apoE-rich High Density Lipoprotein Research Articles

The role of apoE in inhibitory effects of apoE-rich HDL on platelet function

Apolipoprotein E- (ApoE-) rich high-density lipoprotein (HDL), which was prepared from the bound fraction of normolipemic volunteers on heparin-Sepharose and from a hyperalphalipoproteinemic patient, potently inhibited aggregation of human platelets in a dose-dependent fashion. Dimyristoyl phosphatidylcholine liposome with apoE (apoE · DMPC) also inhibited platelet aggregation, and incubation of washed platelets with apoE · DMPC resulted in the release of cholesterol into the supernatant in a time and dose-dependent manner. These results suggest that apoE plays a major role in the inhibitory effect of apoE-rich HDL in platelet function, presumably due to the release of cholesterol from the plasma membrane.

Accumulation of apolipoprotein E-rich high density lipoproteins in hyperalphalipoproteinemic human subjects with plasma cholesteryl ester transfer protein deficiency.

This study characterized the plasma lipoproteins of familial hyperalphalipoproteinemic patients with or without deficiency of cholesteryl ester transfer protein (CETP) activity. The subjects with CETP deficiency have increased levels of apolipoprotein (apo) E. The increased concentration of apo E in these subjects was correlated to the appearance of apo E-rich high density lipoproteins (HDL). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed that these lipoproteins contained predominantly the apo E (82%) and little amount of apo A-I (18%). These apo E-rich HDL displayed a much higher affinity than human LDL in binding to LDL receptors on human fibroblasts. Furthermore, 3.5 times fewer apo E-rich HDL than LDL were required to saturate the receptors on fibroblasts. These data indicated that the apo E-rich HDL in CETP-deficient human subjects contained multiple copies of apo E and bound to the LDL receptor through multiple interactions. The apo E-rich HDL, with similar properties as cholesterol-induced apo E HDLc, were not detectable in normal human subjects or in hyperalphalipoproteinemic subjects with normal CETP activity. The apo E-containing HDL in the latter subjects were smaller and contained only small amounts of apo E (14%). The difference in apo E-containing HDL in these subjects suggests a correlation between CETP level and the appearance of apo E-rich HDL.

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes.

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.

Binding of apoE-rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation.

High density lipoproteins (HDL, d 1.063-1.21 g/ml) are reported to stimulate, to have no effect on, or to inhibit agonist-induced platelet aggregation. We have hypothesized that these conflicting reports might be explained by opposing effects of individual HDL subclasses on platelet aggregability. Physiologic concentrations of HDL3 had little effect on ADP-induced aggregation of washed platelet suspensions, although higher levels were stimulatory. Normal concentrations of HDL2 (0.2-0.4 mg of protein/ml) inhibited aggregation; further fractionation by heparin-Sepharose chromatography identified the particles rich in apolipoprotein E, termed HDL-E, as the major anti-aggregatory subclass. Washed platelets bound radioiodinated HDL-E to a uniform class of saturable sites; they numbered 4,200 per platelet and the KD was 7.9 x 10(-7) M. Binding of HDL-E by platelets, and its anti-aggregatory action, showed a similar rapidity and both occurred within the physiologic concentration range. Moreover, the two processes were independent of the presence of divalent ions and were impaired by chemical modification of the apolipoprotein constituents of HDL-E. We conclude that occupation of cell-surface receptors by HDL-E particles impairs platelet responsiveness to exogenous agonists and that platelet aggregability in the presence of whole HDL may reflect the relative concentrations of the individual subclasses in the particular sample.

Apolipoprotein E-rich HDL binding to liver plasma membranes in copper-deficient rats.

