- Email: [email protected]
180: Effects of OXLDL on the human dendritic cell (DC) phenotype and reception capacity
Journal of Clinical Lipidology, Vol 2, No 5S, October 2008
membrane ruffling and inhibits cellular migration. Treatment with probucol reduced membrane ruffling and restored the capacity of cells to migrate. Using cell lines that have stably knocked down ABCA1 expression (J774-ABCA1-KD) and control cells, we showed that loading with free cholesterol caused a decrease in cell migration by 79.3% in J774-control and by 42.3% in J774ABCA1-KD. Conclusions: Probucol abolishes membrane ruffling and restores cells migration by reducing membrane cholesterol pool. This effect seems to be mediated by ABCA1 and could explain the ability of probucol to reduce cutaneous and tendineous xanthoma, as reported in previous clinical observations.
nitrocellulose immunoblots and agarose gels RtPCR product showed that: i) the intensity of the signal for caveolin-1 in membrane fraction isolated from H hamsters was higher than that in the control group; ii) fluvastatin sodium treatment significantly reduced caveolin-1 expression in all groups; iii) in activated EAhy926 caveolin 1 expression is down regulated as compared with cells grown in standard condition. Conclusions: Caveolin-1 expression is tightly regulated both by hyperlipidemia and statin treatment.
Funding: none
180
179
EFFECTS OF OXLDL ON THE HUMAN DENDRITIC CELL (DC) PHENOTYPE AND RECEPTION CAPACITY A. Monteiro1, A. Azevedo2, J. Barbuto2, M. Gidlund1. 1Laboratory of Immunophisiopathology-USP, São Paulo, Brazil, 2Laboratory of Tumor Immunology-USP, São Paulo, Brazil
EXPRESSION OF CAVEOLIN 1 IN HYPERLIPIDEMIC HAMSTERS AND ENDOTHELIAL DERIVED FOAM CELLS E. Uyy, L. Ivan, R. Haraba, F. Antohe. Institute of Cellular Biology and Patology & Cellular Receptors, Bucharest, Romania Objective: Endothelial permeability plays a major role in lesion formation and is increased in blood vessels with atherosclerotic lesions. The regulation of vascular permeability is one mechanism through which caveolae and caveolins may influence atherosclerotic lesion formation. The aim of this project was to find out if a correlation exists between the caveolin-1, hyperlipidemia and fluvastatin sodium treatment. Methods: Golden Syrian hamsters were divided into two groups fed with standard (N) and hyperlipidemic (H) diet. Each group divided in half was treated with fluvastatin sodium (Nt, Ht) or with culture medium (N, H) as control. Lung tissue was homogenized in buffer with 1% Triton X100 and plasmamembrane enriched fraction was prepared by centrifugation on sucrose gradient. Human endothelial cells (EAhy926) grown in 10% fetal bovine serum (N) were activated by exposure for 48 hours to 1% hyperlipidemic serum (M). The gene and proteins expression of caveolin-1 was assessed comparatively by Rt-PCR and immunoblot techniques. Results: Densitometry performed on
Funding: Romanian Academy and MEdCT grant
Introduction: Two main hypotheses explain the initiation of atherosclerosis: modification of lowdensity lipoproteins (LDL) and an inappropriate immune response to vascular injury. The first step in disease pathogenesis is the monocyte recruitment into the subendothelial space and accumulation of modified forms of LDL (oxLDL). Continued recruitment of monocytes and T cells, and smooth muscle cell migration and proliferation, gives rise to raised fibro-fatty plaques. Recently, also DCs were detected in atherosclerotic plaques. Since these cells are capable of presenting antigens to T cells, whose activation leads to cytokine release, which play an important role in disease progression, we analyzed the relationship between oxLDL with DC differentiation and reception capacity. Methods: DCs were generated from PBMCs obtained from healthy volunteers. Monocytes were cultured in AIM-V medium, containing IL-4 and GM-CSF obtained immature DC (iDC). The mature DCs generation was obtained with add TNF-α. Plasma LDL were isolated by sequential centrifugation and oxidized in CuSO4 (1μM, 37ºC, 18h). On day 6, oxLDL was added and 24h later the cells were harvested for flow cytometry. Results: The
S83
Multiple Risk Factors in Cardiovascular Disease—Abstracts
evaluation reception capacity, the oxLDL capture less mDCs than iDCs. DCs treated with oxLDL had increased expression of CD11c, CD40 and CCR-7 when compared to DCs without treatment oxLDL. Conclusion: mDCs exhibit reduced phagocytic activity, showed decreased oxLDL capture for mDCs. The treatment with oxLDL increases the expression of markers that characterize mature DCs, among which CCR7 was the most affected molecule.
