Lipoprotein-depleted Serum Research Articles

Proliferative and cytotoxic effects of mildly oxidized low-density lipoproteins on vascular smooth-muscle cells.

We have investigated the role of low-density lipoprotein (LDL) oxidation in the proliferative effect of LDLs on cultured bovine aortic smooth-muscle cells and compared it with their effect on bovine aortic endothelial cells. The following conclusions were reached. (1) Non-toxic doses of mildly oxidized LDLs elicit a proliferative effect on smooth-muscle cells significantly higher than that of native LDLs or lipoprotein-depleted serum. The proliferative effect is dependent on time (relatively slow), dose (high doses are cytotoxic) and the level of LDL oxidation. (2) The proliferative effect on smooth-muscle cells is counterbalanced at high concentrations of mildly oxidized LDLs (or at high oxidation levels) by their cytotoxic effect. (3) The same dose of mildly oxidized LDLs exhibits no proliferative effect on endothelial cells but rather a cytotoxic one. Endothelial cells may therefore be intrinsically more susceptible to the cytotoxic effect of mildly oxidized LDLs than are smooth-muscle cells. (4) The proliferative effect of native LDLs on smooth-muscle cells results (at least in part) from cell-induced LDL oxidation during cell culture as suggested by (i) the progressive LDL oxidation over the 3 days of contact between LDLs and smooth-muscle cells and (ii) the concomitant inhibition of LDL oxidation and proliferative effect by butylated hydroxytoluene. The hypothetical mechanisms and potential involvement in atherogenesis are discussed.

Low density lipoproteins induce parasympathetic responsiveness in embryonic chick ventricular myocytes in parallel with a coordinate increase in expression of genes coding for the M2 muscarinic receptor, G alpha i2, and the acetylcholine-sensitive K+ channel.

Growth of chick atrial cells in medium supplemented with lipoprotein-depleted serum has been shown to result in an increase in total cell cholesterol, and an increase in the negative chronotropic response to muscarinic stimulation in parallel with an increase in levels of muscarinic receptors and the G-protein alpha-subunits alpha i and alpha o (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In this study we determined whether growth of chick ventricular cells in medium supplemented with lipoprotein depleted serum could alter levels of muscarinic receptors and G-protein alpha-subunits and induce a negative chronotropic response to muscarinic stimulation. We further determined whether levels of mRNA coding for muscarinic receptors, G-proteins, and the acetylcholine-sensitive K+ channel were coordinately regulated. Growth of embryonic chick ventricular cells from hearts 14 days in ovo in medium supplemented with lipoprotein depleted serum resulted in a 21 +/- 5% (n = 3, +/- S.E.) increase in muscarinic receptor number as demonstrated by [3H]quinuclidinyl benzilate binding and a 4.7 +/- 1.0 (+/- S.E., n = 4)-fold increase in G alpha i2 as demonstrated by Western blot analysis. These changes in receptor and G-protein were associated with a coordinate increase in levels of mRNA coding for the M2 muscarinic receptor, G alpha i2 and the acetylcholine sensitive K+ channel as determined by RNase protection. These increases were reversed by addition of 30 microM mevinolin, an inhibitor of HMG-CoA reductase activity. Carbamylcholine (0.1 mM) had no effect on beat rate in ventricular cells grown in medium supplemented with fetal calf serum. Cells grown in medium supplemented with lipoprotein depleted serum demonstrated a 40 +/- 8% (+/- S.E., n = 10, p < 0.0001) decrease in beat rate in response to 0.1 mM carbamylcholine which was reversed by the addition of 30 microM mevinolin. These data suggest that, during growth in medium supplemented with lipoprotein depleted serum, a component of the cholesterol biosynthetic pathway plays a role in the coordinate induction of mRNAs coding for receptors, G-proteins, and an effector (ion channel) that results in the induction of a parasympathetic response in the ventricular cell characteristic of the atrial phenotype.

