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The prostaglandin e receptor subtype EP4 mediates PGE2-dependent plaque instability in humans
328 THERAPY FIBRATE?
XVII S.I.S.A. National Congress OF
COMBINED
HYPERLIPIDEMIA.
STATIN
OR
A Branchi 1, AM Fiorenza 2, A Torri 2, D Sornmariva 2 1Department of Internal Medicine, University of Milan, Maggiore Hospital IRCCS, Milan; 2Department of Internal Medicine 1, G. Salvini Hospital, Garbagnate Milanese
THE PROSTAGLANDIN E RECEPTOR SUBTYPE EP4 MEDIATES PGE2-DEPENDENT PLAQUE I N S T A B I L I T Y I N HUMANS M Bucci, M Fazia, A I e z z i , B Pini, C Cuccurullo, M Zucchelli, D De Cesare, S Ucchino, F Spigonardo, M De Luca, F Cuccurullo, A Mezzetti, F Cipollone University of Chieti "G. d'Annunzio", Italy
Two classes of drugs can be used in the treatment of combined hyperlipidemia: statins and fibrates. Statins are more powerful than fibrates in decreasing serum cholesterol, but are less effective in reducing serum triglycerides and in increasing HDL cholesterol. Monotherapy is often unsatisfactory and association therapy with a statin and a fibrate may be advisable. However, combination therapy brings about an increased risk of untoward side effects, so that its use must be confined to cases where monotherapy is proven to be unsatisfactory. We have compared the effects of atorvastatin with those of fenofibrate, to assess the best starting therapy of combined hyperlipidemia. Atorvastatin 10 mg was given to 28 patients and micronized fenofibrate 200 mg to 27 comparable patients. In atorvastatin group, after 3 months, serum triglycerides decreased from 3394-17 to 2334-15 mg/dL (-31%) and cholesterol from 3094-8 to 2144-8 mg/dL (-31%). HDL cholesterol achieved a non significant increase by 4%. In fenofibrate group, serum triglycerides decreased from 3394-22 to 1714-13 mg/dL (-50%) and cholesterol from 3104-8 to 2434-7 mg/dL (-22%). HDL cholesterol significantly increased by 9% (from 454-2 to 494-2 mg/dL). Total/HDL cholesterol ratio decreased by 32% in atorvastatin and by 27% in fenofibrate group. The effect of atorvastatin was better than that of fenofibrate in improving the lipid risk profile of patients, suggesting that statin may be the starting drug in combined hyperlipidemia.
We have previously demonstrated that enhanced expression of cyclooxygenase-2/PGE synthase-l(COX-2/mPGES-t) is associated to biosynthesis of PGE2-dependent metalloproteinases (MMPs) leading to plaque instability. However, the specific receptor by which PGE2 may influence NMP generation in plaque is still unknown. The aim of this study was to characterize the expression of PGE2 receptors (EP1-4) in human plaques and to correlate it with inflammatory infiltration, COX2/mPGES-1 and MMP expression. Plaques were obtained from 60 patients who underwent carotid endarterectomy, and were divided into 2 groups (Symptomatic and Asymptomatic) according to recent evidence of stroke. COX-2, mPGES-1, EP1-4, NMP expression was analyzed by RT-PCR, western blot and immunocytochemistry; MMP activity was detected by zymography; CD68+, CD3+ and HI_A-DR+ cells were detected by immunohistochemistry. We observed strong EP4 immunoreactivity, very weak EP2 staining and no expression of EPI and EP3 in atherosclerotic plaques. EP4 was more abundant in COX2/mPGES-1, MNP-rich symptomatic lesions, while there were no differences in EP2 expression. EP4 co-localized with HLA-DR+ macrophages in plaque shoulder, and MMP induction by PGE2 in vitro was inhibited by EP4 antagonist L-161,982 but not by its inactive analog L-162,983 or EP2 antagonist. In conclusion, this study demonstrated the association between EP4 overexpression and PGE2-dependent plaque instability, identifying a new pharmacological target for atherosclerotic plaques stabilization.
