- Email: [email protected]
MS171 REGULATORY MECHANISMS IN CHEMOTAXIS OF DENDRITIC CELLS FROM HEALTHY BLOOD DONORS AND FROM PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
144
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
susceptible one. So PDT will eliminate these cells first of all. However, to slow down atherosclerotic process, it’s not necessary to kill cells as in tumor, but it will be sufficient to decrease their functional activity. Mph are considered to be key cells in vascular pathology because of their lipid accumulation, which is based on phagocytosis. In present study we screened effects of low-fluence PDT on Mph phagocytosis. Methods: Human Mph were obtained and cultured as described elsewhere. 24 h before PDT 10 ug/ml sulfonated aluminium- (Photosens® (PS)) phtalocyanine was added to culture medium. Then cells were washed carefully and illuminated with 675-nm light (0.25−1 J/cm2 ) (AZOR LtD, Russia). Cellular viability was measured with MTT test. To study phagocytosis latex beads were added to Mph culture medium 24 hour after PDT. In 2 hours beads were removed, cells were fixed with methanol. Quantity of phagocytes was determined using Sigma Scan software. Results: PS accumulation alone, laser illumination alone and low-fluence PSPDT (0.25−0.5 J/cm2 ) did not affect Mph. After PDT with 1 J/cm2 reduction of cellular viability was only 10%. Illumination of Al-sPS-loaded cells with 675-nm and 0.25−0.5 J/cm2 fluence decreased phagocytic activity in 1.5 and 2.4 times respectively. Conclusion: Low-fluence PDT can effectively decrease Mph phagocytic activity without reduction of other vascular cell viability. MS171 REGULATORY MECHANISMS IN CHEMOTAXIS OF DENDRITIC CELLS FROM HEALTHY BLOOD DONORS AND FROM PATIENTS WITH ACUTE MYOCARDIAL INFARCTION P. Wolkow1 , A. Gebska1 , J. Godlewski2 , J. Jawien1 , R. Olszanecki1 , M. Jawien1 , K. Zmudka2 , R. Korbut1 . 1 Dept. of Pharmacology, 2 Dept. of Cardiology, Jagiellonian University Collegium Medicum, Krakow, Poland Introduction: Atherosclerotic plaques contain dendritic cells (DCs), capable to initiate T lymphocyte activation. Modifiers of DC chemotaxis may affect immune response and local cytokine production, leading to plaque instability and clinical complications, e.g. myocardial infarction. Aims of the study: 1. To determine effects of protein kinase inhibitors, angiotensin metabolites, and co-culture with thrombocytes on chemotaxis of monocyte − derived DCs from healthy blood donors. 2. To determine the effect of acute myocardial infarction on chemotaxis of peripheral blood DCs and their sensitivity to protein kinase inhibitors. Materials and Methods: Immature dendritic cells were isolated directly from peripheral blood or produced from peripheral blood monocytes by 7-day culture with GM-CSF and varying concentrations of IL-4. 2-day maturation of DCs was induced by prostaglandin E2 with CD40 ligand or thrombocytes. Chemotaxis was induced by CCL19 in Transwell chambers. Studied substances and thrombocytes were added during maturation or shortly before the chemotaxis assay. Results and Conclusions: 1. Decrease of chemotaxis was observed only if src kinase inhibitor was added shortly before the assay, Erk kinase inhibitor during DC maturation, and p38 kinase inhibitor if DCs were cultured with low IL-4 concentration. 2. Angiotensin II inhibited and angiotensin (1−9) stimulated DC chemotaxis. 3. DCs matured in the presence of either thrombocytes or CD40 ligand have similar chemotactic potential if thrombocytes are not physically separated from DCs or treated with aspirin. 4. Chemotaxis of DCs from patients with acute myocardial infarction is impaired and their DCs are more sensitive to protein kinase inhibitors. MS172 SOME ALTERNATIVE DETERMINANTS OF VASCULAR STIFFNESS IN YOUNG INDIVIDUALS AT LOW CARDIOVASCULAR RISK O. Drokina1 , A. Semyonkin1 , G. Nechaeva2 , A. Zhenatov1 , L. Zhivilova1 , E. Lalyukova2 , I. Druk2 , A. Bogdalova1 , T. Miller1 . 