Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice

Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.—Bird, D. A., R. K. Tangirala, J. Fruebis, D. Steinberg, J. L. Witztum, and W. Palinski. Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice. J. Lipid Res. 1998. 39: 1079–1090. Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.—Bird, D. A., R. K. Tangirala, J. Fruebis, D. Steinberg, J. L. Witztum, and W. Palinski. Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice. J. Lipid Res. 1998. 39: 1079–1090. Oxidized lipoproteins may enhance atherosclerosis by a number of mechanisms. These include the recognition of oxidized lipoproteins by macrophage scavenger receptors, their chemotactic and cytotoxic properties, and the modulation of gene expression of vascular cells (reviewed in 1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol. Modifications of low density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Google Scholar, 2Steinberg D. Low density lipoprotein oxidation and its pathobiological significance.J. Biol. Chem. 1997; 272: 20963-20966Google Scholar, 3Berliner J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Atherosclerosis: Basic mechanisms. oxidation, inflammation, and genetics.Circulation. 1995; 91: 2488-2496Google Scholar). The occurrence of oxidized low density lesions D. L. in of Scholar, W. S. S. Parthasarathy S. Carew T.E. D. Witztum J.L. Low density lipoprotein in 1989; Scholar, S. W. Parthasarathy S. Carew T.E. S. Witztum J.L. D. for the of low density lipoprotein in lesions of and 1989; Scholar, A.M. for a by a against LDL in lesions from a J. 1989; Scholar, W. S. S. Witztum J.L. of and apolipoprotein in lesions of from Scholar, and of Biol. 1995; but to the for the of LDL oxidation is the that powerful probucol, and significantly atherosclerosis in and M. K. Probucol the of atherosclerosis in an animal for Scholar, T.E. D. effect of probucol to its that in vivo low density lipoprotein in the of atherosclerosis in the Scholar, L. The antioxidant against Scholar, J. J. Low density lipoprotein is protected from oxidation and the of atherosclerosis is in by the antioxidant Scholar, M. of vascular and to atherosclerosis in Biol. 1995; Scholar, M. Carew T.E. D. R. of atherosclerosis in by probucol. the of atherosclerosis to of LDL to Scholar). The of probucol, strongly plasma LDL against ex vivo However, the mechanisms by probucol and other atherogenesis not Probucol a of cellular effects may or may not due to its antioxidant M. R. of atherosclerosis in the by and Biol. 1995; Scholar, of expression in cells by probucol and Scholar, J. M. W. Effect of probucol treatment on gene expression of and in the aortic of LDL receptor-deficient Biol. 1997; Scholar). probucol gene expression of vascular and macrophage in and the of and J. M. W. Effect of probucol treatment on gene expression of and in the aortic of LDL receptor-deficient Biol. 1997; that atherogenesis J. M. W. Effect of probucol treatment on gene expression of and in the aortic of LDL receptor-deficient Biol. 1997; Scholar, atherogenic a in aortic Scholar, expression of a Scholar). it is that the antiatherogenic effect of may not due or the protection of plasma LDL against in LDL receptor-deficient that the of antioxidant protection of plasma LDL not a of the effect of different J. J. W. of antioxidant protection of plasma LDL is not a of the antiatherogenic effect of Lipid Res. 1997; Scholar). In that a of a probucol and a very low dose (0.025%) of protected plasma LDL to the probucol, failed to a reduction of probucol models for atherogenic mechanisms are to or murine J.L. models for study of lipoprotein metabolism and Scholar, J.L. models of 272: Scholar). murine are in atherosclerosis in the aortic or by the K. J.L. and atherosclerosis in apolipoprotein mice by in Scholar, J.A. and lesions in mice apolipoprotein and the LDL receptor-deficient (LDLR-/-) S. J.L. J. in low density lipoprotein mice and its by gene Scholar). lesions in mice from to and of lesions in other animal models and in in mice of and Scholar, J.L. R. mice lesions of all of atherosclerosis the aortic Scholar). for the of lipoprotein oxidation in the atherogenic in murine models of the of in their lesions and the in their plasma of high of against of W. J.L. D. Witztum J.L. mice are a of lipoprotein oxidation in of in lesions and high of to in Scholar, W. Witztum J.L. against of oxidized LDL in LDL receptor-deficient mice with increased Biol. 1995; Scholar, W. S. Witztum J.L. of to of oxidized lipoproteins from mice. of of oxidized low density lipoprotein in Scholar, S. R. W. Witztum J.L. are against of oxidized recognition of by to of oxidized Scholar). However, to the of lipoprotein oxidation, and the antiatherogenic effect of not in