A Critical Role of PCSK9 in Mediating IL-17-Producing T Cell Responses in Hyperlipidemia

We previously demonstrated that atherogenic Ldlr(−/−)Apobec1(−/−) (LDb) double knockout mice lacking both low-density lipoprotein receptor (LDLR) and apolipoprotein B mRNA-editing catalytic polypeptide-1 (Apobec1) had increased serum IL-17 levels, with T cell programming shifted towards Th17 cells. In this study, we assessed the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in T cell programming and atherogenesis. We deleted the Pcsk9 gene from LDb mice to generate Ldlr(−/−)Apobec1(−/−)Pcsk9(−/−) (LTp) triple knockout mice. Atherosclerosis in the aortic sinus and aorta were quantitated. Lymphoid cells were analyzed by flow cytometry, ELISA and real-time PCR. Despite of dyslipidemia, LTp mice developed barely detectable atherosclerotic lesions. The IL-17, was very low in plasma and barely detectable in the aortic sinus in the LTp mice. In the spleen, the number of CD4⁺CD8⁻ cells and splenocytes were much lower in the LDb mice than LTp mice, whereas, the IL-17-producing cells of γδTCR⁺ T cells and effector memory CD4⁺ T cells (CD44(hi)CD4⁺) in the spleen were significantly higher in the LDb mice than in the LTp mice. The Rorc mRNA expression levels were elevated in LDb mice compared to LTp mice. When re-stimulated with an anti-CD3 Ab, CD44(hi)CD4⁺ T cells from LDb mice secreted more IL-17 than those from LTp mice. T cells from LDb mice (with PCSK9) produce more IL-17 at basal and stimulated conditions when compared with LTp mice (without PCSK9). Despite the dyslipidemic profile and the lack of LDLR, atherogenesis is markedly reduced in LTp mice. These results suggest that PCSK9 is associated with changes in T cell programming that contributes to the development of atherosclerosis.

Genome-wide association study of degenerative mitral valve disease in Maltese dogs

Genome-wide association study (GWAS) is a powerful tool for identifying the genetic causes of various diseases. This study was conducted to identify genomic variation in Maltese dog genomes associated with degenerative mitral valve disease (DMVD) development and to evaluate the association of each biological condition with DMVD in Maltese dogs. DNA was extracted from blood samples obtained from 48 Maltese dogs (32 with DMVD and 16 controls). Genome-wide single nucleotide polymorphism (SNP) genotyping was performed. The top 30 SNPs from each association of various conditions and genetic variations were mapped to their gene locations. A total of 173,662 loci were successfully genotyped, with an overall genotype completion rate of 99.41%. Quality control analysis excluded 46,610 of these SNPs. Manhattan plots were produced using allelic tests with various candidate clinical conditions. A significant peak of association was observed between mitral valve prolapse (MVP) and SNPs on chromosome 17. The present study revealed significant SNPs in several genes associated with cardiac function, including PDZ2, Armadillo repeat protein detected in velo-cardio-facial syndrome, catenin (cadherin-associated protein) alpha 3, low-density lipoprotein receptor class A domain containing protein 4, and sterile alpha motif domain containing protein 3. To our knowledge, this is the first study of a genetic predisposition to DMVD in Maltese dogs. Although only a limited number of cases were analyzed, these data could be the basis for further research on the genetic predisposition to MVP and DMVD in Maltese dogs.

Detection of Familial Hypercholesterolemia Using Next Generation Sequencing in Two Population-Based Cohorts / 전남의대학술지

We aimed to evaluate the prevalence of familial hypercholesterolaemia (FH) in a subject with hypercholesterolaemia from two population-based cohorts in South Korea. A total of 283 subjects with total cholesterol levels of 290 mg/dL (7.5 mmol/L) or higher were selected from the Namwon and Dong-gu Studies. We used next generation sequencing (NGS) to detect mutations in low-density lipoprotein receptors (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. We have confirmed 17 different mutations of the LDLR, APOB and PCSK9 in 23 subjects (8.1%). Eleven LDLR variants and one APOB variant have been previously reported. One LDLR and two PCSK9 rare variants were identified in the variants database, but not in the FH mutation database. Two novel LDLR variants were found, p.Leu680Val, and p.Thr734Phe. No LDLR, APOB or PCSK9 deletions nor insertions were found. When the subjects were restricted to 110 subjects with a total cholesterol ≥310 mg/dL, only 10 variants were found in the 10 subjects (9.1%). These results suggest that given the low prevalence of FH mutations in subjects with high total cholesterol levels, NGS-based testing for a population-based approach to FH detection may not be cost-effective.

