Angeliki Chroni

Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease

Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1- and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.

Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL

The concentration, composition, shape, and size of plasma high-density lipoprotein (HDL) are determined by numerous proteins that influence its biogenesis, remodeling, and catabolism. The discoveries of the HDL receptor (scavenger receptor class B type I, SR-BI) and the ABCA1 (ATP-binding cassette transporter A1) lipid transporter provided two missing links that were necessary to understand the biogenesis and some of the functions of HDL. Existing data indicate that functional interactions between apoA-I and ABCA1 are necessary for the initial lipidation of apoA-I. Through a series of intermediate steps, lipidated apoA-I proceeds to form discoidal HDL particles that can be converted to spherical particles by the action of lecithin:cholesterol acyltransferase (LCAT). Discoidal and spherical HDL can interact functionally with SR-BI and these interactions lead to selective lipid uptake and net efflux of cholesterol and thus remodel HDL. Defective apoA-I/ABCA1 interactions prevent lipidation of apoA-I that is necessary for the formation of HDL particles. In the same way, specific mutations in apoA-I or LCAT prevent the conversion of discoidal to spherical HDL particles. The interactions of lipid-bound apoA-I with SR-BI are affected in vitro by specific mutations in apoA-I or SR-BI. Furthermore, deficiency of SR-BI affects the lipid and apolipoprotein composition of HDL and is associated with increased susceptibility to atherosclerosis. Here we review the current status of the pathway of HDL biogenesis and mutations in apoA-I, ABCA1, and SR-BI that disrupt different steps of the pathway and may lead to dyslipidemia and atherosclerosis in mouse models. The phenotypes generated in experimental mouse models for apoA-I, ABCA1, LCAT, SR-BI, and other proteins of the HDL pathway may facilitate early diagnosis of similar phenotypes in the human population and provide guidance for proper treatment.

The Central Helices of ApoA-I Can Promote ATP-binding Cassette Transporter A1 (ABCA1)-mediated Lipid Efflux AMINO ACID RESIDUES 220–231 OF THE WILD-TYPE ApoA-I ARE REQUIRED FOR LIPID EFFLUX IN VITRO AND HIGH DENSITY LIPOPROTEIN FORMATION IN VIVO

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by ∼80–92% by apoA-I[Δ(185–243)], only by 15% by apoA-I[Δ(1–41)] or apoA-I[Δ(1–59)], and was restored to 75–80% of the wild-type apoA-I control value by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[Δ(232–243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30–40% and 50–65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[Δ(1–41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[Δ(1–41)Δ(185–243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220–231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.

Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer

Purpose of the review This review clarifies the functions of key proteins of the chylomicron and the HDL pathways. Recent findings Adenovirus-mediated gene transfer of several apolipoprotein (apo)E forms in mice showed that the amino-terminal 1-185 domain of apoE can direct receptor-mediated lipoprotein clearance in vivo. Clearance is mediated mainly by the LDL receptor. The carboxyl-terminal 261-299 domain of apoE induces hypertriglyceridemia, because of increased VLDL secretion, diminished lipolysis and inefficient VLDL clearance. Truncated apoE forms, including apoE2-202, have a dominant effect in remnant clearance and may have future therapeutic applications for the correction of remnant removal disorders. Permanent expression of apoE and apoA-I following adenoviral gene transfer protected mice from atherosclerosis. Functional assays, protein cross-linking, and adenovirus-mediated gene transfer of apoA-I mutants in apoA-I deficient mice showed that residues 220-231, as well as the central helices of apoA-I, participate in ATP-binding cassette transporter A1-mediated lipid efflux and HDL biogenesis. Following apoA-I gene transfer, an amino-terminal deletion mutant formed spherical α-HDL, a double amino- and carboxyl-terminal deletion mutant formed discoidal HDL, and a carboxyl-terminal deletion mutant formed only pre-β-HDL. The findings support a model of cholesterol efflux that requires direct physical interactions between apoA-I and ATP-binding cassette transporter A1, and can explain Tangier disease and other HDL deficiencies. Summary New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.

HDL Biogenesis, Remodeling, and Catabolism

In this chapter, we review how HDL is generated, remodeled, and catabolized in plasma. We describe key features of the proteins that participate in these processes, emphasizing how mutations in apolipoprotein A-I (apoA-I) and the other proteins affect HDL metabolism. The biogenesis of HDL initially requires functional interaction of apoA-I with the ATP-binding cassette transporter A1 (ABCA1) and subsequently interactions of the lipidated apoA-I forms with lecithin/cholesterol acyltransferase (LCAT). Mutations in these proteins either prevent or impair the formation and possibly the functionality of HDL. Remodeling and catabolism of HDL is the result of interactions of HDL with cell receptors and other membrane and plasma proteins including hepatic lipase (HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor. Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown to form discrete HDL particles containing these apolipoproteins which may have important but still unexplored functions. Furthermore, several plasma proteins were found associated with HDL and may modulate its biological functions. The effect of these proteins on the functionality of HDL is the topic of ongoing research.

