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P-2 NAD(P)H oxidase inhibitor improves endothelial dysfunction in streptozotocin induced diabetes in rats
DIABETES RESEARCH A N D CLINICAL PRACTICE
Poster presentations Basic Science P-1 NAD(P)H oxidase inhibitor improves endothelial dysfunction and prevents the development of hypertension in fructose fed rats Banappa S. Unger, Basanagouda M. Patil KLES’s College of Pharmacy, Vidya nagar, Hubli, Karnataka, India Introduction: The insulin resistance (IR) state is a major health problem and is associated with vascular dysfunction. Increased oxidative stress has been paid attention to as it is an important causative factor for vascular dysfunction contributing to hypertension. Over the past decade, it has become evident that elevated levels of ROS play a pivotal role in these pathological changes. It is believed that vascular NAD(P)H oxidases are the predominant sources of ROS under these conditions. Increased production of ROS and NAD(P)H oxidase expression and activity has been demonstrated in fructose fed rats, an animal model of insulin resistance. However, there are no reports on the effect of NAD(P)H oxidase inhibitors on endothelial function and blood pressure in fructose fed rats. Aims and objectives: In the present study we examined the acute in-vitro effects of apocynin, (an NAD(P)H oxidase inhibitor,) on ROS (Superoxide, O-2 ) production, NO availability, endothelialdependent relaxation in isolated aorta from fructose fed rats. We measured the same parameters and also the development of hypertension in order to study the chronic in-vivo effects of apocynin on development of hypertension and ROS (Superoxide, O-2 ) production, NO availability and endothelial dependent relaxation in isolated aorta from fructose fed rats Methods: Male SD rats were fed with a fructose (60%) diet for 8 weeks. Systolic blood pressure was measured weekly by tail-cuff method (Harvard apparatus). At the end of the study the rats were sacrificed and the thoracic aorta was isolated and mounted in a tissue bath containing Krebs-Henseleit buffer (pH 7.4) maintained at 37°C and continuously bubbled with 95%O2 +5%CO2 gas mixture. O-2 production was measured in terms of superoxide dismutase mimetic (Tempol)-induced relaxation of aorta pre-contracted with a submaximal dose of phenylephrine. NO production/availability was measured in terms of nitric oxide synthase inhibitor (L-NAME)- induced contraction of aorta pre-contracted with a submaximal dose of phenylephrine. Acetylcholine- induced endothelial- dependent relaxation was measured isometrically by using a force transducer and data acquisition system (Biopac Systems Inc. CA). For study of the acute effects the experiments were carried out in the presence and or absence of apocynin (1x10-4 M) and for chronic in-vivo effects apocynin was administered in drinking water (1.5 mM). All the experiments were carried out according to CPCSEA guidelines and approved by IAEC. Results: There was a significant increase in Tempol- induced relaxation of aorta precontracted with a submaximal dose of phenylephrine, suggesting that increased O-2 production was increased in fructose fed rats compared to controls. There was a significant decrease in L-NAME- induced contraction of aorta precontracted with a submaximal dose of phenylephrine, suggesting that decreased production/availability of NO was decreased in fructose fed rats compared to controls. Endothelial- dependent relaxation induced by acetylcholine was decreased in fructose fed rats compared to controls. There was no change in sodium nitroprusside (SNP)- induced relaxation in fructose fed rats compared to controls. Acute incubation with Apocynin decreased Tempol- induced relaxation, suggesting inhibition of superoxide production, and increased the L-NAME- induced contraction,
79 (2008) S1 – S127
S55
suggesting improved NO production/availability, and also improved the endothelial- dependent relaxation to acetylcholine in the aorta of fructose fed rats. Chronic in-vivo treatment with apocynin completely prevented the elevation of SBP. Further, aortic O-2 production and production/availability of NO became normal and acetylcholine- induced endothelial- dependent relaxation improved. However, acute incubation or chronic in-vivo treatment with apocynin had no effect on SBP, O-2 production, production/availability of NO and endothelial- dependent relaxation in control rat aorta. Discussion and conclusions: 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 that contributes to hypertension in fructose fed rats. The current evidence tends to suggest that NO function is normal, as evidenced by no changes in SNP- induced relaxation, whereas the problem appears to lie with enhanced breakdown of NO. This leads to the logical extension that the endothelial dysfunction associated within insulin resistance may not be directly related to NO, but may be more closely linked with excessive generation of superoxide mediated by the enzyme, NAD(P)H oxidase. In conclusion, the NAD(P)H oxidase inhibitor, apocynin, inhibits excessive superoxide production and improves the blunted endothelial dependent relaxation and prevents the development of hypertension in fructose fed rats. This suggests NAD(P)H oxidase mediated oxidative stress has a crucial role in endothelial dysfunction contributing to the development of hypertension in the insulin resistance state.