Kimchi attenuates fatty streak formation in the aorta of low-density lipoprotein receptor knockout mice via inhibition of endoplasmic reticulum stress and apoptosis

BACKGROUND/OBJECTIVES: Endoplasmic reticulum (ER) stress is positively associated with atherosclerosis via elevating macrophage cell death and plaque formation, in which oxidative stress plays a pivotal role. Antioxidative, lipid-lowering, and anti-atherogenic effects of kimchi, a Korean fermented vegetable, have been established, wherein capsaicin, ascorbic acid, quercetin, 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid, and lactic acids were identified. In this study, mechanisms of action of kimchi methanol extracts (KME) on fatty streak formation via suppression of ER stress and apoptosis in aorta were examined in low-density lipoprotein receptor knockout mice. MATERIALS AND METHODS: Mice fed a high cholesterol diet with an oral administration of KME (KME group, 200 mg·kg-bw⁻¹·day⁻¹) or distilled water (control group) for 8 weeks (n = 20 for group). Plasma lipid and oxidative stress levels were evaluated. Protein expression was measured by western blot assay. Fatty streak lesion size and the degree of apoptosis were examined in the aorta. RESULTS: Compared to the control group, in the KME group, plasma lipids levels were decreased and oxidative stress was alleviated (P < 0.05). Protein expression levels of nuclear factor (erythroid-derived 2)-like 2-mediated antioxidants in aorta were increased whereas those for ER stress markers, glucose regulated protein 78, phospho-protein kinase RNA-like ER kinase, phospho-eukaryotic initiation factor 2 subunit α, X-box binding protein 1, and C/EBP homologous protein were decreased in the KME group (P < 0.05). Moreover, apoptosis was suppressed via downregulation of phospho-c-Jun N-terminal kinase, bcl-2-associated X protein, caspases-9, and -3 with a concomitant upregulation of anti-apoptotic protein, B-cell lymphoma 2 (P < 0.05). Fatty streak lesion size was reduced and the degree of apoptosis was less severe in the KME group (P < 0.05). CONCLUSIONS: In conclusion, antioxidant activity of KME might prevent fatty streak formation through, in part, inhibition of ER stress and apoptosis in aortic sinus where macrophages are harbored.

The Role of Vitamin D in Menopausal Medicine / 고신대학교의과대학학술지

Menopause is the time at which menstruation stops in women. After menopause, women are more susceptible to some diseases, especially osteoporosis and cardiovascular disease. Vitamin D has a protective effect against osteoporosis by facilitating the absorption of calcium and affecting parathyroid hormone. Vitamin D also affects cardiovascular function by lowering the blood pressure, which affects the renin–angiotensin system and alters the low-density lipoprotein receptor activity. This paper discusses supplemental vitamin D in postmenopausal women with osteoporosis and cardiovascular disease.

Corn silk extract improves cholesterol metabolism in C57BL/6J mouse fed high-fat diets

