Atherosclerosis In Familial Hypercholesterolemia Research Articles

MiRNA signature related to atherosclerotic lesion induced shear stress modifications in familial hypercholesterolemia patients with subclinical atherosclerosis: a bioinformatics systems biology study

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Institute of Health Carlos III (ISCIII) Background Patients with familial hypercholesterolemia (FH), even when appropriately treated, are at risk of premature clinical atherosclerotic cardiovascular disease (ASCVD) presentation. Although atherosclerosis (AT) is directly related to hemodynamic disturbances affecting the vascular geometry, there is scarce information on whether there is miRNA-mediated/flow-mediated modulation of atherosclerosis in FH. Purpose To identify a differential plasma miRNA signature related to fluid shear stress (FSS) in well controlled FH-patients with asymptomatic atherosclerosis. Methods FH-patients (N=49) with subclinical atherosclerosis, identified by computed tomographic angiography, and a reference cohort of non-FH subjects (N=32), both from a national FH-Registry, were included in the study. Coronary plaque volume and phenotype was analyzed by means of the QAngio CT imaging system. miRNAs associated to FSS were defined in silico by bioinformatics (KEGG, Cytoscape, GTEX portal, miRWalk, miRBase tools) and validated in plasma samples by RT-qPCR using TaqMan technology. Results By in silico analysis, 7 miRNAs targeting genes involved in features of functions (ROS production, oxidative stress, leukocyte transendothelial/migration-inflammation and vasodilation) of shear stress-induced cellular dysfunction and atherosclerosis (KEGG, hsa05418) were identified by miRWalk and miRBase databases. Of the 7 miRNAs, a 6-miRNA signature showed significantly lower plasma levels in FH-patients than in the non-FH group (P<0.01 for: miR-30e-5p, miR-378a-3p, let-7d-3p, and miR-550-5p; P=0.03 for miR-324-3p). In the FH-group, miR-324-3p, miR-30e-5p, miR-378a-3p and let-7d-3p significantly discriminated patients stratified by the plaque volume of non-calcified lesions (fibro-lipidic phenotype), using the median plaque volume as cut-off value. And even further, a high percentage (65-70%) of FH-subjects with low levels of the 4 miRNA had plaque volumes above the median. Interestingly, this miRNA-signature did not differentiate FH-patients stratified by the volume of calcified plaques. Conclusion FH-patients show a significantly lower content of an atherosclerosis/shear stress signature of 4 miRNAs targeting genes involved in subclinical atherosclerosis progression in FH.

Abstract 15197: Lipopolysaccharide-Nuclear Factor-kappa B Pathway and Lipoprotein Apheresis Effects in Patients With Familial Hypercholesterolemia and Coronary Artery Disease

Introduction: Inflammation may play an important role in atherosclerosis in familial hypercholesterolemia (FH). Lipopolysaccharide (LPS)-nuclear factor-kappa B (NF-κB) pathway is a routine signal process activated in inflammatory status. Hypothesis: We assessed the hypothesis that LPS-NF-κB axis is markedly activated and lipoprotein apheresis has an inhibitory effect on this pathway in patients with FH and coronary artery disease (CAD). Methods: In this matched case-control study a genetically diagnosed FH cohort who presented stable CAD (n=63) was compared with 63 non-FH CAD and 63 non-FH non-CAD controls matched by sex and age. Plasma LPS levels and NF-κB activity were compared among the three groups. In addition, we studied in vitro LPS-induced interleukin-6 production by mononuclear cells from 16 FH cases without previous statin use and compared them with their respective matched control groups. Subsequently, these 16 FH patients underwent lipoprotein apheresis. Blood samples were taken immediately before and regularly after apheresis for measuring LPS and NF-κB. Results: FH plus CAD had higher LPS levels and NF-κB activity than CAD and non-CAD controls (all p <0.01). LPS-induced interleukin-6 production by mononuclear cells of FH plus CAD was also much higher compared with CAD and non-CAD controls (both p <0.01). Moreover, plasma LPS levels ( p <0.001) and NF-κB activity ( p <0.01) were dramatically reduced after apheresis in FH patients. Conclusions: In conclusion, genetically confirmed FH patients with CAD had a marked activation of LPS-NF-κB axis, while lipoprotein apheresis significantly attenuated this key inflammatory pathway, suggesting that inflammation may be an important therapeutic target for FH patients.

