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P-4 Cilostazol inhibits plasminogen activator inhibitor type 1 expression and prevents neointimal hyperplasia
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DIABETES RESEARCH A N D CLINICAL PRACTICE
significant increase in L-NAME induced contraction of aorta precontracted with a submaximal dose of phenylephrine, suggesting increased production of NO in the diabetic rats compared to controls. However, endothelial- dependent relaxation induced by acetylcholine was decreased in diabetic rats compared to controls. There was no change in sodium nitroprusside (SNP)-induced relaxation in diabetic rats compared to controls. Apocynin decreased Tempol- induced relaxation, suggesting inhibition of superoxide production and also increased the LNAME- induced contraction, suggesting improved NO production/availability. Apocynin also improved the endothelial- dependent relaxation to acetylcholine in diabetic rat aorta. However, apocynin had no effect on control rat aorta. Discussion: From the above observations, increased production of O-2 mediated by NAD(P)H oxidase, leading to increased degradation of NO, appears to play a major role in endothelial dysfunction in diabetes. The current evidence tends to suggests that NO function was normal, as evidenced by no change in SNPinduced relaxation and there was no reduction in NO production, whereas the problem appears to lie with enhanced breakdown of NO. This leads to the logical extension that the endothelial dysfunction in diabetes may not be related directly related to NO, but may be more closely linked with excessive generation of superoxide mediated by the enzyme NAD(P)H oxidase. Conclusion: the NAD(P)H oxidase inhibitor, apocynin, inhibits excessive superoxide production and improves the blunted endothelial dependent relaxation. These findings suggest NAD(P)H oxidase mediated oxidative stress has a critical role in endothelial dysfunction associated with diabetes.
P-3 Peroxisome proliferator-activated receptor γ coactivator-1α prevents endothelial apoptosis and dysfunction by increasing adenine nucleotide translocase-1 activity Jong Chul Won 1 , Cheol Young Park 1 , Woo Je Lee 2 , Eun Hee Koh 3 , Min-Seon Kim 3 , Ki-Up Lee 3 , Joong-Yeol Park 3 1 Department of Internal Medicine, Sungkyunkwan University School of Medicine, Kangbuk Samsung Medical Center, Seoul, Republic of Korea, 2 Department of Internal Medicine, Inje University School of Medicine, Seoul Paeik Hospital, Seoul Republic of Korea, 3 Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea Visceral obesity is associated with endothelial dysfunction. The peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α), a transcriptional coactivator playing an important role in energy metabolism, reduces lipid accumulation in cells by increasing fatty acid oxidation (FAO). Here we examined the effect of Ad-PGC-1α in human aortic endothelial cells (HAECs) on apoptosis induced by linoleic acid (LA). PGC-1α increased FAO and reversed LA-induced changes in ∆ψm , adenine nucleotide translocase (ANT) activity, intracellular reactive oxygen species (ROS) and endothelial apoptosis. siRNA against ANT1 partially reversed these effects. In isolated aorta, inhibition of ANT1 also reduced Ad-PGC-1α-induced increases of endothelium-dependent vasorelaxation. These data suggest that PGC-1α functions as a physiologic regulator of ROS generation in endothelial cells. Parts of this effect are mediated by ANT-dependent decreases in ROS generation. Measures to increase PGC-1α expression and ANT in vascular cells may aid the prevention and treatment of atherosclerosis in patients with central obesity.
