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Tu-P7:125 Endothelial dysfunction in diabetic retinopathy
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst part)
212 Tu
123
1
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D I S T I N C T R O L E S F O R PAR1 A N D 2 - M E D I A T E D V A S O M O T O R M O D U L A T I O N IN H U M A N A R T E R I A L A N D V E N O U S C O N D U I T S U S E D IN C O R O N A R Y ARTERY BYPASS SURGERY
R. Ballerio I , M. Brambilla 2 , D. Colnago I , A. Parolaxi I , M. Agrifoglio I , F. Alamanni i M. Camera 1'2 , E. Tremoli 1'2 L. Mussoni 2. 1Centro
Cardiologico Monzino, Milan, Italy." 2Dep Pharmacol Sci, Universi~ of Milan, Milan, Italy The use of internal mammary artery (IMA) in coronary artery bypass grafting (CABG) is associated with improved patency rates in comparison with saphenous vein (GSV); this may be related to endothelial release of vasodilators that antagonise vascular contraction. Recently, a family of protease-activated receptors (PARs) have been shown to modulate endothelium-dependent vasodilatation. Objectives and methods: This study is aimed at evaluating the presence and the functional role of PAR1 and PAR2 in mediating vascular tone in IMAs and GSVs obtained from patients undergoing CABG by means of real time-PCR and isometric tension measurements. Results: PAR1 mRNA levels were higher than PAR2 in both vessels. In pre-contracted rings the selective PARl-activating peptides (PAR1-AP), TFLLR-NH2 (0.001 to 10 Itmol/L), caused an endothelium-dependent relaxation greater in IMAs than GSVs (pD2 values 7.25-4-0.6 and 7.86-4-0.42 respectively, p < 0.05; E m a x values 56.2%-4-17.3 and 29.7%-4-13.4 respectively, p<0.001). PAR2-AR SLIGKV-NH2 (0.01 to 100 Itmol/L), elicited only a slight change in vascular tension in both vessels. Pre-incubation (14 h) of rings with TNFalpha (3 nmol/L), followed by exposure to PAR2-AR increased the relaxation in a significantly manner only in IMAs (p<0.05 vs non stimulated vessels) accompanied by an increase in PAR2 mRNA expression. The relaxations, induced by both PAR-APs, are NO- and endothelium-dependent. Conclusion: These data show that: i) functional active PAR1 and PAR2 axe present in IMAs and GSVs; ii) inflammatory stimuli selectively enhance endothelium-dependent relaxation to PAR2-AP in IMAs.
Tu I P 7 ~124
1
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HIGH GLUCOSE INDUCES TRANSDIFFERENTIATION OF ENDOTHELIAL CELLS INTO SMOOTH MUSCLE CELLS
Y. Liu, X.H. Zu, H.G. LL Q.K. LL M.B. Cad, Y.C. Lv, Z.Q. Wang, Z. Wang, W.D. Yin, Z.B. Wang. Nanhua University, Hengyang, China Objective: Uncontrolled hyperglycemia is the main risk factor in the development of diabetic vascular complications. The endothelial cells are first to be damaged by hyperglycemia due to their unique location as the inner lining of all vessels. The mechanism of endothelial injury by high glucose is still poorly understood. The present study was undertaken to investigate the hypothesis that high glucose can induce endothelial-smooth muscle transdifferentiation in human umbilical vein endothelial cells (HUVECs). Metbods: HUVECs were incubated with 0, 5.5, 11.1, 22.2, 44.4 mmol/L D-glucose or 22.2 mmol/L mannitol for 48 hours, and with 44.4 mmol/L D-glucose for 0, 12, 24, 48, 96 hours or 44.4 mmol/L mannitol for 48 hours respectively. The expression of von willebrand factor (vWF), alpha-smooth muscle actin (SMA) and smooth muscle myosin heavy chains (SM-MHCs) in HUVECs were examined by immunocytochemistry and western blotting. Results: During incubation with graded glucose concentration for 48 hours, HUVECs transferred spindle morphology to a more compact and larger appearance, and expressed SMA and SM-MHCs (as smooth muscle-related markers) in a diffuse cytoplasmic pattern. With graded time in culture, the expression of SMA and SM-MHCs were increased, and expresion of vWF (as a endothelial marker) was decreased. Conclusions: The observation that endothelial-smooth muscle transdifferentiation can take place in HUVECs suggests that endothelium could serve as a potential source of at least some smooth muscle cells in diabetic vascular complications. Funding: Hunan Provincial National Natural Sciences Foundation of China.
