnesium levels inhibited BGP-induced alkaline phosphatase (ALP) activity and decreased the expression of core-binding factor α-1 (Cbfα1). In conclusion, higher magnesium levels prevented BGP-induced calcification in RAVSMCs and inhibited the expression of Cbfα1 and ALP. Thus, magnesium is influencing the expression of Cbfα1 and ALP associated with VC and may have the potential to serve as a role for VC in clinical situations.

Magnesium modulates the expression levels of calcification-associated factors to inhibit calcification in a time-dependent manner.

Vascular calcification, a common complication in patients with chronic kidney disease, involves a variety of mechanisms associated with the regulation of calcification-associated factors. Previous clinical studies have indicated that magnesium is involved in the reduction of vascular calcification; however, the mechanism underlying this process remains unknown. The aim of the present study was to investigate the effects of magnesium on β-glycerophosphate (β-GP)-induced calcification and the underlying mechanisms. Primary rat vascular smooth muscle cells (VSMCs) were exposed to 10 mM β-GP in medium with or without the addition of 3 mM magnesium or 2-aminoethoxy-diphenylborate (2-APB; an inhibitor of magnesium transport), for a 14-day period. Calcium deposition and alkaline phosphatase (ALP) activity were measured by Alizarin red staining, quantification of calcium and enzyme-linked immunosorbent assay. The expression levels of core-binding factor α-1 (Cbfα1), matrix Gla protein (MGP) and osteopontin (OPN) were determined by reverse transcription-polymerase chain reaction or western blot analysis, following incubation for 0, 3, 6, 10 and 14 days with the different media. VSMC calcification and ALP activity was reduced significantly in the high-magnesium medium compared with the calcification medium, during the 14-day incubation. The magnesium-induced changes in the VSMCs included a β-GP-induced downregulation of Cbfα1 by day 3 of incubation, an effect that was gradually enhanced over the 14-day period. By contrast, magnesium produced notable increases in MGP and OPN expression levels, with an opposite pattern to that observed in the Cbfα1 expression levels. However, the addition of 2-APB appeared to inhibit the protective effect of magnesium on the VSMCs. Therefore, magnesium was able to effectively reduce β-GP-induced calcification in rat VSMCs by regulating the expression levels of calcification-associated factors in a time-dependent manner.

Activation of the ERK1/2 Signaling Pathway during the Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Substrates Modified with Various Chemical Groups

The current study examined the influence of culture substrates modified with the functional groups –OH, –COOH, –NH2, and –CH3 using SAMs technology, in conjunction with TAAB control, on the osteogenic differentiation of rabbit BMSCs. The CCK-8 assay revealed that BMSCs exhibited substrate-dependent cell viability. The cells plated on –NH2- and –OH-modified substrates were well spread and homogeneous, but those on the –COOH- and –CH3-modified substrates showed more rounded phenotype. The mRNA expression of BMSCs revealed that –NH2-modified substrate promoted the mRNA expression and osteogenic differentiation of the BMSCs. The contribution of ERK1/2 signaling pathway to the osteogenic differentiation of BMSCs cultured on the –NH2-modified substrate was investigated in vitro. The –NH2-modified substrate promoted the expression of integrins; the activation of FAK and ERK1/2. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked ERK1/2 activation in a dose-dependent manner, as revealed for expression of Cbfα-1 and ALP. Blockade of ERK1/2 phosphorylation in BMSCs by PD98059 suppressed osteogenic differentiation on chemical surfaces. These findings indicate a potential role for ERK in the osteogenic differentiation of BMSCs on surfaces modified by specific chemical functional groups, indicating that the microenvironment affects the differentiation of BMSCs. This observation has important implications for bone tissue engineering.