The effects of two acylcoenzyme a: Cholesterol acyltransferase (ACAT) inhibitors, cyclandelate and a non-hydrolysable ether analogue, benzyl3,3,5-trimethylcyclohexanol on low density lipoprotein metabolism in macrophages and hepatocytes

Cyclandelate (3,3,5,-trimethylcyclohexanylmandelate) caused a dose-dependent decrease in the metabolism of radioiodinated low density lipoprotein [ 125I-LDL] by J774 mouse macrophages. This was probably an indirect effect dur to the inhibition of cholesterol esterification by the cells rather than a direct one one the interaction of LDL with its receptor, since no inhibition was seen in cells which had been cholesterol-depleted by prior incubation with lipoprotein-depleted serum for 48 hr. Cyclandelate also inhibited immediately de novo synthesis of cholesterol from [1- 14C]acetate in J774 cells, suggesting a direct action of the drug on an enzyme of the cholesterol biosynthetic pathway. The drug was an efficient inhibitor of hamster and rat intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) activity in vitro with an ic 50 of 20 μM. Addition of cyclandelate to the diet of meal-fed rats caused a marked inhibition of the rate of appearance of dietary [4- 14C]cholesterol in the plasma. A nonhydrolysable ether analogue of cyclandelate, benzyl3,3,5-trimethylcyclohexanol, was prepared to compare hepatic and extrahepatic actions of the two molecules. The analogue inhibited cholesterol esterification in J774 cells, transformed human macrophages U937 and human umbilical vein endothelial cells with an ic 50 of 20 μM and had effects similar to those of cyclandelate on 125I-LDL metabolism in J774 cells. Differences between the analogue and cyclandelate were seen in hepatocytes and hepatic microsomal fractions, where preincubation with the analogue inhibited cholesterol esterification in both systems while cyclandelate had no inhibitory action in either. Consequently, preincubation of rat hepatocytes with benzyl3,3,5-trimethylcyclohexanol for 17 hr caused a marked decrease in the binding of 125I-LDL to the cells, whereas binding to cells preincubated with cyclandelate was the same as to control cells.

Purification of Human Cholesteryl Ester Transfer Protein by Affinity Chromatography on Immobilized Triazine Dyes

Human cholesteryl ester transfer protein was purified from lipoprotein-depleted serum or plasma in a three-step procedure utilizing commercially available triazine dyes immobilized on agarose. The method used consisted of successive chromatography steps on Reactive Red 120 agarose (Procion Red H-E3B, Cibachron Brilliant Red 4G-E), CM Sepharose, and Reactive Yellow 86 agarose (Procion Yellow M-8G). Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resultant protein preparation displayed two bands of variable intensity. The two components had apparent molecular weights of approximately 64,000 and approximately 65,000, respectively. Both bands reacted strongly to a monoclonal antibody directed against an epitope consisting of the last eight amino acids at the carboxy terminus of human CETP. Yields of cholesteryl ester transfer activity are 10-40% of the activity present in lipoprotein-depleted serum. The activity is approximately 50,000- to 100,000-fold purified relative to the starting material.

Circulating Tumor Gangliosides Enhance Platelet Activation

AbstractGangliosides enhance tumor formation in experimental animals, and high circulating concentrations of gangliosides shed by tumor cells are associated with rapid progression of human neuroblastoma. We studied these shed molecules for effects on platelet function, because platelet activation may play a role in the metastatic process. Preincubation of normal platelets in patient (tumor ganglioside-containing) serum resulted in their aggregation upon exposure to a subthreshold concentration (1 µg/mL) of collagen (up to 34% v <10% in normal serum) and ATP release (up to 1.1 nmol/2.5 × 107 platelets v <0.2 in normal serum). Because circulating shed tumor gangliosides are lipoprotein-associated, we next assessed the effects of the serum lipoprotein fraction on platelet ATP release. The patient serum lipoprotein fraction (d > 1.210) enhanced ATP release (up to 3.1 nmol ATP), whereas the same fraction of normal serum, and both patient and control lipoprotein-depleted serum fractions (d > 1.210), were inactive (<0.2 nmol ATP released). Finally, as little as 0.5 µmol/L patient serum gangliosides (purified from the lipoprotein fraction) caused significantly greater ATP release than did normal serum gangliosides (P < .01) and caused maximal release at 50 µmol/L (up to 3.0 nmol ATP released v ≤0.3 nmol released by platelets exposed to normal serum gangliosides). Purified total human neuroblastoma tumor gangliosides, detected in the patient serum and isolated from LA-N5 cells, were highly active; preincubation of platelets with only 5 µmol/L of these gangliosides resulted in release of 2.5 ± 0.1 nmol ATP. Thus, neuroblastoma patient serum, the lipoprotein fraction, and, specifically, the serum gangliosides enhance platelet activation. This activity appears to reside particularly in the tumor cell gangliosides, which are shed in vivo.

