Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis

Serum paraoxonase (PON1), present on high density lipoprotein, may inhibit low density lipoprotein (LDL) oxidation and protect against atherosclerosis. We generated combined PON1 knockout (KO)/apolipoprotein E (apoE) KO and apoE KO control mice to compare atherogenesis and lipoprotein oxidation. Early lesions were examined in 3-month-old mice fed a chow diet, and advanced lesions were examined in 6-month-old mice fed a high fat diet. In both cases, the PON1 KO/apoE KO mice exhibited significantly more atherosclerosis (50-71% increase) than controls. We examined LDL oxidation and clearance in vivo by injecting human LDL into the mice and following its turnover. LDL clearance was faster in the double KO mice as compared with controls. There was a greater rate of accumulation of oxidized phospholipid epitopes and a greater accumulation of LDL-immunoglobulin complexes in the double KO mice than in controls. Furthermore, the amounts of three bioactive oxidized phospholipids were elevated in the endogenous intermediate density lipoprotein/LDL of double KO mice as compared with the controls. Finally, the expression of heme oxygenase-1, peroxisome proliferator-activated receptor gamma, and oxidized LDL receptors were elevated in the livers of double KO mice as compared with the controls. These data demonstrate that PON1 deficiency promotes LDL oxidation and atherogenesis in apoE KO mice.     

Phosphocholine as a pattern recognition ligand for CD36

We have previously shown that CD36 recognizes oxidation products of phospholipids on oxidized LDL (OxLDL) such as 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). The current study was designed to examine whether the phosphocholine (PC) headgroup in POVPC constitutes an obligatory binding target for CD36. To examine the contribution of PC in the binding of POVPC to CD36, we used well-defined synthetic oxidized phospholipids (OxPLs) cross-linked to BSA or to a hexapeptide. The OxPL adducts were then tested for their ability to bind to CD36-transfected cells and for their ability to inhibit OxLDL binding to CD36. Both POVPC-BSA and POVPC-peptide adducts were high-affinity ligands for CD36 and potent inhibitors of OxLDL binding. Enzymatic removal of the entire PC moiety of the POVPC-peptide, or of the choline headgroup alone, as well as substitution of the choline headgroup by ethanolamine abrogated the inhibitory activity of POVPC. Interestingly, PC by itself or cross-linked to BSA did not show any intrinsic competition activity. In conclusion, our data demonstrate that the PC headgroup of OxPL alone is sufficient for binding to CD36, but only if presented in the correct conformation as in OxPL of OxLDL or as in POVPC-peptide adducts.     

Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.     

LDL receptor deficiency or apoE mutations prevent remnant clearance and induce hypertriglyceridemia in mice

We have used adenovirus-mediated gene transfer and bolus injection of purified apolipoprotein E (apoE) in mice to determine the contribution of LDL receptor family members in the clearance of apoE-containing lipoproteins in vivo and the factors that trigger hypertriglyceridemia. A low dose [5 x 10(8) plaque-forming units (pfu)] of an adenovirus expressing apoE4 did not normalize plasma cholesterol levels of apolipoprotein E-deficient (apoE(-/-)) x low density lipoprotein receptor-deficient (LDLr(-/-)) mice and induced hypertriglyceridemia. A similar phenotype of combined dyslipidemia was induced in apoE(-/-) or apoE(-/-) x LDLr(-/-) mice after infection with a low dose (4 x 10(8) pfu) of an adenovirus expressing the apoE4[R142V/R145V] mutant previously shown to be defective in receptor binding. In contrast, a low dose of 5 x 10(8) pfu of the apoE4-expressing adenovirus corrected hypercholesterolemia in apoE(-/-) mice and did not trigger hypertriglyceridemia. Bolus injection of purified apoE in apoE(-/-) x LDLr(-/-) mice did not clear plasma cholesterol levels and induced mild hypertriglyceridemia. In contrast, similar injection of apoE in apoE(-/-) mice cleared plasma cholesterol and caused transiently mild hypertriglyceridemia. These findings suggest that a) the LDL receptor alone can account for the clearance of apoE-containing lipoproteins in mice, and the contribution of other receptors is minimal, and b) defects in either the LDL receptor or in apoE that affect its interactions with the LDL receptor, increase the sensitivity to apoE-induced hypertriglyceridemia in mice.     

