Moira A. Clay

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.

Factors Influencing the Ability of HDL to Inhibit Expression of Vascular Cell Adhesion Molecule-1 in Endothelial Cells

We have previously reported that high density lipoproteins (HDLs) inhibit the cytokine-induced expression of adhesion molecules in endothelial cells. Here we investigate whether different preparations of HDLs vary in their ability to inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) activated by tumor necrosis factor-alpha (TNF-alpha). HDLs collected from a number of different human subjects all inhibited VCAM-1 expression in a concentration-dependent manner, although the extent of inhibition varied widely between subjects. The inhibitory activities of the HDL2 and HDL3 subfractions isolated from individual subjects also differed. Whether equated for concentrations of apolipoprotein (apo) A-I or cholesterol, the inhibitory activity of HDL3 was superior to that of HDL2. This difference remained apparent even when the HDL subfractions were present only during preincubations with the HUVECs and were removed before activation by TNF-alpha. To determine whether the inhibitory effect of HDL3 was influenced by apolipoprotein composition, preparations of HDL3 were modified by replacing all of their apo A-I with apo A-II. This change in apolipoprotein composition had no effect on the ability of the HDL3 to inhibit endothelial VCAM-1 expression. Thus, it has been shown that different preparations of HDLs differ markedly in their abilities to inhibit VCAM-1 expression in cytokine-activated HUVECs. The mechanism underlying the differences remains to be determined.

Cholesteryl ester transfer protein and hepatic lipase activity promote shedding of apo A-I from HDL and subsequent formation of discoidal HDL.

The effects of lipid transfers and hepatic lipase (HL) on the concentration, composition, particle size distribution and morphology of high density lipoproteins (HDL) have been investigated. Human plasma supplemented with additional very low density lipoproteins (VLDL), cholesteryl ester transfer protein (CETP) and HL has been incubated at 37 degrees C for up to 8 h. The HDL became depleted of cholesteryl esters and reduced in particle size. Within 2 h of such incubation they had also lost about 30% of their apo A-I. However, with extension of the incubations beyond 2 h, the apo A-I returned progressively to the HDL fraction until, after 8 h, the concentration of apo A-I in HDL was identical to that in non-incubated samples. This return of apo A-I to the HDL density range was accompanied by a progressive appearance in electron micrographs of discoidal HDL particles. Thus, the depletion of the core lipid content and the reduction in particle size of HDL promoted by lipid transfers and HL activity in vitro is accompanied by a shedding of apo A-I which forms the nucleus of new discoidal HDL particles. The potential physiological importance of such a process is considerable.

The rabbit as an animal model of hepatic lipase deficiency

A natural deficiency of hepatic lipase in rabbits has been exploited to gain insights into the physiological role of this enzyme in the metabolism of plasma lipoproteins. A comparison of human and rabbit lipoproteins revealed obvious species differences in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL), with the rabbit lipoproteins being relatively enlarged, enriched in triacylglycerol and depleted of cholesteryl ester. To test whether these differences related to the low level of hepatic lipase in rabbits, whole plasma or the total lipoprotein fraction from rabbits was either kept at 4 degrees C or incubated at 37 degrees C for 7 h in (i) the absence of lipase, (ii) the presence of hepatic lipase and (iii) the presence of lipoprotein lipase. Following incubation, the lipoproteins were recovered and subjected to gel permeation chromatography to determine the distribution of lipoprotein components across the entire lipoprotein spectrum. An aliquot of the lipoproteins was subjected also to gradient gel electrophoresis to determine the particle size distribution of the LDL and HDL. Both hepatic lipase and lipoprotein lipase hydrolysed lipoprotein triacylglycerol and to a much lesser extent, also phospholipid. There were, however, obvious differences between the enzymes in terms of substrate specificity. In incubations containing hepatic lipase, there was a preferential hydrolysis of HDL triacylglycerol and a lesser hydrolysis of VLDL triacylglycerol. By contrast, lipoprotein lipase acted primarily on VLDL triacylglycerol. When more enzyme was added, both lipases also acted on LDL triacylglycerol, but in no experiment did lipoprotein lipase hydrolyse the triacylglycerol in HDL. Coincident with the hepatic lipase-induced hydrolysis of LDL and HDL triacylglycerol, there were marked reductions in the particle size of both lipoprotein fractions, which were now comparable to those of human LDL and HDL3, respectively.

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.

Evidence in vitro that hepatic lipase reduces the concentration of apolipoprotein A-I in rabbit high-density lipoproteins

Incubation of rabbit plasma in vitro with hepatic lipase resulted in the hydrolysis of triacylglycerol in high-density lipoproteins (HDL) and a reduction in HDL particle size. These changes were accompanied by a decrease in the concentration of apolipoprotein A-I (apo A-I) in the HDL. The loss of apo A-I was demonstrated independently by ultracentrifugation, size exclusion chromatography and gradient gel-immunoblot analysis. It was unrelated to hydrolysis of HDL phospholipids but did correlate with the reduction in HDL particle size. These studies suggest that the concentration of apo A-I in HDL may be influenced by factors which regulate the metabolism of HDL core lipid constituents.

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.