An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjects with glucose intolerance.

OBJECTIVE: Glucose intolerance is frequently associated with an altered plasma lipid profile and increased cardiovascular disease risk. Nonetheless, lipid metabolism is scarcely studied in normolipidemic glucose-intolerant patients. The aim of this study was to investigate whether important lipid metabolic parameters, such as the kinetics of LDL free and esterified cholesterol and the transfer of lipids to HDL, are altered in glucose-intolerant patients with normal plasma lipids. METHODS: Fourteen glucose-intolerant patients and 15 control patients were studied; none of the patients had cardiovascular disease manifestations, and they were paired for age, sex, race and co-morbidities. A nanoemulsion resembling a LDL lipid composition (LDE) labeled with 14C-cholesteryl ester and ³H-free cholesterol was intravenously injected, and blood samples were collected over a 24-h period to determine the fractional clearance rate of the labels by compartmental analysis. The transfer of free and esterified cholesterol, triglycerides and phospholipids from the LDE to HDL was measured by the incubation of the LDE with plasma and radioactivity counting of the supernatant after chemical precipitation of non-HDL fractions. RESULTS: The levels of LDL, non-HDL and HDL cholesterol, triglycerides, apo A1 and apo B were equal in both groups. The 14C-esterified cholesterol fractional clearance rate was not different between glucose-intolerant and control patients, but the ³H-free-cholesterol fractional clearance rate was greater in glucose-intolerant patients than in control patients. The lipid transfer to HDL was equal in both groups. CONCLUSION: In these glucose-intolerant patients with normal plasma lipids, a faster removal of LDE free cholesterol was the only lipid metabolic alteration detected in our study. This finding suggests that the dissociation of free cholesterol from lipoprotein particles occurs in normolipidemic glucose intolerance and may participate in atherogenic signaling.

Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjects with glucose intolerance

OBJECTIVE: Glucose intolerance is frequently associated with an altered plasma lipid profile and increased cardiovascular disease risk. Nonetheless, lipid metabolism is scarcely studied in normolipidemic glucose-intolerant patients. The aim of this study was to investigate whether important lipid metabolic parameters, such as the kinetics of LDL free and esterified cholesterol and the transfer of lipids to HDL, are altered in glucose-intolerant patients with normal plasma lipids. METHODS: Fourteen glucose-intolerant patients and 15 control patients were studied; none of the patients had cardiovascular disease manifestations, and they were paired for age, sex, race and co-morbidities. A nanoemulsion resembling a LDL lipid composition (LDE) labeled with 14C-cholesteryl ester and ³H-free cholesterol was intravenously injected, and blood samples were collected over a 24-h period to determine the fractional clearance rate of the labels by compartmental analysis. The transfer of free and esterified cholesterol, triglycerides and phospholipids from the LDE to HDL was measured by the incubation of the LDE with plasma and radioactivity counting of the supernatant after chemical precipitation of non-HDL fractions. RESULTS: The levels of LDL, non-HDL and HDL cholesterol, triglycerides, apo A1 and apo B were equal in both groups. The 14C-esterified cholesterol fractional clearance rate was not different between glucose-intolerant and control patients, but the ³H-free-cholesterol fractional clearance rate was greater in glucose-intolerant patients than in control patients. The lipid transfer to HDL was equal in both groups. CONCLUSION: In these glucose-intolerant patients with normal plasma lipids, a faster removal of LDE free cholesterol was the only lipid metabolic alteration detected in our study. This finding suggests that the dissociation of free cholesterol from lipoprotein particles occurs in normolipidemic glucose intolerance and may participate in atherogenic signaling.

Resistance training changes LDL metabolism in normolipidemic subjects: a study with a nanoemulsion mimetic of LDL.

