Effects of cytochalasin B on low-density lipoproteins metabolism by cultured human fibroblasts.

The role of cytoplasmic microfilaments in the metabolism of low-density lipoprotein by human fibroblasts was studied with the aid of cytochalasin B. At concentrations of 5--40 nmol/ml cytochalasin increased the surface binding but decreased the endocytosis of 125I-labelled low-density lipoprotein. Subsequent studies indicated that these changes reflected a reduction of the rate of internalisation of low-density lipoprotein receptors. Independent inhibitory effects were also observed on low-density lipoprotein degradation and on the cellular release of the trichloroacetic acid-soluble degradation products.     

Effect of human plasma lipoproteins on prostacyclin production by cultured endothelial cells

Prostacyclin (PGI2) production by bovine aortic or human umbilical vein endothelial cells increased when either human high density lipoproteins3 (HDL3) or low density lipoproteins (LDL) were added to a serum-free culture medium. At low concentrations and short incubation times, HDL3 produced more PGI2 than LDL, but LDL was just as effective as HDL3 in 18-hr incubations with high concentrations of lipoproteins. Neither lipoprotein was toxic to the cultures as assessed by [3H]leucine incorporation into cell protein. The stimulatory effect of HDL3 and LDL on PGI2 production decreased as growing cultures became confluent. Incubation with lipoproteins neither enhanced arachidonic acid release nor increased PGI2 formation when the cells were stimulated subsequently with ionophore A23187, indicating that the lipoproteins do not affect the intracellular processes involved in PGI2 production. The addition of albumin reduced the amount of PGI2 formation elicited by HDL3 or LDL. As compared with albumin-bound arachidonic acid, from 6- to 13-fold less PGI2 was produced during incubation with the lipoproteins. Furthermore, the amount of PGI2 formation elicited by the lipoproteins in 18 hr was 4-fold less than that produced during incubation with a fatty acid mixture containing only 5% arachidonic acid, and 3-fold less than when the cells were stimulated with the ionophore A23187 for 20 min. Taken together, our results indicate that human HDL and LDL contribute to endothelial PGI2 production only in a modest way and suggest that this process is not specific for either of these two plasma lipoproteins. In view of the greater participation of albumin-bound arachidonic acid in PGI2 production, plasma lipoproteins may not play as important a role in endothelial prostaglandin formation as has been suggested.     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.     

High-density lipoproteins protect endothelial cells from apoptosis induced by oxidized low-density lipoproteins

Summary Endothelial lesion by oxidized low-density liproproteins (LDL) is one of the first stages in the development of atherosclerosis. The effect of these lipoproteins can range from a functional lesion of the endothelium to death of the endothelial cells by apoptosis. High-density lipoproteins (HDL) are one of the factors which can have a protective effect against the development of atheromatous plaques. The aim of this study is to establish whether the death of endothelial cells by apoptosis induced by oxidized LDLs is prevented by HDLs. ECV304 endothelial cells and bovine aorta endothelial cells were incubated with native LDLs, oxidized LDLs, and a combination of both oxidized LDLs and HDLs. Oxidized LDLs caused a significant increase of mortality mainly by apoptosis. However, when HDLs were added together with oxidized LDLs the percentage of total mortality, the degree of lipoprotein oxidation in the medium, and the percentage of cells in apoptosis were all significantly decreased. HDLs protect against the cytotoxicity of oxidized LDLs possibly by preventing the propagation of the oxidative chain in these lipoproteins.Abbreviations LDL low-density lipoproteins - HDL high-density lipoproteins - BAEC bovine aortic endothelial cell - TBARS thiobarbituric acid-reactive substances     

Native and oxidized low-density lipoproteins stimulate endothelin-converting enzyme-1 expression in human endothelial cells

This study addressed the question how different lipoproteins modulate the expression of endothelin-converting enzyme-1 (ECE-1) in human endothelial cells. The effect of native and oxidized low-density lipoproteins (nLDL, oxLDL) on expression of ECE-1, prepro-endothelin-1, and endothelin-1 peptide was studied in primary cultures of human endothelial cells. Native and oxidized LDL increased ECE-1 mRNA after 1 h, reaching its maximum at 100 microg/ml (1.9- and 2.5-fold, respectively). Furthermore, ECE-1 protein expression, prepro-endothelin-1 mRNA, and endothelin-1 peptide release were increased in response to nLDL or oxLDL. Induction of ECE-1 by nLDL and of prepro-endothelin-1 by oxLDL was reduced by protein kinase C inhibition. Increased expression of ECE-1 mRNA by oxLDL and of prepro-endothelin-1 by nLDL was blocked by an angiotensin II receptor type 1 antagonist. Our data provide evidence for a new mechanism how increased LDL plasma levels might contribute to enhanced endothelin-1 release in patients with hypercholesterolemia.     

