Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.

Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.

Hypercholesterolemia is one of the most prevalent, yet underdiagnosed diseases faced by the medical community today. Its prevalence can largely be attributed to diet, lack of exercise, and lifestyle choices such as smoking or drinking, but there is also a genetic component. Familial Hypercholesterolemia is the genetic disorder in which a person is unable to properly eliminate levels of low-density lipoprotein, mostly due to an ineffective receptor in the liver. Hypercholesterolemia has been positively correlated with the prevalence of cardiovascular disease, and patients with the severe homozygous familial hypercholesterolemia typically have abbreviated lifespans. In these situations, and also those less acutely dire, it’s necessary to rely on medication to help maintain one’s cholesterol levels to within low risk ranges. High does statin therapy has been shown to be the most effective therapy for maintaining LDL cholesterol. It has become the standard regardless of the cause of hypercholesterolemia because of its few side effects, its high tolerability, its ease of administration, its safety, and most of all because of its immense efficacy. This has not, however, prevented the exploration into other types of cholesterol therapies that may work in concurrence with statins. Drug classes such as PCSK9 inhibitors, ApoB inhibitors, MTP inhibitors, and thyromimetics have all been explored with varying success. Each of these potential therapies has a separate mechanism of action, allowing for modulation in conjunction with statins. PCSK9 inhibitors and ApoB inhibitors appear to provide the most upside by virtue of LDL lowering capabilities, followed by a drug known as ezetimibe that reduces dietary cholesterol uptake in the gut. MTP inhibitors have been shown to be effective therapies for homozygous familial hypercholesterolemia specifically due to their function of lowering LDL particle creation rather than LDL receptor number or function as statins and PCSK9 inhibitors do. Thyromimetics have yet to yield an effective therapy for cholesterol treatment, but the hope remains alive that this could come to fruition in the future.

The physical properties (i.e., mass, particle diameter and composition) of apolipoprotein B (apoB)-containing lipoproteins (apoB-LP) are a major determinant of atherosclerotic cardiovascular disease (ASCVD) risk. The objective of this research was to investigate how nascent apoB-LP physical properties affect circulating lipoprotein profiles and risk of disease. Relationships between apoB-LP physical properties and arterial plaque formation in four genotypes of mice with apoB isoform specific clearance defects were investigated. Multivariate statistical analysis found that arterial lesions were most closely related to genetic background and apoB concentration related to delayed clearance rate. For defining the dietary effects on circulating lipoprotein profiles, the physical properties of lipoproteins in hamsters fed high-carbohydrate diets containing either 60% fructose or 60% cornstarch for 2 wk were studied. Fructose increased very low-density lipoprotein (VLDL) particle diameter and decreased low-density lipoprotein (LDL) particle diameter. Elevations in all high-density lipoprotein (HDL) fractions were observed in the fructose-fed group. Further investigation was made of whether changes to the physical properties in circulating lipoproteins resulted from changes to nascent particles in the assembly and secretion processes. Intermediate particles used for lipoprotein assembly were isolated from rough endoplasmic reticulum of hamster liver, and nascent VLDL were isolated from plasma after Triton WR-1339 injection of hamsters. A large, TG-rich apoB-deficient particle and a small, lipid-poor apoB-containing particle were isolated in each dietary setting. The diameter of first-step particles was larger in fructose feeding, which indicated that apoB degradation decreases and provides the basis for apoB oversecretion. Fructose feeding significantly increased the concentrations recovered from liver for these two particles and for nascent particles compared with chow or starch feeding. Collectively, these results demonstrate: 1) genetic factors can dictate metabolism, and metabolic conditions can critically affect the physical properties and further atherogenicity of apoB-LP; 2) changes in physical properties of circulating apoB-LP are derived from changes to the nascent particles; and 3) dietary factors can influence the assembly, secretion, and metabolism of apoB-LP. The findings of the research may provide a metabolic basis for the recognition of new targets that could regulate apoB-LP metabolism to prevent and treat ASCVD.