P650Lipopolysaccharide-nuclear factor-kappa B pathway and lipoprotein apheresis effects in patients with familial hypercholesterolemia and coronary artery disease

Abstract Abstract Background Inflammation may play an important role in atherosclerosis in familial hypercholesterolemia (FH). Lipopolysaccharide (LPS)-nuclear factor-kappa B (NF-κB) pathway is a routine signal process activated in inflammatory status. Purpose This study aimed to examine the LPS-NF-κB axis status and the impact of lipoprotein apheresis (LA) on this pathway in patients with FH and coronary artery disease (CAD). Methods In this matched case-control study a genetically diagnosed FH cohort who presented stable CAD (n=63) was compared with 63 non-FH CAD and 63 non-FH non-CAD controls matched by sex and age. Plasma LPS levels and NF-κB activity were compared among the three groups. In addition, we studied in vitro LPS-induced interleukin-6 (IL-6) production by mononuclear cells from 16 FH cases without previous statin use and compared them with their respective matched control groups. Subsequently, these 16 FH patients underwent LA. Blood samples were taken immediately before and regularly after LA for measuring LPS and NF-κB. Results FH plus CAD had higher LPS levels and NF-κB activity than CAD and non-CAD controls (all p values <0.01). LPS-induced IL-6 production by mononuclear cells of FH plus CAD was also much higher compared with CAD and non-CAD controls (both p values <0.01). Moreover, plasma LPS levels (p<0.001) and NF-κB activity (p<0.01) were dramatically reduced after apheresis in FH patients. Conclusion Genetically confirmed FH patients with CAD had a marked activation of LPS-NF-κB axis, while LA significantly attenuated this key inflammatory pathway, suggesting that inflammation may be an important therapeutic target for FH patients.

Lipopolysaccharide-Nuclear Factor-Kappa B Pathway and Lipoprotein Apheresis Effects in Patients with Familial Hypercholesterolemia and Coronary Artery Disease

Background: Inflammation may play an important role in atherosclerosis in familial hypercholesterolemia (FH). Lipopolysaccharide (LPS)-nuclear factor-kappa B (NF-κB) pathway is a routine signal process activated in inflammatory status. However, the LPS-NF-κB axis status and the impact of lipoprotein apheresis (LA) on this pathway in patients with FH and coronary artery disease (CAD) has not been clarified. Methods: A genetically diagnosed FH cohort who presented stable CAD (n=63) was compared with 63 non-FH CAD and 63 non-FH non-CAD controls matched by sex and age. Plasma LPS levels and NF-κB activity were compared among them. In vitro LPS-induced interleukin-6 (IL-6) production by mononuclear cells from 16 FH cases without previous statin use was measured and compared with their respective matched control groups. Subsequently, these 16 FH patients underwent LA. Blood samples were regularly taken for measuring LPS and NF-κB. Findings: FH plus CAD had higher LPS levels and NF-κB activity than CAD and non-CAD controls (all p<0.01). LPS-induced IL-6 production by mononuclear cells of FH plus CAD was also much higher compared with control groups (both p<0.01). Moreover, plasma LPS levels (p<0.001) and NF-κB activity (p<0.01) were dramatically reduced after apheresis in FH patients. Interpretation: Genetically confirmed FH patients with CAD had a marked activation of LPS-NF-κB axis, while LA significantly attenuated this key inflammatory pathway, suggesting that inflammation may be an important therapeutic target for FH patients. Funding Statement: This work was partially supported by the Capital Health Development Fund [201614035] and CAMS Major Collaborative Innovation Project [2016-I2M-1-011] awarded to Dr. J.J.L. Declaration of Interests: R.D.S. has received honoraria related to consulting, speaker and/or research activities from Amgen, Akcea, Astra Zeneca, Biolab, Esperion, Kowa, Novo-Nordisk, Merck and Sanofi/Regeneron. Ethics Approval Statement: The study complied with the Declaration of Helsinki and Title 45, US Code of Federal Regulations, Part 46, Protection of Human Subjects, Revised November 13, 2001, effective December 13, 2001, and was approved by the hospital’s ethical review board (Fu Wai Hospital & National Center for Cardiovascular Diseases, Beijing, China). Each participant provided written, informed consent before enrollment.

