Yifen Liu

Glycation of paraoxonase-1 inhibits its activity and impairs the ability of high-density lipoprotein to metabolize membrane lipid hydroperoxides

Aims  High‐density lipoprotein (HDL) protects against atherosclerosis development. Defective functioning of HDL in Type 2 diabetes may be one cause of increased cardiovascular disease associated with Type 2 diabetes. HDL modulates low‐density lipoprotein and cell membrane oxidation through the action of paraoxonase‐1 (PON1), which is one of the major mechanisms by which HDL is anti‐atherogenic.

Hepatocyte growth factor and c‐Met expression in pericytes: implications for atherosclerotic plaque development

Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c‐Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31‐positive endothelial cells. c‐Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT–PCR, we demonstrated expression of HGF and c‐Met in a rat pericyte cell‐line, TR–PCT1, and in primary pericytes. HGF treatment of TR‐PCT1 cells induced their migration, but not their proliferation, in a dose‐dependent manner (10–100 ng/ml, p < 0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 µM) or LY294002 (10 µM) abolished these effects. This work demonstrates the expression of c‐Met and HGF in human atherosclerotic arteries, in association with SM‐actin‐positive cells and CD‐31‐positive cells, respectively. HGF induces pericyte migration via PI3‐kinase and Akt activation in vitro. HGF and c‐Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage. Copyright

Comparison of the ability of paraoxonases 1 and 3 to attenuate the in vitro oxidation of low-density lipoprotein and reduce macrophage oxidative stress

In light of recent conflicting results regarding the antiatherogenic properties of the paraoxonase (PON) multigene family we have reexamined these properties in vitro. The abilities of recombinant human PON1 and PON3 to retard LDL oxidation, prevent macrophage oxidative stress, and promote macrophage cholesterol efflux were investigated. Both PON1 and PON3 retarded the oxidation of LDL; PON1 was significantly more efficient (50 and 100% at 20 microg PON3 and PON1, respectively (P<0.001)). Neither PON1 nor PON3 were able to prevent macrophage oxidative stress; however, both were able to retard macrophage-induced LDL oxidation (100 and 50% at 20 microg/ml respectively for PON1 and PON3, P<0.05). PON3 promoted macrophage cholesterol efflux (30% at 40 microg/ml, P<0.01); however, PON1 was found to be cytotoxic to the macrophages derived from the human monocyte THP-1 cell line. In conclusion using recombinant proteins we have been able to confirm some but not all of the antiatherosclerotic properties attributed to human PON1 and PON3 but have also discovered a novel cytotoxicity of PON1 toward macrophages derived from the human monocytic THP-1 cell line.

Contribution of VCAF-positive cells to neovascularization and calcification in atherosclerotic plaque development.

Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF‐positive cells were detected within the intima and adventitia in areas where sporadic 3G5‐positive pericytes were localized. Furthermore, VCAF‐positive cells were identified in newly formed microvessels in association with CD34‐positive/CD146‐positive/c‐kit‐positive cells as well as in intact CD31‐positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease. Copyright

Measurement of plasma small-dense LDL concentration by a simplified ultracentrifugation procedure and immunoassay of apolipoprotein B