The physical properties (i.e., mass, particle diameter and composition) of apolipoprotein B (apoB)-containing lipoproteins (apoB-LP) are a major determinant of atherosclerotic cardiovascular disease (ASCVD) risk. The objective of this research was to investigate how nascent apoB-LP physical properties affect circulating lipoprotein profiles and risk of disease. Relationships between apoB-LP physical properties and arterial plaque formation in four genotypes of mice with apoB isoform specific clearance defects were investigated. Multivariate statistical analysis found that arterial lesions were most closely related to genetic background and apoB concentration related to delayed clearance rate. For defining the dietary effects on circulating lipoprotein profiles, the physical properties of lipoproteins in hamsters fed high-carbohydrate diets containing either 60% fructose or 60% cornstarch for 2 wk were studied. Fructose increased very low-density lipoprotein (VLDL) particle diameter and decreased low-density lipoprotein (LDL) particle diameter. Elevations in all high-density lipoprotein (HDL) fractions were observed in the fructose-fed group. Further investigation was made of whether changes to the physical properties in circulating lipoproteins resulted from changes to nascent particles in the assembly and secretion processes. Intermediate particles used for lipoprotein assembly were isolated from rough endoplasmic reticulum of hamster liver, and nascent VLDL were isolated from plasma after Triton WR-1339 injection of hamsters. A large, TG-rich apoB-deficient particle and a small, lipid-poor apoB-containing particle were isolated in each dietary setting. The diameter of first-step particles was larger in fructose feeding, which indicated that apoB degradation decreases and provides the basis for apoB oversecretion. Fructose feeding significantly increased the concentrations recovered from liver for these two particles and for nascent particles compared with chow or starch feeding. Collectively, these results demonstrate: 1) genetic factors can dictate metabolism, and metabolic conditions can critically affect the physical properties and further atherogenicity of apoB-LP; 2) changes in physical properties of circulating apoB-LP are derived from changes to the nascent particles; and 3) dietary factors can influence the assembly, secretion, and metabolism of apoB-LP. The findings of the research may provide a metabolic basis for the recognition of new targets that could regulate apoB-LP metabolism to prevent and treat ASCVD.

OBJECTIVE: Atherosclerotic disease progression is mediated in part, by immunological mechanisms. In recent years, interest has increased towards the prospect of modulating these immune mechanisms through vaccination to ameliorate the course of disease. Patients with lupus are at a significantly higher risk for accelerated atherosclerosis and related complications. The goal of this study was to assess the outcome of immunization in mouse models of lupus, and lupus with accelerated atherosclerosis. MATERIALS/METHODS: Atherosclerosis-prone apoE^-/- mice and autoimmune gld mice were previously crossed to generate the gld.apoE^-/- mouse. Mice were treated with an apoB-100-derived vaccine, Alum (adjuvant control), or PBS control. The antibody response was determined by quantifying the amount of circulating anti-apoB100. Serum triglyceride and cholesterol levels were analyzed. Kidney tissue from gld and gld.apoE^-/- mice was processed and histologically analyzed, using glomerular tuft size as a measure of renal disease and by extension, autoimmune disease severity. Results: Immunization led to a pronounced initial antibody response that was decreased by the endpoint of the study. No significant differences in serum triglyceride or cholesterol were observed regardless of treatment. Similarly, no significant differences were observed in glomerular tuft size. Conclusion: The data suggests that immunization with an apoB-100- derived vaccine neither improves nor worsens autoimmune disease severity in the gld.apoE^-/- mouse model. It also appears that immunization is tolerated in the autoimmune background. While further study is necessary to determine the efficacy of immunization in reducing atherosclerotic disease in this model, this may be a possible therapy to lower incidence of atherosclerosis in lupus patients.

La mortalité cardiovasculaire représente la première cause de mortalité chez les sujets diabétiques de type 2. La dyslipidémie de ces patients caractérisée par le quatuor : hypertriglycéridémie, baisse du HDL-cholestérol, augmentation du nombre de LDL petites et denses et hyperlipidémie postprandiale, constitue un facteur de risque prépondérant. Cette dyslipidémie est en grande partie expliquée par l’accumulation sanguine des lipoparticules riches en triglycérides (TRL) d’origine hépatique (VLDL) et intestinale (chylomicrons). L’hyperproduction des chylomicrons, déjà connue pour les VLDL, est une composante nouvellement reconnue d’insulinorésistance. L’action inhibitrice aigüe de l’insuline sur la production des VLDL est absente chez le sujet diabétique de type 2. Dans notre étude, nous avons montré l’absence d’effet aigu de l’insuline sur la production des chylomicrons chez les sujets diabétiques de type 2 grâce à une étude cinétique utilisant un isotope stable (D3-leucine). / The cardiovascular mortality represents the first cause of mortality in human type 2 diabetes. The typical diabetic dyslipidemia characterized by the quartet: high triglyceride levels, low HDL-cholesterol, increased number of small and dense LDL particles and postprandial hyperlipidemia, is a major cardiovascular risk factor. This dyslipidemia is mainly explained by the accumulation of triglyceride-rich lipoproteins (TRL) from liver (VLDL) and intestine (chylomicrons).The overproduction of chylomicrons, as is known for VLDL, is a newly characteristic of insulin resistant states. The acute inhibitory effect of insulin on VLDL production is absent in type 2 diabetic patients. In our study, we have shown the absence of acute inhibitory effect of insulin on chylomicron production in type 2 diabetic patients, using a kinetic study with stable isotope (D3-leucine).

