Compromised LCAT Function Is Associated With Increased Atherosclerosis
http://www.100md.com
《循环学杂志》
the Departments of Vascular Medicine (G.K.H., A.G.H., W.P., P.R., E.d.G., J.J.P.K., J.A.K.) and Clinical Epidemiology and Biostatistics (B.A.H., A.H.Z.), Academic Medical Center, Amsterdam, the Netherlands
Department of Medicine (A.S.), University Medical Centre Nijmegen, Nijmegen, the Netherlands.
Abstract
Background— Prospective epidemiological studies have shown that low plasma levels of HDL cholesterol (HDL-C) are associated with an increased risk for cardiovascular disease (CVD). Despite nearly 40 years of research, however, it is unclear whether this also holds true for individuals with severely reduced levels of HDL-C due to mutations in the lecithin:cholesterol acyltransferase (LCAT) gene. Better insight into CVD risk in these individuals may provide clues toward the potential of LCAT as a pharmaceutical target to raise HDL-C levels.
Methods and Results— Lipids, lipoproteins, high-sensitivity C-reactive protein (CRP), and carotid artery intima-media thickness (IMT) were assessed in 47 heterozygotes for LCAT gene mutations and 58 family controls. Compared with controls, heterozygotes presented with a mean 36% decrease in HDL-C levels (P<0.0001), a 23% increase in triglyceride levels (P<0.0001), and a 2.1-fold increase in CRP levels (P<0.0001). Mean carotid IMT was significantly increased in heterozygotes compared with family controls (0.623±0.13 versus 0.591±0.08 mm). After adjustment for age, gender, and alcohol use, this difference proved statistically significant (P<0.0015).
Conclusions— The data show that heterozygosity for LCAT gene defects is associated with low HDL-C levels and elevated concentration of triglycerides and CRP in plasma. This phenotype underlies increased IMT in carriers versus controls, which suggests that LCAT protects against atherosclerosis. This in turn indicates that targeting LCAT to raise HDL-C may reduce CVD risk.
Key Words: lipoproteins ; atherosclerosis ; cardiovascular diseases ; cholesterol ; genetics
Introduction
Despite the widespread use of pharmacological agents that can effectively decrease LDL cholesterol (LDL-C), the vast majority of patients at increased risk for atherosclerosis continue to have cardiovascular disease (CVD). This underscores the need for additional therapeutic strategies. In this respect, drugs that increase HDL cholesterol (HDL-C) hold promise. In addition to its pivotal role in reverse cholesterol transport, HDL also exerts antithrombotic, antioxidant, and antiinflammatory properties,1,2 which illustrates that increasing HDL-C levels may result in various beneficial effects.
Hereditary disorders of human HDL metabolism, such as deficiencies of apolipoprotein (apo) A-I, ATP binding cassette A1 (ABCA1), lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP) have been crucial to our understanding of the reverse cholesterol transport pathway. Consequently, they have proven important in the identification of therapeutic targets to increase HDL-C. This is exemplified by the recent materialization of inhibitors of CETP that exhibit strong HDL-C–raising potential in clinical trials.3,4 Also, increasing the function of ABCA1, a transmembrane protein that promotes the efflux of cholesterol and phospholipids, is thought to reduce CVD risk. This assumption is among others that are based on the finding that mutations in the ABCA1 gene are associated with increased risk for atherosclerosis in humans.5 This has led to the search for molecules that selectively enhance ABCA1 function.6 HDL deficiency and increased risk of coronary artery disease have also been described in families with apoA-I mutations.7,8 Supported by convincing epidemiological evidence of the protective role of apoA-I, this has further established this protein as a promising target to reduce coronary artery disease risk. This knowledge has recently prompted the use of infusion of apoA-I–containing phospholipid complexes in patients with acute coronary syndrome, which was shown to induce regression of coronary atheroma.9
LCAT deficiency represents another rare, recessive genetic disorder that underlies HDL deficiency. LCAT is a plasma enzyme that esterifies free cholesterol, primarily at the surface of the HDL particle, after which the cholesteryl ester molecules migrate to the inner core of this lipoprotein. Through this action, LCAT plays a key role in the maturation of HDL particles.10 Norum and Gjone11 first described that LCAT gene mutations underlie familial LCAT deficiency. Later, it was recognized that a less severe clinical phenotype, fish-eye disease, results from mutations in this very gene.12 Familial LCAT deficiency and fish-eye disease are both characterized by HDL deficiency (5% to 10% of normal HDL-C levels). (For a review of the effect of LCAT mutations on lipid metabolism, see Pritchard and Hill.13)
Although LCAT has thus long been known to play an important role in HDL metabolism, its association with atherosclerosis has remained elusive. This is mainly because of the very limited numbers of carriers of LCAT gene mutations. In addition, animal data have provided conflicting results.14,15 We therefore set out to study the relationships between LCAT and atherosclerosis using B-mode ultrasound intima-media thickness (IMT) measurements of the carotid arteries as a validated marker for CVD risk and atherosclerosis in large cohort of LCAT mutation carriers.
Methods
Study Groups
Over the past 14 years, our laboratory has, in collaboration with the laboratory of Dr P.H. Pritchard, characterized 5 families of Dutch Caucasian descent with LCAT deficiency disorders. Families 1,16 2,17,18 and 319 have been described previously. The probands of these 3 families experienced corneal clouding and consulted ophthalmologists before being referred to our Lipid Clinic. More recently, we identified 2 additional probands with LCAT deficiency disorders (families 4 and 5, respectively), but these data were not published. Briefly, the proband of family 4 male, aged 68 years, was referred to our Lipid Clinic for low HDL-C levels. Upon physical examination, corneal opacities were noted, whereas laboratory tests showed an HDL-C level of 0.15 mmol/L. LCAT activity was assessed by using a proteoliposome substrate20 and was severely reduced, which was related to compound heterozygosity for 2 missense mutations (underlying P10Q and V309M, respectively). The proband of family 5, a 20-year-old woman, was referred to our Lipid Clinic for corneal clouding. She had HDL deficiency (HDL-C 0.09 mmol/L), and her plasma LCAT activity was severely reduced due to compound heterozygosity for point mutations that caused amino acid substitutions at positions 123 (T123I) and 309 (V309M) of the mature LCAT protein.
For the present cross-sectional analysis, the probands and their family members of the aforementioned 5 families were invited to participate in an IMT study irrespective of cardiovascular or genetic status; no inclusion or exclusion criteria were used. The recruitment of study individuals was performed as follows: we contacted the index patients of the families who were previously found to be homozygous or compound heterozygous for LCAT gene mutations. Using these individuals as central spokespersons, family members were invited to participate by our physicians and genetic field workers (sometimes through the organization of family reunions). This was done through a letter and/or by telephone. Family members included both first- and second-degree relatives and those who had married into the family.
All LCAT gene defects under study have previously been shown to underlie marked loss of LCAT activity (30% to 42% compared with family controls), which has as its direct consequence a 30% to 38% reduction of HDL-C levels.16,19,21 We therefore only genotyped the participants (using previously described methods16,18,19,21) to distinguish between carriers and noncarriers. Heterozygotes for LCAT gene mutations do not have clinical signs or complaints despite having these marked reductions of HDL-C levels. Thus, importantly, there existed no clinical recruitment bias for the heterozygotes. From families 1, 2, 3, 4, and 5, we recruited 3, 2, 30, 6, and 6 heterozygotes and 6, 0, 45, 3, and 4 controls, respectively.
