Properties Of High-density Lipoprotein Research Articles

Improvements in Methods for Detection of Properties of High Density Lipoprotein

ObjectiveWe have developed an assay in which monocyte migration through filters, determines the level of inflammatory properties of low density lipoprotein (LDL) and high density lipoprotein (HDL) protective capacity. Automated monocyte enumeration demonstrated this property of HDL.MethodsCultured artery wall cells were exposed to either media alone, media plus LDL, or media plus LDL and HDL. The media from the treated cells was assayed in a monocyte chemotaxis assay. Image‐Pro Plus 5 software was used to automate the quantification of migrated monocytes. The accuracy of the data obtained from automated counting of images with 20X objective was compared with manual counting and tested by a two‐tailed paired t‐test. An integrated tool, Macro, was used to perform the automated enumeration on several images with 4X objective (the entire field).Summary of ResultsHDL, when presented to co‐cultures of artery wall cells together with LDL, significantly reduced the increased monocyte transmigration due to LDL alone. The statistical results, comparing the manual and automatic enumeration, demonstrated accuracy and precision (p=0.02, df=11, α= 0.05).ConclusionThe results demonstrate that automated counting can replace manual counting, which should result in higher‐throughput of measurement of monocyte chemotaxis being used to determine the level of HDL proinflammatory or anti inflammatory properties.

Abstract 4870: Mechanisms Leading to Impaired Endothelial-vasoprotective Properties of High Density Lipoprotein (HDL) in Patients With Coronary Disease (CAD)

Background: HDL-raising therapies are currently intensely evaluated as a potential novel therapeutic strategy in patients with CAD. However, vascular effects of HDL have been observed to be highly variable. The aim of the present study was therefore to characterize mechanisms leading to altered endothelial-vasoprotective properties of HDL in patients with CAD. Methods: HDL was isolated from patients with stable CAD (sCAD), an acute coronary syndrome (ACS) and healthy subjects (HS; n=20 –25) by sequential ultracentrifugation. The effects of HDL on endothelial nitric oxide (NO) production, endothelium-dependent, NO-mediated vasodilation, signaling pathways leading to eNOS activation and endothelial superoxide production (ESR spectroscopy analysis) were examined. HDL-associated paraoxonase (PON) activity/content and endothelial binding of HDL (radioactive 125I-labelling of HDL) were characterized. Moreover, the role of eNOS and PON for anti-inflammatory properties of HDL were determined. Results: HDL from HS, but not HDL from sCAD/ACS patients, potently stimulated endothelial NO production. HDL from HS increased endothelial Akt, eNOS-Ser1177-phosphorylation and eNOS-Thr495-dephosphorylation, that was not observed with HDL from sCAD/ACS patients. Endothelial binding capacity of HDL was markedly reduced in patients with CAD. PON activity was reduced (−62.2% and −70.4% vs. HS, P<0.001), whereas PON1 content was increased in HDL from sCAD/ACS patients as compared to HDL from HS, suggesting inactivation of HDL-associated PON in CAD. Inhibition of PON activity prevented the capacity of HDL from HS to stimulate Akt/eNOS phosphorylation, endothelial NO production, NO-dependent vasodilation and to exert anti-inflammatory effects. eNOS siRNA knockdown prevented anti-inflammatory effects of HDL from HS. Conclusion: The present study demonstrates that reduced endothelial binding of HDL and inactivation of HDL-associated paraoxonase are major mechanisms leading to impaired endothelial-vasoprotective and anti-inflammatory effects of HDL in CAD patients. HDL-targeted treatment approaches should therefore not only increase HDL levels, but likely more important, restore endothelial-vasoprotective properties of HDL.

L 001 Changes in the Functional Properties of High-Density Lipoprotein (HDL) and the Cholesteryl Esters and Phospholipids Transfer Proteins Activities in Hamsters Submitted to Hiperlipidemic Diet

L 001 Changes in the Functional Properties of High-Density Lipoprotein (HDL) and the Cholesteryl Esters and Phospholipids Transfer Proteins Activities in Hamsters Submitted to Hiperlipidemic Diet

The role of dysfunctional HDL in atherosclerosis

This review focuses on HDL function in modulating LDL oxidation and LDL-induced inflammation. Dysfunctional HDL has been identified in animal models and humans with chronic inflammatory diseases including atherosclerosis. The loss of antiinflammatory function correlated with a loss of function in reverse cholesterol transport. In animal models and perhaps in humans, dysfunctional HDL can be improved by apoA-I mimetic peptides that bind oxidized lipids with high affinity.