Ethanol increases phosphate‐mediated mineralization and osteoblastic transformation of vascular smooth muscle cells

Vascular calcification is implicated in the pathogenesis of atherosclerosis, diabetes and chronic kidney disease. Human vascular smooth muscle cells (HSMCs) undergo mineralization in response to elevated levels of inorganic phosphate (Pi) in an active and well-regulated process. This process involves increased activity of alkaline phosphatase and increased expression of core binding factor α-1 (CBF-α1), a bone-specific transcription factor, with the subsequent induction of osteocalcin. It has been shown that heavy alcohol consumption is associated with greater calcification in coronary arteries. The goal of our study was to examine whether ethanol alters mineralization of HSMCs provoked by high Pi. Exposure of HSMCs to ethanol increased extracellular matrix calcification in a dose responsive manner, providing a significant additional calcium deposition at concentrations of ≥60 mmol/l. HSMC calcification was accompanied by further enhancement in alkaline phosphatase activity. Ethanol also provoked a significant increase in the synthesis of osteocalcin. Moreover, in cells challenged with ethanol the expression of CBF-α1, a transcription factor involved in the regulation of osteoblastic transformation of HSMCs, was elevated. The observed effects of ethanol were not due to alterations of phosphate uptake by HSMCs. We conclude that ethanol enhances Pi-mediated human vascular smooth muscle calcification and transition of these cells into osteoblast-like cells.

Taurine suppresses osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate disodium, dexamethasone and ascorbic acid via the ERK pathway

Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing β-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.

Taurine inhibits osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate, dexamethasone and ascorbic acid via the ERK pathway

PurposeAortic valve calcification (AVC) has been recognised as an active process characterised by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and has...

Effect of high glucose levels on the calcification of vascular smooth muscle cells by inducing osteoblastic differentiation and intracellular calcium deposition via BMP-2/Cbfα-1 pathway

In this paper, we investigate the effect and the possible mechanism of high glucose levels on the calcification of human aortic smooth muscle cells (HASMCs). HASMCs were divided into four groups: normal glucose group (NG), osmolality control group (OC), high glucose group (HG, HASMCs culture medium containing 30 mmol/L glucose), and high glucose plus recombinant human Noggin protein (bone morphogenetic protein-2 (BMP-2) antagonist) group (HN). The mRNA levels and the protein expressions of BMP-2 and core binding factor alpha-1 (Cbfα-1) were measured by real-time quantitative polymerase chain reaction (PCR) and Western blot. After induced by 10 mmol/L β-glycerol phosphoric acid, cells were harvested for assessments of alkaline phosphatase (ALP) activities at Days 1, 2, and 3, and intracellular calcium contents at Days 7 and 14, respectively. High glucose levels increased the mRNA levels and the protein expressions of BMP-2 and Cbfα-1 (P<0.05). The expression of Cbfα-1 was partially blocked by Noggin protein (P<0.05), while BMP-2 was not (P>0.05). After being induced by β-glycerol phosphoric acid, high glucose levels increased the ALP activity [(48.63±1.03) vs. (41.42±2.28) U/mg protein, Day 3; P<0.05] and the intracellular calcium content [(2.76±0.09) vs. (1.75±0.07) μmol/mg protein, Day 14; P<0.05] in a time-dependent manner when compared with the NG group, while the ALP activity could not be blocked by Noggin protein [(48.63±1.03) vs. (47.37±0.97) U/mg protein, Day 3; P>0.05]. These results show that high glucose levels can evoke the calcification of HASMCs by inducing osteoblastic trans-differentiation and intracellular calcium deposition via the BMP-2/Cbfα-1 pathway, which can be partially blocked by Noggin protein.

Osteogenic differentiation of bone marrow mesenchymal stem cells by adenovirus-mediated expression of leptin

Previous studies demonstrate that leptin has an osteogenic differentiation effect on bone marrow mesenchymal stem cells (MSCs). However, the effect of adenovirus-mediated leptin on MSCs differentiation has not been reported. To explore the mechanism, we constructed a recombinant adenoviral vector Ad-leptin and transfected propagated MSCs in vitro. The effects of Ad-leptin on MSCs growth and osteogenic differentiation were examined. The results showed that Ad-leptin inhibited the transfected MSCs growth significantly, and up-regulated osteocalcin expression and alkaline phosphatase activity. The expression of Cbfα1 and Cbfβ which were the key factors in osteogenic differentiation was also up-regulated. All the findings suggest that genetic engineering of MSCs to express leptin gene may have potential application in the treatment of several genetic diseases and bone reconstruction.