Marta Marchesi

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.

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.

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.

Hypolipidemic effect of dietary pea proteins: Impact on genes regulating hepatic lipid metabolism

Controversial data on the lipid-lowering effect of dietary pea proteins have been provided and the mechanisms behind this effect are not completely understood. The aim of the study was to evaluate a possible hypolipidemic activity of a pea protein isolate and to determine whether pea proteins could affect the hepatic lipid metabolism through regulation of genes involved in cholesterol and fatty acid homeostasis. Rats were fed Naths hypercholesterolemic diets for 28 days, the protein sources being casein or a pea protein isolate from Pisum sativum. After 14 and 28 days of dietary treatment, rats fed pea proteins had markedly lower plasma cholesterol and triglyceride levels than rats fed casein (p<0.05). Pea protein-fed rats displayed higher hepatic mRNA levels of LDL receptor versus those fed casein (p<0.05). Hepatic mRNA concentration of genes involved in fatty acids synthesis, such as fatty acid synthase and stearoyl-CoA desaturase, was lower in pea protein-fed rats than in rats fed casein (p<0.05). In conclusion, the present study demonstrates a marked cholesterol and triglyceride-lowering activity of pea proteins in rats. Moreover, pea proteins appear to affect cellular lipid homeostasis by upregulating genes involved in hepatic cholesterol uptake and by downregulating fatty acid synthesis genes.

The pathway-selective estrogen receptor ligand WAY-169916 reduces infarct size after myocardial ischemia and reperfusion by an estrogen receptor dependent mechanism.

Previous studies have shown that estrogen treatment protects the heart from reperfusion injury. The adverse effects of long-term estrogen treatment limit its clinical use and emphasize the need for the development of specific pharmacological interventions such as pathway-selective estrogen receptor (ER) ligands. Pathway-selective ER ligands are compounds that retain estrogens anti-inflammatory ability, but they are devoid of conventional estrogenic action. In the present study, the pathway-selective ER ligand WAY-169916 was assessed for its cardioprotective potential in an in vivo model of ischemia-reperfusion injury. Anesthetized, ovariectomized rabbits were administered WAY-169916 (1 mg/kg), 17β-estradiol (E2; 20 μg/rabbit), or vehicle intravenously 30 minutes before a 30-minute occlusion and 4 hours of reperfusion. Acute treatment with either WAY-169916 or E2 resulted in a decrease in infarct size, expressed as a percent of area at risk (WAY-169916, 21.2 ± 3.3; P < 0.001 and E2, 18.8 ± 1.7; P < 0.001) compared with vehicle 59.4 ± 5.4). Pretreatment with estrogen receptor antagonist ICI 182,780 significantly limited the infarct size sparing effect of both WAY-169916 and E2 when expressed as a percent of the risk region (WAY 169916, 47.4 ± 4.4; E2, 53.01 ± 5.0). The results demonstrate that WAY-169916 protects the heart against ischemia-reperfusion injury through an ER-dependent mechanism.

The intracellular quality control system down-regulates the secretion of amyloidogenic apolipoprotein A-I variants: A possible impact on the natural history of the disease

Hereditary systemic amyloidosis caused by apolipoprotein A-I variants is a dominantly inherited disease characterised by fibrillar deposits mainly localized in the kidneys, liver, testis and heart. We have previously shown that the apolipoprotein A-I variant circulates in plasma at lower levels than the wild-type form (Mangione et al., 2001; Obici et al., 2004) thus raising the possibility that the amyloid deposits could sequester the circulating amyloidogenic chain or that the intracellular quality control can catch and capture the misfolded amyloidogenic chain before the secretion. In this study we have measured plasma levels of the wild-type and the variant Leu75Pro apolipoprotein A-I in two young heterozygous carriers in which tissue amyloid deposition was still absent. In both cases, the mutant was present at significantly lower levels than the wild-type form, thus indicating that the low plasma concentration of the apolipoprotein A-I variant is not a consequence of the protein entrapment in the amyloid deposits. In order to explore the cell secretion of amyloidogenic apolipoprotein A-I variants, we have studied COS-7 cells expressing either wild-type apolipoprotein A-I or two amyloidogenic mutants: Leu75Pro and Leu174Ser. Quantification of intracellular and extracellular apolipoprotein A-I alongside the intra-cytoplasmatic localization indicates that, unlike the wild-type protein, both variants are retained within the cells and mainly accumulate in the endoplasmic reticulum. The low plasma concentration of amyloidogenic apolipoprotein A-I may therefore be ascribed to the activity of the intracellular quality control that represents a first line of defence against the secretion of pathogenic variants.

