Background and Purpose-Calcium-channel blockers (CCBs) reduce systolic blood pressure and stroke-... more Background and Purpose-Calcium-channel blockers (CCBs) reduce systolic blood pressure and stroke-related mortality in stroke-prone spontaneously hypertensive rats (SPSHR). Brain ischemia is associated with loss of intracellular antioxidants. Increased formation of oxygen radicals and oxidation of LDL may enhance arterial vasoconstriction by various mechanisms. CCBs that also exert antioxidative properties in vitro may therefore be particularly useful. To investigate such antioxidant effects in vivo, we determined several parameters of LDL oxidation in SPSHR treated with two 1,4-dihydropyridine-type (1,4-DHP) CCBs of different lipophilic properties and compared them with antioxidanttreated and untreated controls. We also tested whether these drugs decrease the formation of oxidation-specific epitopes in arteries. Methods-Five groups of 9 to 14 SPSHR each (aged 8 weeks) were treated with 80 mg/kg body wt per day nifedipine, 1 mg or 0.3 mg/kg body wt per day lacidipine, vitamin E (100 IU/d), or carrier for 5 weeks. A group of Wistar-Kyoto rats was used as normotensive control. Plasma samples were taken, and LDL was isolated by ultracentrifugation. Then LDL was exposed to oxygen radicals generated by xanthine/xanthine oxidase reaction (2 mmol/L xanthineϩ100 mU/mL xanthine oxidase), and several parameters of oxidation were determined. The presence of native apolipoprotein B and oxidation-specific epitopes in the carotid and middle cerebral arteries was determined immunocytochemically. Results-1,4-DHP CCBs completely prevented mortality. Normotensive Wistar-Kyoto rats showed less oxidation than control SPSHR. Plasma lipoperoxide levels were 0.87Ϯ0.27 mol/L in control SPSHR, 0.69Ϯ0.19 and 0.63Ϯ0.20 mol/L in the groups treated with 0.3 and 1 mg lacidipine, respectively, and 0.68Ϯ0.23 mol/L in nifedipine-treated animals (PϽ0.05 versus control SPSHR for all values). Both CCBs significantly decreased formation of conjugated dienes and prolonged the lag time in LDL exposed to oxygen radicals. Similarly, lipoperoxides and malondialdehyde were significantly reduced (PϽ0.05). Reduced relative electrophoretic mobility and increased trinitrobenzenesulfonic acid reactivity of LDL from treated rats (PϽ0.01) also indicated that fewer lysine residues of apolipoprotein B were oxidatively modified in the presence of 1,4-DHP CCBs. Finally, these drugs reduced the intimal presence of apolipoprotein B and oxidized LDL (oxidation-specific epitopes) in carotid and middle cerebral arteries. Conclusions-In the SPSHR model, 1,4-DHP CCBs reduce plasma and LDL oxidation and formation of oxidation-specific epitopes and prolong survival independently of blood pressure modifications. Our results support the concept that the in vivo protective effect of these drugs on cerebral ischemia and stroke may in part result from inhibition of oxidative processes. (Stroke. 1999;30:1907-1915
To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into t... more To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more an...
Probucol is a powerful inhibitor of atherosclero- sis in a number of animal models. However, it i... more Probucol is a powerful inhibitor of atherosclero- sis 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 antiox- idants. To investigate whether murine models are suitable to study the antiatherogenic
Background—Oxidized LDL (oxLDL) promotes atherogenesis, and antioxidants reduce lesions in experi... more Background—Oxidized LDL (oxLDL) promotes atherogenesis, and antioxidants reduce lesions in experimental models. OxLDL-mediated effects on c-Myc are poorly characterized, and those on c-Myc nuclear pathways are completely unknown. c-Myc stimulates smooth muscle cell (SMC) proliferation and could be involved in atherosclerosis. We investigated the early effects of oxLDL and a-tocopherol on c-Myc, its binding partner Max, and the carboxy-terminal domain-
Background—Atherosclerotic lesions in intracranial arteries occur later and are less extensive th... more Background—Atherosclerotic lesions in intracranial arteries occur later and are less extensive than in extracranial arteries. To investigate potential mechanisms responsible for this difference, in particular the atherogenic response to hypercholesterolemia and LDL oxidation, we compared the extent of fatty streak formation and the composition of these very early lesions in intracranial arteries of human fetuses from normocholesterolemic and hypercholesterolemic mothers
