Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.     

The high-resolution crystal structure of human LCAT

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.     

Metabolism of cholesterol in normal hamster fibroblasts and those transformed by the SV 40 virus. Effect of low density lipoproteins

The amount of cholesterol and the percentage of esterified cholesterol were increased in transformed cells. The cholesterol synthesis from [14C] sodium acetate was reduced and cholesteryl oleate uptake increased by 3 fold in transformed cells. The activity of acyl coenzyme A-cholesterol-acyltransferase, measured in situ was also increased in transformed cells. Studies with 125I-LDL pointed out an increase of binding, and especially of internalization of LDL by transformed cells. Finally, long term culture in a lipoprotein-deficient medium showed that transformed cells exhibited a higher ability (tested by growth rate and cholesterol synthesis) to adapt themselves to lipid depletion.     

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer’s disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT^−/− mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer’s-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.     

Endotoxin-lipoprotein complex formation as a factor in atherogenesis: associations with hyperlipidemia and with lecithin:cholesterol acyltransferase activity

A potential role of endotoxin–lipoprotein (bacterial lipopolysaccharide–lipoprotein, LPS–LP) complex formation as a pathogenic factor for atherosclerosis has not been studied yet. The aim of this study was to test the hypothesis that in endotoxinemia in humans hyperlipidemia associated with atherosclerosis development can favor an excessive LPS–LP complex formation, and endotoxin presented in blood can inhibit lecithin:cholesterol acyltransferase (LCAT), one of the key enzymes of reverse cholesterol transport. Endotoxin-binding capacity of lipoproteins (LP) in patients with normolipidemia and hyperlipidemia types IIa and IV was estimated from label incorporation into different LP fractions isolated by means of sequential ultracentrifugation following serum preincubation with Salmonella minnesota R595 ¹²⁵I-labeled LPS. The effect of varied concentrations of S. minnesota R595 LPS on LCAT activity was evaluated from the overall esterifying activity of serum using [1,2-³H₂]cholesterol-labeled substrate. The elevation of low density LP (LDL) and very low density LP (VLDL) contents in blood serum in hyperlipidemia types IIa and IV, respectively, resulted in significant elevation of LPS binding to these fractions. LPS added to the blood serum leads to the dose-dependent decrease in LCAT activity. The revealed phenomena of elevated LPS binding to atherogenic LP fractions in hypercholesterolemia and endotoxin-induced LCAT inhibition suggest the pathogenic role of LPS–LP complexes in atherogenesis.     

Plasma lecithin:cholesterol acyltransferase and carotid intima-media thickness in European individuals at high cardiovascular risk

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. LCAT is a major factor in HDL remodeling and metabolism, and it has long been believed to play a critical role in macrophage reverse cholesterol transport (RCT). The effect of LCAT on human atherogenesis is still controversial. In the present study, the plasma LCAT concentration was measured in all subjects (n = 540) not on drug treatment at the time of enrollment in the multicenter, longitudinal, observational IMPROVE study. Mean and maximum intima-media thickness (IMT) of the whole carotid tree was measured by B-mode ultrasonography in all subjects. In the entire cohort, LCAT quartiles were not associated with carotid mean and maximum IMT (P for trend 0.95 and 0.18, respectively), also after adjustment for age, gender, HDL-cholesterol (HDL-C), and triglycerides. No association between carotid IMT and LCAT quartiles was observed in men (P=0.30 and P=0.99 for mean and maximum IMT, respectively), whereas carotid IMT increased with LCAT quartiles in women (P for trend 0.14 and 0.019 for mean and maximum IMT, respectively). The present findings support the concept that LCAT is not required for an efficient reverse cholesterol transport and that a low plasma LCAT concentration and activity is not associated with increased atherosclerosis.     

Decreased activity of lecithin:cholesterol acyltransferase and hepatic lipase in chronic hypothyroid rats: Implications for reverse cholesterol transport

Chronic hypothyroidism is frequently associated with atherosclerosis due to increased cholesterol plasma levels; nevertheless, the contribution of impaired reverse cholesterol transport (RCT) in this process has not been completely elucidated. The aim of this study was to evaluate the effect of thyroidectomy (Htx) upon the main stages of RCT in rats. Plasma lipid alterations induced by thyroidectomy showed a slight, but significant, reduction of total plasma triglycerides, a 300% increase of LDL-cholesterol and a 25% decrease in HDL-cholesterol compared to control rats. We evaluated the first stage of RCT determining ₃H-cholesterol efflux in Fu5AH cells. The capacity of HDL obtained from Htx rats to promote cholesterol efflux was similar to that of controls. Lecithin:cholesterol acyltransferase (LCAT) activity, the second stage and the driving force of RCT was 30% lower in Htx animals compared to controls, as determined by reconstituted HDL used as an external substrate. Lipoproteins are remodeled by hepatic lipase; the mean activity of this enzyme in postheparin plasma of Htx animals was reduced by 30% compared to controls, thus suggesting an impaired HDL remodeling by this enzyme in the hypothyroid status. In contrast, lipoprotein lipase activity in the Htx group was unchanged. In summary, this study demonstrates that chronic hypothyroidism in the rat induced an impaired RCT mainly at the cholesterol esterification, and HDL remodeling mediated by hepatic lipase. The latter probably results in an abnormal HDL structure, i.e. phospholipid enrichment, which contributes to decrease HDL-apo AI fractional catabolic rates.     