Intracellular cholesterol accumulation and coenzyme Q10 deficiency in Familial Hypercholesterolemia

Familial Hypercholesterolemia (FH) is an autosomal co-dominant genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature cardiovascular disease. Here, we examined FH pathophysiology in skin fibroblasts derived from FH patients harboring heterozygous mutations in the LDL-receptor.Fibroblasts from FH patients showed a reduced LDL-uptake associated with increased intracellular cholesterol levels and coenzyme Q10 (CoQ10) deficiency, suggesting dysregulation of the mevalonate pathway.Secondary CoQ10 deficiency was associated with mitochondrial depolarization and mitophagy activation in FH fibroblasts. Persistent mitophagy altered autophagy flux and induced inflammasome activation accompanied by increased production of cytokines by mutant cells. All the pathological alterations in FH fibroblasts were also reproduced in a human endothelial cell line by LDL-receptor gene silencing.Both increased intracellular cholesterol and mitochondrial dysfunction in FH fibroblasts were partially restored by CoQ10 supplementation. Dysregulated mevalonate pathway in FH, including increased expression of cholesterogenic enzymes and decreased expression of CoQ10 biosynthetic enzymes, was also corrected by CoQ10 treatment.Reduced CoQ10 content and mitochondrial dysfunction may play an important role in the pathophysiology of early atherosclerosis in FH. The diagnosis of CoQ10 deficiency and mitochondrial impairment in FH patients may also be important to establish early treatment with CoQ10.

Carotid imt and plasma lipid levels among children and adolescents with familial hypercholesterolemia. A single center experience 2007-2014

Aim: Background: Familial hypercholesterolemia (FH) is an inherited error of lipoprotein metabolism characterized by elevated cholesterol and premature atherosclerotic cardiovascular disease. Current guidelines advocate early evaluation in pediatric carriers of FH. Assessment of carotid intima media thickness (cIMT) is often used to evaluate preclinical atherosclerosis in FH. Early cIMT progression was observed in FH subjects, and statin therapy was shown to slow the progression of IMT.

Epicardial fat is associated with severity of subclinical coronary atherosclerosis in familial hypercholesterolemia

Background and aimsFamilial hypercholesterolemia (FH) is a common genetic disorder characterized by elevated blood cholesterol, increased prevalence of subclinical atherosclerosis and high risk of premature coronary heart disease. However, this risk is not explained solely by elevated LDL-cholesterol concentrations, and other factors may influence atherosclerosis development. There is evidence that increased adiposity may predispose to atherosclerosis in FH. Epicardial fat has been associated with subclinical coronary atherosclerosis in the general population. This study evaluated the association of epicardial fat (EFV) volume with the presence and extent of subclinical coronary atherosclerosis detected by computed tomography angiography in FH patients. MethodsNinety-seven FH subjects (35% male, mean age 45 ± 13 years, LDL-C 281 ± 56 mg/dL, 67% with proven molecular defects) underwent computed tomography angiography and coronary artery calcium (CAC) scoring. EFV was measured in non-contrast images using a semi-automated method. Segment-stenosis score (SSS) and segment-involvement score (SIS) were calculated. Multivariate Poisson regression was utilized to assess an independent association of EFV with coronary atherosclerotic burden. ResultsEFV was positively associated with age, body mass index, waist circumference, blood glucose, the presence of the metabolic syndrome components, but not with LDL-C. After adjusting for confounders and abdominal circumference, an independent association (shown as β coefficients and 95% confidence intervals) of EVF with CAC scores [β = 0.263 (0.234; 0.292), p=0.000], SIS [β = 0.304 (0.141; 0.465) p=0.000] and SSS [β = 0.296 (0.121; 0.471), p=0.001] was found. ConclusionsIn FH, EFV was independently associated with coronary atherosclerotic presence and severity.

Association Between Cholesterol Efflux Capacity and Atherosclerotic Cardiovascular Disease in Patients With Familial Hypercholesterolemia.