Oxidized LDL (oxLDL) is present in atherosclerotic lesions and is believed to play a key role in ... more Oxidized LDL (oxLDL) is present in atherosclerotic lesions and is believed to play a key role in atherogenesis. Mainly on the basis of cell culture studies, oxLDL has been shown to produce many biological effects that influence the atherosclerotic process. To study LDL oxidation in vivo, we have established a model in which Sprague-Dawley rats are given a single injection
Apolipoprotein E-deficient (apoE x/x ) and LDL receptor-deficient (LDLR x/x ) mice develop extens... more Apolipoprotein E-deficient (apoE x/x ) and LDL receptor-deficient (LDLR x/x ) mice develop extensive atherosclerosis, but the occurrence of spontaneous plaque rupture and secondary thrombosis in these models has not been established. The goal of this study was to provide histological evidence of acute complications of atherosclerotic lesions in these mice and to assess their prevalence. Complications of atherosclerosis were initially studied in aortas of control mice which died during previous intervention studies. Coronary arteries and the aortic origin were then systematically assessed in serial sections through the heart of apoE x/x and LDLR x/x mice. Aortic plaque rupture and/or thrombi were seen in 3 of 82 untreated mice from past intervention studies. Screening of heart sections of 33 older apoE x/x mice (age 9-20 months) showed extensive atherosclerosis in one or more coronary arteries of 18 animals. In three coronary arteries, the presence of blood-filled channels within advanced atherosclerotic lesions suggested previous plaque disruption/thrombotic events followed by recanalization. In the aortic origin of the same mice, four deep plaque ruptures (or erosions reaching necrotic core areas) and a large thrombus originating from the core of a disrupted atherosclerotic lesion were observed. Although plaque ruptures/deep erosions were far less frequent than in human populations, these observations demonstrate that spontaneous plaque rupture and secondary thrombosis do occur in apoE x/x and LDLR x/x mice. These mice may therefore be suitable for studying factors contributing to thrombotic complications of atherosclerosis. However, the frequent absence of a clearly defined single fibrous cap in murine coronary lesions limits their usefulness as a model of fibrous cap rupture.
Objective—Lipid-loaded macrophage-derived foam cells populate atherosclerotic lesions and produce... more Objective—Lipid-loaded macrophage-derived foam cells populate atherosclerotic lesions and produce many pro-inflammatory and plaque-destabilizing factors. An excessive accumulation of extensively oxidized low-density lipoprotein (OxLDL) or free cholesterol (FC), both of which are believed to be major lipid components of macrophages in advanced lesions, rapidly induces apoptosis in macrophages. Indeed, there is evidence of macrophage death in lesions, but how the surviving
Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can rea... more Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can react with lipids, apoproteins, and proteins, generating immunogenic neoepitopes. Autoantibodies recognizing model epitopes of oxidized low density lipoprotein, such as malondialdehydelysine, occur in plasma and in atherosclerotic lesions of humans and animals. Because apo E-deficient mice develop particularly high titers of such autoantibodies, we used their spleens to clone 13 monoclonal antibodies to various epitopes of oxidized LDL ("E0 antibodies"). Binding and competitive RIAs demonstrated significant differences in fine specificity even between E0 antibodies initially selected for binding to the same screening antigen. For example, some E0 antibodies selected for binding to malondialdehyde-LDL also recognized copper oxidized LDL, acrolein-LDL, or LDL modified by arachidonic or linoleic acid oxidation products. Circulating IgG and IgM autoantibodies binding to copper-oxidized LDL, 4-hydroxynonenal-LDL, acrolein-LDL, and LDL modified with arachidonic or linoleic acid oxidation products were found in apo E-deficient mice, suggesting that the respective antigens are formed in vivo. Epitopes recognized by some of the E0 monoclonal antibodies were also found on human circulating LDL. Each of the E0 monoclonal antibodies immunostained rabbit and human atherosclerotic lesions, and some of them yielded distinct staining patterns in advanced lesions. Together, this suggests that the natural monoclonal antibodies recognize different epitopes of complex structures formed during oxidation of lipoproteins, or epitopes formed independently at different lesion sites. Our data demonstrate that a profound immunological response to a large number of different epitopes of oxidized lipoproteins occurs in vivo. The availability of "natural" monoclonal autoantibodies should facilitate the identification of specific epitopes inducing this response.