Effects of hyperoxia on DNA synthesis in cultured porcine aortic endothelial cells

The mode of action of hyperoxia on the inhibition of DNA synthesis from thymidine (dThd) was studied in primary cultures of porcine aortic endothelial cells (EC) at confluence. A significant effect of hyperoxia on dThd uptake was detected only after a 48-h exposure to 95% O2. On the other hand, decrease in dThd kinase activity was already observed after a 12-h exposure, and the time course of its reduction followed closely that of the inhibition of dThd incorporation into DNA. The incorporation of dThd triphosphate into DNA in permeabilized EC was unaffected by hyperoxia. Determination of DNA alpha- and beta-polymerase activities showed that hyperoxia reduced the activity of the alpha-polymerase and increased that of the beta-polymerase. We conclude that most of the O2 effects on DNA synthesis from dThd can be attributed to dThd kinase inhibition. The increased activity of DNA beta-polymerase, an enzyme involved in DNA repair, also supports the view that hyperoxia could damage DNA.     

Effect of low-density lipoproteins on the synthesis and secretion of proteoglycans by human endothelial cells in culture.

We studied the effect of low-density lipoproteins (LDL) on the synthesis and secretion of proteoglycans by cultured human umbilical-vein endothelial cells. Confluent cultures were incubated with [35S]sulphate or [3H]glucosamine in lipoprotein-deficient serum in the presence and in the absence (control) of LDL (100-400 micrograms/ml), and metabolically labelled proteoglycans in culture medium and cell layer were analysed. LDL increased accumulation of labelled proteoglycans in medium and cell fractions up to a concentration of 200 micrograms/ml. At this concentration of LDL the accumulations of proteoglycans in medium and cell layer were 65% and 32% respectively above control for 35S-labelled proteoglycans, and 55% and 28% respectively above control for 3H-labelled proteoglycans. At concentrations above this LDL was found to depress the accumulation of proteoglycans in medium and cell layer. Gel filtration on Sepharose CL-4B showed that in both control and LDL-treated cultures the cell layer contained a large (Kav. = 0) and a small (Kav. = 0.35) heparan sulphate proteoglycan, whereas the culture medium contained a large heparan sulphate proteoglycan (Kav. = 0) and a smaller isomeric chondroitin sulphate proteoglycan (control, Kav. = 0.35; LDL-treated, Kav. = 0.17). The relative increase in hydrodynamic size of the isomeric chondroitin sulphate proteoglycan (Mr 150,000 compared with 90,000) in the medium of cultures exposed to LDL was partly attributable to the larger size of the glycosaminoglycan side chains (Mr 39,000 compared with 21,000). The isomeric chondroitin sulphate proteoglycan in LDL-treated culture was relatively enriched in chondroitin 6-sulphate compared with that in control cultures (39% compared with 29%). Pulse-chase studies showed that LDL treatment did not alter the turnover rate of proteoglycans as compared with controls, implying that the elevation in proteoglycan accumulation in LDL-treated cultures was due to enhanced synthesis. These results demonstrate that LDL can modulate proteoglycan synthesis by cultured vascular endothelial cells, resulting in the secretion of a larger isomeric chondroitin sulphate proteoglycan enriched in chondroitin 6-sulphate.     

Morphological studies on the binding of low-density lipoproteins and acetylated low-density lipoproteins to the plasma membrane of cultured monocytes

Binding of low density lipoproteins (LDL) and acetyl-LDL to the plasma membrane of cultured swine monocytes was investigated by immunofluorescent and immunoelectron microscopy. Binding sites for native LDL, visualized on both the light microscopical and the ultrastructural level, were found to be comparable to those of cultured human fibroblasts. These techniques, however, failed to reveal binding of acetyl-LDL to the cell surface. Biochemical experiments showed that both LDL and acetyl-LDL have specific receptors, the acetyl-LDL receptor being distinctly different from the LDL receptor. It is concluded that there are morphological differences in the binding of LDL and acetyl-LDL to cultured monocytes. These differences are supported by biochemical data.     

