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, Nath's 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.

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 Nath's 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.

Rosuvastatin does not affect human apolipoprotein A-I expression in genetically modified mice: a clue to the disputed effect of statins on HDL.

BACKGROUND AND PURPOSE: Besides a significant reduction of low-density lipoprotein (LDL) cholesterol, statins moderately increase high-density lipoprotein (HDL) levels. In vitro studies have indicated that this effect may be the result of an increased expression of apolipoprotein (apo)A-I, the main protein component of HDL. The aim of the present study was to investigate in vivo the effect of rosuvastatin on apoA-I expression and secretion in a transgenic mouse model for human apoA-I. EXPERIMENTAL APPROACH: Human apoA-I transgenic mice were treated for 28 days with 5, 10 or 20 mg·kg(-1) ·day(-1) of rosuvastatin, the most effective statin in raising HDL levels. Possible changes of apoA-I expression by treatment were investigated by quantitative real-time RT-PCR on RNA extracted from mouse livers. The human apoA-I secretion rate was determined in primary hepatocytes isolated from transgenic mice from each group after treatment. KEY RESULTS: Rosuvastatin treatment with 5 and 10 mg·kg(-1) ·day(-1) did not affect apoA-I plasma levels, whereas a significant decrease was observed in mice treated with 20 mg·kg(-1) ·day(-1) of rosuvastatin (-16%, P < 0.01). Neither relative hepatic mRNA concentrations of apoA-I nor apoA-I secretion rates from primary hepatocytes were influenced by rosuvastatin treatment at each tested dose. CONCLUSIONS AND IMPLICATIONS: In human apoA-I transgenic mice, rosuvastatin treatment does not increase either apoA-I transcription and hepatic secretion, or apoA-I plasma levels. These results support the hypothesis that other mechanisms may account for the observed HDL increase induced by statin therapy in humans.

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.

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 Nath's 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.

HDL therapy for the treatment of cardiovascular diseases.

High-density lipoprotein (HDL) therapy is an emerging area of therapeutic development in the cardiovascular field, aimed at supplementing and improving the vascular benefit exerted by current treatments. Several studies have clearly established that HDL-cholesterol (HDL-C) levels are a potent and independent epidemiologic risk factor for cardiovascular diseases; moreover, studies in animal models have established that HDL-C raising interventions, such as over-expression of apolipoprotein A-I (apoA-I), the major HDL protein component, reduce the extent of atherosclerosis. In vitro and in vivo experiments have provided mechanistic explanations for the atheroprotective effects of HDL. HDL not only mediates reverse cholesterol transport, but also exerts antioxidant, anti-inflammatory, antithrombotic and vasodilatory effects. These multiple antiatherosclerotic properties provide an excellent rationale for designing therapeutic interventions targeted at enhancing HDL/apoA-I levels, but also for considering a direct administration of HDL-apoA-I in a variety of cardiovascular diseases. We provide an overview and an update of all therapeutic applications of synthetic HDL tested in animal models or in clinical trials. HDL therapy has proven to be effective in promoting atherosclerosis regression not only in experimental models, but also in humans, whereas applications to other areas of cardiovascular disease have only, up to now, been tested in animal models.

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.

Dose-related effects of repeated ETC-216 (recombinant apolipoprotein A-I Milano/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/POPC complex attenuates post-ischemic ventricular dysfunction in the isolated rabbit heart.

Irreversible myocardial injury is a potential consequence of coronary artery revascularization. Reperfusion leads to the production of oxidized products that can damage myocardium. High-density lipoproteins (HDL) are effective at removing oxidized lipids. We hypothesized that a synthetic HDL preparation, comprising recombinant apolipoprotein A-I(Milano) (apoA-I(M)) complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) (apoA-I(M)/POPC) would protect the heart from reperfusion injury. The ex vivo model consisted of rabbit hearts perfused by the Langendorff method. Hearts were equilibrated with Krebs-Henseleit buffer (10 min), pretreated with either apoA-I(M)/POPC (0.45 mg/mL) or vehicle (10 min), subjected to global ischemia (30 min) and reperfused for 60 min. ApoA-I(M)/POPC (n=7) prevented the left ventricular end-diastolic pressure elevation observed in the vehicle group (n=6) at the end of reperfusion (p<0.05). During reperfusion, coronary artery perfusion pressure increased in the controls (p<0.001), but not with apoA-I(M)/POPC. ApoA-I(M)/POPC reduced the release of creatine kinase at the end of the ischemic period (p<0.001). It also reduced cardiac left ventricle muscle lipid hydroperoxides by 46% (p<0.05). Direct comparison of the antioxidant potential indicated that recombinant apoA-I(M) was much more potent than apoA-I in attenuating low-density lipoprotein oxidation. Electron microscopy showed that apoA-I(M)/POPC prevented mitochondrial granulation, disorganization and sarcomere contraction band formation indicative of reperfusion injury. The apoA-I(M)/POPC complex thus appears to reduce reperfusion injury under global ischemic conditions, and may therefore have therapeutic application in the reduction of myocardial ischemia.

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 estrogen's 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), 17beta-estradiol (E2; 20 microg/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.

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.

Therapeutic use of the high-density lipoprotein protein and peptides.

