Biomedical subjects

K A Rye

Publications and source records attributed to K A Rye.

At least 19 recordsLinked to original sources

PubMed2001

Evidence that apolipoprotein A-I facilitates hepatic lipase-mediated phospholipid hydrolysis in reconstituted HDL containing apolipoprotein A-II.

This study examines hepatic lipase (HL) mediated phospholipid hydrolysis in mixtures of apolipoprotein-specific, spherical reconstituted high-density lipoproteins (rHDL). We have shown previously that apolipoprotein A-I (apoA-I) and apoA-II have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in well-characterized, homogeneous preparations of spherical rHDL [Hime, N. J., Barter, P. J., and Rye, K.-A. (1998) J. Biol. Chem. 273, 27191-27198]. In the present study, phospholipid hydrolysis was assessed in mixtures of rHDL containing either apoA-I only, (A-I)rHDL, apoA-II only, (A-II)rHDL, or both apoA-I and apoA-II, (A-I/A-II)rHDL. The rHDL contained trace amounts of radiolabeled phospholipid, and hydrolysis was measured as the formation of radiolabeled nonesterified fatty acids (NEFA). As predicted from our previous kinetic studies, the (A-II)rHDL acted as competitive inhibitors of HL-mediated phospholipid hydrolysis in (A-I)rHDL. Less expected was the observation that the rate of phospholipid hydrolysis in (A-II)rHDL was enhanced when (A-I)rHDL were also present in the incubation mixture. The rate of phospholipid hydrolysis in (A-I/A-II)rHDL was also greater than in (A-II)rHDL, indicating that apoA-I enhances phospholipid hydrolysis when it is present as a component of (A-I/A-II)rHDL. It is concluded that apoA-I enhances HL-mediated phospholipid hydrolysis in apoA-II containing rHDL, irrespective of whether the apoA-I is present in the same particle as the apoA-II [as in (A-I/A-II)rHDL] or whether it is present as a component of a different particle, such as when (A-I)rHDL are added to incubations of (A-II)rHDL.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

The mechanism of the remodeling of high density lipoproteins by phospholipid transfer protein.

Phospholipid transfer protein (PLTP) remodels high density lipoproteins (HDL) into large and small particles. It also mediates the dissociation of lipid-poor or lipid-free apolipoprotein A-I (apoA-I) from HDL. Remodeling is enhanced markedly in triglyceride (TG)-enriched HDL (Rye, K.-A., Jauhiainen, M., Barter, P. J., and Ehnholm. C. (1998) J. Lipid. Res. 39, 613-622). This study defines the mechanism of the remodeling of HDL by PLTP and determines why it is enhanced in TG-enriched HDL. Homogeneous populations of spherical reconstituted HDL (rHDL) containing apoA-I and either cholesteryl esters only (CE-rHDL; diameter 9.3 nm) or CE and TG in their core (TG-rHDL; diameter 9.5 nm) were used. After 24 h of incubation with PLTP, all of the TG-rHDL, but only a proportion of the CE-rHDL, were converted into large (11.3-nm diameter) and small (7.7-nm diameter) particles. Only small particles were formed during the first 6 h of incubation of CE-rHDL with PLTP. The large particles and dissociated apoA-I were apparent after 12 h. In the case of TG-rHDL, small particles appeared after 1 h of incubation, while dissociated apoA-I and large particles were apparent at 3 h. The composition of the large particles indicated that they were derived from a fusion product. Spectroscopic studies indicated that the apoA-I in TG-rHDL was less stable than the apoA-I in CE-rHDL. In conclusion, these results show that (i) PLTP mediates rHDL fusion, (ii) the fusion product rearranges by two independent processes into small and large particles, and (iii) the more rapid remodeling of TG-rHDL by PLTP may be due to the destabilization of apoA-I.

Carrier Proteins↗

PubMed2001

Time sequence of the inhibition of endothelial adhesion molecule expression by reconstituted high density lipoproteins.

