| BackgroundOld people and patients suffering from type 2 diabetes( TD2) and chronic renal failure( CRF) diseases have significant cardiovascular morbidity and mortality that is in part due to arterial calcification( AC). AC can reduce the compliance of blood vessel wall, rise the systolic pressure and lower the diastolic pressure, which induces cardiomegaly, heart dilation, and the effective coronary artery perfusion is reduced. At the same time, AC as an indicator is used for evaluating prognosis of patients with TD2 and CRF. So with regard to the development and treatment of cardiovascular disease, preventing arterial calcification has important clinical significance. Thus far, there are still no effective methods for AC.AC is a genetic regulated process which shares many similarities with normal bone formation and metabolism. However, recent evidence has shown that apoptosis of vascular smooth muscle cells( VSMCs) also plays a critical role in elevated phosphate induced VSMCs calcification. Proudfoot et al. also showed that VSMCs develop apoptotic bodies before calcium crystals and apoptosis may be the critical first step in calcification process. Apoptotic bodies and phosphatidylserine can generate a potential Ca-binding site and membrane surface suitable for mineral deposition, which derives from apoptotic cells and serves as a calcification nidus.Vitamin K( VK) is a cofactor for the posttranslational carboxylation of glutamate residues. Recently, it has been shown that VK can inhibit warfarin-induced medial elastocalcinosis in rats by activating matrix Gla protein( MGP). To become biologically active, MGP undergoes a post-translational modification by which specific glutamate residues are transformed into gammacarboxy glutamic acid( Gla)-residues by VK, so MGP is known as vitamin K dependent protein( VKDP). Beyond MGP, growth arrest specific gene 6 protein( Gas 6) also is an important VKDP, which produced by many cell types and generates pleiotropic biological functions by binding to different tyrosine kinase receptors( Tyro3, Axl and Mer). Gas 6/Axl interaction has been shown to be involved in the adjustment of cell survival, movement, chemotaxis and phagocytosis. In particular, it is known to protect a range of cell types from apoptosis. Furthermore, Son et al. have shown that the Pi-induced VSMCs apoptosis and subsequent calcification are dependent on down-regulation of the Gas 6/Axl pathway which inhibits apoptosis and increases survival of VSMCs. Thus, is vitamin K dependent on restoring Gas 6/Axl expression and subsequent calcification reversible or not? In addition, the process of carboxylation differs between the organs and cellular. The liver seems to predominantly use plant-synthesized phylloquinones, vitamin K1( VK1), peripheral carboxylation is more dependent on the menaquinones, a structurally distinct group collectively known as vitamin K2( VK2), and Gas 6 was mainly produced by VSMCs in peripheral tissue. Considering these available data from domestic and overseas, our present study was designed to determine ①whether VK2 has any protective effect on VSMCs calcification. ② whether the effect of VK2 is dependent on restoring the Gas 6/Axl/Akt/Bcl-2 pathway.Objective1. To establish rats VSMCs calcification model, and make clearly that the relationship between VSMCs calcification and apoptosis. 2. To explore whether VK2 can protect VSMCs from calcification and apoptosis, and observe the expression of Gas 6/Axl/p-Akt/Bcl-2 anti-apoptosis pathway in VSMCs calcification and adjunction of VK2. 3. To clarify the status of Gas 6/Axl/p-Akt/Bcl-2 pathway in the process of VSMCs calcification, and the mechanism of VK2 retards rats VSMCs calcification.Methods1. Dissectting two male sprague dawley rats( 80- 100 g), VSMCs were isolated from aortas of them under aseptic conditions and identified in the third passages. Cells were used for the experiments between 4 and 8 passages. 2. The VSMCs were randomly divided into control group and calcification group( Ca2++Pi group), and VSMCs in Ca2++Pi group were incubated in medium containing 7.2 m M Ca Cl2 and 10 m M β-Sodium Glycerophosphate( β-GP), and setting up four different time points, that were Ca2+ + Pi 2 d group, Ca2+ + Pi 4 d group, Ca2+ + Pi 6 d group and Ca2+ + Pi 8 d group. Using Alizarin Red S staining method and o-cresolphthalein complexone method to detect calcium nodules and calcium depositions in each group. The osteogenesis markers alkaline phosphatase( ALP) activity and runt-related transcription factor 2( Runx 2) were detected by ALP activity detection kits and immunofluorescence staining. 3. Using immunofluorescent labeling with TUNEL, VSMCs apoptosis was observed with a laser confocal microscope and calculated the rate of VSMCs apoptosis. 4. Incubating VSMCs with calcification medium and applying apoptosis inhibitor( ZVAD.fmk) at the same time, the effects of cell apoptosis on VSMCs calcification were observed by Alizarin Red S staining method and o-cresolphthalein complexone method, Pearson’s correlation was used in analyzing the relation of cell apoptosis to calcification. 