BACKGROUND/OBJECTIVES: Corn silk (CS) extract contains large amounts of maysin, which is a major flavonoid in CS. However, studies regarding the effect of CS extract on cholesterol metabolism is limited. Therefore, the purpose of this study was to determine the effect of CS extract on cholesterol metabolism in C57BL/6J mouse fed high-fat diets. MATERIALS/METHODS: Normal-fat group fed 7% fat diet, high-fat (HF) group fed 25% fat diet, and high-fat with corn silk (HFCS) group were orally administered CS extract (100 mg/kg body weight) daily. Serum and hepatic levels of total lipids, triglycerides, and total cholesterol as well as serum free fatty acid, glucose, and insulin levels were determined. The mRNA expression levels of acyl-CoA: cholesterol acyltransferase (ACAT), cholesterol 7-alpha hydroxylase (CYP7A1), farnesoid X receptor (FXR), lecithin cholesterol acyltransferase (LCAT), low-density lipoprotein receptor, 3-hyroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), adiponectin, leptin, and tumor necrosis factor α were determined. RESULTS: Oral administration of CS extract with HF improved serum glucose and insulin levels as well as attenuated HF-induced fatty liver. CS extracts significantly elevated mRNA expression levels of adipocytokines and reduced mRNA expression levels of HMG-CoA reductase, ACAT, and FXR. The mRNA expression levels of CYP7A1 and LCAT between the HF group and HFCS group were not statistically different. CONCLUSIONS: CS extract supplementation with a high-fat diet improves levels of adipocytokine secretion and glucose homeostasis. CS extract is also effective in decreasing the regulatory pool of hepatic cholesterol, in line with decreased blood and hepatic levels of cholesterol though modulation of mRNA expression levels of HMG-CoA reductase, ACAT, and FXR.

Cholesterol metabolism and homeostasis in the brain

Cholesterol is an essential component for neuronal physiology not only during development stage but also in the adult life. Cholesterol metabolism in brain is independent from that in peripheral tissues due to blood-brain barrier. The content of cholesterol in brain must be accurately maintained in order to keep brain function well. Defects in brain cholesterol metabolism has been shown to be implicated in neurodegenerative diseases, such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), and some cognitive deficits typical of the old age. The brain contains large amount of cholesterol, but the cholesterol metabolism and its complex homeostasis regulation are currently poorly understood. This review will seek to integrate current knowledge about the brain cholesterol metabolism with molecular mechanisms.

Effects of alkaloids from coptidis rhizoma on blood lipid metabolism and low-denstity lipoprotein receptor mRNA in golden hamsters / 中国中药杂志

To study the effects of alkaloids from Coptidis Rhizoma on low-density lipoprotein receptor (LDLR) mRNA expression and antihyperlipedemic levels. The LDLR mRNA expression were detected by real time fluorescence quantitative PCR, and the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in serum were measured at the first and last examination. The results show that, after the drug treatment, compared with the model group, each drug group showed a lipid-lowering effect. Especially, coptisine, palmatine, jatrorrhinze were significantly reduced TC, TG, LDL-c (P < 0.05, P < 0.01), and increased HDL-c (P < 0.01). In addition, they also increased mRNA expression of the LDLR in liver and HepG2 cells. The results showed that alkaloids from Coptidis Rhizoma can regulate lipid metabolism disorder, and coptisine have the best lipid-lowering effect.

Efeitos da quimioterapia neoadjuvante sobre os receptores de lipoproteínas no tecido tumoral em pacientes com carcinoma da mama localmente avançado / Effects of neoadjuvant chemotherapy on lipoprotein receptors in tumor tissues of patients with locally advanced breast cancer

Os tumores malignos apresentam um aumento da expressão dos receptores de lipoproteínas, devido ao aceleramento da proliferação celular com consequente aumento da necessidade de lípides para a síntese das membranas celulares. Esse aumento da expressão dos receptores de LDL no câncer pode ser utilizado para concentrar fármacos de ação antineoplásica em tecido tumoral, utilizando lipoproteínas ou nanoemulsões semelhantes a lipoproteínas como veículo. No presente estudo, foram investigados os efeitos da quimioterapia convencional na expressão dos receptores de LDL e LRP-1 em 16 pacientes com carcinoma de mama estádios II ou III, não candidatas à cirurgia conservadora e com indicação de tratamento quimioterápico neoadjuvante. A expressão dos receptores LDLR e LRP-1 foi avaliada por imunoistoquimica em tecido mamário normal e em tecido neoplásico antes e depois da quimioterapia neoadjuvante. Quatro pacientes que apresentaram resposta completa à quimioterapia foram retiradas da análise da expressão de receptores por não existir tumor no fragmento cirúrgico. Em relação ao LDLR, a expressão desse receptor no tecido neoplásico foi maior em comparação ao tecido normal em 8 das 11 pacientes. Após a quimioterapia, a expressão do receptor de LDL diminuiu em 6, aumentou em 4 e não se alterou em 2 pacientes. Do mesmo modo, a expressão do receptor LRP-1 no tecido tumoral estava aumentada em relação ao tecido normal em 4 pacientes das 12 avaliadas. Em comparação com o tecido tumoral antes da quimioterapia, a expressão do receptor LRP-1 diminuiu em 6, aumentou em 4 e permaneceu inalterada em 2 pacientes após a quimioterapia. Esses dados mostram que o efeito da quimioterapia na expressão dos receptores de lipoproteínas foi heterogêneo. A redução da expressão dos receptores não foi o padrão observado, o que indica que o uso de sistemas de carreamento de fármacos via receptores de LDL para o tratamento do câncer pode ser de grande importância...

