Hypercholesterolemia,low density lipoprotein receptor and proprotein convertase subtilisin/kexin-type 9

Atherosclerotic cardiovascular disease is the main cause of mortality and morbidity in the world. Plasma levels of low density lipoprotein cholesterol （LDL-C） are positively correlated with the risk of atherosclerosis. High plasma LDL concentrations in patients with hypercholesterolemia lead to build-up of LDL in the inner walls of the arteries, which becomes oxidized and promotes the formation of foam cells, consequently initiating atherosclerosis. Plasma LDL is mainly cleared through the LDL receptor （LDLR） pathway. Mutations in the LDLR cause familiar hyperch- olesterolemia and increase the risk of premature coronary heart disease. The expression of LDLR is regulated at the transcriptional level via the sterol regulatory element binding protein 2 （SREBP-2） and at the posttranslational levels mainly through proprotein convertase subtilisin/kexin-type 9 （PCSK9） and inducible degrader of the LDLR （IDOL）. In this review, we summarize the latest advances in the studies of PCSK9.

Influence of Oxidized Low Density Lipoprotein on the Proliferation of Human Artery Smooth Muscle Cells in vitro

The effects of oxidized low density lipoprotein （ox-LDL） on the proliferation of cultured human vascular smooth muscle cells （vSMC） were investigated in vitro. By using NaBr density gradient centrifugation, LDL was isolated and purified from human plasma. Ox-LDL was produced from LDL by being incubated with CuSO4. ox-LDL was then added to the culture medium at different concentrations （35, 60, 85, 110, 135 and 160μg/mL） for 7 days. The influence of ox-LDL on vSMC proliferation was observed in growth curve, mitosis index, and in situ determination of apoptosis. The data were analyzed with SPSS 10.0 software. The results showed that the ox-LDL produced in vitro had a good purity and optimal oxidative degree, which was similar to the intrinsic ox-LDL in atherosclerotic plaque, ox-LDL at a concentration of 35 μg/mL demonstrated the strongest proliferation inducement, and at a concentration of 135 μg/mL, ox-LDL could inhibit the growth of vSMC. ox-LDL at concentrations of 35 and 50 μg/mL presented powerful mitotic trigger, and with the increase of ox-LDL concentration, the mitotic index of vSMC was decreased gradually, ox-LDL at higher concentrations promoted more apoptotic vSMCs, ox-LDL at lower concentrations triggered proliferation of vSMCs, and at higher concentrations induced apoptosis in vSMCs, ox-LDL played a promotional role in the pathogenesis and development of atherosclerosis by affecting vSMC proliferation and apoptosis.

A Modified Chitosan Adsorbent for Selective Removal of Low Density Lipoprotein

A modified chitosan adsorbent was synthesized through a simple preparation procedure, and it demonstrated good adsorption performance for selective removal of low density lipoprotein in human plasma. Phase inversion technique was employed to form chitosan beads, to which epoxy groups were then introduced by reacting with ethyleneglycol diglycidylether, and tryptophan was subsequently coupled to the epoxy-activated beads.

Apolipoprotein B100 quality control and the regulation of hepatic very low density lipoprotein secretion

Apolipoprotein B （apoB） is the main protein component of very low density lipoprotein （VLDL） and is necessary for the assembly and secretion of these triglyceride （TG）-rich particles. Following release from the liver, VLDL is converted to low density lipoprotein （LDL） in the plasma and increased production of VLDL can therefore play a detrimental role in cardiovascular disease. Increasing evidence has helped to establish VLDL assembly as a target for the treatment of dyslipidemias. Multiple factors are involved in the folding of the apoB protein and the formation of a secretion-competent VLDL particle. Failed VLDL assembly can initiate quality control mechanisms in the hepatocyte that target apoB for degradation. ApoB is a substrate for endoplasmic reticulum associated degradation （ERAD） by the ubiquitin proteasome system and for autophagy. Efficient targeting and disposal of apoB is a regu- lated process that modulates VLDL secretion and partitioning of TG. Emerging evidence suggests that significant overlap exists between these degradative pathways. For example, the insulin-mediated targeting of apoB to autop- hagy and postprandial activation of the unfolded protein response （UPR） may employ the same cellular machinery and regulatory cues. Changes in the quality control mechanisms for apoB impact hepatic physiology and pathology states, including insulin resistance and fatty liver. Insulin signaling, lipid metabolism and the hepatic UPR may impact VLDL production, particularly during the postprandial state. In this review we summarize our current understanding of VLDL assembly, apoB degradation, quality control mechanisms and the role of these processes in liver physiology and in pathologic states.

