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The effect of ibuprofen on monocyte function
Wednesday June 28, 2000: Poster Abstracts P: W23 Immune and Inflammatory Mechanisms in Atherosclerosis
198
tween prior infection with C. pneumoniae and complications of atherosclerosis. However, the potential mechanisms underlying such a relationship remain speculative. Since atherosclerotic lesions are of inflammatory nature, we investigated whether C. pneumoniae is capable of inducing antigen-specific stimulation of intraplaque T cells. Methods: T cell lines were generated from atherosclerotic plaques (AP) (carotid endarterectomies, n = 8) and tested for their responsiveness against C. pneumoniae elementary bodies; T cell clones generated from the AP derived T cell lines of 2 patients were tested also. Similarly, the responses of peripheral blood T cells from the same patients were analyzed. Proliferation was measured using 3H-thymidine incorporation and expressed as stimulation index (SI). Results: Four out of eight T cell lines from carotid AP responded to C. pneumoniae. This proliferation could be inhibited with antibodies against HLA-DR. From a total of 57 T cell clones, 17 were C. pneumoniae responsive (3 < SI < 40, mean = 8.0). Of these clones, all except one were Thl-type (secreting high levels of IFN- and low levels of 1L-4 upon stimulation). Interestingly, peripheral blood T lymphocytes from only two patients responded to C. pneumoniae. One of these patients also showed a plaque T cell response, the other not. Concinsions: Our results clearly show that C. pneumoniae responsive T cells are present in the symptomatic AP of 4 out of 8 patients. These results indicate that C. pneumoniae is capable of triggering the plaque inflammatory response in a subgroup of patients, a phenomenon with potential implications for the stability of plaques.
WeP47:W23 [ Induction of CD36 by all-trans retinoic acid I
D.M. Wuttge, A. Romert ] , G.K. Hansson, U. Eriksson I , A. Sirsjt. Center for
Molecular Medicine, Karolinska Hospital; I Ludwig Institute for Cancer Research, Stockholm Branch, Stockholm, Sweden Objective: To investigate the role of the vitamin A derivate all-trans retinoic acid (atRA) in the regulation of CD36, an important scavenger receptor for the uptake of oxidized lipoproteins (oxLDL). CD36 is highly expressed in the human atherosclerotic lesions and has previously been shown to be regulated through retinoic x receptor (RXR). Our study provides evidence, that specific retinoic acid receptor (RAR) agonists also induce expression of CD36. Methods: THP-1 cells were incubated with different RAR- or RXR ligands and atRA with or without specific antagonists. CD36 expression was measured by flow cytometry (FACS) and PCR. Uptake of oxLDL was also analysed by FACS. Results: Addition of atRA to THP-1 cells strongly induced CD36 protein and mRNA after 2 days. This was functionally mirrowed by an increased uptake of oxLDL. Specific RAR agonists increased CD36 expression. RAR antagonist reduced the response of atRA, hut failed to abolish CD36 induction completely. This indicates that a different pathway is additionally involved besides signaling through RAR. Combination of both RAR and RXR ligands gave a stronger upregulation, than RAR ligation alone. Condnsions: The study shows that the induction of CD36 by retinoids are mediated through both RAR and RXR. The upregulation of CD36 by atRA might be important in the pathogensis of atherosclerosis.
I WeP48:W23 [ Vitamin E succinate induces apoptosis in hematopoietic and cancer cell lines: Structural requirements and role of protein kinase J
J. Neuzil I , A. Schroder I , T. Weber I , N. Gellert I , M. Lu 2, M. Sticha3 , R.J. Coffey I , C. Weber I . llnstitutefor Prevention of Cardiovascular
Diseases, Ludwig Maximilians University, Munich, Germany; 2Medical Center North, Vanderbilt University, Nashville, TN, USA; 3Faculty of Science, Charles University, Prague, Czech Republic The vitamin E analogue ot-tocopheryl succinate (a-TOS) can induce apoptosis. We show that the pro-apoptotic activity of ot-TOS in hematopoetic and cancer cell lines involves inhibition of protein kinase C (PKC) and lysosomal/mitochondrial destabilisation. Phorbol myristyl acetate prevented multiple features of ot-TOS-induced apoptosis, while ot-TOS reduced PKC activity and increased protein phosphatase 2A (PP2A) activity. Studies with other effectors inferred that PKCot inhibition is due to PP2A activation. Sensitisation by PKCa antisense oligonucleotides and protection by PKCc~ overexpression confirmed the role of PKCu inhibition in apoptosis. Overexpression of bcl-2 or its gain- or loss-of-function mutants indicated that modulation of bcl-2 by PKC/PP2A is a mechanism mediating ot-TOS-induced apoptosis. Structural analogues of a-TOS revealed that, in addition to a-tocopheryl, the succinyl
moiety is required for effective apoptosis. Notably, the pro-apoptotic effects of ot-TOS were largely restricted to malignant cells, while normal cells were rather resistant. In mice with colon cancer xenografts, ot-TOS suppressed tumour growth. Our findings show that the a-tocopheryl moiety of ot-TOS is involved in dysregulation of PKC/PP2A signalling, among others affecting the function of bcl-2, and the succinyl moiety in lysosmal/mitochondrial destabilisation. Further, they epitomise elimination of malignant cells by a pharmacologically relevant compound.
