Regulation of the activities of thrombin and plasmin by cholesterol sulfate as a physiological inhibitor in human plasma.
(1/2062)
Thrombin and plasmin, both of which are serine proteases in the plasma of vertebrates, play essential roles in blood clotting and fibrinolysis, respectively, and regulation of their activities is important to suppress the excessive reactions within the vascular network and to prevent tissue injury. Along with the peptidic inhibitors belonging to the serpin family, we found that cholesterol sulfate (CS), which is present at the concentration of 2.0+/-1.2 nmol/ml in human plasma, was a potent inhibitor of both plasma thrombin and plasmin. Thrombin, as determined both using a chromogenic substrate and the natural substrate, fibrinogen, was inactivated upon reaction with CS in a dose-dependent manner, but not in the presence of the structurally related steroid sulfates, I3SO3-GalCer and II3NAalpha-LacCer, suggesting that both the sulfate group and the hydrophobic side chain of CS are necessary for the inhibitory activity of CS. Preincubation of thrombin with CS at 37 degrees C for 10 min was required to achieve maximum inhibition, and virtually complete inhibition was achieved at a molar ratio of CS to thrombin of 18:1. CS-treated thrombin had the same Km and a lower Vmax than the original enzyme, and a higher molecular weight. The molecular weight and activity of the original enzyme were not observed on the attempted separation of the CS-treated enzyme by gel permeation chromatography and native PAGE, indicating that the inactivation of thrombin by CS is irreversible. In contrast, CS was readily liberated from the enzyme by SDS-PAGE, suggesting that hydrophobic interactions are involved in the CS-mediated inactivation of thrombin. When acidic lipids were reacted with thrombin after dissolving them in DMSO, I3SO3-GalCer, steroid sulfates and II3NAalpha-LacCer, as well as CS, but not SDS and sodium taurocholate, exhibited inhibitory activity, probably due to micellar formation facilitating interaction between thrombin and negatively charged lipids. On the other hand, plasmin, as determined using a chromogenic substrate, was more susceptible to acidic lipids than thrombin. CS, I3SO3-GalCer and II3NAalpha-LacCer, all of which are present in serum, inhibited the activity of plasmin in aqueous media, as well as in DMSO-mediated lipid solutions. Thus, acidic lipids in plasma were demonstrated to possess regulatory activity as endogenous detergents toward both enzymes for blood clotting and fibrinolysis. (+info)
Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages.
(2/2062)
Foam cells formed from receptor-mediated uptake of lipoprotein cholesterol by macrophages in the arterial intima are critical in the initiation, progression, and stability of atherosclerotic lesions. Macrophages accumulate cholesterol when conditions favor esterification by acyl-CoA:cholesterol acyltransferase (ACAT) over cholesteryl-ester hydrolysis by a neutral cholesteryl-ester hydrolase, such as hormone-sensitive lipase (HSL), and subsequent cholesterol efflux mediated by extracellular acceptors. We recently made stable transfectants of a murine macrophage cell line, RAW 264.7, that overexpressed a rat HSL cDNA and had a 5-fold higher rate of cholesteryl-ester hydrolysis than control cells. The current study examined the effect of macrophage-specific HSL overexpression on susceptibility to diet-induced atherosclerosis in mice. A transgenic line overexpressing the rat HSL cDNA regulated with a macrophage-specific scavenger receptor promoter-enhancer was established by breeding with C57BL/6J mice. Transgenic peritoneal macrophages exhibited macrophage-specific 7-fold overexpression of HSL cholesterol esterase activity. Total plasma cholesterol levels in transgenic mice fed a chow diet were modestly elevated 16% compared to control littermates. After 14 weeks on a high-fat, high-cholesterol diet, total cholesterol increased 3-fold, with no difference between transgenics and controls. However, HSL overexpression resulted in thicker aortic fatty lesions that were 2.5-times larger in transgenic mice. HSL expression in the aortic lesions was shown by immunocytochemistry. Atherosclerosis was more advanced in transgenic mice exhibiting raised lesions involving the aortic wall, along with lipid accumulation in coronary arteries occurring only in transgenics. Thus, increasing cholesteryl-ester hydrolysis, without concomitantly decreasing ACAT activity or increasing cholesterol efflux, is not sufficient to protect against atherosclerosis. hormone-sensitive lipase overexpression in macrophages. (+info)
Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes.
(3/2062)
The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets. These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells. (+info)
Cerebral atherosclerosis in Japanese. Part 4: relationship between lipid content and macroscopic severity of atherosclerosis.
(4/2062)
In order to evaluate chemically the macroscopic scoring methods for severity of arteriosclerosis in the cerebral arteries, concentrations of total lipids, esterified and free cholesterol and lipid phosphorus were compared to the macroscopic severity of lesions in the cerebral arteries obtained from 376 Japanese persons after unexpected death. An increase of cholesterol content was correlated significantly with an increase of Baker's score and/or Gore's atherosclerotic index. The correlation coefficient between Baker's score and total or esterified cholesterol was the highest among the tested correlations (r = 0.82, n = 376). (+info)
Cerebral atherosclerosis in Japanese. Part 5: relationship between cholesterol deposition and glycosaminoglycans.
