High levels of human apolipoprotein A-I in transgenic mice result in increased plasma levels of small high density lipoprotein (HDL) particles comparable to human HDL3

Five lines of transgenic mice, which had integrated the human apolipoprotein (apo) A-I gene and various amounts of flanking sequences, were established. Normally, apoA-I is expressed mainly in liver and intestine, but all of the transgenic lines only expressed apoA-I mRNA in liver, strongly suggesting that 256 base pairs of 5'-flanking sequence was sufficient for liver apoA-I gene expression but that 5.5 kilobase pairs was not sufficient for intestinal expression. Mean plasma levels of human apoA-I varied in different lines from approximately 0.1 to 200% of normal mouse levels. This was not dependent on the amount of flanking sequence. Lipoprotein levels were studied in detail in one of the lines with a significantly increased apoA-I pool size. In one study, the total plasma apoA-I level (mouse plus human) was 381 +/- 43 mg/dl in six animals from this line, compared to 153 +/- 17 mg/dl in matched controls. Total and high density lipoprotein cholesterol (HDL-C) levels were increased 60% in transgenic animals, compared to controls (total cholesterol: 125 +/- 12 versus 78 +/- 13 mg/dl, p = 0.0001; HDL-C 90 +/- 7 versus 55 +/- 11 mg/dl, p = 0.0001). The molar ratio of HDL-C/apoA-I was significantly lower in transgenic animals, 17 +/- 1 versus 25 +/- 2 (p = 0.0001), suggesting the increase was in smaller HDL particles. This was confirmed by native gradient gel electrophoresis. This was not due to aberrant metabolism of human apoA-I in the mouse, since human apoA-I was distributed throughout the HDL particle size range and was catabolized at the same rate as mouse apoA-I. In another study of 23 transgenic mice, HDL-C and human apoA-I levels were highly correlated (r = 0.87, p less than 0.001). The slope of the correlation line also indicated the additional HDL particles were in the smaller size range. We conclude that human apoA-I can be incorporated into mouse HDL, and excessive amounts increase HDL-C levels primarily by increasing smaller HDL particles, comparable to human HDL3 (HDL-C/apoA-I molar ratio = 18).     

Overexpressed lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice.

Lipoprotein lipase (LPL) is known to play a crucial role in lipoprotein metabolism by hydrolyzing triglycerides; however its role in atherogenesis has yet to be determined. We have previously shown that low density lipoprotein receptor knockout mice overexpressing LPL are resistant to diet-induced atherosclerosis due to the suppression of remnant lipoproteins. Plasma lipoproteins and atherosclerosis of apolipoprotein (apo) E knockout mice which overexpress the human LPL transgene (LPL/APOEKO) were compared with those of control apoE knockout mice (APOEKO). On a normal chow diet, LPL/APOEKO mice showed marked suppression of the plasma triglyceride levels compared with APOEKO mice (54 vs. 182 mg/dl), but no significant changes in plasma cholesterol and apoB levels. Non-high density lipoproteins (HDL) from LPL/APOEKO mice had lower triglyceride content, a smaller size, and a more positive charge compared with those from APOEKO mice. Cholesterol, apoA-I, and apoA-IV were increased in HDL. Although both groups developed hypercholesterolemia to a comparable degree in response to an atherogenic diet, the LPL/APOEKO mice developed 2-fold smaller fatty streak lesions in the aortic sinus compared to the APOEKO mice. In conclusion, overproduction of LPL is protective against atherosclerosis even in the absence of apoE.     