Cholesterol-lowering effect of dietary Lupinus angustifolius proteins in adult rats through regulation of genes involved in cholesterol homeostasis

In the absence of a clear indication from previous studies, a rat study was designed to evaluate a possible hypolipidaemic effect of Lupinus angustifolius (blue lupin) proteins. Rats were fed for 28days Naths hypercholesterolaemic diets containing 20% casein or blue lupin proteins. After 14 and 28days of dietary treatment, blue-lupin-fed rats had markedly lower plasma total cholesterol levels than rats fed casein (-53.0% and -55.3%, respectively, p<0.0005). No significant differences were instead observed for triglyceride and HDL-cholesterol levels between the two groups. Lupin-protein-fed rats displayed higher hepatic mRNA levels of SREBP-2, a major transcriptional regulator of intracellular cholesterol levels, and CYP7A1, the rate-limiting enzyme in bile acid biosynthesis (p<0.05). In conclusion, the present study demonstrates a marked cholesterol-lowering activity of proteins from L. angustifolius in rats. Moreover, blue lupin proteins appear to affect cellular lipid homeostasis by up-regulating SREBP-2 and CYP7A1 genes.

Lupin (Lupinus albus) protein isolate (L-ISO) has adequate nutritional value and reduces large intestinal weight in rats after restricted and ad libitum feeding.

Background: A protein isolate from white lupin (Lupinus albus; L-ISO) has potential as a novel human food ingredient, but its nutritional effects are unknown. Methods: We evaluated protein quality and effects on body composition in rats of isoenergic diets of L-ISO, lactalbumin, or casein with both restricted (10-day) and ad libitum (28-day)intake. The diets were equivalent in protein per se, but supplementation was used to balance essential amino acid levels. Results: In both studies, the rats consumed similar amounts of each diet, and no effect of diet on the gain:feed ratio was observed – though gain:N ratio and net protein utilization were slightly lower for the L-ISO diet. Lower large intestinal weights after the L-ISO than after the lactalbumin diet were observed in both studies. The L-ISO diet resulted in lowered body fat percentage in the 10-day study but in an elevated level in the 28-day study. Liver composition (DNA, RNA, glycogen, and fat) and plasma levels of some amino acids (His, Thr, Ala, Pro, Tyr, Val and Met) were affected by diet, but no effects on plasma lipid, glucose, or uric acid were observed. Conclusion: The L-ISO diet did not affect feed intake and has adequate nutritional quality in rats whilst modifying large intestinal weight in a potentially beneficial manner – suggesting potential for this protein in human nutrition.

Effect of the combinations between pea proteins and soluble fibres on cholesterolaemia and cholesterol metabolism in rats

Many functional foods and dietary supplements have been reported to be beneficial for the management of dyslipidaemia, one of the major risk factors for CVD. Soluble fibres and legume proteins are known to be a safe and practical approach for cholesterol reduction. The present study aimed at investigating the hypocholesterolaemic effect of the combinations of these bioactive vegetable ingredients and their possible effects on the expression of genes regulating cholesterol homeostasis. A total of six groups of twelve rats each were fed, for 28 d, Naths hypercholesterolaemic diets, differing in protein and fibre sources, being, respectively, casein and cellulose (control), pea proteins and cellulose (pea), casein and oat fibres (oat), casein and apple pectin (pectin), pea proteins and oat fibres (pea+oat) and pea proteins and apple pectin (pea+pectin). Administration of each vegetable-containing diet was associated with lower total cholesterol concentrations compared with the control. The combinations (pea+oat and pea+pectin) were more efficacious than fibres alone in modulating cholesterolaemia ( - 53 and - 54%, respectively, at 28 d; P< 0·005). In rats fed the diets containing oat fibres or apple pectin, alone or in combination with pea proteins, a lower hepatic cholesterol content (P< 0·005) and higher hepatic mRNA concentrations of CYP7A1 and NTCP were found when compared with the control rats (P< 0·05). In summary, the dietary combinations of pea proteins and oat fibres or apple pectin are extremely effective in lowering plasma cholesterol concentrations in rats and affect cellular cholesterol homeostasis by up-regulating genes involved in hepatic cholesterol turnover.