by Lee Sau Yat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 89-94). / Acknowledgements --- p.I / Abbreviation --- p.II / Abstract --- p.III / Table of content --- p.IV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Historical background in the studies of LDL and LDLR --- p.1 / Chapter 1.2 --- Homeostasis of Cholesterol in Man --- p.4 / Chapter 1.2.1 --- Origin and catabolism of low density lipoprotein --- p.4 / Chapter 1.2.2 --- The LDL receptor --- p.6 / Chapter 1.2.3 --- LDL pathway --- p.7 / Chapter 1.2.4 --- Feedback regulatory action of LDL receptor --- p.10 / Chapter 1.3 --- Gene structure of LDL receptor promoter --- p.11 / Chapter 1.3.1 --- The LDL receptor promoter --- p.11 / Chapter 1.3.2 --- The responsive element in LDL receptor promoter --- p.13 / Chapter 1.4 --- Eukaryotic transcription factor --- p.15 / Chapter 1.5 --- Role of Gel-shifted assay in studying DNA binding protein --- p.17 / Chapter 1.6 --- Objective of the present thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.21 / Chapter 2.1 --- Oligonucleotide synthesis and purification --- p.21 / Chapter 2.1.1 --- Primer construction --- p.21 / Chapter 2.1.2 --- Purification of oligonucleotides --- p.22 / Chapter 2.2 --- Recombinant plasmid construction --- p.24 / Chapter 2.2.1 --- Preparation of competent cell --- p.24 / Chapter 2.2.2 --- Preparation of phage DNA --- p.24 / Chapter 2.2.3 --- Amplification and purification of LDLR-promoter by PCR techniques --- p.26 / Chapter 2.2.3.1 --- Amplification and restriction site construction of LDLR- promoter --- p.26 / Chapter 2.2.3.2 --- Purification of the PCR product --- p.27 / Chapter 2.2.4 --- Preparation of plasmid pGCAT-A --- p.27 / Chapter 2.2.5 --- Recombinant plasmid pLDLRP-GCAT- A construction --- p.29 / Chapter 2.2.6 --- Transformation of DNA to competent cell --- p.29 / Chapter 2.2.7 --- Screening of positive clone pLDLRP- GCAT-A --- p.30 / Chapter 2.3 --- DNA sequencing --- p.31 / Chapter 2.3.1 --- Denaturing the double strand template --- p.31 / Chapter 2.3.2 --- Annealing reaction --- p.31 / Chapter 2.3.3 --- Labeling reaction --- p.32 / Chapter 2.3.4 --- Termination reaction --- p.32 / Chapter 2.3.5 --- Running and fixing the gel --- p.32 / Chapter 2.4 --- Cell culture and passage of different cell lines --- p.34 / Chapter 2.4.1 --- "Routine subculture of HepG 2, CHO, FSF, Cos-7 and FSF" --- p.34 / Chapter 2.4.2 --- "BMN, RTGH-1" --- p.34 / Chapter 2.5 --- Preparation of human LDL and LPDS --- p.36 / Chapter 2.5.1 --- Purification of LDL --- p.36 / Chapter 2.5.2 --- Purification of LPDS --- p.37 / Chapter 2.6 --- DNA transfection and CAT assay --- p.38 / Chapter 2.6.1 --- Transfection of recombinant plasmid to eukaryotic cells --- p.38 / Chapter 2.6.2 --- CAT assay --- p.39 / Chapter 2.7 --- 125I-LDL binding assay --- p.41 / Chapter 2.7.1 --- Radioactive iodination of LDL --- p.41 / Chapter 2.7.2 --- Purification of iodinated LDL --- p.41 / Chapter 2.7.3 --- Down regulation of LDL receptorin different cell lines --- p.41 / Chapter 2.7.4 --- Different Drugs treatment in HepG2 Cell line --- p.42 / Chapter 2.7.5 --- 125I-LDL binding assay --- p.43 / Chapter 2.8 --- Gel-shift mobility assay --- p.44 / Chapter 2.8.1 --- Extraction of crude nuclear extracts --- p.44 / Chapter 2.8.2 --- 5'end-labeling of synthetic oligonucleotides --- p.45 / Chapter 2.8.3 --- Purification of labeled oligonucleotides --- p.45 / Chapter 2.8.4 --- Nuclear protein and DNA binding reaction --- p.46 / Chapter 2.8.5 --- Gel-shift mobility electrophoresis by PhastSystem --- p.46 / Chapter 2.9 --- Construction of λ gt 11 cDNA library