Proliferative tumor cells present a high expression of LDL receptors due to accelerated mitosis rates which takes to increased need of lipids internalization for building new membranes. Upregulation of LDL receptors may be used as a gate to deliver anticancer drugs to tumor tissues using lipoproteins or artificial nanoemulsions as vehicle. This study investigated the effects of conventional chemotherapy on the expression of LDL and LRP-1 receptors in 16 patients with breast cancer in stage II or III who were not candidates to conservative surgery and with indication of neo-adjuvant chemotherapy. Expression of LDL and LRP-1 receptor was evaluated by immunohistochemistry in normal and neoplastic breast tissue before and after chemotherapy. For absence of tumor in the surgical fragments, 4 patients who presented complete response to chemotherapy were excluded from this analysis. In relation of LDLR, the expression in neoplastic tissue was higher than in normal tissue in 8 of 11 patients. After chemotherapy, LDL receptor expression diminished in 6, increased in 4 and unchanged in 2 patients. Expression of LRP-1 in tumor tissue was higher in 4 of 12 patients when compared to normal tissue. After chemotherapy, the expression of LRP-1 diminished in 6, increased in 4 and showed no difference in 2 patients. These data show that the chemotherapy effects on the tumor expression of LDL receptors were very heterogeneous. The diminution of the receptor expression is not the post-chemotherapy pattern, allowing the use of drug carrier systems that target cancer cells via the LDL receptor pathway. These results may contribute for the design of future clinical assays.

Captação celular de uma nanoemulsão semelhante a LDL (LDE): efeito da variação na composição química e expressão de receptores de lipoproteínas / Low density lipoprotein like (LDE) nanoemulsion cell uptake: chemical composition and lipoprotein receptor expression

A nanoemulsão LDE tem composição lipídica semelhante à da LDL natural e é utilizada para estudos do metabolismo da LDL. Estudos anteriores mostraram que a LDE é captada pelas células pelo LDL-r, porém outros receptores podem estar envolvidos nesta captação, como LRP-1, CD36 e CD68. Os objetivos deste estudo foram: investigar a captação da LDE por células endoteliais, fibroblastos, monócitos, macrófagos e H292, identificar os receptores envolvidos na captação da LDE pelas mesmas células e avaliar os efeitos da modificação química da LDE sobre a estabilidade, captação celular, lipoperoxidação celular e citotoxicidade. A LDE marcada com [3H]-colesterol livre e [14C]-éster de colesterol foi incubada por 4 horas com as linhagens celulares. Após a incubação, foram realizados os testes de captação da LDE e competição da LDE com a LDL natural. A expressão dos receptores LDL-r, LRP, CD36 e CD68 foi avaliada pelos métodos de imunocitoquímica, citometria de fluxo e PCR real time. Para investigar os efeitos da modificação da LDE (LDE-CO), o éster de colesterololeato de colesterol (monoinsaturado), foi substituído por linoleato de colesterol (LDE-CL) (poliinsaturado) e por estearato de colesterol (LDE-CE) (saturado). Estas nanoemulsões foram submetidas a testes de estabilidade (tamanho, polidispersão, pH e peroxidação), captação celular, peroxidação lipídica celular e citotoxicidade. Nos resultados, foi observado que todas as células estudadas internalizaram o colesterol livre e éster de colesterol proporcionalmente às concentrações de LDE-CO incubadas com diferença de saturação entre elas, sendo o colesterol livre mais captado que o éster de colesterol da LDE-CO por todas as células estudadas. Além disso, os monócitos (THP-1) demonstraram maior captação de LDE-CO que as demais células. No estudo de competição com a LDL natural ocorreu uma diminuição da captação (r2-0,73), sugerindo que as duas partículas competem pelo mesmo receptor. A LDE-CO foi capaz de inibir a...

