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

Objetivo: O objetivo desse estudo foi avaliar a expressão gênica e a determinação proteica de receptores de LDL (LDL-R e LRP-1) na lesão de endometriose profunda e comparar com o endométrio de mulheres com e sem endometriose, assim como determinar o perfil lipídico de pacientes com e sem endometriose profunda. Pacientes e métodos: Realizamos estudo transversal, caso-controle, exploratório com 39 pacientes, sendo 20 pacientes com diagnóstico histológico de endometriose profunda com comprometimento intestinal e 19 pacientes sem endometriose submetidas à laqueadura tubária laparoscópica. Foram coletadas amostras de sangue periférico no dia do procedimento cirúrgico para análise do perfil lipídico por meio da determinação de Colesterol total, HDL, LDL, VLDL, Triglicérides, APO A I e APO B 100. Foram também coletadas amostras de tecido endometrial com cureta de Pipelle e lesão de endometriose como parte do tratamento cirúrgico laparoscópico. Todas as amostras foram enviadas para análise histológica e submetidas à pesquisa de expressão gênica por PCR Real Time e à determinação proteica por imunoistoquímica dos receptores de LDL (LDL-R e LRP-1). A fase do ciclo menstrual foi determinada no momento do procedimento cirúrgico. Resultados: As pacientes com endometriose profunda apresentaram níveis séricos de LDL-c significativamente inferiores às pacientes sem a doença (119 ± 23 vs 156 ± 35; p=0,001). O mesmo não foi observado com o colesterol total (187 ± 27 vs 194 ± 37; p=0,562), HDL-c (42 ± 9 vs 43 ± 14; p=0,792), Triglicérides (130 ± 40 vs 119 ± 55; p=0,486), APO A I (128,1 ± 33,3 vs 136,5 ± 19,9; p=0,373) e APO B 100 (76 ± 20,9 vs 91,7 ± 30,8; p=0,085). A análise de expressão gênica por PCR Real Time dos receptores de LDL revelou que o LDL-R foi significativamente mais expresso na lesão de endometriose comparado ao endométrio da mesma paciente, mas não ao endométrio de mulheres sem endometriose (0,012 ± 0,009 vs 0,019 ± 0,01 vs 0,027 ± 0,022; p < 0,001) e o LRP-1 foi significativamente mais expresso na lesão de endometriose tanto quando comparado ao endométrio da mesma paciente, quanto quando comparado ao endométrio das pacientes sem a doença (0,089 ± 0,076 vs 0,126 ± 0,072 vs 0,307 ± 0,207; p < 0,001). A análise de determinação proteica por imunoistoquímica dos mesmos receptores revelou que o endométrio de mulheres sem a doença apresentou score de intensidade de marcação de LDL-R significativamente maior que o endométrio e a lesão de mulheres com endometriose (15 ± 78,9 vs 9 ± 45 vs 2 ± 10; p=0,026), porém a marcação para o receptor LRP-1 não apresentou diferença estatisticamente significativa (9 ± 47,4 vs 6 ± 30 vs 0 ± 0; p=0,073). O estudo também demonstrou que houve expressão significativamente maior de RNAm do receptor LDL-R (p=0,001) na fase secretora do ciclo menstrual e o mesmo pode ser observado com relação à expressão de RNAm do receptor LRP-1, que foi superexpresso (p=0,008) no endométrio de mulheres sem a doença. Conclusões: De acordo com os resultados de nossa pesquisa, concluímos que há redução dos níveis séricos de LDL em pacientes com endometriose profunda. Além disso, observamos maior expressão gênica de receptores de LDL em membrana celular de focos de endometriose profunda, comparado ao endométrio, tanto de mulheres sem endometriose quanto de mulheres com endometriose, achado não similar ao observado com a determinação proteica. Houve maior expressão de receptores de LDL em endométrio na fase secretora do ciclo. Este estudo abre oportunidade para viabilização de nanoemulsões lipídicas para acoplamento e direcionamento de drogas antiproliferativas no tratamento da endometriose profunda / Objective: The objective of this study was to evaluate the gene expression and protein determination of LDL receptor (LDL-R and LRP-1) in deep endometriosis lesions and compare with the endometrium of women with and without endometriosis, as well as to profile lipid patients with and without deep endometriosis. Methods: We conducted an transversal, exploratory, case-control study with 39 patients: 20 patients with a histological diagnosis of deep endometriosis with intestinal involvement and 19 women without endometriosis who underwent laparoscopic tubal ligation. Peripheral blood samples were collected