OBJECTIVE: To evaluate the effects of resistance training (RT) on the metabolism of an LDL-like nanoemulsion and on lipid transfer to HDL, an important step of HDL metabolism. METHODS: LDL-like nanoemulsion plasma kinetics was studied in 15 healthy men under regular RT for 1-4 years (age = 25 ± 5 years, VO(2)peak = 50 ± 6 mL/kg/min) and in 15 healthy sedentary men (28 ± 7 years, VO(2)peak = 35 ± 9 mL/kg/min). LDL-like nanoemulsion labeled with (14)C-cholesteryl-ester and (3)H-free-cholesterol was injected intravenously, plasma samples were collected over 24-h to determine decay curves and fractional clearance rates (FCR). Lipid transfer to HDL was determined in vitro by incubating of plasma samples with nanoemulsions (lipid donors) labeled with radioactive free-cholesterol, cholesteryl-ester, triacylglycerols and phospholipids. HDL size, paraoxonase-1 activity and oxidized LDL levels were also determined. RESULTS: The two groups showed similar LDL and HDL-cholesterol and triacylglycerols, but oxidized LDL was lower in RT (30 ± 9 vs. 61 ± 19 U/L, p = 0.0005). In RT, the nanoemulsion (14)C-cholesteryl-ester was removed twice as fast than in sedentary individuals (FCR: 0.068 ± 0.023 vs. 0.037 ± 0.028, p = 0.002), as well as (3)H-free-cholesterol (0.041 ± 0.025 vs. 0.022 ± 0.023, p = 0.04). While both nanoemulsion labels were removed at the same rate in sedentary individuals, RT (3)H-free-cholesterol was removed slower than (14)C-cholesteryl-ester (p = 0.005). HDL size, paraoxonase 1 and the transfer rates to HDL of the four lipids were the same in both groups. CONCLUSIONS: RT accelerated the clearance of LDL-like nanoemulsion, which probably accounts for the oxidized LDL levels reduction in RT. RT also changed the balance of free and esterified cholesterol FCR's. However, RT had no effect on HDL metabolism related parameters.

Effects of metformin plus gliclazide compared with metformin alone on circulating endothelial progenitor cell in type 2 diabetic patients.

Circulating endothelial progenitor cells (EPCs) play an important role in the development and progression of diabetic vascular complications. The aim of this study was to investigate the effects of gliclazide plus metformin (GLIMET) compared with metformin alone (MET) on number and function of circulating EPCs in T2DM patients. Patients with newly diagnosed T2DM were randomly divided into two groups, receiving the following treatments for 16 weeks: MET group (assuming metformin 500-2500 mg/day, n=24) and GLIMET group [assuming gliclazide (modified release, 30-60 mg/day)+metformin (250-1000 mg/day), n=23]. Circulating EPCs were quantified by flow cytometry, and the ability to uptake LDL and stain for lectin were used as another method of characterizing EPCs ex vivo. The functions of circulating EPCs were evaluated by colony-forming units (CFU) and migration. The status of oxidative stress was analyzed by serum-free malonaldehyde (MDA) and superoxide dismutase (SOD). There were no significant differences in clinical characteristics and number and function of circulating EPCs between two groups at baseline. Glycemic responses were similar after treatments. Compared with MET group, GLIMET group was associated with an increase in circulating EPCs number, DiLDL-lectin-positive EPCs, and migration. The mean improvements in MDA and SOD of GLIMET group were more strongly upregulated than those of MET group. This study demonstrated that both metformin mono-treatment and metformin plus gliclazide combination treatment provided with improvements in number and function of circulating EPCs. Compared with metformin mono-treatment, early use of combination therapy with gliclazide plus metformin made more effective improvements in circulating EPCs.

Receptores implicados en la inducción de citoquinas promovida por la LDL electronegativa en monocitos / Receptors involved in cytokine induction promoted by electronegative LDL in monocytes

Introducción La LDL electronegativa (LDL[−]) es una fracción minoritaria de la LDL total que se encuentra en circulación y presenta diferentes propiedades inflamatorias, siendo una de las más relevantes la inducción de citoquinas en células endoteliales y mononucleares. Sin embargo, no se conoce el mecanismo por el cual la LDL(−) ejerce su acción (..) (AU)

Introduction Elecronegative LDL (LDL(−)) is a small fraction of the total circulating LDL and has different inflammatory properties, one of the most important being the induction of cytokines in endotelial cells and monocytes. However, the mechanism by which LDL (−) exercises its action at cellular level is not known. The objective of this study was to evaluate the receptors involved in LDL (−) binding in monocytes, and how the liberation of cytokines (..) (AU)

Exercise training accelerates the removal from plasma of LDL-like nanoemulsion in moderately hypercholesterolemic subjects.