We have used discoidal reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I and dimyristoyl phosphatidylcholine (DMPC) as a tool to investigate the time sequence of the HDL-mediated inhibition of vascular cell adhesion molecule (VCAM)-1 and E-selectin expression in cytokine-activated human umbilical vein endothelial cells (HUVECs). Specifically, we have asked a few questions - (i) how long do the cells need to be exposed to the rHDL before adhesion molecule expression is inhibited and (ii) how long does the inhibition persist after removing the rHDL from the cells. When the cells were not pre-incubated with the rHDL, there was no inhibition. The magnitude of the inhibition increased progressively with increasing duration of pre-incubation up to 16 h. Inhibition did not require the rHDL to be physically present during the activation of adhesion molecule expression by tumour necrosis factor(TNF)-alpha, excluding the possibility that the rHDL was merely interfering with the interaction between TNF-alpha and the cells. When HUVECs were pre-incubated for 16 h with rHDL, the inhibition remained substantial even if the rHDL were removed from the medium up to 8 h prior to addition of TNF-alpha. The HDL-mediated inhibition of VCAM-1 in HUVECs was unaffected by the presence of puromycin, an inhibitor of protein synthesis, excluding the possibility that HDL may have acted by stimulating the synthesis of a cell protein that itself inhibits adhesion molecule expression. These results have important implications in terms of understanding the mechanism(s) of the HDL-mediated inhibition of endothelial adhesion molecule expression.

Cells, Cultured↗

PubMed2001

Cholesteryl ester transfer protein, high density lipoprotein and arterial disease.

The reported relationships between cholesteryl ester transfer protein, high density lipoproteins and arterial disease are confusing and conflicting. Several papers published during the review period add substantially to the evidence base regarding the atherogenicity (or anti-atherogenicity) of cholesteryl ester transfer protein, although none clearly resolves the continuing conflict. These new papers are presented against the backdrop of the previous state of knowledge.

Animals↗

PubMed2000

Oxidation of methionine residues to methionine sulfoxides does not decrease potential antiatherogenic properties of apolipoprotein A-I.

The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.

Apolipoprotein A-I↗

PubMed2000

The conformation of apolipoprotein A-I in high-density lipoproteins is influenced by core lipid composition and particle size: a surface plasmon resonance study.

Plasma high-density lipoproteins (HDL) are a heterogeneous group of particles that vary in size as well as lipid and apoprotein composition. The effect of HDL core lipid composition and particle size on apolipoprotein (apo) A-I structure was studied using surface plasmon resonance (SPR) analysis of the binding of epitope-defined monoclonal antibodies. The association and dissociation rate constants of 12 unique apo A-I-specific monoclonal antibodies for isolated plasma HDL were calculated. In addition, the association rate constants of the antibodies were determined for homogeneous preparations of spherical reconstituted HDL (rHDL) that contained apo A-I as the sole apolipoprotein and differed either in their size or in their core lipid composition. This analysis showed that lipoprotein size affected the conformation of domains dispersed throughout the apo A-I molecule, but the conformation of the central domain between residues 121 and 165 was most consistently modified. In contrast, replacement of core cholesteryl esters with triglyceride in small HDL modified almost the entire molecule, with only two key N-terminal domains of apo A-I being unaffected. This finding suggested that the central and C-terminal domains of apo A-I are in direct contact with rHDL core lipids. This immunochemical analysis has provided valuable insight into how core lipid composition and particle size affect the structure of specific domains of apo A-I on HDL.

Antibodies, Monoclonal↗

PubMed2000

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase.

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.

Apolipoprotein A-I↗

PubMed2000

Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression.

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.

Cells, Cultured↗

PubMed2000

Influence of phospholipid depletion on the size, structure, and remodeling of reconstituted high density lipoproteins.

This study shows that phospholipid depletion has a major impact on the size and structure of spherical, reconstituted high density lipoproteins (rHDL) and their remodeling by cholesteryl ester transfer protein (CETP). Spherical rHDL, 9.2 nm in diameter with a phospholipid/cholesteryl ester/unesterified cholesterol/apolipoprotein A-I (apoA-I) (PL/CE/UC/A-I) molar ratio of 37.3/24.5/4.1/1.0, were depleted progressively of phospholipids by incubation with phospholipase A(2). After 30 min of incubation the PL/CE/UC/A-I molar ratio of the rHDL was 8.0/31.2/4.4/1.0 and their diameter had decreased to 8.0 nm. Comparable changes in rHDL size and composition were also apparent when the incubations were carried out in the presence of other lipoprotein classes and lipoprotein-deficient plasma. The changes in size and composition were not accompanied by the dissociation of apoA-I from the rHDL. Phospholipid depletion did not affect rHDL surface charge or the structure and stability of apoA-I. The remodeling of unmodified and phospholipid-depleted rHDL by CETP was also investigated. When the rHDL were incubated for 3 h with CETP and Intralipid, transfers of core lipids between the phospholipid-depleted rHDL and Intralipid were decreased relative to unmodified rHDL. This difference was no longer apparent when the incubations were extended beyond 3 h. In these incubations apoA-I dissociated from the phospholipid-depleted and unmodified rHDL at 3 and 12 h, respectively. At 24 h the respective diameters of the unmodified rHDL and phospholipid-depleted rHDL were 8.0 and 7.8 nm. In conclusion, phospholipid depletion has a major impact on rHDL size and their remodeling by CETP.

