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Reverse cholesterol transport. A target for innovative treatments against cardiovascular disease
INHIBITION OF ACETYL LDL ENDOCYTOSIS IN MACROPHAGES BY HMG-CoA REDUCTASE INHIBITORS
REVERSE CHOLESTEROL TRANSPORT. A TARGET FOR INNOVATIVE TREATMENTS AGAINST CARDIOVASCULAR DISEASE G. Franceschini Center E. Grossi Paoletti, Institute of Pharmacological Sciences, via Balzaretti 9, 20133 Milano, Italy
F. Bernini#, 5I. Scurati*, G. Bonfadini*, and R. FumagaUi* #Institute of Pharmacology and Pharmacognosy, University of Parma, Via delle Scienze, 43100 Parma, Italy; *Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
Reverse cholesterol transport identifies a series of metabolic events resulting in the transport of excess cholesterol from peripheral tissues, including the arterial wall, to the liver for excretion. High density lipoproteins (HDL) ai'e the vehicle of cholesterol in this reverse transport, a function believed to explain the inverse correlation between plasma HDL levels and the risk for CHD. More recently, low plasma HDL levels have also been associated with an increased rate of restenosis after coronary angioplasty. An attempt to stimulate reverse cholesterol transport may hold promise in the prevention and treatment of arterial diseases. Among drugs affecting lipoprotein metabolism, probucol stimulates the activity of the cholesteryl ester transfer protein and consequently facilitates the reverse transport. Other agents, including certain lipid-lowering drugs, hormones and microsomal enzyme inducers are affective in raising plasma HDL levels. More innovative approaches employ recombinant apolipoproteins or gene therapy. One case, i.e., that of apolipoprotein A-I, is entering the clinical field; the recombinant apoA-IMilano mutant proved effective in animal models, possibly because of a combined cholesterol removing/fibrinolytic activity. In the case of gene therapy, the transfer of foreign DNA to the arterial wall applied successfully to atherosclerotic arteries, achieving biological and therapeutic effects. It is thus time for thinking at future treatments of atherosclerosis, which combine the classical lipid-lowering approach with innovative methods to promote cholesterol removal from the arterial wall.
Mevalonate starvation induced by HMG-CoA reductase inhibitors reduce cholesterol accumulation promoted in macrophages by acetylated LDL (Ac-LDL). In the present study we utilized the new synthetic HMG-CoA reductase inhibitor fluvastatin to investigate the mechanism of this effect. Our results indicate that fluvastatin inhibits more than 125 50%, in a concentration-dependent manner, I-AcLDL degradation by macrophages. This effect was not due to a decrease of lysosomal enzyme activity and it was paralleled by the retention of AcLDL-associated cholesteryl ester in the incubation medium. The ability of fluvastatin to inhibit acetyl LDL degradation was completely overcome by mevalonate, geranylgeraniol, but not by famesol. The inhiNtory effect of fluvastatin on acLDL degradation was shared by another HMG-CoA reductase inhibitor, simvastatin. Evaluation at 4°C of I-AcLDL binding to plasma membrane suggests that the inhibitory effect of fluvastatin on lipoprotein calabolism was not due to a decrease expression of scavenger receptors. Fluorescent microscope analysis of cellular internalization of acetyl-LDL labeled with the fluorochrome DiI demonstrated that fluvastatin inhibits lipoprotein endocytosis: an effect reversed by mevalonate. The ability of fluvastatin to reduce acetyl LDL catabolism and cholesterol esterification was more pronounced in cholesterol enriched cells as compared to normal cells. In conclusion, the present results provide "in vitro" evidence of the involvement of the mevalonate pathway in Ac-LDL endocytosis in macrophages.
