The Role Of Anti-oxidative Transcription Factor Nrf2 In Vascular Calcification And The Underlying Mechanism

Part ? Role of anti-oxidative transcription factor Nrf2 in vascular calcification induced by vitamin D3 and nicotine in ratsObjective:The increased ROS generation and oxidative stress has been recognized as a fundamental cause leading to vascular calcification, while the evidence of cellular antioxidant mechanism in development of vascular calcification is still kept scanty. The objective of this study is to investigate the role of Nrf2 mediated anti-oxidative stress in vascular calcification in vivo.Methods:Twenty male Wistar rats were randomly allocated to Control group and VDN group, the model of VDN induced vascular calcification was established by administration of vitamin D3 (30,0000U/kg) plus nicotine (25mg/kg) at 9 o'clock in the morning, and repeat nicotine once more at 6 o'clock in the afternoon, the thoraco-abdominal aorta was obtained two month later. The occurrence of vascular calcification was detected by vonkossa staining; the calcium content and alkaline phosphatase (ALP) activity were also measured by assay kit; the expression of bone markers including OPN, Runx2, and ? catenin was determined by using Westernblot, the expression of Nrf2 and NQO1 was also simultaneously measured.Results:When compared with Control group, vonkossa staining indicated a strong positive black area among the elastic fibers of the media layer in VDN induced calcified aorta; the calcium content and ALP activity in VDN group were both significantly increased, (p<0.05); the expression of bone markers including OPN, Runx2, and ? catenin were all upregulated (p<0.05); the expression of Nrf2 and its downstream NQO1 were significantly increased.Conclusion:Taken together, the results indicated that Nrf2 and its mediated antioxidative enyzemes NQO1 were increased in calcified aorta in vivo, and Nrf2 may played an important regulatory role in the development of vascular calcification.Part II Inhibitory role of Nrf2 in hyperphosphatemia induced vascular smooth muscle cell calcificationBackground:Oxidative stress, apoptosis, and the osteoblastic differentiation of vascular smooth muscle cells are the main processes that promote the development of vascular calcification. As the key endogenous antioxidant defense transcription factor, nuclear factor E2-related factor 2(Nrf2) regulates a group of antioxidant enzymes and protects cells against oxidative stress and inflammation.In addition, Nrf2 plays a major role in bone metabolism and homeostasis. The aim of this study was to investigate whether the key endogenous antioxidant defense transcription factor, nuclear factor E2-related factor 2 (Nrf2), regulates the calcification of vascular cells and, if so, the underlying mechanisms of this process.Methods:Calcification of primary rat vascular smooth muscle cells (VSMCs) was induced in a hyperphosphatemia medium containing 10 mM ?-glycerophosphate. The expression of bone markers, including ?-catenin, runt-related transcription factor 2(Runx2), bone morphogenetic protein 2(BMP2), and osteopontin (OPN), and apoptosis and oxidative stress were assessed in calcified VSMCs, in cells stimulated by specific the Nrf2 activators, dimethyl fumarate (DMF) and tert-butylhydroquinone (tBHQ), and in cells transfected with Nrf2 siRNA.Results:In this study, the activation of Nrf2 via DMF and tBHQ significantly alleviated VSMC calcification, and significantly decreased the expression of BMP2, Runx2, OPN, and ? catenin. There was also a decrease in the apoptotic marker, cleaved caspase 3, ROS generation, and malondialdehyde content. Inversely, knockdown of Nrf2 significantly promoted osteoblastic differentiation in VSMCs and significantly increased the expression of BMP2, Runx2, osteoprotegerin, and ? catenin.Conclusion:Taken together, these results showed Nrf2's ability to inhibit oxidative stress, apoptosis and osteoblastic differentiation of calcified VSMCs, suggesting a possible new treatment target for vascular calcification.