High-density lipoprotein (HDL) therapy is a novel and emerging area of therapeutic development in the cardiovascular field. It attempts to supplement and improve the vascular benefit exerted by other agents that are active on lipid metabolism, for example, hypolipidaemic drugs. Furthermore, it takes advantage of the novel techniques of coronary evaluation. A number of reports have examined the potential therapeutic properties of the synthetic HDLs prepared by complexing recombinant apolipoprotein (apo) A-I(Milano), a variant form of native apoA-I, with phospholipids. The availability of synthetic HDL complexes containing recombinant apoA-I(Milano) has opened up a new era of therapeutic management for coronary disease. HDL formulations of recombinant apoA-I(Milano)-phospholipid complexes have clearly shown rapid regression of a focal carotid atheroma as well as powerful protection from myocardial infarction in a rabbit model. In a pilot study, ETC-216 showed a significant reduction in coronary plaque burden after five weekly treatments, assessed by intravascular ultrasound in patients with acute coronary syndrome. Other therapeutic options of HDL therapy have recently became available.

High-density lipoproteins induce transforming growth factor-beta2 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)-beta2 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-beta2 mRNA expression and protein release, whereas TGF-beta1 and TGF-beta3 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-beta2 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-beta, is increased in transgenic mice compared with knockout mice. CONCLUSIONS: Because TGF-beta possesses antiinflammatory properties and stabilizes the plaque, the results of the present work suggest a novel target for the antiatherosclerotic effect of HDL.

Apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine complex (ETC-216) protects the in vivo rabbit heart from regional ischemia-reperfusion injury.

Ex vivo studies demonstrated that a synthetic high-density lipoprotein (HDL) comprised of a complex of recombinant apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine protects the isolated rabbit heart from reperfusion injury. Therefore, we sought to determine whether a pharmaceutical preparation of this complex, ETC-216, was cardioprotective in an in vivo model of left anterior descending artery (LAD) occlusion and reperfusion. Initially, ETC-216 (100 mg/kg) was tested in acute (one-treatment) and chronic (two-treatment) i.v. administrations. ETC-216-treated rabbits developed smaller infarcts expressed as percentage of area at risk (p <0.01) compared with vehicle treatments. No differences were noted between chronic and acute administration. Therefore, ETC-216 (10, 3, or 1 mg/kg) or equivalent vehicle volumes were acutely infused. Compared with vehicle, ETC-216 reduced infarct size as a percentage of the area at risk at 10 (p <0.0005) and 3 mg/kg (p <0.05). No significant differences occurred at 1 mg/kg. To determine whether ETC-216 could protect the heart after initiation of ischemia, the synthetic HDL (10 mg/kg) was infused intravenously beginning 5 min before the end of 30 min of LAD occlusion. Infarct size as percentage of the area at risk was 31.6 +/- 3.0 (ETC-216) versus 49.5 +/- 2.5 (vehicle) (p <0.001), and as percentage of left ventricle was 19.7 +/- 1.6 (ETC-216) versus 34.1 +/- 2.3 (vehicle) (p <0.0005). Electron microscopy demonstrated that ETC-216 prevented irreversible cardiac damage as assessed by mitochondrial granulation and sarcomere contraction band formation. These findings suggest ETC-216 reduces reperfusion injury and may have utility for coronary artery revascularization procedures.

17Beta-estradiol as a receptor-mediated cardioprotective agent.

Cardiac tissue that undergoes an ischemic episode exhibits irreversible alterations that become more extensive upon reperfusion. Estrogen treatment has been reported to protect against reperfusion injury, but the mechanism remains unknown. The cardioprotective effects of 17beta-estradiol, a biologically active form of the hormone, and 17alpha-estradiol were assessed in an in vivo occlusion-reperfusion model. Anesthetized, ovariectomized rabbits were administered 17beta-estradiol (20 microg), 17alpha-estradiol (1 mg), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending (LAD) coronary artery followed by 4 h of reperfusion. Infarct size as a percentage of area at risk decreased in the 17beta-estradiol-treated group (18.8 +/- 1.7) compared with 17alpha-estradiol (41.9 +/- 4.8; P < 0.01) or vehicle groups (48 +/- 5.5; P < 0.001). Similar results were obtained when infarct size was expressed as a percentage of total left ventricle. The second objective of the study was to assess fulvestrant (Faslodex, ICI 182,780), an estrogen receptor antagonist, for its effects on infarct size in ovariectomized female rabbits treated with 17beta-estradiol. ICI 182,780 was administered intravenously 1 h before the administration of 17beta-estradiol (20 microg) or vehicle. The hearts were subjected to 30-min LAD coronary artery occlusion and 4 h of reperfusion. Pretreatment with ICI 182,780 significantly limited the infarct size sparing effect of 17beta-estradiol when expressed as a percentage of the risk region (53.0 +/- 5.0). The results indicate that 17beta-estradiol protects the heart against ischemia-reperfusion injury and that the observed cardioprotection is mediated by the estrogen receptor.

Recombinant apolipoprotein A-I(Milano) infusion into rabbit carotid artery rapidly removes lipid from fatty streaks.

Apolipoprotein A-I(Milano) (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.