Postheparin plasma lipoprotein and hepatic lipase are determinants of hypo- and hyperalphalipoproteinemia

To study the role of the two postheparin plasma lipolytic enzymes, lipoprotein lipase (LPL) and hepatic lipase (HL) in high density lipoprotein (HDL) metabolism at a population level, we determined serum lipoproteins, apoproteins A-I, A-II, B, and E, and postheparin plasma LPL and HL activities in 65 subjects with a mean HDL-cholesterol of 34 mg/dl and in 62 subjects with a mean HDL-cholesterol of 87 mg/dl. These two groups represented the highest and lowest 1.4 percentile of a random sample consisting 4,970 subjects. The variation in HDL level was due to a 4.1-fold difference in the HDL2 cholesterol (P less than 0.001) whereas the HDL3 cholesterol level was increased only by 32% (P less than 0.001) in the group with high HDL-cholesterol. Serum apoA-levels were 128 +/- 2.2 mg/dl and 210 +/- 2.8 mg/dl (mean +/- SEM) in hypo- and hyper-HDL cholesterolemia, respectively. Serum apoA-II concentration was elevated by 28% (P less than 0.001) in hyperalphalipoproteinemia. The apoA-I/A-II ratio was elevated only in women with high HDL-cholesterol but not in men, suggesting that elevation of apoA-I is involved in hyperalphalipoproteinemia in females, whereas both apoA proteins are elevated in men with high HDL cholesterol. Serum concentration of apoE and its phenotype distribution were similar in the two groups. The HL activity was reduced in the high HDL-cholesterol group (21.2 +/- 1.5 vs. 38.5 +/- 1.8 mumol/h/ml, P less than 0.001), whereas the LPL activity was elevated in the group with high HDL-cholesterol compared to subjects with low HDL-cholesterol (27.8 +/- 1.3 vs. 19.9 +/- 0.8 mumol/h/ml, P less than 0.001). The HL and LPL activities correlated in opposing ways with the HDL2 cholesterol (r = 0.57, P less than 0.001 and r = 0.51, P less than 0.001, respectively), and this appeared to be independent of the relative ponderosity by multiple correlation analysis. The results demonstrate major influence of both HL and LPL on serum HDL cholesterol concentration at a population level.     

Role of insulin in regulation of high density lipoprotein metabolism

The effect of alloxan-induced insulin deficiency on high density lipoprotein (HDL) metabolism was studied in rabbits. Rabbits with alloxan-induced diabetes had significantly higher (P less than 0.001, mean +/- SEM) plasma concentrations of glucose (541 +/- 13 vs. 130 +/- 2 mg/dl), triglyceride (2851 +/- 332 vs. 101 +/- 10 mg/dl), and total plasma cholesterol (228 +/- 55 vs. 42 +/- 4 mg/dl) than did normal control rabbits. However, diabetic rabbits had lower plasma HDL-cholesterol (7.2 +/- 1 vs. 51.3 +/- 1.3 mg/dl, P less than 0.001) and HDL apoA-I (38.3 +/- 6.0 vs. 87.2 +/- 4.3 mg/dl, P less than 0.001) concentrations. HDL kinetics were compared in diabetic and normal rabbits, using either 125I-labeled HDL or HDL labeled with 125I-labeled apoA-I, and it was demonstrated that HDL fractional catabolic rate (FCR) was slower and residence time was longer in the diabetic rabbits when either tracer was used. The slow FCR and the low apoA-I pool size led to reduced apoA-I/HDL synthetic rate in diabetic rabbits (0.97 +/- 0.11 vs. 0.34 +/- 0.07 mg per kg per hr). Thus, the reduced plasma HDL-cholesterol concentrations seen in rabbits with alloxan-induced insulin deficiency was associated with a lower total apoA-I/HDL synthetic rate. Since insulin treatment restored to normal all of the changes in plasma lipoprotein concentration and kinetics seen in diabetic rabbits, it is unlikely that the phenomena observed were secondary to a nonspecific toxic effect of alloxan. These data strongly support the view that insulin plays an important role in regulation of HDL metabolism.     

Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.     