Nine homozygotes or compound heterozygotes, characterized by near complete HDL-C deficiency, also underwent IMT measurements (average IMT was 0.73±02 mm). However, the low number and the completely different age distribution (60.9±15.6 versus 41.9±16.1 and 42.2±17.4 years in controls and heterozygotes, respectively) unfortunately could not provide the required statistical foundation for solid conclusions with regard to the impact of LCAT on atherosclerosis. The data from this group were therefore not used for further analysis.
Past medical history, presence of cardiovascular risk factors, and use of medication were assessed by questionnaire. The data on clinical events were verified by studying the clinical records. Informed consent was obtained for plasma sampling, storage, genetic analysis, and IMT measurements. The study was approved by the Ethics Committee of the Academic Medical Center in Amsterdam.
Blood Analyses
Blood was collected in EDTA-coated tubes after overnight fasting. Total cholesterol, triglycerides, and HDL-C were measured by established methods, and LDL-C was calculated by the equation of Friedewald.22 Plasma apoA-I and apoB were measured with the Nephelometric BNll System (Dade Behring).23,24 High-sensitivity C-reactive protein (hs-CRP) levels were measured by the Dade Behring method. LCAT activity measurements were performed with an exogenous apoA-I proteoliposome substrate as described previously.20
Carotid Artery IMT and CVD
IMT measurements were performed in a standardized fashion for both the carriers of LCAT gene mutations and the controls, as described previously.25 Briefly, an Acuson 128XP/10v (Acuson Corporation) equipped with a 7.0-MHz linear-array transducer was used to obtain B-mode ultrasound images. The following wall segments were bilaterally scanned over a length of 10 mm: the common carotid artery, the carotid bulb, and the internal carotid artery. Images were saved as JPEG image files, and a reader, blinded for the genetic status of the patient, measured the IMT of the far wall of the respective segments. The mean combined outcome of the 6 segments was used for analysis.
A vascular event was defined by the presence of at least 1 of the following: acute myocardial infarction, percutaneous coronary intervention, coronary artery bypass grafting, and angina pectoris, as well as by the presence of peripheral or cerebrovascular disease. Cardiovascular events were ascertained by use of questionnaires and verified by studying the clinical records.
Statistical Analysis
Results are expressed as means (SD), except for triglycerides, lipoprotein(a), and hs-CRP levels, which are expressed as medians (interquartile range) because of a skewed distribution. These variables were log-transformed before statistical analysis. Differences in terms of demographic and lifestyle characteristics between heterozygotes for LCAT gene mutations and family controls were evaluated with linear or logistic regression analyses with generalized estimating equations in the SAS procedure GENMOD to account for correlations within families. For differences in blood pressure, smoking, and alcohol use, we made adjustments in a multivariate model for age and gender.
To evaluate differences between the 2 groups in biochemical characteristics and mean carotid IMT, we used the same SAS procedure, allowing for clustering within families (due to clustering of genetic and/or environmental factors). For the biochemical characteristics, we adjusted for age, gender and smoking using multivariate models. For mean carotid IMT, the main outcome of this study, a more elaborate procedure was used. We first explored univariately the relation between mean carotid IMT and baseline variables. Hereafter, using multivariate models, we identified independent predictors after stepwise backward selection. For all generalized estimating equation models, the exchangeable correlation structure was used. The difference between controls and LCAT heterozygotes was tested by assessing their interaction (age and group). Probability values <0.05 were considered significant. For statistical analyses the SAS package (release 8.02; SAS Institute Inc) was used.
Results
Genetic and Demographic Characteristics of the Study Groups
A total of 47 heterozygotes for LCAT gene mutations and 58 controls (ascertained by genotyping) were recruited from 5 families of Dutch descent originating from different parts of The Netherlands. Mean average age was nearly identical among the heterozygotes and controls (42.2 versus 41.9 years of age, respectively; Table 1). Males were slightly more prevalent among the heterozygotes (62%) than among controls (47%), but this did not reach statistical significance (P=0.062). Systolic and diastolic blood pressure and alcohol use did not differ among the groups. The percentage of smokers, however, was significantly higher in the heterozygotes than in the controls (21% versus 14%; P=0.015). One heterozygous carrier and 2 subjects in the control group had been prescribed a statin. No patient had diabetes mellitus, defined as use of oral antidiabetic drugs or insulin.
The data on LCAT activity levels represent data from previous publications.16,18,19,21 In these reports, the LCAT mutations in the respective families have been shown to result in 30% to 42% reductions in LCAT activity, with concomitant 30% to 38% reductions in HDL-C levels in the heterozygous carriers compared with family controls.
Lipids, (Apo)lipoproteins, and C-Reactive Protein Levels
IMT and Cardiovascular Events
Mean IMT values were 0.591±0.08 mm for controls and 0.623±0.13 mm for heterozygotes (Table 3). After adjustment for age, gender, use of alcohol, and family, this difference in average IMT proved to be highly statistically significant (P<0.0015). This probability value was obtained with multivariate backward stepwise regression analysis, whereby age, gender, and use of alcohol remained in the model but smoking and blood pressure did not. Although the family controls did not experience cardiovascular events, 1 male heterozygote had angina pectoris at age 50 years and a myocardial infarction at age 54 years, and a second heterozygote had a myocardial infarction at age 58 years.
Discussion
It has long been difficult to assess the risk of atherosclerosis in individuals with genetically determined low HDL-C levels. This knowledge, however, can provide important insight when it comes to targeting HDL metabolism to reduce CVD risk. After almost 40 years of LCAT research, the present study provides evidence that heterozygotes for LCAT gene defects, who present with an average 36% decrease in HDL-C levels, exhibit an increased risk for atherosclerosis as assessed by IMT measurements. These data suggest that intact LCAT function is important in the protection against atherosclerotic vascular disease.
LCAT, Lipids, and Lipoproteins
The present analysis confirms the strong impact of LCAT gene mutations on HDL-C levels. All LCAT gene defects under study have previously been shown to underlie marked loss of LCAT activity (30% to 42% compared with family controls), and as a direct consequence, to result in 30% to 38% reductions in HDL-C levels16,19,21 (for a review, see Pritchard and Hill13). LCAT is primarily active at the phospholipid monolayer of nascent HDL particles, where it converts cholesterol into cholesteryl ester on activation by its cofactor, apoA-I. Even a single change in the amino acid sequence of LCAT has been shown to result in a decreased interaction with apoA-I, with a consequential decrease in LCAT activity26 This will, in turn, cause defective maturation of small HDL into larger, spherical, cholesteryl ester–enriched HDL and hence lower HDL-C levels. The data show that this effect is not counteracted by upregulation of the unaffected LCAT allele in heterozygotes or by other key players in HDL metabolism. Additionally, the present study shows the effect of LCAT mutations on plasma triglycerides levels, as has been reported previously.20,27 Although heterozygotes presented with a moderate 22% increase in triglyceride levels, homozygotes presented with a marked 337% increase in triglyceride levels (data not shown). Mild hypertriglyceridemia is also observed in other genetic HDL deficiencies.28,29 For LCAT, hepatic triglyceride overproduction and reduced lipoprotein lipase activity, as found in LCAT-deficient mice, may explain this phenotype.30 Finally, we identified a small but significant increase in LDL-C levels compared with controls, for which we have no explanation.
LCAT and Atherosclerosis
Despite the fact that LCAT has long been recognized as a key regulator of HDL metabolism, the role of this enzyme in human atherogenesis has remained controversial. Prospective studies that have assessed LCAT activity or LCAT concentration at baseline are nonexistent, whereas cross-sectional observational studies have reported increased and decreased LCAT activity in subjects with CVD.31,32 Initially, the paradoxical finding of complete HDL deficiency and the reported absence of CVD in LCAT-deficient patients has been used to reject the hypothesis that HDL is important in the protection against atherosclerosis. Potential mechanisms to explain these findings were subsequently postulated, such as preferential clearance of HDL fractions that have less atherogenic potential.33 In addition, decreased LDL-C and apoB levels have also been put forward as an explanation for the lack of marked CVD. In the large group of LCAT mutation carriers in the present study, however, LDL-C was clearly not decreased, which refutes this argument.