P-2 NAD(P)H oxidase inhibitor improves endothelial dysfunction in streptozotocin induced diabetes in rats Basanagouda M. Patil, Banappa S. Unger KLES’s College of Pharmacy, Vidya nagar, Hubli, Karnataka, India Introduction: It is well established that there is an increased oxidative stress in diabetes. Endothelial dysfunction, characterized by reduced nitric oxide (NO) production/availability and increased production of reactive oxygen species (ROS) are regarded as crucial and early mechanisms leading to vascular disorders. One of the major sources of ROS in the vasculature is NAD(P)H oxidase. Aims and objectives: In the present study we tested the effect of apocynin (an NAD(P)H oxidase inhibitor), on ROS (superoxide, O-2 ) production and NO availability and endothelial- dependent relaxation in isolated aorta from rats with streptozotocin- induced diabetes. Methods: Diabetes was induced in male Wistar rats by injecting streptozotocin (50mg kg-1 i.p). Eight weeks after induction of diabetes the rats were sacrificed and thoracic aorta was isolated and mounted in a tissue bath containing Krebs-Henseleit buffer (pH 7.4) maintained at 37°C and continuously bubbled with 95%O2 +5%CO2 gas mixture. O-2 production was measured in terms of superoxide dismutase mimetic (Tempol)-induced relaxation of aorta precontracted with a submaximal dose of phenylephrine. NO production/availability was measured in terms of nitric oxide synthase inhibitor (L-NAME)- induced contraction of aorta precontracted with a submaximal dose of phenylephrine. Acetylcholine- induced endothelial- dependent relaxation was measured isometrically in the presence or and absence of apocynin (1×10-4M) by using a force transducer and data acquisition system (Biopac Systems Inc. CA). All the experiments were carried out according to CPCSEA guidelines and approved by IAEC. Results: There was a significant increase in Tempol- induced relaxation of aorta precontracted with a submaximal dose of phenylephrine, suggesting increased O-2 production was increased in diabetic rats compared to controls. There was a
S56
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.
Poster presentations Basic Science P-1 NAD(P)H oxidase inhibitor improves endothelial dysfunction and prevents the development of hypertension in fructose fed rats Banappa S. Unger, Basanagouda M. Patil KLES’s College of Pharmacy, Vidya nagar, Hubli, Karnataka, India Introduction: The insulin resistance (IR) state is a major health problem and is associated with vascular dysfunction. Increased oxidative stress has been paid attention to as it is an important causative factor for vascular dysfunction contributing to hypertension. Over the past decade, it has become evident that elevated levels of ROS play a pivotal role in these pathological changes. It is believed that vascular NAD(P)H oxidases are the predominant sources of ROS under these conditions. Increased production of ROS and NAD(P)H oxidase expression and activity has been demonstrated in fructose fed rats, an animal model of insulin resistance. However, there are no reports on the effect of NAD(P)H oxidase inhibitors on endothelial function and blood pressure in fructose fed rats. Aims and objectives: In the present study we examined the acute in-vitro effects of apocynin, (an NAD(P)H oxidase inhibitor,) on ROS (Superoxide, O-2 ) production, NO availability, endothelialdependent relaxation in isolated aorta from fructose fed rats. We measured the same parameters and also the development of hypertension in order to study the chronic in-vivo effects of apocynin on development of hypertension and ROS (Superoxide, O-2 ) production, NO availability and endothelial dependent relaxation in isolated aorta from fructose fed rats Methods: Male SD rats were fed with a fructose (60%) diet for 8 weeks. Systolic blood pressure was measured weekly by tail-cuff method (Harvard apparatus). At the end of the study the rats were sacrificed and the thoracic aorta was isolated and mounted in a tissue bath containing Krebs-Henseleit buffer (pH 7.4) maintained at 37°C and continuously bubbled with 95%O2 +5%CO2 gas mixture. O-2 production was measured in terms of superoxide dismutase mimetic (Tempol)-induced relaxation of aorta pre-contracted with a submaximal dose of phenylephrine. NO production/availability was measured in terms of nitric oxide synthase inhibitor (L-NAME)- induced contraction of aorta pre-contracted with a submaximal dose of phenylephrine. Acetylcholine- induced endothelial- dependent relaxation was measured isometrically by using a force transducer and data acquisition system (Biopac Systems Inc. CA). For study of the acute effects the experiments were carried out in the presence and or absence of apocynin (1x10-4 M) and for