Effects of Oxidized Low Density Lipoprotein on Transformation of Valvular Myofibroblasts to Osteoblast-like Phenotype

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein（ox-LDL, 50 mg/L）, and divided into four groups according to the ox-LDL treatment time： control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1（DDK-1, 100 μg/L） was added in ox-LDL 72-h group. The expression of α-smooth muscle actin（α-SMA）, bone morphogenetic protein 2（BMP2）, alkaline phosphatase（ALP）, and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of α-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups（P〈0.05 for all）; β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group（P〈0.05）, and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion（P〈0.05）. After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group（P〈0.05）. The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.

Effect of Curcumin on the Gene Expression of Low Density Lipoprotein Receptors

Objective： To investigate the molecular mechanisms and effective target ponits of lipid-lowering drug, Rhizoma Curcumae Longae, and study the effect of curcumin on the expression of low density lipoprotein （LDL） receptors in macrophages in mice. Methods. Macrophages in mice were treated with curcumin, which was purified from the ethanolly extraction of Rhizoma Curcumae Longae for 24 h. The LDL receptors expressed in the macrophages were determined by enzyme-linked immunosorbent assay （ELISA） and assay of Dil labeled LDL uptake by flow cytometer. Results： It was found for the first time that 10 μmol/L-50 μmol/L curcumin could obviously up-regulate the expression of LDL receptor in macrophages in mice, and a dose-effect relationship was demonstrated. Conclusion： One of the lipid-lowering mechanisms of traditional Chinese medicine, Rhizoma Curcumae Longae, was completed by the effect of curcumin through the up-regulation of the expression of LDL receptor.

Effects of Oxidized Low Density Lipoprotein onthe Expression and Function of ABCA1 in Macrophages

Summary:In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65 % cholesterol loaded by acetyl LDL (ac-LDL), and 9.78 % cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with 100 μg /mL ac-LDL or with 100 μg /mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.

Obstructive jaundice leads to accumulation of oxidized low density lipoprotein in human liver tissue

Oxidized low density lipoprotein （ox-LDL） molecule is one of the most important modified lipoproteins produced during the oxidative stress. Modified lipoproteins have been defined as being part of the immune inflammatory mechanisms in association with oxidant stress. We have reported the accumulation of ox-LDL in Balb/c mice liver after bile duct ligation previously. Here, we investigated this finding in human beings with obstructive jaundice. Our study demonstrates that obstructive jaundice results in tremendous accumulation of ox-LDL in the liver tissue of patients.

Expression of Monocyte Chemoattractant Protein-1 in Monocytes and Effects of Native and Oxidized Very Low Density Lipoproteins

Monocyte chemoattractant protein-1(MCP-1), a potent chemoattractant, is thought to play an important role in migration of monocytes into atherosclerotic lesions. The present study was designed to investigate the capacity of human peripheral blood monocytes to express MCP-1 and effects of native very low density lipoprotein (VLDL) and oxidized VLDL(OX-VLDL) on the expression. The total RNA was extracted from cultured monocytes, which were exposed to VLDL and OX-VLDL, and the media conditioned by monocytes were collected. MCP-1 mRNA expression was examined by Northern blot analysis. MCP-1 protein in conditioned media was determined by using sandwich ELISA. The results showed that monocytes can express MCP-1 after a 24 h incubation at 37℃，and the expression was markedly increased by a exposure to OX-VLDL, whereas the expression was slightly increased when exposed to VLDL. It suggests that the capacity of monocytes to produce MCP-1 that recruits and activates circulating monocytes may be of considerable importance in atherogenesis, and oxidation of VLDL enhances its potential to promote atherogenesis.

Function and Significance of Very Low Density Lipoprotein Receptor Subtype Ⅱ

To explore the functions of very low density lipoprotein receptor (VLDL-R) subtype II in lipoprotein metabolism and foam cells formation, the recombinant plasmid with the two subtypes cDNA was constructed respectively, the ldl-A7 cell lines were transfected and two cell lines expressing VLDL-R were obtained: one stably expressing the VLDLR with the O-linked sugar region (type I VLDLR) and the other without the O-linked sugar region (type II VLDLR). In the study on binding of VLDLR to their nuclein labeled natural ligands (VLDL and β-VLDL), it was found that surface binding of 125I-VLDL or 125I-β-VLDL of ldl-A7 cells transfected with type I VLDLR recombinant (ldl-A7-VRI) was more higher than that of ldl-A7 cells transfected with type II VLDLR recombinant (ldl-A7-VRII). After being incubated with VLDL for different time, the contents of triglyceride and total cholesterol in cells were mensurated, and the formation of foam cells and accumulation of lipid in cells was observed by oil-red O staining. The results showed that the contents of triglyceride and total cholesterol in ldl-A7-VR I were much higher than those in ldl-A7-VR II, and ldl-A7-VR I could transform into foam cells notably. It was suggested that type I VLDLR binds with relative higher affinity to VLDL and β-VLDL, and internalizes much more lipoprotein into cells. As a result, we can conclude that type I VLDLR plays a more important role in lipoprotein metabolism and foam cells formation than type II VLDLR.

