Vitamin K Attenuates β-glycerophosphate-induced Rat Vascular Smooth Muscle Cells Calcification In Vitro

Objective: Cardiovascular disease(CVD) is a major cause of death in chronic kidney disease(CKD) patients. Vascular calcification has emerged as an independent risk factor for cardiovascular morbidity and mortality. Medial calcification is typically seen in CKD and understanding the molecular mechanisms that prevent medial calcification in patients with CKD is of great importance in limiting vascular calcification and subsequently mortality. Recently, an inverse relationship between vitamin K2 and vascular calcification has been reported. Treatment of animals with CKD with high doses of vitamin K increases vitamin K tissue concentrations, attenuates development of calcification. However, the role of vitamin K2 on the rat vascular smooth muscle cells(VSMCs) in a β-glycerophosphate(β-GP)-induced vascular calcification model or the target genes in the regulation of VSMC differentiation is unclear. The present study aimed to investigate the effects and mechanisms of vitamin K on β-GP-induced VC at the cellular level using primary VSMC through observing its m RNA expression of Cbfα1, Bone morphogenetic protein 2(BMP-2), SMAD1, SMAD7, Axl, B-cell lymphoma/leukemia-2(Bcl-2)and apoptosis.Methods:1 Primary culture to get vascular smooth muscle cells(VSMCs), and identify the cells by morphology and immune method.2 Experimental and Intervention groups: VSMCs were randomly divided into negative control group, the group of high phosphorus, vitamin K2 intervention group(10μmol/L), vitamin K2 intervention group(25μmol/L) and vitamin K2 intervention group(50μmol/L), negative control group using low glucose DMEM medium containing 10% fetal bovine serum, high phosphorus group on the basis of normal medium added 10mmol/L β-glycerol phosphate for high phosphorus meduim. vitamin K2 intervention group were added vitamin K2 in the medium on the basis of high phosphorus, the final concentration of vitamin K2 were 10 μmol/L, 25 μmol/L and 50 μmol/L.3 Calcification assay: the calcification of VSMCs detected by using Alizarin Red stain of calcification and colorimetric method to determine the calcium content.4 By RT-PCR method to study effects of the different concentration of Cbfα1,Bone morphogenetic protein 2(BMP-2),SMAD1,SMAD7,Anexelekto(Axl), B-cell lymphoma/leukemia-2(Bcl-2) m RNA expression levels.5 By Western-blot method to study effects of the different concentration of Cbfα1 protein expression levels.6 Induction and quantification of apoptosis : Apoptosis was quantified by flow cytometry(Beckman Coulter) after staining with propidium iodide(Sigma) and enumerating the hypodiploid apoptotic cells in the sub-G1 fraction.7 Statistical methods: The experimental data were described by mean±standard deviation( sx ±). Statistical analysis of data using SPSS13.0 software for analysis. Differences among multi-groups by one-way analysis of variance(ANOVA) and means of two subgroups comparison in multi-groups by Student-Newman-Keuls(SNK). With P<0.05 for the difference was statistically significant.Results:1 Primary culture of VSMCs: Use organization block adherent method to obtain the original generation of rat VSMCs, usually about 3-4 d a few cells can climb out from stick wall of artery tissue block, for long fusiform, scattered arrangement, the cell number increased significantly and confluent over time, about 6-7 d cells covered most of the bottle bottom, when the number of cells accounts for the 80%-90% of the total bottom area can be represented. After represented cells were round or oval at first, stick a wall after can be gradually extend into spindle, abundant cytoplasm, nuclear lobes ovoid, crisscross overlap multiple cells growth, is a typical "peak to valley" performance. Detection of smooth muscle cells by immunocytochemistry method specific antibodies-alpha smooth muscle actin(α-SMA actin), that the cell cytoplasm strong expression is positive results, immune reaction products was brown.2 The effect of vitamin K2 on β-GP-induced calcification of VSMCs.To determine the effect of vitamin K2 on vascular calcification, Alizarin Red staining was performed in rat VSMCs after treatment with β-GP in the presence or absence of vitamin K2. β-GP-induced calcification of VSMCs compared with the control, and vitamin K2 inhibited the β-GP-induced VSMC calcification. To further dissect whether vitamin K2 is capable of preventing calcium deposition, the calcium contents were measured by the o-cresolphthalein complexone method. The calcium content was much higher in β-GP-treated VSMCs than in control VSMCs(P<0.01). The β-GP-induced calcium content was remarkably reduced in a dose-dependent manner by treatment with Vitamin K2. These results suggest that vitamin K2 may be capable of preventing the calcification of VSMCs.3 The effect of Vitamin K2 on Cbfα1 Expression in β-GP-induced calcification of VSMCs.The calcification process results from osteochondrocytic differentiation of VSMCs. We chose to assess levels of Cbfα1 as osteochondrocytic differentiation marker. As expected, Cbfα1 levels were significantly increased after 3 days in the presence of β-GP(P<0.01). At 50μM vitamin K2 concentrations, Cbfα1 levels fell back to the control level.4 The effect of vitamin K2 on BMP Signaling in β-GP-induced calcification of VSMCsWe examined the potential involvement of BMP signaling pathway in vitro. BMP-2 m RNA expression showed a significant decrease in response to Vitamin K2 treatment. Additional experiments showed that Smad1 and Smad7 which directly participate in the signal transduction of BMP expression also responds to vitamin K2.5 The effect of vitamin K2 on the β-GP-induced apoptosis of rat VSMCs.To corroborate the effect of vitamin K2 on the prevention of VSMC calcification, VSMC apoptosis was analyzed by flow cytometry using PI staining. When VSMCs were treated with β-GP, the apoptotic rate was increased by 5-fold when compared with untreated control cells. The increased rate of apoptosis was significantly suppressed by treatment with vitamin K2. Axl and Bcl-2 m RNAs was examined in β-GP-treated VSMC using RT-PCR. The m RNA expression of Axl was decreased 2-fold in β-GP-treated VSMC as compared to the untreated control(P<0.01), and this effect was prevented by vitamin K2 treatment. However, β-GP and vitamin K2 had no effect on Bcl-2 expression in rat VSMCs. These data suggest that vitamin K2 exerts anti-apoptotic effects on β-GP-induced VSMC apoptosis, at least in part through the regulation of the Axl expression.Conclusion:1 This subject adopts thoracic aorta tissue block adherent method successfully obtained rat VSMCs, built a platform for VSMCs primary culture.2 Vitamin K2 can be capable of preventing β-GP-induced calcification of VSMCs.3 The mechanisms of this inhibition correlated with not only the suppression of the differentiation of VSMCs to an osteochondrocytic phenotype through abrogating activation of BMP pathway, but also its antiapoptotic activity.