OBJECTIVE: Exercise training improves plasma lipid profile and diminishes risk of coronary heart disease. Previously, we showed that training increases LDL plasma clearance, as tested by an artificial LDL-like nanoemulsion method, presumably by increasing LDL receptor activity. In this study, we investigated whether training could also improve LDL clearance in hypercholesterolemic subjects (HCh) that are exposed to increased risk of cardiovascular events. METHODS: Twenty sedentary HCh and 20 normolipidemic (NL) sedentary volunteers were divided into four groups: 12 HCh submitted to 4-month training program, 8 HCh with no exercise program, 12 NL submitted to 4-month training and 8 NL with no exercise program. An LDL-like nanoemulsion labeled with (14)C-cholesteryl ester was injected intravenously into all subjects and plasma samples were collected during 24 h after injection to determine the fractional clearance rate (FCR, in h(-1)) by compartmental analysis. The study was performed on the first and on the last day of the 4-month study period. RESULTS: In both, trained HCh and NL groups, training increased nanoemulsion FCR by 36% (0.0443+/-0.0126; 0.0602+/-0.0187, p=0.0187 and 0.0503+/-0.0203; 0.0686+/-0.0216, p=0.0827, respectively). After training, LDL cholesterol diminished in both HCh and NL groups. In HCh, but not in NL group, LDL susceptibility to oxidation decreased, but oxidized LDL was unchanged. In both non-trained groups FCR was the same for the last and the 4-month previous evaluation. CONCLUSION: In HCh, exercise training increased the removal of LDL as tested by the nanoemulsion, and this probably accounted for decreased LDL cholesterol and diminished LDL susceptibility to oxidation.

Metabolism of a lipid nanoemulsion resembling low-density lipoprotein in patients with grade iii obesity.

INTRODUCTION: Obesity increases triglyceride levels and decreases high-density lipoprotein concentrations in plasma. Artificial emulsions resembling lipidic plasma lipoprotein structures have been used to evaluate low-density lipoprotein metabolism. In grade III obesity, low density lipoprotein metabolism is poorly understood. OBJECTIVE: To evaluate the kinetics with which a cholesterol-rich emulsion (called a low-density emulsion) binds to low-density lipoprotein receptors in a group of patients with grade III obesity by the fractional clearance rate. METHODS: A low-density emulsion was labeled with [(14)C]-cholesterol ester and [(3)H]-triglycerides and injected intravenously into ten normolipidemic non-diabetic patients with grade III obesity [body mass index higher than 40 kg/m(2)] and into ten non-obese healthy controls. Blood samples were collected over 24 hours to determine the plasma decay curve and to calculate the fractional clearance rate. RESULTS: There was no difference regarding plasma levels of total cholesterol or low-density lipoprotein cholesterol between the two groups. The fractional clearance rate of triglycerides was 0.086 +/- 0.044 in the obese group and 0.122 +/- 0.026 in the controls (p = 0.040), and the fractional clearance rate of cholesterol ester (h(-1)) was 0.052 +/- 0.021 in the obese subjects and 0.058 +/- 0.015 (p = 0.971) in the controls. CONCLUSION: Grade III obese subjects exhibited normal low-density lipoprotein removal from plasma as tested by the nanoemulsion method, but triglyceride removal was slower.

Metabolism of a lipid nanoemulsion resembling low-density lipoprotein in patients with grade III obesity

