betadex and Arteriosclerosis

Oxidized phospholipids, including oxidation products of palmitoyl-arachidonyl-phosphatidyl choline (PAPC), are mediators of inflammation in endothelial cells (ECs) and known to induce several chemokines, including interleukin-8 (IL-8). In this study, we show that oxidized PAPC (OxPAPC), which accumulates in atherosclerotic lesions, paradoxically depletes endothelial cholesterol, causing caveolin-1 internalization from the plasma membrane to the endoplasmic reticulum and Golgi, and activates sterol regulatory element-binding protein (SREBP). Cholesterol loading reversed these effects. SREBP activation resulted in increased transcription of the low-density lipoprotein receptor, a target gene of SREBP. We also provide evidence that cholesterol depletion and SREBP activation are signals for OxPAPC induction of IL-8. Cholesterol depletion by methyl-beta-cyclodextrin induced IL-8 synthesis in a dose-dependent manner. Furthermore, cholesterol loading of ECs by either the cholesterol-cyclodextrin complex or caveolin-1 overexpression inhibited OxPAPC induction of IL-8. These observations suggest that changes in cholesterol level can modulate IL-8 synthesis in ECs. The OxPAPC induction of IL-8 was mediated through the increased binding of SREBP to the IL-8 promoter region, as revealed by mobility shift assays. Overexpression of either dominant-negative SREBP cleavage-activating protein or 25-hydroxycholesterol significantly suppressed the effect of OxPAPC on IL-8 transcription. A role for SREBP activation in atherosclerosis is suggested by the observation that EC nuclei showed strong SREBP staining in human atherosclerotic lesions. The current studies suggest a novel role for endothelial cholesterol depletion and subsequent SREBP activation in inflammatory processes in which phospholipid oxidation products accumulate.

Topics: Animals; Aorta; Arteriosclerosis; beta-Cyclodextrins; Cattle; Caveolin 1; Caveolins; CCAAT-Enhancer-Binding Proteins; Cell Compartmentation; Cell Membrane; Cell Nucleus; Cells, Cultured; Cholesterol; DNA-Binding Proteins; Endoplasmic Reticulum; Endothelial Cells; Endothelium, Vascular; Golgi Apparatus; HeLa Cells; Humans; Hydroxycholesterols; Inflammation; Interleukin-8; Intracellular Signaling Peptides and Proteins; Membrane Lipids; Membrane Proteins; Phosphatidylcholines; Phospholipid Ethers; Recombinant Fusion Proteins; STAT3 Transcription Factor; Sterol Regulatory Element Binding Protein 1; Sterol Regulatory Element Binding Protein 2; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection

In endothelial cells, nitric oxide (NO) is produced by the endothelial isoform of nitric oxide synthase (eNOS), which is localized in the cholesterol-rich plasmalemmal microdomains involved in signal transduction, known as caveolae. The present study was undertaken to evaluate the effect of hypercholesterolemia and fatty streak formation on the endothelial caveolae and on endothelial function, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production.. We first studied the effect of atheroma on endothelial NO production. Fatty streak infiltrated aorta of cholesterol-fed New Zealand White rabbits demonstrated an impairment of acetylcholine-induced relaxation and nearly normal calcium ionophore A23187-induced maximal relaxation. The abundance of caveolae in the endothelium covering the fatty streak, as well as their 'grape-like' clustering, appeared to be decreased. We therefore investigated the effect, on endothelial NO production, of the cholesterol-binding agents 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CD) and filipin, known to alter caveolae structure and/or function. Treatment with either hp-beta-CD (2%) or filipin (4 microg/ml) did not affect contraction to phenylephrine or relaxant responses to A23187 or to the NO donor sodium nitroprusside. In contrast, both treatments impaired acetylcholine-induced relaxation. Cultured bovine aortic endothelial cells (BAEC) similarly treated with hp-beta-CD demonstrated a 50% decrease of total cellular cholesterol and a decreased abundance of caveolae as well as their 'grape-like' clustering. Cholesterol depletion decreased the bradykinin-induced transient peak of free intracellular calcium and subsequent receptor-stimulated NO production (assessed using reporter cells rich in soluble guanylyl cyclase), whereas that elicited by A23187 remained unaltered.. Fatty streak deposit is associated with a decrease in caveolae 'transductosomes' abundance which appears to represent a novel mechanism of endothelial dysfunction.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Aorta, Thoracic; Aortic Diseases; Arteriosclerosis; beta-Cyclodextrins; Cell Culture Techniques; Cell Membrane; Cyclodextrins; Endothelium, Vascular; Filipin; Hypercholesterolemia; Male; Microscopy, Electron; Muscle Contraction; Nitric Oxide; Rabbits