With fat composition similar to natural LDL, the LDE nanoemulsion can be used to study the metabolism of LDL. Other studies have shown that LDE is uptaken by cells by LDL-r receptors. Other receptors such as LRP-1, CD36 and CD38 may also be involved in the uptake. The objectives of this study were to investigate the uptake of LDE by endothelial and tumor cells, fibroblasts, monocytes and macrophages, to identify those receptors involved in this process and to evaluate the effects on LDE uptake by changing its chemical composition. A labeled LDE with [3H]-cholesterol and [14C]- cholesteryl ester was incubated for 4 hours with cells, after which LDE uptake and competition tests were evaluated. LDL-r, LRP, CD36 and CD38 were evaluated by using immunocytochemistry methods, cytometric flow and real time PCR. To investigate the effects of LDE chemical composition modifications, cholesteryl oleate (LDE-CO) was replaced with cholesteryl linoleate (LDE-CL) and cholesterol stearate (LDE-CE). These were then tested for stability, cellular uptake, lipoperoxidation and citotoxitity. Results showed that all cells internalized [3H]-cholesterol and [14C]-cholesteryl ester proportionally to incubated LDE-CO concentrations albeit with some saturation differences. LDE-CO lipid uptake had a higher cholesterol uptake than the cholesteryl ester uptake. Furthermore, monocytes (THP-1) had a higher LDE-CO uptake than other cells. LDE-CO uptake decreased (r2 -0.73) in the presence of natural LDL, suggesting that these two particles may be competing for the same receptors. LDE-CO appeared to inhibit LDL protein receptor expression in HUVEC (3.98 times), in monocytes (6.25 times) and in fibroblasts (3.70 times), as well as the gene expression in monocytes and HUVEC. A decrease in LDL-r expression in both H292 and fibroblasts was also observed. LDE-CO increased the protein expression in HUVEC 3.75 times while in monocytes, it was able to decrease gene and protein expression of LRP-...

Effect of olive antihyperlipidemia capsule on low and high density lipoprotein receptor in rat liver gene expression in hyperlipmia rat liver / 中国中药杂志

<p><b>OBJECTIVE</b>To study on the regulatory mechanism of lipid metabolism disorders in the blood fat of hyperlipemia rat model with Olive Antihyperlipidemia capsule, and do systematic observation on the functions of this medicine on low And high density lipoprotein receptor in rat liver gene expression, and then to clarify the mechanism of action of this medicine on treating hyperlipemia.</p><p><b>METHOD</b>To select SD rat as investigated subject. The hyperlipemia rat models were made with feeding high-fat forage and were randomly divided into six groups based on the total cholesterol level at the ratsfasting for 12 hours: group A, B, C, D, E and group F. The samples in the research were collected and analyzed the changes of LDLR/SR-B1 gene expression in rat's liver by RT-PCR.</p><p><b>RESULT</b>Olive Antihyperlipidemia capsule can markedly enhance LDLR/SR-B1 gene expression in rat's liver and finally accomplish the purpose of reducing blood fat. The experiment shows this medicine has the remarkable effect on hyperlipidemia and proved the theoretical system of treating hyperlipemia for curing the liver is correct.</p><p><b>CONCLUSION</b>Olive Antihyperlipidemia capsule has an applicable value on preventing the cause, enhance LDLR/SR-B1 gene expression in rat's liver and finally accomplish the purpose of reducing blood fat and development of hyperlipemia and its complications.</p>

Efecto de la administración de triiodotironina y glucagón sobre la expresión hepática del receptor de lipoproteínas de alta densidad y el metabolismo del colesterol en el ratón / Effect of triiodothyronine (T3) and glucagon on hepatic expression of SR-BI and lipoprotein cholesterol metabolism in mice