Introduction: Obesity increases triglyceride levels and decreases high-density lipoprotein concentrations in plasma. Artificial emulsions resembling lipidic plasma lipoprotein structures have been used to evaluate low-density lipoprotein metabolism. In grade III obesity, low density lipoprotein metabolism is poorly understood. Objective: To evaluate the kinetics with which a cholesterol-rich emulsion (called a low-density emulsion) binds to low-density lipoprotein receptors in a group of patients with grade III obesity by the fractional clearance rate. Methods: A low-density emulsion was labeled with [14C]-cholesterol ester and [³H]-triglycerides and injected intravenously into ten normolipidemic non-diabetic patients with grade III obesity [body mass index higher than 40 kg/m²] and into ten non-obese healthy controls. Blood samples were collected over 24 hours to determine the plasma decay curve and to calculate the fractional clearance rate. Results: There was no difference regarding plasma levels of total cholesterol or low-density lipoprotein cholesterol between the two groups. The fractional clearance rate of triglycerides was 0.086 ± 0.044 in the obese group and 0.122 ± 0.026 in the controls (p = 0.040), and the fractional clearance rate of cholesterol ester (h-1) was 0.052 ± 0.021 in the obese subjects and 0.058 ± 0.015 (p = 0.971) in the controls. Conclusion: Grade III obese subjects exhibited normal low-density lipoprotein removal from plasma as tested by the nanoemulsion method, but triglyceride removal was slower.

Low density lipoprotein adsorption on sol-gel derived alumina for blood purification therapy.

Among the clinical treatments of Familial Hyper cholesterolemia patients to reduce the concentration of low density lipoprotein (LDL), blood purification therapy is most suitable in which a blood-compatible adsorbent is employed. In the present study, alumina powders were prepared via a sol-gel route to develop a LDL-adsorbent Aluminum tri2-propoxide was hydrolyzed and subsequently calcined up to 1200 degrees C. Surface charge density and pore size distribution were measured, and the phases were identified. The alumina calcined above 400 degrees C had excellent blood compatibility in terms of endogenous clotting parameters, i.e., partial thromboplastin time: (PTT), prothrombin time: (PT), and the amount of fibrinogen: (Fib). The amount of LDL-adsorption (DeltaW(LDL)) increased with the calcining temperature, showing a good linear correlation to surface charge density. The 1200 degrees C sample consisted only of alpha-alumina, and was greatest in DeltaW(LDL). All samples involved pores smaller than 20 nm but not the pores large enough to accommodate LDL molecules (20-25 nm). From those results, it was concluded for the present alumina particles that the surface charge density was the primary factor and that the chemical activity of alpha-alumina also contributed to the excellent LDL-adsorption for the 1200 degrees C sample, while entrapping LDL in the pores was not an active mechanism.

Avaliação do metabolismo de lipídes em diabéticos tipo 1, normolipidêmicos e sem complicações microvasculares e macrovasculares significativas através de nanoemulsão lipídica artificial / Evaluation of lipid metabolism in normolipidemic type 1 diabetes individuals without significant clinical microvascular and macrovascular complications through artificial lipid nanoemulsion

INTRODUÇÃO: portadores de diabetes mellitus tipo 1 (DM1) apresentam, progressivamente, complicações vásculo-neurais. Os fatores que aumentam o risco de coronariopatia - hipertensão, dislipidemia e idade avançada - explicam, em parte, a alta mortalidade cardiovascular, entretanto diabéticos tipo 1 podem morrer de coronariopatia precoce e não apresentar os fatores de risco clássicos para aterosclerose. Modificações estruturais e funcionais nas lipoproteínas, alterando a sua composição e trocas lipídicas poderiam justificar o aumento de eventos vasculares, entretanto estas alterações podem não ser detectadas através das dosagens rotineiras de lípides plasmáticos. OBJETIVOS: através de nanoemulsão lipídica artificial (LDE) que simula a estrutura lipídica da LDL avaliamos, em portadores de DM1, normolipidêmicos, intensivamente tratados e sem complicações significativas da doença, a taxa de esterificação do colesterol, a remoção da nanoemulsão da circulação, o tamanho da partícula HDL e as transferências de lípides entre a nanoemulsão e as partículas HDL. Secundariamente, determinamos a influência do controle glicêmico, resistência à insulina (RI) e insulinização no metabolismo lipídico. MÉTODOS: estudamos 36 indivíduos diabéticos e 37 controles não-diabéticos pareados para idade, sexo e índice de massa corpórea. Nanoemulsão lipídica artificial com marcação radioativa nos lípides éster de colesterol (CE), colesterol livre (CL), triglicérides (TG) e fosfolípides (PL) foi utilizada para os estudos. Nanoemulsão com marcação 14C-CE e 3H-CL foi injetada nos participantes e amostras de sangue foram coletadas durante 24 horas para mensuração da radioatividade. Remoção dos lípides da circulação foi calculada por análise compartimental. A taxa da esterificação do colesterol livre foi calculada após extração e separação de lípides do plasma por cromatografia em camada delgada. Para estudo da transferência de lípides, nanoemulsões com marcação 14C-CE e 3H-CL ou 14C-PL ...