HIGH D E N S I T Y L I P O P R O T E I N S I N D U C E CYCLOOXYGENASE-2 EXPRESSION AND PROSTACYCLIN RELEASE IN HUMAN ENDOTHELIAL CELLS V I A A P38 MAPK/CRE DEPENDENT PAT H W A Y
EFFECT OF L E R C A N I D I P I N E AND ITS ENANTIOMERS ON MACROPHAGE FUNCTIONS I N V O L V E D I N PLAQUE S T A B I L I T Y M Canavesi 1, N Baldini 2, S Bellosta 1, M Monetti 1, A Leonardi 3, F Bernini ~
Callegari E*, Norata G D * , I n o u e H, Catapano A L * . ~Department of Pharmacological Sciences, University of Milan, Italy. Beyond reverse cholesterol transport, HDL possesses several antF atherosclerotic effects, including the induction of prostacyclin (PGI2). HDL supplies endothelial cells with arachidonic acid (AA) leading to the synthesis of prostacyclin. The rate-limiting step in the conversion of AA to eicosanoids is the activity of cyclooxygenase (Cox). In the present study we investigated the molecular mechanisms involved in inducing Cox-2 in endothelial cells by HDL3. Huvec cells were incubated with HDL3 (30~Jg/mL) from 2 to 18 hours. Cox-2 was induced in a time and dose dependent manner and localized mainly in the perinuclear area. HDL3 activated ERK1/2, p38 MAPK and CREB; moreover transient transfection experiments showed that CRE is required for HDL-induced Cox-2 transcription. Using specific inhibitors of ERK1/2 and of p38 MAPK we demonstrated that p38 MAPK activation by HDL3 is involved in Cox2 transcription and mRNA stabilization. As a consequence of Cox-2inducti~ by HDL3 prostacyclin production increased without changes in TXA2 or PGE2 production. Incubation with indomethacin eptyl ester, a Cox-2 selective inhibitor, reverted this effect. We conclude that p38 MAPK is involved in the regulation of Cox-2 mRNA stability and transcription in endothelial cells by HDL3. The understanding of these mechanisms may provide new insights into the antiatherogenic role of HDL.
IDep. of Pharmacological Sciences, University of Milan; 2Dep. of Pharmacological and Biological Sciences and Applied Chemistry, University of Parma; 3Recordati S.p.A., Milan, Italy. Studies in experimental animal models and in human have demonstrated the potential direct antiatherosclerotic effects of calcium antagonists (CA). In this study we evaluated the effect of lercanidipine, a CA of the third generation, and its enantiomers on in vitro macrophages ability to accumulate lipids and to secrete matrix metalloproteinases (MMPs), two major processes involved in atherogenesis. Lercanidipine inhibited up to 90%, in a concentrationdependent manner (10-6-10-5M), cholesterol esterification in macrophages. Cells exposed to the drug had also a reduced content of acetylated LDL derived cholesterol both in the free and esterified form. In addition, lercanidipine inhibited the release of MMP-9 in the extracellular medium. Experiments performed with lercanidipine enantiomers or various dihydropyridine derivatives endowed with different lipophilicity and affinity for calcium channels indicate that the effect could be related to the lipophilic, but not to the calcium channel blockade properties of these molecules. Lercanidipine retained its effect even after 3 days of wash out. Interestingly, in these conditions the active concentration was as low as 10-9M, a concentration similar to that observed in vivo in animals and humans. In conclusion our data demonstrabe that lercanidipine may inhibit macrophage functions involved in atherogenesis and plaque stability with a mechanism independent of calcium channel blockade.
XVII S.I.S.A. National Congress OF
COMBINED
HYPERLIPIDEMIA.
STATIN
OR
A Branchi 1, AM Fiorenza 2, A Torri 2, D Sornmariva 2 1Department of Internal Medicine, University of Milan, Maggiore Hospital IRCCS, Milan; 2Department of Internal Medicine 1, G. Salvini Hospital, Garbagnate Milanese
THE PROSTAGLANDIN E RECEPTOR SUBTYPE EP4 MEDIATES PGE2-DEPENDENT PLAQUE I N S T A B I L I T Y I N HUMANS M Bucci, M Fazia, A I e z z i , B Pini, C Cuccurullo, M Zucchelli, D De Cesare, S Ucchino, F Spigonardo, M De Luca, F Cuccurullo, A Mezzetti, F Cipollone University of Chieti "G. d'Annunzio", Italy
Two classes of drugs can be used in the treatment of combined hyperlipidemia: statins and fibrates. Statins are more powerful than fibrates in decreasing serum cholesterol, but are less effective in reducing serum triglycerides and in increasing HDL cholesterol. Monotherapy is often unsatisfactory and association therapy with a statin and a fibrate may be advisable. However, combination therapy brings about an increased risk of untoward side effects, so that its use must be confined to cases where monotherapy is proven to be unsatisfactory. We have compared the effects of atorvastatin with those of fenofibrate, to assess the best starting therapy of combined hyperlipidemia. Atorvastatin 10 mg was given to 28 patients and micronized fenofibrate 200 mg to 27 comparable patients. In atorvastatin group, after 3 months, serum triglycerides decreased from 3394-17 to 2334-15 mg/dL (-31%) and cholesterol from 3094-8 to 2144-8 mg/dL (-31%). HDL cholesterol achieved a non significant increase by 4%. In fenofibrate group, serum triglycerides decreased from 3394-22 to 1714-13 mg/dL (-50%) and cholesterol from 3104-8 to 2434-7 mg/dL (-22%). HDL cholesterol significantly increased by 9% (from 454-2 to 494-2 mg/dL). Total/HDL cholesterol ratio decreased by 32% in atorvastatin and by 27% in fenofibrate group. The effect of atorvastatin was better than that of fenofibrate in improving the lipid risk profile of patients, suggesting that statin may be the starting drug in combined hyperlipidemia.