Patients with familial hypercholesterolemia (FH) are at high risk for premature atherosclerotic cardiovascular disease (ASCVD), especially because of long-term exposure to high low-density lipoprotein cholesterol levels. It has been reported that low-density lipoprotein-lowering therapy delays the onset of ASCVD. However, it still remains difficult to prevent it. Therefore, novel biomarkers and therapeutic targets are necessary to evaluate and prevent atherosclerosis in FH. The aim of this study was to investigate associations of cholesterol efflux capacity with the presence of ASCVD and clinical features in patients with heterozygous FH. We measured cholesterol efflux capacity in 227 patients with heterozygous FH under pharmaceutical treatment. Seventy-six (33.5%) of them were known to have ASCVD. In a logistic-regression analysis adjusted for risk factors, increased efflux capacity was associated with decreased risk of ASCVD even after the addition of high-density lipoprotein cholesterol level as a covariate (odds ratio per 1-SD increase, 0.95; 95% confidence interval, 0.90-0.99; P<0.05). Decreased cholesterol efflux capacity was associated with the presence of corneal arcus after adjusting for age and sex. In addition, inverse relationships between cholesterol efflux capacity and Achilles tendon thickness, as well as carotid intima-media thickness, were observed after adjustment for age, sex, and traditional cardiovascular risk factors. Cholesterol efflux capacity was independently and inversely associated with the presence of ASCVD in heterozygous FH. In view of residual risks after treatment with statins, cholesterol efflux capacity might be a novel biomarker and a therapeutic target for preventing atherosclerosis in patients with FH.

Elevated atherosclerosis-related gene expression, monocyte activation and microparticle-release are related to increased lipoprotein-associated oxidative stress in familial hypercholesterolemia.

ObjectiveAnimal and in vitro studies have suggested that hypercholesterolemia and increased oxidative stress predisposes to monocyte activation and enhanced accumulation of oxidized LDL cholesterol (oxLDL-C) through a CD36-dependent mechanism. The aim of this study was to investigate the hypothesis that elevated oxLDL-C induce proinflammatory monocytes and increased release of monocyte-derived microparticles (MMPs), as well as up-regulation of CD36, chemokine receptors and proinflammatory factors through CD36-dependent pathways and that this is associated with accelerated atherosclerosis in subjects with heterozygous familial hypercholesterolemia (FH), in particular in the presence of Achilles tendon xanthomas (ATX).Approach and ResultsWe studied thirty FH subjects with and without ATX and twenty-three healthy control subjects. Intima-media thickness (IMT) and Achilles tendon (AT) thickness were measured by ultrasonography. Monocyte classification and MMP analysis were performed by flow cytometry. Monocyte expression of genes involved in atherosclerosis was determined by quantitative PCR. IMT and oxLDL-C were increased in FH subjects, especially in the presence of ATX. In addition, FH subjects had elevated proportions of intermediate CD14++CD16+ monocytes and higher circulating MMP levels. Stepwise linear regression identified oxLDL-C, gender and intermediate monocytes as predictors of MMPs. Monocyte expression of pro-atherogenic and pro-inflammatory genes regulated by oxLDL-C-CD36 interaction was increased in FH, especially in ATX+ subjects. Monocyte chemokine receptor CX3CR1 was identified as an independent contributor to IMT.ConclusionsOur data support that lipoprotein-associated oxidative stress is involved in accelerated atherosclerosis in FH, particularly in the presence of ATX, by inducing pro-inflammatory monocytes and increased release of MMPs along with elevated monocyte expression of oxLDL-C-induced atherosclerosis-related genes.

Detection of subclinical atherosclerosis in familial hypercholesterolemia using non-invasive imaging modalities