Oxidized low density lipoprotein (Ox-LDL) impacts on erythrocyte viscoelasticity and its molecular mechanism

Aim:The oxidized low-density lipoprotein(OxLDL) plays an important role in atherosclerosis yet it remains unclear if it damages circulating erythrocytes. Method: In this study。

EC,ASMC and Macrophage Oxidize Human Low Density Lipoprotein

To investigate the mechanism of LDL oxidation i n vivo , LDL was incubated with endothelium cell (EC),artery smooth muscle cel l (ASMC) and macrophage, and then the change of myeloperoxidase (MPO) activity i n cell and medium and the oxidation of LDL by those three cells were assessed. T he result showed that LDL promoted the activity of cellular and secretive myelop eroxidase which was concentration\|dependent on LDL; with elevation of MPO activ ity, oxidation of LDL intensified, which was expressed by the formation of conju gated dienes and the elevation of thiobarbituric acid teactive substance (TBARS ). Macrophage's MPO activity went up with the increase of LDL at both low and h igh concentration; EC's MPO activity went up with the increase of LDL only at h igh concentration and ASMC's MPO activity wasn't sensitive to LDL concentratio n change. The results suggest that Macrophage might be crucial to the oxidation of LDL in vivo , in which MPO might play an important role.

Relationship between the low density lipoprotein receptor gene polymorphism and serum lipid levels in the Guangxi Bai Ku Yao population

Objectives Bai Ku Yao(White-trousers Yaos)is a special branch of Yao minority in China.They are now living in both Lihu and Baxu villages,Nandan County, Guangxi,China.The population size is about 30,000.The special customs and culture of Bai Ku Yao,including their special clothing,intra-ethnic marriages and alcohol intake are still completely conserved to the present day.In previous epidemiologic studies,we found that the serum lipid levels and the prevalence of hyperlipidaemia were lower in Bai Ku Yao than in Han Chinese from the same region.This ethnic difference in serum lipid profiles is still not well known.We hypothesized that there may be significant differences in some genetic polymorphismsssociation of low density lipoprotein receptor (LDL-R) genepolymorphism and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations.Methods A total of 1024 subjects of Bai Ku Yao and 792 participants of Han Chinese were stud- ied by a stratified randomized cluster sampling.Epidemiological survey was carried out using internationally standardized methods.Information on demographics,socioeconomic status, and lifestyle factors was collected with standardized questionnaires. The height,weight,waist circumference,blood pressure, and serum total cholesterol(TC),triglyceride(TG), high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),apolipoprotein(Apo) A1, and ApoB were measured.Body massindex(BMI,kg/m2) was calculated.Genotyping of the LDL-RAvaⅡwas performed by polymerse chain reaction and restriction fragment length polymorphism combined with gel electrophoresis,and then confirmed by direct sequencing.Results(l)The height,weight,serum TC,HDL-C,LDL-C,ApoAl levels and the ratio of ApoAl to ApoB were lower in Bai Ku Yao than in Han Chinese(P【0.01 for all),whereas the percentage of subjects who consumed alcohol or smoked cigarettes was higher in Bai Ku Yao than in Han Chinese(P【0.01 for each).(2) The frequency of A+ allele in Bai Ku Yao was 34.5%,and the frequencies of A-A-,A-A+ and A+A + genotypes were 42.6%,45.9%and 11.5%;respectively. The frequency of A+ allele in Han Chinese was 19.3%(P【0.001),and the frequencies of A-A-,A-A + and A+A+ genotypes were 64.9%,31.6%and 3.5%(P【0.001);respectively. The frequencies of A-A-,A-A+ and A+A+ genotypes in Bai Ku Yao were significant difference between males and females,between normal TC and high TC subgroup, and between normal LDL-C and high LDL-C subgroup (P【0.05 for all),whereas the frequencies of A- and A+ ? alleles in Han Chinese were significant difference between males and females(P【0.05).(3) Serum LDL-C levels in Bai Ku Yao were significant difference among the A-A-, A-A+ and A+A+ genotypes(P【0.05),the A+ carriers had higher serum LDL-C levels.Serum HDL-C levels in Han Chiese were significant difference among the A-A-,A-A + and A+A+ genotypes(P【0.01),the A+ carriers had higher serum HDL-C levels.(4) After adjusting other factors,the prevalence of LDL-C abnormality was still higher in Han Chiese than in Bai Ku Yao.The prevalence of TC abnormality in Han Chinese was almost twice high as in Bai Ku Yao. The age and diet were common risk factor for TC abnormality. No effect of AvaⅡgenotype or alcohol consumption on the TC abnormality was found,but the combination of geno-type and alcohol consumption can increase the prevalence of TC abnormality[Exp(B) =(1.154)].Age was negatively cor- related with TG level.Conclusions Serum TC and LDL-C levels were lower in Bai Ku Yao than in Han Chinese.There were significant differences in the AvaⅡallele and genotype frequencies between the he A+ carriers in Bai Ku Yao had higher serum LDL-C levels,whereas the A+ carriers in Han had higher serum HDL-C levels.Interactions between alcohol consumption or cigarette smoking and the LDL-R AvaⅡgenotype were also observed.The differences in the serum lipid profiles between the two ethnic groups might partly result from different genotypic frequency of LDL-R AvaⅡpolymorphism or differentgene-enviromental interactions.Bai Ku Yao and Han population,the frequency of A + allele was higher in Bai Ku Yao than in Han.T between the two ethnic groups.Therefore,the aim of the present study was to detect the

