Analysis of Serum Cholesterol Efflux Capacity in a Minipig Model of Nonischemic Heart Failure.

AIM: Circulating levels of high-density lipoprotein cholesterol (HDL-C) are decreased in patients with heart failure (HF). We tested whether HDL-C serum levels are associated with cardiac contractile dysfunction in a minipig HF model. METHODS: Blood samples were collected from 13 adult male minipigs: 1) before pacemaker implantation, 2) 10 days after surgery, and 3) 3 weeks after high-rate LV pacing. Serum cholesterol efflux capacity (CEC), an index of HDL functionality, was assessed through four mechanisms: ATP Binding Cassette transporter A1 (ABCA1), ATP Binding Cassette transporter G1 (ABCG1), Scavenger Receptor-Class B Type I (SR-BI) and Passive Diffusion (PD). RESULTS: HDL-C serum levels significantly decrease in minipigs with HF compared with baseline (p＜0.0001). Serum CEC mediated by PD and SR-BI, but not ABCA1 or ABCG1, significantly decrease in animals with HF (p＜0.05 and p＜0.005, respectively). DISCUSSION: HDL-C serum levels and partial serum CEC reduction may play a pathophysiological role in the cardiac function decay sustained by high-rate LV pacing, opening new avenues to understand of the pathogenesis of nonischemic myocardial remodeling.

Phenotypic variability in 4 homozygous familial hypercholesterolemia siblings compound heterozygous for LDLR mutations.

BACKGROUND: Homozygous familial hypercholesterolemia is a rare clinical phenotype with a variable expression, which is characterized by extremely elevated plasma low-density lipoprotein (LDL), tendon and skin xanthomas, and a progressive atherosclerosis. In 95% of patients, homozygous familial hypercholesterolemia is due to mutations in low-density lipoprotein receptor (LDLR) gene, which abolish (receptor-negative) or greatly reduce (receptor-defective) LDLR function. OBJECTIVE: The objective of the study was the molecular and phenotypic characterization of 4 siblings with severe hypercholesterolemia. METHODS: The major LDL-related genes (LDLR, APOB, PCSK9, ANGPTL3, APOE, and APOC3) were sequenced. LDLR messenger RNA, isolated from leukocytes, was reverse transcribed and sequenced. RESULTS: The index cases were 24-year-old identical twin sisters with long-standing tendon xanthomas and high low-density lipoprotein cholesterol (LDL-C ∼10 mmol/L) but no coronary heart disease. They were carriers of 2 LDLR mutations: (1) a previously reported mutation [p.(G335S)] inherited from the mother who had LDL-C level within normal range; (2) a novel 24 bp deletion in exon 8/intron 8 junction inherited from the hypercholesterolemic (LDL-C 6.1 mmol/L) father. The deletion allele encodes an messenger RNA with a partial deletion of exon 8, whose translation product has an in-frame deletion of 17 amino acids [p.(Glu380_Gly396del)]. Family screening revealed that the 2 siblings of the twin sisters were also compound heterozygotes but had much lower LDL-C levels (8.2 and 7.1 mmol/L). The sequence of potential modifying genes showed that the 2 siblings and the mother of the twin sisters were heterozygous for a rare missense variant of apoB [p.(S2429T)], which might have an LDL-lowering effect. CONCLUSIONS: We report a rare event of 4 siblings found to be compound heterozygotes for 2 LDLR gene mutations but showing a different phenotype severity. The less severely affected siblings were carriers of a rare apoB missense variant.

Detection of biomarkers for inflammatory diseases by an electrochemical immunoassay: the case of neopterin.

An electrochemical immunoassay for neopterin was developed using recently produced specific antibodies immobilized to protein A-coated magnetic beads in combination with differential pulse voltammetry and screen-printed array of electrodes. Neopterin-alkaline phosphatase conjugate was used as label in a competitive assay format. Multiplexed analysis of neopterin was demonstrated by replacing the traditional ELISA with electrochemical detection and the traditional plastic wells with screen-printed array of electrodes. The optimized electrochemical method, based on polyclonal antibodies, reached a limit of detection of 0.008 ng/mL with an average RSD %=10. Serum samples collected from patients with sepsis, healthy volunteers and other patients without a confirmed clinical diagnosis were also analyzed. The obtained results, compared with those of a commercial ELISA kit, had a significant correlation, showing the possibility to distinguish among the serum samples from ill or healthy subjects.

