Giulia Chiesa

Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a).

The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis.

Recombinant Apolipoprotein A-IMilano Infusion Into Rabbit Carotid Artery Rapidly Removes Lipid From Fatty Streaks

Apolipoprotein A-IMilano (AIM), a natural variant of human apolipoprotein A-I, confers to carriers a significant protection against vascular disease. In previous studies, administration of recombinant AIM-phospholipid (AIM-PL) complexes to hypercholesterolemic rabbits markedly inhibited neointimal formation after arterial injury; moreover, repeated injections of AIM-PL in apoE-deficient mice significantly reduced atherosclerosis progression. The objective of the present study was to determine if a single localized infusion of AIM-PL complexes administered directly to atheromatous lesions could promote plaque regression. Lipid-rich, atheromatous plaques were generated at both common carotid arteries of 25 rabbits by applying a perivascular electric injury, followed by 1.5% cholesterol diet for 90 days. Rabbits were infused with either saline, phospholipid vesicles, or 3 different AIM-PL doses (250, 500, or 1000 mg of protein) delivered through an intravascular ultrasound (IVUS) catheter positioned at the origin of the right carotid. The lesions at the left carotid artery were therefore exposed to the agents systemically. Infusion of AIM-PL at the 2 highest doses caused reduction of right carotid artery plaque area by the end a 90-minute infusion as assessed by IVUS analysis. Plaque area regression was confirmed by histology in carotid arteries receiving direct (500 and 1000 mg doses) and systemic (500 mg dose) delivery, 72 hours after the start of the treatment. Plaque lipid content was associated with significant and similar decreases in Oil Red O staining in both arteries. These results suggest AIM-PL complexes enhanced lipid removal from arteries is the mechanism responsible for the observed plaque changes.

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.

Long Pentraxin 3, a Key Component of Innate Immunity, Is Modulated by High-Density Lipoproteins in Endothelial Cells

Objective—High-density lipoproteins (HDL) are endowed with cardiovascular protective activities. In addition to their role in reverse cholesterol transport, HDL exert several beneficial effects on endothelial cells, including the induction of endothelial nitric oxide synthase and prostacyclin release, and the control of the immune and inflammatory response. Methods and Results—To identify possible mechanisms involved in these effects we investigated the modulation of the expression of acute phase proteins of the pentraxin superfamily, such as C-reactive protein (CRP), serum amyloid P component protein (SAP), and the long pentraxin 3 (PTX3) by HDL in human endothelial cells. HDL induced PTX3 mRNA expression and protein release, whereas no effect was observed on CRP and SAP expression. This effect was mainly dependent on the activation of the lysosphingolipids receptors-PI3K/Akt axis and was mimicked by sphingosine 1 phosphate and other S1P mimetics. This observation was confirmed in vivo; indeed an increased expression of PTX3 mRNA was detected in the aorta of transgenic mice overexpressing human apoA-I, compared to apoA-I knock-out mice. Furthermore, plasma levels of PTX3 significantly increased in C57BL/6 mice injected with HDL. Conclusions—These data suggest that part of the atheroprotective effects of HDL could result from the modulation of molecules that act as sensors of the immunoinflammatory balance in the vascular wall.

Sequence polymorphisms in the apolipoprotein (a) gene. Evidence for dissociation between apolipoprotein(a) size and plasma lipoprotein(a) levels.

Apolipoprotein(a) [apo(a)], an apolipoprotein unique to lipoprotein(a) [Lp(a)], is highly polymorphic in size. Previous studies have indicated that the size of the apo(a) gene tends to be inversely correlated with the plasma level of Lp(a). However, several exceptions to this general trend have been identified. Individuals with apo(a) alleles of identical size do not always have similar plasma concentrations of Lp(a). To determine if these differences in plasma Lp(a) concentrations were due to sequence variations in the apo(a) gene, we examined the sequences of apo(a) alleles in 23 individuals homozygous for same-sized apo(a) alleles. We identified four single-strand DNA conformation polymorphisms (SSCPs) in the apo(a) gene. Of the 23 homozygotes, 21 (91%) were heterozygous for at least one of the SSCPs. Analysis of a family in which a parent was homozygous for the same-sized apo(a) allele revealed that each allele, though identical size, segregated with different plasma concentrations of Lp(a). These studies indicate that the apo(a) gene is even more polymorphic in sequence than was previously appreciated, and that sequence variations at the apo(a) locus, other than the number of kringle 4 repeats, contribute to the plasma concentration of Lp(a).

Mechanisms of HDL reduction after probucol. Changes in HDL subfractions and increased reverse cholesteryl ester transfer.

Treatment with probucol, a widely used lipid-lowering agent, is associated with a significant reduction of high density lipoprotein (HDL) cholesterol levels, but with an apparently improved removal of cholesteryl esters from tissues (e.g., from tendon xanthomas). The effects of probucol (500 mg twice daily) on HDL subfraction distribution and cholesteryl ester transfer activity were tested in 12 patients with stable type II hyperlipidemia [low density lipoprotein (LDL) cholesterol greater than 180 mg/dl] after a placebo-controlled cross-over trial. Probucol significantly lowered total cholesterol (-13.8%), LDL cholesterol (-9.1%), and HDL cholesterol (-30%). By rate zonal ultracentrifugation, a marked reduction of HDL2 cholesterol (-68%) was shown, whereas changes in HDL3 were less significant (-21%). These findings were confirmed by polyacrylamide gradient gel electrophoresis, typically showing a reduction or disappearance of HDL2b particles and the prevalence of particles in the HDL3a range. Cholesteryl ester transfer from HDL to lower density lipoproteins was significantly increased (30%) in all patients. These findings suggest that, in addition to the well-documented in vitro changes (prevention of LDL peroxidation and macrophage uptake), probucol characteristically modifies HDL particle distribution in vivo, and is associated with a significant increase of cholesteryl ester transfer activity.