ObjectivesTo investigate the extent of subclinical atherosclerosis in asymptomatic familial hypercholesterolemia (FH) patients using non-invasive images techniques. Patients, methods and resultsThe atherosclerotic burden of 36 molecularly defined FH patients (18 males, 45.7±10.9 years) without evidence of cardiovascular disease receiving lipid-lowering treatment and 19 (47.8±11.3 years) controls was investigated. Descending thoracic aorta magnetic resonance imaging (MRI) was performed in a 1.5T equipment with T1 and T2 sequences to characterize atherosclerotic plaques and to measure aortic wall volumen. Carotid intima-media thickness (cIMT) and presence of plaques were measured using B-mode carotid ultrasound.Mean aortic wall volumen, cIMT and atherosclerotic plaques in aorta were significantly higher in FH cases (P<0.001). A significant correlation between aortic wall volume and cIMT was observed (P<0.01). Aortic MRI detected plaques in 94% and carotid ultrasound in 14% of cases. Lipid-rich plaques were observed only in FH cases (33%) and were associated with family history of premature coronary artery disease (P<0.05). ConclusionsAsymptomatic middle-aged FH patients have significantly higher atherosclerotic burden than controls. cIMT has shown a significant correlation with aortic wall volume and MRI allowed the detection of lipid-rich plaques in FH subjects that were associated with family history of premature coronary artery disease.

Safety and efficacy of therapeutic plasma exchange in a pregnant patient with familial hypercholesterolemia

Dear Sir, Pregnant patients with familial hypercholesterolemia might have an increased risk for premature cardiovascular disease. Treatment such as plasmapheresis appears to be a safe and effective procedure. Plasmapheresis is uncommonly used in pregnancy (1,2), although the technology is widely available. Using modern technology has reduced the complications and morbidity (3). Thus its value in pregnancy is underrated, especially because of the pharmacological limitations of treating many conditions in pregnancy. Because of such limitations, we decided to use plasmapheresis in a patient with familial hypercholesterolemia. To our knowledge, only two patients with this condition have been similarly treated (1,2). Familial hypercholesterolemia is an inherited disorder characterized by excessive amounts of cholesterol in the blood and increased levels of low-density lipoproteins, tendinous xanthomas and premature coronary artery disease. Approximately 1 in 500 patients inherit a single copy of the gene and present with cholesterol levels ranging from 300 to 500 mg/100 ml (4). We present a young woman who had a myocardial infarction of the anterior wall at age 26, significantly increased plasma lipid levels, and a history of angina pectoris. Coronary angiography showed stenosis of the left anterior descending coronary artery. At that time, the diagnosis of familial hypercholesterolemia was made by the presence of hypercholesterolemia in both parents and one sibling (father with three myocardial infarction episodes and a heart transplant, mother with myocardial infarction and bypass grafting, brother with two myocardial infarctions). Seven years later, at age 33, the patient was referred to our hospital at 28 weeks in her first pregnancy. The patient complained of pain in the thoracic cage (angina pectoris), neuralgia of the right facial nerve and the skin of the right lower limb. The electrocardiogram was normal. The serum lipid levels were: total cholesterol 510.6 mg/dl, high-density lipoprotein-C 74.8 mg/dl, low-density lipoprotein-C 383.2 mg/dl, and triglycerides 262.8 mg/dl. Due to the limitation of pharmacological therapy associated with pregnancy, the cardiologist recommended treatment with plasmapheresis with low-density lipoprotein apheresis. Plasmapheresis with a double filtration system using the Autopheresis C System (Fenwal, Lake Zurich –USA) was performed at 30 weeks of pregnancy. The plasma volume removed was 1,980 ml, and the patient received 1,400 ml of 0.9% NaCl, 200 ml of 20% albumins and 400 ml of acid-citrate-dextrose solution. Two days after the procedure, total cholesterol had decreased by 29%, low-density lipoprotein-C by 36%, and triglycerides increased by 14% and the patient's symptoms of angina pectoris subsided. The lipid profile was repeated again at 32 and 34 weeks of gestation and indicated only narrow change (TC-382.0 mg/dl and LDL −264.0 mg/dl). At 34 weeks of gestation the patient was again evaluated by the cardiologist who recommended cesarean section on the indication of active ischemic heart disease and increased risk of myocardial infarction. The patient was delivered at 35 weeks gestation by planned cesarean section (male, birthweight 2,800 g). The cesarean section was performed under spinal/epidural anesthesia at the department of cardiology. After delivery the cardiologist recommended treatment with oral statins, metoprolol, clopidognel bisulphate and acetylsalicylic acid. On postpartum day 5, coronary angiography revealed multiple atherosclerotic changes without significant stenosis. The main concern here was aggravation of coronary insufficiency in the mother, predominantly due to the hemodynamic changes that occur during pregnancy. Thompson et al. showed that if plasmapheresis was repeated every 2–3 weeks, the average cholesterol level would fall by 50% (5). Because of a cardiovascular indication for an early delivery (history and risk of infarction), we could not proceed with another plasmapheresis. However, we think that regular plasma exchange may significantly lower plasma lipid levels, when there is a risk that pregnancy may have deleterious effects on the maternal cardiac status. By temporarily decreasing the plasma lipid levels, the patient's cardiac condition remained stable through the pregnancy and we were able to deliver the patient without too much risk of preterm complications for the baby as well as minimize possible harmful effects for the mother. Plasmapheresis has been shown to cause regression or delay the onset of atherosclerosis in familial hypercholesterolemia and seems to be a safe and effective way to temporally lower the plasma lipid concentrations.