Introducción: El receptor scavenger clase B tipo I (SR-BI) es un elemento clave en el metabolismo de las HDL, donde su expresión ejerce un importante efecto anti-aterogénico controlando la fase hepática del transporte reverso de colesterol. Así, el estudio de la modulación de la expresión de SR-BI permitiría el desarrollo de nuevas alternativas farmacológicas para el tratamiento de la ateroesclerosis. Objetivo: La meta de nuestro estudio fue determinar el efecto de la triiodotironina (T3) y el glucagón sobre el metabolismo del colesterol HDL y la expresión hepática de SR-BI en el ratón, evaluando simultáneamente su impacto sobre el colesterol total y lipoproteico plasmático y la secreción biliar de colesterol. Métodos: Se utilizaron ratones C57BL/6 tratados con T3 (30 nmol/kg/día) o glucagón (80 µg/día) más los respectivos grupos controles. Después del tratamiento, los animales se anestesiaron para recolección de bilis, plasma y tejido hepático. Los niveles totales de colesterol plasmático y biliar fueron medidos por métodos enzimáticos. El colesterol lipoproteico plasmático se evaluó por fraccionamiento cromatográfico del plasma y medición enzimática del colesterol en cada fracción. La expresión hepática de SR-BI se cuantificó mediante western blot. Resultados: El uso de T3 o glucagón disminuyeron significativamente el colesterol plasmático total y aumentaron el colesterol biliar con respecto al grupo control correspondiente. Las fracciones de colesterol VLDL, LDL y HDL disminuyeron en ambos grupos tratados, con un mayor efecto observado en la fracción HDL. La administración de ambas hormonas aumentaron significativamente los niveles hepáticos de SR-BI. Conclusión: Los resultados establecen que T3 y glucagón disminuyen el colesterol plasmático, predominantemente de tipo HDL, y aumentan la secreción de colesterol biliar en el ratón, probablemente como consecuencia del incremento en la expresión hepática...

Introduction: The scavenger receptor class B type I (SR-BI) plays a key role in the metabolism of high-density lipoprotein (HDL) cholesterol. Its expression has an important anti-atherogenic effect by controlling the hepatic phase of the reverse cholesterol transport pathway in vivo. Thus, the study of the modulation of SR-BI expression may allow the development of new pharmacologic approaches for treatment of atherosclerotic cardiovascular disease. Objective: The goal of this study was to determine the effect of triiodothyronine (T3) and glucagon on HDL metabolism and hepatic expression of SR-BI in mice, evaluating also the impact in total and lipoprotein cholesterol as well as biliary cholesterol secretion. Methods: C57BL/6 mice were treated with T3 (30 nmol/kg/día) or glucagon (80 µg/día) in comparison to appropriate control groups. After treatment, bile, plasma and hepatic tissue were collected for analysis. Total plasma and biliary cholesterol levels were measured by enzymatic methods. Lipoprotein cholesterol was also measured enzymatically after chromatographic separation of plasma samples. The hepatic expression of SR-BI protein was quantified by western blotting. Results: The use of T3 or glucagon significantly decreased total plasma cholesterol levels and increased of biliary cholesterol concentrations compared to control groups. Levels of VLDL, LDL and HDL cholesterol were reduced in both treatment groups, with a more important effect observed in the HDL fraction. Both treatments increased hepatic SR-BI protein levels. Conclusions: These results show that T3 and glucagon decrease plasma cholesterol levels, particularly in HDL, and increase biliary cholesterol secretion in mice, probably as a consecuence of higher hepatic expression of SR-BI, which may have led to facilitated HDL cholesterol transport from plasma into bile.