5. The VSMCs were randomly divided into control group, Ca2+ + Pi group and Ca2+ + Pi + VK2 groups. The Ca2+ + Pi + VK2 groups were divided into four groups, that wereCa2+ + Pi + 1 μM VK2 group, Ca2+ + Pi + 5 μM VK2 group, Ca2+ + Pi + 10 μM VK2 group and Ca2+ + Pi + 50 μM VK2 group. Using Alizarin Red S staining and o-cresolphthalein complexone method to detect calcium nodules and calcium depositions in each group. The rate of apoptosis of VSMCs was detected by flow cytometry. 6. Gas 6, Axl, p-Akt and Bcl-2 protein levels were detected by Western blotting. 7. The effects of recombinant mouse Gas 6 protein( rm Gas6) on VSMCs calcification and apoptosis were detected by o-cresolphthalein complexone method and TUNEL staining method. 8. Using Western blotting to screen out the effective concentration of Axl inhibitor( R428). 9. Using R428 to block the Gas 6/Axl pathway, the effects of VK2 on VSMCs calcification and apoptosis were detected by o-cresolphthalein complexone method and TUNEL staining method respectively.Results1. Compared with control group, the calcium nodules and calcium depositions were increased in calcification medium induced VSMCs 6th days( Ca2+ + Pi + 6 d group). And the calcium nodules, calcium depositions, ALP activities and Runx 2 protein level were increased significantly in the Ca2+ + Pi + 8 d group( P < 0.05). 2. The rate of apoptosis was obviously increased by calcification medium in a time-dependent manner. Ca2+ + Pi + 6 d group’s VSMCs apoptotic rate was( 15.98±1.492) %, Ca2+ + Pi + 8 d group’s VSMCs apoptotic rate was( 22.37±2.15) %, they were higher than control group( 6.44±0.89) %,( P < 0.001). 3. Calcium depositions in VSMCs and apoptotic rate were obviously suppressed by caspase inhibitor( ZVAD.fmk) in a dose-dependent manner. 2 μM ZVAD.fmk could reduce calcium depositions and apoptotic rate to( 14.69±2.768) mg/μg and( 3.867±1.55) % respectively. The calcium depositions and apoptotic rate of control group were( 106.2±11.82) mg/μg and( 21.07±3.037) %. There were statistical significance between these two groups( P < 0.05). The result of Pearson’s correlation showed thatthe VSMCs apoptosis is an important impact factor in the process of vascular calcification, R2 = 0.8504, P < 0.001. 4. Calcium depositions in VSMCs and apoptotic rate were obviously suppressed by VK2 in a dose-dependent manner. The calcium depositions and apoptotic rate of Ca2+ + Pi group were( 158±22.62) mg/μg and( 19.87±2.554) %, compare with it, the calcium depositions and apoptotic rate of Ca2+ + Pi + 10 μM VK2 group and Ca2+ + Pi + 50 μM VK2 group were decreased significantly( P < 0.05), that were( 57.33±8.36) mg/μg,( 9±1.21) %,( 53.94±8.742) mg/μg and( 8.1±0.793) %, but there were no statistical significance between Ca2+ + Pi + 10 μM VK2 group and Ca2+ + Pi + 50 μM VK2 group( P > 0.05). 5. The protein expression of Gas 6, Axl, p-Akt and Bcl-2 were shown in grey value ratio, Gas 6, Axl, p-Akt and Bcl-2 expression of control group were respectively( 0.8187±0.096),( 0.679±0.082),( 0.959±0.089) and( 0.867±0.036), and the Ca2+ + Pi group were respectively( 0.301±0.037),( 0.262±0.042),( 0.589±0.092) and( 0.479±0.027). Compare with control group, the protein expression of Gas 6, Axl, p-Akt and Bcl-2 in Ca2+ + Pi group were all reduced( P < 0.05). Gas 6, Axl, p-Akt and Bcl-2 expression of Ca2+ + Pi + 10 μM VK2 group were respectively( 0.816±0.084),( 0.627±0.067),( 1.064±0.11) and( 0.766±0.054). Compare with Ca2+ + Pi group, the protein expression of Gas 6, Axl, p-Akt and Bcl-2 in Ca2+ + Pi +10 μM VK2 group were all increased significantly.( P < 0.05). 6. The calcium depositions and apoptotic rate of Ca2+ + Pi group were( 114.4±11.15) mg/μg and( 17.03±1.01) %, Ca2+ + Pi + 400 n M rm Gas 6 group could reduce them to( 48.18±15.17) mg/μg and( 5±0.66) %, there were statistical significance between these two groups( P < 0.05). 7. Axl inhibitor( 2 μM R428) could block the function of Axl, which inhibited the phosphoralation level of Akt. Compare with the phosphorylated proteins level of Akt in control group( 0.613±0.035), Ca2+ + Pi + 2 μM R428 group could reduce them to( 0.179±0.021), there were statistical significance between these two groups( P < 0.05).8. The calcium depositions and apoptotic rate of Ca2+ + Pi group were( 218.4±18.31) mg/μg and( 28.3±6.86) % respectively. The calcium depositions and apoptotic rate of Ca2+ + Pi + R428 group were( 141.8±27.75) mg/μg and( 17.03±1.01) %, they were obviously reduced compare with Ca2+ + Pi group( P < 0.05). The calcium depositions and apoptotic rate of Ca2+ + Pi + R428 + VK2 group were( 182.3±14.46) mg/μg and( 22.7±2.79) %, there were no statistical significance compare with Ca2+ + Pi group( P > 0.05), Compare with Ca2+ + Pi + R428 group, the calcium depositions and apoptotic rate were reduced in Ca2+ + Pi + R428 + VK2 group, but there were no statistical significance between them( P >0.05).ConclusionThrough our research we find that 7.2 mM CaCl2 and10 mM β-GP could induce rats VSMCs calcification successfully, and clarify the relationship between VSMCs apoptosis and calcification. We find that VK2 could inhibit VSMCs apoptosis and calcification and the Gas 6/Axl/p-Akt/Bcl-2 anti-apoptosis signaling pathway plays an important role in these processes, and illustrate the mechanism of VK2 retarding rats VSMCs calcification. This study shows a new therapeutic target. And it is also an important theory foundation for AC on basis for AC prevention. |