A frequência de Hipercolesterolemia Familial (HF) ainda é desconhecida no Brasil, principalmente pela ausência de estudos com caracterização genotípica associada à fenotípica. Os dados epidemiológicos existentes se baseiam apenas no fenótipos e carecem do diagnóstico molecular confirmatório. O objetivo do presente estudo foi identificar as principais causas genéticas da HF em pacientes diagnosticados fenotipicamente através de um painel exômico com 61 genes a fim de contribuir para um sistema de confirmação do diagnostico molecular em uma amostra da população brasileira. Para isso foram incluídos 141 pacientes, não aparentados, portadores de HF atendidos pelo setor de dislipidemias do Instituto Dante Pazzanese de Cardiologia, Laboratório de Analises Clinicas da Faculdade de Ciências Farmacêuticas da Universidade Federal do Rio Grande do Norte e do Programa Hipercol Brasil do Instituto do Coração. As amostras de sangue periférico foram obtidas para determinações fenotípicas laboratoriais e extração de DNA genômico. A biblioteca de DNA foi construída utilizando o kit Nextera® Rapid Capture Enrichment Custom enriquecendo os éxons de 61 genes que direta ou indiretamente estão relacionados com metabolismo do colesterol. O ultrassequenciamento foi realizado utilizando kit MiSeq Reagent (300 a 500 ciclos) na plataforma MiSeq (Illumina). Os resultados de sequenciamento foram inicialmente alinhados a uma sequência referência e analisados para eliminação de falsos positivos, segundo os parâmetros de qualidade, tais como: cobertura mínima de 30x, frequência do alelo alterado maior que 20% e diferença da distribuição das leituras entre as sequências nucleotídicas menor que 15%. Foram identificadas 472 diferentes variantes em 56 dos genes presentes no painel, sendo 45 consideradas como não descritas. Nos genes APOA1, APOA2, LIPC, RBP4 e TIMP1 não foram observadas variantes dentro dos critérios estabelecidos. Das variantes observadas 25 identificadas em 30 (21,2%) pacientes já tinha sido publicadas em relação à HF nos três principais genes (LDLR, APOB e PCSK9), confirmando o diagnóstico. Foi caracterizado genotipicamente outras dislipidemias primárias em 7 pacientes, sem diagnóstico molecular de HF, através de variantes identificadas no ultrassequenciamento em outros genes. Dos 104 pacientes que não possuíam nenhuma variante já previamente caracterizada, 69 possuíam variantes relacionados com o metabolismo do colesterol. As variantes sem patogenicidade conhecida foram avaliadas através de ferramentas de predição in silico e 22 delas possuíam características sugestivas de patogenicidade em pelo menos 4 das ferramentas utilizadas, duas delas também mostraram alterar a estrutura da proteína segundo análises de docking molecular. Foram identificadas também 223 variantes em região não transcritas (UTR). Quando realizada as análises estatística de todas as variantes identificadas, observamos associação de 13 variantes com concentrações mais elevadas de colesterol da LDL, 5 com concentrações mais elevadas de apolipoproteina B-100, 5 com concentrações mais elevadas de colesterol total, 6 com presença de arco córneo, 2 com manifestação de xantelasmas, 2 com ausência de xantomas e 3 com a presença de doença arterial coronariana. Dessas 6 variantes já haviam sido previamente descritas com HF ou algum outro fenótipo associado e 2 não tinham citação na literatura pesquisada, mas possuíam característica patogênica para a proteína segundo as ferramentas de predição in silico. Este estudo permitiu a identificação das causas genéticas da HF em pacientes brasileiros diagnosticados fenotipicamente, mostrando que a técnica escolhida permitiu caracterizar 21,2% dos pacientes. Além disso, foi possível identificar outras dislipidemias primárias e caracterizar algumas variantes que, apesar de necessitarem serem validadas, indicam uma possível associação com a HF, aumentando o esclarecimento do fenótipo com o genótipo para 74,5%. Este estudo também possibilitou a identificação de novas variantes que devem ser avaliadas para confirmar associação com a doença e utilizar para o diagnóstico propondo um novo painel poligênico. / The frequency of Familial Hypercholesterolemia (FH) is still unknown in Brazil, mainly due to the absence of studies with genotypic characterization associated with phenotype. Existing epidemiological data are based only on the phenotypes and lack the confirmatory molecular diagnosis. The aim of the present study was to identify main genetic causes of FH in patients diagnosed phenotypically through an exomic panel with 61 genes in order to contribute to a system of confirmation molecular diagnosis in a sample of the Brazilian population. To this end, 141 non-related patients with FH treated by the dyslipidemia sector of the Institute Dante Pazzanese of Cardiology, Clinical Analysis Laboratory of the Faculty of Pharmaceutical Sciences of the University Federal of Rio Grande do Norte and the Hipercol Brazil Program of the Heart Institute. Peripheral blood samples were obtained for laboratory phenotypic determinations and extraction of genomic DNA. The DNA library was constructed using the Nextera® Rapid Capture Enrichment Custom kit, enriching with éxons of 61 genes that are directly or indirectly related to cholesterol metabolism. Ultrasequencing was performed using MiSeq Reagent kit (300 to 500 cycles) on the MiSeq platform (Illumina). The sequencing results were initially aligned to a reference sequence and analyzed for false positive elimination according to quality parameters such as: minimum coverage of 30x, altered allele frequency greater than 20%, and difference in the distribution of reads between sequences nucleotides less than 15%. 472 different variants were identified in 56 of the genes present in the panel, of which 45 were considered not described. In the APOA1, APOA2, LIPC, RBP4 and TIMP1 genes no variants were observed within the established criteria. In 25 of the variants observed presents in 30 (21.2%) patients had already been published in relation to FH in the three main genes (LDLR, APOB and PCSK9), confirming the diagnosis. Other primary dyslipidemias were caracterized genotypically in 7 patients, without molecular diagnosis of HF, through variants identified in ultrasequencing in other genes. Of the 104 patients who did not have any previously characterized variant, 69 had variants related to cholesterol metabolism. The variants without known pathogenicity were evaluated using in silico prediction tools and 22 of them had characteristics suggestive of pathogenicity at least 4 of the tools used, two of them also showed to alter the structure of the protein according to molecular docking analyzes. Were also identified 223 non-transcribed region (UTR) variants. Statistical analysis of all the variants identified showed association of 13 variants with higher concentrations of LDL cholesterol, 5 with higher concentrations of apolipoprotein B-100, 5 with higher concentrations of total cholesterol, 6 with presence of an arc corneal, 2 with manifestation of xanthelasms, 2 with absence of xanthomas and 3 with the presence of coronary artery disease. Of these 6 variants had previously been described with HF or some other associated phenotype and 2 had no citation in the researched literature, but had a pathogenic characteristic for the protein according to in silico prediction tools. This study allowed the identification of the genetic causes of FH in Brazilian patients diagnosed phenotypically, showing that the technique chosen allowed to characterize 21.2% of the patients. In addition, it was possible to identify other primary dyslipidemias and to characterize some variants that, although they need to be validated, indicate a possible association with HF, increasing the clarification of the phenotype with the genotype to 74.5%. This study also allowed the identification of new variants that should be evaluated to confirm association with the disease and to use for the diagnosis proposing a new polygenic panel.