(5/2062)
Concentrations of various lipids and glycosaminoglycans (GAG) in the intima of the grossly normal and atherosclerotic cerebral arteries were compared with those of the aorta and coronary arteries. The lowest percentage of esterified cholesterol (EC) in total cholesterol, and of chondroitin sulfate-4/6 (CS-4/6) in total glycosaminoglycans and the highest percentage of heparin sulfate (HS) in total GAG are the characteristic features of the normal intima of normal cerebral arteries when compared with those in the aorta and coronary artery. In the cerebral arterial intimas, but not in the aorta or coronary arteries, there was a significant positive correlation between contents of EC and percentage and total content of CS-4/6. Atherogenesis in cerebral arteries is discussed in comparison to that of the aorta and coronary vessels. (+info)
Lipid transfer inhibitor protein defines the participation of lipoproteins in lipid transfer reactions: CETP has no preference for cholesteryl esters in HDL versus LDL.
(6/2062)
Cholesteryl ester transfer protein (CETP) catalyzes the net transfer of cholesteryl ester (CE) between lipoproteins in exchange for triglyceride (heteroexchange). It is generally held that CETP primarily associates with HDL and preferentially transfers lipids from this lipoprotein fraction. This is illustrated in normal plasma where HDL is the primary donor of the CE transferred to VLDL by CETP. However, in plasma deficient in lipid transfer inhibitor protein (LTIP) activity, HDL and LDL are equivalent donors of CE to VLDL (Arterioscler Thromb Vasc Biol. 1997;17:1716-1724). Thus, we have hypothesized that the preferential transfer of CE from HDL in normal plasma is a consequence of LTIP activity and not caused by a preferential CETP-HDL interaction. We have tested this hypothesis in lipid mass transfer assays with partially purified CETP and LTIP, and isolated lipoproteins. With a physiological mixture of lipoproteins, the preference ratio (PR, ratio of CE mass transferred from a lipoprotein to VLDL versus its CE content) for HDL and LDL in the presence of CETP alone was approximately 1 (ie, no preference). Fourfold variations in the LDL/HDL ratio or in the levels of HDL in the assay did not result in significant preferential transfer from any lipoprotein. On addition of LTIP, the PR for HDL was increased up to 2-fold and that for LDL decreased in a concentration-dependent manner. Under all conditions where LDL and HDL levels were varied, LTIP consistently resulted in a PR >1 for CE transfer from HDL. Short-term experiments with radiolabeled lipoproteins and either partially purified or homogenous CETP confirmed these observations and further demonstrated that CETP has a strong predilection to mediate homoexchange (bidirectional transfer of the same lipid) rather than heteroexchange (CE for TG); LTIP had no effect on the selection of CE or TG by CETP or its mechanism of action. We conclude, in contrast to current opinion, that CETP has no preference for CE in HDL versus LDL, suggesting that the previously reported stable binding of CETP to HDL does not result in selective transfer from this lipoprotein. These data suggest that LTIP is responsible for the preferential transfer of CE from HDL that occurs in plasma. CETP and LTIP cooperatively determine the extent of CETP-mediated remodeling of individual lipoprotein fractions. (+info)
Role of cholesterol ester mass in regulation of secretion of ApoB100 lipoprotein particles by hamster hepatocytes and effects of statins on that relationship.
(7/2062)
Our understanding of the factors that regulate the secretion of apoB100 lipoproteins remains incomplete with considerable debate as to the role, if any, for cholesterol ester in this process. This study examines this issue in primary cultures of hamster hepatocytes, a species in which both cholesterol and apoB100 metabolism are very similar to man. Addition of oleate to medium increased the mass of triglyceride and cholesterol ester within the hepatocyte and also increased the secretion of triglycerides, cholesterol ester, and apoB100 into the medium. Next, the responses of hamster hepatocytes to addition of either an HMG-CoA reductase inhibitor (lovastatin) or an acyl-CoA cholesterol acyltransferase inhibitor (58-035) to the medium, with or without added oleate, were determined. Effects of either agent were only evident in the oleate-supplemented medium in which cholesterol ester mass had been increased above basal. If oleate was not added to the medium, neither agent reduced apoB100 secretion; equally important, over the 24-hour incubation, neither agent, at the concentration used, produced any detectable change in intracellular cholesterol ester mass. However, in contrast to the estimates of mass, which were unchanged, under the same conditions radioisotopic estimates of cholesterol ester synthesis were markedly reduced. Any conclusion as to the relation of cholesterol ester mass to apoB100 secretion would therefore depend on which of the 2 methods was used. Overall, the data indicate a close correlation between the mass of cholesterol ester within the hepatocyte and apoB100 secretion from it and they go far to explain previous apparently contradictory data as to this relation. More importantly, though, taken with other available data, they indicate that the primary response of the liver to increased delivery of lipid is increased secretion rather than decreased uptake. These results point, therefore, to a hierarchy of hepatic responses to increased flux of fatty acids and increased synthesis of cholesterol that in turn suggests a more dynamic model of cholesterol homeostasis in the liver than has been appreciated in the past. (+info)
Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.
(8/2062)
Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100. (+info)