Involvement of RAGE, NADPH Oxidase, and Ras/Raf-1 Pathway in Glycated LDL-Induced Expression of Heat Shock Factor-1 and Plasminogen Activator Inhibitor-1 in Vascular Endothelial Cells

Atherothrombotic cardiovascular diseases are the predominant causes of mortality of diabetic patients. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor for fibrinolysis, and it is also implicated in inflammation and tissue remodeling. Increased levels of PAI-1 and glycated low-density lipoprotein (glyLDL) were detected in patients with diabetes. Previous studies in our laboratory demonstrated that heat shock factor-1 (HSF1) is involved in glyLDL-induced PAI-1 overproduction in vascular endothelial cells (EC). The present study investigated transmembrane signaling mechanisms involved in glyLDL-induced HSF1 and PAI-1 up-regulation in cultured human vascular EC and streptozotocin-induced diabetic mice. Receptor for advanced glycation end products (RAGE) antibody prevented glyLDL-induced increase in the abundance of PAI-1 in EC. GlyLDL significantly increased the translocation of V-Ha-Ras Harvey rat sarcoma viral oncogene homologue (H-Ras) from cytoplasm to membrane compared with LDL. Farnesyltransferase inhibitor-277 or small interference RNA against H-Ras inhibited glyLDL-induced increases in HSF1 and PAI-1 in EC. Treatment with diphenyleneiodonium, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, blocked glyLDL-induced translocation of H-Ras, elevated abundances of HSF1 and PAI-1 in EC, and increased release of hydrogen peroxide from EC. Small interference RNA for p22(phox) prevented glyLDL-induced expression of NOX2, HSF1, and PAI-1 in EC. GlyLDL significantly increased V-raf-1 murine leukemia viral oncogenehomolog 1 (Raf-1) phosphorylation. Treatment with Raf-1 inhibitor blocked glyLDL-induced increase of PAI-1 mRNA in EC. The levels of RAGE, H-Ras, NOX4, HSF1, and PAI-1 were increased in hearts of streptozotocin-diabetic mice and positively correlated with plasma glucose. The results suggest that RAGE, NOX, and H-Ras/Raf-1 are implicated in the up-regulation of HSF1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. (Endocrinology 151: 4455-4466, 2010)