Atherosclerosis, hardening and narrowing of the arteries, is the principal underlying cause of heart attacks, strokes, and peripheral vascular disease, which kills more than 600,000 Americans each year. High plasma levels of low-density lipoproteins (LDL) are linked to the formation of atherosclerotic plaques in arteries. LDL is the last metabolic product of very low-density lipoprotein (VLDL), which is secreted from the liver along with one molecule of apolipoprotein B (apoB). Current therapies to control levels of LDL include: cholesterol synthesis inhibitors or statins, low-fat diets and antisense oligonucleotides to reduce cholesterol levels. Recent studies recommend lower clinical levels of plasma LDL to maintain an individual’s health, especially of those who have already developed atheroscle- rotic plaques. However, existing therapies are often unable to achieve these aggressive limits. Furthermore, patients have shown various levels of intolerance to these treatments. In order to develop new, targeted drugs, that can control LDL levels with minimal side effects, it is imperative to understand, in detail, the process of apoB-containing lipoprotein formation. ApoB is one of the largest human proteins known (4563 residues) and previous attempts to solve the structure have been unsuccessful, mainly due to analyzing the protein as a whole or by large sections. To advance the field we will go by a different approach. I present here a construct that represents roughly 8% of the whole protein, apoB9-17 (residues 430 to 782). This section of the protein is believed to play a pivotal role in the assembly process of LDL. My hypothesis is that this construct will be well-behaved and suitable for structural and functional analysis. The study shows that apoB9-17 can be produced in considerable quantities from bacterial cells and can be purified by means of a 6-histidine tag with a good yield. Furthermore, circular dichroism analysis shows the construct contains the expected secondary structure at room temperature and is stable at a wide temperature range (50 to 70 ◦C) at low concentrations. The construct here described will be useful to test the effect of mutations such as the one found in patients with Familial hypobetalipoproteinemia (FHBL). Furthermore, this construct contains two regions believed to be of vital importance for LDL particle formation: the alpha-helical region (residues 430 to 570) is believed to associate with MTP at the initial stages of LDL formation and the c-sheet (residues 614 to 782), which may form part of the lipid recruiting process. Both essential aspects to ultimately develop therapies that can modulate VLDL particle formation.

No description available.