1 Diagnostics of Internal Diseases, 2 Postgraduate Education, Omsk Medical Academy, Omsk, Russia Objectives: Besides cardiovascular risk factors other abnormalities may influence arterial elasticity such as genetic disorders of connective tissue as seen for example in Marfan’s syndrome. Our aim was to evaluate the alternative determinants of vascular stiffness in young low risk population. Materials and Methods: The study involved 111 young healthy individuals (62 men and 49 women, 18−35 years old). Risk factors screening (blood pressure, lipids, glucose, smoking, obesity) and features of connective tissue disorders (skin, skeletal and tendons abnormalities, myopia) were performed. Stiffness index (SI) after sublingual trinitroglycerin and endothelium dependent vascular dilation (EDVD) to salbutamol were measured using photoplethysmography. Results: SI did not differ in men and women (4.94±0.67 m/s versus 4.80±0.56 m/s, p = 0.29). On multivariate analysis age, mean BP were positively and EDVD − negatively related to SI (p < 0.005, p < 0.001 and p < 0.001, respectively). Among connective tissue disorders features scoliosis, mild chest wall deformities, cutaneous striae and flat feet were positively related to SI (p < 0.05 for each variable). The model including age, mean BP, EDVD
Memory Stick Presentations
and features of connective tissue disorders that were significant on multivariate analysis accounted for 42% of SI variability (R = 0.65, R2 =0.42, p < 0.00001). Conclusions: Thus, as in other populations in low risk young healthy individuals vascular stiffness is determined by age, blood pressure and endothelial function. However, there exist some other factors influencing vascular elasticity, that may be related to inherited connective tissue disorders even in the absence of apparent genetic diseases such as Marfan’s, Ehlers-Danlos syndrome and etc. MS173 EFFECTS OF TUMOR NECROSIS FACTOR (TNF)-ALPHA AND 17-BETA ESTRADIOL (E2 ) ON PATHOLOGICAL CHANGES OF INTIMAL HYPERPLASIA R. Nintasen1 , Y. Maneerat1 , P. Chartiburus2 , C. Punsawad1 , V. Khachansaksumeth1 , P. Viriyavejakul1 , U. Chaisri1 . 1 Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, 2 The Institute of Cardiovascular Diseases, Rajavithi Hospital, Bangkok, Thailand Objectives: To compare between the effects of TNF-alpha and E2 on pathological changes in culture of human saphenous vein with intimal hyperplasia. Methods: A segment of saphenous vein with intimal hyperplasia was obtained from a patient at the time of bypass graft. Two millimeters thick rings of the vessel were cultured in RPMI 1640 with 30% fetal bovine serum (FBS) alone (control), plus rTNF-alpha or E2 in static condition. After 7 days of incubation, the cultured rings were harvested to determine morphology, histopathologic changes and profiles of protein expression involving functions of endothelial cell (EC) using histomorphometric analysis, hematoxylin & eosin staining and immunohistochemistry. Results: The rings treated with 5 ng/ml of TNF-alpha markedly increased the degree of intimal hyperplasia and proliferation of smooth muscle cells (SMC) in both tunica intima and media in comparison to those in the control rings. The damaged endothelial lining increased expression of inducible nitric oxide synthase (iNOS), and adhesion molecules including ICAM-1, VCAM-1 and E-selectin. E2 at the dose of 10 nM could improve morphology of intimal hyperplasia observed in the control rings on day 7 of cultivation. The E2 treated rings possessed wider lumen with perfect contour of EC lining, decreased SMC proliferation in tunica media, and reduced adhesion molecule expressions but increased endothelial nitric oxide synthase (eNOS) in EC. Conclusion: Our