Development of a new high-throughput screening model for human high density lipoprotein receptor (CLA-1) agonists / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells.</p><p><b>METHODS</b>With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized.</p><p><b>RESULTS</b>Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries.</p><p><b>CONCLUSION</b>Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.</p>

Establishment of a drug screening model for identifying up-regulator of human high density lipoprotein receptor / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a new drug screening model based on transcriptional regulation of human high density lipoprotein (HDL) receptor gene CD36 and LIMPII analogous-1 (CLA-1) for discovering up-regulator of this receptor.</p><p><b>METHODS</b>The upstream regulatory sequence of CLA-1 was obtained by polymerase chain reaction. A recombinant reporter plasmid pGL3-CLAP was constructed by inserting the regulatory sequence upstream of luciferase gene of pGL3-Basic. Human hepatoma cell line BEL-7402 was transfected with pGL3-CLAP. Samples were detected by testing luciferase activity of transfected BEL-7402 cells in microtiter wells.</p><p><b>RESULTS</b>The drug screening model was established and optimized. Significant difference was present between pGL3-CLAP and pGL3-Basic transfected BEL-7402 cells (P< 0.001), and coefficient of variation was less than 10%. After primary and secondary screening, 1 compounds and 3 fermentation extracts had up-regulating activities.</p><p><b>CONCLUSION</b>This new drug screening model may be efficiently used to screen up-regulators of human HDL receptor expression, which might become lead compounds for new anti-atherosclerosis drugs.</p>

Functional proteomics of receptor-Ck in the developmental stages of human atherosclerotic arterial wall.

BACKGROUND: The study was undertaken to understand the relationship between the functional proteomics of receptor-Ck and developmental stages of human atherosclerotic aortic wall. METHODS AND RESULTS: Gene expression study of 25 aortas was undertaken and the results revealed a gradual increase in receptor-Ck gene expression paralleled by the regulatory response of its effector genes coding for sterol response element-binding protein, p27, cyclin D, interleukin-6 and CD40 from a normal to atherosclerotic arterial wall (viz. fatty streak and fibrofatty/fibrous plaque). CONCLUSIONS: Based upon this and our earlier studies, we propose that cholesterol-specific receptor-Ck-dependent gene regulation may be of crucial importance in atherogenesis.

Expression and functional activity of receptor-Ck in mononuclear cells of a homozygous hypercholesterolemic family.

The study was addressed to explore the expression and functional activity of a novel cholesterol-specific cell surface receptor-Ck in a typical homozygous familial hypercholesterolemic family. Functional activity of receptor-Ck was characterized by its ability to downregulate Bcl-2 gene expression through a 47 kDa factor having an affinity for the sterol-regulatory element in the promoter region of this gene. The result of such a study revealed normal expression and functional activity of receptor-Ck accompanied by a lack of Apolipoprotein B-specific low-density lipoprotein receptor gene expression in the mononuclear cells derived from these patients. On the basis of these results, it is tempting to speculate that receptor-Ck may be involved in the maintenance of cellular cholesterol homeostasis observed in homozygous familial hypercholesterolemic patients.

Efeito do hormônio de crescimento sobre a expressão gênica do receptor LDL, receptor "scavenger" e da HMG-CoA redutase em células renais: possível correlação com a glomerulosclerose / GH effect on LDL receptor gene expression

Avaliou-se o efeito do GH in vitro sobre a expressão gênica do receptor LDL (RLDL) e da HMG-CoA redutase, bem como sobre a proliferação celular e acúmulo de lípides intracelulares em células mesangiais cultivadas em meio com soro deficiente de lipoproteínas (LPDS) durante 1, 2, 4 e 6 dias. A expressão coordenada entre o RLDL e a HMG-CoA redutase foi observada nas células mesangiais cultivadas em meio com LPDS. O GH aaumentou a proliferação das células mesangiais, dependente da sua concentração. A exposição prolongada ao GH induziu o aumento da expressão de RNAm do RLDL e da HMG-CoA redutase na células mesangiais, bem como o acúmulo de lípides neutros no citoplasma. Nos estudos in vivo...