Early senescence in heterozygous ABCA1 mutation skin fibroblasts: a gene dosage effect beyond HDL deficiency?

PURPOSE: Homozygous ABCA1 gene mutation causes Tangier disease (TD). The effects reported in heterozygous state regard plasma HDL, cell cholesterol efflux and coronary artery disease. We investigated whether in vitro replicative skin fibroblast senescence shown in TD proband (Hom), his father (Het), and in a healthy control might be induced in a "gene-dosage way". METHODS: Senescence was evaluated by staining test for ß-Galactosidase and telomere length (TL) on fibroblast DNA at different replicative stages. ABCG1 and LDLR (low density lipoprotein receptor) gene expression was also evaluated. RESULTS: Hom cells showed early senescent morphology and reduced growth at all passages in vitro. The cell positive percentage for ß-Galactosidase test was highly increased in Hom compared to Het cells at late replicative status (66.1% vs 41.3% respectively). TL was significantly shorter at high stage either in Hom (p<0.0001) or in Het (p<0.005). At early replication cycles ABCG1 gene expression was about 3-fold higher in Hom compared to Het cells (0.44 vs 0.14 arbitrary unit). CONCLUSIONS: ABCA1 gene mutation may have "gene-dosage way" effect on in vitro fibroblast senescence. Furthermore, increased ABCG1 and LDLR gene expression could highlight a role of ABCA1 on cytoskeleton regulation associated to cell cholesterol metabolism.

Inflammation blood and tissue factors of plaque growth in an experimental model evidenced by a systems approach.

PURPOSE: The multifactorial pathogenesis of coronary atherosclerotic lesion formation has been investigated in a swine model of high cholesterol diet induced atherogenesis and data processed by a systems approach. METHODS: Farm pigs were fed on standard or high cholesterol diet of 8 and 16 weeks duration. Plasma assessment of total cholesterol, HDL, LDL, and ELISA of some cytokines and ICAM-1 were performed on baseline and end-diet samples. Segments of the right coronary artery were incubated for 24 h in serum-free medium to collect secreted proteins and their expression analyzed by mass spectrometry. Data of plasma and tissue factors were processed by a statistical systems inference approach: both histologic parameters of coronary intimal thickness (IT) and of lesion area (LA) were chosen as dependent variables (coronary atherosclerotic burden). RESULTS: Relations among plasma adhesion molecules, cytokines, lipoproteins, tissue proteins and histology indexes were integrated in a model regression scheme. Bayesian model averaging (BMA) variable selection was chosen as a method to identify relevant factors associated to atherosclerotic burden: TNFα was identified as an associated plasma marker, oxLDL and HDL as relevant lipoproteins; macrophage function related antioxidant Catalase enzyme, lysosome associated Cathepsin D, S100-A10, and Transforming growth factor-beta-induced protein ig-h3 were identified and selected as associated to atherogenesis outcome. CONCLUSIONS: The results of this systems approach are consistent with the hypothesis that, in high cholesterol diet-induced experimental atherogenesis, the interaction between plasma cytokines, lipoproteins and artery-specific proteins, influences lesion initiation and growth. In particular, some macrophage function related proteins are found significantly and positively associated to atherosclerotic burden, suggesting a novel molecular framework into the atherogenesis-inflammatory disorder.

Cognitive impairment and polidistrectual atherosclerotic disease in chylomicronemia syndrome: a case report.

A case of chylomicronemia syndrome is reported in a 72-year-old male with distinctive features of chronic pancreatic damage, severe hypertriglyceridemia, polidistrectual atherosclerosis and premature cognitive impairment. Although the patient had a positive history for recurrent episodes of pancreatitis the characteristic lesions of the hyperchylomicronemia syndrome, such as eruptive xanthomas and lipemia retinalis, were not present and splenomegaly could not be documented due to a previous post-traumatic splenectomy. Based on clinical phenotype, an apolipoprotein C-II deficiency was excluded by a fresh plasma infusion test, in which clarification of the patient plasma was not obtained. The absence of changes in the lipoprotein electrophoretic plasma after heparin infusion can be secondary to a lipoprotein lipase deficiency, a rare genetic disorder with an incidence of one per million. In relation to the resistance to diet and drugs, plasma exchange therapy was performed. After 3 years of this treatment there was no significant progression of atherosclerosis.