on the day of surgery for analysis of lipid profile by determining total cholesterol, HDL, LDL, VLDL, triglycerides, APO AI and APO B 100. Specimens of endometrial tissue were collected using a Pipelle curette and endometriosis lesion specimens were obtains during therapeutic laparoscopic surgery. All samples were sent for histological evaluation and gene expression analysis by Real Time PCR and protein determination by immunohistochemistry of the LDL receptor (LDL-R and LRP-1). The phase of the menstrual cycle was determined at the time of surgery. Results: Patients with severe endometriosis had serum LDL-C levels significantly lower than the patients without the disease (119 ± 23 vs 156 ± 35; p = 0.001). The same was not observed with total cholesterol (187 ± 27 vs 194 ± 37, p = 0.562), HDL-C (42 ± 9 vs 43 ± 14, p = 0.792), triglycerides (130 ± 40 vs 119 ± 55; p = 0.486), APO AI (128.1 ± 33.3 vs 136.5 ± 19.9; p = 0.373) and APO B 100 (76 ± 20.9 vs 91.7 ± 30.8, p = 0.085). The analysis of gene expression by Real Time PCR of LDL receptors revealed that there was significantly greater expression of LDL-R in endometriosis lesions as compared to the endometrium of the same patient, but not when compared to the endometrium of women without endometriosis (0.012 ± 0.009 vs 0.019 ± 0, 01 vs 0.027 ± 0.022, p &lt;0.001). LRP-1 was significantly expressed in endometriotic lesions both when compared to the endometrium of the same patient as compared to the endometrium of patients without the disease (0.089 ± 0.076 vs 0.126 ± 0.072 vs 0.307 ± 0.207, p &lt;0.001). Protein determination by immunohistochemistry of the same receptors revealed that the endometrium of women without endometriosis had a significantly higher staining intensity score than the endometrium and the lesions of women with endometriosis (15 ± 78.9 vs 9 ± 2 vs 45 ± 10, p = 0.026) but the measurement for the LRP-1 receptor showed no statistically significant difference (9 ± 47.4 vs 6 ± 30 vs 0 ± 0; p = 0.073). The study also demonstrated that there was significantly higher mRNA expression of the LDL-R receptor (p = 0.001) in the secretory phase of the menstrual cycle, and the same can be observed with respect to the mRNA expression of LRP-1 receptor, which was overexpressed (p = 0.008) in the endometrium of women without the disease. Conclusion: Based on the findings of our research, we concluded that there is a reduction of serum LDL levels in patients with deep endometriosis. Moreover, we observed higher gene expression of LDL receptors on the cell membrane of foci of deep endometriosis, compared to the endometrium, both in women without endometriosis and women with endometriosis, a finding unlike that observed with the protein determination. There was greater expression of LDL receptors in the endometrium during the secretory phase of the cycle. These findings suggest the feasibility of using lipid nanoemulsions for coupling and targeted delivery of antiproliferative drugs in the treatment of deep endometriosis

Objetivo: O objetivo desse estudo foi avaliar a expressão gênica e a determinação proteica de receptores de LDL (LDL-R e LRP-1) na lesão de endometriose profunda e comparar com o endométrio de mulheres com e sem endometriose, assim como determinar o perfil lipídico de pacientes com e sem endometriose profunda. Pacientes e métodos: Realizamos estudo transversal, caso-controle, exploratório com 39 pacientes, sendo 20 pacientes com diagnóstico histológico de endometriose profunda com comprometimento intestinal e 19 pacientes sem endometriose submetidas à laqueadura tubária laparoscópica. Foram coletadas amostras de sangue periférico no dia do procedimento cirúrgico para análise do perfil lipídico por meio da determinação de Colesterol total, HDL, LDL, VLDL, Triglicérides, APO A I e APO B 100. Foram também coletadas amostras de tecido endometrial com cureta de Pipelle e lesão de endometriose como parte do tratamento cirúrgico laparoscópico. Todas as amostras foram enviadas para análise histológica e submetidas à pesquisa de expressão gênica por PCR Real Time e à determinação proteica por imunoistoquímica dos receptores de LDL (LDL-R e LRP-1). A fase do ciclo menstrual foi determinada no momento do procedimento cirúrgico. Resultados: As pacientes com endometriose profunda apresentaram níveis