Decreased affinity of low density lipoprotein (LDL) particles for LDL receptors in patients with cholesteryl ester transfer protein deficiency

Abstract. We have reported that the disorder of lipoprotein metabolism in hyperalphalipoproteinae-mic patients with a deficiency of cholesteryl ester transfer protein (CETP) is characterized by the poly-disperse low density lipoprotein (LDL) particles and the accumulation of cholesteryl ester (CE) in high density lipoprotein (HDL) particles, forming cholesterol-induced HDL (HDLc)-like particles. In the present study we have investigated the interaction of these abnormal LDL with LDL receptors of normal human fibroblasts. Since the ultracentrifugally separated LDL fraction (1.019 < d < 1.063 gmL^-1) from the CETP-deficient patients contained HDLc-like particles, these particles were removed by anti-apolipoprotein (apo) A-I immunoaffinity column chromatography. The lipoproteins eluted in the unbound fraction of this column did not contain apo A-I, so this fraction was considered to be authentic LDL. The authentic LDL of the patients were deficient in CE and rich in triglycerides and apo B. The authentic LDL itself showed polydispersity, ranging in size from 23 nm to 30 nm. The affinity of these abnormal LDL particles for LDL receptors was analysed by a competitive assay in which cold LDL from the patients or control compete with ¹²⁵I-labelled LDL for fibroblast LDL receptors. The concentration of LDL particles at which 50% of ¹²⁵I-labelled normal LDL was replaced was two to three times higher for the patients than for the normal control. Therefore, the affinity of patient LDL was thought to be reduced compared to that of control LDL. These results demonstrate that CETP may play an important role in making LDL particles homogeneous and rich in CE. This modulation of LDL by CETP may enhance the affinity of LDL for LDL receptors to deliver cholesterol to peripheral tissues.     

Hydrocortisone decreases the internalization of low density lipoprotein in cultured human fibroblasts and arterial smooth muscle cells

The effect of hydrocortisone on the cellular low density lipoprotein (LDL) pathway was studied in cultured human skin fibroblasts and arterial smooth muscle cells. Hydrocortisone decreased both uptake and degradation of 125I-LDL, LDL binding, measured at 4 C, was not affected by the hormone. Physiological concentrations of hydrocortisone (4 . 1 X 10(-8) mol/l) resulted in a 30% reduction of LDL uptake and degradation which could not be accounted for by an effect of the hormone on macromolecular synthesis, cell protein or cell number. To test whether the decrease in uptake and degradation of 125I-LDL was due to reduced internalization, the effect of hydrocortisone on the internalization of prebound LDL was determined and found to be decreased. Also, preincubation with unlabelled LDL in the presence of hydrocortisone resulted in less down regulation of LDL receptor activity than when no hydrocortisone was present. An effect of the hormone on bulk phase endocytosis has been excluded, since hydrocortisone did not affect either LDL degradation by receptor negative cells or endocytosis of 14C-sucrose by normal skin fibroblasts. Thus, hydrocortisone impairs LDL catabolism by decreasing the internalization of LDL normally bound to its cell surface receptor. These results may be relevant to the pathogenesis of atherosclerosis in conditions associated with reduced cellular LDL catabolism.     

Abnormal regulation of LDL receptor activity and abnormal cellular metabolism of hypertriglyceridaemic low density lipoprotein: normalization with bezafibrate therapy

The regulation of LDL (B,E) receptor activity and of cellular LDL protein metabolism by hypertriglyceridaemic (HTG) low density lipoprotein before and during hypolipidaemic therapy (with bezafibrate (BZ] were determined in cultured human skin fibroblasts. Defective binding and subnormal capacity to regulate LDL receptor activity was found for HTG-LDL. Binding affinity (Kd) of HTG-LDL to the receptor was 4.97 x 10(-8) M and of N-LDL, 1.74 x 10(-8) M. When assayed with normal 125I-LDL, the capacity of HTG-LDL to down-regulate receptor activity was 46-68% less than N-LDL. Both abnormalities reverted towards normal during treatment. The cellular metabolism of HTG-, BZ- and N-LDL in cells grown for 48 h with the respective lipoproteins was determined. In spite of their defective binding to the receptor, the metabolism of HTG-LDL in the regulated cells was accelerated in comparison to N-LDL, and equal to that of BZ-LDL. That observation is explained by the inefficient ability of HTG-LDL to depress LDL receptor activities in the cells.     

A modified conformation sensitive gel electrophoresis (CSGE) method for rapid and accurate detection of low density lipoprotein (LDL) receptor gene mutations in Familial Hypercholesterolemia

OBJECTIVES: A conformation sensitive gel electrophoresis (CSGE) method was modified for detection of LDL receptor gene mutations. DESIGN AND METHODS: Usage of a temperature gradient against running time was tested with 33 identified mutations in the LDL receptor gene. Most of the mutations were missense. RESULTS: 32 mutations were detected using this method (sensitivity: 97%). The duration required for running the test for each of the exons was reduced to 3-4 h. CONCLUSIONS: This modified CSGE method may be used as a screening procedure in genetic diseases where a variety of mutations may cause illness.     