Copper deficiency in rats raises plasma cholesterol concentration while reducing live cholesterol concentration. One consequence of this cholesterol redistribution is the accumulation of a large high-density lipoprotein (HDL) particle rich in apolipoprotein E (apo E). The purpose of this study was to determine, using an in vitro binding assay, if the interaction of apo E-rich HDL with hepatic lipoprotein binding sites may be affected by copper deficiency. Male Sprague-Dawley rats were divided into two dietary treatments (copper-deficient and -adequate) and placed on a dietary regimen for 8 weeks. Subsequent to exsanguination, hepatic plasma membranes were prepared and apo E-rich HDL was isolated from rats of each treatment by ultracentrifugation, agarose column chromatography, and heparin-Sepharose affinity chromatography. Total binding and experimentally derived specific binding of 125I-apo E-rich HDl to hepatic plasma membranes indicated greater binding when lipoproteins and membranes from copper-deficient animals were used in the assay compared to controls. Scatchard analysis of specific binding data indicated that equilibrium binding affinity (Kd) was also affected by copper deficiency. The hepatic binding sites recognizing apo E-rich HDL were not affected by EDTA or pronase, of relatively high capacity, and recognized a variety of other rat lipoproteins.

Plasma high density lipoprotein subfractions in subjects with different coronary risk indices as assessed by plasma lipoprotein concentrations

The distribution of plasma high density lipoprotein (HDL) subfractions was determined in 2 groups of healthy male subjects with different coronary risk indices (CI) as assessed by the ratio of total plasma cholesterol/HDL-cholesterol. The subjects in the 2 groups were of similar age and fitness (as assessed by VO 2max) The higher risk group (CI > 4.0) contained a lower relative concentration of a specific HDL subfraction, HDL 2b, separated by gradient gel electrophoresis, and a lower level of an apo E-rich HDL fraction, isolated by affinity chromatography, than the lower risk group (CI < 4.0). The concentration of total HDL-cholesterol was higher in the lower risk group due to a difference in HDL 2-cholesterol when separation was achieved by polyanion precipitation, but not when separation was made by ultracentrifugation. These observations suggest that the level of these specific HDL subfractions might, when taken in conjunction with plasma cholesterol concentration, provide a better index of coronary risk than that of total HDL as conventionally employed.

Serum lipoproteins in venous blood serum from birth to the end of the first week: feeding influences.

Serum lipoproteins were explored on the first day of life (D1) and on the sixth day (D6) in blood drawn by peripheral venipuncture from 43 normal term newborns, 22 breast-fed and 21 formula-fed, and were compared to those of a control group of 28 young adults. With the exception of apolipoprotein E (Apo E), values of lipoprotein components obtained at D1 were similar, although generally slightly lower than those previously reported for cord blood serum. Total Apo E concentration at D1 (71 +/- 25 mg/l) was much higher than that obtained for the adult group (30 +/- 7 mg/l). Apo E distribution within the lipoprotein spectrum confirmed the presence of an already known Apo E-rich high density lipoprotein subfraction, which was responsible for the high total Apo E level at birth. The rise of lipoproteins of low density from D1 to D6, as evidenced by the increase of very low density lipoprotein + low density lipoprotein cholesterol and low density lipoprotein apolipoprotein B concentrations, was shown to be diet dependent. It was significantly less important in newborns fed a standard formula moderately enriched in unsaturated fatty acids than in breast-fed newborns.

Regulation of bile acid synthesis in cultured rat hepatocytes: stimulation by apoE-rich high density lipoproteins.