Apolipoprotein A-I↗

PubMed1999

High density lipoproteins (HDL) interrupt the sphingosine kinase signaling pathway. A possible mechanism for protection against atherosclerosis by HDL.

The ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been demonstrated in their protective function against the development of atherosclerosis and associated coronary heart disease. A key event in atherogenesis is endothelial activation induced by a variety of stimuli such as tumor necrosis factor-alpha (TNF), resulting in the expression of various adhesion proteins. We have recently reported that sphingosine 1-phosphate, generated by sphingosine kinase activation, is a key molecule in mediating TNF-induced adhesion protein expression. We now show that HDL profoundly inhibit TNF-stimulated sphingosine kinase activity in endothelial cells resulting in a decrease in sphingosine 1-phosphate production and adhesion protein expression. HDL also reduced TNF-mediated activation of extracellular signal-regulated kinases and NF-kappaB signaling cascades. Furthermore, HDL enhanced the cellular levels of ceramide which in turn inhibits endothelial activation. Thus, the regulation of sphingolipid signaling in endothelial cells by HDL provides a novel insight into the mechanism of protection against atherosclerosis.

Arteriosclerosis↗

PubMed1999

Formation of apolipoprotein-specific high-density lipoprotein particles from lipid-free apolipoproteins A-I and A-II.

We have shown previously that apolipoprotein A (apoA)-I-containing high-density lipoprotein (HDL) particles are formed by the conjugation of lipid-free apoA-I with lipids derived from other lipoprotein fractions in a process dependent on non-esterified fatty acids, generated by the lipolysis of very-low-density lipoprotein (VLDL) or provided exogenously. In the present study, we show that this process is also able to generate HDL particles containing apoA-II (A-II HDL) and both apoA-I and apoA-II (A-I/A-II HDL). When lipid-free apoA-II was incubated with either VLDLs and lipoprotein lipase or LDLs and sodium oleate, a significant proportion of the apoA-II was recovered in the HDL density fraction. This was associated with the formation of several populations of HDL-sized particles with pre-beta2 electrophoretic mobility, which contained phospholipids and unesterified cholesterol as their main lipid constituents. When both lipid-free apoA-I and lipid-free apoA-II were incubated with LDL and sodium oleate, both apolipoproteins were recovered in HDLs that contained phospholipids and unesterified cholesterol as their main lipids. Two populations of particles had diameters of 7.4 and 10.8 nm and pre-beta2-migration; there was also a population of pre-beta1-migrating particles of diameter 4.7 nm. ApoA-I and apoA-II were both present in the larger HDLs, whereas only apoA-I was present in the smaller particles. Immunoaffinity chromatography on an anti-(apoA-I)-Sepharose column revealed that 10-20% of the apoA-II resided in particles that also contained apoA-I. The majority of the A-I/A-II HDL were present in a population of pre-beta2 particles of 10.8 nm diameter. These results in vitro illustrate a potential mechanism for the formation of HDLs containing both apoA-I and apoA-II.

Apolipoprotein A-I↗

PubMed1999

Remodelling of high density lipoproteins by plasma factors.

Evidence that the high density lipoproteins (HDL) in human plasma are antiatherogenic has stimulated considerable interest in the factors which regulate their structure and function. Plasma HDL consist of a number of subpopulations of particles of varying size, density and composition. This structural heterogeneity is caused by the continual remodelling of individual HDL subpopulations by various plasma factors. One of the consequences of this remodelling is that the HDL subpopulations in plasma are functionally diverse, particularly in terms of their antiatherogenic properties. This review documents what is currently known about the interaction of HDL with plasma factors and presents an overview of the remodelling of HDL which occurs as a consequence of those interactions.

Apolipoprotein A-I↗

PubMed1999

Oxysterol efflux from macrophage foam cells: the essential role of acceptor phospholipid.