We previously established that probucol de- creases basal expression of VCAM-1 in the aorta of WH... more We previously established that probucol de- creases basal expression of VCAM-1 in the aorta of WHHL rabbits and inhibits the up-regulation of VCAM-1 expres- sion that normally accompanies atherogenesis. To deter- mine whether this effect is shared by other antioxidants in vivo, we now investigated whether a structurally unrelated antioxidant, vitamin E, also inhibits arterial VCAM-1 expres- sion and whether
Proceedings of The National Academy of Sciences, 1998
The peroxisome proliferator-activated receptor gamma (PPARgamma ) is a ligand-dependent transcrip... more The peroxisome proliferator-activated receptor gamma (PPARgamma ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARgamma is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARgamma is expressed in macrophage foam cells
Background and Purpose-Atherosclerosis occurs later and is less extensive in intracranial arterie... more Background and Purpose-Atherosclerosis occurs later and is less extensive in intracranial arteries than in extracranial arteries. However, the mechanisms responsible are poorly understood. A previous study has suggested a better antioxidant protection of intracranial arteries. Methods-To assess the influence of age on arterial activity of antioxidant enzymes and atherogenesis, we compared intracranial and extracranial arteries of humans of different ages who retrospectively lacked confounding classic risk factors (48 premature fetuses aged 6.4Ϯ0.8 months [meanϮSD], 58 children aged 7.9Ϯ3.8 years, 42 adults aged 42.5Ϯ5.1 years, and 40 elderly subjects aged 71.8Ϯ3.4 years; all males). Lesions were quantified by computer-assisted imaging analysis of sections of the middle cerebral and basilar arteries, the left anterior descending coronary artery, the common carotid artery, and the abdominal aorta. Macrophages, apolipoprotein B, oxidized LDL, and matrix metalloproteinase-9 in lesions were determined by immunocytochemistry. The effect of aging on atherogenesis was then compared with that on the activity of 4 antioxidant enzymes in the arterial wall. Results-Atherosclerosis was 6-to 19-fold greater (PϽ0.01) in extracranial arteries than in intracranial arteries, and it increased linearly with age. Intracranial arteries showed significantly greater antioxidant enzyme activities than did extracranial arteries. However, the antioxidant protection of intracranial arteries decreased significantly in older age, coinciding with a marked acceleration of atherogenesis. An increase in matrix metalloproteinase-9 protein expression and in gelatinolytic activity consistent with the degree of intracranial atherosclerosis was also observed. Conclusions-These results suggest that a greater activity of antioxidant enzymes in intracranial arteries may contribute to their greater resistance to atherogenesis and that with increasing age intracranial arteries respond with accelerated atherogenesis when their antioxidant protection decreases relatively more than that of extracranial arteries. (Stroke. 2001;32:2472-2480.)
Proceedings of the National Academy of Sciences, 1997
In vitro studies have identified a number of adhesion molecules and chemokines that may control t... more In vitro studies have identified a number of adhesion molecules and chemokines that may control this process but very little work has been done to evaluate their relative importance in vivo, in part because there have been no methods available of sufficient sensitivity and reliability. This paper proposes a new approach in which advantage is taken of naturally occurring or transgenically induced mutations to ''mark'' donor cells and to follow their fate in recipient animals using highly sensitive PCR methods. The feasibility of the approach is demonstrated by preliminary studies of monocyte recruitment into atherosclerotic lesions. However, the method should in principle be applicable to the study of any of the circulating leukocytes and their rate of entry into any tissue or tissues of interest.
Increasing evidence indicates that several pathogenic mechanisms promoting atherosclerosis are al... more Increasing evidence indicates that several pathogenic mechanisms promoting atherosclerosis are also involved in neurodegenerative diseases, and that insight into the factors determining the susceptibility to, and long-term progression of, atherosclerosis may be of interest for the evolution of diseases such as Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s. Furthermore, atherosclerosis of intracranial arteries or thromboembolic consequences of atherosclerotic extracranial arteries are responsible for most ischemic events in the brain. Age-related changes of cerebrovascular atherosclerosis, and atherosclerosis in general, may therefore be important for stroke and neurodegenerative diseases affecting the elderly. In the following, pathogenic mechanism involving increased lipid peroxidation, oxidative stress, inflammation and immune responses, and fetal programming will be discussed in the context of cerebrovascular disease and aging.
During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low... more During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low-density lipoprotein (oxLDL) and active immunization of hypercholesterolemic mice with oxLDL ameliorates atherogenesis. We unexpectedly found that many autoantibodies to oxLDL derived from 'naive' atherosclerotic mice share complete genetic and structural identity with antibodies from the classic anti-phosphorylcholine B-cell clone, T15, which protect against common infectious pathogens, including pneumococci. To investigate whether in vivo exposure to pneumococci can affect atherogenesis, we immunized Ldlr -/mice with Streptococcus pneumoniae. This induced high circulating levels of oxLDL-specific IgM and a persistent expansion of oxLDLspecific T15 IgM-secreting B cells primarily in the spleen, which were cross-reactive with pneumococcal determinants. Pneumococcal immunization decreased the extent of atherosclerosis, and plasma from these mice had an enhanced capacity to block the binding of oxLDL to macrophages. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have beneficial effects.