Effect of low-density lipoproteins, mevinolin, and G proteins on Ca2+ response in cultured chick atrial cells

Growth of cells from atria of embryonic chick hearts 14 days in ovo in medium supplemented with lipoprotein-depleted serum (LPDS) results in an increase in total cell cholesterol, enhanced parasympathetic responsiveness (7), and decreased sympathetic responsiveness (1). These effects were reversed by the hydroxymethyl glutaryl CoA reductase inhibitor, mevinolin. In these studies, comparison of cell growth in medium supplemented with fetal calf serum (FCS) and LPDS demonstrated that, after growth with LPDS, the ability of Ca2+ and the Ca2+ channel agonist, BAY K 8644, to enhance the amplitude of contraction decreased by 25 and 50%, respectively. These effects of growth in LPDS were reversed by incubation with mevinolin. LPDS had no effect on either Ca2+ channel number as measured by (+)-[5-methyl-3H]PN200-110 binding or Ca2+ current density as measured by the whole cell patch method. Treatment of cells grown in LPDS with pertussis toxin, which inactivates alpha o and alpha i, returned the contractile response to 10(-7) M BAY K 8644 to control levels. Pertussis toxin had no effect on the contractile response or adenosine 3',5'-cyclic monophosphate levels in control cells grown in FCS alone. These data suggest that alterations in the relative levels of alpha o and alpha s in cells grown in LPDS may play a role in regulating the contractile response to Ca2+ channel agonists and to exogenous Ca2+.

The effect of serum on low density lipoprotein metabolism of human monocyte-derived macrophages.

Macrophages oxidatively modify low density lipoprotein (LDL). These cells then recognize and take up oxidized LDL (OxLDL) via the scavenger receptor. In the present study, we examined the effect of lipoprotein depleted serum (LPDS) on the oxidative modification of LDL and its uptake and degradation by human monocyte-derived macrophages. LPDS inhibited LDL oxidation in a concentration-dependent manner and the complete inhibition was observed in the concentration from 2.5 to 10.0%. In the presence of 5% of LPDS, cell-associated 125I-LDL was reduced by 70%, compared with that in the absence of LPDS. In contrast, 125I-LDL degradation increased in the presence of LPDS. The ratio of degradation products to cell-associated radioactivity in the presence of LPDS was five-fold greater than that in the absence of LPDS. In the presence of LPDS, unlabeled LDL competed for about 50% of cellular uptake and degradation of 125I-LDL. In the absence of LPDS, however, unlabeled LDL competed for only 20% of uptake and only 30% of degradation of 125I-LDL. Unlabeled OxLDL competed for about 80% of uptake and degradation in the absence of LPDS. These results suggest that LPDS inhibited the production of OxLDL which was taken up by macrophages via the pathway for the OxLDL.

Lipid lowering activity of P-06139, an inhibitor of acyl-coa: Cholesterol acyltransferase

Cyclandelate (3,3,5,-trimethylcyclohexanylmandelate) caused a dose-dependent decrease in the metabolism of radioiodinated low density lipoprotein [125I-LDL] by J774 mouse macrophages. This was probably an indirect effect dur to the inhibition of cholesterol esterification by the cells rather than a direct one one the interaction of LDL with its receptor, since no inhibition was seen in cells which had been cholesterol-depleted by prior incubation with lipoprotein-depleted serum for 48 hr. Cyclandelate also inhibited immediately de novo synthesis of cholesterol from [1-14C]acetate in J774 cells, suggesting a direct action of the drug on an enzyme of the cholesterol biosynthetic pathway. The drug was an efficient inhibitor of hamster and rat intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) activity in vitro with an ic50 of 20 μM. Addition of cyclandelate to the diet of meal-fed rats caused a marked inhibition of the rate of appearance of dietary [4-14C]cholesterol in the plasma. A nonhydrolysable ether analogue of cyclandelate, benzyl3,3,5-trimethylcyclohexanol, was prepared to compare hepatic and extrahepatic actions of the two molecules. The analogue inhibited cholesterol esterification in J774 cells, transformed human macrophages U937 and human umbilical vein endothelial cells with an ic50 of 20 μM and had effects similar to those of cyclandelate on 125I-LDL metabolism in J774 cells. Differences between the analogue and cyclandelate were seen in hepatocytes and hepatic microsomal fractions, where preincubation with the analogue inhibited cholesterol esterification in both systems while cyclandelate had no inhibitory action in either. Consequently, preincubation of rat hepatocytes with benzyl3,3,5-trimethylcyclohexanol for 17 hr caused a marked decrease in the binding of 125I-LDL to the cells, whereas binding to cells preincubated with cyclandelate was the same as to control cells.