mice. study from that an in murine models. mice with atherosclerosis Witztum J.L. D. W. Effect of the antioxidant on atherogenesis in Biol. 1995; but the effect and may effects In a by of atherosclerosis by probucol treatment in apolipoprotein 1997; that probucol increased atherogenesis in mice. However, the mechanisms atherogenesis in this may due to the of to and other of to study the effects of probucol to its modulation of it to the in the treatment and Probucol a very powerful effect in mice, is not to for in mice, in even on a In and atherosclerosis in LDLR-/- mice is on of a S. J.L. J. in low density lipoprotein mice and its by gene Scholar, W. Witztum J.L. against of oxidized LDL in LDL receptor-deficient mice with increased Biol. 1995; Scholar, S. J.L. J. and atherosclerosis in low density lipoprotein Scholar). This it to plasma in the treatment and groups by the of the to whether murine models are suitable to investigate the mechanisms by probucol and other may carried out a of in LDLR-/- mice. were LDLR-/- mice with a from a from provided by The of experiments is in of of Treatment This received vitamin in to probucol. a This received vitamin in to probucol. in a In female mice were for and plasma The with 0.5% probucol from the This an atherogenic and The a but a of cholesterol. of the probucol a very strong effect in mice, the of the the to plasma to in the treatment In in to the the in both the treatment of the probucol to in the the number of in this to the groups were in to the dose of the effect of probucol and to whether the effect on atherosclerosis with the of antioxidant protection of plasma LDL. male LDLR-/- mice were groups of for and plasma The with 0.5% probucol The did not antioxidant The with a low dose (0.025%) of probucol and a high dose (0.1%) of vitamin E The received only 0.025% probucol in the the study to for the effect of probucol. the of the high probucol from to and that of the lowered from to The of the low probucol lowered from to on and that of the low E to and to 0.5% and In to the in all treatment of the and low E groups and treatment of the low probucol to and that of the high probucol to from the and mice from the low low probucol, and high probucol mice from the low E of unknown carried out to the effect of probucol in male and female mice and to whether the of of different The of In and significantly treatment periods of the high probucol groups were to a the modulation of the by to for the powerful effect of probucol. In in that the of an of the and the the for all In to the the The of atherosclerosis in mice with 0.5% probucol to that in groups of mice a low or high plasma In this female and male mice were three groups The of and males a and cholesterol. The of female and male mice with 0.5% probucol and the cholesterol. The of and males the with cholesterol. 6 6 female mice from were and The mice were for weeks. the mice from the and mice from the probucol The size of the of the study is in plasma and antioxidant in the of the < < < < < to the low dose E < to the of the < to the of the < to the low dose E < to the of the < < < < < < < to the female mice of the < to the of the < to the of the < < < to the of the < to the low dose E < to the of the < < < to the female mice of the and number of mice the of the In the number of mice in 6 female mice were 6 the weeks. plasma plasma not < < to the of the < to the low dose E < < to the female mice of the in a In and number of mice the of the In the number of mice in 6 female mice were 6 the weeks. plasma plasma not and were an J. J. for the of with Chem. Scholar). for were from the of mice and were in high density lipoprotein were by the very low density lipoprotein and LDL with and M. K. R. L. lipoprotein of other lipoproteins by or by Chem. and the plasma The plasma of probucol in plasma from mice or in and an J. D. Carew T.E. of the antiatherogenic effects of probucol and of a of probucol in low density lipoprotein receptor-deficient Scholar). In plasma were with of on a The were with and probucol by the The plasma vitamin E were a with a J. D. Carew T.E. of the antiatherogenic effects of probucol and of a of probucol in low density lipoprotein receptor-deficient Scholar). the vitamin E with and an The and by lipoprotein of and probucol-treated mice were by a plasma of the and were and for and LDL < < from plasma from mice by a with a Witztum J.L. D. W. Effect of the antioxidant on atherogenesis in Biol. 1995; Scholar). HDL < < by from the plasma of LDLR-/- mice a The of LDL to oxidation by the times in the of oxidation, Witztum J.L. D. W. Effect of the antioxidant on atherogenesis in Biol. 1995; Scholar). LDL against to with and a of The of in a the of the to times were the the to the the of the the The in plasma of were