Modulation of cytochrome P450 enzymes in response to continuous or intermittent high-fat diet in pigs.

1. To date, no information has been available on the modulation of cytochrome P450 enzymes (CYPs) following the administration of a hyperlipidemic diet in pigs. 2. We investigated the potential modulation of xenobiotic-metabolizing CYPs in liver, heart and duodenum of pigs subjected to a high-fat/high-cholesterol diet for 2 months continuously (C-HFD) or on alternate weeks (A-HFD). 3. The administration of the high-fat diet resulted in considerably increased plasma cholesterol levels although the animals were still able to manage the lipid overload efficiently, and no sign of effective tissue inflammation occurred in livers. Plasma lipid profile and liver histology indicated a better adaptive response of the A-HFD pigs compared to the C-HFD group. We showed a post-transcriptional induction of hepatic CYP2E1 activity in C-HFD pigs and a transcriptional induction of hepatic CYP3As - especially in the A-HFD group. No further CYP modulation was observed in either liver or extra-hepatic tissues. 4. In conclusion, the administration of a high-fat diet in pigs resulted in limited effects on the drug metabolism system. The better adaptive response of A-HFD pigs compared to C-HFD pigs is a very interesting observation since the intermittent administration of the diet reflects the mode of human behavior more closely.

High density lipoprotein cholesterol in coronary artery disease: when higher means later.

AIM: Although high-density lipoprotein cholesterol (HDL-C) levels are inversely associated with cardiovascular risk, patients with elevated HDL-C also develop coronary artery disease (CAD) and cardiac events. We aimed to draw the clinical profile of CAD patients with elevated HDL-C and to assess the prognostic impact of elevated HDL-C. METHODS: We prospectively examined 2322 patients (age 67±10 years, 79% male) with chronic CAD, defined by >50% coronary stenosis and/or previous myocardial infarction. RESULTS: HDL-C levels were low (<35 mg/dL) in 736 patients (32%), normal (35-60 mg/dL) in 1464 (63%), and high (>60 mg/dL) in 122 (5%). Patients with elevated HDL-C were less frequently male (p<0.0001), smokers (p<0.0001), diabetic (p<0.0001), and obese (p<0.0015) than those with low or normal HDL-C, but were 3 and 5 years older, respectively (p<0.0001). During follow-up (median, 46 months) 143 patients died from cardiac causes and 80 developed a non-fatal infarction. Cardiac event-free survival was lower in patients with low compared to normal HDL-C (p<0.0001), but was not significantly different from that of patients with elevated HDL-C. The prognostic impact of low HDL-C was independent of age, sex, diabetes, LV function, extent of coronary stenoses, low density lipoprotein cholesterol, triglycerides, complete blood count, thyroid and renal function (p<0.0001). Conversely, the prognostic impact of elevated HDL-C disappeared (p>0.10) after adjustment for age. CONCLUSION: Patients with elevated HDL-C develop CAD and cardiac events as do those with low or normal HDL, but at a more advanced age.

Tangier disease: epidemiology, pathophysiology, and management.

