Ability Of Low-density Lipoprotein Research Articles

Interleukin-8 Secretion by Fibroblasts Induced by Low Density Lipoproteins Is p38 MAPK-dependent and Leads to Cell Spreading and Wound Closure

We have previously reported (Dobreva, I., Waeber, G., Mooser, V., James, R. W., and Widmann, C. (2003) J. Lipid Res. 44, 2382-2390) that low density lipoproteins (LDLs) induce activation of the p38 MAPK pathway, resulting in fibroblast spreading and lamellipodia formation. Here, we show that LDL-stimulated fibroblast spreading and wound sealing are due to secretion of a soluble factor. Using an antibody-based human protein array, interleukin-8 (IL-8) was identified as the main cytokine whose concentration was increased in supernatants from LDL-stimulated cells. Incubation of supernatants from LDL-treated cells with an anti-IL-8 blocking antibody completely abolished their ability to induce cell spreading and mediate wound closure. In addition, fibroblasts treated with recombinant IL-8 spread to the same extent as cells incubated with LDL or supernatants from LDL-treated cells. The ability of LDL and IL-8 to induce fibroblast spreading was mediated by the IL-8 receptor type II (CXCR-2). Furthermore, LDL-induced IL-8 production and subsequent wound closure required the activation of the p38 MAPK pathway, because both processes were abrogated by a specific p38 inhibitor. Therefore, the capacity of LDLs to induce fibroblast spreading and accelerate wound closure relies on their ability to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL cholesterol levels, IL-8 production, and extensive remodeling of the vessel wall.

Identification of a domain in apolipoprotein B-100 that inhibits the procoagulant activity of tissue factor.

The ability of low-density lipoprotein (LDL) to inhibit the procoagulant activity of tissue factor is mediated by a direct protein-protein interaction involving apolipoprotein (apo) B-100. A lysine-rich sequence within apo B-100 (residues 3121-3217), which we have termed lysine-rich apo B-100-derived (KRAD)-98 peptide, may be responsible for its activity. Within this region, residues 3147-3160 (KRAD-14) contain an exceptionally high proportion of positive amino acids. Both recombinant KRAD-98 and KRAD-14 peptides inhibited the procoagulant activity of tissue factor by preventing the activation of factor VII. KRAD-14 also inhibited the prothrombinase components, factors Xa and V. In comparison with the parent protein (apo B-100), KRAD-14 peptide displayed a 20-fold enhancement in the rate of inhibition, whereas KRAD-98 peptide exhibited a rate closer to that of apo B-100. Mutational analysis of KRAD-14 peptide revealed three adjacent amino acids, alteration of which greatly reduced the inhibitory potential of this peptide. A peptide derived from tissue factor (residues 58-66) was found to act co-operatively with tissue factor itself, but also augmented the inhibition of tissue-factor activity by apo B-100. In conclusion, LDL may be a physiological regulator of haemostatic mechanisms through the interactions of lysine-rich domains of apo B-100 with tissue factor.

In vivo oxidized low density lipoprotein: degree of lipoprotein oxidation does not correlate with its atherogenic properties.

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.

Low-Density Lipoprotein Receptor-Related Protein/ α2-Macroglobulin Receptor on Murine Peritoneal Macrophages Mediates the Binding and Catabolism of Low-Density Lipoprotein

Low-density lipoprotein receptor-related protein (LRP)/α2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including α2-macroglobulin–proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, andPseudomonasexotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4°C (Kd= 1.8 nM) and competes for the binding of a receptor-recognized form of α2-macroglobulin (α2M*) (Ki= 3 nM). α2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd= 5 nM). LDL can also completely inhibit the binding of α2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with α2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as α2M*. In addition, the ability of α2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.

Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man

Animal studies indicate a possible role for lipid oxidation in the development of atherosclerosis. We set out to investigate whether there was a relation between the ability of low-density lipoprotein (LDL) to resist oxidation in vitro and the severity of coronary atherosclerosis in man. 35 unselected young (mean [SD] age 39·9 [4·2] years) male survivors of myocardial infarction underwent angiography, and LDL was isolated from their plasma by density gradient ultracentrifugation. In-vitro LDL susceptibility to oxidation was assessed by determination of the lag phase for the formation of conjugated dienes in the presence of copper ions. An inverse relation was found between lag phase and quantitative estimates of global coronary atherosclerosis (r=-0·45; p<0·02). Multivariate analysis indicated that the lag phase for oxidative modification of LDL and LDL cholesterol concentration correlated independently with severity of coronary atherosclerosis. The lag phase for oxidation of LDL was also related to the triglyceride content of the LDL fraction (r=-0·55; p<0·002). The finding that susceptibility to LDL oxidation is associated with severity of coronary atherosclerosis may indicate that lipid oxidation promotes premature coronary atherosclerosis and that individuals with an LDL enriched in triglycerides are at particular risk.

Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1-14C]arachidonate (recLDL-[14C]AA-CE), resting monocytes formed three labeled products of the prostaglandin (PG) H synthase pathway: 6-keto-PGF1 alpha, thromboxane B2, and PGE2. The amounts of these eicosanoids in response to recLDL-[14C]AA-CE were comparable to or exceeded those that were produced in response to the addition of 10 microM unesterified [1-14C]AA. By contrast, resting monocytes formed only small amounts of products of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4 from either recLDL-[14C]AA-CE or [14C]AA, indicating preferential utilization of AA in the PGH synthase reaction. However, they converted LDL-derived [14C]AA efficiently into LTB4 and LTC4, when they were first incubated with recLDL-[14C]AA-CE and subsequently stimulated with the chemotactic peptide N-formylmethionylleucylphenylalanine or the Ca2+ ionophore A23187. The classical LDL receptor pathway mediated the synthesis of all of the above eicosanoids from LDL but not from unesterified AA. These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.

Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia.

Familial defective apolipoprotein B-100 is a genetic disorder of apolipoprotein B-100 that causes moderate to severe hypercholesterolemia. A single amino acid mutation in apolipoprotein B diminishes the ability of low density lipoproteins to bind to the low density lipoprotein receptor. Low density lipoproteins accumulate in the plasma because their efficient receptor-mediated catabolism is disrupted. This mutation has been identified in the United States, Canada, and Europe and is estimated to occur at a frequency of approximately 1/500 in these populations. Thus, it appears that this newly described disorder may be a significant genetic cause of hypercholesterolemia in Western societies.

Metabolism of low-density lipoprotein from patients with diabetic hypertriglyceridemia by cultured human skin fibroblasts.

To test whether triglyceride-enriched low-density lipoprotein (LDL) obtained from subjects with diabetic hypertriglyceridemia is metabolized normally by cells, LDL was separated from seven healthy control subjects (fasting plasma glucose [FPG] 91 +/- 10 mg/dl [mean +/- SD], triglyceride [TG] 110 +/- 47 mg/dl), six diabetic normolipidemic patients (FPG 218 +/- 65 mg/dl; TG 139 +/- 75 mg/dl), six diabetic hypertriglyceridemic patients (FPG 214 +/- 71 mg/dl; TG 1915 +/- 1680 mg/dl), and five nondiabetic hypertriglyceridemic patients (FPG 92 +/- 8 mg/dl; TG 2013 +/- 1889 mg/dl). Binding of 125I-labeled LDL from hypertriglyceridemic subjects with and without diabetes to cultured skin fibroblasts was significantly decreased to 74 +/- 19% and 78 +/- 14% of that seen with LDL from normolipidemic nondiabetic subjects and diabetic normolipidemic controls (100 +/- 0%, 101 +/- 25%; P less than 0.005). Unlabeled LDL from hypertriglyceridemic subjects with and without diabetes failed to suppress LDL receptor activity and sterol synthesis from 14C-acetate as efficiently as unlabeled LDL from healthy subjects. The ability of LDL from hypertriglyceridemic subjects, whether diabetic or not, to suppress LDL binding was inversely related to the ratio of triglyceride to protein in LDL (r = 0.71, P less than 0.01) and showed a positive correlation with the LDL cholesterol/protein ratio (0.69, P less than 0.01). Thus, LDL from patients with hypertriglyceridemia, with or without coexistent diabetes, shows impaired binding to LDL receptors and less ability to downregulate LDL receptor activity and sterol synthesis than does LDL from normolipidemic diabetic and nondiabetic subjects. These findings suggest that factors associated with hypertriglyceridemia rather than with diabetes result in altered metabolism of LDL in these disorders.