Tu-P7:125
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E N D O T H E L I A L D Y S F U N C T I O N IN D I A B E T I C RETINOPATHY
"~ A. Lipinska I , M. Puchta 1 , K. Jankowski 1 , D. Korczak I , Z. Lewandowski-, D. Liszewska-Pfejfer I . lDep. of bztetTtal Medicine and Cardiology, Medical
Universi~ of Warsaw, Warsaw, Poland: 2Dep. of Epidemiology, Medical Universi~ of Warsaw, Warsaw, PolatM Endothelial dysfunction and increased leukocyte-endothelial cell adhesion is a key early event in the development of retinopathy in diabetic patients. Endothe-
lial cell adhesion molecules (CAM) mediating adhesion and transendothelial migration of leukocytes play an important role in initiation and development of diabetic retinopathy. The aim of our study was to evaluate the concentration of soluble forms of C A M in men (M) with type 1 diabetes mellitus (DM) without macroangiopathy. The study group consisted of 40 M with DM and retinopathy, mean age 34,14-6,8yrs, duration of disease 14,9-t-4 yrs (group A) and 17 M mean age 31,84-9,2yrs, with DM duration 8,7-t-2,8yrs without vascular diabetic complications (group B). H b A l c concentration was comparable in both groups:8,6-t-l,6%vs 8,94-2,1%(NS). The control group included 55 healthy M, mean age 34,9-t-9yrs(group C). In all groups the levels of soluble isoforms of CAM: E-selectin, ICAM-1,VCAM-1 with ELISA-method were measured. Results: Group A had the highest level of VCAM-1, statistically significant in comparison with group C: 422,6-4-153,5 ng/dl vs 352,3-4-136,6 ng/dl, p<0,05, and non-significantly higher than group B: 422,6-4-153,5 ng/dl vs 364,1-4-114,9ng/dl. E-selectin and ICAM-1 serum concentration did not differ significantly between group A, B and C. Conclusions: 1. patients with DM type 1 and rethinopathy demonstrate the significant increase of VCAM-1 level, 2. our results suggest that increase of VCAM-1 level in this group of patients could be considered as a marker of endothelial dysfunction influencing the development of diabetic rethinopathy.
ITu-P7:1261 DIL-6Y S FCUONRCRTEILOANT EINS YWOIUTNHG EPNODSOTT-HMEYLOICAAL R D I A L INFARCTION PATIENTS B. Erzen, M. Sabovic, M. Sebestjen, P. Poredos, I. Keber. Dept. of Vascular
Diseases, University Medical Centre, Ljubljana, Slovenia Objectives: The estimation of coronary risk based on consideration of classical risk factors is insufficient in young patients with myocardial infarction (MI) who have low expressions of classical risk factors. Endothelial dysfunction (lED) and markers of vascular inflammation may be more appropriate for risk estimation. The relations among ED and inflammation markers in such patients have not yet been explored. Patients a n d Methods: 21 patients (on average 44 years old) in the stable phase after MI, with low expressions of risk factors, were included in the study. The control group consisted of 25 healthy age-matched males. E D was estimated by ultrasound measurement of the endothelium-dependent dilatation of the brachial artery. The following inflammation markers were measured: hs-CRP, IL-6, TNF-alfa, ICAM-1, VCAM-1, selectin-P and selectin-E. Results: Patients had a significantly reduced level of endotheliumdependent vasodilatation (5.6 -4- 3.5 vs. 8.8 -4- 6.5% p<0.05), and an increased level of IL-6 (3.2 [1.5-8.4] vs. 1.4 [0.9-2.3] ng/ml; p<0.01). All other inflammation markers were comparable to controls. We found a significant negative correlation between ED and the levels of IL-6 (r=-0.54, p= 0.012). Conclusion: We found a significant correlation between E D and (increased) I1-6 levels. Thus, it appears that I1-6 is the most valuable circulating marker of ED, and consequently a useful marker of coronary risk. Funding: The Slovenian Ministry of Science and Technology supported the study through grant No. 3311-01-831/463.