Progression of chronic kidney disease in familial LCAT deficiency: a follow-up of the Italian cohort

Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.     

A robust all-atom model for LCAT generated by homology modeling

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A₂ (PLA₂) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA₂. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.     

In vitro modification of the chemical composition of human plasma low density lipoproteins: effects of morphology and thermal properties

The effects of enzymatic action on human low density lipoproteins (LDL) occurring during in vitro incubation of plasma have been studied by chemical analysis, analytical ultracentrifugation, negative stain electron microscopy and X-ray small angle scattering. Chemically, the action of cholesteryl ester exchange and transfer proteins(s) (CEPT) leads to a relative increase in trigylcerides at the expense of cholesteryl esters. Morphologically, the particles maintain their characteristic features detectable by X-ray small angel scattering. Additional action of lecithin/cholesterol acyl transferase (LCAT) causes mainly a decrease in polar lipid contents and a reduction in particle size. The associated changes in the thermotropic transition were found to be strongly correlated to the triglyceride/cholesteryl ester ratio.     

Paraoxonase 1 overexpression in mice and its effect on high-density lipoproteins.

Paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme believed to protect against the early events of atherogenesis by its ability to hydrolyze oxidized phospholipids. A transgenic mouse overexpressing PON1 (mPON1) was developed to address the question of whether overexpression of PON1 is important in protecting HDL function during oxidative stress. Transgenic mice were obtained that have up to a 5-fold increase in mPON1 activity measured as arylesterase activity [52.7 +/- 17.3 U/ml versus 251.7 +/- 25.1 U/ml for wild-type (WT) and mPON1 high expressers, respectively]; this increase in mPON1 activity was reflected by a 5.3-fold increase in relative mass of the enzyme. Excess mPON1 was associated solely with HDL but did not alter HDL composition, size, or charge. Lecithin:cholesterol acyltransferase (LCAT) on HDL is a sensitive indicator of oxidative stress; exposure of plasmas from both WT and mPON1 overexpresser mice to 0.4 mM copper ions for 2 h showed a 30-40% protection of LCAT activity in mPON1 overexpressers compared to WT. Excess mPON1 also inhibited lipid hydroperoxide formation on HDL. These data strongly suggest that overexpression of mPON1 protects HDL integrity and function.     

Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs

Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.     

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse^TM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.     

Lecithin:cholesterol acyltransferase: old friend or foe in atherosclerosis?

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme that catalyzes the esterification of free cholesterol in plasma lipoproteins and plays a critical role in high-density lipoprotein (HDL) metabolism. Deficiency leads to accumulation of nascent preβ-HDL due to impaired maturation of HDL particles, whereas enhanced expression is associated with the formation of large, apoE-rich HDL(1) particles. In addition to its function in HDL metabolism, LCAT was believed to be an important driving force behind macrophage reverse cholesterol transport (RCT) and, therefore, has been a subject of great interest in cardiovascular research since its discovery in 1962. Although half a century has passed, the importance of LCAT for atheroprotection is still under intense debate. This review provides a comprehensive overview of the insights that have been gained in the past 50 years on the biochemistry of LCAT, the role of LCAT in lipoprotein metabolism and the pathogenesis of atherosclerosis in animal models, and its impact on cardiovascular disease in humans.     

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.     

ACAT2 contributes cholesteryl esters to newly secreted VLDL, whereas LCAT adds cholesteryl ester to LDL in mice

The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 +/- 2.1% vs. 3.9 +/- 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 +/- 3.2% vs. 66.7 +/- 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 +/- 4.0% vs. 54.6 +/- 1.9%) and significantly increased the phospholipid percentage (11.2 +/- 0.9% vs. 19.3 +/- 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 +/- 0.8% in controls to 0 +/- 0.7% in ACAT2-deficient mice. In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.     

Plasma lecithin:cholesterol acyltransferase activity modifies the inverse relationship of C-reactive protein with HDL cholesterol in nondiabetic men

Lecithin:cholesterol acyltransferase (LCAT) is instrumental in high-density lipoprotein (HDL) maturation, but high LCAT levels do not predict low cardiovascular risk. LCAT may affect antioxidative or anti-inflammatory properties of HDL. We determined the relationship of plasma high-sensitivity C-reactive protein (CRP) with LCAT activity and evaluated whether LCAT activity modifies the decreasing effect of HDL cholesterol (HDL-C) on CRP, as an estimate of its anti-inflammatory properties. Plasma HDL-C, apolipoprotein (apo) A-I and LCAT activity (exogenous substrate method) were measured in 260 nondiabetic men without cardiovascular disease. CRP was correlated inversely with HDL-C and apo A-I, and positively with LCAT activity (P < 0.01 to 0.001). Multivariate regression analysis demonstrated that age- and smoking-adjusted plasma CRP levels were associated negatively with HDL-C (β = − 0.224, P < 0.001) and positively with LCAT activity (β = 0.119, P = 0.034), as well as with the interaction between HDL-C and LCAT activity (β = 0.123, P = 0.026). There was also an interaction between apo A-I and LCAT activity on CRP (β = 0.159, P = 0.005). These relationships remained similar after adjustment for apo B-containing lipoproteins. In conclusion, the inverse relationship of HDL-C with CRP is attenuated by LCAT activity at higher HDL-C levels. It is hypothesized that LCAT could mitigate HDL's anti-inflammatory or antioxidative properties at higher HDL-C concentrations.     

遗传性LCAT缺陷症与动脉粥样硬化

卵磷脂：胆固醇酰基转移酶（lecithin：cholesterol acyltransferase,LCAT）参与胆固醇酯的合成并在高密度脂蛋白（high density lipoprotein,HDL）的代谢中起重要作用。遗传性LCAT缺陷症是一种以低HDL-胆固醇（HDL-C）为特点的罕见遗传疾病。近年来,LCAT在HDL-C代谢中以及在动脉粥样硬化发生和发展中的作用逐渐被本领域研究者所关注。本文就LCAT缺陷症的遗传学和生化学特点做一综述,重点阐述为何尽管HDL-C水平明显减低,LCAT突变携带者却并未发生早期动脉粥样硬化。     

Analysis of familial hypoalphalipoproteinemia syndromes

Familial hypoalphalipoproteinemias (HA) are a heterogenous group of disorders characterized by various degrees of HDL deficiency. Differential diagnosis involves clinical and biochemical evaluation after intervention designed to correct known secondary causes of low HDL. Two specific HAs are discussed in this report: 1. primary isolated HA (PIHA) is a poorly characterized entity with an apparent autosomal dominant transmission and distinct abnormalities in the structure and function of HDL. 2. Lecithin: cholesterol acyltransferase (LCAT) deficiency syndromes are caused by a number of different genetic defects that lead to at least two distinct clinical presentations i.e. familial LCAT deficiency and fish eye disease. PIHA is an example of a genetic disorder whose diagnosis would greatly be improved by the availability of molecular diagnostic tests. Conversely, the effect of the genetic heterogeneity of LCAT deficiency syndromes on diagnosis is best overcome by utilizing existing biochemical measurement of LCAT activity and the plasma cholesterol esterification rate.     

Cholesterol efflux by acute-phase high density lipoprotein: role of lecithin: cholesterol acyltransferase

HDL plays an initial role in reverse cholesterol transport by mediating cholesterol removal from cells. During infection and inflammation, several changes in HDL composition occur that may affect the function of HDL; therefore, we determined the ability of acute-phase HDL to promote cholesterol removal from cells. Acute-phase HDL was isolated from plasma of Syrian hamsters injected with lipopolysaccharide. Cholesterol removal from J 774 murine macrophages by acute-phase HDL was less efficient than that by control HDL because of both a decrease in cholesterol efflux and an increase in cholesterol influx. LCAT activity of acute-phase HDL was significantly lower than that of control HDL. When LCAT activity of control HDL was inactivated, cholesterol efflux decreased and cholesterol influx increased to the level observed in acute-phase HDL. Inactivation of LCAT had little effect on acute-phase HDL. In GM 3468A human fibroblasts, the ability of acute-phase HDL to remove cholesterol from cells was also lower than that of normal HDL. The impaired cholesterol removal, however, was primarily a result of an increase in cholesterol influx without changes in cholesterol efflux. When control HDL in which LCAT had been inactivated was incubated with fibroblasts, cholesterol influx increased to a level comparable to that of acute-phase HDL, without any change in cholesterol efflux. These results suggest that the ability of acute-phase HDL to mediate cholesterol removal was impaired compared with that of control HDL and the lower LCAT activity in acute-phase HDL may be responsible for this impairment. The decreased ability of acute-phase HDL to remove cholesterol from cells may be one of the mechanisms that account for the well-known relationship between infection/inflammation and atherosclerosis.