séricos de LDL-c significativamente inferiores às pacientes sem a doença (119 ± 23 vs 156 ± 35; p=0,001). O mesmo não foi observado com o colesterol total (187 ± 27 vs 194 ± 37; p=0,562), HDL-c (42 ± 9 vs 43 ± 14; p=0,792), Triglicérides (130 ± 40 vs 119 ± 55; p=0,486), APO A I (128,1 ± 33,3 vs 136,5 ± 19,9; p=0,373) e APO B 100 (76 ± 20,9 vs 91,7 ± 30,8; p=0,085). A análise de expressão gênica por PCR Real Time dos receptores de LDL revelou que o LDL-R foi significativamente mais expresso na lesão de endometriose comparado ao endométrio da mesma paciente, mas não ao endométrio de mulheres sem endometriose (0,012 ± 0,009 vs 0,019 ± 0,01 vs 0,027 ± 0,022; p < 0,001) e o LRP-1 foi significativamente mais expresso na lesão de endometriose tanto quando comparado ao endométrio da mesma paciente, quanto quando comparado ao endométrio das pacientes sem a doença (0,089 ± 0,076 vs 0,126 ± 0,072 vs 0,307 ± 0,207; p < 0,001). A análise de determinação proteica por imunoistoquímica dos mesmos receptores revelou que o endométrio de mulheres sem a doença apresentou score de intensidade de marcação de LDL-R significativamente maior que o endométrio e a lesão de mulheres com endometriose (15 ± 78,9 vs 9 ± 45 vs 2 ± 10; p=0,026), porém a marcação para o receptor LRP-1 não apresentou diferença estatisticamente significativa (9 ± 47,4 vs 6 ± 30 vs 0 ± 0; p=0,073). O estudo também demonstrou que houve expressão significativamente maior de RNAm do receptor LDL-R (p=0,001) na fase secretora do ciclo menstrual e o mesmo pode ser observado com relação à expressão de RNAm do receptor LRP-1, que foi superexpresso (p=0,008) no endométrio de mulheres sem a doença. Conclusões: De acordo com os resultados de nossa pesquisa, concluímos que há redução dos níveis séricos de LDL em pacientes com endometriose profunda. Além disso, observamos maior expressão gênica de receptores de LDL em membrana celular de focos de endometriose profunda, comparado ao endométrio, tanto de mulheres sem endometriose quanto de mulheres com endometriose, achado não similar ao observado com a determinação proteica. Houve maior expressão de receptores de LDL em endométrio na fase secretora do ciclo. Este estudo abre oportunidade para viabilização de nanoemulsões lipídicas para acoplamento e direcionamento de drogas antiproliferativas no tratamento da endometriose profunda / Objective: The objective of this study was to evaluate the gene expression and protein determination of LDL receptor (LDL-R and LRP-1) in deep endometriosis lesions and compare with the endometrium of women with and without endometriosis, as well as to profile lipid patients with and without deep endometriosis. Methods: We conducted an transversal, exploratory, case-control study with 39 patients: 20 patients with a histological diagnosis of deep endometriosis with intestinal involvement and 19 women without endometriosis who underwent laparoscopic tubal ligation. Peripheral blood samples were collected on the day of surgery for analysis of lipid profile by determining total cholesterol, HDL, LDL, VLDL, triglycerides, APO AI and APO B 100. Specimens of endometrial tissue were collected using a Pipelle curette and endometriosis lesion specimens were obtains during therapeutic laparoscopic surgery. All samples were sent for histological evaluation and gene expression analysis by Real Time PCR and protein determination by immunohistochemistry of the LDL receptor (LDL-R and LRP-1). The phase of the menstrual cycle was determined at the time of surgery. Results: Patients with severe endometriosis had serum LDL-C levels significantly lower than the patients without the disease (119 ± 23 vs 156 ± 35; p = 0.001). The same was not observed with total cholesterol (187 ± 27 vs 194 ± 37, p = 0.562), HDL-C (42 ± 9 vs 43 ± 14, p = 0.792), triglycerides (130 ± 40 vs 119 ± 55; p = 0.486), APO AI (128.1 ± 33.3 vs 136.5 ± 19.9; p = 0.373) and APO B 100 (76 ± 20.9 vs 91.7 ± 30.8, p = 0.085). The analysis of gene expression by Real Time PCR of LDL receptors revealed that there was significantly greater expression of LDL-R in endometriosis lesions as compared to the endometrium of the same patient, but not when compared to the endometrium of women without endometriosis (0.012 ± 0.009 vs 0.019 ± 0, 01 vs 0.027 ± 0.022, p &lt;0.001). LRP-1 was significantly