of HepG 2cell --- p.48 / Chapter 2.9.1 --- Purification of mRNA from HepG 2 --- p.48 / Chapter 2.9.2 --- cDNA preparation --- p.48 / Chapter 2.9.3 --- In-vitro packaging of phage --- p.48 / Chapter 2.9.3 --- Screening the expression library --- p.49 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Construction of recombinant plasmid --- p.50 / Chapter 3.1.1 --- hLDLR-promoter λ 34 clone --- p.50 / Chapter 3.1.2 --- Restriction site generation in LDLR- promoter by PCR --- p.52 / Chapter 3.1.3 --- Preparation of pGCAT-A reporter plasmid --- p.55 / Chapter 3.1.4 --- Screening of pGCAT-A-LDLRP recombinant --- p.55 / Chapter 3.1.5 --- Sequencing of pGCAT-A-LDLR- promoter recombinant --- p.57 / Chapter 3.2 --- CAT assay of recombinant plasmid on transfected HepG 2 cell --- p.57 / Chapter 3.3 --- 125I-LDL binding assay --- p.57 / Chapter 3.3.1 --- 125 I - LDL binding assay of different cell lines --- p.57 / Chapter 3.3.2 --- Characterization of cell surface receptor of HepG 2 cell by different drugs treatment --- p.61 / Chapter 3.4 --- Gel shift mobility assay --- p.63 / Chapter 3.4.1 --- Binding effect of Repeat 2 to different cell lines --- p.63 / Chapter 3.4.2 --- Optimizing the binding reactionin HepG 2 cell by poly(dI.dC) --- p.63 / Chapter 3.4.3 --- Specificity of Repeat 2 in binding to HepG 2 cell nuclear protein --- p.66 / Chapter 3.4.4 --- LDL dose response treatment in HepG2 cell --- p.68 / Chapter 3.4.4.1 --- Binding of Repeat 2 to specific nuclear protein --- p.68 / Chapter 3.4.4.2 --- Binding of Repeat 2 to a non- specific cell nuclear protein from cells treated with LDL --- p.68 / Chapter 3.4.5 --- Effect of different drugs on the binding of Repeat 2 to nuclear proteins in HepG 2 cell --- p.71 / Chapter 3.5 --- HepG 2 cell cDNA library screening --- p.73 / Chapter Chapter 4 --- Discussion --- p.77 / Chapter 4.1 --- Strategy on construction of reporter plasmid pLDLRP-GC AT-A --- p.77 / Chapter 4.2 --- The expression of CAT in HepG 2 cell --- p.78 / Chapter 4.3 --- Identification of a DNA binding protein for Repeat 2 in HepG 2 cell --- p.80 / Chapter 4.3.1 --- Binding effect of nuclear protein to Repeat 2 --- p.82 / Chapter 4.3.1.1 --- LDL dose response relationships --- p.82 / Chapter 4.3.1.2 --- Effect of protein inhibitors --- p.83 / Chapter 4.3.1.3 --- Effect of Shanzha --- p.86 / Chapter 4.3.2 --- Screening of the cDNA library of HepG 2 cells --- p.86 / Chapter Chapter 5 --- Further Studies --- p.88 / References --- p.89 / Appendix --- p.95

The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein that mediates the uptake of plasma LDL and thereby provides cholesterol to cells. During its synthesis in the endoplasmic reticulum, the LDL receptor folds and forms disulfide bonds in multiple cysteine-rich repeats. N- and 0-linked oligosaccharide chains are added in the endoplasmic reticulum and processed during passage through the Golgi apparatus, en route to the cell surface. The aim of this thesis was to study the influence of post-translational events on the synthesis of the LDL receptor. Experiments addressed: 1) the necessity of the compartmental organisation of the secretory pathway for the glycosylation of the LDL receptor; 2) the requirements for the formation of disulfide bonds; 3) the role for the chaperone, calnexin, in the folding of the LDL receptor; and 4) the manner in which folding was disrupted by mutations. Experiments were performed in cultured cells that were incubated with [³⁵S]methionine. Biosynthetically-labelled LDL receptor was immunoprecipitated and was analysed by SOS polyacrylamide gel electrophoresis.