by a a In this HDL from LDLR-/- mice by of a of HDL in to of a and on the were by with in for min The were for with of a of the murine and with of a of a were times with and for with of in with of a of to and the were for in the a by in and were the to the HDL in the of by the in the of The of in murine from the HDL from LDLR-/- mice ranging from to To to of the HDL in The of of HDL by and the of the of murine plasma that a on the of the were The and aortic were with and for W. of atherosclerosis in murine lesions in the aortic and in the and differences in the of lesions in LDL receptor-deficient and Lipid Res. 1995; Scholar). size in of the aortic origin, from the of the of the aortic In the the and were in The were of and on W. J.L. D. Witztum J.L. mice are a of lipoprotein oxidation in of in lesions and high of to in Scholar). the with the of the aortic by lesions by W. of atherosclerosis in murine lesions in the aortic and in the and differences in the of lesions in LDL receptor-deficient and Lipid Res. 1995; Scholar). were by of are In to the in the treatment and groups and in of a strong effect of probucol, the probucol a the a only cholesterol. In in LDLR-/- mice, in plasma of W. Witztum J.L. against of oxidized LDL in LDL receptor-deficient mice with increased Biol. 1995; Scholar, W. of atherosclerosis in murine lesions in the aortic and in the and differences in the of lesions in LDL receptor-deficient and Lipid Res. 1995; Scholar). with 0.5% probucol, in the this in plasma of only in the the to The of the to and to the plasma in both groups were significantly different the following in the probucol the plasma were in the and in the high probucol (n.s.) in both groups by the in the probucol to to in the increased in both but were in the probucol the of the Probucol treatment effect on the and animal of the treatment of The effect of probucol on the of atherosclerosis in of the aortic the low plasma and the of the not and did in very lesions in the and W. Witztum J.L. against of oxidized LDL in LDL receptor-deficient mice with increased Biol. 1995; Scholar, W. of atherosclerosis in murine lesions in the aortic and in the and differences in the of lesions in LDL receptor-deficient and Lipid Res. 1995; Scholar). to mice with probucol lesions the not Even though the to the high probucol in this the in and the of the to the that in the in plasma of groups that in the of the were significantly of the high probucol < and that the < the reduction of the of the to and the increase of the of the high probucol to To the the treatment of the high probucol to to to in the The plasma of the low E were to of the the study and the In the of the low probucol the the low E were significantly and the of the < in this to an in all groups in the plasma increased in all but did not significantly groups The of the mice in the groups increased in with differences the of the and low E groups the pro-atherogenic effect of probucol observed in In the high probucol significantly lesions the < The pro-atherogenic effect of probucol The low E and low probucol groups significantly lesions and the (P < but lesions the high probucol of lesions in the only in a but the differences were The of aortic by lesions and in the low low probucol, and high probucol only the high probucol and low probucol groups were significantly different from the (P < in an different in all to in and In both male and female mice, the plasma of the low and the high probucol groups increased the and and in and and in in plasma males and in the treatment the the of male and female groups to However, the plasma and of female mice in the high were in the other groups of female mice (P < 0.001). The plasma and of male mice in the high were even and different from in the male low and high probucol from in female mice in the high (P < 0.001). The plasma of the female mice in all three groups were However, of male mice in the low and high groups were significantly and < of male mice in the high probucol and female mice. in the three female groups the the low and high probucol groups the in the male the of males were significantly of of the pro-atherogenic effect of probucol. In female mice, 6 of the high probucol significantly lesions the low plasma the < 0.05) The high both of atherosclerosis the low but the high probucol of the differences both the high probucol and high groups significantly lesions and the low < 0.05) the size of lesions in the high probucol that in the high the did not of male mice very The high probucol significantly the low < the high significantly lesions < the low the high significantly lesions the high probucol (P < even though the to plasma In the plasma were to the lipoprotein by are in from that received a of to for LDLR-/- mice on