Very low density lipoprotein binding to cultured aortic endothelium

Because of very low density lipoprotein's (VLDL) potential atherogenicity and the demonstration that VLDL can bind to other cells, we examined the interaction of human VLDL with cultured porcine aortic endothelium. The lipoprotein-cell interaction had many properties similar to those seen with the binding of a ligand to a cell surface receptor. It was time and temperature dependent, saturable, and reversible. Scatchard analysis of competition data suggested that there may be more than one class of binding site. The affinity of the low affinity site was similar to that for low density lipoprotein (LDL). Also, the capacity of endothelial cells to bind VLDL was similar to that for LDL, when related to apo B (i.e., particle) concentration. Not only was unlabelled VLDL able to compete for VLDL binding sites, but so was LDL and high density lipoprotein (HDL). The maximal competition either by LDL or by HDL was less than that by VLDL. The maximal competition by HDL was more than by LDL. The VLDL binding was dependent on Ca2+. It was not changed by the content of lipoprotein in the medium in which cells were grown prior to the binding studies. These observations suggest that VLDL binding to endothelial cells is similar in some respects, but not in all, to the binding of LDL. Comparison of the data with endothelial cells to previous data with adipocytes also indicated differences between the interaction of these two cell types with VLDL. It is possible that this binding process may be involved in the formation of atherogenic remnants of triglyceride-rich lipoproteins on the endothelial surface of large blood vessels.     

1 Acyl-2 acetyl-sn-glycero-3 phosphocholine decreases the susceptibility of low-density lipoprotein to oxidative modification by copper ions,monocytes or endothelial cells

The effects of platelet-activating factor (PAF) and its analogue, 1 acyl-2 acetyl-sn-glycero-3 phosphocholine (1 acyl-2 acetyl-GPC), were investigated on the oxidative modification of low-density lipoprotein (LDL) by copper ions, U937 monocyte-like cells or endothelial cells, by determination of the lipid peroxidation end products (TBARS) content and measurement of the electrophoretic mobility of the particle. 1 Acyl-2 acetyl-GPC, in the concentration range 1–5 μg/ml, inhibited LDL oxidation in a dose-dependent manner in the three systems, whereas PAF had no effect. The protective effect of 1 acyl-2 acetyl-GPC was markedly more important when oxidative modification was performed with endothelial cells, leading to total inhibition at 5 μg/ml. At the same concentration, the TBARS production was inhibited by 60% and 20% with monocytes and copper ions, respectively. The degradation by J774 macrophage-like cells of LDL modified by copper ions, U937 monocyte-like cells or endothelial cells was also inhibited when modification was performed in the presence of 1 acyl-2 acetyl-GPC. Furthermore, preincubation of the LDL particle with 1 acyl-2 acetyl-GPC before modification protected the lipoprotein against oxidation, whereas preincubation of the cultured cells with the phospholipid had no effect. Thus 1 acyl-2 acetyl-GPC decreases the susceptibility of the LDL particle to oxidative modification, possibly by intercalation within the lipid phase of the particle. Since LDL oxidation is believed to play an important role in the initiation and progression of atherosclerosis, this inhibitory effect of 1 acyl-2 acetyl-GPC might be of importance in view of the fact that this phospholipid is produced concomitantly with PAF in some inflammatory cells.     

The surface distribution of low density lipoprotein receptors on cultured fibroblasts and endothelial cells. Ultrastructural evidence for dispersed receptors

The distribution of low density lipoprotein (LDL) receptors marked with colloidal gold-conjugated low density lipoproteins has been mapped on the surfaces of cultured human skin fibroblasts and bovine aortic endothelial cells viewed whole in the transmission electron microscope. A dispersed or scattered population of LDL receptors, in addition to and clearly distinct from clustered receptors was detected on the surfaces of both fibroblasts and dividing endothelial cells. No LDL receptors could be detected on contact-inhibited endothelial cells. Clustered receptors imaged in whole-mount preparations were often arranged in rings with an approximate diameter of 250 nm. In ultra-thin sections of marked cells, clustered receptors were localised in coated pits while the few dispersed receptors seen were restricted to non-coated membrane regions. Clustered receptors often appeared localised on the rims of coated pits whose central areas were not marked. The dispersed population of receptors was usually distributed diffusely amongst the clusters on dividing endothelial cells and normal fibroblasts. Only the dispersed population appeared on LDL receptor internalisation-defective mutant fibroblasts. The marginal zones of both fibroblasts and dividing endothelial cells were populated by dispersed receptors. Clusters appeared further "inland" and were rarely seen near the cell margins. These results indicate that LDL receptors on dividing endothelial cells and fibroblasts may be dispersed on the cell surface upon or soon after their insertion during recycling.     