by Ying Tat Mak. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 178-198). / Title --- p.1 / Abstract --- p.2 / Acknowledgments --- p.5 / Contents --- p.6 / Abbreviations --- p.9 / List of Tables --- p.11 / List of Figures --- p.13 / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Cholesterol Metabolism and Atherosclerosis --- p.15 / Chapter 1.1.1 --- Cholesterol and Cholesterol Metabolism --- p.17 / Chapter 1.1.2 --- Cholesterol Transport: Apolipoprotein and Lipoprotein --- p.23 / Chapter 1.1.3 --- Cholesterol and Atherosclerosis --- p.26 / Chapter 1.2 --- Hyperlipidaemia --- p.30 / Chapter 1.2.1 --- Primary and Secondary Hyperlipidaemia --- p.31 / Chapter 1.2.2 --- Mutations leading to Primary Hypercholesterolaemia --- p.36 / Chapter 1.3 --- Familial Hypercholesterolaemia --- p.38 / Chapter 1.3.1 --- Historical Aspects --- p.38 / Chapter 1.3.2 --- Clinical Features - Diagnosis and Consequences --- p.39 / Chapter 1.3.3 --- Population Prevalence --- p.40 / Chapter 1.3.4 --- Mutations in the Low Density Lipoprotein Receptor Gene --- p.41 / Chapter 1.4 --- Methods for Detecting Mutations in LDL Receptor Gene --- p.51 / Chapter 1.4.1 --- Southern Blotting Based Methods --- p.51 / Chapter 1.4.2 --- Polymerase Chain Reaction Based Methods --- p.52 / Chapter 1.4.3 --- Screening Methods for Unknown Mutations in LDL Receptor Gene --- p.56 / Chapter 1.5 --- Mutations of the LDL receptor gene in Chinese --- p.58 / Chapter Chapter 2: --- Objectives --- p.63 / Chapter Chapter 3: --- Materials and Methods / Chapter 3.1 --- Subjects / Chapter 3.1.1 --- Familial Hypercholesterolaemia Patients --- p.65 / Chapter 3.1.2 --- Normocholesterolaemia Subjects --- p.67 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Enzymes --- p.67 / Chapter 3.2.2 --- DNA Markers --- p.68 / Chapter 3.2.3 --- Reagents Kits --- p.68 / Chapter 3.2.4 --- Primers for PCR --- p.68 / Chapter 3.2.5 --- Chemicals and Reagents --- p.69 / Chapter 3.2.6 --- Radioisotopes --- p.70 / Chapter 3.2.7 --- Solutions and Buffers --- p.70 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Blood Collection --- p.71 / Chapter 3.3.2 --- General Biochemistry Tests --- p.72 / Chapter 3.3.3 --- DNA Extraction --- p.72 / Chapter 3.3.4 --- RNA Extraction --- p.73 / Chapter 3.3.5 --- Polymerase Chain Reaction --- p.74 / Chapter 3.3.6 --- Agarose Gel Electrophoresis --- p.76 / Chapter 3.3.7 --- Polyacrylamide Gel Electrophoresis --- p.78 / Chapter 3.3.8 --- Single Strand Conformation Polymorphism --- p.79 / Chapter 3.3.9 --- Reverse Transcription - Polymerase Chain Reaction --- p.79 / Chapter 3.3.10 --- Direct DNA Sequencing --- p.81 / Chapter 3.3.11 --- Haplotyping of the LDL receptor gene --- p.83 / Chapter 3.3.12 --- Restriction Enzyme Digestion --- p.84 / Chapter Chapter 4: --- Results / Chapter 4.1 --- Patients Investigations --- p.88 / Chapter 4.1.1 --- Normal Control Subjects --- p.88 / Chapter 4.1.2 --- Patients --- p.88 / Chapter 4.2 --- PCR-SSCP Analysis of LDL Receptor Gene --- p.90 / Chapter 4.3 --- Summary of Mutations Identified --- p.92 / Chapter 4.4 --- Novel Mutations --- p.94 / Chapter 4.5 --- Previously Reported Mutations --- p.97 / Chapter 4.6 --- Polymorphisms and Silent Mutation --- p.100 / Chapter 4.6.1 --- New Polymorphism --- p.100 / Chapter 4.6.2 --- New Silent Mutation --- p.102 / Chapter 4.6.3 --- Reported Polymorphisms --- p.103 / Chapter 4.7 --- Southern Blotting --- p.103 / Chapter 4.8 --- Haplotypes --- p.104 / (All Figures for Chapter 4) --- p.106 / Chapter Chapter 5: --- Discussions / Chapter 5.1 --- Use of SSCP in Screening for Mutations and Polymorphisms --- p.158 / Chapter 5.2 --- Novel and Reported Mutations --- p.160 / Chapter 5.3 --- Novel Polymorphism and Silent Mutation --- p.170 / Chapter 5.4 --- Common Polymorphisms --- p.171 / Chapter 5.5 --- Possible Common Mutations of the LDL Receptor Gene in Chinese --- p.172 / Chapter 5.6 --- Pattern of LDL Receptor Gene Mutations in Chinese --- p.173 / References --- p.178

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [205]-219).

