Changes in endotoxin-binding proteins during major elective surgery: important role for soluble CD14 in regulation of biological activity of systemic endotoxin.
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Assessment of circulating endotoxin during the perioperative period, which is only demonstrated by the Limulus amebocyte lysate (LAL) test, may be modulated by several endotoxin-binding proteins. Endotoxin-neutralizing capacity (ENC) and the plasma levels of soluble CD14 (sCD14), lipopolysaccharide-binding protein, and bactericidal/permeability-increasing protein (BPI) were determined in 40 patients 6 h prior to skin incision for major abdominal surgery. The bioactivity of plasma endotoxin was tested by the polymyxin B-inhibited stimulatory activity of the plasma samples on healthy monocytes as measured by the release of tumor necrosis factor alpha. Plasma endotoxin levels in almost all patients increased from 0.05 +/- 0.01 to 0.23 +/- 0.03 experimental units (EU) per ml (P < 0.001); more specifically, 17 of 40 samples showed endotoxin levels of greater than 0.2 EU per ml and corresponding reductions in ENC. Soluble CD14 plasma levels were decreased from 5. 6 +/- 0.3 to 4.6 +/- 0.3 microg per ml (P < 0.05). ENC was strongly correlated with the sCD14 plasma concentration throughout the period of observation. The addition of sCD14-neutralizing monoclonal anti-sCD14 antibodies reduced ENC both pre- and postoperatively. No correlation could be established between ENC and the plasma levels of BPI, high-density lipoproteins, or low-density lipoproteins determined by measuring the concentrations of apoprotein A and apoprotein B. Biologically active endotoxin was found in only 6 of 17 samples with endotoxin levels greater than 0.2 EU per ml in the LAL test. These samples could be characterized by their perioperative loss of at least 35% of their sCD14. No change in sCD14 was detected in the remaining 11 samples. The perioperative loss of ENC is partly caused by the loss of sCD14 resulting from its consumption by endotoxin reaching the bloodstream. This study demonstrated the role of sCD14 on the bioactivity of circulating endotoxin in a human model of endotoxemia after major abdominal surgery. (+info)
Effect of plasminogen activators on human recombinant apolipoprotein(a) having the plasminogen activation cleavage site.
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The serine-proteinase domain in human apolipoprotein(a) [apo(a)] and plasminogen exhibit 89% sequence identity including the catalytic triad. Cleavage of the Arg(561)-Val(562) activation site in plasminogen by either tissue- or urokinase-type plasminogen activator results in formation of the fibrinolytic enzyme plasmin. Apo(a) does not contain measurable amidolytic activity nor can it be activated by plasminogen activators. It has been suggested that the latter finding might be explained by the substitution of the plasminogen Arg-Val activation site by Ser-Ile in apo(a). To investigate if introduction of the Arg-Val activation site in apo(a) might result in sensitivity towards plasminogen activators, we expressed wild-type and Arg-Val mutant recombinant apo(a) [r-apo(a)] in human embryonic kidney and hepatocyte cell lines. Free r-apo(a) and lipoprotein-like particles [r-Lp(a)] were obtained in the culture supernatants of transfected 293 and HepG2 cells, respectively. Incubation of mutant r-apo(a)/r-Lp(a) with plasminogen activators produced neither plasmin-like activity nor cleavage at the Arg-Val activation site, even in the presence of various stimulators of plasminogen activation. Our data suggest that the high selectivity of activators for plasminogen activation requires interactions with regions in plasminogen distant from the activation disulfide loop which are not present in apo(a). (+info)
A frequent mutation in the lipoprotein lipase gene (D9N) deteriorates the biochemical and clinical phenotype of familial hypercholesterolemia.
