Wulf Palinski

Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

Autoantibody against oxidised LDL and progression of carotid atherosclerosis

Oxidative modification of LDL renders it immunogenic and autoantibodies to epitopes of oxidised LDL, such as malondialdehyde (MDA)-lysine, are found in serum and recognise material in atheromatous tissue. However, there has been no prospective study to assess the importance of oxidised LDL among patients with vascular disease. We compared the titre of autoantibodies to MDA-modified LDL and native LDL in baseline serum samples of 30 eastern Finnish men with accelerated two-year progression of carotid atherosclerosis and 30 age-matched controls without progression. Neither group had specific antibody binding to native LDL. A titre was defined as a ratio of antibody binding to MDA-LDL/binding to native LDL. Cases had a significantly higher titre to MDA-LDL (2.67 vs 2.06, p = 0.003). Cases also had a greater proportion of smokers (37% vs 3%), higher LDL cholesterol (4.2 mmol/l vs 3.6 mmol/l), and higher serum copper concentration (1.14 mg/l vs 1.04 mg/l). Even after adjusting for these variables and the severity of baseline atherosclerosis, the difference in antibody titre remained significant in a multifactorial logistic model (p = 0.031). Thus, the titre of autoantibodies to MDA-LDL was an independent predictor of the progression of carotid atherosclerosis in these Finnish men. Our data provide further support for a role of oxidatively modified LDL in atherogenesis.

Peroxisome proliferator–activated receptor γ ligands inhibit development of atherosclerosis in LDL receptor–deficient mice

The peroxisome proliferator‐activated receptor γ (PPARγ) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARγ is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARγ-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor‐deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-α and gelatinase B, indicating both systemic and local actions of PPARγ. These findings suggest that PPARγ agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARγ ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities. J. Clin. Invest. 106:523‐531 (2000).

Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions.

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 and larger lesions (758,651+/-87,449 and 451,255+/-37,448 micron2 per section, respectively; mean+/-SD) than aortas from normocholesterolemic mothers (61,862+/-9,555 micron2; P < 0.00005). Serial sections of the arch, thoracic, and abdominal aortas were immunostained for recognized markers of atherosclerosis: macrophages, apo B, and two different oxidation-specific epitopes (malondialdehyde- and 4-hydroxynonenal-lysine). Of the atherogenic sites that showed positive immunostaining for at least one of these markers, 58.6% were established lesions containing both macrophage/foam cells and oxidized LDL (OxLDL). 17.3% of all sites contained only native LDL, and 13.3% contained only OxLDL without monocyte/ macrophages. In contrast, only 4.3% of sites contained isolated monocytes in the absence of native or oxidized LDL. In addition, 6.3% of sites contained LDL and macrophages but few oxidation-specific epitopes. These results demonstrate that LDL oxidation and formation of fatty streaks occurs already during fetal development, and that both phenomena are greatly enhanced by maternal hypercholesterolemia. The fact that in very early lesions LDL and OxLDL are frequently found in the absence of monocyte/macrophages, whereas the opposite is rare, suggests that intimal LDL accumulation and oxidation contributes to monocyte recruitment in vivo.

Angiogenesis Inhibitors Endostatin or TNP-470 Reduce Intimal Neovascularization and Plaque Growth in Apolipoprotein E–Deficient Mice

BACKGROUND Neovascularization within the intima of human atherosclerotic lesions is well described, but its role in the progression of atherosclerosis is unknown. In this report, we first demonstrate that intimal vessels occur in advanced lesions of apolipoprotein E-deficient (apoE -/-) mice. To test the hypothesis that intimal vessels promote atherosclerosis, we investigated the effect of angiogenesis inhibitors on plaque growth in apoE -/- mice. METHODS AND RESULTS ApoE -/- mice were fed a 0.15% cholesterol diet. At age 20 weeks, mice were divided into 3 groups and treated for 16 weeks as follows: group 1, recombinant mouse endostatin, 20 mg. kg-1. d-1; group 2, fumagillin analogue TNP-470, 30 mg/kg every other day; and group 3, control animals that received a similar volume of buffer. Average cholesterol levels were similar in all groups. Plaque areas were quantified at the aortic origin. Median plaque area before treatment was 0.250 mm2 (range, 0.170 to 0.348; n=10). Median plaque areas were 0.321 (0.238 to 0.412; n=10), 0.402 (0.248 to 0.533; n=15), and 0.751 mm2 (0.503 to 0.838; n=12) for the endostatin, TNP-470, and control groups, respectively (P</=0.0001). Therefore, endostatin and TNP-470 inhibited plaque growth during the treatment period by 85% and 70%. Intimal smooth muscle cell contents of plaques from control and treated mice were similar. CONCLUSIONS Prolonged treatment with either angiogenesis inhibitor reduced plaque growth and intimal neovascularization in apoE -/- mice. Although the mechanism of plaque inhibition induced by these agents is not established, these results suggest that intimal neovascularization may promote plaque development.

Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein.

Increasing evidence indicates that low density lipoprotein (LDL) has to be modified to induce foam cell formation. One such modification, oxidation of LDL, generates a number of highly reactive short chain-length aldehydic fragments of oxidized fatty acids capable of conjugating with lysine residues of apoprotein B. By immunizing animals with homologous malondialdehyde-modified LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu+(+)-oxidized LDL, we developed polyvalent and monoclonal antibodies against three epitopes found in oxidatively modified LDL. The present article characterizes an antiserum and monoclonal antibody (MAL-2 and MDA2, respectively) specific for MDA-lysine, and an antiserum and monoclonal antibody (HNE-6 and NA59, respectively) specific for 4-HNE-lysine. In addition, a monoclonal antibody (OLF4-3C10) was developed against an as yet undefined epitope generated during Cu++ oxidation of LDL. With these antibodies, we demonstrated that MDA-lysine and 4-HNE-lysine adducts develop on apo-lipoprotein B during copper-induced oxidation of LDL in vitro. The application of these antibodies for immunocytochemical demonstration of oxidized lipoproteins in atherosclerotic lesions of progressive severity is described in the companion article. These antibodies should prove useful in studying the role of oxidatively modified lipoproteins as well as other oxidatively modified proteins in atherogenesis.

Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARα, β/δ, and γ

PPARα, β/δ, and γ regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor–ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARα, β, and γ agonists on foam-cell formation and atherosclerosis in male LDL receptor–deficient (LDLR–/–) mice. Like the PPARγ agonist, a PPARα-specific agonist strongly inhibited atherosclerosis, whereas a PPARβ-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARα and PPARγ agonists, but not the PPARβ agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARα and PPARγ agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARα required LXRs, activation of PPARγ reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.

Influence of maternal hypercholesterolaemia during pregnancy on progression of early atherosclerotic lesions in childhood: Fate of Early Lesions in Children (FELIC) study

BACKGROUND 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. METHODS 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<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<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. INTERPRETATION 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.

Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL.

Atherosclerotic lesions contain relatively large quantities of IgG. We have previously shown that both human and rabbit sera contain autoantibodies against epitopes of oxidized (Ox) low-density lipoprotein (LDL) and that LDL isolated from atherosclerotic lesions contains small amounts of tightly bound IgG. However, it is not known whether IgG isolated from atherosclerotic lesions recognizes epitopes present in native LDL or Ox-LDL. IgG was isolated from Watanabe heritable hyperlipidemic (WHHL) rabbit atherosclerotic lesions by sequential salt extractions, purified by fast protein liquid chromatography on protein G, and used in a solid-phase radioimmunoassay. IgG and immune complexes were also isolated from the saline extracts of human lesions by adsorption onto latex beads coated with anti-human IgG antibodies or protein A. IgG isolated from rabbit lesions showed significant titers against malondialdehyde (MDA)-modified LDL and LDL oxidized by copper ions for 4 and 18 hours but not against native LDL. On Western blots, lesion IgG stained MDA-LDL and fragments of Ox-LDL. Western blots of immune complexes isolated from human lesions revealed the presence in the isolated complexes of both apoprotein B and apoprotein B fragments, which reacted with antibodies to MDA-lysine. Furthermore, rabbit lesion IgG immunostained epitopes of Ox-LDL present in human atherosclerotic lesions. Immunostains obtained with rabbit lesion IgG were similar to those obtained with a monoclonal antibody specific for MDA-lysine. The results show that human and rabbit atherosclerotic lesions contain IgG that recognizes epitopes characteristic of Ox-LDL. These data suggest that immunologic processes may be an important component of the atherogenic process.

Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits.

Antisera and monoclonal antibodies generated against autologous malondialdehyde-conjugated low density lipoprotein (MDA-LDL), 4-hydroxynonenal conjugated LDL (4-HNE-LDL), and the protein fragments of apoprotein B resulting from the copper oxidation of LDL, as well as antibodies against apoprotein B, were used to immunostain atherosclerotic lesions of varying severity from Watanabe heritable hyperlipemic rabbits. In macrophage-rich fatty streaks and transitional lesions, all of the antibodies recognizing oxidation specific epitopes exhibited predominantly cell-associated staining in particulate and annular patterns. This is in contrast to the limited, extracellular, diffuse staining obtained with the antibodies recognizing apoprotein B. In more advanced lesions containing areas with reduced numbers of cells, there was increased extracellular, diffuse staining with the antibodies against oxidation specific epitopes and co-localization with apoprotein B. In addition, there were annular staining patterns associated with the necrotic core and increased staining of intimal and medial smooth muscle cells. We interpret these data as suggesting that in areas of lesions rich in macrophages, LDL is oxidized and taken up by the cells. In more advanced lesions that are relatively devoid of macrophages, both native and oxidized LDL, as well as oxidation products released from dead and decaying cells, are trapped in the matrix, out of reach of those cells capable of accumulating oxidized LDL.