I W e P 4 9 : W 2 3 ] The progression of atherosclerotic lesions in
hypercholesterolemic rabbits is delayed by chronic treatment with the iNOS inhibitor, L-NIL T.J. Verbeuren I , D. Behr-Roussel2, S. S imonet J , E. Bonhomme j , S. Coumailleau 1, A. Cordi 1, B. Serkiz I , J.-N. Fabiani 2, A. Rupin j . IServier
Research Institute; 2Suresnes, Broussais Hospital, Paris, France Objective: The presence of an active inducible nitric oxide synthase (iNOS) in advanced atherosclerotic lesions is now well-accepted but its impact on the development of atherosclerosis is not yet known. The aim of this study was to examine the implication of iNOS in the progression of atherosclerosis. Methods: Rabbits were fed a 0.3% cholesterol diet for 24 weeks. One group of 9 rabbits was then examined (BASELINE). The remaining animals were kept on the diet for another 12 weeks and treated with L-NIL by mini-osmotic pumps delivering the drug intravenously (5 mg/kg/day, L-NIL, n = 8), its vehicle (SALINE, n = 9), or L-arginine (2.25% in drinking water, L-ARG, n = 8). Results: Most biological parameters (i.e. cholesterol levels, blood pressure) were not affected by the treatments. L-NIL delayed the progression of atherosclerosis on the thoracic aorta (the intima/media ratio increased from 0.98 4- 0.04 (BASELINE) to 1.63 4- 0.17 (p < 0.05) in the SALINE group but not in the L-NIL group (1.32 4- 0.23; NS) and the coronary arteries (the intima/media ratio decreased between the SALINE group and the L-NIL group (0.96 4- 0.19 versus 0.34 4- 0.19, p < 0.05). Lesion development was partially limited in the abdominal aorta by both L-NIL or L-arginine. Inflammatory and smooth muscle cell infiltration were not modified by the treatments but the significant increase in the intimal collagen content of thoracic aortas, from 21.8 4- 4.5%, BASELINE to 61.7 4- 7.0%, SALINE and 57.2 4- 7.2%, L-ARG groups was not found in the L-NIL group (40.75 4- 10.6%, NS). Conclusions: In conclusion, while L-arginine treatment does not appear to have a beneficial role in severe atherosclerotic lesion progression, chronic treatment with the iNOS inhibitor, L-NIL, limits the progression of severe pre-existing atherosclerotic lesions.
I WeP50:W23 I The effect of ibuprofen on monocyte function D. Zapolska-Downar, A. Zapolski-Downar, M. Markiewski, H. Bukowska, B. Millo, M. Naruszewicz. Pomeranian Medical University, Szczecin, Poland Tobacco smoking is a major risk factor of atherosclerosis and a useful model for studying chronic inflammation. We compared monocyte function and lipid profiles in smokers and nonsmokers, before and after oral ibuprofen intake. The adhesion of peripheral blood monocytes (PBM) to native and TNF~-stimulated human umbilical vein endothelial cells (HUVEC), as well as superoxide (O~) levels and reactive oxygen species (ROS) production in resting and PMA-stimulated PBM were determined. A group of 10 smokers without any other coronary risk factor was compared with an age-matched group of 10 nonsmokers. Tests were performed before and after a two-week course of oral ibuprofen (600 mg/day). In the smokers group before ibuprofen, monocyte adhesion to native and TNFc~-stimulated HUVEC was increased (p < 0.001), as were O~ levels in native and PMA-stimulated PBM (p _< 0.01). Ibuprofen reduced the adhesion of native and stimulated monocytes to HUVEC (p _< 0.001) and O] generation by resting and PMA-stimulated cells (p _< 0.01) in both groups. ROS production by resting and PMA-stimulated monocytes was reduced in smokers (p _< 0.001 and ns, respectively) and nonsmokers (p < 0.001 and p _< 0.05, respectively). Interestingly, ibuprofen increased HDL-C levels in smokers (p < 0.05) and nonsmokers (p < 0.01 ). In conclusion, oral administration of ibuprofen reduced the adhesion of monocytes to HUVEC, suppressed oxidative stress and increased HDL-C levels in smokers and nonsmokers.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
198