Effects of oxidized low density lipoprotein on the growth of human artery smooth muscle cells

Background Studies have shown that oxidized low density lipoprotein （ox-LDL） promotes the pathogenesis and development of atherosclerosis （AS）, and that the proliferation, migration and phenotype alteration of vascular smooth muscle cells （vSMCs） into foam cells are critical changes in AS. It is proposed that ox-LDL might play a novel role in the pathologic process of vSMCs. The present study was performed ex vivo to investigate the effects of ox-LDL on the growth of cultured human vSMCs.Methods Using NaBr density gradient centrifugation, LDL from human plasma was isolated and purified. ox-LDL was produced from LDL after being incubated with CuSO4. ox-LDL was then added to the culture medium at different concentrations （25μg/ml, 50μg/ml, 75 μg/ml, 100μg/ml, 125μg/ml, and 150μg/ ml） for 7 days. The influence of ox-LDL on vSMC growth was observed from several aspects as growth curve, mitosis index, lipid staining, and in situ determination of apoptosis. The digital results were analyzed with SPSS 10.0.Results The ox-LDL produced ex vivo had a good purity and optimal oxidative degree, which was similar to the intrinsic ox-LDL in atherosclerotic plaque, ox-LDL at a concentration of 25 μg,/ml demonstrated the strongest proliferation. At the concentration of 125 μg,/ml, ox-LDL suppressed the growth of vSMCs. At concentrations of 25 μg/ml and 50 μg/ml, ox-LDL presented powerful mitotic trigger. When the concentration of ox-LDL increased, the mitotic index of vSMCs decreased gradually, ox-LDL induced more foam cells from vSMCs with rich intracellular lipid accumulation at concentrations of 25μg/ml and 50μg/ml, ox-LDL at higher concentrations induced more apoptotic vSMCs.Conclusions ox-LDL at lower concentrations may trigger proliferation and phenotype alteration into foam cells of vSMCs, and at higher concentrations it may induce apoptosis in vSMCs, ox-LDL plays an important role in the pathogenesis and development of atherosclerosis by its effect on vSMCs proliferation, phenotype alteration and apoptosis.

Effects of Curcuma Longa on proliferation of cultured bovine smooth muscle cells and on expression of low density lipoprotein receptor in cells

Objective To investigate the inhibitory effects of aqueous turmeric extract (AqT) and serum of rats orally treated with ethanol extract of turmeric (SeT) on proliferation of vascular smooth muscle cells (VSMC) and its effects on the expression of low density lipoprotein receptor (LDL R) antigen on the surface of smooth muscle cells. Methods Enzyme linked immunosorbent assay (ELISA) for the expression of LDL R protein and thiazolyl blue (MTT) assay for the proliferation of VSMC were used in this study. Results Both aqueous turmeric extract (AqT) and serum of rats orally treated with ethanol extract of turmeric (SeT) could inhibit 10% serum activated proliferation of VSMC. The inhibition shown in both experiments was dose dependent with an inhibitory rate of 18.9% at 20 mg/ml AqT and rate of 20.1% at 10% SeT respectively. AqT up regulated the expression of LDL R protein with a highest rate at 5 mg/ml AqT in 3% lipoprotein deficient serum (LPDS). SeT did not show significant effect on the expression of LDL R on the surface of VSMC. Conclusion The extracts of turmeric may be extended to decrease the risk of atherosclerosis (AS).