Single repeat deletion in ApoA-I blocks cholesterol esterification and results in rapid catabolism of delta6 and wild-type ApoA-I in transgenic mice

The deletion mutation Delta6 apolipoprotein A-I lacks residues 143-164 or repeat 6 in the mature apoA-I protein. In vitro studies show this mutation dramatically reduces the rate of lecithin:cholesterol acyltransferase (LCAT) catalyzed cholesterol esterification. The present study was initiated to investigate the effect of this mutation on in vivo high density lipoprotein (HDL) cholesterol esterification and metabolism. Transgenic mice expressing human Delta6 apoA-I (TgDelta6 +/+) were created and then crossed with apoA-I knockout mice (-/-) to generate mice expressing only human Delta6 apoA-I (TgDelta6 -/-). Human Delta6 apoA-I was associated with homogeneous sized alpha-HDL, when wild-type mouse apoA-I was present (in TgDelta6 +/+ and +/- mice). However, in the absence of endogenous mouse apoA-I, Delta6 apoA-I was found exclusively in cholesterol ester-poor HDL, and lipid-free HDL fractions. This observation coincides with the 6-fold lower cholesterol ester mass in TgDelta6 -/- mouse plasma compared with control. Structural studies show that despite the structural perturbation of a domain extending from repeat 5 to repeat 8 (137-178), Delta6 apoA-I binds to spherical unilamellar vesicles with only 2-fold less binding affinity. In summary, these data show a domain corresponding to apoA-I repeat 6 is responsible for providing an essential conformation for LCAT catalyzed generation of cholesterol esters. Deletion of apoA-I repeat 6 not only blocks normal levels of cholesterol esterification but also exerts a dominant inhibition on the ability of wild-type apoA-I to activate LCAT in vivo.     

Impact of hydrogenated fat on high density lipoprotein subfractions and metabolism

Relative to saturated fatty acids, trans-fatty acids/hydrogenated fat-enriched diets have been reported to increase low density lipoprotein (LDL) cholesterol levels and either decrease or have no effect on high density lipoprotein (HDL) cholesterol levels. To better understand the effect of trans-fatty acids/hydrogenated fat on HDL cholesterol levels and metabolism, 36 subjects (female, n = 18; male, n = 18) were provided with each of three diets containing, as the major sources of fat, vegetable oil-based semiliquid margarine, traditional stick margarine, or butter for 35-day periods. LDL cholesterol levels were 155 +/- 27, 168 +/- 30, and 177 +/- 32 mg/dl after subjects followed the semiliquid margarine, stick margarine, and butter-enriched diets, respectively. HDL cholesterol levels were 43 +/- 10, 42 +/- 9, and 45 +/- 10 mg/dl, respectively. Dietary response in apolipoprotein (apo) A-I levels was similar to that in HDL cholesterol levels. HDL(2) cholesterol levels were 12 +/- 7, 11 +/- 6, and 14 +/- 7 mg/dl, respectively. There was virtually no effect of dietary fat on HDL3 cholesterol levels. The dietary perturbations had a larger effect on particles containing apoA-I only (Lp A-I) than apoA-I and A-II (Lp A-I/A-II). Cholesterol ester transfer protein (CETP) activity was 13.28 +/- 5.76, 15.74 +/- 5.41, and 14.35 +/- 4.77 mmol x h(-1) x ml(-1), respectively. Differences in CETP, phospholipid transfer protein activity, or the fractional esterification rate of cholesterol in HDL did not account for the differences observed in HDL cholesterol levels.These data suggest that the saturated fatty acid component, rather than the trans- or polyunsaturated fatty acid component, of the diets was the putative factor in modulating HDL cholesterol response.     

Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR(-/-)) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR(-/-) mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR(-/-) mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.     

