Phospholipid liposomes are synthetic mediators of "reverse" cholesterol transport from peripheral tissue to liver in vivo and can shrink atherosclerotic lesions in animals. Hepatic disposal of this cholesterol, however, has not been examined. We compared hepatic effects of large (approximately equal to 120-nm) and small (approximately equal to 35-nm) unilamellar vesicles (LUVs and SUVs), both of which mediate reverse cholesterol transport in vivo but were previously shown to be targeted to different cell types within the liver. On days 1, 3, and 5, rabbits were intravenously injected with 300 mg phosphatidylcholine (LUVs or SUVs) per kilogram body weight or with the equivalent volume of saline. After each injection, LUV- and SUV-injected animals showed large increases in plasma concentrations of unesterified cholesterol, indicating mobilization of tissue stores. After hepatic uptake of this cholesterol, however, SUV-treated animals developed persistently elevated plasma LDL concentrations, which by day 6 had increased to more than four times the values in saline-treated controls. In contrast, LUV-treated animals showed normal LDL levels. By RNase protection assay, SUVs suppressed hepatic LDL receptor mRNA at day 6 (to 61 +/- 4% of control, mean +/- SEM), whereas LUVs caused a statistically insignificant stimulation. Hepatic HMG-CoA reductase message was also significantly suppressed with SUV, but not LUV treatment, and hepatic 7 alpha-hydroxylase message showed a similar trend. These data on hepatic mRNA levels indicate that SUVs, but not LUVs, substantially perturbed liver cholesterol homeostasis. We conclude that LUVs and SUVs mobilize peripheral tissue cholesterol and deliver it to the liver, but to distinct metabolic pools that exert different regulatory effects. The effects of one of these artificial particles, SUVs, suggest that reverse cholesterol transport may not always be benign. In contrast, LUVs may be a suitable therapeutic agent, because they mobilize peripheral cholesterol to the liver without suppressing hepatic LDL receptor mRNA and without provoking a subsequent rise in plasma LDL levels.

Abstract-Endothelial dysfunction is a major pathophysiological consequence of hypercholesterolemia and other conditions. We examined whether a synthetic mediator of lipid transport from peripheral tissues to the liver (ie, the "reverse" pathway) could restore normal endothelial function in vivo. Using assays of macrovascular and microvascular function, we found that genetically hypercholesterolemic apolipoprotein E knockout mice exhibited key endothelial impairments. Treatment of the mice for 1 week with daily intravenous bolus injections of large "empty" phospholipid vesicles, which accelerate the reverse pathway in vivo, restored endothelium-dependent relaxation, leukocyte adherence, and endothelial expression of vascular cell adhesion molecule-1 to normal or nearly normal levels. These changes occurred despite the long-standing hyperlipidemia of the animals and the persistence of high serum concentrations of cholesterol-rich atherogenic lipoproteins during the treatment. Our results indicate that dysfunctional macrovascular and microvascular endothelium in apolipoprotein E knockout mice can recover relatively quickly in vivo and that accelerated reverse lipid transport may be a useful therapy. Key Words: cell adhesion molecules Ⅲ endothelium-derived factors Ⅲ hypercholesterolemia Ⅲ leukocytes Ⅲ vasodilation I n hypercholesterolemic animals and humans, the endothelium of medium-sized muscular arteries, such as the coronary arteries, loses its ability to release biologically available nitric oxide, a vasodilator required for endotheliumdependent relaxation of vessels in response to ADP, acetylcholine, hypoxia, and other stimuli. [1][2][3][4][5][6] Furthermore, in hypercholesterolemia, the luminal surface of vascular endothelium displays abnormally high levels of P-selectin and vascular cell adhesion molecule-1 (VCAM-1). On the venous but not the arterial side, 6,7 these endothelial cell adhesion molecules mediate leukocyte rolling (tethering) and adherence (immobilization), respectively, by binding to counterreceptors on the leukocytes. 8 -10 These changes in the macrovasculature and microvasculature occur early, within 1 to 2 weeks after the onset of atherogenic hypercholesterolemia, 3,6,8 -10 well before overt atherosclerosis has developed. In humans, lowering the plasma concentration of LDL and other cholesterol-rich atherogenic lipoproteins that deliver harmful lipids into the vessel wall 11,12 has been reported to improve endothelium-dependent vasomotor regulation after several months, 4,5,13,14 although full normalization of endothelium-dependent relaxation is uncommon 4,5,13 and may require the simultaneous administration of antioxidants 15,16 or extreme measures to lower LDL levels. 17,18 Similarly, elevated plasma concentrations of soluble cell adhesion molecules seen in human hyperlipidemias respond only minimally to aggressive drug treatments to lower plasma lipid levels. 19 The therapeutic approach of LDL lowering, with or without the coadministration of antioxidants, presumably attenuates further...

Chondroitin 6-sulfotransferase (C6ST) is the key enzyme in the biosynthesis of chondroitin 6-sulfate, a glycosaminoglycan implicated in chondrogenesis, neoplasia, atherosclerosis, and other processes. C6ST catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to carbon 6 of the N-acetylgalactosamine residues of chondroitin. Based on the previously published avian sequence, we searched the database of expressed sequence tags (dbEST) and obtained partial-length cDNAs that we completed by 5'-RACE using human chondrosarcoma and endothelial-cell RNA as template. Stable transfection of our full-length expression construct into CHO-K1 cells resulted in marked increases in C6ST and keratan sulfate sulfotransferase (KSST) enzymatic activities in cell homogenates. The predicted 411 amino acid sequence of human C6ST contains an N-terminal hydrophobic domain consistent with membrane insertion, four potential sites for N-linked glycosylation, several consensus sequences for protein phosphorylation, and one RGD sequence. The human and chick C6ST cDNA share 51% nucleotide identity, 40% amino acyl identity, and 75% amino acyl conservation. The human C6ST gene structure has been elucidated and exhibits an intron-less coding region, and the gene has been mapped to human chromosome 11 by radiation hybrid panel mapping.