Modification of human serum low density lipoprotein by oxidation--characterization and pathophysiological implications

Plasma low density lipoprotein (LDL) can undergo free radical oxidation either catalyzed by divalent cations, such as Cu2+ or Fe2+ or promoted by incubation with cultured cells such as endothelial cells, smooth muscle cells and monocytes. The content of vitamin E, beta-carotene and unsaturated fatty acids is decreased in oxidized LDL. A breakdown of apolipoprotein-B (apoB), hydrolysis of the phospholipids, an increase of thiobarbituric acid reactive substances and the generation of aldehydes also occur. Changes in the ratio of lipid to protein, the electrophoretic mobility and the fluorescent properties have also been reported to accompany oxidation of this lipoprotein. The functional changes of oxidized LDL include its recognition by the scavenger receptor on macrophages, its cytotoxicity especially to proliferating cells, its chemotactic properties with respect to monocyte-macrophages and its regulation of platelet-derived growth factor-like protein (PDGFc) production by endothelial cells. In this article we summarize some of the contributions to this topic and present speculations relating oxidized LDL to pathological conditions such as atherosclerosis.     

Role of endothelial cells and their products in the modification of low-density lipoproteins

A majority of the LDL preparations from various donors could be modified by incubation with endothelial cells from human arteries, veins and microvessels. These alterations comprise changes in electrophoretic mobility, buoyant density and lipid composition of LDL, the generation of thiobarbituric acid reactive substances in the medium, and a decrease in primary amino groups of LDL. Furthermore, the association of endothelial cell proteins with LDL was demonstrated by [35S]methionine incorporation and trichloroacetic acid precipitation of reisolated endothelial cell-modified LDL. After SDS-polyacrylamide gel electrophoresis of the reisolated modified LDL particles, radioactivity was mainly found at a molecular mass of 48 kDa and at one or two bands with a molecular mass of more than 100 kDa. The 48 kDa protein was identified as a latent plasminogen activator inhibitor. Cell viability was necessary for the cell-mediated LDL modification, which indicates that endothelial cells are actively involved in this process. The Ca2+ ionophore A23187 and monensin did not influence LDL modification. LDL modification was markedly inhibited by antioxidants. It was not prevented by cyclooxygenase and lipoxygenase inhibitors, which indicates that non-enzymatic lipid peroxidation is involved. Transition metal- (copper-) induced lipid peroxidation results in similar physiochemical alterations of the LDL particle as found with endothelial cells; it is prevented by the presence of superoxide dismutase. In contrast, endothelial cell LDL modification was not influenced by superoxide dismutase. Catalase or singlet oxygen and hydroxyl radical scavengers also did not affect it. We suggest that yet unidentified radicals or lipid peroxides are generated in the cells or on the cell membrane and that these reactive molecule(s) will react with LDL after leaving the cell. HDL and lipoprotein-depleted serum prevented LDL modification markedly, and to a larger extent than that by copper ions. We speculate that LDL modification by endothelial cells will only occur under those conditions in which the balance between the generation of reactive oxygen molecules and the cellular protection against these reactive species is disturbed.     

Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a)

Apo(a), the distinguishing protein component of lipoprotein(a) [Lp(a)], exhibits sequence similarity to plasminogen and can inhibit binding of plasminogen to cell surfaces. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. The ability of apo(a) to inhibit pericellular plasminogen activation on vascular cells was therefore evaluated. Two isoforms of apo(a), 12K and 17K, were found to significantly decrease tissue-type plasminogen activator-mediated plasminogen activation on human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes and macrophages. Lp(a) purified from human plasma decreased plasminogen activation on THP-1 monocytes and HUVECs but not on THP-1 macrophages. Removal of kringle V or the strong lysine binding site in kringle IV₁₀ completely abolished the inhibitory effect of apo(a). Treatment with carboxypeptidase B to assess the roles of carboxyl-terminal lysines in cellular receptors leads in most cases to decreases in plasminogen activation as well as plasminogen and apo(a) binding; however, inhibition of plasminogen activation by apo(a) was unaffected. Our findings directly demonstrate that apo(a) inhibits pericellular plasminogen activation in all three cell types, although binding of apo(a) to cell-surface receptors containing carboxyl-terminal lysines does not appear to play a major role in the inhibition mechanism.     

Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells.

The uptake and transport of cholesterol-carrying low density lipoprotein (LDL) by the arterial wall is a continuous dynamic process, contributing to the cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R). In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membranes in endothelial cells; (ii) the role of LDL receptors in the control of cholesterol homeostasis and (iii) the translocation of LDL receptor across the EC. To this purpose bovine aortic EC were cultured on filters in a double-chamber system, in Dulbecco's medium supplemented either with 10% fetal calf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS). The cells were exposed for 3h to 13H]acetate (40 microCi) added to both compartments of the cell culture inserts. The newly synthesized [3H]cholesterol was detected by thin layer chromatography and quantified by liquid scintillation counting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular pathway of LDL-R was examined by electron microscopy using a complex made of protein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor antibody (Au-PA-C7). To evaluate the distribution and the transport of LDL-R from one cell surface to the other, EC grown in LPDS were radioiodinated either on the apical or on the basolateral surface, incubated on the same surface with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunoprecipitated with anti-LDL-R antibody or with mouse IgG (as control). The eluted antigen-antibody complexes were precipitated with streptavidin-agarose beads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the complex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lysosomes, irrespective of the cell surface exposed to the complex; (c) biochemical assays indicated that upon ligand binding, the LDL-R were translocated preferentially from the apical to the basal plasma membrane.     

Electronegative LDL induces MMP-9 and TIMP-1 release in monocytes through CD14 activation: Inhibitory effect of glycosaminoglycan sulodexide

Objective

Electronegative LDL (LDL(?)) is involved in atherosclerosis through the activation of the TLR4/CD14 inflammatory pathway in monocytes. Matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]) are also crucially involved in atherosclerosis, but their modulation by LDL(?) has never been investigated. The aim of this study was to examine the ability of LDL(?) to release MMPs and TIMPs in human monocytes and to determine whether sulodexide (SDX), a glycosaminoglycan-based drug, was able to affect their secretion.

Alpha-tocopherol protects against monocyte Mac-1 (CD11b/CD18) expression and Mac-1-dependent adhesion to endothelial cells induced by oxidized low-density lipoprotein

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.     

Ibuprofen protects low density lipoproteins against oxidative modification

Oxidative modification of LDL by vascular cells has been proposed as the mechanism by which LDL become atherogenic. The effect of ibuprofen on LDL modification by copper ions, monocytes and endothelial cells was studied by measuring lipid peroxidation products. Ibuprofen inhibited LDL oxidation in a dose-dependent manner over a concentration range of 0.1 to 2.0 mM. Ibuprofen (2 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during 2 and 6 h incubation in the presence of copper ions by 52 and 28%, respectively. Weak free radical scavenging activity of ibuprofen was observed in the DPPH test. The protective effect of ibuprofen was more marked when oxidation was induced by monocytes or endothelial cells. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides generated in LDL during monocyte-mediated oxidation by 40%. HUVEC-mediated oxidation of LDL in the absence and presence of Cu2+ was reduced by 32 and 39%, respectively. More lipid peroxides appeared when endothelial cells were stimulated by IL-1beta or TNFalpha and the inhibitory effect of ibuprofen in this case was more pronounced. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during incubation of LDL with IL-1beta-stimulated HUVEC by 43%. The figures in the absence and presence of Cu2+ for HUVEC stimulated with TNFalpha were 56 and 59%, respectively. To assess the possibility that ibuprofen acts by lowering the production rate of reactive oxygen species, the intracellular concentration of H2O2 was measured. Ibuprofen (1 mM) reduced intracellular production of hydrogen peroxide in PMA-stimulated mononuclear cells by 69%. When HUVEC were stimulated by IL-1beta or TNFalpha the reduction was 62% and 66%, respectively.     