Plasma turnover of HDL apoC-I,apoC-III,and apoE in humans: in vivo evidence for a link between HDL apoC-III and apoA-I metabolism

Numerous factors are known to affect the plasma metabolism of HDL, including lipoprotein receptors, lipid transfer protein, lipolytic enzymes and HDL apolipoproteins. In order to better define the role of HDL apolipoproteins in determining plasma HDL concentrations, the aims of the present study were: a) to compare the in vivo rate of plasma turnover of HDL apolipoproteins [i.e., apolipoprotein A-I (apoA-I), apoC-I, apoC-III, and apoE], and b) to investigate to what extent these metabolic parameters are related to plasma HDL levels. We thus studied 16 individuals with HDL cholesterol levels ranging from 0.56-1.66 mmol/l and HDL apoA-I levels ranging from 89-149 mg/dl. Plasma kinetics of HDL apolipoproteins were investigated using a primed constant (12 h) infusion of deuterated leucine. Plasma HDL apolipoprotein levels were 41.8 +/- 1.5, 9.7 +/- 0.5, 4.9 +/- 0.5, and 0.7 +/- 0.1 micromol/l for apoA-I, apoC-I, apoC-III and apoE. Plasma transport rates (TRs) were 388.6 +/- 24.7, 131.5 +/- 12.5, 66.5 +/- 9.1, and 31.4 +/- 3.3 nmol.kg-1.day-1; and residence times (RTs) were 5.1 +/- 0.4, 3.7 +/- 0.3, 3.6 +/- 0.3, and 1.1 +/- 0.1 days, respectively. HDL cholesterol and apoA-I levels were significantly correlated with HDL apoA-I RT (r = 0.69 and r = 0.56), and were not significantly correlated with HDL apoA-I TR. In contrast, HDL apoC-I, apoC-III, and apoB levels were all positively related to their TRs and not their RTs. HDL apoC-III TR was positively correlated with levels of HDL apoC-III (r = 0.73, P < 0.01), and with those of HDL cholesterol and apoA-I (r = 0.54 and r = 0.53, P < 0.05, respectively). HDL apoC-III TR was in turn related to HDL apoA-I RT (r = 0.51, P < 0.05). Together, these results provide in vivo evidence for a link between the metabolism of HDL apoC-III and apoA-I, and suggest a role for apoC-III in the regulation of plasma HDL levels.     

The effects of apolipoprotein F deficiency on high density lipoprotein cholesterol metabolism in mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20-25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/-0.9 mg/dl vs. WT: 1.2+/-0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls.     

Reduction of plasma triglycerides in apolipoprotein C-II transgenic mice overexpressing lipoprotein lipase in muscle

LPL and its specific physiological activator, apolipoprotein C-II (apoC-II), regulate the hydrolysis of triglycerides (TGs) from circulating TG-rich lipoproteins. Previously, we developed a skeletal muscle-specific LPL transgenic mouse that had lower plasma TG levels. ApoC-II transgenic mice develop hypertriglyceridemia attributed to delayed clearance. To investigate whether overexpression of LPL could correct this apoC-II-induced hypertriglyceridemia, mice with overexpression of human apoC-II (CII) were cross-bred with mice with two levels of muscle-specific human LPL overexpression (LPL-L or LPL-H). Plasma TG levels were 319 +/- 39 mg/dl in CII mice and 39 +/- 5 mg/dl in wild-type mice. Compared with CII mice, apoC-II transgenic mice with the higher level of LPL overexpression (CIILPL-H) had a 50% reduction in plasma TG levels (P = 0.013). Heart LPL activity was reduced by approximately 30% in mice with the human apoC-II transgene, which accompanied a more modest 10% decrease in total LPL protein. Overexpression of human LPL in skeletal muscle resulted in dose-dependent reduction of plasma TGs in apoC-II transgenic mice. Along with plasma apoC-II concentrations, heart and skeletal muscle LPL activities were predictors of plasma TGs. These data suggest that mice with the human apoC-II transgene may have alterations in the expression/activity of endogenous LPL in the heart. Furthermore, the decrease of LPL activity in the heart, along with the inhibitory effects of excess apoC-II, may contribute to the hypertriglyceridemia observed in apoC-II transgenic mice.     

Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice

Helical apolipoprotein(apo)s generate pre-beta-high density lipoprotein (HDL) by removing cellular cholesterol and phospholipid upon the interaction with cells. To investigate its physiological relevance, we studied the effect of an in vitro inhibitor of this reaction, probucol, in mice on the cell-apo interaction and plasma HDL levels. Plasma HDL severely dropped in a few days with probucol-containing chow while low density protein decreased more mildly over a few weeks. The peritoneal macrophages were assayed for apoA-I binding, apoA-I-mediated release of cellular cholesterol and phospholipid and the reduction by apoA-I of the ACAT-available intracellular cholesterol pool. All of these parameters were strongly suppressed in the probucol-fed mice. In contrast, the mRNA levels of the potential regulatory proteins of the HDL level such as apoA-I, apoE, LCAT, PLTP, SRB1 and ABC1 did not change with probucol. The fractional clearance rate of plasma HDL-cholesteryl ester was uninfluenced by probucol, but that of the HDL-apoprotein was slightly increased. No measurable CETP activity was detected either in the control or probucol-fed mice plasma. The change in these functional parameters is consistent with that observed in the Tangier disease patients. We thus concluded that generation of HDL by apo-cell interaction is a major source of plasma HDL in mice.     

A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels

Pre-beta1-HDL, a putative discoid-shaped high density lipoprotein (HDL) of approximately 67-kDa mass that migrates with pre-beta mobility in agarose gel electrophoresis, contains apolipoprotein A-I (apoA-I), phospholipids, and unesterified cholesterol. It participates in the retrieval of cholesterol from peripheral tissues. In this study we established a new sandwich enzyme immunoassay (EIA) for measuring plasma pre-beta1-HDL using mouse anti-human pre-beta1-HDL monoclonal antibody (MAb 55201) and goat anti-human apoA-I polyclonal antibody. MAb 55201 reacted with apoA-I in lipoprotein [A-I] with molecular mass less than 67 kDa, and with pre-beta1-HDL separated by nondenaturing two-dimensional electrophoresis, whereas it did not react with apoA-I in alpha-HDL. Pre-beta1-HDL levels measured by this method declined when incubated at 37 degrees C for 2 h, whereas this decrease was not observed in the presence of 2 mM lecithin:cholesterol acyltransferase inhibitor 5,5'-dithiobis (2-nitrobenzoic acid). To clarify the clinical significance of measuring pre-beta1-HDL by this method, 47 hyperlipidemic subjects [male/female 22/25; age 55 +/- 14 years; body mass index 25 +/- 4.5 kg/m(2); total cholesterol (TC) 245 +/- 64 mg/dl; triglyceride (TG) 232 +/- 280 mg/dl; HDL cholesterol (HDL-C) 51 +/- 23 mg/dl] and 25 volunteers (male/female 15/10; age 36 +/- 9.3 years; body mass index 23 +/- 3.5 kg/m(2); TC 183 +/- 28 mg/dl; TG 80 +/- 34 mg/dl; HDL-C 62 +/- 15 mg/dl) were involved. Plasma pre-beta1-HDL levels were significantly higher in hyperlipidemic subjects than in volunteers (39.3 +/- 10.1 vs. 22.5 +/- 7.5 mg/ml, P < 0.001) whereas plasma apoA-I levels did not differ (144.2 +/- 28.4 vs. 145.3 +/- 16.3 mg/dl).These results indicate that this sandwich EIA method specifically recognizes apoA-I associated with pre-beta1-HDL.     