Exploration of the Relationship between Phlegm-Dampness Constitution and Polymorphism of Low Density Lipoprotein Receptor Genes Pvu Ⅱand AvaⅡ

Objective： To explore the polymorphism of low density lipoprotein receptor （LDL-R） genes Pvu Ⅱ end Ave Ⅱ in e population with phlegm-dampness constitution （PDC）. Methods： Polymorphism of LDL-R genes at Pvu Ⅱ end Ave Ⅱ of 48 persons with gentle constitution （GC） end 61 with PDC were analyzed with PCR-RELP technique, end their serum contents of lipids end glucose were determined end compared as well. Results： The A＋ ellelic end P- ellelic frequency were higher end the P＋ ellelic frequency was lower in subjects with PDC then those in subjects with GC, which were 0.3083 vs 0.1771, 0.9098 vs 0.7708 end 0.0902 vs 0.2292, respectively, ell showing significant difference between the two groups （P〈0.05）. Comparison of the two groups in serum levels of triglyceride （TG）, fasting blood glucose, 2 h postprandial blood glucose, end 2 h postprandial insulin showed that ell the parameters were higher in subjects with PDC then in subjects with GC respectively, showing significant difference （P〈0.05）. Conclusion： PDC is related with the P- end A＋ ellelic frequency of higher LDL-R genes at Pvu Ⅱ end Ave Ⅱ, therefore, the polymorphism of LDL-R genes could be taken as one of the genetic markers for PDC, end humans with PDC ere more liable to suffer from blood lipids end glucose disorder then those with GC.

Influence of glycated low density lipoprotein on the proliferation, expression of intercellular adhesion molecule-l, von Willebrand factor of human umbilical endothelial cells

Diabetes mellitus known as its macro- and microangiopathy has caused thousands of mortality per year. Recent researches showed that hyperglycemia, advanced glycation end products （AGEs） and some other factors acted on the process of atherogenesis. AGEs can combine with receptors of AGEs （RAGEs）, which exist on the vascular endothelium, smooth muscle cells, macrophage, lymphocyte and so on. They can stimulate series of signal transduction systems including nuclear factor κB （NF-κB） pathway, finally promote the secretion of inflammatory factor such as interleukin-1 （IL-1）, interleukin-6 （IL-6）, tumor necrosis factor-α （TNF-α）, intercellular adhesion molecule-1 （ICAM-1）,1＇2 as well as increase the synthesis and secretion of coagulant modulatory factors such as vascular cell adhesion molecule- 1 （VCAM- 1） and von Willebrand factor （vWF）.3

Inhibition of Oxidative Modification of Human Low Density Lipoprotein by Resveratrol

To further investigate whether resveratrol, a polyphenolic compound in red wine, affects the oxidation of human low density lipoprotein (LDL), LDL purified from normolipidemic subjects was subjected to Cu\+\{2+\}induced or azo compoundinitiated oxidative modification. The extent of LDL modification was assessed by measuring the formation of thiobarbituric acid reactive substances (TBARS) and the relative electrophoretic mobilities (REM) of LDL. Resveratrol (50 mol/L) reduced TBARS and REM of LDL during Cu\+\{2+\}induced oxidation by 70.5% and 42.3%, respectively (P<0.01), and prolonged the lag phase associated with the oxidative modification of LDL by copper ion or azo compound. These in vitro results suggest that resveratrol may afford LDL protection against oxidative modification, possibly by acting as a free radical scavenger.

BLOOD PLATELETS AND OXIDIZED LOW DENSITY LIPOPROTEIN

Functional morphological alterations of human blood platelets induced by oxidatively modified low density lipoprotein (LDL) were studied with an in vitro model by means of electron microscopy, reflection contrast microscopy and quantitative image analysis. The oxidized LDL (50-300 μg/ml) induced the disc-sphere transformation of platelets, the formation of pseudopodia, centralization of granules and degranulation. Platelet plasma membrane was damaged by oxidized LDL leading to a lower electron density of he cytoplasm compared to controls. In an incubation chamber, oxidized LDL-treated platelets sedimented onto the