Erythropoietin attenuated high glucose-induced apoptosis in cultured human aortic endothelial cells

High glucose-induced apoptosis in vascular endothelial cells may contribute to the acceleration of atherosclerosis associated with diabetes. Here, we show that erythropoietin attenuates high glucose-induced apoptosis in cultured human aortic endothelial cells (HAECs). Exposure of HAECs to high glucose level for 72h significantly increased the number of apoptotic cells compared with normal glucose level, as evaluated by TUNEL assay. Simultaneous addition of erythropoietin (100 U/ml) significantly attenuated high glucose-induced apoptosis. In parallel, exposure to high glucose level induced caspase-3 activation and erythropoietin also prevented it. Erythropoietin stimulated Akt phosphorylation in a dose-dependent manner (1-100 U/ml). PI3 kinase inhibitor, wortmannin or LY294002 eliminated erythropoietin's inhibitory effect on caspase-3 activity. In conclusion, erythropoietin may attenuate high glucose-induced endothelial cell apoptosis via PI-3 kinase pathway. Replacing therapy with erythropoietin is often used for correction of renal anemia, but may have potential in preventing atherosclerosis in diabetic patients with end-stage renal failure.     

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.     

Effect of superoxide and lipid peroxide on cytosolic free calcium concentration in cultured pig aortic endothelial cells

The effect of reactive oxygen on cytosolic free calcium concentration [( Ca++]i) in pig aortic endothelial cells (ECs) was studied. Linoleate hydroperoxide (LHO) and superoxide radicals generated from xanthine with xanthine oxidase (X-XO) were used as sources of reactive oxygen. [Ca++]i in ECs was measured with quin 2 and the value for quiescent ECs was 112 +/- 11 nM. Both LHO and X-XO increased [Ca++]i in a dose-dependent manner without accompanying the significant cellular damage. Nifedipine suppressed the increase in [Ca++]i provoked by LHO and X-XO. Thus, the biological effects of reactive oxygen might be mediated, at least in part, by the activation of voltage-dependent calcium channels in ECs.     

Characterization of cultured rat aortic endothelial cells.

We describe a new method to obtain rat aortic endothelial cells without contamination by vascular smooth muscle cells. The endothelial cells were characterized up to the 20th passage by low density lipoprotein incorporation, the absence of alpha-smooth muscle actin, the production of endothelium derived relaxing factor, and an elevation in intracellular free calcium concentration in response to bradykinin and ATP but not to AMP and angiotensin II.     

Mechanisms of low-density lipoprotein-induced expression of connective tissue growth factor in human aortic endothelial cells

Hyperlipidemia is a recognized risk factor for atherosclerotic vascular disease. The underlying mechanisms that link lipoproteins and vascular disease are undefined. Connective tissue growth factor (CTGF) is emerging as a key determinant of progressive fibrotic diseases, and its expression is upregulated by diabetes. To define the mechanisms through which low-density lipoproteins (LDL) promote vascular injury, we evaluated whether LDL can modulate the expression of CTGF and collagen IV in human aortic endothelial cells (HAECs). Treatment of HAECs with LDL (50 microg/ml) for 24 h produced a significant increase in the mRNA and the protein levels of CTGF and collagen IV compared with unstimulated controls. To explore the mechanisms by which LDL regulates CTGF and collagen IV expression in HAECs, we determined first if CTGF and collagen IV are downstream targets for regulation by transforming growth factor-beta (TGF-beta). The results demonstrated that TGF-beta produced a concentration-dependent increase in the protein levels of CTGF. To assess whether the induction of CTGF in response to LDL is mediated via autocrine activation of TGF-beta, HAECs were treated with LDL for 24 h in the presence and absence of anti-TGF-beta neutralizing antibodies (anti-TGF-beta NA). The results demonstrated that the increase in CTGF induced by LDL was significantly inhibited by the anti-TGF-beta NA. To investigate the upstream mediators of TGF-beta on activity of CTGF in response to LDL, HAECs were treated with LDL for 24 h in the presence and absence of cell-permeable MAPK inhibitors. Inhibition of p38(mapk) activities did not affect LDL-induced TGF-beta1, CTGF, and collagen IV expression. On the other hand, SP-600125, a specific inhibitor of c-Jun NH(2)-terminal kinase, suppressed LDL-induced TGF-beta, CTGF, and collagen IV expression, and PD-98059, a selective inhibitor of p44/42(mapk), suppressed LDL-induced TGF-beta and CTGF expression. These findings are the first to implicate the MAPK pathway and TGF-beta as key players in LDL signaling, leading to CTGF and collagen IV expression in HAECs. The data also point to a potential mechanistic pathway through which lipoproteins may promote vascular injury.     