I Tu-P7:127 I R E A C T I V E
OXYGEN SPECIES SIGNAL FOR C Y C L O O X Y G E N A S E - 2 I N D U C T I O N IN H U M A N E N D O T H E L I A L C E L L S : R O L E IN T H E R E P A I R O F WOUND
~ S. Eligini 1, I. Arenaz ] , S.S. Baxbieri 1, M.L. Faleri 3 , E. Tremoh• 1 "~, S. Colli 1.
1Dept. Pharmacological Sciences, Univ. of Milan, Milan, Italy: 2Centro Cardiologico Monzino, Univ. of Milan, Milan, Italy: 3Div. Anatomia Patologica, Osp. Niguarda Ca' Gramla, Milan, Italy Objective: This study examines the signaling pathways driven by reactive oxygen species (ROS), either from exogenous source (H202) or endogenously generated by the prototypical inflammatory cytokine TNFalpha, that lead to cyclooxygenase-2 (Cox-2) induction in human endothelial cells. In addition, it addresses the role of ROS-induced Cox-2 in the repair of mechanically wounded endothelium monolayer. Metbods: Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to H 2 0 2 or to TNFalpha; Cox-2 protein and mRNA levels were evaluated by Western blot and by RT-PCR. Prostaglandin E2 (PGE2) and 6-keto prostaglandin F l a l p h a (6-keto PGFlalpha) levels were measured in cell medium as index of Cox-2 activity. Repair of wounded HUVEC monolayer was microscopically determined by the extent of cell migration into denuded areas of confluent cell monolayers.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
212 Tu
123
1
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D I S T I N C T R O L E S F O R PAR1 A N D 2 - M E D I A T E D V A S O M O T O R M O D U L A T I O N IN H U M A N A R T E R I A L A N D V E N O U S C O N D U I T S U S E D IN C O R O N A R Y ARTERY BYPASS SURGERY
R. Ballerio I , M. Brambilla 2 , D. Colnago I , A. Parolaxi I , M. Agrifoglio I , F. Alamanni i M. Camera 1'2 , E. Tremoli 1'2 L. Mussoni 2. 1Centro
Cardiologico Monzino, Milan, Italy." 2Dep Pharmacol Sci, Universi~ of Milan, Milan, Italy The use of internal mammary artery (IMA) in coronary artery bypass grafting (CABG) is associated with improved patency rates in comparison with saphenous vein (GSV); this may be related to endothelial release of vasodilators that antagonise vascular contraction. Recently, a family of protease-activated receptors (PARs) have been shown to modulate endothelium-dependent vasodilatation. Objectives and methods: This study is aimed at evaluating the presence and the functional role of PAR1 and PAR2 in mediating vascular tone in IMAs and GSVs obtained from patients undergoing CABG by means of real time-PCR and isometric tension measurements. Results: PAR1 mRNA levels were higher than PAR2 in both vessels. In pre-contracted rings the selective PARl-activating peptides (PAR1-AP), TFLLR-NH2 (0.001 to 10 Itmol/L), caused an endothelium-dependent relaxation greater in IMAs than GSVs (pD2 values 7.25-4-0.6 and 7.86-4-0.42 respectively, p < 0.05; E m a x values 56.2%-4-17.3 and 29.7%-4-13.4 respectively, p<0.001). PAR2-AR SLIGKV-NH2 (0.01 to 100 Itmol/L), elicited only a slight change in vascular tension in both vessels. Pre-incubation (14 h) of rings with TNFalpha (3 nmol/L), followed by exposure to PAR2-AR increased the relaxation in a significantly manner only in IMAs (p<0.05 vs non stimulated vessels) accompanied by an increase in PAR2 mRNA expression. The relaxations, induced by both PAR-APs, are NO- and endothelium-dependent. Conclusion: These data show that: i) functional active PAR1 and PAR2 axe present in IMAs and GSVs; ii) inflammatory stimuli selectively enhance endothelium-dependent relaxation to PAR2-AP in IMAs.
