OBJECTIVE: To screen for LDLR gene mutations in 9 patients with familial hypercholesterolemia (FH). METHODS: All exons of the LDLR gene and flanking intronic sequences were amplified by PCR and subjected to automatic DNA sequencing. For patients with homozygous or compound heterozygous mutations, parental DNA sequencing or T cloning sequencing was carried out to determine the parental origin of the mutant alleles. RESULTS: Direct sequencing of PCR products revealed 8 LDLR variants in 7 patients, which included c.259T>G, c.513delC, c.530C>T, c.682G>T, c.763C>T, c.1187-10G>A, c.1948delG, and c.1730G>A, among which c.1948delG was novel. Four patients have carried heterozygous mutations, two carried homozygous mutations, and one carried compound heterozygous mutations. The patients with biallelic mutations presented with a more severe phenotype compared those carrying heterozygous mutations. CONCLUSION LDLR mutations were identified in 7 out of 9 patients with FH. Among the 8 identified LDLR mutations, c.1948delG was firstly reported. Above findings have expanded the mutation spectrum of LDLR gene.
DNA Mutational Analysis ; Genetic Testing ; Humans ; Hyperlipoproteinemia Type II ; genetics ; Mutation ; Phenotype ; Receptors, LDL ; genetics

Hyperlipidemia (HLP) is the No.1 risk factor for patients with atherosclerosis (AS) and is directly related to the occurrence of coronary artery disease (CAD) and cerebrovascular disease. Therefore, prevention and treatment of AS is of great importance and of practical significance in controlling the incidence and mortality of CAD. With its peculiar syndrome-dependent therapy, traditional Chinese medicine (TCM) has accumulated abundant practical experiences in this field and good clinical effects have been achieved. Chinese herbal medicine, with its particularly unique advantages and high potentials yet to be tapped, displays its huge strength in HLP prevention and treatment. The progress of studies concerning prevention and treatment of HLP by Chinese herbal medicines, in the form of monomers or compound recipes, is reviewed in this paper.
Cholesterol ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Hyperlipidemias ; drug therapy ; Lipid Metabolism ; Lipid Peroxidation ; Receptors, LDL ; analysis

OBJECTIVETo determine the genomic structure of low density lipoprotein receptor related protein 5 (LRP5) gene.

METHODScDNA sequence encoding LRP5 was used to screen genomic clones containing LRP5 gene by computer hybridization approach. By comparing the cDNA sequence of LRP5 with the genomic sequences, the genomic structure of LRP5 was determined, and then it was conformed by amplifying and sequencing the sequences of exons and splicing junction.

RESULTSThe genomic sequence of LRP5 gene was 131.6 kb in length, containing 23 exons and 22 introns. Three single nucleotide polymorphisms were detected within the coding sequences of LRP5 gene, namely A459G in exon 2, C2220T in exon 10 and G4416C in exon 21. Four polymorphic markers, D11S1917, D11S4087, D11S1337 and D11S4178, located in the 5' flank sequence, introns 1, 4, and 13 of the LRP5 gene, respectively.

CONCLUSIONThe characterization of genomic structure of LRP5 gene allows the investigators to detect disease-causing mutation within the gene and further study the function of LRP5 gene.

Base Sequence ; DNA ; chemistry ; genetics ; Exons ; Genes ; genetics ; Humans ; Introns ; LDL-Receptor Related Proteins ; Low Density Lipoprotein Receptor-Related Protein-5 ; Polymorphism, Single Nucleotide ; Receptors, LDL ; genetics ; Sequence Analysis, DNA

BACKGROUNDLipid abnormalities are often complicated by renal dysfunction. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are the first-line choice for lowering cholesterol levels. The present study was designed to investigate whether statins could prevent and invert the development of renal injury in cholesterol-fed rabbits and to find the possible mechanism of their effects by detecting gene and protein expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the renal artery.