Protein kinase C-beta mediates lipoprotein-induced generation of PAI-1 from vascular endothelial cells

Elevated levels of low-density lipoproteins (LDL) and lipoprotein(a) [Lp(a)] have been considered strong risk factors for atherosclerotic cardiovascular disease. Increased production of plasminogen activator inhibitor-1 (PAI-1) has been implicated in the development of thrombosis and atherosclerosis. Previous studies by our group and others demonstrated that oxidation enhances LDL- and Lp(a)-induced production of PAI-1 in human umbilical vein endothelial cells (HUVEC). The present study examined the involvement of protein kinase C (PKC) and its isoform in vascular endothelial cells (EC) induced by native or oxidized LDL and Lp(a). Treatment with Lp(a) or LDL transiently increased PKC activity at 15 min and 5.5 h after the start of lipoprotein treatment in EC. Copper-oxidized LDL and Lp(a) induced greater PKC activation in EC compared with comparable forms of those lipoproteins. Additions of 1 microM calphostin C, a PKC-specific inhibitor, at the beginning or > or =5 h, but not > or = 9 h, after the initiation of lipoprotein treatment, blocked native and oxidized LDL- or Lp(a)-induced increases in PKC activity and PAI-1 production. Treatment of LDL, Lp(a), or their oxidized forms was induced in translocation of PKC-beta1 from cytosol to membrane in HUVEC. Treatments with 60 nM 379196, a PKC-beta-specific inhibitor, effectively prevented PAI-1 production induced by LDL, Lp(a), or their oxidized forms in HUVEC and human coronary artery EC. The results suggest that activation of PKC-beta may mediate the production of PAI-1 in cultured arterial and venous EC induced by LDL, Lp(a), or their oxidized forms.     

Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration

Conditioned medium (CM) derived from co-cultures of bovine aortic endothelial cells (BAECs) and bovine smooth muscle cells (BSMCs) contains transforming growth factor-beta (TGF-beta) formed via a plasmin-dependent activation of latent TGF-beta (LTGF beta), which occurs in heterotypic but not in homotypic cultures (Sato, Y., and D. B. Rifkin. 1989. J. Cell Biol. 107: 1199-1205). The TGF-beta formed is able to block the migration of BSMCs or BAECs. We have found that the simultaneous addition to heterotypic culture medium of plasminogen and the atherogenic lipoprotein, lipoprotein (a) (Lp(a)), which contains plasminogen-like kringles, inhibits the activation of LTGF-beta in a dose-dependent manner. The inclusion of LDL in the culture medium did not show such an effect. Control experiments indicated that Lp(a) does not interfere with the basal level of cell migration, the activity of exogenous added TGF-beta, the release of LTGF-beta from cells, the activation of LTGF-beta either by plasmin or by transient acidification, or the activity of plasminogen activator. The addition of Lp(a) to the culture medium decreased the amount of plasmin found in BAECs/BSMCs cultures. Similar results were obtained using CM derived from cocultures of human umbilical vein endothelial cells and human foreskin fibroblasts. These results suggest that Lp(a) can inhibit the activation of LTGF-beta by competing with the binding of plasminogen to cell or matrix surfaces. Therefore, high plasma levels of Lp(a) might enhance smooth muscle cell migration by decreasing the levels of the migration inhibitor TGF-beta thus contributing to generation of the atheromatous lesions.     

Effect of clonal senescence on low density lipoprotein-receptor activity of bovine arterial endothelial cells

Senescent and young bovine arterial endothelial cells derived from the same parental cell clone were compared to test the effect of in vitro endothelial cell senescence on low density lipoprotein (LDL) and modified LDL-receptor activities. Low density lipoprotein binding and degradation were both increased in cells that underwent a larger number of population doublings, whereas acetyl LDL binding and degradation were unchanged. The increased LDL-receptor activity associated with endothelial cell senescence remained significant after variation of cell number among senescent and young clones was taken into account. Thus, aging endothelial cells seem capable of continuing to process LDL and modified LDL, which could play a role in the arterial wall changes that occur with age in vivo.     

