Inflammatory transcription factors in atherosclerosis
Sammanfattning: Transcription factors such as nuclear factor-kB (NF-kB), activator protein-1 (AP-1) and peroxisome proliferator-activated receptors (PPARs) may play important roles in atherogenesis, by regulating chronic subclinical vascular inflammation. In the first study, we showed that VLDL activates the proinflammatory transcription factor NF-kB in endothelial cells both in vitro and in vivo. Oxidation of VLDL reduced its capacity to activate NF-kB in vitro, whereas free fatty acids such as linoleic and oleic acid activated NF-kB to the same extent as VLDL. Intravenous injection of human VLDL (6 mg protein per kg) into rats resulted in arterial activation of NF-kB as assessed by electrophoretic mobility shift assay. Aortic endothelial cells showed positive nuclear staining for the activated RelA (p65) subunit of NF-kB at 6 to 24 hours after injection. There was also a parallel expression of the adhesion molecules ICAM-1 and VCAM-1, as well as the cytokine TNF-alpha Pretreatment of the rats with diet containing 1% of the antioxidant probucol for 8 weeks did not inhibit arterial activation of NF-kB in response to injection of VLDL. Moreover, injection of triglycerides (10% Intralipid, 5 ml/kg) activated arterial expression of NF-kB to the same extent as VLDL. The upregulation of endothelial VCAM-1 expression by linoleic acid in vitro was shown to be NF-kB dependent (study 2), as assessed by cells tranfected with a nonphosphorylatable IkB construct (IkB-alpha deletion mutant lacking amino acids 1-37). These results suggest that linoleic acid derived from VLDL particles promotes the development of atherosclerotic lesions by activation of the proinflammatory transcription factor NF-kB. VLDL enhances PAI-1 production in cultured endothelial cells, and there is also a close relation between high plasma levels of PAI-1 and hypertriglyceridemia. In the third study, we showed that acute VLDL elevation in the circulation stimulates PAI-1 production in the vascular cells also in vivo. Both endothelial cells and medial smooth muscle cells appeared to be responsible for increased PAI-1 production due to accumulated VLDL particles. Within the rat PAI-1 promoter we identified a sequence (-589 to -571) with 74% homology with the recently described VLDL responsive element in the human PAI-1 promoter (-675 to -657) and adjacent to a 4G motif presumably corresponding to the human 4G/5G polymorphism. Transient transfection studies showed that VLDL exerts its stimulatory effects on rat PAI-1 gene expression in vascular cells by interaction with promoter sequences located within bp –656 and –505. Electrophoretic mobility shift assays showed that VLDL stimulates the binding of unidentified protein(s) to this element. PAI-1 produced by endothelial cells predisposes to thrombosis, the hallmark of acute coronary syndroms. PAI-1 produced by vascular smooth muscle cells (SMCs) inhibits proteolysis by decreasing plasmin-dependent activation of metalloproteinases. The later effect increases matrix accumulation and reduces matrix degradation and inhibits SMC migration from the media into developing atherosclerotic lesions. In the fourth study, oxidized LDL was characterized as a possible candidate responsible for the increased PAI-1 production of SMCs within the plaque. Lysophosphatidylcholine formed during the oxidation of LDL appeared to be the mediating substance, by activating PAI-1 transcription through increased AP-1 activity. Alpha 1-Antitrypsin (AAT), the main physiological inhibitor of proteinases, undergoes conformational changes after inactivation of a target enzyme, yielding a cleaved hydrophobic C-terminal peptide (C-36) which forms amyloid fibrils. In study 5, this C-36 peptide but not native AAT nor related fibrils was found to activate PPAR-alpha, PPAR-gamma, NF-kB and AP-1 in human monocytes. Furthermore, C-36 peptide was detected in carotid atherosclerotic plaques.
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