Studies focusing on the role of LCAT in human atherosclerosis have thus far been hampered by the paucity of clinical events in small numbers of individuals with LCAT mutations. The advent of validated surrogate markers for atherosclerosis has proved very useful in this context. IMT measurements have been used to show that carriers of ABCA1 and apoA-I defects are at increased risk for coronary artery disease,8,34,35 which illustrates the power of this tool to study small, interesting groups of patients. The present study now shows that heterozygotes for LCAT mutations also have increased atherosclerosis compared with family controls. This finding was not based on a few heterozygous outliers with a very thick carotid intima-media complex but on solid IMT data. This is illustrated by the fact that the 30 heterozygotes of the largest family (family 3) presented with a thicker IMT than their 45 family control members (0.614 versus 0.589 mm). This effect, however, did not reach statistical significance, which illustrates the need to recruit and study more families with the same rare genetic disorder to answer our primary research question. During the preparation of this report, Ayyobi et al36 also reported vascular abnormalities in a small cohort of 9 heterozygotes for 1 specific LCAT mutation. The investigators could not, however, study family controls, and because of the small number of study subjects, these investigators could not draw conclusions regarding the risk for atherosclerosis.36
A potential but dangerous drawback of studying atherosclerosis in relatively small cohorts, such as investigated here, is the referral basis of the index patients. In this respect, we would like to emphasize that the probands of the currently studied families came to our attention because they experienced corneal opacifications, or they were referred because of previously identified low HDL-C levels. This excludes a bias for selecting those families with LCAT mutations with an increased or established risk for CVD. Exactly how reduced LCAT function affects IMT, however, cannot be deduced from the present study. LCAT mutations may directly compromise reverse cholesterol transport by reducing the flux of cholesterol from cholesterol-loaded peripheral macrophages to the HDL fraction. The markedly reduced HDL-C levels in carriers also may affect endothelial function directly.37 Alternatively, loss of LCAT activity may cause enhanced oxidation of LDL.38
LCAT and Inflammation
Elevated levels of CRP are an established predictor for coronary artery disease,39 but there is much debate about whether this plasma factor plays an active role in atherosclerosis or whether it merely represents an innocent bystander. We were surprised to find that hs-CRP levels were 2.2-fold increased in the heterozygotes. From the present study, however, it can not be appreciated whether increased levels of CRP are a direct consequence of reduced LCAT activity or of the consequentially reduced HDL-C levels. Pirro and colleagues40 also reported significantly higher CRP levels in other subjects with hypoalphalipoproteinemia, which suggests a proinflammatory status in subjects with low HDL-C levels per se.
Concluding Remarks
Upregulation of LCAT function has been proposed as an HDL-C increasing therapy, but its atheroprotective effects have been questioned.41 The present study suggests that increasing LCAT activity may reduce atherosclerosis progression, at least in subjects with subnormal levels of LCAT. Efforts to develop LCAT protein therapy have failed, but LCAT gene therapy protocols are more promising, as recently illustrated by decreased atherosclerosis in dyslipidemic obese mice42 after adenovirus-mediated transfer of the LCAT gene. Also, Zhang et al43 recently showed that a similar strategy increased reverse cholesterol transport in hamsters but, interestingly, not in mice. The present report indicates that increasing LCAT activity in plasma might be speculated to result not only in increased HDL-C levels but also in decreased concentrations of triglycerides and hs-CRP. Moreover, increased LCAT activity might also result in an augmented capacity of HDL to exhibit antioxidative, antiinflammatory,44 and antithrombotic effects.
Acknowledgments
We thank all participants of this study for their cooperation. Moreover, we thank Joris Butzelaar for his work as a reader of the IMT files, and Kobie Los, who drew blood samples from the participants.
References
Nofer JR, Kehrel B, Fobker M, Levkau B, Assmann G, von Eckardstein A. HDL and arteriosclerosis: beyond reverse cholesterol transport. Atherosclerosis. 2002; 161: 1–16.
Stein O, Stein Y. Atheroprotective mechanisms of HDL. Atherosclerosis. 1999; 144: 285–301.
Brousseau ME, Schaefer EJ, Wolfe ML, Bloedon LT, Digenio AG, Clark RW, Mancuso JP, Rader DJ. Effects of an inhibitor of cholesteryl ester transfer protein on HDL cholesterol. N Engl J Med. 2004; 350: 1505–1515.
de Grooth GJ, Kuivenhoven JA, Stalenhoef AF, de Graaf J, Zwinderman AH, Posma JL, van Tol A, Kastelein JJP. Efficacy and safety of a novel cholesteryl ester transfer protein inhibitor, JTT-705, in humans: a randomized phase II dose-response study. Circulation. 2002; 105: 2159–2165.
Singaraja RR, Brunham LR, Visscher H, Kastelein JJ, Hayden MR. Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene. Arterioscler Thromb Vasc Biol. 2003; 23: 1322–1332.
Sparrow CP, Baffic J, Lam MH, Lund EG, Adams AD, Fu X, Hayes N, Jones AB, Macnaul KL, Ondeyka J, Singh S, Wang J, Zhou G, Moller DE, Wright SD, Menke JG. A potent synthetic LXR agonist is more effective than cholesterol loading at inducing ABCA1 mRNA and stimulating cholesterol efflux. J Biol Chem. 2002; 277: 10021–10027.
Ikewaki K, Matsunaga A, Han H, Watanabe H, Endo A, Tohyama J, Kuno M, Mogi J, Sugimoto K, Tada N, Sasaki J, Mochizuki S. A novel two nucleotide deletion in the apolipoprotein A-I gene, apoA-I Shinbashi, associated with high density lipoprotein deficiency, corneal opacities, planar xanthomas, and premature coronary artery disease. Atherosclerosis. 2004; 172: 39–45.
Hovingh GK, Brownlie A, Bisoendial RJ, Dube MP, Levels JHM, Petersen W, Dullaart RPF, Stroes ESG, Zwinderman AH, de Grooth E, Hayden MR, Kuivenhoven JA, Kastelein JJP. A novel ApoA-I mutation (L178P) leads to endothelial dysfunction, increased arterial wall thickness and premature coronary artery disease. J Am Coll Cardiol. 2004; 44: 1429–1435.
Nissen SE, Tsunoda T, Tuzcu EM, Schoenhagen P, Cooper CJ, Yasin M, Eaton GM, Lauer MA, Sheldon WS, Grines CL, Halpern S, Crow T, Blankenship JC, Kerensky R. Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA. 2003; 290: 2292–2300.
Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res. 1968; 9: 155–167.
Norum KR, Gjone E. Familial serum-cholesterol esterification failure: a new inborn error of metabolism. Biochim Biophys Acta. 1967; 144: 698–700.
Carlson LA, Philipson B. Fish-eye disease: a new familial condition with massive corneal opacities and dyslipoproteinaemia. Lancet. 1979; 2: 922–924.
Pritchard PH, Hill JS. Genetic disorders of lecithin:cholesterol acyltransferase. In: Betteridge DJ, Illingworth DR, Shepherd J, eds. Lipoproteins in Health and Disease. 1st ed. London, UK: Arnold; 1999: 799–814.
Furbee JW Jr, Parks JS. Transgenic overexpression of human lecithin:cholesterol acyltransferase (LCAT) in mice does not increase aortic cholesterol deposition. Atherosclerosis. 2002; 165: 89–100.
