Association of lysolecithin acyltransferase with the high density lipoproteins and its activation by the low density lipoproteins in normal human plasma.

The lysolecithin acyltransferase of human plasma is shown to be associated with the high-density lipoprotein fraction. Although the low density lipoproteins do not have intrinsic enzyme activity, their presence activated the enzyme 3--7-fold. This activation is not affected by heat-treatment of the low density lipoproteins, but is abolished by the addition of heparin.     

High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture.

Endothelial cells, explanted from human umbilical veins and cultured, maintained morphological characteristics of vascular endothelium. When exposed to human serum lipoproteins, the cells bound and took up low density lipoproteins in preference to high density lipoproteins. High density lipoproteins reduced markedly the uptake of low density lipoproteins and affected surface binding to a lesser extent. These data suggest that the different levels of high density lipoprotein encountered in normal plasma of males and females could modulate differently the transendothelial transport of low density lipoproteins and provide a possible explanation for the lesser severity of atheromatosis in the aortic intima of premenopausal females.     

Binding of low density lipoproteins (LDL) to platelets

Binding of LDL to platelets was studied by two independent methods, radioactive and flow cytofluorimetry, using 125I- and fluorescently labelled RITC-LDL. Saturation of 125I- and RITC-LDL binding to platelets, inhibition of binding by unlabelled LDL and a lower inhibitory effect of unlabelled HDL evidence the existence of a limited number of binding sites specific for LDL in platelets. Unlike nuclear cells platelets do not degrade LDL. The binding of LDL to platelets is reversible and independent of Ca2+. The decrease of total binding level at 4 degrees and the absence of heparin effect on the release of bound LDL suggest LDL incorporation into platelets.     

Promotion of human T lymphocyte activation and proliferation by fatty acids in low density and high density lipoproteins

Mitogen-induced lymphocyte DNA synthesis measured by [3H]thymidine incorporation and lymphocyte proliferation assessed by counting the number of cells were reduced by greater than 95% when cells were cultured at low density in the absence of serum. Supplementation with either transferrin or lipoprotein alone only partially restored lymphocyte responses. Addition of both transferrin and lipoproteins of each major subclass permitted mitogen-induced lymphocyte DNA synthesis and proliferation equal to that observed in serum-containing medium. The degree of enhancement was dependent on the concentration of the lipoprotein added and could not be explained by the nonspecific addition of protein to the defined medium. The mechanisms of growth promotion by various lipoprotein fractions did not appear to be explained by provision of cholesterol to the cells. Neither cholesterol nor cholesteryl ester from endogenous sources or supplied exogenously was able to enhance mitogen-induced lymphocyte responses. In contrast, fatty acids, phospholipid, and triglyceride alone supported lymphocyte responses. Furthermore, lipoproteins retained the capacity to enhance lymphocyte responses following extraction of neutral lipid. Both low density lipoprotein and high density lipoprotein, subclass 3, increased the number of cells initially activated by mitogenic stimulation and supported the subsequent continued growth of the activated cells. Low density lipoprotein was more efficient than high density lipoprotein, subclass 3, in this latter regard. These results indicate that lipoproteins can promote maximal growth of mitogen-activated lymphocytes in transferrin-containing medium by providing growth factors other than cholesterol necessary for initial activation and required for continued lymphocyte proliferation.     

Recycling of vitamin E in human low density lipoproteins.

Oxidative modification of low density lipoproteins (LDL) and their unrestricted scavenger receptor-dependent uptake is believed to account for cholesterol deposition in macrophage-derived foam cells. It has been suggested that vitamin E that is transported by LDL plays a critical role in protecting against LDL oxidation. We hypothesize that the maintenance of sufficiently high vitamin E concentrations in LDL can be achieved by reducing its chromanoxyl radicals, i.e., by vitamin E recycling. In this study we demonstrate that: i) chromanoxyl radicals of endogenous vitamin E and of exogenously added alpha-tocotrienol, alpha-tocopherol or its synthetic homologue with a 6-carbon side-chain, chromanol-alpha-C6, can be directly generated in human LDL by ultraviolet (UV) light, or by interaction with peroxyl radicals produced either by an enzymic oxidation system (lipoxygenase + linolenic acid) or by an azo-initiator, 2,2'-azo-bis(2,4-dimethylvaleronitrile) (AMVN; ii) ascorbate can recycle endogenous vitamin E and exogenously added chromanols by direct reduction of chromanoxyl radicals in LDL; iii) dihydrolipoic acid is not efficient in direct reduction of chromanoxyl radicals but recycles vitamin E by synergistically interacting with ascorbate (reduces dehydroascorbate thus maintaining the steady-state concentration of ascorbate); and iv) beta-carotene is not active in vitamin E recycling but may itself be protected against oxidative destruction by the reductants of chromanoxyl radicals. We suggest that the recycling of vitamin E and other phenolic antioxidants by plasma reductants may be an important mechanism for the enhanced antioxidant protection of LDL.     

Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation.

Very low density lipoproteins (VLDL) have been isolated and subfractionated on the basis of their differing flotation rates. The procedure consists of a single 45-min zonal ultracentrifugation step using a linear density gradient of d = 1.00 to 1.15 g/ml. Appropriate fractions of the zonal rotor effluent containing the entire VLDL spectrum were characterized by analytical ultracentrifugation, gel filtration chromatography, and complete chemical analysis. Flotation rates of VLDL subspecies from hypertriglyceridemic and normolipemic plasmas correlated directly with their Stokes radii and triglyceride content and inversely with their proportion of cholesterol, cholesteryl esters, phospholipids, and total protein. There was also an inverse correlation of flotation rate with the fraction of tetramethylurea-insoluble protein. This procedure provides a reliable methodology for a rapid isolation of VLDL subfractions and the accurate determination of their flotation rates.     

Heterogeneity of human very low density lipoproteins by gel filtration chromatography

Very low density lipoproteins were separated by gel filtration on Sepharose 4B. A decrease in mean particle diameter and flotation rate was seen with increasing elution volumes. The smaller lipoproteins had relatively more protein and phospholipid and less triglyceride than the larger ones. No differences were noted in the relative contents of the various phospholipids or partial glycerides between small and large lipoproteins. Fatty acid patterns of triglycerides and cholesteryl esters were also similar for the various lipoproteins. Relatively more lecithin containing linoleoyl acyl groups was found in smaller lipoproteins of some subjects. More of the protein of smaller lipoproteins was apo-LDL protein. Apo-HDL peptide was lost from the very low density lipoprotein as a consequence of the gel filtration.     

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.     

Secondary structure in very low density and intermediate density lipoproteins of human serum.

We have studies the secondary structures of the protein moieties of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) of human serum by circular dichroism (CD). Two potential complications in the application of this technique to lipoproteins have been evaluated. First, using chronographic potentiometry in CD measurements of VLDL fractions of different mean particle diameters, we have analyzed statistically the CD signals in order to define the limits imposed by light scattering with respect to both particle diameter and wavelength. We found that CD measurements can be made to as low as 210 nm on particles of 520 A or smaller, and to 194 nm on particles of 450 A and below. Second, we have evaluated the CD contribution of lipid chromophores. Despite the high ratio of lipid to protein, the relative CD effect of the lipids is smaller than for low density lipoproteins (LDL). due to the extremely small ellipticity of natural VLDL triglycerides. Thus, CD measurements can be obtained with confidence on the preponderant bulk of normal VLDL. For the first time we report the CD spectra of human VLDL and IDL. In contrast with human LDL and the lipoproteins of the hypercholesterolemic rabbit, the entire CD SPECTRUM OF HUMAN VLDL shows increased ellipticity with decreasing temperature, which is completely reversible. We have found that the protein moieties of human VLDL and IDL contain substantially more helix (approximately 50%) than does that of human LDL.     

Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets.

Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

Properties of human serum low density lipoproteins after modification by succinic anhydride

Human serum low density lipoprotein of d 1.019-1.063 (LDL(2)) treated with succinic anhydride at pH 7.5-8.0 showed the same chemical composition, hydrodynamic properties (flotation and sedimentation coefficients, intrinsic viscosity) and optical properties (circular dichroism) as untreated LDL(2). However, in contrast to LDL(2), the succinylated product (s-LDL(2)) failed to react with rabbit anti-LDL(2) antisera. Extraction with ethanol-ether 3:1 yielded the succinylated apoprotein (s-apo-LDL(2)), which was, unlike untreated apoprotein, soluble in aqueous buffers. Succinylated apoprotein, which was also immunologically unreactive, appeared to differ in structure from s-LDL(2), as assessed by the parameters of intrinsic viscosity and circular dichroism. The molecular weights of both LDL(2) and s-LDL(2) obtained by the technique of sedimentation equilibrium were 2.1-2.3 x 10(6). By the same method, s-apo-LDL(2) gave an uncorrected figure of 3.95-4.15 x 10(4) and, after correction for succinyl functions, of 3.60-3.80 x 10(4). Because of the assumptions made in the computations, the latter figure was considered approximate. The marked differences in molecular weight between s-apo-LDL(2) and whole apo-LDL(2) ( approximately 5 x 10(5)) were taken to support the subunit structure of apo-LDL(2), which is envisaged as an aggregate of about 12 subunits which dissociate upon succinylation. Further, the large percentage (about 90%) of the free amino groups of LDL(2) found to react with succinic anhydride suggests that these groups are at the surface of the molecule.     