in HLA-DR and CD11c positive cells frequency when compared with DCs treated with oxLDL. The evaluation of oxLDL reception capacity, showed that DCs treated with E2 capture less than DCs without E2. Conclusions: The results suggest that E2 increases the HLA-DR+ and CD11c+ cells and oxLDL incorporation. This could have implications for DC function within the atherosclerotic plaque. Funding: none
Funding: none 182 181 EFECTS OF ESTRADIOL IN HLA-DR AND CD11C EXPRESSION AND PHAGOCYTIC ACTIVITY BY HUMAN MONOCYTE-DERIVED DENDRITIC CELLS TREATED WITH OXLDL A. Azevedo1, A. Monteiro2, M. Gidlund2, J. Barbuto1. 1Laboratory of Tumor ImmunologyUSP, São Paulo, Brazil, 2Laboratory of Immunophisiopathology-USP, São Paulo, Brazil Introduction: The female steroidal hormone, 17hestradiol (E2), triggers a wide variety of biological effects in a number of target systems. E2 is implicated in many vascular disorders.The modification of low-density lipoproteins (oxLDL) and an inappropriate immune response to vascular injury could cause atherosclerosis. Dendritic cells (DCs) were detected in atherosclerotic plaques. The DCs are the most efficient antigen presenting cells, having a distinct role as initiators and modulators of the immune response. The presence of estradiol (E2) receptors on DCs provides the possibility that E2 modulates DC functions directly, which then influence T cell responses. In the present study, we analyzed the HLA-DR and CD11c expression in DC differentiation from blood precursors. Methods: DCs were generated from peripheral blood mononuclear cells obtained from healthy volunteers. Monocytes were cultured in AIM-V medium, containing IL-4 and GM-CSF. Cultures were also supplemented or not with E2 (1mM), on day 0, and oxLDL, on 6 day. On the 7 day, we were analyzed the expression of anti-HLADR and anti-CD11c and oxLDL phagocytic activity assay by flow cytometry. Results: The DCs treated with E2 and oxLDL showed increased
S84
TADALAFIL REDUCES ENDOTHELIAL CELLS INFLAMMATORY RESPONSE INDUCED BY MYELOPEROXIDASE MODIFIED LDL (MOX-LDL) K. Zouaoui Boudjeltia1, T. Roumeguere1,2, A. Rousseau1, V. Nuyens1, P. Van Antwerpen4, J. Ducobu1, M. Vanhaeverbeek1, D. Brohee1, E. Wespes3. 1Laboratory of Experimental Medicine, Unit 222, ISPPC CHU Charleroi, Vesale Hospital, Montigny-le-Tilleul, Belgium, 2University Clinics of Brussels, Department of Urology-Erasme Hospital, Brussels, Belgium, 3Deparment of Urology, ISPPC CHU Charleroi, Charleroi, Belgium, 4Laboratory of Pharmaceutical Chemistry, Intitute of Pharmacy, Free University of Brussels, Brussels, Belgium Objective: Endothelial dysfunction and the disruption of the nitric oxide (NO)-cGMP pathway have been considered as the early common mechanism for the development of both erectile dysfunction (ED) and cardiovascular diseases. Myeloperoxidase modified LDL (Mox-LDL) can trigger inflammatory response, involved in atherosclerosis, through increasing the release of IL-8 by endothelial cells (EC). Mox-LDL have been found in the human atherosclerotic lesions and penis. Phosphodiesterases-5 (PDE-5) inhibitors are used in the treatment of ED. Inhibition of PDE 5, 6 and 9 (zaprinast) negatively attenuated NFêB activation. We aimed to test the possible anti-inflammatory effect of Tadalafil (an inhibitor of PDE-5 and PDE-11 used in the ED treatment) on EC. Methods: EC (EA.hy926) were incubated in the presence of either TNF-alpha (100 pg/ml) or Mox-LDL (200 μg/ml) or both in combination with Tadalafil (1, 3 and 5 μM). IL-8
membrane ruffling and inhibits cellular migration. Treatment with probucol reduced membrane ruffling and restored the capacity of cells to migrate. Using cell lines that have stably knocked down ABCA1 expression (J774-ABCA1-KD) and control cells, we showed that loading with free cholesterol caused a decrease in cell migration by 79.3% in J774-control and by 42.3% in J774ABCA1-KD. Conclusions: Probucol abolishes membrane ruffling and restores cells migration by reducing membrane cholesterol pool. This effect seems to be mediated by ABCA1 and could explain the ability of probucol to reduce cutaneous and tendineous xanthoma, as reported in previous clinical observations.