Cultured rat hepatocytes obtained by liver perfusion with collagenase in the presence of soybean trypsin inhibitor were used to examine the role of high density lipoproteins (HDL) in supplying cholesterol to the hepatocyte for bile acid synthesis. Within 6 hr of adding HDL (d 1.07-1.21 g/ml) obtained from rat serum there was a significant stimulation of bile acid synthesis and secretion that reached 2-fold after 24 hr. The stimulation by HDL occurred at normal plasma concentrations (i.e., 500 micrograms/ml) and showed further stimulation in a dose-dependent manner reaching a maximum stimulation of 2- to 2.5-fold. The stimulation of bile acid synthesis was dependent on the cholesteryl ester content of the HDL. Several lines of evidence show that the HDL is taken up by a receptor-mediated process dependent on apoE. These include: 1) at the same concentration (500 micrograms/ml) apoE-poor HDL (not retained by heparin affinity chromatography of HDL isolated from the plasma of rats fasted for 72 hr stimulated bile acid synthesis by 48%, whereas apoE-rich HDL stimulated bile acid synthesis by 110%; 2) reductive methylation totally blocked the stimulation of bile acid synthesis by HDL; 3) HDLC, which contained apoE as its major protein component, also maximally stimulated bile acid synthesis; and 4) human HDL, which contained no detectable apoE, failed to stimulate bile acid synthesis. Additional studies showed that apoE-enriched HDL and HDLC both inhibited cholesterol synthesis (determined by the incorporation of 3H2O) and caused a net accumulation of cholesteryl esters in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper Deficiency-Induced Hypercholesterolemia: Effects on HDL Subfractions and Hepatic Lipoprotein Receptor Activity in the Rat

Male Sprague-Dawley rats (10 per group) were fed diets adequate (control) or deficient (CuDef) in copper for 6 wk. In the CuDef group, plasma total cholesterol, high density lipoprotein (HDL) cholesterol and apoA-I levels were significantly higher than in controls. Apolipoprotein analysis of the HDL fractions revealed a relative enrichment of apoE in the CuDef group. Size analysis of 1.21 density lipoproteins by nondenaturing gradient gel electrophoresis demonstrated the presence of more material migrating in the size range of HDL1 in the CuDef group. Separation of HDL into apoE-rich and apoA-I-rich fractions by heparin-affinity chromatography confirmed the presence of increased apoE-rich HDL in the CuDef group. To determine the mechanism responsible for higher apoE-rich HDL in the CuDef group, the lipoprotein receptor binding activity in hepatic membranes from the control and CuDef group was assayed. Kinetic analysis of the binding data revealed that the lipoprotein binding assay was primarily measuring the activity of the HDL receptor, which is not apoE mediated. When corrected for differential enrichment of plasma membrane, hepatic membranes from the CuDef group bound significantly fewer lipoproteins than did controls. Furthermore, the hepatic receptor binding activity was negatively correlated with the proportion of HDL enriched with apoE.

Enrichment of endothelial cell arachidonate by lipid transfer from high density lipoproteins: relationship to prostaglandin I2 synthesis.

We have previously shown that plasma high density lipoproteins (HDL) stimulate release of prostacyclin, measured as its stable metabolite, 6-keto-PGF1 alpha, by cultured porcine aortic endothelial cells. The present experiments were designed to elucidate the contribution of HDL lipids to endothelial cellular phospholipid pools and to prostacyclin synthesis. In experiments with reconstituted HDL, both the lipid and protein moieties were required to stimulate prostacyclin release in amounts equivalent to the native HDL particle. Endothelial cells incorporated label from reconstituted HDL containing cholesteryl [1-14C]arachidonate into the cellular neutral and phospholipid pools as well as into 6-keto-PGF1 alpha and PGE2. Labeled arachidonate incorporated into endothelial cell lipids from reconstituted HDL containing cholesteryl [1-14C]arachidonate was also metabolized to prostaglandins after the cells were exposed to the calcium ionophore, A-23187. Both rat and human HDL which stimulated 6-keto-PGF1 alpha release (rat greater than human) increased the weight percentage of arachidonate in endothelial cell phospholipids; phospholipid arachidonate in the enriched cells fell after exposure to the phospholipase activator, A-23187, with release of 6-keto-PGF1 alpha which was greater than in control cells. Rat HDL that was depleted of cholesteryl arachidonate (achieved by incubation with human low density lipoproteins (LDL) in the presence of cholesteryl ester transfer protein) stimulated 6-keto-PGF1 alpha release less than native rat HDL. LDL enriched in cholesteryl arachidonate stimulated 6-keto-PGF1 alpha release more than native LDL. ApoE-depleted HDL also stimulated 6-keto-PGF1 alpha release more than apoE-rich HDL suggesting the apoE receptor was not involved in the response.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Copper Deficiency on the Apolipoprotein E-Rich High Density Lipoproteins in Rats