Multiple actions of high-density lipoprotein

Purpose of review The most accepted property of high-density lipoprotein is reverse cholesterol transport. However, other beneficial actions may contribute to the antiatherogenic role of high-density lipoprotein. This review addresses the action of high-density lipoprotein beyond reverse cholesterol transport. Recent findings High-density lipoprotein cholesterol levels are inversely associated with coronary heart disease and other forms of vascular disease. Apart from transferring excess cholesterol to the liver, high-density lipoprotein exhibits favorable effects on oxidation, inflammation, thrombosis and endothelial function. Some of these actions are at least in part attributed to high-density lipoprotein-associated enzymes, such as paraoxonase and platelet-activating factor acetylhydrolase. However, high-density lipoprotein can become dysfunctional and proatherogenic under certain circumstances. Summary Current data suggest that high-density lipoprotein possesses various properties beyond reverse cholesterol transport. However, many issues on the exact role of high-density lipoprotein remain unknown. Future research is needed.

Abstract 1316: Preclinical Atherosclerosis in Primary Antiphospholipid Syndrome: The Impact of Oxidative Properties of High Density Lipopoprotein

Background: Antiphospholipid syndrome (APS) is characterised by increased thrombogenicity and/or pregnancy morbidity in the presence of raised levels of antiphospholipid antibodies (aPL). Increased oxidative properties of high density lipoprotein (HDL)(decreased activity of paraoxonase (PON)) is associated with increased risk for atherosclerosis and has been described in APS. The impact of PON on atherosclerotic disease progression in APS is unclear. We therefore examined the effect of PON on intima media thickness (IMT), and pulse wave velocity (PWV) in patients with positive aPL. Methods: We studied 77 women with positive aPL (aPL) aged 46.6±1.2 yrs (mean±SE) and a control group of 77 women aged 47.5±1.2 yrs matched for traditional cardiovascular risk factors. High resolution ultrasound was used to determine carotid IMT. Arterial stiffness was assessed non-invasively by carotid-radial PWV. PON activity was assessed by measuring p-nitrophenol formation and activity expressed as nmoles p-nitrophenol/ml serum/minute. Results: APL patients had significantly increased IMT and PWV compared to controls (0.75±0.02mm vs 0.65±0.01mm, p<0.001 and 9.14±0.18 m/s vs 8.56±0.21m/s, p<0.05 respectively). PON activity was significantly reduced in aPL compared to controls (91.5[64.3, 05.1]mmol/ml/min, median[IQR] vs 103.1[80.4, 111.5] mmol/ml/min, p<0.006). Although PON activity was not associated with vascular measures in controls, an inverse association was noted in aPL patients (r=−0.26 [cIMT] and r=−0.23 [PWV], both p<0.05). In multivariate analysis, accounting for cardiovascular risk factors, PON activity (β=−0.42, p<0.001), age (β=0.33, p<0.001) and systolic blood pressure (β=0.24, p<0.05) were independent determinants of cIMT while PON activity (β=−0.32, p<0.01) and systolic blood pressure (β=0.28, p<0.05) remained the only independent predictors of PWV in aPL positive patients. Conclusions: APS is associated with increased arterial stiffness and carotid intimal thickening. Paraoxonase activity is inversely associated with IMT and PWV in ApL positve patients. These findings indicate that oxidative stress may play an important role in the development of atherosclerosis in patients with primary antiphospholipid syndrome.

Transient Triglyceridaemia Reduces the Anti-inflammatory Properties of High-density Lipoprotein

Transient Triglyceridaemia Reduces the Anti-inflammatory Properties of High-density Lipoprotein

2P-0467 Enhanced anti-inflammatory property of high density lipoprotein following infusion of chylomicron-like emulsions

2P-0467 Enhanced anti-inflammatory property of high density lipoprotein following infusion of chylomicron-like emulsions