nitrocellulose immunoblots and agarose gels RtPCR product showed that: i) the intensity of the signal for caveolin-1 in membrane fraction isolated from H hamsters was higher than that in the control group; ii) fluvastatin sodium treatment significantly reduced caveolin-1 expression in all groups; iii) in activated EAhy926 caveolin 1 expression is down regulated as compared with cells grown in standard condition. Conclusions: Caveolin-1 expression is tightly regulated both by hyperlipidemia and statin treatment.
Funding: none
180
179
EFFECTS OF OXLDL ON THE HUMAN DENDRITIC CELL (DC) PHENOTYPE AND RECEPTION CAPACITY A. Monteiro1, A. Azevedo2, J. Barbuto2, M. Gidlund1. 1Laboratory of Immunophisiopathology-USP, São Paulo, Brazil, 2Laboratory of Tumor Immunology-USP, São Paulo, Brazil
EXPRESSION OF CAVEOLIN 1 IN HYPERLIPIDEMIC HAMSTERS AND ENDOTHELIAL DERIVED FOAM CELLS E. Uyy, L. Ivan, R. Haraba, F. Antohe. Institute of Cellular Biology and Patology & Cellular Receptors, Bucharest, Romania Objective: Endothelial permeability plays a major role in lesion formation and is increased in blood vessels with atherosclerotic lesions. The regulation of vascular permeability is one mechanism through which caveolae and caveolins may influence atherosclerotic lesion formation. The aim of this project was to find out if a correlation exists between the caveolin-1, hyperlipidemia and fluvastatin sodium treatment. Methods: Golden Syrian hamsters were divided into two groups fed with standard (N) and hyperlipidemic (H) diet. Each group divided in half was treated with fluvastatin sodium (Nt, Ht) or with culture medium (N, H) as control. Lung tissue was homogenized in buffer with 1% Triton X100 and plasmamembrane enriched fraction was prepared by centrifugation on sucrose gradient. Human endothelial cells (EAhy926) grown in 10% fetal bovine serum (N) were activated by exposure for 48 hours to 1% hyperlipidemic serum (M). The gene and proteins expression of caveolin-1 was assessed comparatively by Rt-PCR and immunoblot techniques. Results: Densitometry performed on
Funding: Romanian Academy and MEdCT grant
Introduction: Two main hypotheses explain the initiation of atherosclerosis: modification of lowdensity lipoproteins (LDL) and an inappropriate immune response to vascular injury. The first step in disease pathogenesis is the monocyte recruitment into the subendothelial space and accumulation of modified forms of LDL (oxLDL). Continued recruitment of monocytes and T cells, and smooth muscle cell migration and proliferation, gives rise to raised fibro-fatty plaques. Recently, also DCs were detected in atherosclerotic plaques. Since these cells are capable of presenting antigens to T cells, whose activation leads to cytokine release, which play an important role in disease progression, we analyzed the relationship between oxLDL with DC differentiation and reception capacity. Methods: DCs were generated from PBMCs obtained from healthy volunteers. Monocytes were cultured in AIM-V medium, containing IL-4 and GM-CSF obtained immature DC (iDC). The mature DCs generation was obtained with add TNF-α. Plasma LDL were isolated by sequential centrifugation and oxidized in CuSO4 (1μM, 37ºC, 18h). On day 6, oxLDL was added and 24h later the cells were harvested for flow cytometry. Results: The
S83
Multiple Risk Factors in Cardiovascular Disease—Abstracts
evaluation reception capacity, the oxLDL capture less mDCs than iDCs. DCs treated with oxLDL had increased expression of CD11c, CD40 and CCR-7 when compared to DCs without treatment oxLDL. Conclusion: mDCs exhibit reduced phagocytic activity, showed decreased oxLDL capture for mDCs. The treatment with oxLDL increases the expression of markers that characterize mature DCs, among which CCR7 was the most affected molecule.