79 (2008) S1 – S127
P-4 Cilostazol inhibits plasminogen activator inhibitor type 1 expression and prevents neointimal hyperplasia Kyeong-Min Lee 1 , Keun-Gyu Park 2 , Hye-Soon Kim 2 , Ho-Chan Cho 2 , Mi-kyung Kim 2 , Young-Yun Jang 2 , Yong Deuk Kim 1 , Jung-Guk Kim 1 , Bo-wan Kim 1 , Ju-Young Lee 1 , Min-Ho Song 3 , Hueng-Sik Choi 4 , In-kyu Lee 1 1 Department of Internal medicine, and Biochemistry and Cell Biology, Kyungpook National University School of Medicine Daegu, 2 Department of Internal Medicine, Keimyung University School of Medicine, Daegu, 3 Department of Internal medicine, Chungnam National University School of Medicine, Daejeon, 4 Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Kwangju, Republic of Korea Aims: Patients with diabetes mellitus have higher restenosis rates after coronary intervention than nondiabetic patients. The plasma concentration of plasminogen activator inhibitor-1 (PAI1) is increased in these patients. Increased PAI-1 expression in VSMCs promotes neointimal hyperplasia and thrombosis which leads to restenosis after vascular intervention. Cilostazol, a selective type 3 phosphodiesterase inhibitor, is currently used to treat patients with diabetic vascular complications. Previous studies showed that cilostazol reduced the restenosis rate including neointimal formation and in stent thrombosis after vascular intervention. However, the precise mechanism underlying this effect of cilostazol on this process is not fully understood. Here, we examined whether cilostazol inhibits PAI-1 expression in VSMCs and prevents neointima hyperplasia. Methods: The effects of cilostaozol on high glucose, Ang II and TGF-β-stimulated PAI-1 expression in primary cultured VSMCs and vascular injury-induced PAI-1 expression in neointimal area were measured. To investigate the mechanism by which cilostazol inhibits PAI-1 expression, we examined the effects of cilostazol on the TGF-β/Smad3 signaling pathway, as well as AP-1 binding activity. Results: Cilostozol inhibited PAI-1 and TGF-β expression in the neointimal region after vascular injury, as well as high glucose, Ang II and TGF-β-stimulated PAI-1 expression in primary cultured VSMCs. Cilostazol inhibited TGF-β- and Smad3/ALK5stimulated PAI-1 promoter activity. Cilostazol inhibited TGFβ-induced Smad3 phosphorylation and nuclear localization of phosphorylated Smad3, but increased TGF-β-suppression of Smad7. Additionally, cilostazol inhibited high glucose and Ang II-stimulated AP1 activity. Comments/conclusions: Our data showed that cilostazol effectively attenuated PAI-1 expression in the neointimal region and neointimal formation after vascular injury. Cilostazol appears to inhibit PAI-1 expression by multiple mechanisms including downregulation of the TGF-β/Smad3 signaling pathway and AP1 activity. In addition to the antiproliferative effect of cilostazol on VSMCs, this study reveals a molecular mechanism by which cilostazol inhibits PAI-1 gene expression and may help explain how cilostazol exerts its antithrombogenic effects after arterial intervention. This work was supported by the Brain Korea 21 project.
DIABETES RESEARCH A N D CLINICAL PRACTICE
significant increase in L-NAME induced contraction of aorta precontracted with a submaximal dose of phenylephrine, suggesting increased production of NO in the diabetic rats compared to controls. However, endothelial- dependent relaxation induced by acetylcholine was decreased in diabetic rats compared to controls. There was no change in sodium nitroprusside (SNP)-induced relaxation in diabetic rats compared to controls. Apocynin decreased Tempol- induced relaxation, suggesting inhibition of superoxide production and also increased the LNAME- induced contraction, suggesting improved NO production/availability. Apocynin also improved the endothelial- dependent relaxation to acetylcholine in diabetic rat aorta. However, apocynin had no effect on control rat aorta. Discussion: From the above observations, increased production of O-2 mediated by NAD(P)H oxidase, leading to increased degradation of NO, appears to play a major role in endothelial dysfunction in diabetes. The current evidence tends to suggests that NO function was normal, as evidenced by no change in SNPinduced relaxation and there was no reduction in NO production, whereas the problem appears to lie with enhanced breakdown of NO. This leads to the logical extension that the endothelial dysfunction in diabetes may not be related directly related to NO, but may be more closely linked with excessive generation of superoxide mediated by the enzyme NAD(P)H oxidase. Conclusion: the NAD(P)H oxidase inhibitor, apocynin, inhibits excessive superoxide production and improves the blunted endothelial dependent relaxation. These findings suggest NAD(P)H oxidase mediated oxidative stress has a critical role in endothelial dysfunction associated with diabetes.