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The D9N substitution is a common mutation in the lipoprotein lipase (LPL) gene. This mutation has been associated with reduced levels of HDL cholesterol and elevated triglycerides (TG) in a wide variety of patients. We investigated the influence of this D9N mutation on lipid and lipoprotein levels and risk for cardiovascular disease (CVD) in patients with familial hypercholesterolemia (FH). A total of 2091 FH heterozygotes, all of Dutch extraction, were screened for the D9N mutation using standard polymerase chain reaction techniques, followed by specific enzyme digestion. A total of 94 FH subjects carried the D9N mutation at a carrier frequency of 4.5%. Carriers of other common LPL mutations, such as the N291S and the S447X were excluded. Clinical data on 80 FH individuals carrying the D9N were available and were compared with a FH control group matched for age, sex, and body mass index (n=203). Analysis revealed significantly higher TG (P=0.01) and lower HDL-cholesterol levels (P=0.002). Dyslipidemia was more pronounced in D9N carriers with higher body mass index. Moreover, FH patients carrying this common LPL mutation were at higher risk for CVD, (odds ratio=2.8; 95% CI, 1.43 to 5.32; P=0.002). The common D9N LPL mutation leads to increased TG and decreased HDL plasma levels in patients with FH. These effects are most apparent in those FH heterozygotes with an increased body mass index. Furthermore, this mutation, present in 4.5% of Dutch FH heterozygotes, leads to increased risk for CVD. (+info)
Severe hyperlipidemia in apolipoprotein E2 homozygotes due to a combined effect of hyperinsulinemia and an SstI polymorphism.
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More than 90% of patients with type III hyperlipoproteinemia are homozygous carriers of the apolipoprotein (apo) E*2 allele. The great majority of these apoE2(Arg158-->Cys) homozygotes in the general population, however, are normolipidemic. Apparently, expression of the hyperlipidemic state requires additional genetic and/or environmental factors, suggesting a multifactorial etiology. To elucidate these additional risk factors, we analyzed normolipidemic and hyperlipidemic apoE2 homozygotes. Hyperinsulinemia was observed in 27 of 49 apoE2 homozygotes and associated with elevated lipid levels: hyperinsulinemic apoE2 homozygotes had type III hyperlipoproteinemia 6 times more often than apoE2 homozygotes with normal insulin levels (odds ratio 6.2, P=0.02). We screened the normolipidemic and hyperlipidemic apoE2 homozygotes for common variants in candidate genes involved in lipolysis-the APOA1-C3-A4 gene cluster, lipoprotein lipase, and hepatic lipase-and analyzed for associations with the expression of hyperlipidemia. In the hyperinsulinemic group, the 7 carriers of the SstI polymorphism (S2) in the APOC3 gene displayed severely elevated VLDL cholesterol (P(insulin by SstI)<0.001) and VLDL triglyceride (P(insulin by SstI)<0.01) and low levels of HDL (P(insulin by SstI)<0.02). In the normoinsulinemic group, no such relation of the SstI polymorphism with hyperlipidemia was observed. These data provide the first evidence for a combined effect of hyperinsulinemia and the SstI polymorphism on the expression of hyperlipidemia in apoE2 homozygotes. (+info)
Linkage of a candidate gene locus to familial combined hyperlipidemia: lecithin:cholesterol acyltransferase on 16q.
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Familial combined hyperlipidemia (FCHL) is a common lipid disorder characterized by elevated levels of plasma cholesterol and triglycerides that is present in 10% to 20% of patients with premature coronary artery disease. To study the pathophysiological basis and genetics of FCHL, we previously reported recruitment of 18 large families. We now report linkage studies of 14 candidate genes selected for their potential involvement in the aspects of lipid and lipoprotein metabolism that are altered in FCHL. We used highly polymorphic markers linked to the candidate genes, and these markers were analyzed using several complementary, nonparametric statistical allele-sharing linkage methodologies. This current sample has been extended over the one in which we identified an association with the apolipoprotein (apo) AI-CIII-AIV gene cluster. We observed evidence for linkage of this region and FCHL (P<0.001), providing additional support for its involvement in FCHL. We also identified a new locus showing significant evidence of linkage to the disorder: the lecithin:cholesterol acyltransferase (LCAT) locus (P<0.0006) on chromosome 16. In addition, analysis of the manganese superoxide dismutase locus on chromosome 6 revealed a suggestive linkage result in this sample (P<0.006). Quantitative traits related to FCHL also provided some evidence of linkage to these regions. No evidence of linkage to the lipoprotein lipase gene, the microsomal triglyceride transfer protein gene, or several other genes involved in lipid metabolism was observed. The data suggest that the lecithin:cholesterol acyltransferase and apolipoprotein AI-CIII-AIV loci may act as modifying genes contributing to the expression of FCHL. (+info)
Stimulation of jejunal synthesis of apolipoprotein A-IV by ileal lipid infusion is blocked by vagotomy.