INTRODUTION: people with type 1 diabetes mellitus (DM1) have progressively neuro-vascular complications. Factors that increase the risk of coronary artery disease hypertension, dislipidemia and advanced age explains part of increased cardiovascular mortality, however some DM1 died of early coronary artery disease and often do not have atherosclerosis classical risk factors. Structural and functional changes in lipoproteins, altering their composition and activities of lipid exchange could justify the increase in vascular events but these changes are generally not detected by routine clinical laboratory plasma lipid exams. OBJETIVES: in normolipidemic DM1, intensively treated and without significant complications of disease we evaluated, by an artificial lipid nanoemulsion that resembles the lipid structure of LDL, rates of cholesterol esterification, nanoemulsion removal of the circulation, HDL particle size and lipid transfer from nanoemulsion to HDL. Secondarily, we determine the influence of glycemic control, insulin resistance (IR) and insulinization on lipid metabolism. METHODS: we studied 36 diabetics and 37 non-diabetic controls paired by age, sex and body mass index. Artificial lipid nanoemulsion labeled with radioactive lipids cholesterol ester (CE), cholesterol (CL), phospholipids (PL) and triglycerides (TG) was used for studies. Intravenous infusion of nanoemulsion 14C-CE e 3H-CL was injected in participants and blood was sampled over 24 hours for radioactivity measurement. Circulation lipid removal was calculated through compartmental analysis. Rate of cholesterol esterification was calculated after lipid extraction and separation by thin-layer chromatography. Nanoemulsion was incubated with plasma and radioactivity of lipids 14C-EC, 3H-CL, 14C-PL and 3H-TG transferred to the HDL was quantified after the precipitation of other apoB lipoproteins. The HDL diameter was measured by laser light scattering. The insulin resistance in diabetic ...

Effects of lipoprotein lipase and statins on cholesterol uptake into heart and skeletal muscle.

Regulation of cholesterol metabolism in cultured cells and in the liver is dependent on actions of the LDL receptor. However, nonhepatic tissues have multiple pathways of cholesterol uptake. One possible pathway is mediated by LPL, an enzyme that primarily hydrolyzes plasma triglyceride into fatty acids. In this study, LDL uptake and tissue cholesterol levels in heart and skeletal muscle of wild-type and transgenic mice with alterations in LPL expression were assessed. Overexpression of a myocyte-anchored form of LPL in heart muscle led to increased uptake of LDL and greater heart cholesterol levels. Loss of LDL receptors did not alter LDL uptake into heart or skeletal muscle. To induce LDL receptors, mice were treated with simvastatin. Statin treatment increased LDL receptor expression and LDL uptake by liver and skeletal muscle but not heart muscle. Plasma creatinine phosphokinase as well as muscle mitochondria, cholesterol, and lipid droplet levels were increased in statin-treated mice overexpressing LPL in skeletal muscle. Thus, pathways affecting cholesterol balance in heart and skeletal muscle differ.

A single common portal for clathrin-mediated endocytosis of distinct cargo governed by cargo-selective adaptors.

Sorting of transmembrane cargo into clathrin-coated vesicles requires endocytic adaptors, yet RNA interference (RNAi)-mediated gene silencing of the AP-2 adaptor complex only disrupts internalization of a subset of clathrin-dependent cargo. This suggests alternate clathrin-associated sorting proteins participate in cargo capture at the cell surface, and a provocative recent proposal is that discrete endocytic cargo are sorted into compositionally and functionally distinct clathrin coats. We show here that the FXNPXY-type internalization signal within cytosolic domain of the LDL receptor is recognized redundantly by two phosphotyrosine-binding domain proteins, Dab2 and ARH; diminishing both proteins by RNAi leads to conspicuous LDL receptor accumulation at the cell surface. AP-2-dependent uptake of transferrin ensues relatively normally in the absence of Dab2 and ARH, clearly revealing delegation of sorting operations at the bud site. AP-2, Dab2, ARH, transferrin, and LDL receptors are all present within the vast majority of clathrin structures at the surface, challenging the general existence of specialized clathrin coats for segregated internalization of constitutively internalized cargo. However, Dab2 expression is exceptionally low in hepatocytes, likely accounting for the pathological hypercholesterolemia that accompanies ARH loss.