Lipoprotein binding and metabolism in actively-dividing (subconfluent) and quiescent (postconfluent) bovine aortic endothelial cells (ECs) were qualitatively investigated by fluorescence microscopy using dioctadecylindocarbocyanine-labelled lipoproteins and by indirect immunofluorescence microscopy. LDL and acetylated-LDL (AcLDL) were seen bound to the surfaces of subconfluent ECs (at 4°C or at 37°C), as a random distribution of punctate foci. ECs therefore closely resembled fibroblasts in the distribution of LDL receptors on their surfaces. No binding of LDL was seen on postconfluent EC surfaces by either direct or indirect fluorescence microscopy. The patterns of AcLDL binding on postconfluent ECs resembled those on subconfluent ECs. Intracellular LDL and AcLDL occurred as perinuclear accumulations of large fluorescent disc-shaped profiles in subconfluent ECs. These accumulations were shown to arise from surface-bound material by pulse-chase experiments. Intracellular LDL was absent in the majority of postconfluent ECs, while AcLDL accumulation was massive. "Wounding" of cultures allowed simultaneous assessment of lipoprotein metabolism in quiescent and actively-dividing areas of the same culture. Quantitative assessments of the above-mentioned phenomena were made using ¹²⁵I-labelled lipoproteins. Receptor-mediated binding of LDL decreased five to ten-fold as the cultures modulated from subconfluent to postconfluent morphology. No receptor-bound LDL was detected in postconfluent ECs. Conversely, the amount of AcLDL bound increased at least fivefold during EC growth in parallel cultures. The amounts of lipoproteins endocytosed and metabolised were generally related proportionately to the amounts bound in each case. The distribution of LDL receptors on cultured cells was also investigated at the ultrastructural level using colloidal gold-conjugated LDL as a probe, and similarly labelled antibodies as probes. Whole-mounted cells with receptor probes bound to them were examined directly in the transmission electron microscope. The topographical distribution of LDL receptors has not been investigated by these techniques before. A novel method of preparing cytochemically-labelled, whole-mounted cells from styrene culture dishes was developed and used in this study. LDL Receptors expressed on the surfaces of human skin fibroblasts served to standardise these colloidal gold techniques and fortuitously led to new information on receptor distribution. Normal (FGo) and LDL receptor-negative mutant fibroblasts (GM 2000) acted as positive and negative controls respectively. Normal fibroblast LDL receptors were grouped into clusters consistent in size with coated pits (200 - 500 nm in diameter). A novel finding was the presence of a diffuse population of receptors scattered randomly amongst the clustered receptors. Another mutant fibroblast, GM 2408A, known to have an aberrant LDL receptor distribution, was also examined. Its receptors were shown to be dispersed singly, and in occasional groups of two and three, at random over the cell surfaces. No clusters were detected. The receptor-negative GM 2000 bound virtually no probes. While not as sensitive as the colloidal gold-conjugated LDL probe, an antireceptor monoclonal antibody (IgG-C7), localised by indirect immunogold labelling, gave similar results when applied to the above cells. This was taken as strong corroborative evidence that the LDL receptor distributions as determined by colloidal gold-conjugated LDL were correct. It is suggested that the dispersed population of receptors on normal fibroblasts may represent newly-emerged recycling receptors which have yet to cluster in coated pits. A further new finding reported here is the existence of the same two patterns of LD L receptors, dispersed and clustered, on the surface of subconfluent ECs. It was noted, from the study of whole-mounted and thin-sectioned cells, that the receptors were preferentially arranged in rings following the circumference of coated pit areas on the cell surface. Often these rings associated in groups or even coalesced into compound clusters. The significance of these groupings is not yet understood. In sharp contrast to the situation on subconfluent ECs, no LDL receptors (probed with the extremely sensitive colloidal-gold conjugated LDL) could be detected at the EM level on the surface of postconfluent ECs. Active cells in wounded postconfluent monolayers expressed abundant receptors detected at the EM level. It is concluded that postconfluent quiescent bovine aortic ECs in vitro metabolise virtually no LDL via the LDL-receptor pathway due to a vanishingly low number of LDL receptors. This contrasts with the ability of postconfluent cells to metabolise relatively large amounts of AcLDL via a receptor-mediated mechanism. The significance of these conclusions is discussed with respect to the interaction of plasma lipoproteins with the endothelium in vivo.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: The regulation of LRP in the CNS. Includes bibliographical references. Also available online through Digital Dissertations.