A unique protease-sensitive high density lipoprotein particle containing the apolipoprotein A-I(Milano) dimer effectively promotes ATP-binding Cassette A1-mediated cell cholesterol efflux

Carriers of the apolipoprotein A-I(Milano) (A-I(M)) variant present with severe reductions of plasma HDL levels, not associated with premature coronary heart disease (CHD). Sera from 14 A-I(M) carriers and matched controls were compared for their ability to promote ABCA1-driven cholesterol efflux from J774 macrophages and human fibroblasts. When both cell types are stimulated to express ABCA1, the efflux of cholesterol through this pathway is greater with A-I(M) than control sera (3.4 +/- 1.0% versus 2.3 +/- 1.0% in macrophages; 5.2 +/- 2.4% versus 1.9 +/- 0.1% in fibroblasts). A-I(M) and control sera are instead equally effective in removing cholesterol from unstimulated cells and from fibroblasts not expressing ABCA1. The A-I(M) sera contain normal amounts of apoA-I-containing prebeta-HDL and varying concentrations of a unique small HDL particle containing a single molecule of the A-I(M) dimer; chymase treatment of serum degrades both particles and abolishes ABCA1-mediated cholesterol efflux. The serum content of chymase-sensitive HDL correlates strongly and significantly with ABCA1-mediated cholesterol efflux (r = 0.542, p = 0.004). The enhanced capacity of A-I(M) serum for ABCA1 cholesterol efflux is thus explained by the combined occurrence in serum of normal amounts of apoA-I-containing prebeta-HDL, together with a unique protease-sensitive, small HDL particle containing the A-I(M) dimer, both effective in removing cell cholesterol via ABCA1.     

Hypertriglyceridemia is associated with prebeta-HDL concentrations in subjects with familial low HDL

Prebeta-HDL particles act as the primary acceptors of cellular cholesterol in reverse cholesterol transport (RCT). An impairment of RCT may be the reason for the increased risk of coronary heart disease (CHD) in subjects with familial low HDL. We studied the levels of serum prebeta-HDL and the major regulating factors of HDL metabolism in 67 subjects with familial low HDL and in 64 normolipidemic subjects. We report that the subjects with familial low HDL had markedly reduced prebeta-HDL concentrations compared with the normolipidemic subjects (17.4 +/- 7.2 vs. 23.4 +/- 7.8 mg apolipoprotein A-I/dl; P < 0.001). A positive correlation was observed between prebeta-HDL concentration and serum triglyceride (TG) level (r = 0.334, P = 0.006). In addition, serum TG level was found to be the strongest predictor of prebeta-HDL concentration in subjects with familial low HDL. The activities of cholesteryl ester transfer protein and hepatic lipase were markedly increased in subjects with familial low HDL without a significant correlation to prebeta-HDL concentration. Our results support the hypothesis that impaired RCT is one mechanism behind the increased risk for CHD in subjects with familial low HDL.     

Differential involvement of thrombin receptors in Ca2+ release from two different intracellular stores in human platelets

In the present study we have used adenovirus-mediated gene transfer of apoA-I (apolipoprotein A-I) mutants in apoA-I-/- mice to investigate how structural mutations in apoA-I affect the biogenesis and the plasma levels of HDL (high-density lipoprotein). The natural mutants apoA-I(R151C)Paris, apoA-I(R160L)Oslo and the bioengineered mutant apoA-I(R149A) were secreted efficiently from cells in culture. Their capacity to activate LCAT (lecithin:cholesterol acyltransferase) in vitro was greatly reduced, and their ability to promote ABCA1 (ATP-binding cassette transporter A1)-mediated cholesterol efflux was similar to that of WT (wild-type) apoA-I. Gene transfer of the three mutants in apoA-I-/- mice generated aberrant HDL phenotypes. The total plasma cholesterol of mice expressing the apoA-I(R160L)Oslo, apoA-I(R149A) and apoA-I(R151C)Paris mutants was reduced by 78, 59 and 61% and the apoA-I levels were reduced by 68, 64 and 55% respectively, as compared with mice expressing the WT apoA-I. The CE (cholesteryl ester)/TC (total cholesterol) ratio of HDL was decreased and the apoA-I was distributed in the HDL3 region. apoA-I(R160L)Oslo and apoA-I(R149A) promoted the formation of prebeta1 and alpha4-HDL subpopulations and gave a mixture of discoidal and spherical particles. apoA-I(R151C)Paris generated subpopulations of different sizes that migrate between prebeta and alpha-HDL and formed mostly spherical and a few discoidal particles. Simultaneous treatment of mice with adenovirus expressing any of the three mutants and human LCAT normalized plasma apoA-I, HDL cholesterol levels and the CE/TC ratio. It also led to the formation of spherical HDL particles consisting mostly of alpha-HDL subpopulations of larger size. The correction of the aberrant HDL phenotypes by treatment with LCAT suggests a potential therapeutic intervention for HDL abnormalities that result from specific mutations in apoA-I.     