PHARMACOKINETIC-PHARMACODYNAMIC MODELLING OFTHE ANTI-LIPOLYTIC EFFECTS OF THE ADENOSINE A1-RECEPTOR AGONIST N%(P-SULFOPHENYL)ADENOSINE (SPA) IN RATS
PLATELET-RELEASED NAP-2 VARIANTS INDUCE PMN LEUKOCYTE I N T R A C E L L U L A R CALCIUM CHANGES ([Ca2+]i). Paola Piccardoni, Virgilio Evangelista, Antonio Piccoli, Alfred Walz and Chiara Cerletti. "G.Bizzozero" Laboratory of Platelet and Leucocyte Pharmacology, Istituto Rioerche Farmacol. Mario Negri, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy and Theodor Kocher Institut, Bern, Switzerland
E.A. van Schaick, H.J.M.M. de Greef, M.W.E. Langemeijer, A.P. IJzerman and M. Danhof Divisions of Pharmacology and Medicinal Chemistry, Leiden~Amsterdam Center for Drug Research, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands.
Activated platelets may stimulate PMN leukocyte function by releasing soluble products. The aim of this study was to investigate the effect of supernatants of thrombin-activated platelets on PMN [Ca2~]i and to characterize the platelet release product(s) responsible for PMN stimulation. Platelet supernatants induced a concentration-dependent [Ca2+]i increase in Fura-2qoaded PMN (0.5x107/ml) from a resting value of 169.7+19.7 nM to 1993 + 284 nM (n= 4) in the presence of 10~ platelets/ml, Complete ATP removal by apyrase treatment only partially reduced PMN stimulatory effect of platelet supernatant, suggesting the presence of other active compounds. Heat (100 ° C, 15 min) and trypsin or elastase treatment of platelet supernatants significantly reduced their maximal activity by more than 50%; cathepsin G on the other hand increased platelet supernatants activity. FPLC and HPLC procedures allowed isolation of proteins (of about 7.500 d) responsible for the PMN stimulatory activity released in platelet supernatants. The N-terminal sequence analysis of purified active fractions revealed the presence of two peptides of 73 and 74 amino acids, respectively; both are truncated forms of CTAP-III, corresponding to NAP-2, but having 3 or 4 additional amino acids at the N-terminus: Asp-Leu-Tyr... and Ser-AspLeu-Tyr... The treatment of platelets with different protease inhibitors (EDTA, PMSF, leupeptin, iodacetamide) did not significantly modify the calcium stimulatory activity of supernatants, ruling out the possibility that NAP-2 variants may be generated from inactive precursors by platelet proteases released upon thrombin stimulation. Moreover, lysates obtained bE platelet sonication treated with apyrase also induced PMN [CaZ+]i increase, suggesting that active NAP-2 variants are preformed in platelets. In conclusion, our study indicates that NAP-2 variants are released by activated platelets and induce PMN [Ca2+]i.
For therapeutic use of adenosine analogues, it is important to develop compounds with selectivity of action. In this study tissue-selectivity of the adenosine A~-receptor agonist SPA was investigated by quantification of the hemodynamic and anti-lipolytic effects on the basis of an integrated pharmacokinetic-pharmacodynamic (PK/PD) model. Pharmacoldnetics and pharmacodynamics were determined after intravenous administration of N 6(p-sulfophenyl)adenosine (SPA) to male Wistar rats. Arterial blood samples were drawn frequenOy for the determination of blood SPA concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. For each individual rat the relationship between the SPA concentrations and the NEFA lowering effect could be described by the indirect suppression model. Administration of SPA at different rates and doses (0.06 mg/kg in 5 rain, 0.06 mg/kg in 15 min and, 0.12 mg/kg in 60 min) revealed uniform pharmacodynamic parameter estimates. The averaged parameters (mean + SEM, n=19) were Em~x: 0.8 ± 0.03, ECs0:23 ± 2 ng/ml and Hill coefficient: 2.3 ± 0.3. In addition the model estimated the elimination rate constant of NEFAs (0.097 + 0.007 rain-l). In another group, which received 0.40 mg/kg SPA in 15 min, pharmacodynamic parameters for both heart rate and antilipolytic effect were derived within the same animal. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The ECs0 values (mean ± SEM, fi=6) were 131 + 31 ng/ml and 20 _+ 2.9 rig/m1 for heart rate and NEFA lowering effect, respectively. In conclusion, the relationship between blood concentrations of SPA and antilipolytic effect could be described by the indirect suppression model, yielding estimates of potency and activity of the drug in vivo. The results showed a 6-fold difference in potency of SpA between the effects on heart rate and NEFAs. On the basis of preclinical PK/PD modelling selectivity of action of newly developed purinergic drugs can be assessed in vivo.