METHODSTwenty-four male New Zealand white rabbits were divided into three groups: (1) control group, regular granules chow; (2) HC-diet group, granules chow with 1% cholesterol and 5% lard oil; and (3) fluvastatin group, 1% cholesterol and 5% lard oil diet plus fluvastatin [10 mg.kg(-1).d(-1)]. After 16 weeks, serum total cholesterol (TC), low-density lipoprotein (LDL) and creatinine (Cr) levels were measured. Renal hemodynamics and function, mainly including glomerular filtration rate (GFR) in vivo were quantified using (99m)Tc-DTPA single photon emission computed tomograph ((99m)Tc-DTPA SPECT). The thickness of the renal artery intima was quantitated in HE-stained segments by histomorphometry. Gene expression of LOX-1 in the renal artery was examined by semi-quantitative RT-PCR and its protein expression was evaluated by immunohistochemistry.

RESULTSHigh cholesterol diet induced hypercholesterolemia (HC) complicated by renal dysfunction with increased levels of serum lipid and Cr, decreased GFR and delayed excretion and extensively thickened renal arterial intima in the HC-diet group. Rabbits in the control group showed a minimal LOX-1 expression (mRNA and protein) in the endothelium and neointima of the renal artery. Intimal proliferation of the renal artery in the HC-diet group was associated with a marked increase of LOX-1 expression (protein and mRNA). Treatment with fluvastatin improved renal function, attenuated intimal proliferation of the renal artery and markedly decreased the enhanced LOX-1 expression in the endothelium and neointima of the renal artery in rabbits.

CONCLUSIONSFluvastatin treatment could prevent the development of renal injury in patients with HC and early atherosclerosis (AS). This beneficial effect might be mediated by its pleiotropic effects including a decrease in total cholesterol exposure level and prevention of LOX-1 expression in atherosclerotic arteries.

Animals ; Cholesterol ; blood ; Creatinine ; blood ; Fatty Acids, Monounsaturated ; pharmacology ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Hypercholesterolemia ; drug therapy ; metabolism ; pathology ; Immunohistochemistry ; Indoles ; pharmacology ; Kidney ; drug effects ; pathology ; Male ; RNA, Messenger ; analysis ; Rabbits ; Receptors, LDL ; analysis ; genetics ; Receptors, Oxidized LDL ; Scavenger Receptors, Class E ; Tomography, Emission-Computed, Single-Photon

BACKGROUNDFamilial hypercholesterolemia (FH) is a type of dominant autosomal disease that causes high levels of plasma low-density lipoprotein cholesterol (LDL-C). In the past years, molecular data related to FH were limited in China. Now, to gain more information about FH, we analyzed one proband with a severe FH phenotype as well as his relatives.

METHODSAfter the entire coding sequence and the intron-exon junctions of the low-density lipoprotein receptor (LDLR) gene were amplified using PCR, we sequenced the LDLR gene of a Chinese FH family. RT-PCR was used to detect changes in the mRNA.

RESULTSTwo novel mutations were identified in the LDLR gene of this family. One, W165X, was a G > A substitution at the third nucleotide of codon 165. The other, IVS5-1G > A, was also a G > A substitution at the acceptor splice site of intron 5. The most striking discovery is that the proband was heterozygous for W165X but homozygous for IVS5-1G > A. The cDNA sequencing showed that the IVS5-1G > A mutation caused the insertion of 10 nucleotides, namely GCTCTCACAA, between exon 5 and exon 6.

CONCLUSIONSThe two nucleotide variations are thought to be the FH-causing mutations because the co-segregation of the mutant allele with the phenotype of FH has been shown in this Chinese family. These data show an increase in the mutational spectrum of FH in China and verify a scarce mutational form in the LDLR gene.

Adult ; Child ; DNA, Complementary ; analysis ; Female ; Humans ; Hyperlipoproteinemia Type II ; genetics ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Receptors, LDL ; genetics

OBJECTIVETo observe the change of lipid metabolism and vascular endothelium as well as morphology of heart tissue in rats who were long-time exposed to moxa smoke with different concentrations in order to provide reference for safety assessment of moxa smoke on cardiovascular system.

METHODSOne hundred and sixty-eighty Wistar rats were randomly divided into a control group, a low-concentration group, a median-concentration group and a high-concentration group, 42 rats in each one. The rats were exposed to moxa smoke with concentration of 0%, 10%, 40% and 70%, respectively, for 20 min per day. After continuous intervention for six months, enzyme-linked immunosorbent assay (ELISA) was applied to measure the level of low density lipoprotein-receptor (LDL-r) and intercellular adhesion molecule-1 (ICAM-1) in blood serum in each group; the slices of heart tissue were stained with hematoxylin-eosin staining method to observe morphology change of heart tissue.

