Browsing by Subject "atherosclerosis"
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Item Dysregulation of nuclear factor kappa B activity and osteopontin expression in oxidant-induced atherogenesis(Texas A&M University, 2004-09-30) Williams, Edward SpencerNF-?B activity is critical in the regulation of atherosclerotic vascular smooth muscle cell (vSMC) phenotypes induced following oxidative injury by allylamine. The present studies were designed to detail dysregulation of NF-?B activity in these altered phenotypes, and to assess the importance of NF-?B in the regulation of osteopontin, a cytokine which modulates atherosclerosis. Increased degradation of I?B? was observed in allylamine-induced atherosclerotic vSMC phenotypes (henceforth referred to as allylamine cells). Enhanced phosphorylation of I-?-kinases was observed by Western immunoblotting. NF-?B DNA binding activity as assessed by electrophoretic mobility shift assay demonstrated changes in the kinetics and magnitude of induction of binding. Enhancement of NF-?B binding activity was evident in allylamine cells compared to controls when seeded on plastic, fibronectin, and laminin, but not collagen I. Posttranscriptional alterations in Rel protein expression and nuclear localization partly account for changes in NF-?B DNA binding activity. Promoter-specific NF-?B binding profiles suggest altered dimer prevalence as a consequence of the changes in Rel protein expression. The expression of NF-?B regulated genes osteopontin and MMP-2 was enhanced in allylamine-treated aortas, while cyclin D1 and MMP-9 were unchanged. As the importance of osteopontin in atherosclerosis has been described in several models, subsequent studies were designed to assess osteopontin promoter activity. Activity of the osteopontin promoter was significantly reduced in allylamine cells compared to controls as assessed using a luciferase reporter. Deletion analysis suggested the presence of inhibitory cis-acting elements in the regulatory region of the gene. Mutation of these elements, including VDRE, AP-1, NF-?B, and USF1, indicated that NF-?B and USF1 mediate suppression of osteopontin promoter activity in allylamine cells. Decreased serine phosphorylation of immunoprecipitated RelA/p65 was observed in allylamine cells, indicating decreased ability of this protein to transactive gene promoters. NF-?B was found to play a role in suppression of osteopontin promoter activity by collagen I-mediated integrin signaling. These findings suggest that enhancements in NF-?B activity suppress osteopontin promoter activity in oxidant-activated vSMC cultures. Dysregulation of NF-?B activity occurs as a result of altered matrix and intracellular signaling upstream of the nucleus and possibly differential dimer assembly leading to cell-specific profiles of NF-?B-dependent gene regulation.Item Identification and molecular characterization of novel genomic targets in oxidant-induced vascular injury(Texas A&M University, 2007-04-25) Partridge, Charles RandalGene expression was examined in vascular smooth muscle cells to study the complex interaction between oxidative injury and the pathogenesis of vascular disease. Extensive vascular remodeling coupled to increased production of 8-epi-PGF2???? nuclear localization of NF????B, and alterations in glutathione homeostasis were identified as major responses of the vascular wall to oxidative stress. Transcriptional profiling studies, supported by immunohistochemistry and in situ hybridization measurements, identified genes involved in adhesion and extracellular matrix deposition (????1 integrin, collagen), cytoskeletal rearrangements (????-smooth muscle actin, ????-tropomyosin), and signal transduction (NF????B, osteopontin, and LINE) as targets of oxidant injury. In the case of osteopontin (OPN), elevation of OPN levels in vSMCs was shown to be mediated by redox-regulated transcriptional mechanisms. A 200bp region located in the 5' UTR of the osteopontin promoter was found to be responsive to oxidative stress. This regulatory region contained two distinct cis acting elements involved in promoter inducibility. These elements were tentatively identified as NFKB and TIEG-1 binding sites and shown to be highly responsive to hydrogen peroxide and chemical antioxidants. Collectively these studies answer central questions regarding the mechanisms underlying the vascular response to oxidative stress and the involvement of OPN in diseases of the vascular wall.