finding supports previous studies in animal models that E2 may have good efficiency to diminish degree of neointimal hyperplasia in patients who have coronary bypass graft. MS174 CAN NEUROECTODERMAL ORIGIN OF SMOOTH MUSCLE CELLS IN AORTIC ATHEROSCLEROSIS BE A PREDISPOSING FACTOR FOR THE ACCUMULATION OF LIPIDS? I. Tanaskovic. Department of Histology and Embryology, Medical Faculty, University of Kragujevac, Kragujevac, Serbia Aims of this study was to examine the origine and the phenotype status of VSMCs in aortic intima. Methods: We immunocytochemicaly examined 20 samples of atherosclerotically changed parts of the aorta descendens, at the fatty streak stage. Immunocytochemical staining (LSAB+/HRP) was performed to identify a-SMA, vimentin, and S-100 protein. Results: In the intima of the samples analyzed in the present study, there was a substantial number of VSMCs that showed immunoreactivity to a-SMA and vimentin, some of which contain lipid inclusions in the cytoplasm (foam cells). In addition to those VSMCs, there is a presence of VSMCs that showed immunoreactivity a-SMA and S-100 protein that also look as if they are at different stages of phenotype transformations to foam cells. Conclusions: All of the analyzed cells show a a-SMA-immunoreactivity, which indicates that we are dealing with VSMCs. Vimentin-immunoreactivity in most VSMCs point out to their syntetic/proliferative activity and also to their mesenchymal origin. It is well-known that VSMCs can also express scavenger receptors and that they transform to the foam cells. Nevertheless, in the analyzed parts of the aorta, there is a presence of foam cells that originated from VSMCs that express a-SMA and S-100 protein. The expession of S-100 protein indicates that these cells are of a neuroectodermal origine. As this protein shows a large affinity to binding unsaturated lipid acids, it has been assumed that the neuroectodermal origin of VSMCs of the coronary arteries can be a predisposing factor for the accumulation of lipids.
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
susceptible one. So PDT will eliminate these cells first of all. However, to slow down atherosclerotic process, it’s not necessary to kill cells as in tumor, but it will be sufficient to decrease their functional activity. Mph are considered to be key cells in vascular pathology because of their lipid accumulation, which is based on phagocytosis. In present study we screened effects of low-fluence PDT on Mph phagocytosis. Methods: Human Mph were obtained and cultured as described elsewhere. 24 h before PDT 10 ug/ml sulfonated aluminium- (Photosens® (PS)) phtalocyanine was added to culture medium. Then cells were washed carefully and illuminated with 675-nm light (0.25−1 J/cm2 ) (AZOR LtD, Russia). Cellular viability was measured with MTT test. To study phagocytosis latex beads were added to Mph culture medium 24 hour after PDT. In 2 hours beads were removed, cells were fixed with methanol. Quantity of phagocytes was determined using Sigma Scan software. Results: PS accumulation alone, laser illumination alone and low-fluence PSPDT (0.25−0.5 J/cm2 ) did not affect Mph. After PDT with 1 J/cm2 reduction of cellular viability was only 10%. Illumination of Al-sPS-loaded cells with 675-nm and 0.25−0.5 J/cm2 fluence decreased phagocytic activity in 1.5 and 2.4 times respectively. Conclusion: Low-fluence PDT can effectively decrease Mph phagocytic activity without reduction of other vascular cell viability. MS171 REGULATORY MECHANISMS IN CHEMOTAXIS OF DENDRITIC CELLS FROM HEALTHY BLOOD DONORS AND FROM PATIENTS WITH ACUTE MYOCARDIAL INFARCTION P. Wolkow1 , A. Gebska1 , J. Godlewski2 , J. Jawien1 , R. Olszanecki1 , M. Jawien1 , K. Zmudka2 , R. Korbut1 . 