BACKGROUND Existing methods for detecting small-dense low-density lipoprotein (SD-LDL) are either semiquantitative (e.g., gradient gel electrophoresis) or require specialised laboratory methods (e.g., density-gradient ultracentrifugation, DGU). METHODS We report a method in which plasma was adjusted to a density (D) of 1.044 and 1.060 g/ml, respectively, in two tubes, both of which underwent ultracentrifugation (UC). A measure of SD-LDL apolipoprotein B (apo B) was obtained by subtraction of the apo B concentration in D>1.060 g/ml lipoproteins from that in D>1.044 g/ml lipoproteins to correct for apo B associated with lipoprotein (a) [Lp(a)]. This procedure was evaluated in paired plasma samples in healthy men (n=62) and in age-matched healthy women (n=74) and in age-matched primary dyslipidaemic men (n=72) and women (n=29) and compared with an established density-gradient ultracentrifugation (DGU) method. RESULTS The dyslipidaemic patients had either decreased high-density lipoprotein cholesterol (HDL-C) and/or increased triglycerides. In dyslipidaemic men, SD-LDL apo B level (23 [5-77] mg/dl) was significantly higher than in healthy men (P<0.001). In dyslipidaemic women, the SD-LDL apo B levels (11 [4-71] mg/dl) were significantly higher than in healthy women (7 [1-45] mg/dl; P<0.005). The concentration of SD-LDL apo B correlated inversely with HDL-C in both women (r=-0.280: P<0.005) and men (r=-0.464; P<0.0001) and positively with triglyceride concentration in both women (r=0.213; P<0.05) and men (r=0.592: P<0.0001). Correction for apo B in Lp(a) increased the analytical variation, which was 12% for apo B at D=1.044-1.060 g/ml and 9% for apo B measured at D>1.044 g/ml. Although the correlation between the new method and DGU results was high (r=0.830; P<0.0001, n=43), the concentration of apo B at D>1.044 g/ml correlated strongly with both corrected results (r=0.978; P<0.0001; n=237) and also with SD-LDL isolated using the DGU method (r=0.832; P<0.0001). Results at D>1.044 g/ml showed the expected correlations both with HDL-C (r=-0.465: P<0.0001) and triglycerides (r=0.526; P<0.0001). CONCLUSIONS The new method gave results consistent with earlier published findings using other techniques. Further simplification of the method using a single-density spin at D>1.044 g/ml appears feasible and may provide an easier quantitative method for clinical use.

Effect of Extended‐Release Niacin on High‐Density Lipoprotein (HDL) Functionality, Lipoprotein Metabolism, and Mediators of Vascular Inflammation in Statin‐Treated Patients

Background The aim of this study was to explore the influence of extended-release niacin/laropiprant (ERN/LRP) versus placebo on high-density lipoprotein (HDL) antioxidant function, cholesterol efflux, apolipoprotein B100 (apoB)-containing lipoproteins, and mediators of vascular inflammation associated with 15% increase in high-density lipoprotein cholesterol (HDL-C). Study patients had persistent dyslipidemia despite receiving high-dose statin treatment. Methods and Results In a randomized double-blind, placebo-controlled, crossover trial, we compared the effect of ERN/LRP with placebo in 27 statin-treated dyslipidemic patients who had not achieved National Cholesterol Education Program-ATP III targets for low-density lipoprotein cholesterol (LDL-C). We measured fasting lipid profile, apolipoproteins, cholesteryl ester transfer protein (CETP) activity, paraoxonase 1 (PON1) activity, small dense LDL apoB (sdLDL-apoB), oxidized LDL (oxLDL), glycated apoB (glyc-apoB), lipoprotein phospholipase A2 (Lp-PLA2), lysophosphatidyl choline (lyso-PC), macrophage chemoattractant protein (MCP1), serum amyloid A (SAA) and myeloperoxidase (MPO). We also examined the capacity of HDL to protect LDL from in vitro oxidation and the percentage cholesterol efflux mediated by apoB depleted serum. ERN/LRP was associated with an 18% increase in HDL-C levels compared to placebo (1.55 versus 1.31 mmol/L, P<0.0001). There were significant reductions in total cholesterol, triglycerides, LDL cholesterol, total serum apoB, lipoprotein (a), CETP activity, oxLDL, Lp-PLA2, lyso-PC, MCP1, and SAA, but no significant changes in glyc-apoB or sdLDL-apoB concentration. There was a modest increase in cholesterol efflux function of HDL (19.5%, P=0.045), but no change in the antioxidant capacity of HDL in vitro or PON1 activity. Conclusions ERN/LRP reduces LDL-associated mediators of vascular inflammation, but has varied effects on HDL functionality and LDL quality, which may counter its HDL-C-raising effect. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT01054508.

How HDL protects LDL against atherogenic modification: paraoxonase 1 and other dramatis personae.

Purpose of review To summarize the current evidence about how HDL impedes the oxidative and glycative atherogenic modification of LDL. Recent findings Paraoxonase 1 (PON1) is located on HDL. Meta-analysis of clinical epidemiological investigations reveals a substantial association of low serum PON1 activity with coronary heart disease incidence independent of other risk factors including HDL cholesterol and apolipoprotein AI (apoAI). Transgenic animal models also indicate an antiatherosclerotic role for PON1. However, highly purified and recombinant PON1 do not retain their antioxidant properties. Summary The therapeutic potential of PON1 should be recognized in preventing atherosclerosis and combating infection and organophosphate toxicity. In unleashing this potential, it is important to consider that both highly purified and recombinant PON1 are dissociated from the lipid phase and other components of HDL, such as apoAI and apoM, all of which may be required for HDL (through its PON1 component) to hydrolyze more lipophilic substrates.