J.A. and lesions in mice apolipoprotein of the in the LDL that the HDL-cholesterol is that in LDLR-/- mice of probucol-treated mice of for a consistently HDL-cholesterol the effect of probucol treatment failed to in of HDL-cholesterol in in the HDL-cholesterol in mice of the high probucol were significantly in the low a and of the high in both male and female mice for (P < to < 0.001). in plasma from all in were by The plasma of to in probucol-treated mice. the did not due to a three of the plasma In in a reduction of the plasma in both male and female mice for with 0.5% probucol, to in the low (P < 0.05) that treatment with 0.5% probucol to a reduction of plasma HDL in LDLR-/- mice, with in other mice J.L. Probucol apolipoprotein and high density lipoprotein in and apolipoprotein and in J.L. D. D. R. J. of with probucol, and 1989; Scholar, Parthasarathy S. Witztum J.L. Effect of probucol on plasma and lipoprotein and on protection of low density lipoprotein against in oxidation in Scholar, J. L. L. S. J. of by probucol the of the to atherosclerosis in with Probucol Biol. 1995; Scholar). In the probucol in the plasma from mice from the high probucol The probucol in LDL from the plasma This to the plasma probucol of female mice 0.5% probucol in However, male mice the dose of probucol in and significantly of plasma probucol and < The plasma vitamin E in the high probucol groups of and were significantly of the groups (P < plasma of vitamin E observed in the low probucol of (P < even though the plasma of probucol in this in the high probucol an increase in plasma vitamin E in the low E to the but the increase that the were a high dose of vitamin E of the the plasma of probucol in the low E 6 to in the low probucol that received the dose of the plasma of vitamin E and probucol J. D. Carew T.E. of the antiatherogenic effects of probucol and of a of probucol in low density lipoprotein receptor-deficient and that probucol treatment of L. K. J. J. Probucol treatment of Biol. 1995; Scholar). The of protection of LDL against in oxidation by the times in the of a oxidation M. Effect of on of Med. Scholar). from are in In all three experiments, the times of LDL from the groups were to from the high probucol groups of 1400 The of antioxidant protection in study to that by probucol in J. D. Carew T.E. of the antiatherogenic effects of probucol and of a of probucol in low density lipoprotein receptor-deficient probucol a reduction of Treatment with low-dose probucol a of The of times in the low E and low probucol groups in were to min and to The of times in mice only the low dose of probucol, to mice the dose of probucol plus a high dose of vitamin by the that the plasma of probucol in the low probucol that of the low E the increase in plasma vitamin E in the The study that probucol not only failed to atherogenesis in LDLR-/- mice, but it in a dose in both male and female Even the of a high dose of vitamin E to a very low dose (0.025%) of probucol did not the atherogenic effect of probucol, the increase in plasma of vitamin E in LDLR-/- mice are with in mice by of atherosclerosis by probucol treatment in apolipoprotein 1997; found in size ranging from to in male and female mice with 0.5% probucol for In probucol treatment of LDLR-/- mice in a of the The that probucol even though it strongly increased the of plasma LDL to ex vivo oxidation, that the antiatherogenic effect of may not by the of plasma LDL to oxidation ex vivo J. J. W. of antioxidant protection of plasma LDL is not a of the antiatherogenic effect of Lipid Res. 1997; Scholar). In and the powerful effect of probucol only for by the of the of the treatment and it in the to in all groups by the treatment of the probucol This to a in of the probucol-treated in the high probucol of on the of the the of and plasma that both the of and the of to a on This may not or it may not the differences in are it to that the increase in atherosclerosis by probucol in and may not the effect of probucol on but a of the In other it is that the to a of plasma in the probucol atherosclerosis the to a in the this out and provided for the of probucol in LDLR-/- mice. In this the and the of atherosclerosis in the treatment to that in different of a plasma to in the treatment the The a in plasma the probucol consistently lesions of both in male and female mice and different for the pro-atherogenic effect of probucol in LDLR-/- mice may its effect on that probucol consistently HDL-cholesterol and plasma This is with the of HDL in but not all on probucol-treated J.L. D. D. R. J. of with probucol, and 1989; Scholar, Parthasarathy S. Witztum J.L. Effect of probucol on plasma and lipoprotein and on protection of low density lipoprotein against in oxidation in Scholar, J. L. L. S. J. of by probucol