Neovascular responses induced by cultured aortic endothelial cells

Neovascularization was studied in the chorioallantoic membrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscle cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2-fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor--stimulation of cellular migration and proliferation--can also be demonstrated using endothelial cell-conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In addition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.     

Ethanolamine metabolism in cultured bovine aortic endothelial cells

The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells (0.62 +/- 0.07 nmol/mg of protein versus 0.27 +/- 0.05 nmol/mg of protein, respectively). Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine (25 microM) decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine. The results show that an extracellular source of ethanolamine significantly influences the phospholipid metabolism of cultured bovine aortic endothelial cells.     

The effect of serum lipoproteins on cholesterol content and sterol exchange in cultured human endothelial cells.

Cultured human endothelial cells preincubated with the infranatant of human serum increased their content of cholesterol when subsequently exposed to low density lipoproteins (LDL) as compared to control cultures further incubated in the presence of infranatant only. Replacing LDL with high density lipoproteins (HDL) resulted in no change in the cellular cholesterol content compared to the control. The addition of HDL did not influence the increase in cellular cholesterol content mediated by LDL. HDL stimulated the efflux of endogenously synthesized 14C-labelled sterols compared to the infranatant fraction, whereas LDL had only a slight effect. Cells preincubated with whole serum did not change their cholesterol content when subsequently exposed to LDL, compared to cultures further incubated in presence of whole serum. Replacing whole serum (during the final incubation) with infranatant, resulted in a decrease of the cellular cholesterol content, which was not influenced by further addition of HDL.     

Effects of oxidized low-density lipoproteins on the hepatic microvasculature

Oxidized low-density lipoproteins (oxLDLs) are involved in proinflammatory and cytotoxic events in different microcirculatory systems. The liver is an important scavenger organ for circulating oxLDLs. However, the interaction of oxLDL with the hepatic microcirculation has been poorly investigated. The present study was conducted to examine the effects of differently modified oxLDLs on the hepatic microvasculature. C57Bl/6J mice were injected intravenously with low-density lipoprotein (LDL), or LDL oxidized for 3 h (oxLDL(3)) or 24 h (oxLDL(24)), at doses resembling oxLDL plasma levels in cardiovascular disease patients. Radioiodinated ligands were used to measure blood decay and organ distribution, and nonlabeled ligands to evaluate microcirculatory responses, examined by in vivo microscopy 30-60 min after ligand injection, immunohistochemistry, and scanning and transmission electron microscopy. Mildly oxLDL (oxLDL(3)) was cleared from blood at a markedly slower rate than heavily oxLDL (oxLDL(24)), but significantly faster than LDL (P < 0.01). Injected oxLDLs distributed to liver. OxLDL effects were most pronounced in central areas of the liver lobules where oxLDL(3) elicited a significant (P < 0.05) reduction in perfused sinusoids, and both oxLDL(3) and oxLDL(24) significantly increased the numbers of swollen endothelial cells and adherent leukocytes compared with LDL (P < 0.05). OxLDL-treated livers also exhibited increased intercellular adhesion molecule (ICAM)-1 centrilobular staining. Electron microscopy showed a 30% increased thickness of the liver sinusoidal endothelium in the oxLDL(3) group (P < 0.05) and a reduced sinusoidal fenestration in centrilobular areas with increased oxidation of LDL (P for linear trend <0.05). In conclusion, OxLDL induced several acute changes in the liver microvasculature, which may lead to sinusoidal endothelial dysfunction.