P-3 Peroxisome proliferator-activated receptor γ coactivator-1α prevents endothelial apoptosis and dysfunction by increasing adenine nucleotide translocase-1 activity Jong Chul Won 1 , Cheol Young Park 1 , Woo Je Lee 2 , Eun Hee Koh 3 , Min-Seon Kim 3 , Ki-Up Lee 3 , Joong-Yeol Park 3 1 Department of Internal Medicine, Sungkyunkwan University School of Medicine, Kangbuk Samsung Medical Center, Seoul, Republic of Korea, 2 Department of Internal Medicine, Inje University School of Medicine, Seoul Paeik Hospital, Seoul Republic of Korea, 3 Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea Visceral obesity is associated with endothelial dysfunction. The peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α), a transcriptional coactivator playing an important role in energy metabolism, reduces lipid accumulation in cells by increasing fatty acid oxidation (FAO). Here we examined the effect of Ad-PGC-1α in human aortic endothelial cells (HAECs) on apoptosis induced by linoleic acid (LA). PGC-1α increased FAO and reversed LA-induced changes in ∆ψm , adenine nucleotide translocase (ANT) activity, intracellular reactive oxygen species (ROS) and endothelial apoptosis. siRNA against ANT1 partially reversed these effects. In isolated aorta, inhibition of ANT1 also reduced Ad-PGC-1α-induced increases of endothelium-dependent vasorelaxation. These data suggest that PGC-1α functions as a physiologic regulator of ROS generation in endothelial cells. Parts of this effect are mediated by ANT-dependent decreases in ROS generation. Measures to increase PGC-1α expression and ANT in vascular cells may aid the prevention and treatment of atherosclerosis in patients with central obesity.
79 (2008) S1 – S127
P-4 Cilostazol inhibits plasminogen activator inhibitor type 1 expression and prevents neointimal hyperplasia Kyeong-Min Lee 1 , Keun-Gyu Park 2 , Hye-Soon Kim 2 , Ho-Chan Cho 2 , Mi-kyung Kim 2 , Young-Yun Jang 2 , Yong Deuk Kim 1 , Jung-Guk Kim 1 , Bo-wan Kim 1 , Ju-Young Lee 1 , Min-Ho Song 3 , Hueng-Sik Choi 4 , In-kyu Lee 1 1 Department of Internal medicine, and Biochemistry and Cell Biology, Kyungpook National University School of Medicine Daegu, 2 Department of Internal Medicine, Keimyung University School of Medicine, Daegu, 3 Department of Internal medicine, Chungnam National University School of Medicine, Daejeon, 4 Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Kwangju, Republic of Korea Aims: Patients with diabetes mellitus have higher restenosis rates after coronary intervention than nondiabetic patients. The plasma concentration of plasminogen activator inhibitor-1 (PAI1) is increased in these patients. Increased PAI-1 expression in VSMCs promotes neointimal hyperplasia and thrombosis which leads to restenosis after vascular intervention. Cilostazol, a selective type 3 phosphodiesterase inhibitor, is currently used to treat patients with diabetic vascular complications. Previous studies showed that cilostazol reduced the restenosis rate including neointimal formation and in stent thrombosis after vascular intervention. However, the precise mechanism underlying this effect of cilostazol on this process is not fully understood. Here, we examined whether cilostazol inhibits PAI-1 expression in VSMCs and prevents neointima hyperplasia. Methods: The effects of cilostaozol on high glucose, Ang II and TGF-β-stimulated PAI-1 expression in primary cultured VSMCs and vascular injury-induced PAI-1 expression in neointimal area were measured. To investigate the mechanism by which cilostazol inhibits PAI-1 expression, we examined the effects of cilostazol on the TGF-β/Smad3 signaling pathway, as well as AP-1 binding activity. Results: Cilostozol inhibited PAI-1 and TGF-β expression in the neointimal region after vascular injury, as well as high glucose, Ang II and TGF-β-stimulated PAI-1 expression in primary cultured VSMCs. Cilostazol inhibited TGF-β- and Smad3/ALK5stimulated PAI-1 promoter activity. Cilostazol inhibited TGFβ-induced Smad3 phosphorylation and nuclear localization of phosphorylated Smad3, but increased TGF-β-suppression of Smad7. Additionally, cilostazol inhibited high glucose and Ang II-stimulated AP1 activity. Comments/conclusions: Our data showed that cilostazol effectively attenuated PAI-1 expression in the neointimal region and neointimal formation after vascular injury. Cilostazol appears to inhibit PAI-1 expression by multiple mechanisms including downregulation of the TGF-β/Smad3 signaling pathway and AP1 activity. In addition to the antiproliferative effect of cilostazol on VSMCs, this study reveals a molecular mechanism by which cilostazol inhibits PAI-1 gene expression and may help explain how cilostazol exerts its antithrombogenic effects after arterial intervention. This work was supported by the Brain Korea 21 project.