the of the to atherosclerosis in with Probucol Biol. 1995; Scholar). HDL effect in probucol-treated and M. Carew T.E. D. R. of atherosclerosis in by probucol. the of atherosclerosis to of LDL to Scholar, J. D. Carew T.E. of the antiatherogenic effects of probucol and of a of probucol in low density lipoprotein receptor-deficient Scholar). The that probucol in atherosclerosis in animal even though it lowered HDL to against this the other mice and the lipoprotein of LDLR-/- mice on a is very different from that of other and is by a very HDL LDLR-/- mice are an atherogenic HDL its the of and the HDL in to the and LDL S. J.L. J. in low density lipoprotein mice and its by gene Scholar). HDL an in the oxidation of LDL and in oxidized A.D. J.A. Fogelman A.M. Navab M. effect of high density lipoprotein of the of oxidized low density 1995; Scholar). it is that of the by the atherogenic of HDL by probucol a powerful atherogenic However, the of HDL in murine atherosclerosis is from of atherosclerosis by probucol treatment in apolipoprotein 1997; that mice for both and plasma HDL-cholesterol but the of atherosclerosis mice. the other of protected mice from atherosclerosis J. apolipoprotein E atherosclerosis in Scholar, J.L. apolipoprotein gene expression high density lipoprotein and atherosclerosis in the apolipoprotein 91: Scholar). for the increased atherosclerosis in the treatment that the effects of probucol the cellular are different in mice in and is that atherogenesis may by by vascular and J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Atherosclerosis: Basic mechanisms. oxidation, inflammation, and genetics.Circulation. 1995; 91: 2488-2496Google Scholar, in 1995; Scholar). of gene expression of may and may by a in the intracellular or even by LDL oxidized to an that not to recognition by scavenger the expression of chemotactic and in cells and cells J.A. A.J. Navab M. R. Fogelman A.M. low density lipoprotein chemotactic in cells and Scholar, J.A. Navab M. Fogelman A.M. Lusis A.J. of expression of and macrophage by low density Scholar, of the gene by LDL. of Biol. 1995; Scholar, Fogelman A.M. Lusis A.J. of gene and in to an atherogenic in 91: Scholar, Fogelman A.M. Lusis A.J. for a gene and aortic in Scholar). and mechanisms by the of expression of and in vascular is that may the intracellular and cellular in in to lipoproteins against it is that probucol, are of the cellular expression of atherogenic In both and probucol treatment the cellular of lesions M. R. of atherosclerosis in the by and Biol. 1995; Scholar, K. Probucol treatment the cellular but not low density lipoprotein of from and that probucol of M. R. of atherosclerosis in the by and Biol. 1995; Scholar). the effects of probucol on the expression of that are to and and the of atherogenesis J. M. W. Effect of probucol treatment on gene expression of and in the aortic of LDL receptor-deficient Biol. 1997; Scholar). In this probucol in to of and the of gene and expression that Probucol the of expression found in of and In probucol did not expression of J. M. W. Effect of probucol treatment on gene expression of and in the aortic of LDL receptor-deficient Biol. 1997; Scholar). These provided the in vivo for an effect of probucol on and that an of the antiatherogenic effect of probucol in models may a of However, it is unknown whether the expression is due to an of expression of lipoprotein or whether it from antioxidant or other effects of probucol the intracellular The that probucol increased may that the cellular antiatherogenic effects of probucol in and not only are in LDLR-/- mice, but that probucol actively atherogenic mechanisms. the other it out that differences in the and/or of probucol in murine vascular cells may the antiatherogenic effect that probucol in other and that the increase in atherosclerosis is a of HDL a cellular effect of probucol. of of atherosclerosis by probucol treatment in apolipoprotein 1997; against the of in that in are in in mice. that treatment of mice with a antioxidant that not atherosclerosis by Witztum J.L. D. W. Effect of the antioxidant on atherogenesis in Biol. 1995; and a suggests that of vitamin E and other in LDLR-/- mice of LDL oxidation and atherogenesis by in LDL mice. 1997; Scholar). The of murine models to the of in to However, the that probucol is antiatherogenic in and but pro-atherogenic in LDLR-/- mice may an to the mechanisms by probucol atherosclerosis in the models. the effect of probucol on the expression of in mice to that in or not only the of probucol, but on are for the of for and for the and for the of the plasma and These were by and of in and J. D. and W. apolipoprotein high density lipoprotein low density lipoprotein LDL receptor-deficient chemotactic macrophage oxidized LDL vascular