Twenty-four male weanling Sprague-Dawley rats were randomly divided into two treatments, namely a copper-adequate (8 mg Cu/kg diet) or a copper-deficient (0.85 mg Cu/kg diet) group. Feed and distilled-demineralized water were provided ad libitum. After 7 wk, plasma high density lipoproteins (HDL) were isolated by ultracentrifugation and agarose column chromatography. A heparin-Sepharose affinity column chromatographic method was used to subfractionate HDL into apolipoprotein E (apoE) -rich and apoE-poor subfractions. Significantly higher protein and cholesterol contents of HDL and the second retained (RII) subfraction (apoE-rich) were observed in copper-deficient than in control rats. The apoE contents of HDL and the apoE-rich (RII) subfraction were about threefold higher in copper-deficient rats. The increase in apoE in the HDL fraction was due primarily to the increased apoE concentration of the RII subfraction. In addition to a higher apoE concentration, apolipoprotein A-I concentration was also higher in the RII subfraction of the copper-deficient rats as compared to controls. However, the percentage of these apolipoproteins remained unaltered by copper deficiency. The present data suggest that the hypercholesterolemia observed in copper deficiency was associated with marked increases in cholesterol and apolipoprotein contents of the apoE-rich HDL.

Rat plasma lipoproteins and apolipoproteins in experimental hypothyroidism.

Hyperlipidemia associated with hypothyroidism is well documented in man and several animal species. The effect of hypothyroidism on apolipoprotein metabolism in the absence of complicating factors such as high cholesterol or fat content in the diet is virtually unknown. Hypothyroidism was therefore induced in male Sprague-Dawley rats by radiothyroidectomy (RTx-treated) or treatment with propylthiouracil (PTU-treated). Both treatments resulted in an over 90% decrease in circulating thyroid hormone concentrations accompanied by a 50-100% increase in plasma cholesterol and a 20-40% reduction in plasma triglyceride concentrations. Plasma apo E and apo B concentrations increased by 100% in the PTU-treated group and 40-50% in the RTx-group. Apo A-I increased 10 and 30% in the RTx- and PTU-treated rats, respectively, while the concentration of apo A-IV was not altered. A large increase in the low-density (LDL) and high-density lipoprotein (HDL) protein was observed and accompanied by a marked reduction of very low density lipoprotein (VLDL) in the hypothyroid rats. The electrophoretic pattern of plasma lipoproteins in the hypothyroid rats was changed by the appearance of a slow pre-beta band shown to be beta-VLDL. A redistribution of apo B occurred within the lipoprotein fractions. Apo B content in the VLDL fraction decreased and a large increase was noted in LDL. The major portion of the apo E and apo A-I increment was recovered in the HDL and to a lesser degree in LDL. An accumulation of apo E-rich larger HDL particles, resembling HDLc in apolipoprotein composition and distinct from the apo A-I-containing species, was observed by column chromatography. The results presented are consistent with the hypothesis that hypothyroidism in the rat may induce an accelerated production of VLDL catabolic remnants, including LDL, but at the same time reduce the rate of removal of these lipoproteins from the circulation.

The uptake of high density lipoprotein cholesteryl ester in the perfused rat liver.

The hepatic uptake of high density lipoprotein cholesteryl ester was determine in a nonrecycling isolated rat liver perfusion. High density lipoprotein rich in the E apoprotein (apoE) showed about 10 times more uptake of the ester on a single pass than the bulk of the high density lipoproteins rich in the A-I protein. The apoportein recoveries in the liver paralleled the ester for both lipoproteins. The uptake appeared to occur primarily in the hepatic parenchymal cell. Addition of human C-III-1 apoprotein to the rat apoE-rich high density lipoprotein inhibited the hepatic uptake of its constituents.