High-density lipoprotein cholesterol raising: Does it matter?

Purpose of review Cardiovascular disease (CVD) is the leading cause of morbidity and premature mortality in Europe and the United States, and is increasingly common in developing countries. High-density lipoprotein cholesterol (HDL-C) is an independent risk factor for CVD and is superior to low-density lipoprotein cholesterol (LDL-C) as a predictor of cardiovascular events. The residual risk conferred by low HDL-C in patients with a satisfactory LDL-C was recently highlighted by the European Atherosclerosis Society. Despite the lack of randomized controlled trials, it has been suggested that raising the level of HDL-C should be considered as a therapeutic strategy in high-risk patients because of the strong epidemiological evidence, compelling biological plausibility, and both experimental and clinical research supporting its cardioprotective effects. Recent findings Three recent large randomized clinical trials investigating the effect of HDL-C raising with niacin and dalcetrapib in statin-treated patients failed to demonstrate an improvement in cardiovascular outcomes. Summary There is evidence to support the view that HDL functionality and the mechanism by which a therapeutic agent raises HDL-C are more important than plasma HDL-C levels. Future therapeutic agents will be required to improve this functionality rather than simply raising the cholesterol cargo.

Impairment of high-density lipoprotein resistance to lipid peroxidation and adipose tissue inflammation in obesity complicated by obstructive sleep apnea.

CONTEXT Obstructive sleep apnea (OSA) complicates morbid obesity and is associated with increased cardiovascular disease incidence. An increase in the circulating markers of chronic inflammation and dysfunctional high-density lipoprotein (HDL) occur in severe obesity. OBJECTIVE The objective of the study was to establish whether the effects of obesity on inflammation and HDL dysfunction are more marked when complicated by OSA. DESIGN AND PATIENTS Morbidly obese patients (n = 41) were divided into those whose apnea-hypoapnea index (AHI) was more or less than the median value and on the presence of OSA [OSA and no OSA (nOSA) groups]. We studied the antioxidant function of HDL and measured serum paraoxonase 1 (PON1) activity, TNFα, and intercellular adhesion molecule 1 (ICAM-1) levels in these patients. In a subset of 19 patients, we immunostained gluteal sc adipose tissue (SAT) for TNFα, macrophages, and measured adipocyte size. RESULTS HDL lipid peroxide levels were higher and serum PON1 activity was lower in the high AHI group vs the low AHI group (P < .05 and P < .0001, respectively) and in the OSA group vs the nOSA group (P = .005 and P < .05, respectively). Serum TNFα and ICAM-1 levels and TNFα immunostaining in SAT increased with the severity of OSA. Serum PON1 activity was inversely correlated with AHI (r = -0.41, P < .03) in the OSA group. TNFα expression in SAT directly correlated with AHI (r = 0.53, P < .03) in the subset of 19 patients from whom a biopsy was obtained. CONCLUSION Increased serum TNFα, ICAM-1, and TNFα expression in SAT provide a mechanistic basis for enhanced inflammation in patients with OSA. Decreased serum PON1 activity, impaired HDL antioxidant function, and increased adipose tissue inflammation in these patients could be a mechanism for HDL and endothelial dysfunction.

A review of paradoxical HDL-C responses to fenofibrate, illustrated by a case report

High-density lipoprotein cholesterol (HDL-C) concentration is an independent risk factor for cardiovascular disease. Fibrates are widely used in the management of atherogenic dyslipidemia, principally for their triglyceride-lowering and HDL-C-raising effects. Fibrates may cause paradoxical reductions in HDL-C. These reductions are usually modest, but significant reductions have been observed. The molecular mechanism for these paradoxical reductions remains unexplained despite advances in our understanding of lipid metabolism. This review considers possible mechanisms for this effect, illustrated by a patient with an observed reduction in HDL-C of 88% after introduction of fenofibrate.