LDL-receptor mutations and familial defective apolipoprotein B-100 (codon 3500) (FOB), the known causes of monogenic hypercholesterolemia (MH), have similar clinical features. The nature of the mutations responsible for MH in South Africans of Indian origin was previously unknown. Similarly, the mutations in the LDL-receptor gene of a South African Black FH homozygote had also not been characterised. The aim of this thesis was to identify and analyse the LDL-receptor mutations in the Indian homozygotes NS, D, AV and AA and in the Black homozygote JL. In addition, the possible importance of FOB as a cause of MH in South Africans was also assessed. The patient NS was characterized as having two "Null" LDL-receptor alleles. His skin fibroblasts expressed no detectable LDL-receptor protein and very low levels of LDL-receptor mRNA of approximately normal size. Since NS' s LDLreceptor promoter sequence was normal, his alleles are likely to harbour exonic point mutations or minor rearrangements that cause premature stop codons. The patient D was found to be a heteroallelic homozygote. Two new point mutations in the LDL receptor, Asp₆₉ -Tyr and Glu₁₁₉-Lys, were identified. D's fibroblasts expressed about 30% of the normal surf ace complement of receptors that bound LDL poorly. This low number could at least be partially explained by their decreased stability. These mutations were not identified in any other Indian FH or hypercholesterolemic patients. Patients AV and AA were both shown to be homoallelic homozygotes for the Pro₆₆₄ -Leu mutation. This mutation was identified in 4 unrelated Muslim families of Gujerati origin suggesting that the mutation arose from this area in India. Contrary to previous reports (Knight et al. 1990, Soutar et al. 1989), neither LOL nor β-VLDL binding were shown to be affected by this mutation. These mutant receptors were rapidly degraded. Thus the disease FH in these subjects is presumably due to the low steady-state level of mature receptors that are functionally normal but exhibit accelerated turnover. The Pedi FH homozygote, JL, expressed very few LOL receptors due to decreased receptor synthesis associated with low mRNA levels and not due to enhanced degradation. One of JL's LOL receptor alleles has a 3 b.p. deletion in repeat 1 of the promoter (G. Zuilani, H. Hobbs and L.F. de Waal, personal communication). The nature of the defect in his other allele is unknown. The importance of FOB as a cause of monogenic hypercholesterolemia in the South African Indian, "Coloured" and Afrikaner populations was determined by screening hypercholesterolemic subjects with or without xanthomata. The absence of FOB in such patients, in whom the relevant common or founder South African mutations were excluded, suggested that this disorder was rarer in these groups than in North America and Europe. FOB was identified in two different families of mixed British and Afrikaner ancestry. One family contained individuals who were heterozygous for the FOB mutation, as well as the FH Afrikaner-1 and the FH Afrikaner-2 LOL-receptor mutations. In addition, 4 compound heterozygotes, who had both FOB and the FH Afrikaner-1 mutation and one individual whu inherited all 3 defects, were identified. This family allowed us to characterise the compound heterozygotes with one mutant LOLreceptor allele and FOB as having a condition that was probably intermediate in severity between the FH heterozygote and homozygote states.