Induction of neonatal tolerance to oxidized lipoprotein reduces atherosclerosis in ApoE knockout mice

BACKGROUND: In the course of atherosclerosis, humans and apolipoprotein (apoE) Knockout (KO) mice exhibit an active cell-mediated and humoral immune process, both at the systemic level and within atheromata. Low density lipoproteins (LDL) infiltrate the vascular wall, where they are oxidatively modified. This oxidative modification may generate new epitopes for which tolerance is not achieved during ontogenesis. Such epitopes could constitute new targets for autoreactive immune responses that may have a physiopathological role in disease development. MATERIALS AND METHODS: Exposing mice to high dose of antigens during thymic T-cell education induces immunological tolerance to the administered antigens. We injected newborn apoE KO mice with oxidized LDL. They were fed a cholesterol-rich diet and aortic atherosclerosis, cell-mediated immune response, and T-cell repertoire were analyzed after 5 months. RESULTS: Injection of oxidized LDL at birth reduced not only the immune response to oxidized LDL, but also susceptibility to atherosclerosis in apoE mice. Injection of oxidized LDL induced T-cell tolerance due to clonal deletion, rather than anergy of the reactive T cells. The T-cell repertoire of apoE KO mice was affected by the development of the disease, whereas tolerization normalized it. CONCLUSIONS: This study demonstrates that the immune response against oxidized LDL has a deleterious role in atherogenesis and that a fine-tuning of this response could modify the course of the disease.     

A novel function of lipoprotein [a] as a preferential carrier of oxidized phospholipids in human plasma

Oxidized phospholipids (OxPLs) on apolipoprotein B-100 (apoB-100) particles are strongly associated with lipoprotein [a] (Lp[a]). In this study, we evaluated whether Lp[a] is preferentially the carrier of OxPL in human plasma. The content of OxPL on apoB-100 particles was measured with monoclonal antibody E06, which recognizes the phosphocholine (PC) headgroup of oxidized but not native phospholipids. To assess whether OxPLs were preferentially bound by Lp[a] as opposed to other lipoproteins, immunoprecipitation and ultracentrifugation experiments, in vitro transfer studies, and chemiluminescent ELISAs were performed. Immunoprecipitation of Lp[a] from human plasma with an apolipoprotein [a] (apo[a])-specific antibody demonstrated that more than 85% of E06 reactivity (i.e., OxPL) coimmunoprecipitated with Lp[a]. Ultracentrifugation experiments showed that nearly all OxPLs were found in fractions containing apo[a], as opposed to other apolipoproteins. In vitro transfer studies showed that oxidized LDL preferentially donates OxPLs to Lp[a], as opposed to LDL, in a time- and temperature-dependent manner, even in aqueous buffer. Approximately 50% of E06 immunoreactivity could be extracted from isolated Lp[a] following exposure of plasma to various lipid solvents. These data demonstrate that Lp[a] is the preferential carrier of PC-containing OxPL in human plasma. This unique property of Lp[a] suggests novel insights into its physiological function and mechanisms of atherogenicity.     

Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors

Previous studies have shown that macrophage receptors for oxidized LDL (OxLDL) recognize both the lipid and protein moieties, and that a monoclonal antibody against OxLDL, EO6, also recognizes both species. The present studies show directly that during LDL oxidation phospholipids become covalently attached to apolipoprotein B (apoB). After exhaustive extraction of lipids, apoB of native LDL contained 4 +/- 3 moles of phosphorus/mole protein. In contrast, apoB of OxLDL contained approximately 75 moles of phosphorus/mole protein. Saponification of this apoB released phosphorus, choline, and saturated fatty acids in a molar ratio of 1.0:0.98:0.84. When LDL was reductively methylated prior to oxidation, the amount of phospholipid covalently bound was reduced by about 80%, indicating that the phospholipids attach at lysine epsilon amino groups. Progressive decreases in the phospholipid associated with apoB of OxLDL decreased the ability of the protein to compete for binding to macrophage scavenger receptors and decreased its reactivity with antibody EO6.We postulate that some oxidized phospholipids containing fatty acid aldehydes at the sn-2 position bind to lysine residues of apoB while others remain unreacted within the lipid phase. This would account for the interchangeability of lipid and apolipoprotein of OxLDL with respect to receptor binding and antibody recognition.     