Differences between the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and low density lipoprotein receptor in human hepatoma cells and fibroblasts reside primarily at the translational and post-translational levels.

We have carried out parallel analyses of the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGr) and low density lipoprotein receptor (LDLr) in two highly differentiated human hepatoma cell lines, HepG2 and Hep3B, and primary cultures of human fibroblasts. Analyses of the levels of HMGr and LDLr mRNAs under a variety of culture conditions that perturb intracellular sterol metabolism, or which differ in the levels of extracellular sterols, indicated that the hepatoma cells and fibroblasts responded similarly in terms of the repression or induction ratios of both mRNAs. However, the absolute levels of the mRNAs were severalfold higher in the hepatoma cells. The major difference between the responses of the hepatoma cells and fibroblasts involved the increase in expression of LDLr which occurred upon shifting the cells from complete to lipoprotein-depleted serum. Under these conditions, the 3-fold increase in rate of synthesis of LDLr in the hepatomas was closely matched by increases in the level of its mRNA. In the case of fibroblasts, a 10-fold increase in translational efficiency was required to explain the 30-fold change in rate of synthesis of LDLr. Polysome profiles from both hepatoma cells and fibroblasts suggest that the rate of elongation or termination on LDLr mRNA is relatively low in the presence of reconstituted complete serum, and that it increases in fibroblasts upon lipoprotein depletion, but not in the hepatoma cells. These data indicate that hepatic expression of LDLr may be relatively refractory to induction by decreased circulating levels of lipoprotein when compared with peripheral tissues.

Serum lipoproteins are required for multiplication of Trypanosoma brucei brucei under axenic culture conditions

Bloodstream form Trypanosoma brucei brucei IL3201 and IL3202, derived from two different serodemes, were adapted to grow in a semi-defined medium under axenic culture conditions. The organisms required low- or high-density lipoproteins, isolated from foetal bovine serum (FBS) in addition to components of lipoprotein-depleted serum, to multiply in vitro. Low- and high-density lipoproteins, isolated from a number of different species, were equally efficient at supporting in vitro growth of the parasites in medium supplemented with lipoprotein-depleted FBS. Chylomicrons and very-low-density lipoproteins did not support multiplication of the T. b. brucei and did not influence the capacity of low- or high-density lipoproteins to support parasite multiplication. Removal of the lipoprotein-lipids abrogated the capacity of low- or high-density lipoproteins to support T. b. brucei multiplication.

Modifications of LDL-receptor-mediated endocytosis rates in CEM lymphoblastic cells grown in lipoprotein-depleted fetal calf serum

The efficiency of supplying cholesterol by the LDL endocytic pathway of lymphoblastic T CEM cells was compared when incubated in the presence of either fetal calf serum (FCS) or lipoprotein-depleted fetal calf serum (LDFCS). In the presence of FCS, there were 8600 +/- 2000 LDL receptors/cell with a Kd of (2.2 +/- 0.8).10(-8) M and a receptor cycling time of about 7 min; about 90% of the internalized LDL was degraded. LDL degradation produced 98% of total cellular cholesterol and only 2% came from endogenous synthesis. The absence of LDL in the culture medium of lymphoblastic CEM cells deeply modified certain metabolic and structural characteristics of the cells. Their cholesterol content decreased; the total number of LDL receptors increased 6-fold, whereas their affinity for the ligand decreased by the same factor (Kd = (1.2 +/- 0.2).10(-7) M); the receptor cycling time increased 3-fold. Finally, LDL degraded by cholesterol-depleted CEM cells amounted to about 40% of that degraded by untreated CEM cells.

Effects of Low Density Lipoproteins and Mevinolin on Sympathetic Responsiveness in Cultured Chick Atrial Cells: Regulation of β-adrenergic receptors and αs

