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1P-0157 Peroxisome proliferator-activator receptor-γ ligands inhibits TGF-β1-induced fibronectin expression in glomerular mesangial cells
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Monday September 29, 2003: Poster Session Diabetic angiopathy
study was to investigate the effects of Ang II on retinal vascular endothelial cell survival. Methods: To quantitate the antiapoptotic effects of Ang II, TUNEL assay and caspase-3 activity of porcine retinal endothelial cells (PRECs) incubated with serum-free medium for 24h. Western blot analysis for detection of the effects of Ang II on Akt and ERK1/2 phosphorylation and survivin expression was performed. The retinal vascular area of wild type and Ang II type 1a receptor knock out (AT1a KO) mice exposed to 75% O2 from postnatal day 7 (P7) to P12 was evaluated. Results: Ang II dose-dependently inhibited apoptosis and caspase-3 activation of PRECs under a serum-deprived state. 100nM of Ang II significantly inhibited the apoptotic cell rates (50%, p<0.0001) and caspase-3 activity (60%, p<0.05) for 24h. AT1 antagonist and LY294002, PI 3-kinase inhibitor, reversed the Ang II-induced anti-apoptotic effect, Akt and ERK1/2 phosphorylation and caspase-3 inactivation. Ang II induced a significant increase of survivin expression in a time- and dose-dependent manner, with a maximal 7-fold increase after 24-h stimulation (p<0.01). Expression of a dominant-negative form of Akt blocked the Ang II-induced survivin expression, antiapoptotic effect and caspase-3 inactivity. AT1a KO mice showed 50.5% avascular area whereas wild type mice 38.6% (p<0.0001). Conclusions: The results indicate that Ang II can be a survival factor for retinal endothelial cells through AT1 activation and PI 3-kinase-Akt pathway. The finding could be persuasive for the treatment of diabetic retinopathy with ACEI or ARB in inducing apoptosis in angiogenic retinal vascular cells. Conversely, it might call a little caution by its possibility to deteriorate retinal endothelial consistency. 1P-0155
Prognostic significance of flow-mediated dilatation for fatal and nonfatal vascular events in elderly diabetics
Y. Ohike 1 , M. Hashimoto 2 , K. Kozaki 1 , J. Ako 1 , K. Iijima 1 , T. Watanabe 1 , T. Kojima 1 , Y. Ouchi 1 . 1 Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Tokyo; 2 Department of General Internal Medicine, Kobe University School of Medicine, Japan Objective: Diabetics are often affected from macrovascular complications, therefore are subject to vascular events. We previously reported that impaired flow-mediated vasodilatation (FMD) could be an early marker for atherosclerosis and accumulation of coronary risk factors is associated with impaired FMD. In this study, we examined FMD in elderly diabetics and followed vascular events and mortality. Design: Forty-five elderly diabetics (HbA1c<10.0) aged 60 or over were enrolled. We performed ultrasound measurement of FMD in the brachial artery. Thereafter, patients were followed 66.6±18.3 months (mean±SD) for vascular (cardiac, cerebral and peripheral vascular) events. Results: Patients were divided into two groups, %FMD (flow-mediated increase in FMD over the rest) <3.5 (Low FMD, n=19) and %FMD >3.5 (High FMD, n=26). There were no significant differences in age, sex, BP, lipid parameters, and HbA1c between the two groups. Smoker ratio and fasting plasma glucose were higher in the Low FMD group (58% vs 27% and 131.3±34.2 vs 98.6±16.5 mg/dl, respectively). After 66.6±18.3 months, ten Low FMD subjects had vascular events, while eight High FMD subjects had vascular events, reaching statistical significance by the Logrank analysis (p=0.005). Simple regression analysis showed significant difference just in body mass Index (BMI, p=0.048) between subjects affected from vascular events and subjects who were not. Logistic regression analysis showed that %FMD (p=0.039), TC (p=0.038), and BMI (p=0.012) remained significant after adjustment with variables shown above. In addition, four subjects died from cardiovascular or cerebrovascular events in Low FMD group, while none died in High FMD group (p=0.026). Conclusions: Our results indicate that FMD could be a good marker for high-risk diabetics who develop fatal and nonfatal vascular events. 1P-0156
[TRL]), in arterial vessels is increased in rabbit and rodent models of insulin deficient diabetes. In this study we investigated the presence of select extracellular matrix proteins and corresponding retention of cholesterol-rich chylomicron-remnants (CRM’s) in STZ-induced diabetic rodents. Methods: Chylomicron remnants were fluorescently labelled (Cy5) and perfused through the common carotid artery under physiological conditions of pressure and flow rate. Chylomicron-remnant cholesterol associated with carotid arteries was detected and quantified in three dimensions using digital confocal microscopy. Co-localization assessment of CRM’s with selected arterial proteoglycans and advanced glycation endproducts (AGE) were made. Results: Both visual and quantitative data from these studies demonstrate focal and enhanced accumulation of CRM’s in the intima of arterial vessels from diabetic animals compared to controls (p<0.05). Analysis of CRM’s indicated significant co-localization (as measured by Pearson’s co-efficient) with AGEs (0.78±0.13) and with biglycan proteoglycan (0.71±0.18).