Oxidized forms of cholesterol (oxysterols) are present in atherosclerotic lesions and may play an active role in lesion development. For example, 7-ketocholesterol (7KC) inhibits cholesterol efflux from macrophage foam cells induced by apolipoprotein A-I (apoA-I). Such oxysterols may promote foam cell formation in atherosclerotic lesions by preventing effective clearance of excess cholesterol. ApoA-I also induces phospholipid (PL) export from foam cells and it has been suggested that cholesterol efflux is dependent upon PL association with the apolipoprotein. In the current study, the effect of oxysterol enrichment of foam cells on phospholipid efflux was measured. Export of cellular PL to apoA-I from 7KC-enriched foam cells was inhibited to the same extent as cholesterol, indicating that the reduced cholesterol export may be a consequence of a decline in the capacity of the foam cells to generate PL/apoA-I particles capable of accepting cellular cholesterol. Incubation of foam cells with pre-formed PL/apoA-I discs increased cholesterol export from 7KC-enriched cells to levels seen in 7KC-free cells. Foam cells produced by uptake of oxidized LDL, which contain similar amounts of 7KC plus other oxidation products, expressed a more profound inhibition of PL export to apoA-I. Cholesterol efflux from these cells improved only partially by provision of PL-containing acceptors. Efflux of 7KC from both foam cell types occurred to PL/apoA-I discs but was only minimal to lipid-free apoA-I, indicating that export of this oxysterol is more dependent than cholesterol upon the presence of extracellular phospholipid.

Animals↗

PubMed1999

Ability of reconstituted high density lipoproteins to inhibit cytokine-induced expression of vascular cell adhesion molecule-1 in human umbilical vein endothelial cells.

Previous studies have shown that both high density lipoproteins (HDL) isolated from human plasma and reconstituted HDL (rHDL) are effective inhibitors of adhesion molecule expression in human endothelial cells. In this study rHDL have been used to investigate whether HDL particle shape, size, apolipoprotein composition or lipid composition are important determinants of the ability of HDL to inhibit the TNF-alpha induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). On the basis of these studies it is possible to draw several firm conclusions. i) Neither phospholipid-containing vesicles nor lipid-free apolipoprotein (apo) A-I inhibit VCAM-1 expression in HUVECs. ii) Simple discoidal complexes containing only phospholipid and apoA-I (discoidal (A-I)rHDL) are sufficient to inhibit the TNF-alpha-induced expression of VCAM-1 in HUVECs. iii) Spherical apoA-I-containing rHDL (spherical (A-I)rHDL) are superior to discoidal (A-I)rHDL as inhibitors. iv) The particle size of spherical (A-I)rHDL has no influence on the inhibition. v) Spherical rHDL that contain apoA-I are superior as inhibitors of VCAM-1 to those containing apoA-II when the rHDL preparations are equated for apolipoprotein molarity. However, when compared at equivalent particle molarities, this difference is no longer apparent. vi) Replacement of cholesteryl esters with triglyceride in the core of spherical (A-I)rHDL has no effect on the ability of these particles to inhibit VCAM-1 expression. From these results it is tempting to speculate that variations in inhibitory activity may contribute to the variations observed in the anti-atherogenicity of different HDL subpopulations.-Baker, P. W., K-A. Rye, J. R. Gamble, M. A. Vadas, and P. J. Barter. Ability of reconstituted high density lipoproteins to inhibit cytokine-induced expression of vascular cell adhesion molecule-1 in human umbilical vein endothelial cells.

Analysis of Variance↗

PubMed1998

Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway.

The signaling pathways that couple tumor necrosis factor-alpha (TNFalpha) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFalpha induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFalpha resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFalpha on endothelial cells leading to extracellular signal-regulated kinases and NF-kappaB activation, whereas ceramide or sphingosine was not. Furthermore, N, N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFalpha-induced extracellular signal-regulated kinases and NF-kappaB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFalpha-induced endothelial cell activation.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

The influence of apolipoproteins on the hepatic lipase-mediated hydrolysis of high density lipoprotein phospholipid and triacylglycerol.