Tangier disease is one of the most severe forms of familial high-density lipoprotein (HDL) deficiency. Since its discovery it has been diagnosed in about 100 patients and is characterized by severe plasma deficiency or absence of HDL, apolipoprotein A-I (apoA-I, the major HDL apolipoprotein) and by accumulation of cholesteryl esters in many tissues throughout the body. The biochemical signs of this condition are plasma HDL concentrations less than 5 mg/dL, low total plasma cholesterol (below 150 mg/dL), and normal or high plasma triglycerides. Tangier disease is caused by mutations in the 'ATP-Binding Cassette transporter A1' (ABCA1) gene, which encodes the membrane transporter ABCA1. This transporter plays a key role in the first step of reverse cholesterol transport, through which the efflux of free cholesterol from peripheral cells is transferred to lipid-poor apoA-I. The Tangier disease clinical phenotype is inherited as an autosomal recessive trait, the biochemical phenotype is inherited as an autosomal co-dominant trait. Nearly all the children affected by Tangier disease were identified on the basis of large, yellow-orange tonsils, while half of the adult patients affected by Tangier disease came to medical attention because of symptoms of neuropathy. Diagnosis in the remaining subjects was related to the clinical features of hepatomegaly, splenomegaly, premature myocardial infarction (about 30% of Tangier disease cases) or stroke, thrombocytopenia, anemia, gastrointestinal disorders, corneal opacities, hypocholesterolemia, low HDL cholesterol, or following a familial screening of Tangier patients. To date there is no specific treatment for Tangier disease. Old and recently designed drugs, known to increase HDL levels, have been shown to be ineffective in Tangier patients. The possible and more realistic therapeutic strategy should be designed to obtain a selective increase of mature HDL concentration to restore cholesterol efflux. Recently designed drugs like the cholesteryl ester transfer protein (CETP) inhibitors dalcetrapib and anacetrapib and reconstituted forms of HDL could be considered until the development of gene therapy.

Myeloperoxidase modulation by LDL apheresis in familial hypercholesterolemia.

BACKGROUND: Myeloperoxidase (MPO) is a marker of plaque vulnerability and a mechanistic bridge between inflammation and cardiovascular disease, and thus is a suitable target for therapeutic strategy against cardiovascular disease. METHODS: Since hypercholesterolemia is associated with atherosclerosis and inflammation, we tested whether MPO serum levels were up-regulated in Familial Hypercholesterolemia (FH) and whether acute reduction of total cholesterol (TC) would also reduce MPO concentration. FH subjects undergoing LDL-apheresis (LDL-A) treatment are a paradigmatic clinical model where TC rapidly plunges from extremely high to extremely low levels after selective LDL removal, and then spontaneously rebounds to baseline conditions. This clinical setting allows multiple intra-patient observations at different plasma TC concentrations. We measured MPO levels in serum by ELISA tests, and in peripheral leukocytes by immunofluorescence, to learn whether they were affected by the changes in TC levels. Serum MPO was measured before and serially up to the 14th day following LDL-A. RESULTS: In both serum and peripheral leukocytes, MPO concentrations were i) higher than in sex- and age-matched healthy controls (p < 0.01); ii) decreased with TC reduction; iii) parallel with TC time course; iv) correlated with plasma TC. At regression analysis, plasma TC was the only variable considered that influenced MPO serum levels (ß 0.022 ± 0.010, p < 0.0001). CONCLUSIONS: In FH the MPO serum levels were modulated through changes in the TC concentrations carried out by LDL-A. Further study is needed to determine whether reduced MPO levels obtained by LDL-A could have any therapeutic impact.

Low high-density lipoprotein predicts death in patients with mild left ventricular dysfunction regardless of coronary atherosclerosis.

OBJECTIVE: Low plasma high-density lipoprotein (HDL)-cholesterol is a major risk factor for cardiovascular diseases. We investigated whether HDL-cholesterol levels had a prognostic value in patients with mild left ventricular dysfunction, irrespective of the presence of coronary disease. METHODS: The study included 686 consecutive patients hospitalized between January 2002 and December 2006 because of left ventricular dysfunction. All patients completed a lipid profile and underwent coronary angiography. Patients were followed for a mean period of 23 months, during which major events were recorded. RESULTS: Seventy-three percent of patients were New York Heart Association (NYHA) class I-II, with the mean values of left ventricular ejection fraction and left ventricular end-diastolic diameter being, respectively, 36.3 +/- 8.6% and 58.3 +/- 7.9 mm. Half of the patients (52%) had HDL values less than 40 mg/dl, 28% presented with HDL less than 35 mg/dl. In multivariable analysis, patients with HDL-cholesterol concentration less than 40 mg/dl showed higher risk for cumulative mortality (HR 1.77, P < 0.05) and for cardiac death (HR 2.06, P < 0.05). This higher risk was also observed in patients with low HDL-cholesterol levels but without significant coronary stenosis. The inclusion of the C-reactive protein (CRP) inflammation marker into the model highly improved the power of death prediction. CONCLUSION: In patients with left ventricular dysfunction, regardless of the presence of coronary atherosclerosis, lower HDL-cholesterol was a strong and independent predictor of both cardiac and all-cause death.

