Painupong A., Suwanprasert K.,Ph.D .

1. Intravascular Oxidative Stress Through Lectin-like Oxidized Low Density Lipoprotein Receptor-1(LOX-1) Induces eNOS Uncoupling Painupong A., SuwanprasertK.,Ph.D. Faculty of Medicine, Thammasat University, Pathumthani, Thailand 12120 Summary: Vascular inflammation caused by oxidative stress was an initial step in atherosclerosis. We hypothesized that eNOS uncoupling was induced by activated LOX-1 through ROS/RNS. In this study, cultured umbilical artery was activated by 40 ng/ml TNF-α and followed by 5 μMVit C as stabilizing BH4. mRNA LOX-1 and eNOS expression were activated by TNF-α through ROS. Lower NO and SOD inactivity indicate less amount of superoxide anion available. An increase in nitrotyrosine, peroxynitrite product, was evident. This findings point out the influences of ROS and RNS overwhelm coupled eNOS and induce uncoupling process. Introduction: During development of atherosclerosis, eNOS uncoupling is proposed as superoxide source resulting endothelial dysfunction and vascular inflammation (1) Tumor necrosis factor-α (TNF-α) promotes free radicals through NADPH oxidase and also activates overexpression of LOX-1 (2) . Recently, direct activation of mildly, moderately and fully oxidized LDL through LOX-1 generates reactive oxygen species (ROS) that is in turn loop and damages to superoxide dismutase (SOD) and eNOS as dose dependent response (3). Moreover, desferosamine (DFO) is not capable of preventing ROS formation indicating an enzymatic source such as endothelial NADPH oxidase (3, 4) plays a critical role rather than transition metal driven ROS path. We conclude that biomolecular damage effect such as SOD is from ROS and somehow, RNS especially peroxynitrite. Hence, in this study, we hypothesize that overwhelmed ROS/RNS generated during vascular inflammation of TNF-α stimulation may induce eNOS uncoupling. Methods: * Cultured Umbilical artery Results: 40 ng/ml TNF-α treatment for 2-24 hours resulted reduction of NO as shown in figure 1. Inducible LOX-1 was significantly upregulated after treated with 40 ng/ml TNF-α for 16 and 24 hours which corresponding with profound decrease in nitric oxide level (fig 2). Nitric oxide was gradually lowered and corresponded with a high level of nitrotyrosine. eNOS expression was upregulated and reached significant level (fig 3). Figure 2.Overexpression of LOX-1 was demonstrated at 16th and 24th hours experiment *, p<0.05 compared with control. #, p<0.05 compared among groups (n=9) RNA extraction cDNA synthesis PCR Figure 3. Expression of eNOS after pretreatment with TNF-α for 16 hours and treated with Vit C for 2-24 hours. *, p<0.05 compared with control. #, p<0.05 compared among groups (n=9) NO measurement Meanwhile, SOD activity was lower than control after treated with TNF-α for 16 hours and profound inactivity even though Vit C administration for 2-24 hours (fig 4). Reduced SOD activity may indicate low superoxide anion remained. It may be possible that peroxynitrite was produced from fast reaction of NO and superoxide. Nitrotyrosine, peroxynitrite product, was elevated after TNF-α treatment for 16 hours and still high after treated with Vit C for 2-24 hours (fig 5). This finding indicates uncoupled eNOS induced by overwhelmed ROS from TNF-α mediated LOX-1. Conclusion: We concluded that oxidative stress caused by ROS through TNF-α stimulated NADPH oxidase is capable of upregulating LOX-1 and peroxynitrite formation from superoxide and nitric oxide. Endothelial dysfunction caused by ROS is observed as low nitric oxide production. Activated LOX-1 through ROS from major source such as NADPH oxidase produces more peroxynitrite as seen in low SOD activity and reduced eNOS expression. Enhanced eNOS activity by Vit C which stabilize BH 4 is still incomplete process. This findings indicate the influence of ROS and RNS overwhelms a coupling of eNOS and eventually induce uncoupling process. Acknowledgements: This work was supported by the National Research University Project of Thailand Office of Higher Education Commission and program Strategic Scholarships for Frontier Research Network for the Ph.D. Program Thai Doctoral degree Nitrotyrosine ELISA assay SOD assay Figure 5. Increased nitrotyrosine concentration after 16 hours TNF-α pretreatment and 2-24 hours Vit C treatment. *, p<0.05 compared with control. #, p<0.05 compared among groups (n=9) Figure 4. SOD activity after 16 hours TNF-α pretreatment and 2-24 hours Vit C treatment. *, p<0.05 compared with control. #, p<0.05 compared among groups (n=9) Figure 1. Reduction of NO after treated with 40 ng/ml of TNF-α at 2-24 hours. *, p<0.05 compared with control. #, p<0.05 compared among groups (n=9) References 1. adamanchi NR., Vendrov A., Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 2005, 25(1), 29-38. 2. Zhang H. et al Role of TNF-α in vascular dysfunction. Clinical Science 2009, 116, 219-230. 3. Chatchanayeunyong R. and Suwanprasert K. Vascular Oxidative Stress through Superoxide Dismutase (SOD) and Intracellular Reactive Oxygen Species activated by ox LDL mediated LOX-1 receptor expression. Thammasat Medical Journal 2009, 9(2), 164-173. 4. Channon KM. Tetrahydrobiopterin: regulator of endothelial nitric oxide synthase in vascular disease. Trends Cardiovasc Med. 2004, 14(8), 323-327.