Tu I P 7 ~124
1
/
HIGH GLUCOSE INDUCES TRANSDIFFERENTIATION OF ENDOTHELIAL CELLS INTO SMOOTH MUSCLE CELLS
Y. Liu, X.H. Zu, H.G. LL Q.K. LL M.B. Cad, Y.C. Lv, Z.Q. Wang, Z. Wang, W.D. Yin, Z.B. Wang. Nanhua University, Hengyang, China Objective: Uncontrolled hyperglycemia is the main risk factor in the development of diabetic vascular complications. The endothelial cells are first to be damaged by hyperglycemia due to their unique location as the inner lining of all vessels. The mechanism of endothelial injury by high glucose is still poorly understood. The present study was undertaken to investigate the hypothesis that high glucose can induce endothelial-smooth muscle transdifferentiation in human umbilical vein endothelial cells (HUVECs). Metbods: HUVECs were incubated with 0, 5.5, 11.1, 22.2, 44.4 mmol/L D-glucose or 22.2 mmol/L mannitol for 48 hours, and with 44.4 mmol/L D-glucose for 0, 12, 24, 48, 96 hours or 44.4 mmol/L mannitol for 48 hours respectively. The expression of von willebrand factor (vWF), alpha-smooth muscle actin (SMA) and smooth muscle myosin heavy chains (SM-MHCs) in HUVECs were examined by immunocytochemistry and western blotting. Results: During incubation with graded glucose concentration for 48 hours, HUVECs transferred spindle morphology to a more compact and larger appearance, and expressed SMA and SM-MHCs (as smooth muscle-related markers) in a diffuse cytoplasmic pattern. With graded time in culture, the expression of SMA and SM-MHCs were increased, and expresion of vWF (as a endothelial marker) was decreased. Conclusions: The observation that endothelial-smooth muscle transdifferentiation can take place in HUVECs suggests that endothelium could serve as a potential source of at least some smooth muscle cells in diabetic vascular complications. Funding: Hunan Provincial National Natural Sciences Foundation of China.
Tu-P7:125
3
/
E N D O T H E L I A L D Y S F U N C T I O N IN D I A B E T I C RETINOPATHY
"~ A. Lipinska I , M. Puchta 1 , K. Jankowski 1 , D. Korczak I , Z. Lewandowski-, D. Liszewska-Pfejfer I . lDep. of bztetTtal Medicine and Cardiology, Medical
Universi~ of Warsaw, Warsaw, Poland: 2Dep. of Epidemiology, Medical Universi~ of Warsaw, Warsaw, PolatM Endothelial dysfunction and increased leukocyte-endothelial cell adhesion is a key early event in the development of retinopathy in diabetic patients. Endothe-
lial cell adhesion molecules (CAM) mediating adhesion and transendothelial migration of leukocytes play an important role in initiation and development of diabetic retinopathy. The aim of our study was to evaluate the concentration of soluble forms of C A M in men (M) with type 1 diabetes mellitus (DM) without macroangiopathy. The study group consisted of 40 M with DM and retinopathy, mean age 34,14-6,8yrs, duration of disease 14,9-t-4 yrs (group A) and 17 M mean age 31,84-9,2yrs, with DM duration 8,7-t-2,8yrs without vascular diabetic complications (group B). H b A l c concentration was comparable in both groups:8,6-t-l,6%vs 8,94-2,1%(NS). The control group included 55 healthy M, mean age 34,9-t-9yrs(group C). In all groups the levels of soluble isoforms of CAM: E-selectin, ICAM-1,VCAM-1 with ELISA-method were measured. Results: Group A had the highest level of VCAM-1, statistically significant in comparison with group C: 422,6-4-153,5 ng/dl vs 352,3-4-136,6 ng/dl, p<0,05, and non-significantly higher than group B: 422,6-4-153,5 ng/dl vs 364,1-4-114,9ng/dl. E-selectin and ICAM-1 serum concentration did not differ significantly between group A, B and C. Conclusions: 1. patients with DM type 1 and rethinopathy demonstrate the significant increase of VCAM-1 level, 2. our results suggest that increase of VCAM-1 level in this group of patients could be considered as a marker of endothelial dysfunction influencing the development of diabetic rethinopathy.