The cellular degradation of the low density lipoprotein (LDL) receptor, and its ligand, LDL, were investigated in order to clarify certain mechanistic aspects of these important processes. Long-term lymphoblastoid cell lines and cultured human skin fibroblasts were used to examine the fate of ¹²⁵I-LDL subsequent to its uptake via receptor-mediated endocytosis. In both cases, binding activity was saturable, depended on the presence of calcium ions in the medium, and was calculated to have an equilibrium dissociation constant at 4ᵒC of 2 μg ¹²⁵I-LDL/ml. No high-affinity binding was detected when the ligand was modified by acetylation. After incubating the monolayers at 37°C LDL/LDL receptor complexes were internalized, and the receptors were recycled back to the surface within about 10 minutes. Apolipo-protein B in the LDL particles was largely degraded to the amino acid level: chloroquine, a lysosomotropic agent, inhibited the formation of the ¹²⁵I-LDL degradation products. Cells obtained from a number of heterozygous and homozygous familial hypercholesterolemic patients, as expected, bound markedly reduced amounts of ligand. The half-life of ¹²⁵I-LDL was measured after it had been introduced into cultured fibroblasts by one of the following processes: (i) uptake via receptor-mediated endocytosis in human skin fibroblasts with normal LDL receptors, or (ii) incorporation via scrape-loading into fibroblasts defective in LDL receptor content. The half-lives obtained were about 1 hour and 50 hours, respectively, indicating that efficient degradation of LDL occurred only when it was deIivered to lysosomes via receptor-mediated endocytosis.

As doenças cardiovasculares ainda são as principais causas de mortalidade e morbidade em todo o mundo. E a aterosclerose é uma das principais precursoras de vários desfechos clínicos como isquemias e infarto do miocárdio. As placas ateroscleróticas se desenvolvem preferencialmente em regiões de bifurcação ou curvatura dos vasos, onde o shear stress (SS) encontra-se diminuído ou perturbado. A expressão de proteínas pró-aterogênicas em regiões de baixo SS e ateroprotetoras em regiões de SS alto foram relatadas na literatura, porém o mecanismo completo carece de elucidação. Este trabalho teve por objetivo integrar proteômica e metabolômica para um melhor entendimento das alterações moleculares que acontecem nas células endoteliais em situações de alto e baixo SS, que podem resultar no desenvolvimento de lesões e placas ateroscleróticas. Para esta finalidade, células endoteliais foram submetidas a alto e baixo SS em sistema cone plate, seguido de análise proteômica e metabolômica por espectrometria de massas. Nossos dados demonstraram que o metabolismo de lipídio e metabolismo de modificações pós-traducionais de proteínas (N-glicosilações) estavam diminuídos em baixo SS. Em relação ao metabolismo de lipídio, foi identificada diminuição na concentração de ácidos graxos e na expressão de enzimas e proteínas transportadoras de lipídios em células sob baixo SS. O receptor de LDL, proteína importante para a homeostase do colesterol, foi identificado em menor concentração na membrana, bem como com alteração no seu perfil de glicosilação em células após baixo SS. As células submetidas a baixo SS e, portanto, aquelas com perfil pró-aterogênico, quando tratadas com estatina para o aumento da expressão de LDLR, aproximaram seu fenótipo ao de células submetidas a alto SS, adquirindo parte de um fenótipo ateroprotetor, com recuperação dos níveis de aminoácidos, lipídios, açúcares e ácidos carboxílicos. Os dados deste trabalho sugerem que o metabolismo de lipídios é um processo importante na manutenção do perfil ateroprotetor de células submetidas a alto SS. Além disso, as evidências demonstraram que estatinas apresentam uma atividade protetora, não apenas sistêmica, com diminuição do LDL circulante, mas também no microambiente vascular, contribuindo para o bom funcionamento das células endoteliais / Cardiovascular diseases are the main cause of the mortality and morbidity worldwide. Atherosclerotic plaque development is closely associated to the hemodynamic forces applied to endothelial cells (EC). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. EC under low and/or oscillatory SS (LSS) have upregulation of inflammatory proteins, adhesion and cellular permeability molecules. On the contrary, cells under high/laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind the SS regulating an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes suffered by endothelial cells under LSS and HSS that promote the atheroprone and atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism and post translational modification protein metabolism were downregulated in cells under LSS. About lipid metabolism, we found the LDLR, one important protein in cholesterol homeostasis, showed significant alterations both at the quantitative expression level, as well as regarding post-translational modifications. Under LSS, LDLR was seem at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of an HSS metabolic phenotype in EC under LSS. The phenotype was recovery based on increasing of amino acids, lipids, sugars and carboxylic acids. Altogether, our data suggest lipid metabolism is important in atheroprotective phenotype of endothelial cells under HSS. Statins showed benefits not only systemic, decreasing cholesterol level in blood, but also in vascular environment, contributing for protector phenotype of endothelial cells