Structural and functional properties of human plasma high density-sized lipoprotein containing only apoE particles

To investigate the metabolism of HDL-apolipoprotein E (apoE) particles in human plasma, we isolated a fraction of plasma HDL-apoEs that lack apoA-I (HDL-LpE) from subjects with apoE3/3 phenotype by immunoaffinity. Plasma HDL-LpE had a particle size ranging from 9 nm to 18.5 nm in diameter and was characterized by two-dimensional nondenaturing gradient gel electrophoresis as having either gamma-, prebeta1-, prebeta2-, or alpha-electrophoretic mobility. HDL-LpE was also present in the medium of cultured human hepatoma cell lines and monocyte-derived macrophages. The majority of apoE3 was found as a monomeric form in HDL-LpE and floated at density d > 1.21 g/ml. Plasma levels of HDL-LpE in normolipidemic, CETP-deficient, and ABCA1-deficient subjects were 0.72 +/- 0.15 mg/dl (n = 12), 1.77 +/- 0.75 mg/dl (n = 3), and 0.55 +/- 0.11 mg/dl (n = 3), respectively. The ratio of HDL-apoE containing apoA-I to HDL-LpE was significantly higher 4 h after a fat load, representing a 35 +/- 9% increase (n = 3). Isolated plasma HDL-LpE3 was as effective as apoE3, reconstituted HDL particles, or apoA-I in promoting cellular cholesterol efflux. These results demonstrate that 1) plasma HDL-LpE may have hepatogenous and macrophagic origins; 2) HDL-LpE was preserved even with large reductions in apoA-I-containing lipoproteins; 3) HDL-LpE was active in the transfer of apoE to triglyceride-rich lipoproteins, and 4) HDL-LpEs efficiently take up cell-derived cholesterol.     

In vivo studies of HDL assembly and metabolism using adenovirus-mediated transfer of ApoA-I mutants in ApoA-I-deficient mice.

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.     

Cholesterol efflux from macrophage foam cells is enhanced by active phospholipid transfer protein through generation of two types of acceptor particles

Phospholipid transfer protein (PLTP) is expressed by macrophage-derived foam cells in human atherosclerotic lesions, suggesting a regulatory role for PLTP in cellular cholesterol homeostasis. However, the exact role of PLTP in the reverse cholesterol transport pathway is not known. PLTP is present in plasma as two forms, a highly active (HA-PLTP) and a lowly active (LA-PLTP) form. In this study we clarify the role of the two forms of PLTP in cholesterol efflux from [3H]cholesterol oleate-acetyl-LDL-loaded THP-1 macrophages. Incubation of HDL in the presence of HA-PLTP resulted in the formation of two types of acceptor particles, prebeta-HDL and large fused HDL. HA-PLTP increased prebeta-HDL formation and caused a 42% increase in [3H]cholesterol efflux to HDL, while LA-PLTP neither formed prebeta-HDL nor increased cholesterol efflux. Removal of the formed prebeta-HDL by immunoprecipitation decreased cholesterol efflux by 47%. Neither HA- nor LA-PLTP enhanced cholesterol efflux to lipid-free apoA-I. Importantly, also the large fused HDL particles formed during incubation of HDL with HA-PLTP acted as efficient cholesterol acceptors. These observations demonstrate that only HA-PLTP increases macrophage cholesterol efflux, via formation of efficient cholesterol acceptors, prebeta-HDL and large fused HDL particles.