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We examined the role of vagal innervation in lipid-stimulated increases in expression and synthesis of intestinal apolipoprotein A-IV (apoA-IV). In rats with duodenal cannulas and superior mesenteric lymph fistulas given duodenal infusions of lipid emulsion, vagotomy had no effect on either intestinal lipid transport, lymphatic apoA-IV output, or jejunal mucosal apoA-IV synthesis. In rats with jejunal Thiry-Vella fistulas, ileal lipid infusion elicited a twofold stimulation of apoA-IV synthesis without affecting apoA-IV mRNA levels; vagotomy blocked this increase in apoA-IV synthesis. Direct perfusion of jejunal Thiry-Vella fistulas produced 2- to 2.5-fold increases in both apoA-IV synthesis and mRNA levels in the Thiry-Vella segment; these effects were not influenced by vagal denervation. These results suggest two mechanisms whereby lipid stimulates intestinal apoA-IV production: 1) a vagal-dependent stimulation of jejunal apoA-IV synthesis by distal gut lipid that is independent of changes in apoA-IV mRNA levels and 2) a direct stimulatory effect of proximal gut lipid on both synthesis and mRNA levels of jejunal apoA-IV that is independent of vagal innervation. (+info)
Aspirin reduces apolipoprotein(a) (apo(a)) production in human hepatocytes by suppression of apo(a) gene transcription.
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High serum lipoprotein(a) (Lp(a)) is a risk factor for vascular disorders. Our preliminary observations suggest that, in some patients with coronary heart disease with high serum Lp(a) levels, administration of aspirin reduced Lp(a) levels. Therefore, we aimed to analyze the effects of aspirin on the production of apo(a), the expression of apolipoprotein(a) (apo(a)) mRNA and the transcriptional activity of apo(a) gene promoter. Aspirin (5 mM) reduced the apo(a) levels in culture medium of human hepatocytes and suppressed apo(a) mRNA expression to 73% and 85% of the controls, respectively. Aspirin also reduced the transcriptional activity of apo(a) gene transfected into HepG2 hepatoma cells in a dose-dependent manner, with a maximal effect at 5 mM (44.3 +/- 1.5% of the control). Sodium salicylate (5 mM) also reduced apo(a) gene transcription, whereas indomethacin (10 microM) had no effect. Deletion analysis of apo(a) gene promoter showed that promoter region extending from -30 to +138 is critical for the effect of aspirin. Furthermore, enhanced production, mRNA expression, and gene transcription of apo(a) by interleukin-6 were also inhibited by aspirin. These results demonstrate that aspirin reduces apo(a) production from hepatocytes via reduction of the transcriptional activity of apo(a) gene with suppression of apo(a) mRNA expression. The suppression of apo(a) production by aspirin may at least in part play a role in the anti-atherogenic effect of aspirin in vascular disorders. (+info)
Human apolipoprotein A-IV reduces gastric acid secretion and diminishes ulcer formation in transgenic mice.
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We have investigated the involvement of human apolipoprotein A-IV (apoA-IV) in gastric acid secretion and ulcer formation in recently generated apoA-IV transgenic mice. Compared to control littermates, transgenic animals showed a gastric acid secretion decreased by 43-77% whereas only slight variations were observed in the different cell population densities within the gastric mucosa. In addition, no variation in gastrin levels was observed. Transgenics were protected against indomethacin-induced ulcer formation, with lesions diminishing by 45 to 64% compared to controls. These results indicate that endogenous apoA-IV expression can regulate gastric acid secretion and ulcer development. (+info)