乳腺导管原位癌的X线影像特征及病理核分级与人表皮生长因子受体2、雌激素受体及孕激素受体表达的相关性

目的 探讨乳腺导管原位癌(DCIS)的X线影像特征及病理核分级与雌激素受体(ER)、孕激素受体(PR)及人表皮生长因子受体2(HER2)表达的相关性。方法 回顾经手术病理证实为DCIS的患者105例,共107个病灶,术前均接受乳腺X线摄影检查,系统描述病变的X线影像学特征。分析不同病理核分级及其影像特征与不同肿瘤因子表达(HER2阳性组、ER阳性组和三阴组)的相关性。结果 钙化为DCIS的主要影像学特征(74/107,69.15%),HER2阳性组、ER阳性组、三阴组间钙化形态及分布类型的差异均有统计学意义(P均<0.05),HER2阳性组以区段样分布(23/45,51.11%),线状分枝状钙化(18/45,40.00%)表现为主,主要为高级别DCIS(38/55,69.09%)。ER阳性组以区域性分布(23/45,51.11%)、多形性钙化(12/23,52.17%)表现为主,以非高级别为主(22/42,52.38%)。结论 DCIS患者的乳腺X线表现特征与肿瘤的一些分子生物学表达存在相关性,可以间接反映肿瘤的某些生物学行为。     

An interaction between the human cholesteryl ester transfer protein (CETP) and apolipoprotein A-I genes in transgenic mice results in a profound CETP-mediated depression of high density lipoprotein cholesterol levels.

We have previously described two transgenic mouse lines, one heterozygous for the human apo A-I gene and the other heterozygous for a human cholesteryl ester transfer protein (CETP) minigene driven by the mouse metallothionein-I gene promoter. In the current study, these two lines were crossed producing control, HuCETPTg, HuAITg, and HuAICETPTg mice to study the influence of CETP on HDL cholesterol levels, particle size distribution, and metabolism in animals with mouse and human-like HDL. In the HuCETPTg and HuAICETPTg animals, zinc induction approximately doubled plasma CETP activity, with no activity in plasma from the control and HuAITg animals. The only significant effect of CETP on lipoprotein subfraction cholesterol concentrations was for HDL-C. Compared to control animals, HuCETPTg animals had lower HDL-C, 20% before and 35% after Zn induction, and compared to HuAITg animals, HuAICETPTg animals had lower HDL-C, 35% before and 66% after Zn induction. Control and HuCETPTg HDL consist primarily of a single size population with a mean diameter of 10.00 +/- 0.10 nm and 9.71 +/- 0.05 nm, respectively. HuAITg HDL consists primarily of three distinct HDL size subpopulations with peak diameters of 10.35 +/- 0.08 nm, 8.80 +/- 0.06 nm, 7.40 +/- 0.10 nm, and HuAICETPTg HDL also consists primarily of three distinct HDL size subpopulations with peak diameters of 9.87 +/- 0.05 nm, 8.60 +/- 0.10 nm, 7.30 +/- 0.15 nm before, and 9.71 +/- 0.08 nm, 8.50 +/- 0.11 nm, 7.27 +/- 0.15 nm after zinc induction, respectively. Western blotting analysis of nondenaturing gradient gels of plasma with a monoclonal antibody to CETP indicated that in HuCETPTg and HuAICETPTg mice, 22 and 100%, respectively, of the CETP was HDL associated. Turnover studies with HDL doubly labeled with 125I apo A-I and 3H cholesteryl linoleate indicated that the CETP-induced fall in HDL-C was associated with increased HDL-cholesterol ester fractional catabolic rate in both the absence and presence of human apo A-I, suggesting CETP-mediated transfer of HDL-cholesterol ester to apo B-containing lipoproteins. In summary, these studies suggest that CETP has a much more profound effect on HDL cholesterol levels in transgenic animals expressing human apo A-I. This may be due to an enhanced interaction of CETP with human compared to mouse apo A-I or to the HDL particles they produce.     

Association between reduced low density lipoprotein oxidation and inhibition of monocyte chemoattractant protein-1 production in statin-treated subjects

Monocyte chemoattractant protein-1 (MCP-1) is essential in atherogenesis. Oxidized lipids regulate MCP-1 expression and release from mononuclear cells. In this study we investigated (1) whether statin therapy reduces lipopolysaccharide (LPS)-stimulated MCP-1 production in human whole-blood samples and (2) the relationships between in vitro low-density lipoprotein (LDL) oxidation and MCP-1 production. Fasting blood samples were obtained from 55 healthy nonsmoking adults with moderate hypercholesterolemia who were participating in a randomized double-blind 8-week trial comparing the effects of statin therapy with those of placebo on cytokine production. Samples were analyzed for resistance to copper-mediated LDL oxidation (lag time in minutes), as well as MCP-1- and interleukin-8 (IL-8)-stimulated production. Statin therapy reduced MCP-1 production (mean +/- SD) -161 +/- 399 pg/mL/mm 3 white cells) compared with 267 +/- 985 pg/mL/mm 3 in the placebo group, but changes were not different between active and placebo groups ( P = .13). Statin therapy also increased lag times (median [interquartile range]; 20.5 [7.0-51.2] minutes vs -17.0 [-5.3-16.5] minutes; P = .067 for group difference). Inhibition of MCP-1 production correlated with prolongation of lag time ( r = .46, P = .0056) in statin-treated subjects. Statin therapy reduced MCP-1 production in the whole blood of human subjects and these changes were correlated with improvement in LDL oxidative resistance.