Topology of Scavenger Receptor Class B Type I (SR-BI) on Brush Border Membrane

Both hydropathy plot and in vitro translation results predict the topology of SR-BI; the receptor is an integral membrane protein of 509 amino acids, consisting of a short cytoplasmic N-terminus of 9 amino acids followed by a first transmembrane domain of 22 amino acids, the extracellular domain of 408 amino acids, the second transmembrane domain of 22 amino acids, and the cytoplasmic C-terminus of 47 amino acids. The immunoblot of rBBMV in the presence or absence of pAb589 peptide antigen (the C-terminal 22 amino acid residues of SR-BI) confirmed that the bands at apparent molecular weight of 140 and 210 kDa are SR-BI related protein which might be multimeric forms of SR-BI. 125I apo A-I overlay analysis showed that SR-BI can bind to its ligand, apo A-I, only when it is thoroughly matured - glycosylated and dimerized. The antibody which was generated against extracellular domain of SR-BI (pAb230) not only prevented 125I-labeled apo A-I from binding to 140 kDa band but also inhibited the esterified cholesterol uptake of rabbit BBMV with its IC50 value of 40 microgram/ml of IgG. In contrast, the antibody generated against the C-terminal domain of SR-BI (pAb589) did not show any effect either on cholesterol uptake of rabbit BBMV or 125I-labeled apo A-I binding to 140 kDa band. Overall results show that the ligand binding site of SR-BI in rabbit BBMV is located in extracellular domain, and SR-BI is only functional when it is part of dimeric forms which rationalize the previously found cooperative nature of the binding interaction and maybe a fundamental finding towards the so far poorly understood mechanism of SR-BI function.

Molecular basis of cholesterol feedback lesion in CNS tumours.

An important feature of malignant transformation of tumours is the loss of cholesterol feedback inhibition mechanism (cholesterol-feedback lesion) that regulates mevalonate pathway recognized to play a crucial role in cellular growth, death and differentiation. Recently, it was shown that Receptor-C(k)-dependent signalling regulates genes involved in maintaining cellular cholesterol homeostasis through a transcription factor sterol response element binding protein (SREBP) having affinity for sterol regulatory element (SRE) present in the promoter region of these genes. The present study revealed that CNS tumours exhibit overexpression of Receptor-C(k) gene product which was accompanied by their inability to express SREBP gene product and this phenomenon has the inherent capacity to initiate the cholesterol feedback lesion in these tumours. Based upon these and our earlier studies, we propose for the first time that this loss of cholesterol feedback control may be responsible for the initiation of these tumours.

The usefulness of the genetic markers at the low-density lipoprotein receptor gene locus for the genetic diagnosis of familial hypercholesterolemia / 대한내과학회지

BACKGROUND: Familial hypercholesterolemia(FH) is an autosomal dominant metabolic disorder caused by the mutation in low density lipoprotein receptor(LDLR) gene. However, direct genetic diagnosis of LDLR gene mutation is not easily available because more than 300 mutations have been described in LDLR gene of FH patients. Therefore indirect genetic diagnosis using the genetic markers can be used to follow the inheritance of defective gene in FH families. The purpose of this study was to evaluate the usefulness of indirect genetic markers for detecting identical-by-descent LDLR gene abnormalities in FH families. METHODS: We examined the allele frequency, heterozygosity, polymorphism information content(PIC) of each genetic markers(D19S394, Taq I, Hinc II, Ava II, ATn, D19S221) in 94 unrelated healthy subjects. The genetic polymorphic haplotypes in 3 FH families were also determined. RESULTS: The heterozygosity and PIC values of RFLP's(Taq I, Hinc II, Ava II) were 0.51/0.344, 0.25/0.223, 0.28/0.233 and microsatellite markers(D19S394, ATn, D19S221) were 0.64/0.558, 0.56/0.455, 0.60/0.475. Hinc II and Ava II were significantly linked(|D|=0.72, p< 0.05). The cumulative PIC values of Taq I+Hinc II, Taq I+Hinc II+ATn, D19S394+ATn were 0.520, 0.814, 0.813, respectively. When applied in the FH pedigree, the genetic diagnosis using only one marker was not available in most cases. However, combination of two or more genetic markers could successfully discriminate the affected and unaffected members in FH families. Among the several combinations of the genetic markers, the combination of D19S394 and ATn was supposed to be the most effective and informative. Because one case of recombination was suspected in D19S221 allele, it was thought to be carefully used for genetic diagnosis of FH. CONCLUSION: We concluded that indirect genetic diagnosis using intragenic or extragenic genetic markers was useful for detecting identical-by-descent LDLR gene abnormalities in FH families and the most effective and informative combination of genetic marker seemed to be D19S394 and ATn.