chronic in-vivo effects apocynin was administered in drinking water (1.5 mM). All the experiments were carried out according to CPCSEA guidelines and approved by IAEC. Results: There was a significant increase in Tempol- induced relaxation of aorta precontracted with a submaximal dose of phenylephrine, suggesting that increased O-2 production was increased in fructose fed rats compared to controls. There was a significant decrease in L-NAME- induced contraction of aorta precontracted with a submaximal dose of phenylephrine, suggesting that decreased production/availability of NO was decreased in fructose fed rats compared to controls. Endothelial- dependent relaxation induced by acetylcholine was decreased in fructose fed rats compared to controls. There was no change in sodium nitroprusside (SNP)- induced relaxation in fructose fed rats compared to controls. Acute incubation with Apocynin decreased Tempol- induced relaxation, suggesting inhibition of superoxide production, and increased the L-NAME- induced contraction,
79 (2008) S1 – S127
S55
suggesting improved NO production/availability, and also improved the endothelial- dependent relaxation to acetylcholine in the aorta of fructose fed rats. Chronic in-vivo treatment with apocynin completely prevented the elevation of SBP. Further, aortic O-2 production and production/availability of NO became normal and acetylcholine- induced endothelial- dependent relaxation improved. However, acute incubation or chronic in-vivo treatment with apocynin had no effect on SBP, O-2 production, production/availability of NO and endothelial- dependent relaxation in control rat aorta. Discussion and conclusions: 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 that contributes to hypertension in fructose fed rats. The current evidence tends to suggest that NO function is normal, as evidenced by no changes in SNP- induced relaxation, whereas the problem appears to lie with enhanced breakdown of NO. This leads to the logical extension that the endothelial dysfunction associated within insulin resistance may not be directly related to NO, but may be more closely linked with excessive generation of superoxide mediated by the enzyme, NAD(P)H oxidase. In conclusion, the NAD(P)H oxidase inhibitor, apocynin, inhibits excessive superoxide production and improves the blunted endothelial dependent relaxation and prevents the development of hypertension in fructose fed rats. This suggests NAD(P)H oxidase mediated oxidative stress has a crucial role in endothelial dysfunction contributing to the development of hypertension in the insulin resistance state.
P-2 NAD(P)H oxidase inhibitor improves endothelial dysfunction in streptozotocin induced diabetes in rats Basanagouda M. Patil, Banappa S. Unger KLES’s College of Pharmacy, Vidya nagar, Hubli, Karnataka, India Introduction: It is well established that there is an increased oxidative stress in diabetes. Endothelial dysfunction, characterized by reduced nitric oxide (NO) production/availability and increased production of reactive oxygen species (ROS) are regarded as crucial and early mechanisms leading to vascular disorders. One of the major sources of ROS in the vasculature is NAD(P)H oxidase. Aims and objectives: In the present study we tested the effect of apocynin (an NAD(P)H oxidase inhibitor), on ROS (superoxide, O-2 ) production and NO availability and endothelial- dependent relaxation in isolated aorta from rats with streptozotocin- induced diabetes. Methods: Diabetes was induced in male Wistar rats by injecting streptozotocin (50mg kg-1 i.p). Eight weeks after induction of diabetes the rats were sacrificed and thoracic aorta was isolated and mounted in a tissue bath containing Krebs-Henseleit buffer (pH 7.4) maintained at 37°C and continuously bubbled with 95%O2 +5%CO2 gas mixture. O-2 production was measured in terms of superoxide dismutase mimetic (Tempol)-induced relaxation of aorta precontracted with a submaximal dose of phenylephrine. NO production/availability was measured in terms of nitric oxide synthase inhibitor (L-NAME)- induced contraction of aorta precontracted with a submaximal dose of phenylephrine. Acetylcholine- induced endothelial- dependent relaxation was measured isometrically in the presence or and absence of apocynin (1×10-4M) by using a force transducer and data acquisition system (Biopac Systems Inc. CA). All the experiments were carried out according to CPCSEA guidelines and approved by IAEC. Results: There was a significant increase in Tempol- induced relaxation of aorta precontracted with a submaximal dose of phenylephrine, suggesting increased O-2 production was increased in diabetic rats compared to controls. There was a
S56
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.