Accumulation of intestinal cholesterol-rich remnant lipoproteins within arterial vessels may be exacerbated by advanced glycation endproducts and the co-localization of proteoglycans in diabetes
Conclusions: Our observations confirm that modulation of the arterial extracellular matrix prevalent during insulin deficiency and hyperglycemia may help to explain the exacerbated retention of CRM’s in diabetes. 1P-0157
Peroxisome proliferator-activator receptor-γ ligands inhibit TGF-β1-induced fibronectin expression in glomerular mesangial cells
B. Guo, D. Koya, A. Kashiwagi, M. Haneda. Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Japan The thiazolidinedione (TZD) class of antidiabetic drugs, which are ligands for peroxisome proliferator-activated receptor-γ (PPAR-γ), has been shown to possess potent anti-inflammatory and anti-neoplastic action in various systems. Here, we show in mesangial cells that PPAR-γ agonists inhibit fibronectin (FN) expression induced by transforming growth factor-β1 (TGF-β1), a key cytokine in mediating extracellular matrix (ECM) accumulation in diabetic nephropathy. TGF-β1 stimulated FN mRNA expression, and this enhancement was abrogated by a treatment with pioglitazone. Electrophoretic mobility shift assay (EMSA) identified that pioglitazone inhibited TGF-β1-induced DNA binding activity of activator protein-1 (AP-1). Furthermore, pioglitazone inhibited AP-1 reporter activity but not Smad binding elements (SBEs) reporter activity without affecting TGF-β1-induced activation of mitogen-activated protein kinases (MAPKs) and Smad2. Similarly to pioglitazone, PPAR-γ overexpression also inhibited TGF-β1-induced FN mRNA expression as well as the activation of AP-1. 15d-PGJ2, a natural PPAR-γ ligand, also inhibited TGF-β1-induced FN expression by suppressing AP-1 reporter activity and AP-1 DNA binding activity. Alternatively, 15d-PGJ2 inhibited the MAPKs activation induced by TGF-β1. The overexpression of dominant negative PPAR-γ completely abrogated the inhibitory effect of pioglitazone, while partial prevention was observed in the inhibitory effect of 15d-PGJ2 on TGF-β1-induced AP-1 reporter activity. In conclusion, pioglitazone inhibits TGF-β1-induced FN expression by inhibiting AP-1 activation dependent of PPAR-γ activation, while 15d-PGJ2 acts through a dual mechanism independent and dependent of PPAR-γ activation in mouse mesangial cells.