Children generally have low cholesterol and no clinical manifestations of atherosclerosis, but fa... more Children generally have low cholesterol and no clinical manifestations of atherosclerosis, but fatty-streak formation begins in fetuses and is greatly increased by maternal hypercholesterolaemia during pregnancy. In the FELIC study we assessed the evolution of such lesions during childhood. Computer-assisted imaging was used to measure the area of the largest individual lesion and the cumulative lesion area per section in serial cross-sections through the entire aortic arch and abdominal aorta of 156 normocholesterolaemic children aged 1-13 years, who died of trauma and other causes. Children were classified by whether their mother had been normocholesterolaemic (n=97) or hypercholesterolaemic (n=59) during pregnancy. Atherosclerosis was correlated with 13 established or potential risk factors. Findings The largest fatty streaks in the aortic arch of children younger than 3 years of hypercholesterolaemic mothers were 64% smaller than those previously found in corresponding fetuses (p&lt;0.0001), which suggests that fetal fatty streaks may regress after birth. In the two groups, lesion size in the aortic arch and abdominal aorta increased linearly with age (r=0.87-0.98). However, lesions progressed strikingly faster in children of hypercholesterolaemic mothers than in those of normocholesterolaemic mothers (p&lt;0.0001). Conventional risk factors for atherosclerosis in children or mothers correlated with lesion size, but did not account for the faster progression of atherogenesis in normocholesterolaemic children of hypercholesterolaemic mothers. Our results suggest that maternal hypercholesterolaemia during pregnancy induces changes in the fetal aorta that determine the long-term susceptibility of children to fatty-streak formation and subsequent atherosclerosis. If so, cholesterol-lowering interventions in hypercholesterolaemic mothers during pregnancy may decrease atherogenesis in children.
Oxidatively modified low-density lipoprotein (LDL) is present in atherosclerotic but not normal a... more Oxidatively modified low-density lipoprotein (LDL) is present in atherosclerotic but not normal arteries and plays a crucial role in the pathogenesis and adverse consequences of atherosclerotic lesions. We previously generated a series of monoclonal antibodies (MoAb) against oxidation-specific neo-epitopes formed during the oxidative modification of LDL. MDA2, a prototype MoAb, recognizes malondialdehyde-lysine epitopes (eg, in malondi-aldehyde-modified LDL) within atherosclerotic lesions. We describe the in vivo characteristics of MDA2 and initial noninvasive imaging studies of atherosclerosis in rabbits. To assess the in vivo specificity of MDA2 for atherosclerotic lesions, iodine 125-MDA2 was intravenously injected into 7 LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) and 2 normal New Zealand white (NZW) rabbits, and the aortic plaque uptake was evaluated 24 hours later. 125I-Halb, an isotype-matched irrelevant MoAb that binds to human albumin, was injected into 5 WHHL and 2 NZW rabbits as a control. Aortic autoradiography was performed, and the mean uptake of MoAbs was measured as the percent injected dose per gram aortic tissue. Gamma camera imaging was then carried out in 7 WHHL rabbits and 2 NZW rabbits with 99mTc-MDA2. Imaging was carried out at 10 minutes and at 12 or 24 hours. Malondialdehyde-LDL was then injected to clear the blood pool signal, and final images were obtained 2 hours later. Mean uptake of 125I-MDA2 in the entire aorta was 17.4-fold higher in WHHL than in NZW aortas (P &lt; .001), and 2.8-fold higher than 125I-Halb in WHHL aortas. 125I-MDA2 also had higher specificity for lesioned areas than 125I-Halb (plaque/normal ratio 6.3 vs 2.9, P &lt; .001). Autoradiograph of aortas of 125I-MDA2-injected WHHL rabbits revealed uptake in lipid-stained lesions with absence of signal in adjacent normal arterial tissue. Immunostaining of WHHL lesions, which accumulated MDA2 as noted on autoradiography, revealed that uptake was highest in areas with abundant foam cells and in lipid-rich necrotic core areas. Autoradiograph of aortas from NZW rabbits injected with 125I-MDA2 did not yield any visible signal. Planar gamma camera in vivo scintigraphy revealed a visible signal in 4/7 WHHL rabbits, which was confirmed by aortic Sudan staining. Radiolabeled MDA2 shows excellent in vivo uptake and specificity for atherosclerotic lesions containing abundant oxidation-specific epitopes. The in vivo imaging studies suggest that noninvasive imaging of oxidation-rich atherosclerotic lesions with radiolabeled MDA2 may be feasible in human beings with optimization of the imaging methods.
