| Backgrounds: Uric acid(UA) is the product of purine metabolism in human. The uricase gene mutation and function inactivation in the process of human evolution lead to the serum uric acid level significantly higher than that of other mammals. A large number of clinical investigations and evidence based medicine practice find that hyperuricemia is an independent risk factor of cardiovascular diseases. Vascular calcification can lead to vascular fragility, increase stiffness and the risk of atherosclerotic plaque and vascular rupture or aneurysm formation. Vascular calcification is the main risk of myocardial infarction, stroke and other cardiovascular events. Whether high UA is engaged in the occurrence and development of artery atherosclerosis by inducing vascular calcification is worth studying. Several studies have suggested a significant role of the phenotypic transition of vascular smooth muscle cells(VSMCs) into osteoblast-like cells in vascular calcification. Numerous studies confirm that uric acid can induce vascular smooth muscle cell proliferation and oxidative stress. Whether UA can induce vascular smooth muscle cells into osteoblast like cells and contribute to vascular calcification is not yet clear and worthy to carrying on further investigation. In the prophase work, the research group found direct evidence of early arteriosclerosis characterized by vascular calcification in the animal model of hyperuricemia for the first time in the world. Based on these findings, this project aims to recover the mechanism why hyperuricemia can lead to vascular calcification.Part 1 Uric acid induced rat primary vascular smooth muscle cells osteoblast differentiationObjective: To explore whether uric acid can induce primary vascular smooth muscle cells osteoblast differentiation in general culture environment.Methods: Isolate and culture rat aortic smooth muscle cells. VSMCs were identified by detecting alpha-smooth muscle actin through immunohistochemistry.(1)Culture the VSMCs for 24 h, 48 h and 72 h in different concentrations of uric acid(0μmol/L, 200μmol/L, 400μmol/L, 800μmol/L). The proliferation of VSMCs was determined by CCK-8 method.(2)Culture VSMCs in different concentrations of uric acid(0 μmol/L, 200μmol/L, 400 μmol/L, 800 μmol/L) for 3 days, 7 days and 14 days. The activity of ALP was determined by ELISA kit and normalized to total protein.(3)The rat aortic smooth muscle cells were seeded in 24 well-plate and cultured indifferent concentrations of uric acid(0 μmol/L, 200 μmol/L, 400 μmol/L, 800μmol/L) for 14 days. A mineralized nodule staining was done by Alizarin red.( 4) The VSMCs were seeded in 6 well-plate and cultured in different concentrations of uric acid(0 μmol/L, 200 μmol/L, 400 μmol/L, 800 μmol/L) for 14 days. Total cellular RNA was extracted. Real-Time PCR was conducted to detect osteoblast markers Runx2, BMP-2, OPN m RNA expressions and smooth muscle phenotype markers of SM22αm RNA expression.( 5) The VSMCs were seeded in 6 well-plate and cultured in different concentrations of uric acid(0 μmol/L, 200 μmol/L, 400 μmol/L, 800 μmol/L) for 14 days. Total cellular protein was extracted and the protein expression of Runx2 was detected by Western Blotting.Results:(1)Vascular smooth muscle cells proliferation was in a concentration and time dependent manner at uric acid concentration of 200 μmol/L and 400 μmol/L. The cell proliferation attenuated at uric acid concentration of 800μmol/L compared to400 μmol/L.(2)ALP activity increased significantly in a time dependent manner at the 7th day and 14 day with uric acid concentration of 800μmol/L. The activity of ALP increased not obviously with the time with uric acid concentration of 200μmol/L and400μmol/L.(3)Alizarin red staining showed no obvious mineralized nodule formation.(4)Osteoblast cells markers Runx2, BMP-2, OPN m RNA expression increased significantly in uric acid concentration of 800μmol/L. The smooth muscle cells phenotype marker SM22α showed no obvious difference in different groups.(5)Runx2 protein expression was significantly increased in uric acid concentration of 800μmol/L, and the increase of Runx2 protein expression was not obvious in other groups.Conclusion: High concentration of uric acid can induce VSMC proliferation. High concentration of uric acid can induce osteoblast cell markers Runx2, BMP-2 and OPN m RNA expression, which suggests that uric acid can induce rat aortic smooth muscle cells osteoblast differentiation.Part 2 Uric acid promotes rat aortic vascular smooth muscle cells osteoblast differentiation and mineralizationObjective: To explore the capacity of uric acid promoting VSMCs osteoblast differentiation and mineralization under the existence of osteogenic inducer.Methods:(1)Culture the VSMCs for 24 h, 48 h and 72 h in different concentrations of uric acid(0μmol/L, 200μmol/L, 400μmol/L, 800μmol/L)+osteogenic inducer. The proliferation of VSMCs was determined by CCK-8 method.(2)Culture VSMCs in different concentrations of uric acid(0 μmol/L, 200μmol/L, 400 μmol/L, 800 μmol/L) +osteogenic inducer for 14 days. The activity of ALP was determined by ELISA kit and normalized to total protein.