RESULTS(1) After the intervention of moxa smoke, the levels of LDL-r and ICAM-1 in the low-concentration group were not statistically different from those in the control group (both P > 0.05); the level of LDL-r in the median-concentration group was significantly increased, which was statistically different from that in the control group [(3.87 +/- 0.27) mg/mL vs (2.12 +/- 0.13) mg/mL, P < 0.01], however, the content of ICAM-1 was not obviously changed; although the level of LDL-r in the high-concentration group was presented with an escalating trend, it was not statistically different from that in the control group (P > 0.05) while the level of ICAM-1 was obviously increased (P < 0.01). (2) Under the light microscope, the abnormalities of cardiac muscle fibers and myocardial cell in each group were not been observed.

CONCLUSIONThe long-time intervention of low-concentration moxa smoke has no significant effects on lipid metabolism and vascular endothelium of rats, indicating that clinical application of low-concentration moxa smoke is relatively safe. The long-time intervention of moderate-concentration moxa smoke could significantly increase the clearance rate of cholesterol, implying the beneficial regulation of moxa smoke on lipid metabolism. The high-concentration moxa smoke could induce certain damage to vascular endothelium but its mechanism is in need of further research. The pathologic change of heart tissue could not be induced by moxa smoke with any concentration.

Animals ; Heart ; anatomy & histology ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipid Metabolism ; Male ; Moxibustion ; adverse effects ; Myocardium ; pathology ; Rats ; Rats, Wistar ; Receptors, LDL ; metabolism ; Smoke ; adverse effects ; analysis

BACKGROUNDLow-density lipoprotein (LDL) receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line (HepG2) cells under physiological and inflammatory conditions.

METHODSIntracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum (ER) to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies.

RESULTSLDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1 beta (IL-1 beta) further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1 beta caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium.

CONCLUSIONSIL-1 beta disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.

Cell Line, Tumor ; Cholesterol ; analysis ; Endoplasmic Reticulum ; metabolism ; Feedback, Physiological ; Humans ; Interleukin-1beta ; pharmacology ; Intracellular Signaling Peptides and Proteins ; analysis ; genetics ; Membrane Proteins ; analysis ; genetics ; Protein Transport ; RNA, Messenger ; analysis ; Receptors, LDL ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sterol Regulatory Element Binding Protein 2 ; analysis ; genetics

Familial hypercholesterolemia(FH) is a disease based on defects of low-density lipoprotein receptors(LDL-R). To interrupt and control the natural course of this disease, early identification of these patients is important. The routine lipid profile tests for hypercholesterolemia can not differentiate objectively FH from secondary hypercholesterolemia. The exact diagnosis of FH heterozygotes is especially essential because it is easier to develop premature coronary heart diseases compared with secondary hyper-cholesterolemia. A simplified rapid and precise method for the mass screening of FH patients and the differentiation between FH heterozygote and secondary hyperlipidemia was needed. For the test, lymphocytes were used as target cells in LDL-R assay. After a 5 day culture with anti-CD3 Ab as a mitogen, indirect immunofluorescence stain and flow cytometric analysis were applied. The results were as follows; 74 +/- 9% of the stimulated lymphoblasts from normal controls expressed LDL-R activity. Cultured, but unstimulated, lymphocytes of normal controls showed 27 +/- 8% positivity and total cultured lymphocytes showed positivity of 46 +/- 11% positivity. Lymphoblasts, unstimulated lymphocytes, and total cultured lymphocytes from hyper-cholesterolemia without FH showed 74 +/- 10%, 25 +/- 10% and 50 +/- 17%, respectively, which showed no significant differences from normal control groups. FH Heterozygotes showed LDL-R positivity, 21 +/- 11% in lymphoblasts, 11 +/- 6% in unstimulated lymphocytes and 18 +/- 7% in total cultured lymphocytes. These data imply that adequately stimulated lymphocytes might be used for detecting defects in LDL-R and used to differentiate FH from secondary hypercholesterolemia.
Adult ; Antibodies/*pharmacology ; Antigens, CD3/*immunology ; Female ; Human ; Hypercholesterolemia, Familial/*blood ; Lipids/blood ; Lymphocyte Activation/drug effects ; Lymphocytes/drug effects/*ultrastructure ; Male ; Middle Age ; Phytohemagglutinins/pharmacology ; Receptors, LDL/*analysis ; Support, Non-U.S. Gov't