HDL: the 'new' target of cardiovascular medicine.

Clinical, experimental and epidemiological research has shown the undeniable causal relationship between low HDL plasma concentrations and cardiovascular disease. Low HDL levels are present in about 10% of the general population and represent the most frequent form of dyslipidemia in patients with coronary disease. Reduced HDL concentrations seem to be unable to eliminate efficiently the cholesterol excess at vascular wall level, contributing to the onset of the inflammatory response that typically occurs in the pathogenesis of atherosclerosis right from its earliest stages. The results of numerous studies quite convincingly suggest that HDL is capable of exerting anti-inflammatory activity either directly or by modulating the expression of a number of acute phase proteins. Although the therapeutic options currently available for raising HDL levels still show modest efficacy, both in experimental and pre-clinical fields, genetic investigation and specifically aimed pharmacological treatment have produced more encouraging results, shedding some light on the concrete possibility of being able to treat this disease in the very near future.

HDL inflammation and atherosclerosis: current and future perspectives.

The role of high-density lipoprotein (HDL) in the genesis and evolution of cardiovascular disease is a topical and interesting issue. Reduced HDL concentrations appear to be unable to efficiently eliminate the cholesterol excess at the vascular wall level, contributing to the onset of the inflammatory response that typically occurs in the pathogenesis of atherosclerosis from its earliest stages. In the last decade, many studies have explored the possibility of reducing cardiovascular risk through modulation of HDL levels, glimpsing new fascinating therapeutic horizons. This review summarizes recent findings on HDL and cardiovascular disease, mainly with an educational objective, considering the biochemical, cellular and molecular aspects of these particles.

Inflammatory markers and serum lipids in idiopathic dilated cardiomyopathy.

Coronary microcirculation is impaired in idiopathic dilated cardiomyopathy (IDC), possibly because of endothelial dysfunction. High-density lipoproteins (HDLs) have the potential to regulate endothelial function and modulate inflammation and the innate immune response. This study investigated whether reduced HDLs, concomitantly with the activation of inflammation, are associated with IDC. Fifty-five patients with IDC, without evidence of other organ or systemic, chronic, or recurrent diseases, were compared with 55 healthy controls for HDLs and complete lipid profiles, C-reactive protein, C3 and C4 complement fractions, soluble intercellular adhesion molecule-1 and soluble endothelial leukocyte adhesion molecule-1, haptoglobin, and ceruloplasmin. Patients with IDC differed from controls, with lower HDL levels, lower apolipoprotein A-I and A-II levels, and higher triglyceride levels, but not on total and low-density lipoprotein cholesterol, apolipoprotein B, or lipoprotein(a). In addition, all measured inflammation markers were significantly greater in patients with IDC than in controls and were negatively correlated with HDLs. A strong and independent association with IDC was found for age, soluble intercellular adhesion molecule-1, and HDLs that, when categorized as <40 or >40 mg/dl, showed the strongest association (prevalence odds ratio 0.10, p <0.0005) with the disease. In conclusion, the data here reported on reduced HDLs and increased endothelial inflammatory activation and the linear negative correlation between HDLs and inflammation markers, particularly soluble intercellular adhesion molecule-1, could suggest a role for HDLs in the endothelial-microvascular dysfunction seen in IDC.

[High-density lipoproteins: the "new" target of cardiovascular medicine]. / Lipoproteine ad alta densità: il "nuovo" target della medicina cardiovascolare.