Arachidonic acid and colorectal carcinogenesis

Vascular lesion development is associated with an accumulation of extracellular matrix proteins within the vessel wall. Matrix metalloproteinases (MMPs) degrade these proteins. Conversely, oxidized low density lipoprotein (LDL) is implicated in atherogenesis through, amongst other cellular effects, a stimulation of the deposition of collagen within the vascular lesion. The present study investigated the potential for an interaction between oxidized LDL and MMP levels. Within the vessel wall fibroblasts, smooth muscle, endothelial and infiltrating cells have been reported to secrete MMPs into the extracellular space to effect remodeling of the extracellular matrix. A consequence of angioplasty and atherosclerotic disease is the loss of endothelial cells or endothelial function, respectively. We have investigated the effects of chronic incubation of cultured vascular smooth muscle cells from rabbit thoracic aorta with oxidized LDL and its influence on MMP levels in the extracellular space. Our data indicate that a low concentration of minimally oxidized LDL (0.005 mg/mL) significantly depressed the levels of MMP-2 and MMP-9 present in the culture medium. Native LDL exerted the same effect but exhibited reduced potency. The effects were not attributable to cytotoxicity exerted by the oxidized LDL. The reduction in MMP secretion into the extracellular medium was a result of decreased enzyme synthesis within the smooth muscle cell. Our results demonstrate that an important atherogenic moiety, oxidized LDL, can reduce MMP activity and hence has the potential to increase the deposition of extracellular matrix proteins within SMC-rich vascular lesions.     

Binding of long-term glycated low density lipoprotein and AGE-albumin by peripheral monocytes and endothelial cells

Modification of low density lipoprotein (LDL) and plasma or tissue proteins by non-enzymatic glycation culminating in the formation of advanced glycation endproducts (AGEs) is one of the essential pathomechanisms leading to diabetes-associated long-term complications. We compared binding of glycated, glycoxidated and oxidated LDL by peripheral monocytes in activated and quiescent form. Interaction via specific receptors was different for glycated as compared to (glyc)oxidated LDL-modifications. In addition, binding of glycated LDL to quiescent and activated human umbilical vein endothelial cells was studied. In patients with insulin-dependent diabetes mellitus (IDDM), AGE-binding was significantly increased as compared to healthy individuals. Specific and non-specific monocyte binding mechanisms were detected, and both were significantly increased in IDDM patients. Specific and non-specific binding strategies possibly act in concert to eliminate circulating AGEs, which are instrumental in the development and progress of microangiopathic and macroangiopathic complications of diabetes mellitus.     

Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism

A microcarrier co-culture system for aortic endothelial cells and smooth muscle cells (SMCs) was developed as a model for metabolic interactions between cells of the vessel wall. Low density lipoprotein (LDL) metabolism in SMCs was significantly influenced by co-culture with endothelium. The numbers of high affinity receptors for LDL was increased more than twofold (range, 2.1-5.6), with concomitant increases in LDL receptor-mediated endocytosis and degradation. These effects reached a plateau at an endothelial cell/SMC ratio of 1. Kinetic analysis of the endocytic pathway for LDL in SMCs indicated that, in co-culture with endothelium, there was no alteration in the binding affinity of LDL to its receptors but that the internalization rate constant declined and the rate constant for degradation increased. This analysis suggested that the formation and migration of endocytic vesicles was the rate-limiting step of enhanced LDL metabolism under co-culture conditions. Two mechanisms by which endothelial cells influenced smooth muscle LDL metabolism were identified. First, mitogen(s) derived from endothelial cells stimulated entry of SMCs into the growth cycle, and the changes in LDL metabolism occurred as a consequence of G1-S transition. Second, SMC lipoprotein metabolism was stimulated in the absence of mitogens by a low molecular weight (less than 3,500) factor or factors. Co-culture was a required condition for the latter effect, suggesting that the mediator(s) may be unstable or that cell-cell communication was necessary for expression. These results (a) demonstrate that vascular cell interactions can modify LDL metabolism in SMCs, (b) provide some insights into the mechanisms responsible, and (c) identify co-culture as an experimental approach appropriate to certain aspects of vascular cell biology.     