tween prior infection with C. pneumoniae and complications of atherosclerosis. However, the potential mechanisms underlying such a relationship remain speculative. Since atherosclerotic lesions are of inflammatory nature, we investigated whether C. pneumoniae is capable of inducing antigen-specific stimulation of intraplaque T cells. Methods: T cell lines were generated from atherosclerotic plaques (AP) (carotid endarterectomies, n = 8) and tested for their responsiveness against C. pneumoniae elementary bodies; T cell clones generated from the AP derived T cell lines of 2 patients were tested also. Similarly, the responses of peripheral blood T cells from the same patients were analyzed. Proliferation was measured using 3H-thymidine incorporation and expressed as stimulation index (SI). Results: Four out of eight T cell lines from carotid AP responded to C. pneumoniae. This proliferation could be inhibited with antibodies against HLA-DR. From a total of 57 T cell clones, 17 were C. pneumoniae responsive (3 < SI < 40, mean = 8.0). Of these clones, all except one were Thl-type (secreting high levels of IFN- and low levels of 1L-4 upon stimulation). Interestingly, peripheral blood T lymphocytes from only two patients responded to C. pneumoniae. One of these patients also showed a plaque T cell response, the other not. Concinsions: Our results clearly show that C. pneumoniae responsive T cells are present in the symptomatic AP of 4 out of 8 patients. These results indicate that C. pneumoniae is capable of triggering the plaque inflammatory response in a subgroup of patients, a phenomenon with potential implications for the stability of plaques.
WeP47:W23 [ Induction of CD36 by all-trans retinoic acid I
D.M. Wuttge, A. Romert ] , G.K. Hansson, U. Eriksson I , A. Sirsjt. Center for
Molecular Medicine, Karolinska Hospital; I Ludwig Institute for Cancer Research, Stockholm Branch, Stockholm, Sweden Objective: To investigate the role of the vitamin A derivate all-trans retinoic acid (atRA) in the regulation of CD36, an important scavenger receptor for the uptake of oxidized lipoproteins (oxLDL). CD36 is highly expressed in the human atherosclerotic lesions and has previously been shown to be regulated through retinoic x receptor (RXR). Our study provides evidence, that specific retinoic acid receptor (RAR) agonists also induce expression of CD36. Methods: THP-1 cells were incubated with different RAR- or RXR ligands and atRA with or without specific antagonists. CD36 expression was measured by flow cytometry (FACS) and PCR. Uptake of oxLDL was also analysed by FACS. Results: Addition of atRA to THP-1 cells strongly induced CD36 protein and mRNA after 2 days. This was functionally mirrowed by an increased uptake of oxLDL. Specific RAR agonists increased CD36 expression. RAR antagonist reduced the response of atRA, hut failed to abolish CD36 induction completely. This indicates that a different pathway is additionally involved besides signaling through RAR. Combination of both RAR and RXR ligands gave a stronger upregulation, than RAR ligation alone. Condnsions: The study shows that the induction of CD36 by retinoids are mediated through both RAR and RXR. The upregulation of CD36 by atRA might be important in the pathogensis of atherosclerosis.
I WeP48:W23 [ Vitamin E succinate induces apoptosis in hematopoietic and cancer cell lines: Structural requirements and role of protein kinase J
J. Neuzil I , A. Schroder I , T. Weber I , N. Gellert I , M. Lu 2, M. Sticha3 , R.J. Coffey I , C. Weber I . llnstitutefor Prevention of Cardiovascular
Diseases, Ludwig Maximilians University, Munich, Germany; 2Medical Center North, Vanderbilt University, Nashville, TN, USA; 3Faculty of Science, Charles University, Prague, Czech Republic The vitamin E analogue ot-tocopheryl succinate (a-TOS) can induce apoptosis. We show that the pro-apoptotic activity of ot-TOS in hematopoetic and cancer cell lines involves inhibition of protein kinase C (PKC) and lysosomal/mitochondrial destabilisation. Phorbol myristyl acetate prevented multiple features of ot-TOS-induced apoptosis, while ot-TOS reduced PKC activity and increased protein phosphatase 2A (PP2A) activity. Studies with other effectors inferred that PKCot inhibition is due to PP2A activation. Sensitisation by PKCa antisense oligonucleotides and protection by PKCc~ overexpression confirmed the role of PKCu inhibition in apoptosis. Overexpression of bcl-2 or its gain- or loss-of-function mutants indicated that modulation of bcl-2 by PKC/PP2A is a mechanism mediating ot-TOS-induced apoptosis. Structural analogues of a-TOS revealed that, in addition to a-tocopheryl, the succinyl
moiety is required for effective apoptosis. Notably, the pro-apoptotic effects of ot-TOS were largely restricted to malignant cells, while normal cells were rather resistant. In mice with colon cancer xenografts, ot-TOS suppressed tumour growth. Our findings show that the a-tocopheryl moiety of ot-TOS is involved in dysregulation of PKC/PP2A signalling, among others affecting the function of bcl-2, and the succinyl moiety in lysosmal/mitochondrial destabilisation. Further, they epitomise elimination of malignant cells by a pharmacologically relevant compound.