We have previously demonstrated that cultures of myocytes from embryonic chick atria grown in media supplemented with fetal calf serum from which lipoproteins have been removed demonstrate a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared with cells grown with control medium. This increased response to carbamylcholine was associated with a 1.4-fold increase in total cell cholesterol, a 2-fold increase in the number of muscarinic receptors which bind agonist with high affinity, and a 2-fold increase in the levels of the alpha subunits of Go and Gi (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In the studies reported here, we determined the responsiveness of cells grown in lipoprotein-depleted serum (LPDS) to beta-adrenergic stimulation. Isoproterenol stimulated a contractile response of 58% measured as an increase in amplitude of contraction with a half-maximal effect at 3 x 10(-7) M for cells grown in fetal calf serum, but had no significant effect on amplitude of contraction on cells grown in LPDS. In cells grown in media supplemented with fetal calf serum, isoproterenol (1 x 10(-3) M) stimulated adenylate cyclase activity 100% over basal with an EC50 of 7 x 10(-6) M compared with an increase of 32% in cells grown in media supplemented with LPDS. beta-Adrenergic receptor number as measured by the binding of 125I-pindolol decreased from 24 +/- 3 (+/- S.E., n = 6) fmol/mg protein in cells grown under control conditions to 12 +/- 2 (n = 6) fmol/mg protein in media supplemented with LPDS. The level of alpha s as measured both by ADP-ribosylation with cholera toxin in the presence of 32P-NAD and by immunoblotting with specific antibody to alpha s decreased by 3-fold in cells grown in media supplemented with LPDS compared with control. All of these effects of growth of cells in LPDS were reversed by incubating cells with LPDS plus 30 microM mevinolin, an inhibitor of endogenous cholesterol synthesis. These studies indicate that growth of cells in media supplemented with LPDS results in a coordinate decrease in the levels of beta-adrenergic receptors and alpha s. Taken together with our previous studies these data support the hypothesis that the receptors and guanine nucleotide-binding proteins which mediate sympathetic and parasympathetic responsiveness in the heart are reciprocally regulated.

Oxidized low density lipoprotein stimulates prostacyclin production by adult human vascular endothelial cells.

Interactions between vascular endothelium and low density lipoprotein (LDL) have been implicated in the development of atherosclerosis. The effect of normal and oxidized LDL (Ox-LDL) on prostaglandin release by cultured adult human saphenous vein endothelial cells was investigated. Ox-LDL induced a rapid release of prostacyclin (PGI2) to levels which were several-fold higher than those observed with control LDL. PGI2 release was concentration-dependent and was biphasic, with a first peak occurring within 30 minutes (followed by a decrease), and a second peak occurring after several hours of incubation. PGI2 production was inhibited by lipoprotein-depleted serum and by indomethacin, an antagonist of cyclooxygenase activity. These cells produced mainly PGF2 alpha, with some PGE2 and PGI2 when stimulated by the ionophore A23187 at confluency. However, among these prostanoids, mainly PGI2 was produced in response to Ox-LDL. The data indicate that Ox-LDL induces the production of PGI2 by human vascular endothelial cells. Since Ox-LDL is cytotoxic, this phenomenon may be a manifestation of an early response to injury.

Effects of low density lipoproteins and mevinolin on cholesterol content and muscarinic cholinergic responsiveness in cultured chick atrial cells. Regulation of levels of muscarinic receptors and guanine nucleotide regulatory proteins.

Cultures of myocytes from embryonic chick atria grown in medium supplemented with fetal calf serum from which lipoproteins had been removed demonstrated a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared to cells grown with control serum. This effect was reversed by growth of cells in medium supplemented with lipoprotein-depleted serum (LPDS) reconstituted with the low density lipoprotein fraction from fetal calf serum. In cells grown in LPDS, total cell cholesterol was increased 32% over control levels and returned to control levels in cells grown with LPDS reconstituted with low density lipoprotein. Growth of cells in LPDS plus mevinolin, an inhibitor of endogenous cholesterol synthesis, also reversed the effects of LPDS on cholesterol content and sensitivity of beating to carbamylcholine. The ability of mevinolin (30 microM) to reverse the effect of LPDS on sensitivity of beating to carbamylcholine was inhibited by mevalonic acid, a metabolic precursor to cholesterol, with an IC50 of 7 x 10(-5) M. These data suggest that mevinolin reverses the effects of LPDS by altering cellular cholesterol levels. Enhanced responsiveness of embryonic chick heart cells to muscarinic stimulation was associated with a 2-fold increase in the number of muscarinic receptors with high affinity for agonist from 82 +/- 10 fmol/mg protein in media containing fetal calf serum to 175 +/- 12 fmol/mg protein in cells grown in the presence of LPDS. The distribution of receptors between high affinity (RH) and low affinity (RL) forms changed from 41% RH and 59% RL in cells grown in control serum to 66.5% RH and 33.5% RL in cells grown in LPDS. Quantitation of the effect of growth in LPDS on the levels of guanine nucleotide regulatory proteins No and Ni which couple the muscarinic receptor to a physiologic response, demonstrated that the relative levels of the 39-kDa alpha subunits of No and 41-kDa alpha subunits of Ni determined by ADP ribosylation with pertussis toxin and immunoblotting increased 2-fold compared to control cells grown with fetal calf serum. Growth of cells with medium supplemented with LPDS plus mevinolin reduced the levels of alpha 39 and alpha 41 to below the levels in control cells. Levels of the beta subunit of No and Ni were unaffected by growth with LPDS.(ABSTRACT TRUNCATED AT 400 WORDS)

Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line

Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control cultures.