Effect of Aluminium on Lipid Peroxidation of Human High Density Lipoproteins

We investigated the effect of aluminium (Al 3+ ) on lipid peroxidation and physico-chemical properties of high density lipoproteins (HDL) isolated from human plasma. Our results demonstrated that Al 3+ enhances lipid peroxidation of human HDL as shown by the significant increase in lipid hydroperoxides in Al-treated HDL with respect to control HDL. The oxidative effect was higher at acid pH (pH 5.5) with respect to pH 7.4. Moreover, a stimulating effect of Al 3+ on iron-induced lipid peroxidation of HDL was demonstrated. The study of the effect of Al 3+ on the physico-chemical properties of HDL, using the fluorescence polarization (Pf) of the probes TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide) and DPH (1,6-diphenyl-1,3,5-hexatriene), showed a significant decrease of Pf in Al-treated HDL with respect to control. These results suggest that Al 3+ induces a decrease of molecular order at the lipoprotein surface. Moreover, the study of tryptophan (Trp) fluorescence demonstrated that aluminium induces structural modifications of HDL apoproteins and on HDL physico-chemical properties. The effect of Al 3+ on lipid peroxidation of HDL was observed at aluminium concentrations similar to those observed in the brain of patients affected by neurological diseases. Aluminium-induced oxidative damage of HDL could be involved in the development of neurological diseases.

Hypochlorite-modified High Density Lipoprotein, a High Affinity Ligand to Scavenger Receptor Class B, Type I, Impairs High Density Lipoprotein-dependent Selective Lipid Uptake and Reverse Cholesterol Transport

Hypochlorous acid/hypochlorite (HOCl/OCl(-)), a potent oxidant generated in vivo by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells. Binding of HOCl-HDL was saturable, and the degree of HOCl modification was the determining factor for increased binding affinity to SR-BI. Competition experiments further confirmed that HOCl-HDL binds with increased affinity to the same or overlapping domain(s) of SR-BI as does native HDL. Furthermore, SR-BI-mediated selective HDL-cholesteryl ester association as well as time- and concentration-dependent cholesterol efflux from SR-BI overexpressing Chinese hamster ovary cells were, depending on the degree of HOCl modification of HDL, markedly impaired. The most significant findings of this study were that the presence of very low concentrations of HOCl-HDL severely impaired SR-BI-mediated bidirectional cholesterol flux mediated by native HDL. The colocalization of immunoreactive HOCl-modified epitopes with apolipoprotein A-I along with deposits of lipids in serial sections of human atheroma shown here indicates that the myeloperoxidase-H(2)O(2)-halide system contributes to oxidative damage of HDL in vivo.

Effect of HDL, from Japanese white rabbit administered a new cholesteryl ester transfer protein inhibitor JTT-705, on cholesteryl ester accumulation induced by acetylated low density lipoprotein in J774 macrophage

We have previously reported a potent and specific cholesteryl ester transfer protein (CETP) inhibitor JTT-705 was a potentially anti-atherogenic compound (Nature 406 (2000) 203). In the present study, we investigated in vitro how this compound affects properties of high density lipoprotein (HDL) in Japanese white (JW) rabbits in terms of reverse cholesterol transport in J774 macrophages. Plasma HDL-cholesterol (C) level was significantly higher in the rabbits administered JTT-705 than in control rabbits on days 3 and 7. Both HDL 2 and HDL 3-C levels were also significantly higher in JTT-705-administered rabbits than in control rabbits. During this period, plasma CETP activity was kept lower in JTT-705-administered rabbits than in controls. To determine how this compound affects the property of HDL particles, we investigated the C efflux induced by HDL from JTT-705-administered and control rabbits in J774 macrophages. Cholesterol ester (CE) concentration in J774 macrophages was reduced in proportion with increasing concentration of the added HDL to the culture media for J774 macrophages in both groups, suggesting that the HDL from JTT-705-administered rabbits was able to reduce CE concentration in J774 macrophages as efficiently as that from control rabbits. This result, together with the finding that the absolute HDL concentration increased in JW rabbits administered this CETP inhibitor, suggests that treatment with this new compound causes a beneficial effect on lipid metabolism in terms of anti-atherogenicity.

Cerivastatin modulates antiatherogenic properties of high-density lipoproteins in patients with coronary heart disease and hyperlipidemia

The effects of cerivastatin on antiatherogenic properties of high-density lipoproteins were studied in patients with coronary heart disease and hyperlipidemia. Apart from hypolipidemic effects, cerivastatin changed the phospholipid composition of high-density lipoproteins and improved their cholesterol-acceptor properties. This effect was most pronounced in the serum from patients with low content of high-density lipoprotein cholesterol. These data indicate that cerivastatin modulates antiatherogenic properties of high-density lipoproteins.