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306
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REVERSE CHOLESTEROL TRANSPORT. A TARGET FOR INNOVATIVE TREATMENTS AGAINST CARDIOVASCULAR DISEASE G. Franceschini Center E. Grossi Paoletti, Institute of Pharmacological Sciences, via Balzaretti 9, 20133 Milano, Italy
F. Bernini#, 5I. Scurati*, G. Bonfadini*, and R. FumagaUi* #Institute of Pharmacology and Pharmacognosy, University of Parma, Via delle Scienze, 43100 Parma, Italy; *Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
Reverse cholesterol transport identifies a series of metabolic events resulting in the transport of excess cholesterol from peripheral tissues, including the arterial wall, to the liver for excretion. High density lipoproteins (HDL) ai'e the vehicle of cholesterol in this reverse transport, a function believed to explain the inverse correlation between plasma HDL levels and the risk for CHD. More recently, low plasma HDL levels have also been associated with an increased rate of restenosis after coronary angioplasty. An attempt to stimulate reverse cholesterol transport may hold promise in the prevention and treatment of arterial diseases. Among drugs affecting lipoprotein metabolism, probucol stimulates the activity of the cholesteryl ester transfer protein and consequently facilitates the reverse transport. Other agents, including certain lipid-lowering drugs, hormones and microsomal enzyme inducers are affective in raising plasma HDL levels. More innovative approaches employ recombinant apolipoproteins or gene therapy. One case, i.e., that of apolipoprotein A-I, is entering the clinical field; the recombinant apoA-IMilano mutant proved effective in animal models, possibly because of a combined cholesterol removing/fibrinolytic activity. In the case of gene therapy, the transfer of foreign DNA to the arterial wall applied successfully to atherosclerotic arteries, achieving biological and therapeutic effects. It is thus time for thinking at future treatments of atherosclerosis, which combine the classical lipid-lowering approach with innovative methods to promote cholesterol removal from the arterial wall.
Mevalonate starvation induced by HMG-CoA reductase inhibitors reduce cholesterol accumulation promoted in macrophages by acetylated LDL (Ac-LDL). In the present study we utilized the new synthetic HMG-CoA reductase inhibitor fluvastatin to investigate the mechanism of this effect. Our results indicate that fluvastatin inhibits more than 125 50%, in a concentration-dependent manner, I-AcLDL degradation by macrophages. This effect was not due to a decrease of lysosomal enzyme activity and it was paralleled by the retention of AcLDL-associated cholesteryl ester in the incubation medium. The ability of fluvastatin to inhibit acetyl LDL degradation was completely overcome by mevalonate, geranylgeraniol, but not by famesol. The inhiNtory effect of fluvastatin on acLDL degradation was shared by another HMG-CoA reductase inhibitor, simvastatin. Evaluation at 4°C of I-AcLDL binding to plasma membrane suggests that the inhibitory effect of fluvastatin on lipoprotein calabolism was not due to a decrease expression of scavenger receptors. Fluorescent microscope analysis of cellular internalization of acetyl-LDL labeled with the fluorochrome DiI demonstrated that fluvastatin inhibits lipoprotein endocytosis: an effect reversed by mevalonate. The ability of fluvastatin to reduce acetyl LDL catabolism and cholesterol esterification was more pronounced in cholesterol enriched cells as compared to normal cells. In conclusion, the present results provide "in vitro" evidence of the involvement of the mevalonate pathway in Ac-LDL endocytosis in macrophages.
PHARMACOKINETIC-PHARMACODYNAMIC MODELLING OFTHE ANTI-LIPOLYTIC EFFECTS OF THE ADENOSINE A1-RECEPTOR AGONIST N%(P-SULFOPHENYL)ADENOSINE (SPA) IN RATS
PLATELET-RELEASED NAP-2 VARIANTS INDUCE PMN LEUKOCYTE I N T R A C E L L U L A R CALCIUM CHANGES ([Ca2+]i). Paola Piccardoni, Virgilio Evangelista, Antonio Piccoli, Alfred Walz and Chiara Cerletti. "G.Bizzozero" Laboratory of Platelet and Leucocyte Pharmacology, Istituto Rioerche Farmacol. Mario Negri, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy and Theodor Kocher Institut, Bern, Switzerland
E.A. van Schaick, H.J.M.M. de Greef, M.W.E. Langemeijer, A.P. IJzerman and M. Danhof Divisions of Pharmacology and Medicinal Chemistry, Leiden~Amsterdam Center for Drug Research, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands.