Atherosclerosis involves the arterial accumulation of lipid-laden "foam cells" containing oxidized and unoxidized sterols and their esters (Mattsson-Hulten, L., Lindmark, H., Diczfalusy, U., Bjorkhem, I., Ottosson, M., Liu, Y., Bondjers, G., and Wiklund, O. (1996) J. Clin. Invest. 97, 461-8). Oxidized sterols are probably critical to atherogenesis because they inhibit cholesterol removal from cells and are cytotoxic. We recently reported that there is deficient induction of cellular cholesterol efflux by apolipoprotein A-I, the main initial acceptor of cellular cholesterol from macrophages loaded in vitro with oxidized low density lipoprotein (Kritharides, L., Jessup, W., Mander, E., and Dean, R. T. (1995) Arterioscler. Thromb. 15, 276-289). There was an even more marked impairment of the release of 7-ketocholesterol which is a major oxysterol in these cells and in human atherosclerotic lesions. Here we show that hydroxypropyl-beta-cyclodextrin can induce selective efflux of 7-ketocholesterol. Efflux of 7-ketocholesterol was time and concentration dependent, and the rate of its removal was 50-fold greater for hydroxypropyl-beta-cyclodextrin than for apolipoprotein A-I. Over a defined range of concentrations (0-5 mg/ml), efflux of 7-ketocholesterol was preferred over that of cholesterol and occurred without cell toxicity. Efflux of free 7-ketocholesterol was associated with decreased intracellular free and esterified 7-ketocholesterol. Hydroxypropyl-beta-cyclodextrin also enhanced efflux of other oxysterols. The physical solubilization of 7-ketocholesterol by the cyclodextrin was much greater than that of cholesterol, in accordance with its differential effects on efflux. These data highlight the importance of extracellular sterol solubilization in the efflux of cellular oxysterols and the mobilization of intracellular free and esterified oxysterol pools in macrophages loaded with oxidized low density lipoprotein. Synthetic sterol-solubilizing agents such as hydroxypropyl-beta-cyclodextrin are thus potential prototypes for the further development of oxysterol-removing agents.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Apolipoprotein A-I; Arteriosclerosis; beta-Cyclodextrins; Cell Survival; Cells, Cultured; Cholesterol; Chromatography, High Pressure Liquid; Cyclodextrins; Humans; Ketocholesterols; Kinetics; Lipoproteins, LDL; Macrophages, Peritoneal; Mice; Mice, Inbred Strains

Heparin inhibits smooth muscle cell proliferation in vitro, a property that makes it potentially useful in preventing restenosis after angioplasty. Its utility in this setting is limited by the inability to use high doses (secondary to anticoagulant effects) and the need for subcutaneous administration. We tested the ability of beta-cyclodextrin tetradecasulfate (CDT), a nonanticoagulant synthetic heparin mimic, to inhibit smooth muscle cell proliferation in vitro and tested its efficacy when orally administered for the prevention of angioplasty restenosis in a rabbit atherosclerosis model. Vascular smooth muscle cells were cultured from rabbit aortas by the explant technique. Passaged cells were plated at low density in microtiter plates in the presence or absence of varying concentrations of heparin or CDT in culture medium containing 10% fetal calf serum. Using both 3H-thymidine incorporation and total protein assays, both heparin and CDT caused a similar dose-dependent inhibition of proliferation. We next tested the effect of orally administered CDT in the prevention of restenosis in focal femoral artery arteriosclerotic lesions created in hypercholesterolemic New Zealand White rabbits by air-dessication endothelial injury and subsequent peripheral angioplasty. Animals were followed up for 1 month and were fed normal chow supplemented by tap water with or without CDT. In animals receiving the highest concentration of CDT (2 mg/mL drinking water), the percentage of arterial cross-sectional area with intimal hyperplasia decreased from 50.5 +/- 1.7% (control) to 26.9 +/- 2.2% (p < 0.001), with the intimal/medial ratio being decreased from 1.4 +/- 0.4 to 0.5 +/- 0.2 (p = 0.056).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Angioplasty; Animals; Arteriosclerosis; beta-Cyclodextrins; Cell Division; Constriction, Pathologic; Cyclodextrins; Disease Models, Animal; Muscle, Smooth, Vascular; Rabbits; Vascular Diseases