IMMUNOSUPPRESSION BY NEONATAL APOLIPOPROTEIN E

Recent reports have suggested that in adults certain lipoproteins, particularly those carrying apolipoprotein (apo) E and apo B, play a role in suppression of the immune system. We have investigated the ability of lipoproteins from umbilical cord blood (CB) to suppress immune response. CB lipoprotein concentrations are lower than those of adult, i.e., the low density lipoprotein (LDL) level in CB is 30% that of adult while the high density lipoprotein (HDL) level is 50% of adult. Apo E concentration in CB is 2-fold higher than adult (5.8±2.5 mg/dl vs. 3.1±0.9 mg/dl). The ability of LDL and HDL to inhibit mitogen-stimulated 3H-thymidine uptake in adult peripheral blood mononuclear cells (PMC) was used as an in vitro test system to study immunosuppression. Relative to adult lipoproteins, CB LDL and HDL were 2 to 4 times more potent in inhibiting PMC proliferation. Radioimmunoassay showed a strong correlation between amount of apo E in CB LDL and HDL and PMC inhibition. Selective removal of apo E-containing lipoproteins decreased significantly the inhibitory effect in CB LDL and eliminated almost completely inhibition by HDL. Results indicate that CB lipoproteins containing apo E in association with apo B and AI are capable of suppressing the immune response. Since the fetus is an allograft to its mother, the relatively high apo E levels may have a functional significance in the establishment of self and maintenance of the fetus in utero.

Serum lipid transport systems: recent advances.

AbstractLipids circulating in the plasma are transported in water soluble form as lipid‐protein complexes. Lipoproteins can be classified according to size, density, electrophoretic mobility and protein composition. The ability of low density lipoproteins and very low density lipoproteins (VLDL) to form complexes with different polyanions has been also used as a method for separation and study of serum lipoproteins. Even within classes of lipoproteins closely related otherwise, the amount of different lipids and their ratios to each other and to protein are variable. Two enzymatic systems seem to be at least partly responsible for the different lipid compositions of serum lipoproteins: lipoprotein lipase and lecithin‐cholesterol acyltransferase (LCAT). LCAT, which seems to be associated with α‐lipoproteins, is responsible for the formation of the bulk of cholesteryl‐esters in human serum. Changes in activity of this enzyme may explain the observed changes with age and disease of serum cholesteryl‐ester fatty acids (CEFA). Differences in CEFA pattern are found between newborn and adult animals, including man. The activity of serum LCAT was observed to increase with age in animals and to decrease markedly in patients with liver cirrhosis. These patients show abnormal serum CEFA patterns and abnormally low proportions of pre‐β‐ (VLDL) and α‐ (high density) lipoproteins.

Transport sanguin et mode d’action d’un médicament hypolipidémiant apparenté au Clofibrate: Le G P 45.699

G P 45.699 is a chemical hypolipidemic agent related to Clofibrate. The present study, achieved in man during a clinical assay, shows that the drug, after its intestinal absorption, is carried in blood by serum albumin, and also by low density lipoproteins, chiefly density 1.006–1.063 lipoproteins. In 4 normal subjects, the drug is found to be bound to albumin for 93–98%; to low density lipoproteins for only 2–6%. But when molecular weights are considered, the mole ratio of the drug per mole of albumin is less than 1, whereas per mole of lipoprotein it reaches 13 and even 25. Similar results were obtained in 4 hyperlipidemic subjects. This ability of low density lipoproteins to transport G P 45.699 &lt;i&gt;in vivo&lt;/i&gt; was confirmed &lt;i&gt;in vitro&lt;/i&gt; by the ability of purified &lt;i&gt;β&lt;/i&gt; lipoproteins to bind the drug in saline. The presence of the drug on low density lipoproteins allows a new hypothesis concerning the hypolipidemic action of G P 45.699 and of related chemical compounds, Clofibrate being the best known. According to this hypothesis, the drug when binding with peptide B (apo-&lt;i&gt;β&lt;/i&gt;-lipoprotein) within the mucosal cell, would reduce its ability to bind and transport triglycerides and cholesterol. A direct interaction between the drug and lipoproteins would fit better with its effects on lipidemia. Its interaction with albumin being responsible for some other effects, such an interference with several antivitamine-K anticoagulants.