Background and Purpose-Calcium-channel blockers (CCBs) reduce systolic blood pressure and stroke-... more Background and Purpose-Calcium-channel blockers (CCBs) reduce systolic blood pressure and stroke-related mortality in stroke-prone spontaneously hypertensive rats (SPSHR). Brain ischemia is associated with loss of intracellular antioxidants. Increased formation of oxygen radicals and oxidation of LDL may enhance arterial vasoconstriction by various mechanisms. CCBs that also exert antioxidative properties in vitro may therefore be particularly useful. To investigate such antioxidant effects in vivo, we determined several parameters of LDL oxidation in SPSHR treated with two 1,4-dihydropyridine-type (1,4-DHP) CCBs of different lipophilic properties and compared them with antioxidanttreated and untreated controls. We also tested whether these drugs decrease the formation of oxidation-specific epitopes in arteries. Methods-Five groups of 9 to 14 SPSHR each (aged 8 weeks) were treated with 80 mg/kg body wt per day nifedipine, 1 mg or 0.3 mg/kg body wt per day lacidipine, vitamin E (100 IU/d), or carrier for 5 weeks. A group of Wistar-Kyoto rats was used as normotensive control. Plasma samples were taken, and LDL was isolated by ultracentrifugation. Then LDL was exposed to oxygen radicals generated by xanthine/xanthine oxidase reaction (2 mmol/L xanthineϩ100 mU/mL xanthine oxidase), and several parameters of oxidation were determined. The presence of native apolipoprotein B and oxidation-specific epitopes in the carotid and middle cerebral arteries was determined immunocytochemically. Results-1,4-DHP CCBs completely prevented mortality. Normotensive Wistar-Kyoto rats showed less oxidation than control SPSHR. Plasma lipoperoxide levels were 0.87Ϯ0.27 mol/L in control SPSHR, 0.69Ϯ0.19 and 0.63Ϯ0.20 mol/L in the groups treated with 0.3 and 1 mg lacidipine, respectively, and 0.68Ϯ0.23 mol/L in nifedipine-treated animals (PϽ0.05 versus control SPSHR for all values). Both CCBs significantly decreased formation of conjugated dienes and prolonged the lag time in LDL exposed to oxygen radicals. Similarly, lipoperoxides and malondialdehyde were significantly reduced (PϽ0.05). Reduced relative electrophoretic mobility and increased trinitrobenzenesulfonic acid reactivity of LDL from treated rats (PϽ0.01) also indicated that fewer lysine residues of apolipoprotein B were oxidatively modified in the presence of 1,4-DHP CCBs. Finally, these drugs reduced the intimal presence of apolipoprotein B and oxidized LDL (oxidation-specific epitopes) in carotid and middle cerebral arteries. Conclusions-In the SPSHR model, 1,4-DHP CCBs reduce plasma and LDL oxidation and formation of oxidation-specific epitopes and prolong survival independently of blood pressure modifications. Our results support the concept that the in vivo protective effect of these drugs on cerebral ischemia and stroke may in part result from inhibition of oxidative processes. (Stroke. 1999;30:1907-1915
To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into t... more To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more an...
Probucol is a powerful inhibitor of atherosclero- sis in a number of animal models. However, it i... more Probucol is a powerful inhibitor of atherosclero- sis 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 antiox- idants. To investigate whether murine models are suitable to study the antiatherogenic
Background—Oxidized LDL (oxLDL) promotes atherogenesis, and antioxidants reduce lesions in experi... more Background—Oxidized LDL (oxLDL) promotes atherogenesis, and antioxidants reduce lesions in experimental models. OxLDL-mediated effects on c-Myc are poorly characterized, and those on c-Myc nuclear pathways are completely unknown. c-Myc stimulates smooth muscle cell (SMC) proliferation and could be involved in atherosclerosis. We investigated the early effects of oxLDL and a-tocopherol on c-Myc, its binding partner Max, and the carboxy-terminal domain-
Background—Atherosclerotic lesions in intracranial arteries occur later and are less extensive th... more Background—Atherosclerotic lesions in intracranial arteries occur later and are less extensive than in extracranial arteries. To investigate potential mechanisms responsible for this difference, in particular the atherogenic response to hypercholesterolemia and LDL oxidation, we compared the extent of fatty streak formation and the composition of these very early lesions in intracranial arteries of human fetuses from normocholesterolemic and hypercholesterolemic mothers