Immunological responses to oxidized LDL

Considerable evidence now points to an important role for the immune system in experimental models of atherosclerosis. We have reviewed the growing body of evidence that oxidation of LDL generates a wide variety of neoself determinants that lead to cellular and humoral immune responses. In particular, we have demonstrated that at least some of the oxidation-specific epitopes generated on the oxidized LDL particle, such as oxidized phospholipid epitopes, are also generated on apoptotic cells and are also present on the surface of some bacteria. Many of these same epitopes serve as important ligands mediating the binding and clearance of oxidatively damaged lipoprotein particles and apoptotic cells, and the innate immune response to these epitopes can be seen as a conserted response to effect their removal. In addition, other epitopes of OxLDL also undoubtedly play a role in the immune activation that characterizes the progressive atherosclerotic plaque. It will be of great importance to define the importance of the role of these responses and to understand which are beneficial and which deleterious. Such information could lead one day to novel therapeutic approaches to inhibit atherogenesis that take advantage of the ability to manipulate the immune response.     

ApoE-/-Fas-/- C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia

To establish a mouse model of accelerated atherosclerosis in lupus, we generated apolipoprotein E-deficient (apoE(-/-)) and Fas(lpr/lpr) (Fas(-/-)) C57BL/6 mice. On a normal chow diet, 5 month old apoE(-/-)Fas(-/-) mice had enlarged glomerular tuft areas, severe proteinuria, increased circulating autoantibody levels, and increased apoptotic cells in renal and vascular lesions compared with either single knockout mice. Also, double knockout mice developed increased atherosclerotic lesions but decreased serum levels of total and non-HDL cholesterol compared with apoE(-/-)Fas(+/+) littermates. Moreover, female apoE(-/-)Fas(-/-) mice had lower vertebral bone mineral density (BMD) and bone volume density (BV/TV) than age-matched female apoE(-/-)Fas(+/+) mice. Compared with apoE(-/-)Fas(+/+) and apoE(+/+)Fas(-/-) mice, apoE(-/-)Fas(-/-) mice had decreased circulating oxidized phospholipid (OxPL) content on apoB-100 containing lipoprotein particles and increased serum IgG antibodies to OxPL, which were significantly correlated with aortic lesion areas (r = 0.58), glomerular tuft areas (r = 0.87), BMD (r = -0.57), and BV/TV (r = -0.72). These results suggest that the apoE(-/-)Fas(-/-) mouse model might be used to study atherosclerosis and osteopenia in lupus. Correlations of IgG anti-OxPL with lupus-like disease, atherosclerosis, and bone loss suggested a shared pathway of these disease processes.     

Deficiency of inducible NO synthase reduces advanced but not early atherosclerosis in apolipoprotein E-deficient mice

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.     

Molecular mechanisms of type III hyperlipoproteinemia: The contribution of the carboxy-terminal domain of ApoE can account for the dyslipidemia that is associated with the E2/E2 phenotype

Apolipoprotein E2, which has an R158 for C substitution, has reduced affinity for the LDL receptor and is associated with type III hyperlipoproteinemia in humans. Consistent with these observations, we have found that following adenovirus-mediated gene transfer, full-length apoE2 aggravates the hypercholesterolemia and induces hypertriglyceridemia in E-deficient mice and induces combined hyperlipidemia in C57BL/6 mice. Unexpectedly, the truncated apoE2-202 form that has an R158 for C substitution when expressed at levels similar to those of the full-length apoE2 normalized the cholesterol levels of E-deficient mice without induction of hypertriglyceridemia. The apoE2 truncation increased the affinity of POPC-apoE particles for the LDL receptor, and the full-length apoE2 had a dominant effect in VLDL triglyceride secretion. Hyperlipidemia in normal C57BL/6 mice was prevented by coinfection with equal doses of each, the apoE2 and the apoE2-202-expressing adenoviruses, indicating that truncated apoE forms have a dominant effect in remnant clearance. Hypertriglyceridemia was completely corrected by coinfection of mice with an adenovirus-expressing wild-type lipoprotein lipase, whereas an inactive lipoprotein lipase had a smaller effect. The findings suggest that the apoE2-induced dyslipidemia is not merely the result of substitution of R158 for C but results from increased secretion of a triglyceride-enriched VLDL that cannot undergo lipolysis, inhibition of LpL activity, and impaired clearance of chylomicron remnants. Infection of E(-)(/)(-)xLDLr(-)(/)(-) double-deficient mice with apoE2-202 did not affect the plasma cholesterol levels, and also did not induce hypertriglyceridemia. In contrast, apoE2 exacerbated the hypercholesterolemia and induced hypertriglyceridemia, suggesting that the LDL receptor is the predominant receptor in remnant clearance.     

Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.     

Autoantibody titers against OxLDL are correlated with Achilles tendon thickness in patients with familial hypercholesterolemia

Achilles tendon xanthomas are associated with increased cardiovascular risk in patients with familial hypercholesterolemia (FH). Oxidized low density lipoprotein (OxLDL), the antibodies against OxLDL, and the LDL-associated phospholipase A(2) (Lp-PLA(2)) may play important roles in atherogenesis. We investigated the possible association between plasma levels of OxLDL, Lp-PLA(2) activity, and autoantibody titers against various types of mildly OxLDL with Achilles tendon thickness (ATT). ATT was determined by sonography in 80 unrelated heterozygous FH patients. Three different types of mildly OxLDL were prepared: OxLDL(L), OxLDL(P), and OxLDL(D), at the end of the lag, propagation, and decomposition phases of oxidation, respectively. Similar types of OxLDL were also prepared after inactivation of the LDL-associated Lp-PLA(2). These types were denoted OxLDL(-)(L), OxLDL(-)(P), and OxLDL(-)(D). FH patients exhibited significantly higher plasma OxLDL levels and serum IgG titers against OxLDL(P) and OxLDL(D) compared with 40 normolipidemic apparently healthy controls. ATT values were positively correlated with autoantibody titers against OxLDL(P) and OxLDL(D); however, in multiple regression analysis, ATT was independently associated only with the autoantibody titers against OxLDL(D). We conclude that the IgG autoantibody titers against OxLDL(D) but not OxLDL or Lp-PLA(2) may play an important role in the pathogenesis of Achilles tendon xanthomas in FH patients.     

Definition of the immunogenic forms of modified human LDL recognized by human autoantibodies and by rabbit hyperimmune antibodies

Humans and laboratory animals recognize human modified LDL as immunogenic. Immune complexes (ICs) isolated from human sera contain malondialdehyde-modified LDL (MDA-LDL) and N (epsilon)(carboxymethyl)lysine-modified LDL (CML-LDL) as well as antibodies reacting with MDA-LDL, copper-oxidized LDL (OxLDL), CML-LDL, and advanced glycosylation end product (AGE)-modified LDL. OxLDL and AGE-LDL antibodies isolated from human sera recognize the same LDL modifications and do not react with modified non-LDL proteins. Rabbit antibodies have different reactivity patterns: MDA-LDL antibodies react strongly with MDA-LDL and MDA-BSA but weakly with OxLDL; OxLDL antibodies react strongly with OxLDL and weakly with MDA-LDL; CML-LDL antibodies react with CML-LDL > CML-BSA > AGE-LDL > OxLDL; AGE-LDL antibodies react strongly with AGE-LDL, react weakly with OxLDL, and do not react with CML-LDL. Thus, human and rabbit antibodies seem to recognize different epitopes. Capture assays carried out with all rabbit antibodies showed binding of apolipoprotein B-rich lipoproteins isolated from ICs, suggesting that laboratory-generated epitopes are expressed by in vivo-modified LDL, although they are not necessarily recognized by the human immune system. Thus, the definition of immunogenic forms of modified LDL eliciting human autoimmune responses requires the isolation and characterization of autoantibodies and modified LDL from human samples, whereas rabbit antibodies can be used to detect in vivo-modified human LDL.     