S. Proctor 1 , J.M. Forbes 2 , J.C.L. Mamo 1 . 1 Dept. Nutrition, Curtin University, Perth; 2 Baker Heart Research Institute, Melbourne, Australia Objectives: The mechanism(s) underlying the accelerated progression of atherosclerosis, particularly in type-1 diabetes remains unclear. We have demonstrated previously that the accumulation of lipoproteins rich in cholesterol (such as LDL and remnants of intestinal triglyceride-rich lipoproteins XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
Monday September 29, 2003: Poster Session Diabetic angiopathy
study was to investigate the effects of Ang II on retinal vascular endothelial cell survival. Methods: To quantitate the antiapoptotic effects of Ang II, TUNEL assay and caspase-3 activity of porcine retinal endothelial cells (PRECs) incubated with serum-free medium for 24h. Western blot analysis for detection of the effects of Ang II on Akt and ERK1/2 phosphorylation and survivin expression was performed. The retinal vascular area of wild type and Ang II type 1a receptor knock out (AT1a KO) mice exposed to 75% O2 from postnatal day 7 (P7) to P12 was evaluated. Results: Ang II dose-dependently inhibited apoptosis and caspase-3 activation of PRECs under a serum-deprived state. 100nM of Ang II significantly inhibited the apoptotic cell rates (50%, p<0.0001) and caspase-3 activity (60%, p<0.05) for 24h. AT1 antagonist and LY294002, PI 3-kinase inhibitor, reversed the Ang II-induced anti-apoptotic effect, Akt and ERK1/2 phosphorylation and caspase-3 inactivation. Ang II induced a significant increase of survivin expression in a time- and dose-dependent manner, with a maximal 7-fold increase after 24-h stimulation (p<0.01). Expression of a dominant-negative form of Akt blocked the Ang II-induced survivin expression, antiapoptotic effect and caspase-3 inactivity. AT1a KO mice showed 50.5% avascular area whereas wild type mice 38.6% (p<0.0001). Conclusions: The results indicate that Ang II can be a survival factor for retinal endothelial cells through AT1 activation and PI 3-kinase-Akt pathway. The finding could be persuasive for the treatment of diabetic retinopathy with ACEI or ARB in inducing apoptosis in angiogenic retinal vascular cells. Conversely, it might call a little caution by its possibility to deteriorate retinal endothelial consistency. 1P-0155
Prognostic significance of flow-mediated dilatation for fatal and nonfatal vascular events in elderly diabetics
Y. Ohike 1 , M. Hashimoto 2 , K. Kozaki 1 , J. Ako 1 , K. Iijima 1 , T. Watanabe 1 , T. Kojima 1 , Y. Ouchi 1 . 1 Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Tokyo; 2 Department of General Internal Medicine, Kobe University School of Medicine, Japan Objective: Diabetics are often affected from macrovascular complications, therefore are subject to vascular events. We previously reported that impaired flow-mediated vasodilatation (FMD) could be an early marker for atherosclerosis and accumulation of coronary risk factors is associated with impaired FMD. In this study, we examined FMD in elderly diabetics and followed vascular events and mortality. Design: Forty-five elderly diabetics (HbA1c<10.0) aged 60 or over were enrolled. We performed ultrasound measurement of FMD in the brachial artery. Thereafter, patients were followed 66.6±18.3 months (mean±SD) for vascular (cardiac, cerebral and peripheral vascular) events. Results: Patients were divided into two groups, %FMD (flow-mediated increase in FMD over the rest) <3.5 (Low FMD, n=19) and %FMD >3.5 (High FMD, n=26). There were no significant differences in age, sex, BP, lipid parameters, and HbA1c between the two groups. Smoker ratio and fasting plasma glucose were higher in the Low FMD group (58% vs 27% and 131.3±34.2 vs 98.6±16.5 mg/dl, respectively). After 66.6±18.3 months, ten Low FMD subjects had vascular events, while eight High FMD subjects had vascular events, reaching statistical significance by the Logrank analysis (p=0.005). Simple regression analysis showed significant difference just in body mass Index (BMI, p=0.048) between subjects affected from vascular events and subjects who were not. Logistic regression analysis showed that %FMD (p=0.039), TC (p=0.038), and BMI (p=0.012) remained significant after adjustment with variables shown above. In addition, four subjects died from cardiovascular or cerebrovascular events in Low FMD group, while none died in High FMD group (p=0.026). Conclusions: Our results indicate that FMD could be a good marker for high-risk diabetics who develop fatal and nonfatal vascular events. 1P-0156
[TRL]), in arterial vessels is increased in rabbit and rodent models of insulin deficient diabetes. In this study we investigated the presence of select extracellular matrix proteins and corresponding retention of cholesterol-rich chylomicron-remnants (CRM’s) in STZ-induced diabetic rodents. Methods: Chylomicron remnants were fluorescently labelled (Cy5) and perfused through the common carotid artery under physiological conditions of pressure and flow rate. Chylomicron-remnant cholesterol associated with carotid arteries was detected and quantified in three dimensions using digital confocal microscopy. Co-localization assessment of CRM’s with selected arterial proteoglycans and advanced glycation endproducts (AGE) were made. Results: Both visual and quantitative data from these studies demonstrate focal and enhanced accumulation of CRM’s in the intima of arterial vessels from diabetic animals compared to controls (p<0.05). Analysis of CRM’s indicated significant co-localization (as measured by Pearson’s co-efficient) with AGEs (0.78±0.13) and with biglycan proteoglycan (0.71±0.18).