Leapfrogging Data

Properly designed and conducted randomized controlled clinical trials (RCTs) are the premier tool for both testing mechanistic hypotheses and critically ascertaining the risks and benefits of a therapy or strategy for clinical care. The sample size of a trial is mainly a function of the rates of its primary objectives and the presumed influence of the intervention. Trials focusing on a primary outcome variable that can be readily quantified in each subject, such as blood pressure or plasma cholesterol levels, require substantially fewer participants and shorter durations to determine whether their predefined measurement is altered compared with a morbidity and mortality trial. Trials designed to determine whether clinical prognosis is altered by an intervention depend on the proportion of patients experiencing the predefined adverse clinical event(s) and often require 100s-fold–greater patient-time exposures to test their primary hypothesis and provide even modest information about safety. These resource-intense morbidity and mortality trials are generally only performed when information from observational studies as well as smaller mechanistic and surrogate- outcomes RCTs are so highly supportive of a favorable outcome that they justify the effort. Despite this understandable stacking of the cards with the best available information, many of the morbidity and mortality trials conducted to test for a potential favorable impact of an intervention conclude by not supporting the prestudy hypothesis-generating data.1 The lessons in humility offered by these neutral or negative outcomes trials underscore the importance of obtaining crucial risk–benefit data before widespread adoption of even an apparently favorable therapy.2 Articles pp 2506 and 2515 For rational therapeutic decision making, we would ideally like to have both a framework of reliable mechanistic information and robust clinical outcomes and safety data. Sometimes major clinical outcomes trials are designed with a complement of embedded ancillary trials to generate a more complete picture …

Correlating corneal arcus with atherosclerosis in familial hypercholesterolemia.

BackgroundA relationship between corneal arcus and atherosclerosis has long been suspected but is controversial. The homozygous familial hypercholesterolemia patients in this study present a unique opportunity to assess this issue. They have both advanced atherosclerosis and corneal arcus.MethodsThis is a cross-sectional study of 17 patients homozygous for familial hypercholesterolemia presenting to the Clinical Center of the National Institutes of Health. Plasma lipoproteins, circumferential extent of arcus, thoracic aorta and coronary calcific atherosclerosis score, and Achilles tendon width were measured at the National Institutes of Health.ResultsPatients with corneal arcus had higher scores for calcific atherosclerosis (mean 2865 compared to 412), cholesterol-year score (mean 11830 mg-yr/dl compared to 5707 mg-yr/dl), and Achilles tendon width (mean 2.54 cm compared to 1.41 cm) than those without. Corneal arcus and Achilles tendon width were strongly correlated and predictive of each other. Although corneal arcus was correlated with calcific atherosclerosis (r = 0.67; p = 0.004), it was not as highly correlated as was the Achilles tendon width (r = 0.855; p < 0.001).ConclusionCorneal arcus reflects widespread tissue lipid deposition and is correlated with both calcific atherosclerosis and xanthomatosis in these patients. Patients with more severe arcus tend to have more severe calcific atherosclerosis. Corneal arcus is not as good an indicator of calcific atherosclerosis as Achilles tendon thickness, but its presence suggests increased atherosclerosis in these hypercholesterolemic patients.