Cationic domain 141-150 of apoE covalently linked to a class A amphipathic helix enhances atherogenic lipoprotein metabolism in vitro and in vivo

We previously showed 1 that a peptide, Ac-hE18A-NH(2), in which the arginine-rich heparin-binding domain of apolipoprotein E (apoE) [residues 141;-150] (LRKLRKRLLR), covalently linked to 18A (DWLKAFYDKVAEKLKEAF; a class A amphipathic helix with high lipid affinity), enhanced LDL uptake and clearance. Because VLDL and remnants contain more cholesterol per particle than LDL, enhanced hepatic clearance of VLDL could lead to an effective lowering of plasma cholesterol. Therefore, in the present article we compared the ability of this peptide to mediate/facilitate the uptake and degradation of LDL and VLDL in HepG2 cells. The peptide Ac-hE18A-NH(2), but not Ac-18A-NH(2), enhanced the uptake of LDL by HepG2 cells 5-fold and its degradation 2-fold. The association of the peptides with VLDL resulted in the displacement of native apoE; however, only Ac-hE18A-NH(2) but not Ac-18A-NH(2) caused markedly enhanced uptake (6-fold) and degradation (3-fold) of VLDL. Ac-hE18A-NH(2) also enhanced the uptake (15-fold) and degradation (2-fold) of trypsinized VLDL Sf 100;-400 (containing no immuno-detectable apoE), indicating that the peptide restored the cellular interaction of VLDL in the absence of its essential native ligand (apoE). Pretreatment of HepG2s with heparinase and heparitinase abrogated all peptide-mediated enhanced cellular activity, implicating a role for cell-surface heparan sulfate proteoglycans (HSPG). Intravenous administration of Ac-hE18A-NH(2) into apoE gene knockout mice reduced plasma cholesterol by 88% at 6 h and 30% at 24 h after injection. We conclude that this dual-domain peptide associates with LDL and VLDL and results in rapid hepatic uptake via a HSPG-facilitated pathway.     

Infection induces a positive acute phase apolipoprotein E response from a negative acute phase gene: role of hepatic LDL receptors

Apolipoprotein E (apoE) plays important roles in lipid homeostasis, anti-inflammation, and host defense. Since tissue apoE mRNA levels have been reported to decrease during inflammatory responses, we were surprised to find that plasma apoE levels were significantly elevated during septic infections in both humans and mice. This apparent paradox was also observed during lipopolysaccharide-induced acute inflammation in mice: plasma levels of apoE increased up to 4-fold despite sharply decreased apoE gene expression in the liver, macrophages, and extrahepatic tissues. We hypothesized that apoE levels were augmented by decreased plasma clearance. Our analysis revealed that apoE associated principally with HDL in mice and that apoE was cleared from the circulation principally via LDL receptors. The acute inflammatory response decreased LDL receptor expression in the liver and significantly reduced the rate of apoE clearance. In contrast, the same inflammatory stimuli increased LDL receptor expression in macrophages. Our results define a novel acute phase mechanism that increases circulating apoE levels as apoE production decreases. Diminished hepatic LDL receptor expression may thus cooperate with elevated LDL receptor expression in macrophages to facilitate the forward transport of apoE and its associated lipids to these key defense cells.     

Intact endothelial autophagy is required to maintain vascular lipid homeostasis

The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular ¹²⁵I‐LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE^?/?mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age‐dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.     

Evaluation of low density lipoprotein oxidation in a course of hypothyroidism

INTRODUCTION: The aim of this study was to evaluate the influence of hypothyroidism on oxidative modification of low density lipoprotein (LDL). MATERIAL AND METHODS: 24 patients with overt hypothyroidism and 10 patients with mild hypothyroidism were enrolled to the study. The control group consisted of 24 healthy subjects with normal serum TSH. Plasma level of oxidized LDL (oxLDL) and serum level of antibodies against oxidized LDL (anti-oxLDL) determined lipoprotein oxidation. RESULTS: Significantly increased plasma oxLDL levels were found in patients with overt hypothyroidism in comparison to patients with mild hypothyroidism and control group. Anti-oxLDL levels in patients with overt or mild hypothyroidism and in the control group showed no significant differences. OxLDL plasma levels in patients with hypothyroidism inversely correlated with FT(4) levels and positively correlated with TSH, total cholesterol, LDL cholesterol and triglycerides levels. CONCLUSIONS: The presented study indicates increased lipoprotein oxidation in patients with hypothyroidism which depends on the degree of hypothyroidism and changes in lipid profile. Elevated cholesterol and triglycerides levels are the factors increasing lipoprotein oxidation. Plasma oxLDL levels may constitute a useful marker indicating the risk for atherosclerosis in hypothyroidism.     