Santamarina-Fojo S, Lambert G, Hoeg JM, Brewer HB Jr. Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis. Curr Opin Lipidol. 2000; 11: 267–275.
Kuivenhoven JA, Wiebusch H, Pritchard PH, Funke H, Benne R, Assmann G, Kastelein JJP. An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease). J Clin Invest. 1996; 98: 358–364.
Funke H, von Eckardstein A, Pritchard PH, Albers JJ, Kastelein JJ, Droste C, Assmann G. A molecular defect causing fish eye disease: an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of alpha-LCAT activity. Proc Natl Acad Sci U S A. 1991; 88: 4855–4859.
Kastelein JJ, Pritchard PH, Erkelens DW, Kuivenhoven JA, Albers JJ, Frohlich JJ. Familial high-density-lipoprotein deficiency causing corneal opacities (fish eye disease) in a family of Dutch descent. J Intern Med. 1992; 231: 413–419.
Kuivenhoven JA, van Voorst tot Voorst EJ, Wiebusch H, Marcovina SM, Funke H, Assmann G, Pritchard PH, Kastelein JJP. A unique genetic and biochemical presentation of fish-eye disease. J Clin Invest. 1995; 96: 2783–2791.
Frohlich J, McLeod R, Pritchard PH, Fesmire J, McConathy W. Plasma lipoprotein abnormalities in heterozygotes for familial lecithin:cholesterol acyltransferase deficiency. Metabolism. 1988; 37: 3–8.
Kuivenhoven JA, Stalenhoef AF, Hill JS, Demacker PN, Errami A, Kastelein JJ, Pritchard PH. Two novel molecular defects in the LCAT gene are associated with fish eye disease. Arterioscler Thromb Vasc Biol. 1996; 16: 294–303.
Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972; 18: 499–502.
Pruvot I, Fievet C, Durieux C, Vu DN, Fruchart JC. Electroimmuno- and immunonephelometric assays of apolipoprotein A-I by using a mixture of monoclonal antibodies. Clin Chem. 1988; 34: 2048–2052.
Heuck CC, Schlierf G. Nephelometry of apolipoprotein B in human serum. Clin Chem. 1979; 25: 221–226.
de Groot E, Jukema JW, Montauban van Swijndregt AD, Zwinderman AH, Ackerstaff RG, van der Steen AF, Bom N, Lie KI, Bruschke AV. B-mode ultrasound assessment of pravastatin treatment effect on carotid and femoral artery walls and its correlations with coronary arteriographic findings: a report of the Regression Growth Evaluation Statin Study (REGRESS). J Am Coll Cardiol. 1998; 31: 1561–1567.
Peelman F, Vandekerckhove J, Rosseneu M. Structure and function of lecithin cholesterol acyl transferase: new insights from structural predictions and animal models. Curr Opin Lipidol. 2000; 11: 155–160.
Miettinen HE, Gylling H, Tenhunen J, Virtamo J, Jauhiainen M, Huttunen JK, Kantola I, Miettinen TA, Kontula K. Molecular genetic study of Finns with hypoalphalipoproteinemia and hyperalphalipoproteinemia: a novel Gly230 Arg mutation (LCAT[Fin]) of lecithin:cholesterol acyltransferase (LCAT) accounts for 5% of cases with very low serum HDL cholesterol levels. Arterioscler Thromb Vasc Biol. 1998; 18: 591–598.
Wang CS, Alaupovic P, Gregg RE, Brewer HB Jr. Studies on the mechanism of hypertriglyceridemia in Tangier disease: determination of plasma lipolytic activities, k1 values and apolipoprotein composition of the major lipoprotein density classes. Biochim Biophys Acta. 1987; 920: 9–19.
Ng DS, Leiter LA, Vezina C, Connelly PW, Hegele RA. Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia. J Clin Invest. 1994; 93: 223–229.
Ng DS, Xie C, Maguire GF, Zhu X, Ugwu F, Lam E, Connelly PW. Hypertriglyceridemia in lecithin-cholesterol acyltransferase-deficient mice is associated with hepatic overproduction of triglycerides, increased lipogenesis, and improved glucose tolerance. J Biol Chem. 2004; 279: 7636–7642.
Solajic-Bozicevic N, Stavljenic A, Sesto M. Lecithin:cholesterol acyltransferase activity in patients with acute myocardial infarction and coronary heart disease. Artery. 1991; 18: 326–340.
Wells IC, Peitzmeier G, Vincent JK. Lecithin:cholesterol acyltransferase and lysolecithin in coronary atherosclerosis. Exp Mol Pathol. 1986; 45: 303–310.
Rader DJ, Ikewaki K, Duverger N, Schmidt H, Pritchard H, Frohlich J, Clerc M, Dumon MF, Fairwell T, Zech L. Markedly accelerated catabolism of apolipoprotein A-II (ApoA-II) and high density lipoproteins containing ApoA-II in classic lecithin:cholesterol acyltransferase deficiency and fish-eye disease. J Clin Invest. 1994; 93: 321–330.
van Dam MJ, de Groot E, Clee SM, Hovingh GK, Roelants R, Brooks-Wilson A, Zwinderman AH, Smit AJ, Smelt AH, Groen AK, Hayden MR, Kastelein JJP. Association between increased arterial-wall thickness and impairment in ABCA1-driven cholesterol efflux: an observational study. Lancet. 2002; 359: 37–42.
Hovingh GK, de Groot E, van der Steeg WA, Boekholdt SM, Hutten BA, Kuivenhoven JA, Kastelein JJP. Inherited disorders of HDL metabolism and atherosclerosis. Curr Opin Lipidol. 2005; 16: 139–145.
Ayyobi AF, McGladdery SH, Chan S, Mancin J, Hil JS, Frohlich JJ. Lecithin:cholesterol acyltransferase (LCAT) deficiency and risk of vascular disease: 25 year follow-up. Atherosclerosis. 2004; 177: 361–366.
Bisoendial RJ, Hovingh GK, Levels JH, Lerch PH, Andresen I, Hayden MR, Kastelein JJ, Stroes ESG. Restoration of endothelial function by increasing high-density lipoprotein in subjects with isolated low high-density lipoprotein. Circulation. 2003; 107: 2944–2948.
Vohl MC, Neville TA, Kumarathasan R, Braschi S, Sparks DL. A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation. Biochemistry. 1999; 38: 5976–5981.
Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000; 342: 836–843.
Pirro M, Siepi D, Lupattelli G, Roscini AR, Schillaci G, Gemelli F, Vaudo G, Marchesi S, Pasqualini L, Mannorino E. Plasma C-reactive protein in subjects with hypo/hyperalphalipoproteinemias. Metabolism. 2003; 52: 432–436.
Rader DJ, Maugeais C. Genes influencing HDL metabolism: new perspectives and implications for atherosclerosis prevention. Mol Med Today. 2000; 6: 170–175.
Mertens A, Verhamme P, Bielicki JK, Phillips MC, Quarck R, Verreth W, Stengel D, Ninoi E, Navab M, Mackness B, Mackness M, Holvoet P. Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis. Circulation. 2003; 107: 1640–1646.
Zhang A-H, Gao S, Fan J, Huang W, Zhao T-Q, Liu G. Increased plasma HDL cholesterol levels and biliary cholesterol excretion in hamster by LCAT overexpression. FEBS Lett. 2004; 570: 25–29.
Nicholls SJ, Dusting GJ, Cutri B, Bao S, Drummond GR, Rye KA, Barter PJ. Reconstituted high-density lipoproteins inhibit the acute pro-oxidant and proinflammatory vascular changes induced by a periarterial collar in normocholesterolemic rabbits. Circulation. 2005; 111: 1543–1550.(Compromised LCAT Function)
Department of Medicine (A.S.), University Medical Centre Nijmegen, Nijmegen, the Netherlands.
Abstract
Background— Prospective epidemiological studies have shown that low plasma levels of HDL cholesterol (HDL-C) are associated with an increased risk for cardiovascular disease (CVD). Despite nearly 40 years of research, however, it is unclear whether this also holds true for individuals with severely reduced levels of HDL-C due to mutations in the lecithin:cholesterol acyltransferase (LCAT) gene. Better insight into CVD risk in these individuals may provide clues toward the potential of LCAT as a pharmaceutical target to raise HDL-C levels.
Methods and Results— Lipids, lipoproteins, high-sensitivity C-reactive protein (CRP), and carotid artery intima-media thickness (IMT) were assessed in 47 heterozygotes for LCAT gene mutations and 58 family controls. Compared with controls, heterozygotes presented with a mean 36% decrease in HDL-C levels (P<0.0001), a 23% increase in triglyceride levels (P<0.0001), and a 2.1-fold increase in CRP levels (P<0.0001). Mean carotid IMT was significantly increased in heterozygotes compared with family controls (0.623±0.13 versus 0.591±0.08 mm). After adjustment for age, gender, and alcohol use, this difference proved statistically significant (P<0.0015).
Conclusions— The data show that heterozygosity for LCAT gene defects is associated with low HDL-C levels and elevated concentration of triglycerides and CRP in plasma. This phenotype underlies increased IMT in carriers versus controls, which suggests that LCAT protects against atherosclerosis. This in turn indicates that targeting LCAT to raise HDL-C may reduce CVD risk.
Key Words: lipoproteins ; atherosclerosis ; cardiovascular diseases ; cholesterol ; genetics
Introduction
Despite the widespread use of pharmacological agents that can effectively decrease LDL cholesterol (LDL-C), the vast majority of patients at increased risk for atherosclerosis continue to have cardiovascular disease (CVD). This underscores the need for additional therapeutic strategies. In this respect, drugs that increase HDL cholesterol (HDL-C) hold promise. In addition to its pivotal role in reverse cholesterol transport, HDL also exerts antithrombotic, antioxidant, and antiinflammatory properties,1,2 which illustrates that increasing HDL-C levels may result in various beneficial effects.
Hereditary disorders of human HDL metabolism, such as deficiencies of apolipoprotein (apo) A-I, ATP binding cassette A1 (ABCA1), lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP) have been crucial to our understanding of the reverse cholesterol transport pathway. Consequently, they have proven important in the identification of therapeutic targets to increase HDL-C. This is exemplified by the recent materialization of inhibitors of CETP that exhibit strong HDL-C–raising potential in clinical trials.3,4 Also, increasing the function of ABCA1, a transmembrane protein that promotes the efflux of cholesterol and phospholipids, is thought to reduce CVD risk. This assumption is among others that are based on the finding that mutations in the ABCA1 gene are associated with increased risk for atherosclerosis in humans.5 This has led to the search for molecules that selectively enhance ABCA1 function.6 HDL deficiency and increased risk of coronary artery disease have also been described in families with apoA-I mutations.7,8 Supported by convincing epidemiological evidence of the protective role of apoA-I, this has further established this protein as a promising target to reduce coronary artery disease risk. This knowledge has recently prompted the use of infusion of apoA-I–containing phospholipid complexes in patients with acute coronary syndrome, which was shown to induce regression of coronary atheroma.9
LCAT deficiency represents another rare, recessive genetic disorder that underlies HDL deficiency. LCAT is a plasma enzyme that esterifies free cholesterol, primarily at the surface of the HDL particle, after which the cholesteryl ester molecules migrate to the inner core of this lipoprotein. Through this action, LCAT plays a key role in the maturation of HDL particles.10 Norum and Gjone11 first described that LCAT gene mutations underlie familial LCAT deficiency. Later, it was recognized that a less severe clinical phenotype, fish-eye disease, results from mutations in this very gene.12 Familial LCAT deficiency and fish-eye disease are both characterized by HDL deficiency (5% to 10% of normal HDL-C levels). (For a review of the effect of LCAT mutations on lipid metabolism, see Pritchard and Hill.13)
Although LCAT has thus long been known to play an important role in HDL metabolism, its association with atherosclerosis has remained elusive. This is mainly because of the very limited numbers of carriers of LCAT gene mutations. In addition, animal data have provided conflicting results.14,15 We therefore set out to study the relationships between LCAT and atherosclerosis using B-mode ultrasound intima-media thickness (IMT) measurements of the carotid arteries as a validated marker for CVD risk and atherosclerosis in large cohort of LCAT mutation carriers.
Methods
Study Groups
Over the past 14 years, our laboratory has, in collaboration with the laboratory of Dr P.H. Pritchard, characterized 5 families of Dutch Caucasian descent with LCAT deficiency disorders. Families 1,16 2,17,18 and 319 have been described previously. The probands of these 3 families experienced corneal clouding and consulted ophthalmologists before being referred to our Lipid Clinic. More recently, we identified 2 additional probands with LCAT deficiency disorders (families 4 and 5, respectively), but these data were not published. Briefly, the proband of family 4 male, aged 68 years, was referred to our Lipid Clinic for low HDL-C levels. Upon physical examination, corneal opacities were noted, whereas laboratory tests showed an HDL-C level of 0.15 mmol/L. LCAT activity was assessed by using a proteoliposome substrate20 and was severely reduced, which was related to compound heterozygosity for 2 missense mutations (underlying P10Q and V309M, respectively). The proband of family 5, a 20-year-old woman, was referred to our Lipid Clinic for corneal clouding. She had HDL deficiency (HDL-C 0.09 mmol/L), and her plasma LCAT activity was severely reduced due to compound heterozygosity for point mutations that caused amino acid substitutions at positions 123 (T123I) and 309 (V309M) of the mature LCAT protein.
For the present cross-sectional analysis, the probands and their family members of the aforementioned 5 families were invited to participate in an IMT study irrespective of cardiovascular or genetic status; no inclusion or exclusion criteria were used. The recruitment of study individuals was performed as follows: we contacted the index patients of the families who were previously found to be homozygous or compound heterozygous for LCAT gene mutations. Using these individuals as central spokespersons, family members were invited to participate by our physicians and genetic field workers (sometimes through the organization of family reunions). This was done through a letter and/or by telephone. Family members included both first- and second-degree relatives and those who had married into the family.
All LCAT gene defects under study have previously been shown to underlie marked loss of LCAT activity (30% to 42% compared with family controls), which has as its direct consequence a 30% to 38% reduction of HDL-C levels.16,19,21 We therefore only genotyped the participants (using previously described methods16,18,19,21) to distinguish between carriers and noncarriers. Heterozygotes for LCAT gene mutations do not have clinical signs or complaints despite having these marked reductions of HDL-C levels. Thus, importantly, there existed no clinical recruitment bias for the heterozygotes. From families 1, 2, 3, 4, and 5, we recruited 3, 2, 30, 6, and 6 heterozygotes and 6, 0, 45, 3, and 4 controls, respectively.
Nine homozygotes or compound heterozygotes, characterized by near complete HDL-C deficiency, also underwent IMT measurements (average IMT was 0.73±02 mm). However, the low number and the completely different age distribution (60.9±15.6 versus 41.9±16.1 and 42.2±17.4 years in controls and heterozygotes, respectively) unfortunately could not provide the required statistical foundation for solid conclusions with regard to the impact of LCAT on atherosclerosis. The data from this group were therefore not used for further analysis.
Past medical history, presence of cardiovascular risk factors, and use of medication were assessed by questionnaire. The data on clinical events were verified by studying the clinical records. Informed consent was obtained for plasma sampling, storage, genetic analysis, and IMT measurements. The study was approved by the Ethics Committee of the Academic Medical Center in Amsterdam.
Blood Analyses
Blood was collected in EDTA-coated tubes after overnight fasting. Total cholesterol, triglycerides, and HDL-C were measured by established methods, and LDL-C was calculated by the equation of Friedewald.22 Plasma apoA-I and apoB were measured with the Nephelometric BNll System (Dade Behring).23,24 High-sensitivity C-reactive protein (hs-CRP) levels were measured by the Dade Behring method. LCAT activity measurements were performed with an exogenous apoA-I proteoliposome substrate as described previously.20
Carotid Artery IMT and CVD
IMT measurements were performed in a standardized fashion for both the carriers of LCAT gene mutations and the controls, as described previously.25 Briefly, an Acuson 128XP/10v (Acuson Corporation) equipped with a 7.0-MHz linear-array transducer was used to obtain B-mode ultrasound images. The following wall segments were bilaterally scanned over a length of 10 mm: the common carotid artery, the carotid bulb, and the internal carotid artery. Images were saved as JPEG image files, and a reader, blinded for the genetic status of the patient, measured the IMT of the far wall of the respective segments. The mean combined outcome of the 6 segments was used for analysis.
A vascular event was defined by the presence of at least 1 of the following: acute myocardial infarction, percutaneous coronary intervention, coronary artery bypass grafting, and angina pectoris, as well as by the presence of peripheral or cerebrovascular disease. Cardiovascular events were ascertained by use of questionnaires and verified by studying the clinical records.
Statistical Analysis
Results are expressed as means (SD), except for triglycerides, lipoprotein(a), and hs-CRP levels, which are expressed as medians (interquartile range) because of a skewed distribution. These variables were log-transformed before statistical analysis. Differences in terms of demographic and lifestyle characteristics between heterozygotes for LCAT gene mutations and family controls were evaluated with linear or logistic regression analyses with generalized estimating equations in the SAS procedure GENMOD to account for correlations within families. For differences in blood pressure, smoking, and alcohol use, we made adjustments in a multivariate model for age and gender.
To evaluate differences between the 2 groups in biochemical characteristics and mean carotid IMT, we used the same SAS procedure, allowing for clustering within families (due to clustering of genetic and/or environmental factors). For the biochemical characteristics, we adjusted for age, gender and smoking using multivariate models. For mean carotid IMT, the main outcome of this study, a more elaborate procedure was used. We first explored univariately the relation between mean carotid IMT and baseline variables. Hereafter, using multivariate models, we identified independent predictors after stepwise backward selection. For all generalized estimating equation models, the exchangeable correlation structure was used. The difference between controls and LCAT heterozygotes was tested by assessing their interaction (age and group). Probability values <0.05 were considered significant. For statistical analyses the SAS package (release 8.02; SAS Institute Inc) was used.
Results
Genetic and Demographic Characteristics of the Study Groups
A total of 47 heterozygotes for LCAT gene mutations and 58 controls (ascertained by genotyping) were recruited from 5 families of Dutch descent originating from different parts of The Netherlands. Mean average age was nearly identical among the heterozygotes and controls (42.2 versus 41.9 years of age, respectively; Table 1). Males were slightly more prevalent among the heterozygotes (62%) than among controls (47%), but this did not reach statistical significance (P=0.062). Systolic and diastolic blood pressure and alcohol use did not differ among the groups. The percentage of smokers, however, was significantly higher in the heterozygotes than in the controls (21% versus 14%; P=0.015). One heterozygous carrier and 2 subjects in the control group had been prescribed a statin. No patient had diabetes mellitus, defined as use of oral antidiabetic drugs or insulin.
The data on LCAT activity levels represent data from previous publications.16,18,19,21 In these reports, the LCAT mutations in the respective families have been shown to result in 30% to 42% reductions in LCAT activity, with concomitant 30% to 38% reductions in HDL-C levels in the heterozygous carriers compared with family controls.
Lipids, (Apo)lipoproteins, and C-Reactive Protein Levels
IMT and Cardiovascular Events
Mean IMT values were 0.591±0.08 mm for controls and 0.623±0.13 mm for heterozygotes (Table 3). After adjustment for age, gender, use of alcohol, and family, this difference in average IMT proved to be highly statistically significant (P<0.0015). This probability value was obtained with multivariate backward stepwise regression analysis, whereby age, gender, and use of alcohol remained in the model but smoking and blood pressure did not. Although the family controls did not experience cardiovascular events, 1 male heterozygote had angina pectoris at age 50 years and a myocardial infarction at age 54 years, and a second heterozygote had a myocardial infarction at age 58 years.
Discussion
It has long been difficult to assess the risk of atherosclerosis in individuals with genetically determined low HDL-C levels. This knowledge, however, can provide important insight when it comes to targeting HDL metabolism to reduce CVD risk. After almost 40 years of LCAT research, the present study provides evidence that heterozygotes for LCAT gene defects, who present with an average 36% decrease in HDL-C levels, exhibit an increased risk for atherosclerosis as assessed by IMT measurements. These data suggest that intact LCAT function is important in the protection against atherosclerotic vascular disease.
LCAT, Lipids, and Lipoproteins
The present analysis confirms the strong impact of LCAT gene mutations on HDL-C levels. All LCAT gene defects under study have previously been shown to underlie marked loss of LCAT activity (30% to 42% compared with family controls), and as a direct consequence, to result in 30% to 38% reductions in HDL-C levels16,19,21 (for a review, see Pritchard and Hill13). LCAT is primarily active at the phospholipid monolayer of nascent HDL particles, where it converts cholesterol into cholesteryl ester on activation by its cofactor, apoA-I. Even a single change in the amino acid sequence of LCAT has been shown to result in a decreased interaction with apoA-I, with a consequential decrease in LCAT activity26 This will, in turn, cause defective maturation of small HDL into larger, spherical, cholesteryl ester–enriched HDL and hence lower HDL-C levels. The data show that this effect is not counteracted by upregulation of the unaffected LCAT allele in heterozygotes or by other key players in HDL metabolism. Additionally, the present study shows the effect of LCAT mutations on plasma triglycerides levels, as has been reported previously.20,27 Although heterozygotes presented with a moderate 22% increase in triglyceride levels, homozygotes presented with a marked 337% increase in triglyceride levels (data not shown). Mild hypertriglyceridemia is also observed in other genetic HDL deficiencies.28,29 For LCAT, hepatic triglyceride overproduction and reduced lipoprotein lipase activity, as found in LCAT-deficient mice, may explain this phenotype.30 Finally, we identified a small but significant increase in LDL-C levels compared with controls, for which we have no explanation.
LCAT and Atherosclerosis
Despite the fact that LCAT has long been recognized as a key regulator of HDL metabolism, the role of this enzyme in human atherogenesis has remained controversial. Prospective studies that have assessed LCAT activity or LCAT concentration at baseline are nonexistent, whereas cross-sectional observational studies have reported increased and decreased LCAT activity in subjects with CVD.31,32 Initially, the paradoxical finding of complete HDL deficiency and the reported absence of CVD in LCAT-deficient patients has been used to reject the hypothesis that HDL is important in the protection against atherosclerosis. Potential mechanisms to explain these findings were subsequently postulated, such as preferential clearance of HDL fractions that have less atherogenic potential.33 In addition, decreased LDL-C and apoB levels have also been put forward as an explanation for the lack of marked CVD. In the large group of LCAT mutation carriers in the present study, however, LDL-C was clearly not decreased, which refutes this argument.
Studies focusing on the role of LCAT in human atherosclerosis have thus far been hampered by the paucity of clinical events in small numbers of individuals with LCAT mutations. The advent of validated surrogate markers for atherosclerosis has proved very useful in this context. IMT measurements have been used to show that carriers of ABCA1 and apoA-I defects are at increased risk for coronary artery disease,8,34,35 which illustrates the power of this tool to study small, interesting groups of patients. The present study now shows that heterozygotes for LCAT mutations also have increased atherosclerosis compared with family controls. This finding was not based on a few heterozygous outliers with a very thick carotid intima-media complex but on solid IMT data. This is illustrated by the fact that the 30 heterozygotes of the largest family (family 3) presented with a thicker IMT than their 45 family control members (0.614 versus 0.589 mm). This effect, however, did not reach statistical significance, which illustrates the need to recruit and study more families with the same rare genetic disorder to answer our primary research question. During the preparation of this report, Ayyobi et al36 also reported vascular abnormalities in a small cohort of 9 heterozygotes for 1 specific LCAT mutation. The investigators could not, however, study family controls, and because of the small number of study subjects, these investigators could not draw conclusions regarding the risk for atherosclerosis.36
A potential but dangerous drawback of studying atherosclerosis in relatively small cohorts, such as investigated here, is the referral basis of the index patients. In this respect, we would like to emphasize that the probands of the currently studied families came to our attention because they experienced corneal opacifications, or they were referred because of previously identified low HDL-C levels. This excludes a bias for selecting those families with LCAT mutations with an increased or established risk for CVD. Exactly how reduced LCAT function affects IMT, however, cannot be deduced from the present study. LCAT mutations may directly compromise reverse cholesterol transport by reducing the flux of cholesterol from cholesterol-loaded peripheral macrophages to the HDL fraction. The markedly reduced HDL-C levels in carriers also may affect endothelial function directly.37 Alternatively, loss of LCAT activity may cause enhanced oxidation of LDL.38
LCAT and Inflammation
Elevated levels of CRP are an established predictor for coronary artery disease,39 but there is much debate about whether this plasma factor plays an active role in atherosclerosis or whether it merely represents an innocent bystander. We were surprised to find that hs-CRP levels were 2.2-fold increased in the heterozygotes. From the present study, however, it can not be appreciated whether increased levels of CRP are a direct consequence of reduced LCAT activity or of the consequentially reduced HDL-C levels. Pirro and colleagues40 also reported significantly higher CRP levels in other subjects with hypoalphalipoproteinemia, which suggests a proinflammatory status in subjects with low HDL-C levels per se.
Concluding Remarks
Upregulation of LCAT function has been proposed as an HDL-C increasing therapy, but its atheroprotective effects have been questioned.41 The present study suggests that increasing LCAT activity may reduce atherosclerosis progression, at least in subjects with subnormal levels of LCAT. Efforts to develop LCAT protein therapy have failed, but LCAT gene therapy protocols are more promising, as recently illustrated by decreased atherosclerosis in dyslipidemic obese mice42 after adenovirus-mediated transfer of the LCAT gene. Also, Zhang et al43 recently showed that a similar strategy increased reverse cholesterol transport in hamsters but, interestingly, not in mice. The present report indicates that increasing LCAT activity in plasma might be speculated to result not only in increased HDL-C levels but also in decreased concentrations of triglycerides and hs-CRP. Moreover, increased LCAT activity might also result in an augmented capacity of HDL to exhibit antioxidative, antiinflammatory,44 and antithrombotic effects.
Acknowledgments
We thank all participants of this study for their cooperation. Moreover, we thank Joris Butzelaar for his work as a reader of the IMT files, and Kobie Los, who drew blood samples from the participants.
References
Nofer JR, Kehrel B, Fobker M, Levkau B, Assmann G, von Eckardstein A. HDL and arteriosclerosis: beyond reverse cholesterol transport. Atherosclerosis. 2002; 161: 1–16.
Stein O, Stein Y. Atheroprotective mechanisms of HDL. Atherosclerosis. 1999; 144: 285–301.
Brousseau ME, Schaefer EJ, Wolfe ML, Bloedon LT, Digenio AG, Clark RW, Mancuso JP, Rader DJ. Effects of an inhibitor of cholesteryl ester transfer protein on HDL cholesterol. N Engl J Med. 2004; 350: 1505–1515.
de Grooth GJ, Kuivenhoven JA, Stalenhoef AF, de Graaf J, Zwinderman AH, Posma JL, van Tol A, Kastelein JJP. Efficacy and safety of a novel cholesteryl ester transfer protein inhibitor, JTT-705, in humans: a randomized phase II dose-response study. Circulation. 2002; 105: 2159–2165.
Singaraja RR, Brunham LR, Visscher H, Kastelein JJ, Hayden MR. Efflux and atherosclerosis: the clinical and biochemical impact of variations in the ABCA1 gene. Arterioscler Thromb Vasc Biol. 2003; 23: 1322–1332.
Sparrow CP, Baffic J, Lam MH, Lund EG, Adams AD, Fu X, Hayes N, Jones AB, Macnaul KL, Ondeyka J, Singh S, Wang J, Zhou G, Moller DE, Wright SD, Menke JG. A potent synthetic LXR agonist is more effective than cholesterol loading at inducing ABCA1 mRNA and stimulating cholesterol efflux. J Biol Chem. 2002; 277: 10021–10027.
Ikewaki K, Matsunaga A, Han H, Watanabe H, Endo A, Tohyama J, Kuno M, Mogi J, Sugimoto K, Tada N, Sasaki J, Mochizuki S. A novel two nucleotide deletion in the apolipoprotein A-I gene, apoA-I Shinbashi, associated with high density lipoprotein deficiency, corneal opacities, planar xanthomas, and premature coronary artery disease. Atherosclerosis. 2004; 172: 39–45.
Hovingh GK, Brownlie A, Bisoendial RJ, Dube MP, Levels JHM, Petersen W, Dullaart RPF, Stroes ESG, Zwinderman AH, de Grooth E, Hayden MR, Kuivenhoven JA, Kastelein JJP. A novel ApoA-I mutation (L178P) leads to endothelial dysfunction, increased arterial wall thickness and premature coronary artery disease. J Am Coll Cardiol. 2004; 44: 1429–1435.
Nissen SE, Tsunoda T, Tuzcu EM, Schoenhagen P, Cooper CJ, Yasin M, Eaton GM, Lauer MA, Sheldon WS, Grines CL, Halpern S, Crow T, Blankenship JC, Kerensky R. Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA. 2003; 290: 2292–2300.
Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res. 1968; 9: 155–167.
Norum KR, Gjone E. Familial serum-cholesterol esterification failure: a new inborn error of metabolism. Biochim Biophys Acta. 1967; 144: 698–700.
Carlson LA, Philipson B. Fish-eye disease: a new familial condition with massive corneal opacities and dyslipoproteinaemia. Lancet. 1979; 2: 922–924.
Pritchard PH, Hill JS. Genetic disorders of lecithin:cholesterol acyltransferase. In: Betteridge DJ, Illingworth DR, Shepherd J, eds. Lipoproteins in Health and Disease. 1st ed. London, UK: Arnold; 1999: 799–814.
Furbee JW Jr, Parks JS. Transgenic overexpression of human lecithin:cholesterol acyltransferase (LCAT) in mice does not increase aortic cholesterol deposition. Atherosclerosis. 2002; 165: 89–100.
Santamarina-Fojo S, Lambert G, Hoeg JM, Brewer HB Jr. Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis. Curr Opin Lipidol. 2000; 11: 267–275.
Kuivenhoven JA, Wiebusch H, Pritchard PH, Funke H, Benne R, Assmann G, Kastelein JJP. An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease). J Clin Invest. 1996; 98: 358–364.
Funke H, von Eckardstein A, Pritchard PH, Albers JJ, Kastelein JJ, Droste C, Assmann G. A molecular defect causing fish eye disease: an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of alpha-LCAT activity. Proc Natl Acad Sci U S A. 1991; 88: 4855–4859.
Kastelein JJ, Pritchard PH, Erkelens DW, Kuivenhoven JA, Albers JJ, Frohlich JJ. Familial high-density-lipoprotein deficiency causing corneal opacities (fish eye disease) in a family of Dutch descent. J Intern Med. 1992; 231: 413–419.
Kuivenhoven JA, van Voorst tot Voorst EJ, Wiebusch H, Marcovina SM, Funke H, Assmann G, Pritchard PH, Kastelein JJP. A unique genetic and biochemical presentation of fish-eye disease. J Clin Invest. 1995; 96: 2783–2791.
Frohlich J, McLeod R, Pritchard PH, Fesmire J, McConathy W. Plasma lipoprotein abnormalities in heterozygotes for familial lecithin:cholesterol acyltransferase deficiency. Metabolism. 1988; 37: 3–8.
Kuivenhoven JA, Stalenhoef AF, Hill JS, Demacker PN, Errami A, Kastelein JJ, Pritchard PH. Two novel molecular defects in the LCAT gene are associated with fish eye disease. Arterioscler Thromb Vasc Biol. 1996; 16: 294–303.
Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972; 18: 499–502.
Pruvot I, Fievet C, Durieux C, Vu DN, Fruchart JC. Electroimmuno- and immunonephelometric assays of apolipoprotein A-I by using a mixture of monoclonal antibodies. Clin Chem. 1988; 34: 2048–2052.
Heuck CC, Schlierf G. Nephelometry of apolipoprotein B in human serum. Clin Chem. 1979; 25: 221–226.
de Groot E, Jukema JW, Montauban van Swijndregt AD, Zwinderman AH, Ackerstaff RG, van der Steen AF, Bom N, Lie KI, Bruschke AV. B-mode ultrasound assessment of pravastatin treatment effect on carotid and femoral artery walls and its correlations with coronary arteriographic findings: a report of the Regression Growth Evaluation Statin Study (REGRESS). J Am Coll Cardiol. 1998; 31: 1561–1567.
Peelman F, Vandekerckhove J, Rosseneu M. Structure and function of lecithin cholesterol acyl transferase: new insights from structural predictions and animal models. Curr Opin Lipidol. 2000; 11: 155–160.
Miettinen HE, Gylling H, Tenhunen J, Virtamo J, Jauhiainen M, Huttunen JK, Kantola I, Miettinen TA, Kontula K. Molecular genetic study of Finns with hypoalphalipoproteinemia and hyperalphalipoproteinemia: a novel Gly230 Arg mutation (LCAT[Fin]) of lecithin:cholesterol acyltransferase (LCAT) accounts for 5% of cases with very low serum HDL cholesterol levels. Arterioscler Thromb Vasc Biol. 1998; 18: 591–598.
Wang CS, Alaupovic P, Gregg RE, Brewer HB Jr. Studies on the mechanism of hypertriglyceridemia in Tangier disease: determination of plasma lipolytic activities, k1 values and apolipoprotein composition of the major lipoprotein density classes. Biochim Biophys Acta. 1987; 920: 9–19.
Ng DS, Leiter LA, Vezina C, Connelly PW, Hegele RA. Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia. J Clin Invest. 1994; 93: 223–229.
Ng DS, Xie C, Maguire GF, Zhu X, Ugwu F, Lam E, Connelly PW. Hypertriglyceridemia in lecithin-cholesterol acyltransferase-deficient mice is associated with hepatic overproduction of triglycerides, increased lipogenesis, and improved glucose tolerance. J Biol Chem. 2004; 279: 7636–7642.
Solajic-Bozicevic N, Stavljenic A, Sesto M. Lecithin:cholesterol acyltransferase activity in patients with acute myocardial infarction and coronary heart disease. Artery. 1991; 18: 326–340.
Wells IC, Peitzmeier G, Vincent JK. Lecithin:cholesterol acyltransferase and lysolecithin in coronary atherosclerosis. Exp Mol Pathol. 1986; 45: 303–310.
Rader DJ, Ikewaki K, Duverger N, Schmidt H, Pritchard H, Frohlich J, Clerc M, Dumon MF, Fairwell T, Zech L. Markedly accelerated catabolism of apolipoprotein A-II (ApoA-II) and high density lipoproteins containing ApoA-II in classic lecithin:cholesterol acyltransferase deficiency and fish-eye disease. J Clin Invest. 1994; 93: 321–330.
van Dam MJ, de Groot E, Clee SM, Hovingh GK, Roelants R, Brooks-Wilson A, Zwinderman AH, Smit AJ, Smelt AH, Groen AK, Hayden MR, Kastelein JJP. Association between increased arterial-wall thickness and impairment in ABCA1-driven cholesterol efflux: an observational study. Lancet. 2002; 359: 37–42.
Hovingh GK, de Groot E, van der Steeg WA, Boekholdt SM, Hutten BA, Kuivenhoven JA, Kastelein JJP. Inherited disorders of HDL metabolism and atherosclerosis. Curr Opin Lipidol. 2005; 16: 139–145.
Ayyobi AF, McGladdery SH, Chan S, Mancin J, Hil JS, Frohlich JJ. Lecithin:cholesterol acyltransferase (LCAT) deficiency and risk of vascular disease: 25 year follow-up. Atherosclerosis. 2004; 177: 361–366.
Bisoendial RJ, Hovingh GK, Levels JH, Lerch PH, Andresen I, Hayden MR, Kastelein JJ, Stroes ESG. Restoration of endothelial function by increasing high-density lipoprotein in subjects with isolated low high-density lipoprotein. Circulation. 2003; 107: 2944–2948.
Vohl MC, Neville TA, Kumarathasan R, Braschi S, Sparks DL. A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation. Biochemistry. 1999; 38: 5976–5981.
Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000; 342: 836–843.
Pirro M, Siepi D, Lupattelli G, Roscini AR, Schillaci G, Gemelli F, Vaudo G, Marchesi S, Pasqualini L, Mannorino E. Plasma C-reactive protein in subjects with hypo/hyperalphalipoproteinemias. Metabolism. 2003; 52: 432–436.
Rader DJ, Maugeais C. Genes influencing HDL metabolism: new perspectives and implications for atherosclerosis prevention. Mol Med Today. 2000; 6: 170–175.
Mertens A, Verhamme P, Bielicki JK, Phillips MC, Quarck R, Verreth W, Stengel D, Ninoi E, Navab M, Mackness B, Mackness M, Holvoet P. Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis. Circulation. 2003; 107: 1640–1646.
Zhang A-H, Gao S, Fan J, Huang W, Zhao T-Q, Liu G. Increased plasma HDL cholesterol levels and biliary cholesterol excretion in hamster by LCAT overexpression. FEBS Lett. 2004; 570: 25–29.
Nicholls SJ, Dusting GJ, Cutri B, Bao S, Drummond GR, Rye KA, Barter PJ. Reconstituted high-density lipoproteins inhibit the acute pro-oxidant and proinflammatory vascular changes induced by a periarterial collar in normocholesterolemic rabbits. Circulation. 2005; 111: 1543–1550.(Compromised LCAT Function)