Reactive oxygen species (ROS) play an important but not yet fully defined role in the expression of inflammatory genes such as monocyte chemoattractant protein (MCP)-1. We used complementary molecular and biochemical approaches to explore the roles of specific ROS and their molecular linkage to inflammatory signaling in endothelial cells. Adenovirus-mediated expression of superoxide dismutase and catalase inhibited TNF-alpha-induced MCP-1 gene expression, suggesting important roles of superoxide (O(2)(-).) and H(2)O(2) in MCP-1 gene activation. In addition, the iron chelator 1,2-dimethyl-3-hydroxypyridin-4-one and the hydroxyl radical scavengers dimethylthiourea and dimethyl sulfoxide inhibited TNF-alpha-induced MCP-1 expression, suggesting important roles of iron and hydroxyl radicals in inflammatory signal activation. In contrast, scavenging of peroxynitrite with 5,10,15,20-tetrakis-(4-sulfonatophenyl)prophyrinato iron (III) chloride had no effect on TNF-alpha-induced MCP-1 expression. Inhibition of NADPH oxidase, the major oxidase responsible for O(2)(-). generation, with diphenylene iodonium suppressed TNF-alpha-induced MCP-1 mRNA accumulation. Rac1 is an upstream signaling molecule for the activation of NADPH oxidase and O(2)(-). generation. Expression of dominant negative N17Rac1 by adenovirus suppressed TNF-alpha-induced MCP-1 mRNA levels and MCP-1 protein secretion. Expression of N17Rac1 inhibited TNF-alpha-induced MCP-1 and NF-kappaB transcriptional activity. These data suggest that ROS such as superoxide and H(2)O(2) derived from Rac1-activated NADPH oxidase mediate TNF-alpha-induced MCP-1 expression in endothelial cells.
. The present study demonstrated that LDL oxidized by copper, iron, or 3-morpholinosydnonimine increased the expression of NADPH oxidase (NOX) 2, PAI-1, and heat shock factor-1 (HSF1) in human umbilical vein EC or coronary artery EC compared with LDL or vehicle. Diphenyleneiodonium, a NOX inhibitor, prevented the increases of the expression of HSF1 and PAI-1 in EC induced by oxidized LDLs. Small-interference RNA (siRNA) for p22 phox , an essential subunit of NOX, prevented oxidized LDL-induced expression of NOX2, HSF1, and PAI-1 in EC. HSF1 siRNA inhibited oxidized LDL-induced expression of PAI-1 and HSF1, but not NOX2, in EC. The binding of HSF1 to PAI-1 promoter and the activity of PAI-1 promoter in EC were enhanced by oxidized LDL. Butylated hydroxytulene, a potent antioxidant, inhibited oxidized LDL-induced release of hydrogen peroxide (H2O2) and the expression of NOX2, HSF1, and PAI-1 in EC. Treatment with H2O2 increased the abundance of NOX2, HSF1, and PAI-1 in EC. The results of the present study indicate that oxidized LDL-induced expression of NOX may lead to the elevated release of reactive oxygen species, the activation of HSF1, and the enhancement of the transcription of PAI-1 gene in cultured vascular EC.
Coronary artery disease is the predominant cause of death in diabetic patients. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of plasminogen activators. Heat shock protein (Hsp) was upregulated in uncontrolled diabetic patients. Our previous studies demonstrated that glycated LDL stimulated the generation of PAI-1 from vascular endothelial cells. The present study examined the effect of glycated LDL on the expression of heat shock factor-1 (HSF1), a physiological transcription factor of Hsp, and the involvement of HSF-1 in glycated LDL-induced production of PAI-1 in cultured human umbilical vein endothelial cells ( T he incidence of diabetes in North America has rapidly increased during the last three decades, and the trend is expected to continue (1). The most common cause of death in diabetic patients is coronary artery disease (CAD). Acute coronary syndrome is often associated with thrombosis at the lesions of atherosclerotic plaques (2,3). Thrombogenesis depends on an imbalance between coagulation and fibrinolysis in local blood circulation. Attenuated fibrinolytic activity has been detected in peripheral circulation of type 1 or type 2 diabetic patients (4,5). Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor for fibrinolysis, which modulates the activity of tissue and urokinase plasminogen activators on the formation of plasmin. PAI-1 is also implicated in inflammation, endothelial dysfunction, and extracellular matrix remodeling (6). An elevated level of PAI-1 in plasma has been considered as a nontraditional risk factor for CAD and a marker of endothelial dysfunction (7).Hyperglycemia and dyslipoproteinemia are two major biochemical markers of diabetes. Elevated LDL is a classical risk factor for atherosclerotic cardiovascular disease. LDL clearance via the LDL receptor is attenuated by glycation (8). Elevated levels of small, dense LDL and glycated LDL were frequently detected in diabetic patients (9 -11). Previous studies in our laboratory demonstrated that glycated LDL increased the production of PAI-1 in cultured venous or arterial endothelial cells. LDL isolated from diabetic patients or glycated LDL modified in vitro enhanced the activity of PAI-1 promoter in endothelial cells (12)(13)(14)(15). Glycated LDL stimulated the generation of reactive oxygen species (ROS) and decreased the abundance of reduced glutathione in endothelial cells (16). The findings imply that glycated LDL may induce oxidative stress in vasculature. Heat shock, mechanical shear, or oxidative stress induces stress responses in cells, which are mediated by heat shock proteins (Hsps). The transcription of Hsp is mediated by heat shock factor (HSF) (17). HSF1 is the most widely distributed form of HSF in human body (18 -20). The activation of HSF1 is detected during embryo growth (21) or in diet-induced atherosclerotic animal models (22). The levels of Hsp-70 were increased in the peripheral circulation of diabetic patients with ketoacidosis (23). Neither the impact of ...
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