I W e P 4 9 : W 2 3 ] The progression of atherosclerotic lesions in
hypercholesterolemic rabbits is delayed by chronic treatment with the iNOS inhibitor, L-NIL T.J. Verbeuren I , D. Behr-Roussel2, S. S imonet J , E. Bonhomme j , S. Coumailleau 1, A. Cordi 1, B. Serkiz I , J.-N. Fabiani 2, A. Rupin j . IServier
Research Institute; 2Suresnes, Broussais Hospital, Paris, France Objective: The presence of an active inducible nitric oxide synthase (iNOS) in advanced atherosclerotic lesions is now well-accepted but its impact on the development of atherosclerosis is not yet known. The aim of this study was to examine the implication of iNOS in the progression of atherosclerosis. Methods: Rabbits were fed a 0.3% cholesterol diet for 24 weeks. One group of 9 rabbits was then examined (BASELINE). The remaining animals were kept on the diet for another 12 weeks and treated with L-NIL by mini-osmotic pumps delivering the drug intravenously (5 mg/kg/day, L-NIL, n = 8), its vehicle (SALINE, n = 9), or L-arginine (2.25% in drinking water, L-ARG, n = 8). Results: Most biological parameters (i.e. cholesterol levels, blood pressure) were not affected by the treatments. L-NIL delayed the progression of atherosclerosis on the thoracic aorta (the intima/media ratio increased from 0.98 4- 0.04 (BASELINE) to 1.63 4- 0.17 (p < 0.05) in the SALINE group but not in the L-NIL group (1.32 4- 0.23; NS) and the coronary arteries (the intima/media ratio decreased between the SALINE group and the L-NIL group (0.96 4- 0.19 versus 0.34 4- 0.19, p < 0.05). Lesion development was partially limited in the abdominal aorta by both L-NIL or L-arginine. Inflammatory and smooth muscle cell infiltration were not modified by the treatments but the significant increase in the intimal collagen content of thoracic aortas, from 21.8 4- 4.5%, BASELINE to 61.7 4- 7.0%, SALINE and 57.2 4- 7.2%, L-ARG groups was not found in the L-NIL group (40.75 4- 10.6%, NS). Conclusions: In conclusion, while L-arginine treatment does not appear to have a beneficial role in severe atherosclerotic lesion progression, chronic treatment with the iNOS inhibitor, L-NIL, limits the progression of severe pre-existing atherosclerotic lesions.
I WeP50:W23 I The effect of ibuprofen on monocyte function D. Zapolska-Downar, A. Zapolski-Downar, M. Markiewski, H. Bukowska, B. Millo, M. Naruszewicz. Pomeranian Medical University, Szczecin, Poland Tobacco smoking is a major risk factor of atherosclerosis and a useful model for studying chronic inflammation. We compared monocyte function and lipid profiles in smokers and nonsmokers, before and after oral ibuprofen intake. The adhesion of peripheral blood monocytes (PBM) to native and TNF~-stimulated human umbilical vein endothelial cells (HUVEC), as well as superoxide (O~) levels and reactive oxygen species (ROS) production in resting and PMA-stimulated PBM were determined. A group of 10 smokers without any other coronary risk factor was compared with an age-matched group of 10 nonsmokers. Tests were performed before and after a two-week course of oral ibuprofen (600 mg/day). In the smokers group before ibuprofen, monocyte adhesion to native and TNFc~-stimulated HUVEC was increased (p < 0.001), as were O~ levels in native and PMA-stimulated PBM (p _< 0.01). Ibuprofen reduced the adhesion of native and stimulated monocytes to HUVEC (p _< 0.001) and O] generation by resting and PMA-stimulated cells (p _< 0.01) in both groups. ROS production by resting and PMA-stimulated monocytes was reduced in smokers (p _< 0.001 and ns, respectively) and nonsmokers (p < 0.001 and p _< 0.05, respectively). Interestingly, ibuprofen increased HDL-C levels in smokers (p < 0.05) and nonsmokers (p < 0.01 ). In conclusion, oral administration of ibuprofen reduced the adhesion of monocytes to HUVEC, suppressed oxidative stress and increased HDL-C levels in smokers and nonsmokers.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000