Measurement of the Reflectivity of the Intima-Medial Layer of the Common Carotid Artery Improves the Discriminatory Value of Intima-Medial Thickness Measurement as a Predictor of Risk of Atherosclerotic Disease

Our aim was to assess the predictive value of a measurement of intima-medial layer (IML) reflectivity in the differentiation of pathological from physiological increases in intima-medial thickness (IMT). Both common carotid arteries (CCA) of familial hypercholesterolemia (FH) patients and age- and sex-matched controls (no cardiovascular risk factors) were imaged using a 10- to 15-MHz linear array transducer ( n = 30). Images of the CCA far wall were analyzed in the IMT “plug-in” of “HDI Lab.” The IML reflectivity, averaged over an 8- to 12-mm length of arterial wall, was expressed as a ratio of reflectivity at a point 0.21-mm deep to the intima-medial interface divided by the reflectivity at the intima-medial interface, termed the intima-medial reflectivity index (IMRI). The risk of atherosclerosis was assessed in terms of IMT alone and IMT coupled with IMRI. Defining high risk of atherosclerosis in FH, in terms of both IMT alone and IMT coupled with IMRI, produced an appropriate, when compared with cholesterol-years score, statistically significant stratification ( p < 0.01 and p < 0.005). Analysis of the low-risk subjects revealed a tendency to define a subject as “high risk” based on a physiological increase in IMT, but when IMRI is included in the assessment, all controls are correctly identified as low risk. This method of quantifying the reflectivity of the IML improved the discriminatory performance of IMT increase as an indicator of atherosclerotic risk by enabling a smaller, therefore earlier, increase in IMT to be considered pathologic when accompanied by an increase in IMRI. (E-mail: paul.sidhu@kch.nhs.uk)

Early diagnosis of carotid atherosclerosis in familial hypercholesterolemia caused by Padua-1 and Padua-4 mutations

Early diagnosis of carotid atherosclerosis in familial hypercholesterolemia caused by Padua-1 and Padua-4 mutations

Positive family history for coronary heart disease and ‘midband lipoproteins’ are potential risk factors of carotid atherosclerosis in familial hypercholesterolemia

Patients with heterozygous familial hypercholesterolemia (FH) were examined with B-mode ultrasound in order to determine intima-media thickness (IMT) in the common carotid artery, and to uncover potential risk factors responsible for the development of IMT. Ninety seven FH subjects and 132 non FH type IIa hyperlipidemic subjects were involved in the present study. Age was found to correlate positively with IMT in both FH and non FH groups. FH individuals showed a higher IMT, along with elevated low density lipoprotein (LDL) cholesterol levels, compared with age-matched non FH individuals. To clarify potential factors contributing to the formation and development of carotid atherosclerosis, we divided the FH subjects into two subgroups, namely FH with high IMT group (HIG), and those with low IMT group (LIG). We investigated those two subgroups on the presence of angiographically documented coronary heart disease (CHD), of family history of CHD and of ‘midband lipoproteins’ by polyacrylamide gel electrophoresis (PAGE) analysis, by matching for age and LDL-cholesterol (LDL-C) level. Fifty percent of FH men in HIG was found to have CHD, whereas only 14% of those in LIG had CHD ( P<0.05). Thirty-three percent of FH women in HIG was found to have CHD, whereas only 12% of those in LIG had CHD ( P<0.05). Fifty percent of FH men in HIG was found to have ‘midband lipoproteins’, whereas only 7% of those in LIG had ‘midband lipoproteins’ ( P<0.01). Seventy-three percent of FH women in HIG had ‘midband lipoproteins’, whereas only 21% of those in LIG had ‘midband lipoproteins’ ( P<0.0005). Fifty-five percent of FH men in HIG was had positive family history for CHD, whereas only 14% of those in LIG had positive family history for CHD ( P<0.05). Sixty-three percent of FH women in HIG was found to have positive family history for CHD, whereas only 29% of those in LIG had positive family history for CHD ( P<0.05). Based on these findings, we propose that, besides age and elevated levels of LDL-C, positive family history for CHD and ‘midband lipoproteins’ are important determinants for the development of carotid atherosclerosis in FH individuals in Japanese population.