expressed in endometriotic lesions both when compared to the endometrium of the same patient as compared to the endometrium of patients without the disease (0.089 ± 0.076 vs 0.126 ± 0.072 vs 0.307 ± 0.207, p &lt;0.001). Protein determination by immunohistochemistry of the same receptors revealed that the endometrium of women without endometriosis had a significantly higher staining intensity score than the endometrium and the lesions of women with endometriosis (15 ± 78.9 vs 9 ± 2 vs 45 ± 10, p = 0.026) but the measurement for the LRP-1 receptor showed no statistically significant difference (9 ± 47.4 vs 6 ± 30 vs 0 ± 0; p = 0.073). The study also demonstrated that there was significantly higher mRNA expression of the LDL-R receptor (p = 0.001) in the secretory phase of the menstrual cycle, and the same can be observed with respect to the mRNA expression of LRP-1 receptor, which was overexpressed (p = 0.008) in the endometrium of women without the disease. Conclusion: Based on the findings of our research, we concluded that there is a reduction of serum LDL levels in patients with deep endometriosis. Moreover, we observed higher gene expression of LDL receptors on the cell membrane of foci of deep endometriosis, compared to the endometrium, both in women without endometriosis and women with endometriosis, a finding unlike that observed with the protein determination. There was greater expression of LDL receptors in the endometrium during the secretory phase of the cycle. These findings suggest the feasibility of using lipid nanoemulsions for coupling and targeted delivery of antiproliferative drugs in the treatment of deep endometriosis

Les maladies cardiovasculaires représentent la première cause de mortalité en France avec une prévalence encore plus importante à La Réunion. La plupart des décès sont attribuables aux cardiopathies ischémiques due à la rupture d'une plaque d'athérosclérose et à la formation d'un caillot entrainant une ischémie cardiaque. L'oxydation des lipoprotéines de basse densité (LDL) et la dysfonction de l'endothélium représentent des étapes importantes dans la déstabilisation de la plaque, d'où l’intérêt de rechercher un traitement capable de diminuer l'oxydation des LDL ou de protéger les cellules endothéliales de leurs effets cytotoxiques. Les polyphénols sont des molécules antioxydantes très présentes dans le règne végétal. Des études préliminaires du laboratoire sur des adipocytes humain on montré que des extraits riches en polyphénols d'Antirhea borbonica et de Doratoxylon apetalum, deux espèces respectivement endémique et indigène de l'Île de La Réunion, possédaient des propriétés antioxydantes et anti-inflammatoires (Marimoutou, M et al. 2015). Le But de cette thèse était, dans un premier temps, de tester les capacités antioxydantes et cytoprotectrices d'extraits riches en polyphénols de ces deux espèces sur des cellules endothéliales. Puis dans un second temps nous avons testé l'extrait de Doratoxylon apetalum sur un modèle murin d'athérosclérose (souris ApoE KO) pour évaluer son effet anti-inflammatoire sur les plaques d'athérome. / Cardiovascular diseases are the leading cause of death in France with an even higher prevalence in Reunion. Most deaths are due to ischemic heart disease because of atherosclerotic plaque rupture and thrombus formation leading to cardiac ischemia. The oxidation of low density lipoproteins (LDL) and endothelial dysfunction represent important steps in the destabilization of plaque, hence the interest of a treatment capable of reducing the oxidation of LDL or of protecting endothelial cells from their cytotoxic effects. Polyphenols are antioxidant molecules very present in the plant kingdom. Preliminary studies on human adipocytes have shown that polyphenol-rich extracts of Antirhea borbonica and Doratoxylon apetalum, respectively endemic and indigenous species of Reunion Island, had antioxidant and anti-inflammatory properties (Marimoutou , M et al., 2015). The aim of this thesis was, first, to test the antioxidant and cytoprotective capacities of polyphenol rich extracts of these two species on endothelial cells. Then in a second time we tested the extract of Doratoxylon apetalum on a murine model of atherosclerosis (ApoE KO mice) to evaluate its anti-inflammatory effect on atheroma plaques.