Characterization of antioxidant/anti-inflammatory properties and apoA-I-containing subpopulations of HDL from family subjects with monogenic low HDL disorders

BACKGROUND Genetic factors regulate both high-density lipoprotein (HDL) levels and functionality, thus affecting HDL antiatherogenic properties. We characterized the HDL antioxidant/anti-inflammatory properties and apoA-I-containing subpopulations in families with monogenic low HDL disorders. METHODS Subjects with mutations in apolipoprotein A-I (apoA-I), ATP-binding cassette transporter A1 (ABCA1) or lecithin:cholesterol acyltransferase (LCAT) and family controls were studied. HDL antioxidant/anti-inflammatory properties were assayed by an in vitro fluorometric method and HDL-associated paraoxonase-1 (PON1), platelet activating factor-acetylhydrolase (PAF-AH), LCAT, malondialdehyde (MDA), PAF and serum amyloid A (SAA) were measured. ApoA-I-containing HDL subpopulations were analyzed by two-dimensional non-denaturing gel electrophoresis. RESULTS ApoA-I heterozygotes and subjects with partial or complete ABCA1 or LCAT deficiency had HDL with reduced antioxidant/anti-inflammatory properties and increased MDA levels. HDL-PON1 activity was reduced in apoA-I heterozygotes and in subjects with complete ABCA1 deficiency. HDL-PAF-AH activity was reduced in subjects with partial or complete ABCA1 deficiency or complete LCAT deficiency. HDL-LCAT activity was reduced in all LCAT mutation carriers. HDL-PAF levels were increased in apoA-I heterozygotes. HDL-SAA levels were increased in subjects with complete ABCA1 deficiency. ApoA-I, ABCA1 and LCAT heterozygotes were depleted of the large α1 HDL subpopulation. Subjects with complete LCAT deficiency showed mostly the small α4 HDL subpopulation and subjects with complete ABCA1 deficiency the α4 and preβ HDL subpopulations. CONCLUSIONS This study shows that mutations in apoA-I, ABCA1 and LCAT have direct effect on the antioxidant/anti-inflammatory properties of HDL. Furthermore, our study shows the effect of specific mutations on the apoA-I-containing HDL subpopulation profiles.

An apolipoprotein E4 fragment can promote intracellular accumulation of amyloid peptide beta 42

J. Neurochem. (2010) 115, 873–884.

Biophysical Analysis of Progressive C-Terminal Truncations of Human Apolipoprotein E4: Insights into Secondary Structure and Unfolding Properties

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimers disease and has been associated with a variety of neuropathological processes. ApoE4 C-terminally truncated forms have been found in brains of Alzheimers disease patients. Structural rearrangements in apoE4 are known to be key to its physiological functions. To understand the effect of C-terminal truncations on apoE4 lipid-free structure, we produced a series of recombinant apoE4 forms with progressive C-terminal deletions between residues 166 and 299. Circular dichroism measurements show a dramatic loss in helicity upon removal of the last 40 C-terminal residues, whereas further truncations of residues 203-259 lead to recovery of helical content. Further deletion of residues 186-202 leads to a small increase in helical content. Thermal denaturation indicated that removal of residues 260-299 leads to an increase in melting temperature but truncations down to residue 186 did not further affect the melting temperature. The progressive C-terminal truncations, however, gradually increased the cooperativity of thermal unfolding. Chemical denaturation of the apoE4 forms revealed a two-step process with a clear intermediate stage that is progressively lost as the C-terminus is truncated down to residue 230. Hydrophobic fluorescent probe binding suggested that regions 260-299 and 186-202 contain hydrophobic sites, the former being solvent accessible in the wild-type molecule and the latter being accessible only upon truncation. Taken together, our results show an important but complex role of apoE4 C-terminal segments in secondary structure stability and unfolding and suggest that interactions mediated by the C-terminal segments are important for the structural integrity and conformational changes of apoE4.

Discrete roles of apoA-I and apoE in the biogenesis of HDL species: lessons learned from gene transfer studies in different mouse models.

Using adenovirus‐mediated gene transfer in apolipoprotein A‐I (apoA‐I)‐deficient mice, we have established that apoA‐I mutations inhibit discrete steps in a pathway that leads to the biogenesis and remodeling of high‐density lipoprotein (HDL). To this point, five discrete categories of apoA‐I mutants have been characterized that may affect the interactions of apoA‐I with ATP‐binding cassette superfamily A, member 1 (ABCA1) or lecithin:cholesterol acyl transferase (LCAT) or may influence the plasma phospholipid transfer protein activity or may cause various forms of dyslipidemia. Biogenesis of HDL is not a unique property of apoA‐I. Using adenovirus‐mediated gene transfer of apoE in apoA‐I‐ or ABCA1‐deficient mice, we have established that apolipoprotein E (apoE) also participates in a novel pathway of biogenesis of apoE‐containing HDL particles. This process requires the functions of the ABCA1 lipid transporter and LCAT, and it is promoted by substitution of hydrophobic residues in the 261 to 269 region of apoE by Ala. The apoE‐containing HDL particles formed in the circulation may have atheroprotective properties. ApoE‐containing HDL may also have important biological functions in the brain that confer protection from Alzheimers disease.

Naturally occurring and bioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL: the abnormal HDL phenotypes can be corrected by treatment with LCAT

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