We have previously demonstrated that enhanced expression of cyclooxygenase-2/PGE synthase-l(COX-2/mPGES-t) is associated to biosynthesis of PGE2-dependent metalloproteinases (MMPs) leading to plaque instability. However, the specific receptor by which PGE2 may influence NMP generation in plaque is still unknown. The aim of this study was to characterize the expression of PGE2 receptors (EP1-4) in human plaques and to correlate it with inflammatory infiltration, COX2/mPGES-1 and MMP expression. Plaques were obtained from 60 patients who underwent carotid endarterectomy, and were divided into 2 groups (Symptomatic and Asymptomatic) according to recent evidence of stroke. COX-2, mPGES-1, EP1-4, NMP expression was analyzed by RT-PCR, western blot and immunocytochemistry; MMP activity was detected by zymography; CD68+, CD3+ and HI_A-DR+ cells were detected by immunohistochemistry. We observed strong EP4 immunoreactivity, very weak EP2 staining and no expression of EPI and EP3 in atherosclerotic plaques. EP4 was more abundant in COX2/mPGES-1, MNP-rich symptomatic lesions, while there were no differences in EP2 expression. EP4 co-localized with HLA-DR+ macrophages in plaque shoulder, and MMP induction by PGE2 in vitro was inhibited by EP4 antagonist L-161,982 but not by its inactive analog L-162,983 or EP2 antagonist. In conclusion, this study demonstrated the association between EP4 overexpression and PGE2-dependent plaque instability, identifying a new pharmacological target for atherosclerotic plaques stabilization.
HIGH D E N S I T Y L I P O P R O T E I N S I N D U C E CYCLOOXYGENASE-2 EXPRESSION AND PROSTACYCLIN RELEASE IN HUMAN ENDOTHELIAL CELLS V I A A P38 MAPK/CRE DEPENDENT PAT H W A Y
EFFECT OF L E R C A N I D I P I N E AND ITS ENANTIOMERS ON MACROPHAGE FUNCTIONS I N V O L V E D I N PLAQUE S T A B I L I T Y M Canavesi 1, N Baldini 2, S Bellosta 1, M Monetti 1, A Leonardi 3, F Bernini ~
Callegari E*, Norata G D * , I n o u e H, Catapano A L * . ~Department of Pharmacological Sciences, University of Milan, Italy. Beyond reverse cholesterol transport, HDL possesses several antF atherosclerotic effects, including the induction of prostacyclin (PGI2). HDL supplies endothelial cells with arachidonic acid (AA) leading to the synthesis of prostacyclin. The rate-limiting step in the conversion of AA to eicosanoids is the activity of cyclooxygenase (Cox). In the present study we investigated the molecular mechanisms involved in inducing Cox-2 in endothelial cells by HDL3. Huvec cells were incubated with HDL3 (30~Jg/mL) from 2 to 18 hours. Cox-2 was induced in a time and dose dependent manner and localized mainly in the perinuclear area. HDL3 activated ERK1/2, p38 MAPK and CREB; moreover transient transfection experiments showed that CRE is required for HDL-induced Cox-2 transcription. Using specific inhibitors of ERK1/2 and of p38 MAPK we demonstrated that p38 MAPK activation by HDL3 is involved in Cox2 transcription and mRNA stabilization. As a consequence of Cox-2inducti~ by HDL3 prostacyclin production increased without changes in TXA2 or PGE2 production. Incubation with indomethacin eptyl ester, a Cox-2 selective inhibitor, reverted this effect. We conclude that p38 MAPK is involved in the regulation of Cox-2 mRNA stability and transcription in endothelial cells by HDL3. The understanding of these mechanisms may provide new insights into the antiatherogenic role of HDL.
IDep. of Pharmacological Sciences, University of Milan; 2Dep. of Pharmacological and Biological Sciences and Applied Chemistry, University of Parma; 3Recordati S.p.A., Milan, Italy. Studies in experimental animal models and in human have demonstrated the potential direct antiatherosclerotic effects of calcium antagonists (CA). In this study we evaluated the effect of lercanidipine, a CA of the third generation, and its enantiomers on in vitro macrophages ability to accumulate lipids and to secrete matrix metalloproteinases (MMPs), two major processes involved in atherogenesis. Lercanidipine inhibited up to 90%, in a concentrationdependent manner (10-6-10-5M), cholesterol esterification in macrophages. Cells exposed to the drug had also a reduced content of acetylated LDL derived cholesterol both in the free and esterified form. In addition, lercanidipine inhibited the release of MMP-9 in the extracellular medium. Experiments performed with lercanidipine enantiomers or various dihydropyridine derivatives endowed with different lipophilicity and affinity for calcium channels indicate that the effect could be related to the lipophilic, but not to the calcium channel blockade properties of these molecules. Lercanidipine retained its effect even after 3 days of wash out. Interestingly, in these conditions the active concentration was as low as 10-9M, a concentration similar to that observed in vivo in animals and humans. In conclusion our data demonstrabe that lercanidipine may inhibit macrophage functions involved in atherogenesis and plaque stability with a mechanism independent of calcium channel blockade.