Effect of human apolipoprotein A-I gene on vasoactive properties of high density lipoproteins in rats of different age

Vasodilatory activity of high density lipoproteins and the content of apolipoprotein A-I in these particles decreases in senescent rats in comparison with adult animals. Implantation of human apolipoprotein A-I gene increases the content of this apolipoprotein in high density lipoproteins and improves their vasodilatory effect.

Effect of the surface lipid composition of reconstituted LPA-I on apolipoprotein A-I structure and lecithin:cholesterol acyltransferase activity

Characterization of the factors that regulate plasma cholesterol esterification shows that the increased activity of lecithin:cholesterol acyltransferase (LCAT) in the plasma of hyperlipidemic subjects is due to enhanced interactions with a preferred substrate. The details of how the physical properties of high density lipoproteins (HDL) may affect their ability to stimulate cholesterol esterification by LCAT have been investigated in homogeneous reconstituted HDL particles containing two molecules of apolipoprotein (apo) A-I (Lp2A-I) and palmitoyl-oleoyl phosphatidylcholine (POPC). Increasing the POPC or sphingomyelin (SPH) content in an Lp2A-I complex increases particle size and stability but decreases the negative surface charge of apoA-I. Increasing Lp2A-I POPC or SPH content also significantly inhibits cholesterol esterification by LCAT. Increase in the maximum rate of CE production ( V max) by LCAT is directly related to an increased negative charge on the different Lp2A-I particles and to a reduced amount and stability of amphipathic α-helices in apoA-I. In contrast, increasing the Lp2A-I complex negative charge directly by addition of a charged lipid, phosphatidylinositol (PI), has minimal effect on apoA-I conformation and LCAT activation. While variations in Lp2A-I PI content have little effect on the interfacial binding of LCAT, increasing POPC content appears to directly increase the binding affinity of LCAT for the different Lp2A-I particles. These results show that LCAT is stimulated by an apoA-I conformation-dependent increase in negative charge but is less sensitive to electrostatic changes in the lipid interface of discoidal Lp2A-I. The activation of LCAT appears to be dependent on the exposure of both central (residues 98–132) and N-terminal (residues 2–8) domains in apoA-I. A strong relationship between the immunoreactivity of two specific mAbs, 4H1 and A11, and LCAT reactivity suggests that the N-terminus of apoA-I may interact with a central domain in a manner that may regulate the accessibility of LCAT to the edge of the disc. This indicates that the conformation and charge of apoA-I are sensitive to the surface-lipid composition of HDL particles and play a central role in regulating LCAT activation. Since alterations in the surface lipid composition of HDL particles from hyperlipidemic subjects also modify the charge and structure of these particles, this may stimulate the rates of cholesterol esterification by making these lipoproteins preferred LCAT substrates.

Effects of Reagent and Enzymatically Generated Hypochlorite on Physicochemical and Metabolic Properties of High Density Lipoproteins

Myeloperoxidase (MPO), a protein secreted by activated phagocytes, may be a potential candidate for the generation of modified/oxidized lipoproteins in vivo via intermediate formation of HOCl, a powerful oxidant. During the present study, the effects of reagent NaOCl and OCl- generated by the MPO/H2O2/Cl- system on physicochemical and metabolic properties of high density lipoprotein (HDL) subclass 3 (HDL3) were investigated. Up to a molar oxidant:lipoprotein ratio of approximately 30:1, apolipoprotein A-I (apoA-I), the major HDL3 apolipoprotein component, represented the preferential target for OCl- attack (consuming 35-76% of the oxidant), thereby protecting HDL3 fatty acids (consuming between 17 and 30% of the oxidant) against OCl--mediated modification. At molar oxidant:HDL3 ratios >/= 60:1, we have observed pronounced consumption of HDL3 unsaturated fatty acids with concomitant formation of fatty acid chlorohydrins. Modification of HDL3 in the presence of the MPO/H2O2/Cl- system resulted in amino acid oxidation in a manner comparable with that found with reagent NaOCl only. Treatment of HDL3 with reagent NaOCl as well as modification by the MPO/H2O2/Cl- system resulted in significantly enhanced turnover rates of HDL3 by mouse peritoneal macrophages, an effect that was not a result of HDL3 aggregation as judged by dynamic and static light-scattering experiments. In comparison with native HDL3, the degradation by macrophages was enhanced by 4- and 15-fold when HDL3 was modified with reagent NaOCl or the MPO/H2O2/Cl- system. Finally, the ability of HDL3 to promote cellular cholesterol efflux from macrophages was significantly diminished after modification with reagent NaOCl. Collectively, these results demonstrate that the modification of HDL3 by hypochlorite (added as reagent or generated by the MPO/H2O2/Cl- system) transformed an antiatherogenic lipoprotein particle into a modified lipoprotein with characteristics similar to lipoproteins commonly thought to initiate foam cell formation in vivo.