Effect on endothelial cell gene expression of shear stress, oxygen concentration, and low-density lipoprotein as studied by a novel flow cell culture system

A new cell culture system has been developed that reflects the vascular microenvironment. By means of this system the cultured cells are exposed not only to shear stress by the circulating culture medium, but also to an oxygen concentration gradient and certain critical blood components such as low-density lipoprotein (LDL) and monocytes. DNA microarray analysis was performed for human umbilical vein endothelial cells cultured in this system in the absence and presence of laminar flow at a low shear stress, 0.2 dyn/cm(2). In addition to shear stress, either an oxygen concentration gradient, or LDL (1 mg/ml), or both were applied. Many Nrf-2-regulating genes, such as heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, solute carrier family 7 No. 11, and glutamate-cysteine ligase modifier subunit, were induced by laminar flow at very low shear stress regardless of the additional conditions. Certain genes were specifically affected by exposure to the oxygen gradient and/or LDL under shear stress, but the degree was very low. These results suggest that shear stress is the most critical factor affecting gene expression in endothelial cells and that Nrf-2-regulating proteins may contribute to protecting endothelial cells against other vascular stress. This system should provide highly relevant and useful information about both vascular physiology and pathology, in the latter on such urgent matters as the specific steps involved in atherogenesis.     

Epigenetic upregulation of p66shc mediates low-density lipoprotein cholesterol-induced endothelial cell dysfunction

Hypercholesterolemia characterized by elevation of low-density lipoprotein (LDL) cholesterol is a major risk factor for atherosclerotic vascular disease. p66shc mediates hypercholesterolemia-induced endothelial dysfunction and atheromatous plaque formation. We asked if LDL upregulates endothelial p66shc via changes in the epigenome and examined the role of p66shc in LDL-stimulated endothelial cell dysfunction. Human LDL stimulates human p66shc promoter activity and p66shc expression in human endothelial cells. LDL leads to hypomethylation of two CpG dinucleotides and acetylation of histone 3 in the human p66shc promoter. These two CpG dinucleotides mediate LDL-stimulated p66shc promoter activity. Inhibition or knock down of DNA methyltransferases negates LDL-induced endothelial p66shc expression. p66shc mediates LDL-stimulated increase in expression of endothelial intercellular adhesion molecule-1 (ICAM1) and decrease in expression of thrombomodulin (TM). Mirroring these changes in ICAM1 and TM expression, p66shc mediates LDL-stimulated adhesion of monocytes to endothelial cells and plasma coagulation on endothelial cells. These findings indicate that LDL cholesterol upregulates human endothelial p66shc expression via hypomethylation of CpG dinucleotides in the p66shc promoter. Moreover, they show that LDL-stimulated p66shc expression mediates a dysfunctional endothelial cell surface, with proadhesive and procoagulant features.     

猴肝细胞膜脂蛋白(a)受体的研究

猴肝细胞膜蛋白经 SDS- PAGE和蛋白转移电泳后用免疫印迹法测定 ,硝酸纤维素膜分别与抗牛肾上腺皮质 LDL受体的抗体、LDL、Lp( a)和 PLG温育后见有 3条不同的蛋白条带 ,分子量分别为 370 kd、2 90 kd和 80 kd.用酶标法测定猴肝细胞膜蛋白 ,发现经 Lp( a) + PLG温育后用 Lp( a)抗体反应的结果与 Lp( a)温育后用 Lp( a)抗体反应的结果比较无显著差异 ,而经 Lp( a) + PLG温育后用 PLG抗体反应的结果比单独用 PLG温育后用 PLG抗体反应的作用强 ;同样经 Lp( a) +LDL温育后用 Lp( a)抗体反应的结果与经 Lp( a)温育后用 Lp( a)抗体反应的结果比较无显著差异 ,而经 Lp( a) + LDL温育后用 LDL抗体反应比单独经 LDL温育后用 LDL抗体的反应强 ,排除Lp( a)与 PLG和 LDL的交叉反应 ,实验认为猴肝细胞膜上可能同时存在 LDL受体、PLG受体和Lp( a)受体     

Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.