1 Dept. of Pharmacology, 2 Dept. of Cardiology, Jagiellonian University Collegium Medicum, Krakow, Poland Introduction: Atherosclerotic plaques contain dendritic cells (DCs), capable to initiate T lymphocyte activation. Modifiers of DC chemotaxis may affect immune response and local cytokine production, leading to plaque instability and clinical complications, e.g. myocardial infarction. Aims of the study: 1. To determine effects of protein kinase inhibitors, angiotensin metabolites, and co-culture with thrombocytes on chemotaxis of monocyte − derived DCs from healthy blood donors. 2. To determine the effect of acute myocardial infarction on chemotaxis of peripheral blood DCs and their sensitivity to protein kinase inhibitors. Materials and Methods: Immature dendritic cells were isolated directly from peripheral blood or produced from peripheral blood monocytes by 7-day culture with GM-CSF and varying concentrations of IL-4. 2-day maturation of DCs was induced by prostaglandin E2 with CD40 ligand or thrombocytes. Chemotaxis was induced by CCL19 in Transwell chambers. Studied substances and thrombocytes were added during maturation or shortly before the chemotaxis assay. Results and Conclusions: 1. Decrease of chemotaxis was observed only if src kinase inhibitor was added shortly before the assay, Erk kinase inhibitor during DC maturation, and p38 kinase inhibitor if DCs were cultured with low IL-4 concentration. 2. Angiotensin II inhibited and angiotensin (1−9) stimulated DC chemotaxis. 3. DCs matured in the presence of either thrombocytes or CD40 ligand have similar chemotactic potential if thrombocytes are not physically separated from DCs or treated with aspirin. 4. Chemotaxis of DCs from patients with acute myocardial infarction is impaired and their DCs are more sensitive to protein kinase inhibitors. MS172 SOME ALTERNATIVE DETERMINANTS OF VASCULAR STIFFNESS IN YOUNG INDIVIDUALS AT LOW CARDIOVASCULAR RISK O. Drokina1 , A. Semyonkin1 , G. Nechaeva2 , A. Zhenatov1 , L. Zhivilova1 , E. Lalyukova2 , I. Druk2 , A. Bogdalova1 , T. Miller1 . 1 Diagnostics of Internal Diseases, 2 Postgraduate Education, Omsk Medical Academy, Omsk, Russia Objectives: Besides cardiovascular risk factors other abnormalities may influence arterial elasticity such as genetic disorders of connective tissue as seen for example in Marfan’s syndrome. Our aim was to evaluate the alternative determinants of vascular stiffness in young low risk population. Materials and Methods: The study involved 111 young healthy individuals (62 men and 49 women, 18−35 years old). Risk factors screening (blood pressure, lipids, glucose, smoking, obesity) and features of connective tissue disorders (skin, skeletal and tendons abnormalities, myopia) were performed. Stiffness index (SI) after sublingual trinitroglycerin and endothelium dependent vascular dilation (EDVD) to salbutamol were measured using photoplethysmography. Results: SI did not differ in men and women (4.94±0.67 m/s versus 4.80±0.56 m/s, p = 0.29). On multivariate analysis age, mean BP were positively and EDVD − negatively related to SI (p < 0.005, p < 0.001 and p < 0.001, respectively). Among connective tissue disorders features scoliosis, mild chest wall deformities, cutaneous striae and flat feet were positively related to SI (p < 0.05 for each variable). The model including age, mean BP, EDVD
Memory Stick Presentations