Structure and interactions of lipids in human plasma low density lipoproteins.

Temperature-dependent techniques (differential scanning calorimetry, polarizing microscopy, and x-ray scattering and diffraction techniques) were used to compare the properties of human plasma low density lipoproteins (LDL) with its extracted lipid classes. Three types of thermal transitions were characterized: (a) a reversible transition in intact LDL near body temperature associated with a liquid crystalline order-disorder phase change of cholesterol esters within the particles; (b) an irreversible high temperature transition (approximately 70-90 degrees) associated with LDL denaturation and release of cholesterol esters from the disrupted particles; and (c) low temperature transitions related to liquid crystalline and crystalline phase changes in these released esters. The temperature of the reversible transition in intact LDL varies among individual donors. Correlation analysis shows that the temperature of this transition negatively correlates with the amount of triglyceride relative to cholesterol ester in LDL. Studies on mixtures of cholesterol esters and triglycerides isolated from LDL show a similar effect, increasing amounts of triglycerides decreasing the temperature of the liquid leads to smectic liquid crystalline transition of the isolated esters. Thus, the amount of triglyceride in LDL influences the fluidity of the cholesterol esters in LDL. The enthalpy of the reversible transition in intact LDL is 0.69 cal/g of LDL cholesterol ester. This compares with 0.89 cal/g for the liquid leads to liquid crystalline transition of the cholesterol esters released from denatured LDL and 1.01 cal/g for the same transition in the extracted esters. Unlike the cholesterol esters released from denatured LDL, or isolated LDL esters, cholesterol ester in the intact LDL particle does not crystallize. These findings suggest that the behavior of cholesterol esters in intact LDL is constrained relative to their behavior when freed from the restrictions of the particle. These results together with experiments on partitioning of the individual lipid classes of LDL allow us to define the distribution and interaction of lipids in the intact LDL particle.     

The catabolism of human and rat very low density lipoproteins by perfused rat hearts.

The catabolism of human and rat 125I-labelled very low density lipoproteins (VLDL) was compared by perfusing the lipoproteins through beating rat hearts. Triacylglycerol was removed from the VLDL to a greater extent than the protein moiety, leaving remnants containing relatively more apo-B and less apo-C. The change in apo-C content of the remnants correlated with the loss of triacylglycerol. The extent of removal of triacylglycerol from the rat and human VLDL was similar and in most cases appeared to saturate the heart lipoprotein lipase. The remnants were slightly smaller in size than the VLDL, and included particles which appeared to be partially emptied. In addition to remnants of d less than 1.019 g/ml, iodinated lipoproteins derived from rat and human VLDL were recovered at d 1.019-1.063 and 1.063-1.21 g/ml. The former contained largely cholesterol and cholesteryl esters, while phospholipids were the dominant lipid in the latter. An average of 40% of the 125I-labelled apoprotein lost from the VLDL was associated with the perfused hearts. Very little d 1.019-1.063 g/ml lipoprotein was produced from low (physiological) concentrations of rat VLDL, most of the lipoprotein being removed by the heart. However, lipoproteins of density 1.019-1.063 g/ml were formed from human VLDL at all concentrations in the perfusate, as well as from higher concentrations of the rat VLDL. Agarose gel filtration of lipoproteins following heart perfusion with human VLDL revealed large aggregates containing particles which resemble low density lipoproteins (LDL) in electron microscopic appearance and apoprotein composition, since they contain largely apo-B. These data suggest that at normal concentrations rat VLDL are almost completely catabolised and taken up by the heart without the formation of LDL, while LDL is produced from human VLDL at all concentrations.     

Interactions of native and modified human low density lipoproteins with human skin fibroblasts.

125I-labeled low density lipoprotein (LDL) covalently bonded to Sepharose beads was not degraded by normal human fibroblasts nor did it trigger inhibition of sterol synthesis. The Sepharose beads loaded with LDL bound very tightly to the surface both of normal fibroblasts and fibroblasts from a subject with homozygous familial hypercholesterolemia; control Sepharose beads (activated sites covered with glycine) did not adhere to either cell type. LDL was extracted by a modification of the method of Gustafson (Gustafson, A. (1965) J. Lipid Res. 6, 512-517), so as to remove essentially all cholesterol, cholesterol ester and triglyceride. This modified LDL was bound, internalized and degraded as well as or better than native LDL. However, it failed to suppress sterol synthesis. These results provide additional evidence that the sterol moiety of the LDL is the key component affecting sterol synthesis. They also imply that the neutral lipids of LDL play a minor role in the binding of LDL to cell membranes and that the apoprotein rather than molecular size and shape is the critical factor.