People with diabetes suffer from an increased incidence of atherosclerosis, possibly due to the hyperglycaemia associated with this disease. Glucose may covalently modify proteins via glycation and glycoxidation reactions. Reactive aldehydes (e.g. methylglyoxal and glycolaldehyde) generated from these glycation and glycoxidation reactions, lipid peroxidation and other metabolic pathways may also modify proteins in glycation and glycoxidation reactions. These reactions can result in the formation of advanced glycation end-products, which are increased in diabetes and associated complications such as atherosclerosis. Low-density lipoproteins (LDLs) are the main source of lipid in atherosclerotic plaques, and the lipid-laden foam cells contained within. Modification of the single protein in LDL, apolipoprotein B-100 (apo B) by glucose and aldehydes may result in recognition of these altered LDL particles by macrophage scavenger receptors and cellular accumulation of cholesteryl esters; such accumulation is characteristic of atherosclerotic foam cells. The extent and nature of the modifications of LDLs that give rise to this behaviour have been poorly characterised, especially in regards to modification/oxidation of protein versus lipid components induced by glucose and low-molecular-mass aldehydes. Therefore the aims of this project were to: 1) characterise LDL modification by glucose, methylglyoxal and glycolaldehyde; 2) examine the effect of these modified LDLs on arterial cells by monitoring cellular viability, proliferation and cholesterol and cholesteryl ester levels; and 3) examine macrophage handling of apo B from these modified LDLs. Glycolaldehyde induced more rapid and more extensive changes to LDL than methylglyoxal, which was significantly more modified than LDL exposed to glucose, in the presence or absence of Cu2+. LDL was modified by glycolaldehyde and methylglyoxal in a time- and concentration-dependent manner. These aldehyde-modified LDLs were significantly more negatively charged relative (determined by changes in relative electrophoretic mobility), more aggregated (by SDS-PAGE) and lost more Arg, Lys and Trp residues (assessed by fluorescence-based assays) than glucose-modified and control LDLs. Glucose-modified LDL had more modest increases in net negative charge, aggregation and only significantly lost Arg residues. Under the conditions examined none of the modified LDLs contained significant levels of the protein oxidation products DOPA and o-tyrosine, the lipid oxidation products 7-ketocholesterol and cholesteryl ester hydro(pero)oxides, nor marked depletion of the major antioxidant &alpha;-tocopherol or significant radical formation (EPR spectroscopy). Therefore these LDLs were glycated, but not (glyc)oxidised, and so allowed the cellular uptake of glycated LDL, rather than glycoxidised LDL, to be examined. These glycated LDLs had no effect on the cellular viability (assessed by LDH release), cell protein (BCA assay), and cholesterol and cholesteryl ester levels (quantified by reverse-phase HPLC) of endothelial and smooth muscle cells. The glycated LDLs also had no effects on human and mouse macrophage viability, protein and free cholesterol levels. However, exposure of macrophages to some of the glycated LDLs resulted in significant accumulation of cholesteryl esters and apo B. The greatest cellular accumulation of cholesteryl esters was in cells exposed to glycolaldehyde-modified LDL, which occurred in a time- and concentration-dependent manner. Less cholesteryl ester accumulation was observed in cells exposed to methylglyoxal-modified LDL, but some conditions resulted in significantly more cellular cholesteryl esters as compared to control LDLs, unlike glucose-modified LDL. Macrophages endocytosed significantly more apo B from glycolaldehyde-modified LDL labelled with 125I on the apo B, than methylglyoxal-modified 125I-LDL. Apo B from methylglyoxal-modified 125I-LDL was also endocytosed and degraded in greater amounts than control 125I-LDLs, unlike glucose-modified 125I-LDLs. The glycation of LDL by some low-molecular-mass aldehydes have been shown to result in model foam cell formation as characterised by cholesteryl ester and apo B accumulation. This accumulation correlated with increases in net negative charge, aggregation and loss of Lys and Trp residues of the apo B in glycated LDL particles. However, the differences in cellular uptake of glycolaldehyde- versus methylglyoxal-modified LDL were not completely resolved and it is postulated that this may arise from the extent or type of products formed on key amino acid residues, resulting in differential uptake by macrophage scavenger receptors, rather than loss of particular amino acids per se. Therefore these studies provide a potential mechanism to explain the increased atherosclerosis in people with diabetes, and a suitable model to examine the potential inhibition of the effects of glycated LDLs. This could provide potential therapeutic interventions to reduce diabetes-induced atherosclerosis.