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 Ca 2+ 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 physicochemical 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.

Cholesterol ester concentrations and the ester hydrolases in cultured glioblastoma cells: Effect of lipoprotein depleted serum

We have investigated the effects of substituting lipoprotein depleted serum (LPDS) for normal fetal calf serum (FCS) in culture media on cholesterol ester concentrations and the activity of the ester hydrolases in cultured glioblastoma (C-6 glial) cells. Glial cells grown in media supplemented with 10% FCS contained 16-23% of total cholesterol as esterified sterol. Total sterol content of the cells cultured in media supplemented with LPDS was reduced by 55-75% as compared to cells cultured in FCS media and none of this sterol was in esterified form. Cholesterol ester hydrolase activity was maximal at pH values of 4.5 and 6.5 and required Triton X-100 for optimal activity. Cholesterol ester hydrolase activity at pH 4.5 was significantly higher in cells grown in FCS media than in cells cultured in LPDS media, but the activity at pH 6.5 was not significantly different. The protein: DNA ratio of cells cultured in FCS was higher than in cells cultured in LPDS. These findings indicate that the increase in cholesterol ester concentrations in cells is accompanied by increased activity of lysosomal cholesterol ester hydrolase; and suggest that, in cells cultured in FCS, the availability of free cholesterol for incorporation into cellular membranes is regulated by cholesterol ester hydrolase. The findings also indicate that changes in growth and differentiation of cells cultured in LPDS may be related to reduced availability of exogenous cholesterol.

Proline-rich protein (PRP)の臨床的意義: 脂質, リポ蛋白およびアポ蛋白との相関

Proline-rich protein (PRP) is present in chylomicron and d>1.21 fraction of human plasma. PRP was separated from lipoprotein-depleted serum by absorption to Intralipid. Antiserum to PRP was raised by rabbit and the serum concentration was assayed by single immunodiffusion. PRP was correlated to serum total cholesterol and triglyceride, VLDL-cholesterol and VLDL-trigly-ceride and Apo B, C-II, C-III. These data suggested that PRP relates to the metabolism of TG-rich lipoproteins.

Modulation by lipoproteins of amphotericin B-induced immunostimulation

Previous reports indicate that amphotericin B (AmB) and amphotericin methyl ester (AME) are potent adjuvants and polyclonal B-cell activators, and that most mouse strains can be classified as high or low responders to AmB and AME. In the present study, an inbred strain with very high plasma cholesterol concentration (HC strain) proved to be a low responder. Responses of HC mice to other immune stimuli were normal, suggesting that HC lymphoid cells expressed selectively weak responses to AmB and AME. Plasma levels of low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), and high-density lipoprotein subfraction (HDL 2) were very much higher in HC mice than in AKR mice, an AmB-high responder strain. Low responses in vitro to AME were observed with lymphoid cells from HC mice and from AKR → HC bone marrow chimeras, i.e., AmB-high responder strain lymphocytes from a low responder host. However, enhanced AME responses were induced by a 2- or 48-hr preincubation of splenocytes from either HC or AKR mice in medium containing lipoprotein-depleted fetal calf serum. Taken together these studies indicate that plasma lipoproteins can inhibit lymphocyte responses to AME; this seems to account for the low AmB and AME responses of the HC strain. The mechanism of this lipoprotein-induced inhibition remains obscure, but it cannot be accounted for by competitive binding of AmB by lipoproteins.

各種疾患における血清「Proline-rich protein」値について

PRP is present in chylomicron and d>1.21 fraction of human plasma. PRP was separated from lipoprotein-depleted serum by absorption to Intralipid. Antiserum to PRP was raised by rabbit and the serum concentration was assayed by single immunodiffusion. PRP levels in acute hepatitis and hepatic cirrhosis were lower than controls. PRP levels in fatty liver and nephrotic syndrome were higher. Also PRP levels in ischemic heart disease, diabetes mellitus, cerebrovasculer disease and hyperlipidemia were higher. Furthermore, patients with inflammatory disease had higher PRP levels. These data suggested that liver may be involved in PRP synthesis and possible relation to atherosclerotic disease and inflammatory disease, indicated.