Atherosclerotic Lesions Are Associated with Increased Immunoreactivity for Inducible Nitric Oxide Synthase and Endothelin-1 in Thoracic Aortic Intimal Cells of Hyperlipidemic Watanabe Rabbits

The development and progression of atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits is associated with increases in inducible nitric oxide synthase (NOS2) and endothelin-1 (ET-1) immunoreactivity. In contrast, there is a reduction of immunoreactivity for neuronal NOS (NOS1) in aortic endothelial cells, but no change in endothelial NOS (NOS3) immunoreactivity. However, subendothelial macrophages and smooth muscle showed a different pattern of immunoreactivity of NADPH-diaphorase (NADPH-d), NOS2, ET-1, and NOS1. The lipid-rich macrophages in the intima were positively labeled for NADPH-d, NOS1, NOS2, NOS3, and ET-1. Smooth muscle cells in the subendothelium and the medial layers of the vascular wall were also positive for these markers. These results are consistent with the reduction of endothelium-dependent vasorelaxation that is known to occur during the development and progression of atherosclerosis in familial hypercholesterolemia. The data suggest a key role for vasoactive substances in the development of atherosclerosis.

Premature coronary and extracoronary atherosclerosis in familial hypercholesterolemia caused by padua-1 mutation

Premature coronary and extracoronary atherosclerosis in familial hypercholesterolemia caused by padua-1 mutation

Paraoxonase gene polymorphisms are associated with carotid arterial wall thickness in subjects with familial hypercholesterolemia

Human serum paraoxonase (PON) is a high density lipoprotein (HDL) associated enzyme capable of hydrolyzing lipid peroxides in vitro. PON has recently attracted attention as a protective factor against oxidative modification of LDL and may therefore play an important role in the prevention of the atherosclerotic process. Two frequent mutations at the paraoxonase gene locus ( PON1) are the leucine (L allele)→methionine (M allele) and the glutamine (Q allele)→arginine (R allele) substitutions at residues 55 and 192, respectively. We have examined the influence of these two polymorphisms on carotid atherosclerosis in familial hypercholesterolemia (FH) patients. The allele frequencies of these two polymorphisms were determined by PCR and restriction fragment analysis, for both the FH population and healthy controls. High resolution B-mode ultrasound was used to assess intima-media wall thickness (IMT) of the carotid artery. No differences were found in allele frequencies between the FH and the control population. In FH patients, the LL, LM and MM genotypes at position 55 occurred in 86 (46.0%), 78 (41.7%) and 23 (12.3%) subjects, respectively, whereas the QQ, QR and RR genotypes at position 192 were found in 90 (48.1%), 79 (42.2%) and 18 (9.6%) individuals. When both polymorphisms were considered separately, no different carotid IMTs were found between the genotype groups. However, our data did show a significant association between the various genotypes of the combined polymorphisms at position 55 and 192 of PON1 and the carotid artery IMT in FH subjects. Subjects with the homozygous wildtype LL/QQ for paraoxonase had the highest mean carotid IMTs when compared to other genotypes, combined. Multiple regression analysis demonstrated age ( β=0.34, P<0.0001), total plasma cholesterol ( β=0.17, P=0.0109) and the LL/QQ genotype of the PON1 gene ( β=0.22, P=0.0018) to be significant risk factors for carotid atherosclerosis in subjects with FH. The LL/QQ genotype could explain 5.3% of total variance of carotid IMT. In conclusion, this is the first study to report an independent association between the combined PON1 polymorphism genotypes and carotid wall thickness. The homozygous wildtype LL/QQ for PON1 may represent an additional risk factor for carotid atherosclerosis in subjects with FH.

Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia : a study of patients with familial hypercholesterolemia from Sardinia.

One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the alpha-globin gene (alpha-thalassemia trait) and that 6% to 17% are beta-thalassemia carriers. In this population, a single mutation of beta-globin gene (Q39X, beta(0) 39) accounts for >95% of beta-thalassemia cases. Because previous studies have shown that Sardinian beta-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the beta-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76+/-1.08 mmol/L in subjects with beta(0)-thalassemia trait and 8.25+/-1.66 mmol/L in subjects without this trait (P<0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had beta(0)-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of beta(0)-thalassemia trait emerged also when we pooled the data from all FH subjects with and without beta(0)-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of beta(0)-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B-containing lipoproteins. The observation that our FH subjects with beta(0)-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of beta(0)-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.