This study describes the influence of apolipoproteins on the hepatic lipase (HL)-mediated hydrolysis of phospholipids and triacylglycerol in high density lipoproteins (HDL). HL-mediated hydrolysis was assessed in well characterized, homogeneous preparations of spherical reconstituted high density lipoproteins (rHDL). The rHDL were comparable in size and lipid composition and contained either apoA-I ((A-I)rHDL) or apoA-II ((A-II)rHDL) as their sole apolipoprotein constituent. Preparations of rHDL containing only cholesteryl esters (CE) in their core, (A-I/CE)rHDL and (A-II/CE)rHDL, were used to assess phospholipid hydrolysis. Preparations of rHDL that contained triacylglycerol as their predominant core lipid, (A-I/TG)rHDL and (A-II/TG)rHDL, were used to assess both triacylglycerol and phospholipid hydrolysis. The rHDL contained trace amounts of either radiolabeled phospholipid or radiolabeled triacylglycerol. Hydrolysis was measured as the release of radiolabeled nonesterified fatty acids (NEFA) from the rHDL. Kinetic analysis showed that HL had a greater affinity for the phospholipids in (A-II/CE)rHDL (Km(app) = 0.2 mM) than in (A-I/CE)rHDL (Km(app) = 3.1 mM). This was also evident when hydrolysis was measured directly by quantitating NEFA mass. HL also had a greater affinity for the phospholipids and triacylglycerol in (A-II/TG)rHDL than in (A-I/TG)rHDL. The Vmax for phospholipid hydrolysis was, by contrast, greater for (A-I/CE)rHDL than for (A-II/CE)rHDL: 309.3 versus 49.1 nmol of NEFA formed/ml of HL/h. Comparable Vmax values were obtained for the hydrolysis of the phospholipids in (A-II/TG)rHDL and (A-I/TG)rHDL. In the case of triacylglycerol hydrolysis, the respective Vmax values for (A-I/TG)rHDL and (A-II/TG)rHDL were 1154.8 and 240.2 nmol of NEFA formed/ml of HL/h. These results show that apolipoproteins have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL.

Apolipoprotein A-I↗

PubMed1998

Oxidation of high density lipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.

Human high density lipoproteins (HDL) can reduce cholesteryl ester hydroperoxides to the corresponding hydroxides (Sattler W., Christison J. K., and Stocker, R. (1995) Free Radical Biol. & Med. 18, 421-429). Here we demonstrate that this reducing activity extended to hydroperoxides of phosphatidylcholine, was similar in HDL2 and HDL3, was independent of arylesterase and lecithin:cholesteryl acyltransferase activity, was unaffected by sulfhydryl reagents, and was expressed by reconstituted particles containing apoAI or apoAII only, as well as isolated human apoAI. Concomitant with the reduction of lipid hydroperoxides specific oxidized forms of apoAI and apoAII formed in blood-derived and reconstituted HDL. Similarly, specific oxidized forms of apoAI accumulated upon treatment of isolated apoAI with authentic cholesteryl linoleate hydroperoxide. These specific oxidized forms of apoAI and apoAII have been shown previously to contain Met sulfoxide (Met(O)) at Met residues and are also formed when HDL is exposed to Cu2+ or soybean lipoxygenase. Lipid hydroperoxide reduction and the associated formation of specific oxidized forms of apoAI and apoAII were inhibited by solubilizing HDL with SDS or by pretreatment of HDL with chloramine T. The inhibitory effect of chloramine T was dose-dependent and accompanied by the conversion of specific Met residues of apoAI and apoAII into Met(O). Canine HDL, which contains apoAI as the predominant apolipoprotein and which lacks the oxidation-sensitive Met residues Met112 and Met148, showed much weaker lipid hydroperoxide reducing activity and lower extents of formation of oxidized forms of apoAI than human HDL. We conclude that the oxidation of specific Met residues of apoAI and apoAII to Met(O) plays a significant role in the 2-electron reduction of hydroperoxides of cholesteryl esters and phosphatidylcholine associated with human HDL.

Acyltransferases↗

PubMed1998

Low density lipoprotein of synovial fluid in inflammatory joint disease is mildly oxidized.

Oxidatively modified low density lipoprotein (LDL) has many biological activities which could contribute to the pathology of the atherosclerotic lesion. Because atherosclerosis has an inflammatory component, there has been much interest in the extent to which LDL could be oxidatively modified in vivo by inflammation. The present study examined LDL present in an accessible inflammatory site, the inflamed synovial joint, for evidence of compositional change and oxidative modification. LDL was isolated from knee joint synovial fluid (SF) from subjects with inflammatory arthropathies and also from matched plasma samples. SF and plasma LDL had similar free cholesterol and alpha-tocopherol content, but SF LDL had a lower content of esterified cholesterol. On electrophoresis, SF LDL was slightly more electronegative than LDL from matched plasma samples, but the changes were much less than those resulting from Cu2+-treatment of LDL. Oxidized cholesterol was not detected in any samples, but cholesterol ester hydroperoxide levels were greater in SF than in plasma LDL. When samples from three subjects were incubated with macrophages, the SF LDL did not cause significant loading of the cells with cholesterol or cholesterol esters, in contrast to the situation with acetylated LDL. Overall, the SF LDL displayed evidence of slightly increased oxidation by comparison with matched plasma samples. Despite their isolation from an environment with active inflammation, changes were modest compared with those resulting from Cu2+ treatment. Thus, extensive LDL oxidation is not a necessary correlate of location in a chronic inflammatory site, even though it is characteristic of atherosclerotic lesions.

Animals↗