Dose-Related Effects of Repeated ETC-216 (Recombinant Apolipoprotein A-IMilano/1-Palmitoyl-2-Oleoyl Phosphatidylcholine Complexes) Administrations on Rabbit Lipid-Rich Soft Plaques. In Vivo Assessment by Intravascular Ultrasound and Magnetic Resonance Imaging

OBJECTIVES This study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-I(Milano) phospholipid complex (recombinant apoA-I(Milano) and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques. BACKGROUND A single high dose of recombinant apoA-I(Milano) has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216. METHODS Thirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg. RESULTS Atheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216-treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to -6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p < 0.0001). The MRI evaluations after the second infusion established that a significant regression was achieved with only 2 administrations of the highest dose. CONCLUSIONS These results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume.

Apolipoprotein A-IMilano: current perspectives

Purpose of review Strategies to increase HDL are among the major targets of clinical research in atherosclerosis prevention. The mutant apolipoprotein A-IMilano has been associated with a reduced incidence of coronary disease in carriers. Furthermore, recombinant apolipoprotein A-IMilano has displayed remarkable atheroprotective activities and the possibility of directly reducing the burden of atherosclerosis in experimental models. This review is aimed at providing an update on the experimental studies in which apolipoprotein A-IMilano, produced as a recombinant protein, has displayed important effects in the treatment of vascular diseases. Recent findings In the past year, two reports have appeared, indicating that a single-dose administration of recombinant apolipoprotein A-IMilano dimers formulated into liposomes can reduce atheromas in models such as the apolipoprotein E-deficient mice and a rabbit model of carotid focal lesion, in which a direct 90 min infusion of the product reduced atheroma up to 30%. This finding was associated with an increase in HDL free cholesterol and the permanence of the recombinant product in the lesion for over 72 h. Summary Recombinant apolipoprotein A-IMilano, formulated as synthetic HDL with phospholipids, appears to exert a direct removing effect on arterial cholesterol. This is well evident in experimental animals and, more recently in clinical findings, as indicated by a dramatic increase in HDL free cholesterol after the infusion of different doses of the agent. As the product appears to be well tolerated and non-immunogenic, ongoing phase II studies in patients are being awaited with interest to obtain a ‘proof of principle’ for ‘HDL therapy’.

High-Density Lipoproteins Induce Transforming Growth Factor-β2 Expression in Endothelial Cells

Background—HDL is endowed with cardiovascular protective activities. In addition to its role in reverse cholesterol transport, HDL influences different functions of endothelial cells. In the present study, we investigated in endothelial cells the genes involved in inflammation modulated by HDL. Methods and Results—Through cDNA array analysis, transforming growth factor (TGF)-&bgr;2 appeared to be a gene responsive to HDL treatment in endothelial cells. Quantitative real-time polymerase chain reaction confirmed that HDL subfraction 3 selectively induces TGF-&bgr;2 mRNA expression and protein release, whereas TGF-&bgr;1 and TGF-&bgr;3 were not affected. This effect was mainly PI3K/Akt dependent. Lysosphingolipids present in HDL such as sphingosine 1 phosphate and sphingosylphosphorylcholine mimicked the effects of the whole HDL. These results were confirmed in vivo in transgenic mice overexpressing human apolipoprotein (apo) A-I. Compared with apoA-I–knockout mice, phospho-Akt, phospho-ERK1/2, and TGF-&bgr;2 expression was increased in the aorta of transgenic mice overexpressing human apoA-I. In addition, the expression of phospho-Smad2/3, the transcription factor activated by TGF-&bgr;, is increased in transgenic mice compared with knockout mice. Conclusions—Because TGF-&bgr; possesses antiinflammatory properties and stabilizes the plaque, the results of the present work suggest a novel target for the antiatherosclerotic effect of HDL.

Hypolipidaemic and anti-atherosclerotic effects of lupin proteins in a rabbit model

The biological activities of a protein isolate from lupin (Lupinus albus) were studied in a rabbit model of atherosclerosis. Focal plaque development was induced at both common carotid arteries by perivascular injury. After surgery, animals were fed three different diets for 90 d, all with 1% cholesterol, 15% SFA and 20% protein; the protein source was casein (CAS), lupin proteins (LUP) or 50% CAS+50% LUP (CAS+LUP). Lower cholesterolaemia was detected in the LUP v. the CAS group at 60 and 90 d of treatment (-40·3 and -33·5%, respectively; P<0·05). Cryosection analyses of the carotids indicated a significant reduction in focal lesion progression in the LUP v. the CAS group (-37·4%; P<0·05). In summary, in a rabbit model of atherosclerosis, a protein isolate from L. albus reduced cholesterolaemia and exerted a remarkable protective activity against atherosclerosis progression.