Oxidized LDL (oxLDL) is present in atherosclerotic lesions and is believed to play a key role in ... more Oxidized LDL (oxLDL) is present in atherosclerotic lesions and is believed to play a key role in atherogenesis. Mainly on the basis of cell culture studies, oxLDL has been shown to produce many biological effects that influence the atherosclerotic process. To study LDL oxidation in vivo, we have established a model in which Sprague-Dawley rats are given a single injection
Apolipoprotein E-deficient (apoE x/x ) and LDL receptor-deficient (LDLR x/x ) mice develop extens... more Apolipoprotein E-deficient (apoE x/x ) and LDL receptor-deficient (LDLR x/x ) mice develop extensive atherosclerosis, but the occurrence of spontaneous plaque rupture and secondary thrombosis in these models has not been established. The goal of this study was to provide histological evidence of acute complications of atherosclerotic lesions in these mice and to assess their prevalence. Complications of atherosclerosis were initially studied in aortas of control mice which died during previous intervention studies. Coronary arteries and the aortic origin were then systematically assessed in serial sections through the heart of apoE x/x and LDLR x/x mice. Aortic plaque rupture and/or thrombi were seen in 3 of 82 untreated mice from past intervention studies. Screening of heart sections of 33 older apoE x/x mice (age 9-20 months) showed extensive atherosclerosis in one or more coronary arteries of 18 animals. In three coronary arteries, the presence of blood-filled channels within advanced atherosclerotic lesions suggested previous plaque disruption/thrombotic events followed by recanalization. In the aortic origin of the same mice, four deep plaque ruptures (or erosions reaching necrotic core areas) and a large thrombus originating from the core of a disrupted atherosclerotic lesion were observed. Although plaque ruptures/deep erosions were far less frequent than in human populations, these observations demonstrate that spontaneous plaque rupture and secondary thrombosis do occur in apoE x/x and LDLR x/x mice. These mice may therefore be suitable for studying factors contributing to thrombotic complications of atherosclerosis. However, the frequent absence of a clearly defined single fibrous cap in murine coronary lesions limits their usefulness as a model of fibrous cap rupture.
Objective—Lipid-loaded macrophage-derived foam cells populate atherosclerotic lesions and produce... more Objective—Lipid-loaded macrophage-derived foam cells populate atherosclerotic lesions and produce many pro-inflammatory and plaque-destabilizing factors. An excessive accumulation of extensively oxidized low-density lipoprotein (OxLDL) or free cholesterol (FC), both of which are believed to be major lipid components of macrophages in advanced lesions, rapidly induces apoptosis in macrophages. Indeed, there is evidence of macrophage death in lesions, but how the surviving
Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can rea... more Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can react with lipids, apoproteins, and proteins, generating immunogenic neoepitopes. Autoantibodies recognizing model epitopes of oxidized low density lipoprotein, such as malondialdehydelysine, occur in plasma and in atherosclerotic lesions of humans and animals. Because apo E-deficient mice develop particularly high titers of such autoantibodies, we used their spleens to clone 13 monoclonal antibodies to various epitopes of oxidized LDL ("E0 antibodies"). Binding and competitive RIAs demonstrated significant differences in fine specificity even between E0 antibodies initially selected for binding to the same screening antigen. For example, some E0 antibodies selected for binding to malondialdehyde-LDL also recognized copper oxidized LDL, acrolein-LDL, or LDL modified by arachidonic or linoleic acid oxidation products. Circulating IgG and IgM autoantibodies binding to copper-oxidized LDL, 4-hydroxynonenal-LDL, acrolein-LDL, and LDL modified with arachidonic or linoleic acid oxidation products were found in apo E-deficient mice, suggesting that the respective antigens are formed in vivo. Epitopes recognized by some of the E0 monoclonal antibodies were also found on human circulating LDL. Each of the E0 monoclonal antibodies immunostained rabbit and human atherosclerotic lesions, and some of them yielded distinct staining patterns in advanced lesions. Together, this suggests that the natural monoclonal antibodies recognize different epitopes of complex structures formed during oxidation of lipoproteins, or epitopes formed independently at different lesion sites. Our data demonstrate that a profound immunological response to a large number of different epitopes of oxidized lipoproteins occurs in vivo. The availability of "natural" monoclonal autoantibodies should facilitate the identification of specific epitopes inducing this response.
