Spherical Reconstituted High Density Lipoproteins Research Articles

Influence of Fatty Acid Modification on Uptake of Lovastatin-Loaded Reconstituted High Density Lipoprotein by Foam Cells.

Spherical reconstituted high density lipoprotein (rHDL) can target atherosclerotic lesions by the very low density lipoprotein (VLDL) receptor, which is seldom expressed in liver. By promoting this pathway, the targeting efficiency was hyphothesized to be improved due to avoiding undesired uptake in liver mediated by the scavenger receptor class B type I (SR-BI). In this study, how fatty acid modification in spherical rHDL influenced the VLDL receptor-mediated endocytosis pathway was investigated. Stearic acid (SA) and arachidonic acid (AA) with different saturation levels were utilized to modify the lovastatin-loaded rHDL (LS-rHDL). Phagocytosis test on foam cells with or without cholesteryl ester transfer protein (CETP) expression was conducted to observe the cellular uptake of the SA or AA modified rHDL and the non-modified one. Raman spectroscopy, guanidine hydrochloride (Gdn-HCl) denaturation experiment and in vitro evaluation of drug release were used to analyze the related mechanism. In comparison with the non-modified rHDL, AA modification could reduce the packing order of the rHDL phospholipid acyl chains, leading to the decreased apoA-I binding extent with lipid and the increased drug release, while the opposite was true for SA modification. The AA-modified rHDL exhibited a higher uptake of foam cells expressing CETP than the non-modified one, while the SA-modified one showed the lowest cellular uptake among the three rHDLs. Increased unsaturation level can facilitate lipid-interchange process where the cargo in rHDL core may transfer to VLDL more easily, and then promote the endocytosis mediated by the VLDL receptor.

Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.

N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins

Endothelial lipase (EL) is a member of the triglyceride lipase gene family with high phospholipase and low triacylglycerol lipase activities and a distinct preference for hydrolyzing phospholipids in HDL. EL has five potential N-glycosylation sites, four of which are glycosylated. The aim of this study was to determine how glycosylation affects the phospholipase activity of EL in physiologically relevant substrates. Site-directed mutants of EL were generated by replacing asparagine (N) 62, 118, 375, and 473 with alanine (A). These glycan-deficient mutants were used to investigate the kinetics of phospholipid hydrolysis in fully characterized preparations of spherical reconstituted high density lipoprotein (rHDL) containing apolipoprotein E2 (apoE2) [(E2)rHDL], apoE3 [(E3)rHDL], apoE4 [(E4)rHDL], or apoA-I [(A-I)rHDL] as the sole apolipoprotein. Wild-type EL hydrolyzed the phospholipids in (A-I)rHDL, (E2)rHDL, (E3)rHDL, and (E4)rHDL to similar extents. The phospholipase activities of EL N118A, EL N375A, and EL N473A were significantly diminished relative to that of wild-type EL, with the greatest reduction being apparent for (E3)rHDL. The phospholipase activity of EL N62A was increased up to 6-fold relative to that of wild-type EL, with the greatest enhancement of activity being observed for (E2)rHDL. These data show that individual N-linked glycans have unique and important effects on the phospholipase activity and substrate specificity of EL.

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.

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.

4.P.375 Effect of spherical reconstituted high density lipoprotein apolipoproteins on expression of vascular cell adhesion molecule-1

4.P.375 Effect of spherical reconstituted high density lipoprotein apolipoproteins on expression of vascular cell adhesion molecule-1

The Influence of Sphingomyelin on the Structure and Function of Reconstituted High Density Lipoproteins

The effect of sphingomyelin (SPM) on the structure and function of discoidal and spherical reconstituted high density lipoproteins (rHDL) has been studied. Three preparations of discoidal rHDL with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SPM/unesterified cholesterol (UC)/apolipoprotein (apo)A-I molar ratios of 99.6/0. 0/10.2/1.0, 86.0/13.6/10.8/1.0, and 72.5/26.3/11.4/1.0 were prepared by cholate dialysis. SPM did not affect discoidal rHDL size or surface charge. Esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT) was inhibited in the SPM-containing discoidal rHDL. When the discoidal rHDL of POPC/SPM/UC/apoA-I molar ratio 99.6/0.0/10.2/1.0 were incubated with low density lipoproteins (LDL) and LCAT, SPM transferred spontaneously from the LDL to the rHDL (t1/2 = 0.8 h) and spherical particles with a POPC/SPM/UC/CE/apoA-I molar ratio of 24.6/4.9/3. 6/24.9/1.0 were formed. Depleting the spherical rHDL of SPM head groups by incubation with sphingomyelinase increased the negative charge on the surface, but did not change their size. Cholesteryl ester transfer protein (CETP)-mediated transfers of cholesteryl esters and triglyceride between spherical rHDL and Intralipid were not affected by SPM head group depletion. The effect of SPM on rHDL structure was assessed spectroscopically. SPM increased POPC acyl chain and head group packing in the discoidal rHDL. When the spherical rHDL were depleted of SPM head groups, POPC acyl chain packing order decreased, but head group packing order was not affected. SPM inhibited the lipid-water interfacial hydration of discoidal rHDL. This parameter was not affected when the spherical rHDL were depleted of SPM head groups. The SPM molecule and the SPM head group, respectively, inhibited the unfolding of apoA-I in discoidal and spherical rHDL. It is concluded that (i) SPM influences the structure of discoidal and spherical rHDL, (ii) SPM inhibits the LCAT reaction in discoidal rHDL, and (iii) the SPM head group does not affect CETP-mediated lipid transfers into or out of spherical rHDL.

The use of spherical reconstituted high density lipoprotein for the measurement of cholesteryl ester transfer protein activity in human plasma.

The use of spherical reconstituted high density lipoprotein for the measurement of cholesteryl ester transfer protein activity in human plasma.

Apolipoprotein A-I structure and lipid properties in homogeneous, reconstituted spherical and discoidal high density lipoproteins.

We prepared a spherical reconstituted high density lipoprotein (rHDL) particle in pure form and compared it with its homogeneous discoidal rHDL precursors, in terms of the structure and stability of the apolipoprotein A-I (apoA-I) component, the dynamics of the surface lipids, and the relative reactivity with lecithin-cholesterol acyltransferase. The apoA-I-structure was examined in the rHDL particles by circular dichroism and fluorescence spectroscopic methods, and the binding of monoclonal antibodies specific for apoA-I epitopes. The stability of apoA-I on the rHDL particles was assessed by the effects of guanidine hydrochloride on the wavelength of maximum intrinsic fluorescence of the apolipoprotein. Lipid dynamics in the acyl chain region and the polarity of the lipid-water interface were investigated by means of fluorescence probes. The conformation of apoA-I in the spherical 93-A rHDL particles was found to be very similar to that in the 96-A rHDL discs but distinct from the apoA-I structure in the 78-A rHDL discs. The stability of apoA-I to denaturation by guanidine hydrochloride was highest in the 93-A rHDL spheres. The experiments on the lipids indicate somewhat more ordered and motionally restricted acyl chains in the spheres, relative to the discs, but a similar surface polarity. These results suggest that the folding and organization of apoA-I on the three particles include protein domains consisting of interacting alpha-helical segments in the carboxyl-terminal region and a globular domain in the amino-terminal region of each apoA-I molecule. The reactivity with lecithin-cholesterol acyltransferase was highest for the 96-A rHDL disc, and 16- and 34-fold lower for the 78-A rHDL disc and the 93-A rHDL sphere, respectively, possibly as a result of differences in apoA-I structure and product inhibition in these particles.