Diane W. Morel

Monocytes and Neutrophils Oxidize Low Density Lipoprotein Making It Cytotoxic

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte‐derived free radicals. This is the first demonstration that short‐lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.

Carotenoid uptake and secretion by CaCo-2 cells β-carotene isomer selectivity and carotenoid interactions

In presence of oleate and taurocholate, differentiated CaCo-2 cell monolayers on membranes were able to assemble and secrete chylomicrons. Under these conditions, both cellular uptake and secretion into chylomicrons of β-carotene (β-C) were curvilinear, time-dependent (2–16 h), saturable, and concentration-dependent (apparent Km of 7–10 μM) processes. Under linear concentration conditions at 16 h incubation, the extent of absorption of all-trans β-C was 11% (80% in chylomicrons), while those of 9-cis- and 13-cis-β-C were significantly lower (2–3%). The preferential uptake of the all-trans isomer was also shown in hepatic stellate HSC-T6 cells and in a cell-free system from rat liver (microsomes), but not in endothelial EAHY cells or U937 monocyte-macrophages. Moreover, extents of absorption of α-carotene (α-C), lutein (LUT), and lycopene (LYC) in CaCo-2 cells were 10%, 7%, and 2.5%, respectively. Marked carotenoid interactions were observed between LYC/β-C and β-C/α-C. The present results indicate that β-C conformation plays a major role in its intestinal absorption and that cis isomer discrimination is at the levels of cellular uptake and incorporation into chylomicrons. Moreover, the kinetics of cellular uptake and secretion of β-C, the inhibition of the intestinal absorption of one carotenoid by another, and the cellular specificity of isomer discrimination all suggest that carotenoid uptake by intestinal cells is a facilitated process.

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

Distribution of oxysterols in human serum: Characterization of 25-hydroxycholesterol association with serum albumin

Abstract Oxysterols, derived either from cholesterol autoxidation in vitro or through lipoprotein peroxidation in vivo are biologically active compounds implicated in the pathogenesis of atherosclerosis. Using 25-hydroxycholesterol (25OHC) as a model and radiolabeled 25OHC to trace mass, in this study we examined the transport of oxysterols in human serum. In contrast to cholesterol, which is associated exclusively with serum lipoproteins, 55 to 75% of 25OHC added to serum in vitro was associated with the lipoprotein-deficient fraction of serum (LPDS, density > 1.21 g/mL) over a wide concentration range. Upon sequential gel filtration, ion-exchange chromatography, and nondenaturing polyacrylamide gel electrophoresis, 25OHC added to LPDS eluted in a single peak corresponding to a protein peak of molecular weight about 67 kD. Immunoprecipitation of serum albumin from LPDS also precipitated 25OHC. 25OHC added to albumin-depleted LPDS did not associate with any of the remaining serum proteins, suggesting that albumin is the sole protein with which 25OHC associates to any significant extent in LPDS. Bovine serum albumin (BSA) was used to characterize the association of 25OHC with albumin. Other oxysterols, including 19-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, effectively competed with 25OHC for association with BSA, suggesting that they also associate with albumin in serum. The association of 25OHC with albumin in serum is selective but of low affinity as calculated from Scatchard analysis. However, given the high concentration of albumin in serum a significant amount of serum oxysterols may be delivered to or removed from various tissues via albumin.