Platelet-activating factor (PAF) increase intracellular lipid accumulation by increasing both LDL and scavenger receptors in human mesangial cells.

Intra- and extracellular lipid accumulation and the production of inflammatory mediators by renal and accessory cells may play an important role in the initiation and progression of these lesions. Platelet activating factor (PAF) is a biologically active phospholipid that is produced by various cells upon activation by different stimuli. It has been suggested that PAF plays a role in atherogenesis, and several studies indicated its participation in the pathogenesis of renal diseases. The aim of this study is to investigate the role of PAF on intracellular lipid accumulation and gene regulation of lipoprotein receptors in human mesangial cells (HMCs). A human mesangial cell line (HMC) was used to study the effects of PAF on foam cell formation by Oil red O staining and on the expression of LDLr, SR-AI, and PAF-R mRNA using RT-PCR. Native LDL caused foam cell formation in HMC in the presence of PAF. PAF enhanced LDLr expression and overrode LDL receptor suppression induced by a high concentration of LDL. Moreover, it enhanced SR-AI expression. PAF also caused increase in PAF-R expression. The above data suggest that PAF enhances its own receptor expression and then increases lipid accumulation by dysregulating LDL receptor regulation and inducing scavenger receptor expression in HMCs. These results suggest that PAF has a potential role in lipid mediated renal injury.

The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was identified as a major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells. LOX-1 critically mediates the endothelial dysfunction and the progression of atherosclerosis by oxLDL stimulation. It might be an important target for vascular endothelium. In order to obtain human LOX-1 and identify its mimic ligand for facilitating the study of LOX-1 function, a recombinant plasmid pPIC9K-His-hLOX-1 was structured and expressed human LOX-1 in Pichia pastoris GS115. Western blot analysis ensured the expressed recombinant human LOX-1 protein and a receptor-ligand binding assay showed that it had high binding affinity with oxLDL. With this receptor protein, a competitive fluorescence polarization-based high throughput screening method was established in a 384-well microplate to isolate the mimic ligands of human LOX-1. The evaluating parameter Z' value of 0.72 for this method showed that fluorescence polarization-based high throughput screening assay was robust and the results had a high reliability. By the fluorescence polarization-based high throughput screening assay, a total of 20,316 chemicals were screened, and 2 chemicals were identified that they have a high affinity with human LOX-1. Competitive uptake DiI-oxLDL assay by human LOX-1 transfected CHO-K1 cells further confirmed that two chemicals block the uptake of DiI-oxLDL. And the preliminary results indicated that isolated mimic ligands may act as a function of antagonist. The discovery of human LOX-1 mimic ligand would benefit to further study the function of LOX-1 and identify a novel avenue for prevention and treatment atherosclerosis.

In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells.

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.

Cholesteryl esters of aggregated LDL are internalized by selective uptake in human vascular smooth muscle cells.

OBJECTIVE: Low-density lipoprotein (LDL) receptor-related protein (LRP1) mediates the internalization of aggregated LDL (agLDL)-LDL trapped in the arterial intima bound to proteoglycans-into human vascular smooth muscle cells (VSMC). LRP1-mediated agLDL uptake induces high-intracellular cholesteryl ester (CE) accumulation. The aim of this study was to characterize the mechanism of agLDL internalization in human VSMC. METHODS AND RESULTS: The lipidic component of LDL was labeled with [3H] and the apolipoprotein component with [(125)I]. We found that >90% of intracellular CE derived from agLDL uptake was not associated with apoB100 degradation but was selectively taken up from agLDL. The inhibition of LRP1 expression by small interfering RNA treatment led to a decrease of 80+/-0.05% in agLDL-CE selective uptake. AgLDL induced intracellular CE accumulation without a concomitant CE synthesis. Cytosolic and cytoskeletal proteins were not required for CE transport. Electron and confocal microscopy experiments indicate that CE derived from agLDL accumulated in adipophilin-stained lipid droplets that were not removable by high-density lipoprotein. CONCLUSIONS: Taken together, these results demonstrate that LRP1 mediates the selective uptake of CE from agLDL and that CE derived from agLDL is not intracellularly processed but stored in lipid droplets in human VSMC.