Experimental, clinical and epidemiological researches have shown the incontestable causal relationship between low high-density lipoprotein (HDL) plasma concentrations and cardiovascular pathology on an atherosclerotic basis. Low HDL levels characterize about 10% of the general population and they represent the most frequent dyslipidemia in patients with coronary artery disease. Reduced HDL concentrations would be unable to effectively eliminate the cholesterol excess at the vascular wall, contributing to the inflammatory phenomena that characterize the pathogenesis of atherosclerosis since its initial phases. Results of numerous studies reasonably allow to suppose that HDL are able to exert, also directly, anti-inflammatory actions through the modulation of expression of diverse acute phase proteins. Although the today available therapeutic options aiming to increase HDL levels still show a modest effectiveness, in the experimental and pre-clinical field, the results of genetic investigations and pharmacological interventions have given more encouraging results, making nearer the possibility of treating this pathology concrete.

Human myotonic dystrophy protein kinase effect in S. cerevisiae.

Human myotonic dystrophy protein kinase (DMPK), the product of the myotonic dystrophy (DM) locus, is a member of a novel class of multidomain serine-threonine protein kinases, which interacts with members of the Rho family of small GTPases. DMPK has been shown to affect the cell growth, size and shape in different organisms, from fission yeast to man, but its physiological role is still unclear. We examined the effect of the overexpression of two forms of human DMPK, full-length (DMFL) and a C-terminal truncated form (DMT) on the growth and cell morphology of S. cerevisiae, which possesses a DMPK homologous gene (CBK1) important for polarized growth and cell division. We report that the overexpression of either forms of human DMPK did not complement the CBK1 function in the haploid strain WR208-1a, deleted for CBK1. The truncated form, but not the full length one, slowed down growth rate and induced elongation of the haploid wild type strain CBK1. Similar results were obtained in the diploid wild type strain RS112 of S. cerevisiae where also the full-length form was effective. These effects were abolished when either DMFL or DMT were mutated in the ATP binding site (K100R mutation), suggesting that the kinase activity of DMPK is required. Interestingly, DMPK localization in yeast is similar to that of Cbk1 protein suggesting that it might affect a pathway, which regulates cell morphogenesis and progression through cell cycle, possibly involving CBK1.

[Prognostic value of high-density lipoproteins in the cardiology unit]. / Valore prognostico delle lipoproteine ad alta densità nella corsia cardiologica.

BACKGROUND: Clinical and epidemiological studies have shown an inverse and independent association between high-density lipoproteins (HDL) and risk of developing coronary artery disease. The aim of this study was to estimate the prevalence of the phenotype characterized by low HDL values and to assess the impact of HDL plasma levels on the prognosis of patients hospitalized in the cardiologic unit of our Institute, during 30 months of follow-up. METHODS: Between February 1999 and February 2002, 1169 patients (778 men and 391 women) who had undergone hospitalization, were enrolled in a cardiovascular registry. The lipid profile was evaluated for all patients; a subgroup (n = 626) underwent coronary angiography. Patients were followed up for a mean period of 30 months, during which the frequency of revascularization procedures (coronary angioplasty or bypass), myocardial infarction and death were recorded. RESULTS: Nearly half of the subjects (45%, of which 77% men) showed HDL values < 40 mg/dl, and 25% of the population (80% men) were characterized by extremely reduced HDL concentrations (< or = 35 mg/dl). Patients with coronary atherosclerosis showed HDL levels lower than those of subjects with negative angiography (41.0 +/- 10.8 vs 46.6 +/- 10.9 mg/dl, p < 0.0005). An inverse relationship was found between HDL and coronary artery disease: the risk of developing the illness decreased by 4% for each increase of 1 mg/dl in HDL (p < 0.005); in males < 60 years and females < 65 years, the coronary artery disease risk association increased by 3% (p < 0.005). The risk of interventions decreased by 2% for each increase of 1 mg/dl in HDL (p < 0.01), both in the total population and in males < 60 years and females < 65 years. Finally, patients with HDL < 40 mg/dl showed a higher risk of revascularization interventions (+25%, p < 0.01) in comparison to the subjects with HDL > 40 mg/dl. CONCLUSIONS: This study demonstrates: a) the high prevalence of the phenotype characterized by low plasma concentrations of HDL among patients hospitalized in a cardiologic unit; b) lower HDL values in subjects with coronary atherosclerosis compared to subjects with normal coronary angiography; c) the inverse relationship between HDL and coronary atherosclerosis; d) the prognostic value of HDL as a predictor of surgical revascularization.