Fluorometric density gradient ultracentrifugation is used to measure the lipoprotein density profile for cardiovascular disease risk assessment. The work presented establishes the effectiveness of using a single-spin separation as both an analytical tool and a preparative tool, while yielding valuable density information. This research expands on the analytical power of density gradient ultracentrifugation (DGU) by combining novel ethylenediaminetetraacetic acid (EDTA) gradient solutions, a fluorescent probe for analysis, and modern statistical methods for classification of heart disease risk. Sub-classes of lipoproteins are analyzed based on their density from the fluorescent lipoprotein density profile. The application of linear discriminant analysis (LDA) and sliced average variance estimation (SAVE) to the fluorometric DGU data yields a powerful classification tool. This method is capable of determining differences between control and cardiovascular disease patients that do not exhibit the traditional risk factors. The combination of these methods has great potential to serve as analytical tools for researchers in understanding the mechanisms of disease development and as a diagnostic tool for clinicians.

Background. The published reports of the effectiveness of simvastatin in treatment of hypercholesterolemia were mostly conducted in western populations, and only few studies in Asian or domestic populations have ever been reported. By regulations from Bureau of National Health Insurance, the effective dosage of lipid lowering agents should be started from lower dose, such as 20 mg of simvastatin per day. Whether this dosage of simvastatin is effective for treatment of patients with hypercholesterolemia and the efficacy and safety of such dosage are the objects of this study. Materials and Methods. After the approval of IRB in a medical center located at north Taiwan, a randomized 12-week study was conducted to evaluate the efficacy and safety of 20mg/day simvastatin treatment on hypercholesterolemia. By randomization 65 patients, followed up at cardiovascular outpatient department under the diagnosis of primary hypercholesterolemia, were enrolled into a 4-week washout period, and finally 49 intent-to-treat patients entered a 12-week treatment with 20mg simvastatin per day, given through oral routine in the evening. Demographic and laboratory data were obtained before and after treatment. The primary efficacy measure was the changes from baseline in lipid parameters. Tolerability was assessed in terms of the overall incidence of adverse experiences and the most commonly reported adverse experiences. Results. The per-protocol analyses included 39 hypercholesterolemic patients whom completed 12 weeks of therapy. Total cholesterol and LDL cholesterol at the end of the study period showed significant reductions by 22.5¢H (p<0.001) and 29.8¢H(p<0.001), respectively. Triglyceride also showed a significant reduction by 31.8% (p=0.006), whereas total alkaline phosphatase and calcium showed a weak and insignificant change over the study period. The female group had significantly greater reduction in triglyceride than that in the male group, and the non-smoking group also had significantly greater reduction in triglyceride than that in smoking group after 12-week treatment. There were 17 studied cases (34.7%) had minor transient but clinical insignificant increases in serum aspartate aminotransferase and alanine aminotransferase, and 7 cases (14.3%) experienced symptom of painful muscle, of whom 3 cases (6.1%) dropped out this study. Conclusion. Our results, although obtained from a small scale of hypercholesterolemic patients, suggest a probable positive efficacy and good tolerability with only few minor side effects of simvastatin on blood lipids.

No description available.