State of the art of low-density lipoprotein apheresis in the year 2003.

Low-density lipoprotein (LDL) apheresis is a last-resort treatment for hypercholesterolemic patients resistant to conservative lipid-lowering therapy. In the extracorporeal circuit, LDL, Lp(a) and coagulation factors are selectively eliminated, while the beneficial proteins like high-density lipoprotein, albumin and immunoglobulins are returned to the patient. Clinical effects of LDL apheresis comprise improvement of symptoms like angina and exercise tolerance, reduction of clinical coronary events like unstable angina, need for angioplasty or bypass operation, myocardial infarction and ultimately coronary mortality. The reduction of atherogenic lipoproteins and of coagulation factors by LDL apheresis (LA) positively influences hemorheology, endothelial function and coronary reserve. In the controlled LAARS, LA significantly improved the electrocardiographic signs of myocardial ischemia in the treadmill test. In angiographically controlled trials such as LARS and L-CAPS, a reduction of progression of coronary lesions was observed; in favorable cases, regression of the stenoses could be documented. In addition, in the LDL apheresis coronary morphology trial, LA decreased the coronary plaque area. The Hokuriku trial documented a 72% decrease of coronary events (MACE) in the LA group vs. controls treated only by statins. In longitudinal studies, the incidence of MACE after regular LA decreased compared with the preapheresis period in the same patients. Apart from coronary heart disease, recent studies indicate a positive effect of LA also on carotid artery stenoses and peripheral vascular disease. Prospective randomized studies showed the beneficial effects of cascade filtration on age-related macular degeneration and of heparin-induced LDL precipitation apheresis on acute inner ear deafness.

Cells derived from regenerated endothelium of the porcine coronary artery contain more oxidized forms of apolipoprotein-B-100 without a modification in the uptake of oxidized LDL.

Increased accumulation of lipoproteins and cholesterol within cells from regenerated endothelium may be responsible for their reported dysfunction. This study compared the presence and uptake of oxidized forms of low-density lipoprotein (LDL) in cells derived from native and regenerated endothelium. Four weeks after balloon denudation, primary cultures of native and regenerated endothelial cells were prepared from porcine coronary arteries. Regenerated endothelium stained more strongly using an antibody against oxidized lipoproteins. The increase in oxidized forms of apolipoprotein-B-100 exhibited by cells from regenerated endothelium was not due to an increase in extracellular-induced oxidation of native LDL, measured as the production of thiobarbituric-acid-reactive substances, being identical in both cell types. Intracellular cholesterol and cholesterol ester content were unchanged in regenerated cells. Using flow cytometry, accumulation of oxidized LDL was investigated further by quantifying the uptake of a mildly oxidized preparation of 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate-labelled LDL. The parameters of uptake, EC(50) and E(max), were not different between cells from native and regenerated endothelium suggesting that the number of LOX-1 receptors was identical in the two cell types. Moreover, a negative correlation between the increased uptake of acetylated LDL and decreased cGMP production in response to bradykinin was observed in cells from regenerated endothelium. Thus, the increased incorporation of modified LDL and their intracellular oxidation could be responsible for the alteration in NO production. The presence of oxidized forms of LDL may be a marker of endothelium regeneration and could be involved in the endothelial dysfunction of pig coronary arteries 4 weeks after balloon denudation.

Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers.

BACKGROUND: Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of human telomerase reverse transcriptase (hTERT). METHODS: Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3-TWNT-1 co-culture system. RESULTS: TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retrovirally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis. CONCLUSIONS: TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.