Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.     

Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.     

Macrophage apoAI protects against dyslipidemia-induced dermatitis and atherosclerosis without affecting HDL

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor^−/−/apoAI^−/− mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4⁺ T-cell levels (−39.8%), lesion size (−25%), and necrotic core area (−31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4⁺ T-cell levels. Macrophage apoAI also reduced CD4⁺ T-cell levels (−32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4⁺ T-cell levels, without affecting serum HDL or tissue macrophage levels.     

Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [³H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [³H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.     

Hepatic overexpression of methionine sulfoxide reductase A reduces atherosclerosis in apolipoprotein E-deficient mice

Methionine sulfoxide reductase A (MsrA), a specific enzyme that converts methionine-S-sulfoxide to methionine, plays an important role in the regulation of protein function and the maintenance of redox homeostasis. In this study, we examined the impact of hepatic MsrA overexpression on lipid metabolism and atherosclerosis in apoE-deficient (apoE^−/−) mice. In vitro study showed that in HepG2 cells, lentivirus-mediated human MsrA (hMsrA) overexpression upregulated the expression levels of several key lipoprotein-metabolism-related genes such as liver X receptor α, scavenger receptor class B type I, and ABCA1. ApoE^−/− mice were intravenously injected with lentivirus to achieve high-level hMsrA expression predominantly in the liver. We found that hepatic hMsrA expression significantly reduced plasma VLDL/LDL levels, improved plasma superoxide dismutase, and paraoxonase-1 activities, and decreased plasma serum amyloid A level in apoE^−/− mice fed a Western diet, by significantly altering the expression of several genes in the liver involving cholesterol selective uptake, conversion and excretion into bile, TG biosynthesis, and inflammation. Moreover, overexpression of hMsrA resulted in reduced hepatic steatosis and aortic atherosclerosis. These results suggest that hepatic MsrA may be an effective therapeutic target for ameliorating dyslipidemia and reducing atherosclerosis-related cardiovascular diseases.     

Levels of atherogenic lipoproteins are unexpectedly reduced in interstitial fluid from type 2 diabetes patients

At a given level of serum cholesterol, patients with T2D have an increased risk of developing atherosclerosis compared with nondiabetic subjects. We hypothesized that T2D patients have an increased interstitial fluid (IF)-to-serum gradient ratio for LDL, due to leakage over the vascular wall. Therefore, lipoprotein profiles in serum and IF from 35 T2D patients and 35 healthy controls were assayed using fast performance liquid chromatography. The IF-to-serum gradients for VLDL and LDL cholesterol, as well as for apoB, were clearly reduced in T2D patients compared with healthy controls. No such differences were observed for HDL cholesterol. Contrary to our hypothesis, the atherogenic VLDL and LDL particles were not increased in IF from diabetic patients. Instead, they were relatively sparser than in healthy controls. The most probable explanation to our unexpected finding is that these lipoproteins are more susceptible to retainment in the extravascular space of these patients, reflecting a more active uptake by, or adhesion to, tissue cells, including macrophages in the vascular wall. Further studies are warranted to further characterize the mechanisms underlying these observations, which may be highly relevant for the understanding of why the propensity to develop atherosclerosis is increased in T2D.