We previously established that probucol de- creases basal expression of VCAM-1 in the aorta of WH... more We previously established that probucol de- creases basal expression of VCAM-1 in the aorta of WHHL rabbits and inhibits the up-regulation of VCAM-1 expres- sion that normally accompanies atherogenesis. To deter- mine whether this effect is shared by other antioxidants in vivo, we now investigated whether a structurally unrelated antioxidant, vitamin E, also inhibits arterial VCAM-1 expres- sion and whether
Proceedings of The National Academy of Sciences, 1998
The peroxisome proliferator-activated receptor gamma (PPARgamma ) is a ligand-dependent transcrip... more The peroxisome proliferator-activated receptor gamma (PPARgamma ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARgamma is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARgamma is expressed in macrophage foam cells
Background and Purpose-Atherosclerosis occurs later and is less extensive in intracranial arterie... more Background and Purpose-Atherosclerosis occurs later and is less extensive in intracranial arteries than in extracranial arteries. However, the mechanisms responsible are poorly understood. A previous study has suggested a better antioxidant protection of intracranial arteries. Methods-To assess the influence of age on arterial activity of antioxidant enzymes and atherogenesis, we compared intracranial and extracranial arteries of humans of different ages who retrospectively lacked confounding classic risk factors (48 premature fetuses aged 6.4Ϯ0.8 months [meanϮSD], 58 children aged 7.9Ϯ3.8 years, 42 adults aged 42.5Ϯ5.1 years, and 40 elderly subjects aged 71.8Ϯ3.4 years; all males). Lesions were quantified by computer-assisted imaging analysis of sections of the middle cerebral and basilar arteries, the left anterior descending coronary artery, the common carotid artery, and the abdominal aorta. Macrophages, apolipoprotein B, oxidized LDL, and matrix metalloproteinase-9 in lesions were determined by immunocytochemistry. The effect of aging on atherogenesis was then compared with that on the activity of 4 antioxidant enzymes in the arterial wall. Results-Atherosclerosis was 6-to 19-fold greater (PϽ0.01) in extracranial arteries than in intracranial arteries, and it increased linearly with age. Intracranial arteries showed significantly greater antioxidant enzyme activities than did extracranial arteries. However, the antioxidant protection of intracranial arteries decreased significantly in older age, coinciding with a marked acceleration of atherogenesis. An increase in matrix metalloproteinase-9 protein expression and in gelatinolytic activity consistent with the degree of intracranial atherosclerosis was also observed. Conclusions-These results suggest that a greater activity of antioxidant enzymes in intracranial arteries may contribute to their greater resistance to atherogenesis and that with increasing age intracranial arteries respond with accelerated atherogenesis when their antioxidant protection decreases relatively more than that of extracranial arteries. (Stroke. 2001;32:2472-2480.)
Proceedings of the National Academy of Sciences, 1997
In vitro studies have identified a number of adhesion molecules and chemokines that may control t... more In vitro studies have identified a number of adhesion molecules and chemokines that may control this process but very little work has been done to evaluate their relative importance in vivo, in part because there have been no methods available of sufficient sensitivity and reliability. This paper proposes a new approach in which advantage is taken of naturally occurring or transgenically induced mutations to ''mark'' donor cells and to follow their fate in recipient animals using highly sensitive PCR methods. The feasibility of the approach is demonstrated by preliminary studies of monocyte recruitment into atherosclerotic lesions. However, the method should in principle be applicable to the study of any of the circulating leukocytes and their rate of entry into any tissue or tissues of interest.
Increasing evidence indicates that several pathogenic mechanisms promoting atherosclerosis are al... more Increasing evidence indicates that several pathogenic mechanisms promoting atherosclerosis are also involved in neurodegenerative diseases, and that insight into the factors determining the susceptibility to, and long-term progression of, atherosclerosis may be of interest for the evolution of diseases such as Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s. Furthermore, atherosclerosis of intracranial arteries or thromboembolic consequences of atherosclerotic extracranial arteries are responsible for most ischemic events in the brain. Age-related changes of cerebrovascular atherosclerosis, and atherosclerosis in general, may therefore be important for stroke and neurodegenerative diseases affecting the elderly. In the following, pathogenic mechanism involving increased lipid peroxidation, oxidative stress, inflammation and immune responses, and fetal programming will be discussed in the context of cerebrovascular disease and aging.
During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low... more During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low-density lipoprotein (oxLDL) and active immunization of hypercholesterolemic mice with oxLDL ameliorates atherogenesis. We unexpectedly found that many autoantibodies to oxLDL derived from 'naive' atherosclerotic mice share complete genetic and structural identity with antibodies from the classic anti-phosphorylcholine B-cell clone, T15, which protect against common infectious pathogens, including pneumococci. To investigate whether in vivo exposure to pneumococci can affect atherogenesis, we immunized Ldlr -/mice with Streptococcus pneumoniae. This induced high circulating levels of oxLDL-specific IgM and a persistent expansion of oxLDLspecific T15 IgM-secreting B cells primarily in the spleen, which were cross-reactive with pneumococcal determinants. Pneumococcal immunization decreased the extent of atherosclerosis, and plasma from these mice had an enhanced capacity to block the binding of oxLDL to macrophages. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have beneficial effects.