in HLA-DR and CD11c positive cells frequency when compared with DCs treated with oxLDL. The evaluation of oxLDL reception capacity, showed that DCs treated with E2 capture less than DCs without E2. Conclusions: The results suggest that E2 increases the HLA-DR+ and CD11c+ cells and oxLDL incorporation. This could have implications for DC function within the atherosclerotic plaque. Funding: none
Funding: none 182 181 EFECTS OF ESTRADIOL IN HLA-DR AND CD11C EXPRESSION AND PHAGOCYTIC ACTIVITY BY HUMAN MONOCYTE-DERIVED DENDRITIC CELLS TREATED WITH OXLDL A. Azevedo1, A. Monteiro2, M. Gidlund2, J. Barbuto1. 1Laboratory of Tumor ImmunologyUSP, São Paulo, Brazil, 2Laboratory of Immunophisiopathology-USP, São Paulo, Brazil Introduction: The female steroidal hormone, 17hestradiol (E2), triggers a wide variety of biological effects in a number of target systems. E2 is implicated in many vascular disorders.The modification of low-density lipoproteins (oxLDL) and an inappropriate immune response to vascular injury could cause atherosclerosis. Dendritic cells (DCs) were detected in atherosclerotic plaques. The DCs are the most efficient antigen presenting cells, having a distinct role as initiators and modulators of the immune response. The presence of estradiol (E2) receptors on DCs provides the possibility that E2 modulates DC functions directly, which then influence T cell responses. In the present study, we analyzed the HLA-DR and CD11c expression in DC differentiation from blood precursors. Methods: DCs were generated from peripheral blood mononuclear cells obtained from healthy volunteers. Monocytes were cultured in AIM-V medium, containing IL-4 and GM-CSF. Cultures were also supplemented or not with E2 (1mM), on day 0, and oxLDL, on 6 day. On the 7 day, we were analyzed the expression of anti-HLADR and anti-CD11c and oxLDL phagocytic activity assay by flow cytometry. Results: The DCs treated with E2 and oxLDL showed increased
S84
TADALAFIL REDUCES ENDOTHELIAL CELLS INFLAMMATORY RESPONSE INDUCED BY MYELOPEROXIDASE MODIFIED LDL (MOX-LDL) K. Zouaoui Boudjeltia1, T. Roumeguere1,2, A. Rousseau1, V. Nuyens1, P. Van Antwerpen4, J. Ducobu1, M. Vanhaeverbeek1, D. Brohee1, E. Wespes3. 1Laboratory of Experimental Medicine, Unit 222, ISPPC CHU Charleroi, Vesale Hospital, Montigny-le-Tilleul, Belgium, 2University Clinics of Brussels, Department of Urology-Erasme Hospital, Brussels, Belgium, 3Deparment of Urology, ISPPC CHU Charleroi, Charleroi, Belgium, 4Laboratory of Pharmaceutical Chemistry, Intitute of Pharmacy, Free University of Brussels, Brussels, Belgium Objective: Endothelial dysfunction and the disruption of the nitric oxide (NO)-cGMP pathway have been considered as the early common mechanism for the development of both erectile dysfunction (ED) and cardiovascular diseases. Myeloperoxidase modified LDL (Mox-LDL) can trigger inflammatory response, involved in atherosclerosis, through increasing the release of IL-8 by endothelial cells (EC). Mox-LDL have been found in the human atherosclerotic lesions and penis. Phosphodiesterases-5 (PDE-5) inhibitors are used in the treatment of ED. Inhibition of PDE 5, 6 and 9 (zaprinast) negatively attenuated NFêB activation. We aimed to test the possible anti-inflammatory effect of Tadalafil (an inhibitor of PDE-5 and PDE-11 used in the ED treatment) on EC. Methods: EC (EA.hy926) were incubated in the presence of either TNF-alpha (100 pg/ml) or Mox-LDL (200 μg/ml) or both in combination with Tadalafil (1, 3 and 5 μM). IL-8