People with diabetes suffer from an increased incidence of atherosclerosis, possibly due to the hyperglycaemia associated with this disease. Glucose may covalently modify proteins via glycation and glycoxidation reactions. Reactive aldehydes (e.g. methylglyoxal and glycolaldehyde) generated from these glycation and glycoxidation reactions, lipid peroxidation and other metabolic pathways may also modify proteins in glycation and glycoxidation reactions. These reactions can result in the formation of advanced glycation end-products, which are increased in diabetes and associated complications such as atherosclerosis. Low-density lipoproteins (LDLs) are the main source of lipid in atherosclerotic plaques, and the lipid-laden foam cells contained within. Modification of the single protein in LDL, apolipoprotein B-100 (apo B) by glucose and aldehydes may result in recognition of these altered LDL particles by macrophage scavenger receptors and cellular accumulation of cholesteryl esters; such accumulation is characteristic of atherosclerotic foam cells. The extent and nature of the modifications of LDLs that give rise to this behaviour have been poorly characterised, especially in regards to modification/oxidation of protein versus lipid components induced by glucose and low-molecular-mass aldehydes. Therefore the aims of this project were to: 1) characterise LDL modification by glucose, methylglyoxal and glycolaldehyde; 2) examine the effect of these modified LDLs on arterial cells by monitoring cellular viability, proliferation and cholesterol and cholesteryl ester levels; and 3) examine macrophage handling of apo B from these modified LDLs. Glycolaldehyde induced more rapid and more extensive changes to LDL than methylglyoxal, which was significantly more modified than LDL exposed to glucose, in the presence or absence of Cu2+. LDL was modified by glycolaldehyde and methylglyoxal in a time- and concentration-dependent manner. These aldehyde-modified LDLs were significantly more negatively charged relative (determined by changes in relative electrophoretic mobility), more aggregated (by SDS-PAGE) and lost more Arg, Lys and Trp residues (assessed by fluorescence-based assays) than glucose-modified and control LDLs. Glucose-modified LDL had more modest increases in net negative charge, aggregation and only significantly lost Arg residues. Under the conditions examined none of the modified LDLs contained significant levels of the protein oxidation products DOPA and o-tyrosine, the lipid oxidation products 7-ketocholesterol and cholesteryl ester hydro(pero)oxides, nor marked depletion of the major antioxidant &alpha;-tocopherol or significant radical formation (EPR spectroscopy). Therefore these LDLs were glycated, but not (glyc)oxidised, and so allowed the cellular uptake of glycated LDL, rather than glycoxidised LDL, to be examined. These glycated LDLs had no effect on the cellular viability (assessed by LDH release), cell protein (BCA assay), and cholesterol and cholesteryl ester levels (quantified by reverse-phase HPLC) of endothelial and smooth muscle cells. The glycated LDLs also had no effects on human and mouse macrophage viability, protein and free cholesterol levels. However, exposure of macrophages to some of the glycated LDLs resulted in significant accumulation of cholesteryl esters and apo B. The greatest cellular accumulation of cholesteryl esters was in cells exposed to glycolaldehyde-modified LDL, which occurred in a time- and concentration-dependent manner. Less cholesteryl ester accumulation was observed in cells exposed to methylglyoxal-modified LDL, but some conditions resulted in significantly more cellular cholesteryl esters as compared to control LDLs, unlike glucose-modified LDL. Macrophages endocytosed significantly more apo B from glycolaldehyde-modified LDL labelled with 125I on the apo B, than methylglyoxal-modified 125I-LDL. Apo B from methylglyoxal-modified 125I-LDL was also endocytosed and degraded in greater amounts than control 125I-LDLs, unlike glucose-modified 125I-LDLs. The glycation of LDL by some low-molecular-mass aldehydes have been shown to result in model foam cell formation as characterised by cholesteryl ester and apo B accumulation. This accumulation correlated with increases in net negative charge, aggregation and loss of Lys and Trp residues of the apo B in glycated LDL particles. However, the differences in cellular uptake of glycolaldehyde- versus methylglyoxal-modified LDL were not completely resolved and it is postulated that this may arise from the extent or type of products formed on key amino acid residues, resulting in differential uptake by macrophage scavenger receptors, rather than loss of particular amino acids per se. Therefore these studies provide a potential mechanism to explain the increased atherosclerosis in people with diabetes, and a suitable model to examine the potential inhibition of the effects of glycated LDLs. This could provide potential therapeutic interventions to reduce diabetes-induced atherosclerosis.

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