Children generally have low cholesterol and no clinical manifestations of atherosclerosis, but fa... more Children generally have low cholesterol and no clinical manifestations of atherosclerosis, but fatty-streak formation begins in fetuses and is greatly increased by maternal hypercholesterolaemia during pregnancy. In the FELIC study we assessed the evolution of such lesions during childhood. Computer-assisted imaging was used to measure the area of the largest individual lesion and the cumulative lesion area per section in serial cross-sections through the entire aortic arch and abdominal aorta of 156 normocholesterolaemic children aged 1-13 years, who died of trauma and other causes. Children were classified by whether their mother had been normocholesterolaemic (n=97) or hypercholesterolaemic (n=59) during pregnancy. Atherosclerosis was correlated with 13 established or potential risk factors. Findings The largest fatty streaks in the aortic arch of children younger than 3 years of hypercholesterolaemic mothers were 64% smaller than those previously found in corresponding fetuses (p&lt;0.0001), which suggests that fetal fatty streaks may regress after birth. In the two groups, lesion size in the aortic arch and abdominal aorta increased linearly with age (r=0.87-0.98). However, lesions progressed strikingly faster in children of hypercholesterolaemic mothers than in those of normocholesterolaemic mothers (p&lt;0.0001). Conventional risk factors for atherosclerosis in children or mothers correlated with lesion size, but did not account for the faster progression of atherogenesis in normocholesterolaemic children of hypercholesterolaemic mothers. Our results suggest that maternal hypercholesterolaemia during pregnancy induces changes in the fetal aorta that determine the long-term susceptibility of children to fatty-streak formation and subsequent atherosclerosis. If so, cholesterol-lowering interventions in hypercholesterolaemic mothers during pregnancy may decrease atherogenesis in children.
Oxidatively modified low-density lipoprotein (LDL) is present in atherosclerotic but not normal a... more Oxidatively modified low-density lipoprotein (LDL) is present in atherosclerotic but not normal arteries and plays a crucial role in the pathogenesis and adverse consequences of atherosclerotic lesions. We previously generated a series of monoclonal antibodies (MoAb) against oxidation-specific neo-epitopes formed during the oxidative modification of LDL. MDA2, a prototype MoAb, recognizes malondialdehyde-lysine epitopes (eg, in malondi-aldehyde-modified LDL) within atherosclerotic lesions. We describe the in vivo characteristics of MDA2 and initial noninvasive imaging studies of atherosclerosis in rabbits. To assess the in vivo specificity of MDA2 for atherosclerotic lesions, iodine 125-MDA2 was intravenously injected into 7 LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) and 2 normal New Zealand white (NZW) rabbits, and the aortic plaque uptake was evaluated 24 hours later. 125I-Halb, an isotype-matched irrelevant MoAb that binds to human albumin, was injected into 5 WHHL and 2 NZW rabbits as a control. Aortic autoradiography was performed, and the mean uptake of MoAbs was measured as the percent injected dose per gram aortic tissue. Gamma camera imaging was then carried out in 7 WHHL rabbits and 2 NZW rabbits with 99mTc-MDA2. Imaging was carried out at 10 minutes and at 12 or 24 hours. Malondialdehyde-LDL was then injected to clear the blood pool signal, and final images were obtained 2 hours later. Mean uptake of 125I-MDA2 in the entire aorta was 17.4-fold higher in WHHL than in NZW aortas (P &lt; .001), and 2.8-fold higher than 125I-Halb in WHHL aortas. 125I-MDA2 also had higher specificity for lesioned areas than 125I-Halb (plaque/normal ratio 6.3 vs 2.9, P &lt; .001). Autoradiograph of aortas of 125I-MDA2-injected WHHL rabbits revealed uptake in lipid-stained lesions with absence of signal in adjacent normal arterial tissue. Immunostaining of WHHL lesions, which accumulated MDA2 as noted on autoradiography, revealed that uptake was highest in areas with abundant foam cells and in lipid-rich necrotic core areas. Autoradiograph of aortas from NZW rabbits injected with 125I-MDA2 did not yield any visible signal. Planar gamma camera in vivo scintigraphy revealed a visible signal in 4/7 WHHL rabbits, which was confirmed by aortic Sudan staining. Radiolabeled MDA2 shows excellent in vivo uptake and specificity for atherosclerotic lesions containing abundant oxidation-specific epitopes. The in vivo imaging studies suggest that noninvasive imaging of oxidation-rich atherosclerotic lesions with radiolabeled MDA2 may be feasible in human beings with optimization of the imaging methods.
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Papers by Wulf Palinski