4.P.364 Effects of hypochlorite-modification on physico-chemical and biological properties of high density lipoproteins

4.P.364 Effects of hypochlorite-modification on physico-chemical and biological properties of high density lipoproteins

Lipoproteins, Coagulation States, and Risk of Thrombotic Events

To the Editor. —The results of clinical studies correlating lipoprotein levels with risk for thrombotic events like myocardial infarction are inconsistent and not sensitive indicators of risk. 1 One may assume that these thrombotic events are causally related to hypercoagulability and that protection from these events is associated with relative hypocoagulability. Regrettably, there is no coagulation test to detect a prothrombotic (high-risk) state or antithrombotic (low-risk) state as a marker of the effects of lipoproteins. We here describe the utilization of a modified recalcification time (MRT) test to document the adverse coagulation-accelerating effect of low-density lipoproteins (LDLs) and the beneficial coagulation-retarding property of high-density lipoproteins (HDLs). This test was described in detail in a recent article. 2 Unlike other tests, by using whole blood, the MRT incorporates the role of both cellular and chemical participants in coagulation. Also, incubating blood with an activator like a lipoprotein induces the monocyte to

High density lipoprotein-binding proteins in porcine liver. Isolation and histological localization.

The antiatherogenic properties of high density lipoproteins (HDLs) are thought to reside in their involvement in the reverse cholesterol transport pathway. Specific HDL-binding proteins could play a key role in this process. Two HDL-binding proteins of approximately 90 and 180 kd were identified in porcine liver by ligand blotting and were purified to apparent homogeneity by a combination of protein extraction, DEAE-cellulose chromatography, Con A-Sepharose chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of 125I-HDL by these proteins could be actively competed for by unlabeled HDL but not by low density lipoprotein. Polyclonal antisera have been raised against these two proteins. Each antiserum recognized only one of the HDL-binding proteins, indicating that they are not immunologically related. Moreover, striking differences in localization were observed in immunohistochemical studies. The 90-kd protein is located within the hepatocellular plates, while the 180-kd protein is present along the lining of the sinusoids. These results suggest functional differences between the two HDL-binding proteins described.

Effect of recent alcohol intake on acceptor properties of high-density lipoproteins and their interaction with liver cells

Effect of recent alcohol intake on acceptor properties of high-density lipoproteins and their interaction with liver cells

Lipoprotein separation and low density lipoprotein molecular weight determination using high performance gel-filtration chromatography

Plasma lipoproteins were isolated at d less than 1.225 g/ml from nonhuman primates of three species, cynomolgus, rhesus, and African green (vervet) monkeys. Individual lipoprotein classes were separated by high performance gelfiltration chromatography and low density lipoprotein (LDL) molecular weight was determined. A comparison was made using column configurations including TSK 3000 SW, 4000 SW, and 5000 PW columns. Due to its relative simplicity, stability, and economy, a single 5000 PW column was selected for most of the work. The recovery of lipoprotein cholesterol from the column averaged 91 +/- 2.5%. A comparison of the immunologic, chemical, and electrophoretic properties of high density lipoproteins (HDL) and LDL isolated by this technique with those of HDL and LDL isolated by conventional agarose column chromatography indicated that lipoproteins isolated by high performance gel-filtration chromatography were intact and reasonably free of cross contamination. A standard preparation of 125I-labeled LDL was added to the d less than 1.225 g/ml lipoprotein fraction just prior to separation and a relative size index, r1, was determined. When r1 values for a large number of samples were compared with the log of the LDL molecular weight (determined by agarose column chromatography) a linear relationship was found with a correlation coefficient, r = 0.85. The regression equation for this relationship could be used to calculate LDL molecular weights from the r1 value. These values agreed with LDL molecular weight determined by flotation equilibrium analysis in the analytical ultracentrifuge. We conclude that high performance gel-filtration chromatography using the TSK 5000 PW column provides an analytical and preparative technique for simultaneous separation of individual lipoproteins and determination of LDL molecular weight.