(3)The rat aortic smooth muscle cells were seeded in 24 well-plate and cultured in different concentrations of uric acid(0 μmol/L, 200 μmol/L, 400 μmol/L, 800μmol/L) +osteogenic inducer for 14 days. The deposition of calcium salt staining was conducted with Von Kossa staining kit.(4)The rat aortic smooth muscle cells were seeded in 24 well-plate and cultured in different concentrations of uric acid(0 μmol/L, 200 μmol/L, 400 μmol/L, 800μmol/L)+osteogenic inducer for 14 days. Mineralized nodules staining by Alizarin red and quantitative analysis of calcium salt were done.( 5) The VSMCs were seeded in 6 well-plate and cultured in different concentrations of uric acid(0 μmol/L, 200 μmol/L, 400 μmol/L, 800μmol/L)+osteogenic inducer for 14 days. Total cellular RNA was extracted.Real-Time PCR was conducted to detect osteoblast markers Runx2, BMP-2, OPN m RNA expressions and smooth muscle phenotype markers of SM22α m RNA expression.( 6) The VSMCs were seeded in 6 well-plate and cultured in different concentrations of uric acid(0 μmol/L, 200 μmol/L, 400 μmol/L, 800μmol/L)+osteogenic inducer for 14 days. Total cellular protein was extracted and the protein expression of Runx2 was detected by Western Blotting.Results:(1)Vascular smooth muscle cells proliferation was in a concentration and time dependent manner at uric acid concentration of 200 μmol/L, 400 μmol/L and 800μmol/L in the presence of osteogenic inducer.(2)The ALP activity in uric acid concentration of 400 μmol/L, 800 μmol/L were significantly increased and showed a concentration dependent manner.(3)Von Kossa staining showed deposition of calcium salt was more in uric acid(200 mol/L, 400 mol/L, 800 mol/L) + osteogenic inducer groups than in the controlgroup in naked eye evaluation.( 4) Alizarin red staining showed orange precipitate in all groups, and UA800μmol/L+ osteogenic inducer group was the most obvious group. The result of quantitative analysis of calcium salt deposition was in a uric acid concentration dependent growth.( 5) Osteoblast cell markers Runx2, BMP-2 and OPN m RNA expression were significantly increased in uric acid concentration of 400μmol/L+osteogenic inducer and 800 μmol/L+ osteogenic inducer in a concentration dependent manner. Smooth muscle cell phenotypic markers of SM22α in 800 μmol/L+osteogenic inducer group decreased significantly compared with the control group.(6)Runx2 protein expression was significantly increased in uric acid concentration of 400μmol/L+osteogenic inducer and 800μmol/L+osteogenic inducer group in a concentration dependent manner.Conclusion: High concentration of uric acid can significantly promote the rat aortic smooth muscle cells osteoblast differentiation and mineralization in the presence of osteogenic inducer.Part 3 Uric acid promotes vascular smooth muscle cells osteoblast differentiation by activation of Wnt/β-catenin pathwayObjective: To explore the mechanism of uric acid promoting rat VSMC cells osteoblast differentiation.Methods:(1)Culture the VSMCs for 24 h and 48 h in different concentrations of uric acid(0μmol/L, 200μmol/L, 400μmol/L, 800μmol/L)+osteogenic inducer. ROS fluorescence probe-DHE staining and DAPI staining of nuclei were done.(2)Experimental groups: uric acid(0μmol/L, 200μmol/L, 400μmol/L,800μmol/L)+osteogenic inducer, UA800μmol/L+osteogenic inducer+Dkk-1,UA800μmol/L+ osteogenic inducer+CHIR98014. Culture the VSMCs for 14 days and ALP activity was detected.(3)Experimental groups: uric acid(0μmol/L, 200μmol/L, 400μmol/L,800μmol/L)+osteogenic inducer, UA800μmol/L+osteogenic inducer+Dkk-1,UA800μmol/L+ osteogenic inducer+CHIR98014. Culture the VSMCs for 14 days in6 well-plate. Total cellular RNA was extracted. Real-Time PCR was conducted to detect osteoblast markers Runx2, BMP-2, OPN m RNA expressions.(4)Experimental groups: uric acid(0μmol/L, 200μmol/L, 400μmol/L,800μmol/L)+osteogenic inducer, UA800μmol/L+osteogenic inducer+Dkk-1,UA800μmol/L+ osteogenic inducer+CHIR98014. Culture the VSMCs for 14 days in6 well-plate. Total cellular protein was extracted. Western blotting was conducted to detect β-catenin, Runx2 protein expressions.Results:(1)DHE staining showed that with the increase of the concentration of uric acid the bright of red fluorescence in VSMCs gradually increased, suggesting that uric acid can induce rat aortic smooth muscle cells ROS production, and the effect was in a concentration and time dependent manner.( 2) ALP activity decreased significantly in the UA800μmol/L+ osteogenic inducer+Dkk-1 group and increased slightly in the UA800μmol/L+ osteogenic inducer+CHIR group.(3)The m RNA expressions of Runx2, BMP-2 and OPN decreased significantly in the UA800μmol/L+ osteogenic inducer+Dkk-1 group.(4)The protein expressions of β-catenin and Runx2 decreased significantly in the UA800μmol/L+ osteogenic inducer+Dkk-1 group and increased significantly in the UA800μmol/L+ osteogenic inducer+CHIR group.Conclusion: Uric acid promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells partly by activating Wnt/β-catenin pathway...
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