In vivo clearance of low-density lipoproteins and beta-very-low-density lipoproteins in normal and hypercholesterolemic White Carneau pigeons

Low-density lipoproteins (hLDL) and beta-migrating-very-low-density lipoproteins (beta-VLDL) were isolated from the plasma of cholesterol-fed White Carneau (WC) pigeons and low-density lipoproteins (nLDL) were isolated from the plasma of grain-fed WC pigeons. The lipoproteins were radiolabeled with 125I or 131I and injected into normocholesterolemic or hypercholesterolemic WC pigeons to determine their rate of clearance from the plasma. The fractional catabolic rate (FCR) of nLDL and hLDL in normocholesterolemic pigeons averaged 0.202 and 0.206 pools/h.respectively. beta-VLDL was cleared at a significantly slower rate of 0.155 pools/h. The FCR of the same lipoproteins injected into hypercholesterolemic pigeons was reduced by 17% for nLDL, 50% for hLDL and 57% for beta-VLDL, indicating that the effect of hypercholesterolemia on clearance in vivo was different for the three lipoproteins. The FCR of reductively methylated pigeon LDL (MeLDL), which gives a measure of receptor-independent clearance of LDL, was shown previously to be 0.037 pools/h. These studies suggest therefore that LDL and beta-VLDL are cleared from the plasma of normocholesterolemic and hypercholesterolemic pigeons at a rate substantially greater than that predicted for non-specific processes. Despite the reduction in the clearance rate of hLDL and beta-VLDL due to cholesterol feeding, the absolute amount of cholesterol that was cleared from the plasma by these lipoproteins was increased from approx. 200 mg/kg body weight per day in the normocholesterolemic pigeons to greater than 1000 mg/kg body weight per day in the hypercholesterolemic pigeons. This is due principally to the enrichment in cholesterol relative to protein of the lipoproteins isolated from cholesterol-fed pigeons and the failure of hypercholesterolemia to completely inhibit receptor-dependent clearance of LDL and beta-VLDL. The lower rate of clearance of beta-VLDL relative to LDL is in marked contrast to mammalian beta-VLDL, which is cleared much faster than LDL, but is consistent with the lack of apo E on pigeon lipoproteins. Apo E is the apoprotein that is thought to be responsible for the rapid clearance of beta-VLDL in normocholesterolemic mammals. The low rate of beta-VLDL clearance in pigeons also suggests that pigeons lack an apolipoprotein that function like mammalian apo E.     

Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4

We have used adenovirus-mediated gene transfer in apolipoprotein (apo)E^−/− mice to elucidate the molecular etiology of a dominant form of type III hyperlipoproteinemia (HLP) caused by the R142C substitution in apoE4. It was found that low doses of adenovirus expressing apoE4 cleared cholesterol, whereas comparable doses of apoE4[R142C] greatly increased plasma cholesterol, triglyceride, and apoE levels, caused accumulation of apoE in VLDL/IDL/LDL region, and promoted the formation of discoidal HDL. Co-expression of apoE4[R142C] with lecithin cholesterol acyltransferase (LCAT) or lipoprotein lipase (LPL) in apoE^−/− mice partially corrected the apoE4[R142C]-induced dyslipidemia. High doses of C-terminally truncated apoE4[R142C]-202 partially cleared cholesterol in apoE^−/− mice and promoted formation of discoidal HDL. The findings establish that apoE4[R142C] causes accumulation of apoE in VLDL/IDL/LDL region and affects in vivo the activity of LCAT and LPL, the maturation of HDL, and the clearance of triglyceride-rich lipoproteins. The prevention of apoE4[R142C]-induced dyslipidemia by deletion of the 203-299 residues suggests that, in the full-length protein, the R142C substitution may have altered the conformation of apoE bound to VLDL/IDL/LDL in ways that prevent triglyceride hydrolysis, cholesterol esterification, and receptor-mediated clearance in vivo.