Accumulation of intestinal cholesterol-rich remnant lipoproteins within arterial vessels may be exacerbated by advanced glycation endproducts and the co-localization of proteoglycans in diabetes
Conclusions: Our observations confirm that modulation of the arterial extracellular matrix prevalent during insulin deficiency and hyperglycemia may help to explain the exacerbated retention of CRM’s in diabetes. 1P-0157
Peroxisome proliferator-activator receptor-γ ligands inhibit TGF-β1-induced fibronectin expression in glomerular mesangial cells
B. Guo, D. Koya, A. Kashiwagi, M. Haneda. Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Japan The thiazolidinedione (TZD) class of antidiabetic drugs, which are ligands for peroxisome proliferator-activated receptor-γ (PPAR-γ), has been shown to possess potent anti-inflammatory and anti-neoplastic action in various systems. Here, we show in mesangial cells that PPAR-γ agonists inhibit fibronectin (FN) expression induced by transforming growth factor-β1 (TGF-β1), a key cytokine in mediating extracellular matrix (ECM) accumulation in diabetic nephropathy. TGF-β1 stimulated FN mRNA expression, and this enhancement was abrogated by a treatment with pioglitazone. Electrophoretic mobility shift assay (EMSA) identified that pioglitazone inhibited TGF-β1-induced DNA binding activity of activator protein-1 (AP-1). Furthermore, pioglitazone inhibited AP-1 reporter activity but not Smad binding elements (SBEs) reporter activity without affecting TGF-β1-induced activation of mitogen-activated protein kinases (MAPKs) and Smad2. Similarly to pioglitazone, PPAR-γ overexpression also inhibited TGF-β1-induced FN mRNA expression as well as the activation of AP-1. 15d-PGJ2, a natural PPAR-γ ligand, also inhibited TGF-β1-induced FN expression by suppressing AP-1 reporter activity and AP-1 DNA binding activity. Alternatively, 15d-PGJ2 inhibited the MAPKs activation induced by TGF-β1. The overexpression of dominant negative PPAR-γ completely abrogated the inhibitory effect of pioglitazone, while partial prevention was observed in the inhibitory effect of 15d-PGJ2 on TGF-β1-induced AP-1 reporter activity. In conclusion, pioglitazone inhibits TGF-β1-induced FN expression by inhibiting AP-1 activation dependent of PPAR-γ activation, while 15d-PGJ2 acts through a dual mechanism independent and dependent of PPAR-γ activation in mouse mesangial cells.
S. Proctor 1 , J.M. Forbes 2 , J.C.L. Mamo 1 . 1 Dept. Nutrition, Curtin University, Perth; 2 Baker Heart Research Institute, Melbourne, Australia Objectives: The mechanism(s) underlying the accelerated progression of atherosclerosis, particularly in type-1 diabetes remains unclear. We have demonstrated previously that the accumulation of lipoproteins rich in cholesterol (such as LDL and remnants of intestinal triglyceride-rich lipoproteins XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan