Laura Calabresi

Endothelial Protection by High-Density Lipoproteins. From Bench to Bedside

Abstract—There are several potential mechanisms by which HDLs protect against the development of vascular disease. One relates to the unique ability of these lipoproteins to remove cholesterol from the arterial wall. Another is the ability of HDL to prevent and eventually correct endothelial dysfunction, a key variable in the pathogenesis of atherosclerosis and its complications. HDLs help maintain endothelial integrity, facilitate vascular relaxation, inhibit blood cell adhesion to vascular endothelium, reduce platelet aggregability and coagulation, and may favor fibrinolysis. These functions of HDLs complement their activity in arterial cholesterol removal by providing an excellent rationale for favorably influencing pathological processes underlying a variety of clinical conditions, such as accelerated atherosclerosis, acute coronary syndromes, and restenosis after coronary angioplasty, through a chronic or acute elevation of plasma HDL concentration.

Triglycerides Are Major Determinants of Cholesterol Esterification/Transfer and HDL Remodeling in Human Plasma

Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are responsible for the esterification of cell-derived cholesterol and for the transfer of newly synthesized cholesteryl esters (CE) from HDL to apoB-containing lipoproteins in human plasma. LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles with a high capacity for cell cholesterol uptake are generated in plasma. In the present study, cholesterol esterification and transfer were evaluated in 60 patients with isolated hypercholesterolemia (HC, n = 20) and isolated (HTG, n = 20) or mixed hypertriglyceridemia (MHTG, n = 20) and in 20 normolipidemic healthy individuals (NL). Cholesterol esterification rate (CER) and net CE transfer rate (CETR) were measured in whole plasma. LCAT and CETP concentrations were determined by specific immunoassays. HDL remodeling was analyzed by monitoring changes in HDL particle size distribution during incubation of whole plasma at 37 degrees C. Mean CER and CETR were 48% and 73% higher, respectively, in hypertriglyceridemic (HTG + MHTG) versus normotriglyceridemic individuals. HDL remodeling was also significantly accelerated in plasma from hypertriglyceridemic patients. Strong positive correlations were found in the total sample between plasma and VLDL triglyceride levels and CER (r = .722 and r = .642, respectively), CETR (r = .510 and r = .491, respectively), and HDL remodeling (r = .625 and r = .620, respectively). No differences in plasma LCAT and CETP concentrations were found among the various groups except for a tendency toward higher CETP levels in hypercholesterolemic patients (+51% in MHTG and +20% in HC) versus control subjects (NL). By stepwise regression analysis, VLDL triglyceride level was the sole significant predictor of CER and CETR and contributed significantly together with baseline HDL particle distribution to HDL remodeling. These results indicate that plasma triglyceride level is a major factor in the regulation of cholesterol esterification/transfer and HDL remodeling in human plasma, whereas LCAT/CETP concentrations play a minor role in the modulation of reverse cholesterol transport.

Endothelial Protection by High-Density Lipoproteins

Abstract—There are several potential mechanisms by which HDLs protect against the development of vascular disease. One relates to the unique ability of these lipoproteins to remove cholesterol from the arterial wall. Another is the ability of HDL to prevent and eventually correct endothelial dysfunction, a key variable in the pathogenesis of atherosclerosis and its complications. HDLs help maintain endothelial integrity, facilitate vascular relaxation, inhibit blood cell adhesion to vascular endothelium, reduce platelet aggregability and coagulation, and may favor fibrinolysis. These functions of HDLs complement their activity in arterial cholesterol removal by providing an excellent rationale for favorably influencing pathological processes underlying a variety of clinical conditions, such as accelerated atherosclerosis, acute coronary syndromes, and restenosis after coronary angioplasty, through a chronic or acute elevation of plasma HDL concentration.

The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families.

Objective—To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene. Methods and Results—Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-&bgr; high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL3 particle size were reduced in a gene–dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former. Conclusion—In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene–dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED.

Increased Cholesterol Efflux Potential of Sera From ApoA-IMilano Carriers and Transgenic Mice

The ability of HDL to remove cholesterol from peripheral cells and drive it to the liver for excretion is believed to explain most of the strong inverse correlation between plasma HDL cholesterol levels and coronary heart disease. Carriers of the ApoA-IMilano (A-IM) mutant have a severe hypoalphalipoproteinemia but are not at increased risk for premature of coronary heart disease. To explain this apparent paradox, we compared the capacity of serum from A-IM and control subjects to extract cholesterol from Fu5AH cells. Because the A-IM carriers are all heterozygotes for the mutation, we also compared the cholesterol efflux capacity of serum from transgenic mice expressing A-IM or wild-type ApoA-I (A-IWT), in the absence of murine ApoA-I. In the whole series of human or mouse sera, cholesterol efflux was significantly correlated with several HDL-related parameters; after adjustment for concomitant variables, the only parameter that remained significantly correlated with cholesterol efflux was the serum ApoA-I concentration (r2=0.85 in humans and 0.84 in mice). The same was true when samples from control subjects, A-IM carriers, A-IWT or A-IM mice were analyzed separately. Cholesterol efflux to sera from the A-IM carriers was only reduced slightly compared with control sera (25.0+/-4.2% versus 30.4+/-3.3%), although there was a large reduction (-45%) in the serum ApoA-I concentration in the former. Cholesterol efflux was also lower to sera from A-IM than A-IWT mice (15.6+/-3.8% versus 30. 1+/-7.1%), but less than expected from the 70% reduction in serum ApoA-I concentration. A relative efflux potential of serum was calculated in each group as the slope of the regression line fitting cholesterol efflux to ApoA-I concentrations. Therefore, the relative efflux potential reflects the relative efficiency of ApoA-I in determining cell cholesterol efflux. The relative efflux potential of mouse and human sera was in the following order: A-IM mice>A-IM carriers>A-IWT mice=control subjects, suggesting a gene-dosage effect of the A-IM mutation on the efficiency of serum to extract cholesterol from cells. The high efficiency of A-IM-containing HDL for cell cholesterol uptake would result in an improved reverse cholesterol transport in the A-IM carriers, possibly explaining the low susceptibility to atherosclerosis development.

Role of LCAT in HDL remodeling : investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (−77% and −13%), and LCAT activities (−99% and −39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated preβ-1 and α-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with α-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, preβ-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with preβ-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of preβ-1 into α-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.

Omacor in familial combined hyperlipidemia: effects on lipids and low density lipoprotein subclasses.

Elevations of plasma cholesterol and/or triglycerides, and the prevalence of small, dense LDL particles remarkably increase coronary risk in patients with familial combined hyperlipidemia (FCHL). A total of 14 FCHL patients were studied, to investigate the ability of Omacor, a drug containing the n-3 fatty acids eicosapentaenoic and docosahexaenoic acid (EPA and DHA), to favorably correct plasma lipid/lipoprotein levels and LDL particle distribution. The patients received four capsules daily of Omacor (providing 3.4 g EPA+DHA per day) or placebo for 8 weeks in a randomized, double-blind, cross-over study. Omacor significantly lowered plasma triglycerides and VLDL-cholesterol levels, by 27 and 18%, respectively. Total cholesterol did not change but LDL-cholesterol and apolipoprotein B (apoB) concentrations increased by 21 and 6%. As expected, LDL particles were small (diameter=24.9+/-0.3 nm) and apoB-rich (LDL-cholesterol/apoB ratio=1.27+/-0.26) in the selected subjects. After Omacor treatment LDL became enriched in cholesterol (LDL-cholesterol/apoB ratio=1.40+/-0.17), mainly cholesteryl esters, indicating accumulation in plasma of more buoyant and core enriched LDL particles. Indeed, the separation of LDL subclasses by rate zonal ultracentrifugation showed an increase of the plasma concentration of IDL and of the more buoyant, fast floating LDL-1 and LDL-2 subclasses after Omacor, with a parallel decrease in the concentration of the denser, slow floating LDL-3 subclass. However, the average LDL size did not change after Omacor (25.0+/-0.3 nm). The resistance of the small LDL pattern to drug-induced modifications implies that a maximal lipid-lowering effect must be achieved to reduce coronary risk in FCHL patients.

Intestinal specific LXR activation stimulates reverse cholesterol transport and protects from atherosclerosis

Several steps of the HDL-mediated reverse cholesterol transport (RCT) are transcriptionally regulated by the nuclear receptors LXRs in the macrophages, liver, and intestine. Systemic LXR activation via synthetic ligands induces RCT but also causes increased hepatic fatty acid synthesis and steatosis, limiting the potential therapeutic use of LXR agonists. During the last few years, the participation of the intestine in the control of RCT has appeared more evident. Here we show that while hepatic-specific LXR activation does not contribute to RCT, intestinal-specific LXR activation leads to decreased intestinal cholesterol absorption, improved lipoprotein profile, and increased RCT in vivo in the absence of hepatic steatosis. These events protect against atherosclerosis in the background of the LDLR-deficient mice. Our study fully characterizes the molecular and metabolic scenario that elects the intestine as a key player in the LXR-driven protective environment against cardiovascular disease.

Functional Lecithin: Cholesterol Acyltransferase Is Not Required for Efficient Atheroprotection in Humans

Background— Mutations in the LCAT gene cause lecithin:cholesterol acyltransferase (LCAT) deficiency, a very rare metabolic disorder with 2 hypoalphalipoproteinemia syndromes: classic familial LCAT deficiency (Online Mendelian Inheritance in Man No. 245900), characterized by complete lack of enzyme activity, and fish-eye disease (Online Mendelian Inheritance in Man No. 136120), with a partially defective enzyme. Theoretically, hypoalphalipoproteinemia cases with LCAT deficiency should be at increased cardiovascular risk because of high-density lipoprotein deficiency and defective reverse cholesterol transport. Methods and Results— The extent of preclinical atherosclerosis was assessed in 40 carriers of LCAT gene mutations from 13 Italian families and 80 healthy controls by measuring carotid intima-media thickness (IMT). The average and maximum IMT values in the carriers were 0.07 and 0.21 mm smaller than in controls (P=0.0003 and P=0.0027), respectively. Moreover, the inheritance of a mutated LCAT genotype had a remarkable gene-dose–dependent effect in reducing carotid IMT (P=0.0003 for average IMT; P=0.001 for maximum IMT). Finally, no significant difference in carotid IMT was found between carriers of LCAT gene mutations that cause total or partial LCAT deficiency (ie, familial LCAT deficiency or fish-eye disease). Conclusions— Genetically determined low LCAT activity in Italian families is not associated with enhanced preclinical atherosclerosis despite low high-density lipoprotein cholesterol levels. This finding challenges the notion that LCAT is required for effective atheroprotection and suggests that elevating LCAT expression or activity is not a promising therapeutic strategy to reduce cardiovascular risk.

Small Discoidal Pre-β1 HDL Particles Are Efficient Acceptors of Cell Cholesterol via ABCA1 and ABCG1

The aim of this study was to correlate the lipid content and size of discoidal reconstituted HDL particles with their ability to promote cellular cholesterol efflux. Homogeneous discoidal rHDL particles containing apoA-I and POPC, with diameters of 7.8, 9.6, 10.8, 12.5, and 17.0 nm, were prepared by the cholate dialysis technique. Cholesterol efflux to rHDL was evaluated in pathway-specific cell models for ABCA1-, ABCG1-, and SR-BI-mediated efflux. ABCA1-mediated efflux was efficiently promoted by the 7.8 nm rHDL containing 82 POPC molecules per particle. This rHDL also promoted ABCG1, but not SR-BI, cholesterol efflux. All large and lipid-rich rHDLs, with a diameter of >or=9.6 nm and a phospholipid content of >/=202 molecules per particle, promoted both SR-BI- and ABCG1-mediated efflux. Our results indicated that the ABCA1-mediated cell cholesterol efflux can be efficiently driven not only by monomolecular lipid free/poor apoA-I but also by a small discoidal phospholipid-containing particle resembling plasma pre-beta1 HDL. This same particle also promotes ABCG1- but not SR-BI-mediated efflux. These results help to clarify the role of plasma pre-beta1 HDL in reverse cholesterol transport.