Background-The cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) are secreted by the different cell populations of the vascular wall and have been suggested to promote atherosclerosis. Methods and Results-Their respective roles in fatty-streak formation in apolipoprotein E-deficient mice were investigated by use of IL-1 receptor antagonist and TNF binding protein. Estradiol-17 was used as a positive control. Blocking TNF seemed to be active in female animals but not in males. IL-1 receptor antagonist was as effective as or more effective than estradiol in both sexes. Conclusions-IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model, and blocking the activity of this cytokine should be considered as a therapeutic possibility. (Circulation. 1998;97:242-244.)Key Words: atherosclerosis Ⅲ apolipoproteins Ⅲ interleukins Ⅲ tumor necrosis factor I t is known that IL-1␣ and -1 and TNF-␣ and - are secreted in the vascular wall by endothelial and smooth muscle cells as well as by monocytes/macrophages. 1,2 These cytokines have been shown to increase permeability of the endothelial cell barrier, 3 induce the expression of surface leukocyte adhesion molecules, 1,4,5 and enhance the production of other cytokines and growth factors, such as IL-6 6 and macrophage colony-stimulating factor, 7-9 all such activities being considered to promote atherosclerosis. The objective of the present study was therefore to clarify the role of IL-1 and TNF in the initial steps of the atherosclerotic process, ie, fatty-streak formation, using apo E KO mice as an animal model of atherosclerosis 10,11 and human IL-1ra and TNFbp as the specific cytokine antagonists. IL-1ra is a recombinant 17-kD protein, which binds to IL-1 receptors and competes with both IL-1␣ and IL-1 without detectable IL-1 agonistic effects. 12,13 TNFbp is a specific TNF inhibitor consisting of two molecules of the extracellular domain of the human type 1 TNF receptor added to both ends of a molecule of polyethylene glycol. TNFbp binds with equal affinity to TNF-␣ and TNF-.14 -16 E 2 treatment was used as a positive control, because we and others have shown that this hormone prevents fatty-streak formation in the apo E KO mouse animal model. 17,18 The data obtained showed that TNFbp was active in female animals but not in males. Like E 2 , IL-1ra was active in both sexes, suggesting that IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model. Methods Study ProtocolApo E KO mice, originally obtained from the Jackson Laboratory, Bar Harbor, Me (sixth generation of backcross from 129/B6 F1 heterozygous to C57BL/6), were housed as previously described 18 and fed normal laboratory mouse chow containing 4.3% fat and 0.02% cholesterol. Four-week-old animals were gonadectomized under general anesthesia. At 2 months of age, these animals were given 0.2 mg 60-day time-release E 2 pellets (Innovative Research of America), a dose that was found to exert a maximal effect on fatty-strea...
The reality of the atheroprotective effect of estrogens is still a matter of debate, and its unknown mechanisms could involve favorable changes in blood lipids and lipoproteins and/or direct action at the level of the arterial wall. We used the recently developed animal model of atherosclerosis constituted by apolipoprotein E-deficient mice in an attempt to clarify these issues. Male and female animals, fed a low-fat chow diet, were treated with increasing doses of 17 beta-estradiol (E2) after castration and compared with testosterone treated and uncastrated (intact) animals. Total serum cholesterol, LDL-cholesterol, and HDL-cholesterol concentrations decreased under E2 treatment in each sex and were weakly correlated with lesion area. However, a highly significant correlation between lesion area and serum E2 levels also suggested a direct action of E2 on cells of the vascular wall. A dose-response curve analysis revealed that these activities were sex-dependent, with females being nearly twice as sensitive to E2 as males. It also revealed that the atheroprotective activity was recruited at higher E2 concentrations than those needed by other E2 target tissues such as uterus or functions such as apoA-1 and LDL production and/or clearance rates.
Oxidized LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotin nick end labeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.
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