ITu-P7:1261 DIL-6Y S FCUONRCRTEILOANT EINS YWOIUTNHG EPNODSOTT-HMEYLOICAAL R D I A L INFARCTION PATIENTS B. Erzen, M. Sabovic, M. Sebestjen, P. Poredos, I. Keber. Dept. of Vascular
Diseases, University Medical Centre, Ljubljana, Slovenia Objectives: The estimation of coronary risk based on consideration of classical risk factors is insufficient in young patients with myocardial infarction (MI) who have low expressions of classical risk factors. Endothelial dysfunction (lED) and markers of vascular inflammation may be more appropriate for risk estimation. The relations among ED and inflammation markers in such patients have not yet been explored. Patients a n d Methods: 21 patients (on average 44 years old) in the stable phase after MI, with low expressions of risk factors, were included in the study. The control group consisted of 25 healthy age-matched males. E D was estimated by ultrasound measurement of the endothelium-dependent dilatation of the brachial artery. The following inflammation markers were measured: hs-CRP, IL-6, TNF-alfa, ICAM-1, VCAM-1, selectin-P and selectin-E. Results: Patients had a significantly reduced level of endotheliumdependent vasodilatation (5.6 -4- 3.5 vs. 8.8 -4- 6.5% p<0.05), and an increased level of IL-6 (3.2 [1.5-8.4] vs. 1.4 [0.9-2.3] ng/ml; p<0.01). All other inflammation markers were comparable to controls. We found a significant negative correlation between ED and the levels of IL-6 (r=-0.54, p= 0.012). Conclusion: We found a significant correlation between E D and (increased) I1-6 levels. Thus, it appears that I1-6 is the most valuable circulating marker of ED, and consequently a useful marker of coronary risk. Funding: The Slovenian Ministry of Science and Technology supported the study through grant No. 3311-01-831/463.
I Tu-P7:127 I R E A C T I V E
OXYGEN SPECIES SIGNAL FOR C Y C L O O X Y G E N A S E - 2 I N D U C T I O N IN H U M A N E N D O T H E L I A L C E L L S : R O L E IN T H E R E P A I R O F WOUND
~ S. Eligini 1, I. Arenaz ] , S.S. Baxbieri 1, M.L. Faleri 3 , E. Tremoh• 1 "~, S. Colli 1.
1Dept. Pharmacological Sciences, Univ. of Milan, Milan, Italy: 2Centro Cardiologico Monzino, Univ. of Milan, Milan, Italy: 3Div. Anatomia Patologica, Osp. Niguarda Ca' Gramla, Milan, Italy Objective: This study examines the signaling pathways driven by reactive oxygen species (ROS), either from exogenous source (H202) or endogenously generated by the prototypical inflammatory cytokine TNFalpha, that lead to cyclooxygenase-2 (Cox-2) induction in human endothelial cells. In addition, it addresses the role of ROS-induced Cox-2 in the repair of mechanically wounded endothelium monolayer. Metbods: Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to H 2 0 2 or to TNFalpha; Cox-2 protein and mRNA levels were evaluated by Western blot and by RT-PCR. Prostaglandin E2 (PGE2) and 6-keto prostaglandin F l a l p h a (6-keto PGFlalpha) levels were measured in cell medium as index of Cox-2 activity. Repair of wounded HUVEC monolayer was microscopically determined by the extent of cell migration into denuded areas of confluent cell monolayers.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006