and features of connective tissue disorders that were significant on multivariate analysis accounted for 42% of SI variability (R = 0.65, R2 =0.42, p < 0.00001). Conclusions: Thus, as in other populations in low risk young healthy individuals vascular stiffness is determined by age, blood pressure and endothelial function. However, there exist some other factors influencing vascular elasticity, that may be related to inherited connective tissue disorders even in the absence of apparent genetic diseases such as Marfan’s, Ehlers-Danlos syndrome and etc. MS173 EFFECTS OF TUMOR NECROSIS FACTOR (TNF)-ALPHA AND 17-BETA ESTRADIOL (E2 ) ON PATHOLOGICAL CHANGES OF INTIMAL HYPERPLASIA R. Nintasen1 , Y. Maneerat1 , P. Chartiburus2 , C. Punsawad1 , V. Khachansaksumeth1 , P. Viriyavejakul1 , U. Chaisri1 . 1 Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, 2 The Institute of Cardiovascular Diseases, Rajavithi Hospital, Bangkok, Thailand Objectives: To compare between the effects of TNF-alpha and E2 on pathological changes in culture of human saphenous vein with intimal hyperplasia. Methods: A segment of saphenous vein with intimal hyperplasia was obtained from a patient at the time of bypass graft. Two millimeters thick rings of the vessel were cultured in RPMI 1640 with 30% fetal bovine serum (FBS) alone (control), plus rTNF-alpha or E2 in static condition. After 7 days of incubation, the cultured rings were harvested to determine morphology, histopathologic changes and profiles of protein expression involving functions of endothelial cell (EC) using histomorphometric analysis, hematoxylin & eosin staining and immunohistochemistry. Results: The rings treated with 5 ng/ml of TNF-alpha markedly increased the degree of intimal hyperplasia and proliferation of smooth muscle cells (SMC) in both tunica intima and media in comparison to those in the control rings. The damaged endothelial lining increased expression of inducible nitric oxide synthase (iNOS), and adhesion molecules including ICAM-1, VCAM-1 and E-selectin. E2 at the dose of 10 nM could improve morphology of intimal hyperplasia observed in the control rings on day 7 of cultivation. The E2 treated rings possessed wider lumen with perfect contour of EC lining, decreased SMC proliferation in tunica media, and reduced adhesion molecule expressions but increased endothelial nitric oxide synthase (eNOS) in EC. Conclusion: Our finding supports previous studies in animal models that E2 may have good efficiency to diminish degree of neointimal hyperplasia in patients who have coronary bypass graft. MS174 CAN NEUROECTODERMAL ORIGIN OF SMOOTH MUSCLE CELLS IN AORTIC ATHEROSCLEROSIS BE A PREDISPOSING FACTOR FOR THE ACCUMULATION OF LIPIDS? I. Tanaskovic. Department of Histology and Embryology, Medical Faculty, University of Kragujevac, Kragujevac, Serbia Aims of this study was to examine the origine and the phenotype status of VSMCs in aortic intima. Methods: We immunocytochemicaly examined 20 samples of atherosclerotically changed parts of the aorta descendens, at the fatty streak stage. Immunocytochemical staining (LSAB+/HRP) was performed to identify a-SMA, vimentin, and S-100 protein. Results: In the intima of the samples analyzed in the present study, there was a substantial number of VSMCs that showed immunoreactivity to a-SMA and vimentin, some of which contain lipid inclusions in the cytoplasm (foam cells). In addition to those VSMCs, there is a presence of VSMCs that showed immunoreactivity a-SMA and S-100 protein that also look as if they are at different stages of phenotype transformations to foam cells. Conclusions: All of the analyzed cells show a a-SMA-immunoreactivity, which indicates that we are dealing with VSMCs. Vimentin-immunoreactivity in most VSMCs point out to their syntetic/proliferative activity and also to their mesenchymal origin. It is well-known that VSMCs can also express scavenger receptors and that they transform to the foam cells. Nevertheless, in the analyzed parts of the aorta, there is a presence of foam cells that originated from VSMCs that express a-SMA and S-100 protein. The expession of S-100 protein indicates that these cells are of a neuroectodermal origine. As this protein shows a large affinity to binding unsaturated lipid acids, it has been assumed that the neuroectodermal origin of VSMCs of the coronary arteries can be a predisposing factor for the accumulation of lipids.