Family hypercholesterolemia (FH) is a genetic disorder caused by mutation in the low density lipoprotein receptor (LDLR) gene. It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to tendon xanthenes and premature coronary artery disease. We studied a FH family ,which was diagnosed by clinical features and blood lipid tests. The Total cholesterol level of the family was 19.05 mmol/L and the LDL level was 17.06 mmol/L in the proband homozygous FH subjects, while the total cholesterol was 7.96 mmol/L and LDL was 5.55 mmol/L in the heterozygous FH subjects. DNA segments amplified with PCR were sequenced in heterozygous and homozygous FH patients. Two novel identical mutation alleles of GAG683GCG, which caused an amino acid change from Glu to Ala, were detected in Exon4 of LDL receptor gene in homozygous proband. DNA sequencing revealed that the proband's parents were heterozygotes with the same mutational alleles as the proband. These results are in coincidence with the clinical diagnoses. Moreover Epstein-Barr virus transformed lymphocytes (EBV-Ls) were derived by routine virus infection transforming protocol. The cells bounded with the fluorescently conjugated LDL were measured by fluorescence flow cytometry. The ratios of functional LDLR in EBV-Ls originated from homozygous FH, heterozygous FH and normal control were 7.02%, 62.64% and 84.69%, respectively. As a result, the homozygous FH patient's LDLR had 8.29% and the heterozygous FH patient's LDLR had 73.96% of the activity of the control. It is apparent that LDL receptor activity of homozygous FH subject is significantly lower than normal control. The data from fluorescence flow cytometry analysis of EBV-Ls strongly support the clinical diagnoses and the results of DNA sequencing. In accordance with the updated version of UMD-LDLR, the mutant GAG683GCG in Exon4 of LDLR gene which we have identified is a novel mutation of the LDLR gene in human with hypercholesterolemia.
Base Sequence ; DNA ; genetics ; DNA Mutational Analysis ; Exons ; Female ; Heterozygote ; Homozygote ; Humans ; Hyperlipoproteinemia Type II ; genetics ; Male ; Molecular Sequence Data ; Pedigree ; Phenotype ; Point Mutation ; Polymorphism, Single-Stranded Conformational ; Receptors, LDL ; genetics

OBJECTIVETo investigate the relationship between atrial fibrillation (AF) and serum soluble CD163.

METHODSA total of 336 patients with heart valve disease were included in this study, including 167 with AF and 169 with sinus rhythm. The clinical data were compared between the two grops, and Logistic regression analysis was used to identify the risk factors associated with AF.

RESULTSThe levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), tumor necrosis factor (TNF), interleukin-6 (IL - 6), high-sensitivity C-reactive protein (hs-CRP) and left atrial diameter (LAD) all differed significantly between the two groups (P<0.05). Serum soluble CD163 levels in AF patients were significantly higher than those in patients with sinus rhythm (P<0.05). Serum soluble CD163 was positively correlated with TNF (r=0.244, P=0.244), IL-6 (r=0.186, P=0.186), hs-CRP (r=0.183, P=0.183) and LAD (r=0.194, P=0.194) in patients with AF. Logistic regression analysis showed that LAD, IL-6, TNF, hs-CRP and CD163 were all associated with AF. ROC curve analysis showed that the area under curve of serum soluble CD163 was 0.861 in patients with AF (CI 95%: 0.820-0.901, P<0.01) with a sensitivity and a specificity of 80.8 and 76.9%, respectively.

CONCLUSIONSerum soluble CD163 level may be a risk factor for AF, and an increased soluble CD163 level may indicate active inflammation in AF patients.

Antigens, CD ; blood ; Antigens, Differentiation, Myelomonocytic ; blood ; Atrial Fibrillation ; blood ; C-Reactive Protein ; analysis ; Heart Atria ; pathology ; Humans ; Inflammation ; blood ; Interleukin-6 ; blood ; Lipoproteins, HDL ; blood ; Lipoproteins, LDL ; blood ; Receptors, Cell Surface ; blood ; Risk Factors ; Tumor Necrosis Factor-alpha ; blood