Activated platelets may stimulate PMN leukocyte function by releasing soluble products. The aim of this study was to investigate the effect of supernatants of thrombin-activated platelets on PMN [Ca2~]i and to characterize the platelet release product(s) responsible for PMN stimulation. Platelet supernatants induced a concentration-dependent [Ca2+]i increase in Fura-2qoaded PMN (0.5x107/ml) from a resting value of 169.7+19.7 nM to 1993 + 284 nM (n= 4) in the presence of 10~ platelets/ml, Complete ATP removal by apyrase treatment only partially reduced PMN stimulatory effect of platelet supernatant, suggesting the presence of other active compounds. Heat (100 ° C, 15 min) and trypsin or elastase treatment of platelet supernatants significantly reduced their maximal activity by more than 50%; cathepsin G on the other hand increased platelet supernatants activity. FPLC and HPLC procedures allowed isolation of proteins (of about 7.500 d) responsible for the PMN stimulatory activity released in platelet supernatants. The N-terminal sequence analysis of purified active fractions revealed the presence of two peptides of 73 and 74 amino acids, respectively; both are truncated forms of CTAP-III, corresponding to NAP-2, but having 3 or 4 additional amino acids at the N-terminus: Asp-Leu-Tyr... and Ser-AspLeu-Tyr... The treatment of platelets with different protease inhibitors (EDTA, PMSF, leupeptin, iodacetamide) did not significantly modify the calcium stimulatory activity of supernatants, ruling out the possibility that NAP-2 variants may be generated from inactive precursors by platelet proteases released upon thrombin stimulation. Moreover, lysates obtained bE platelet sonication treated with apyrase also induced PMN [CaZ+]i increase, suggesting that active NAP-2 variants are preformed in platelets. In conclusion, our study indicates that NAP-2 variants are released by activated platelets and induce PMN [Ca2+]i.
For therapeutic use of adenosine analogues, it is important to develop compounds with selectivity of action. In this study tissue-selectivity of the adenosine A~-receptor agonist SPA was investigated by quantification of the hemodynamic and anti-lipolytic effects on the basis of an integrated pharmacokinetic-pharmacodynamic (PK/PD) model. Pharmacoldnetics and pharmacodynamics were determined after intravenous administration of N 6(p-sulfophenyl)adenosine (SPA) to male Wistar rats. Arterial blood samples were drawn frequenOy for the determination of blood SPA concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. For each individual rat the relationship between the SPA concentrations and the NEFA lowering effect could be described by the indirect suppression model. Administration of SPA at different rates and doses (0.06 mg/kg in 5 rain, 0.06 mg/kg in 15 min and, 0.12 mg/kg in 60 min) revealed uniform pharmacodynamic parameter estimates. The averaged parameters (mean + SEM, n=19) were Em~x: 0.8 ± 0.03, ECs0:23 ± 2 ng/ml and Hill coefficient: 2.3 ± 0.3. In addition the model estimated the elimination rate constant of NEFAs (0.097 + 0.007 rain-l). In another group, which received 0.40 mg/kg SPA in 15 min, pharmacodynamic parameters for both heart rate and antilipolytic effect were derived within the same animal. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The ECs0 values (mean ± SEM, fi=6) were 131 + 31 ng/ml and 20 _+ 2.9 rig/m1 for heart rate and NEFA lowering effect, respectively. In conclusion, the relationship between blood concentrations of SPA and antilipolytic effect could be described by the indirect suppression model, yielding estimates of potency and activity of the drug in vivo. The results showed a 6-fold difference in potency of SpA between the effects on heart rate and NEFAs. On the basis of preclinical PK/PD modelling selectivity of action of newly developed purinergic drugs can be assessed in vivo.
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306
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