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OMGKRP is one of various Karuho poison mysteriously used by unscrupulous individuals to kill people during conflict and animals in Goma City, in DRC. The symptoms and signs of most cases are usually confused with many chronic diseases like tuberculosis and HIV/AIDS; with renal, hepatic and cardiac manifestations as well as blood chemistry changes. The study investigated the toxic effect of OMGKRP poison on blood chemistry, serum enzymes and organ toxicity including the kidney, lung, liver and heart of Wistar albino rats. A laboratory-based experimental study was conducted. Fifty animals in 5 groups each with 10 animals were dosed daily for 28 days with 1.0 mg, 5.0 mg, 20.0 mg and 5000.0 mg/Kg body weight of OMGKRP and normal saline as control group. International standard guidelines, OECD 407 and NIH 2011 were followed during the study period. The blood chemistry analysis, relative organ weight and histopathological changes in the kidney, lung, liver and heart were performed. The findings showed that OMGKRP was associated with increased blood chemistry parameters including total proteins, creatinine, urea, K+ levels, direct albumin levels, a decrease in Cl− levels and albumin levels. Histopathological findings showed an increased relative weight and tissue damages of the lung, kidney, liver and heart. Therefore, OMGKRP Karuho poison caused toxicity on blood chemistry, serum enzymes as well as histopathological changes in the lung, renal, hepatic and cardiac tissue damages in Wistar albino rats.

Background: M. oleifera is a highly valued medicinal plant used widely from time immemorial to treat various ailments. However, with continued un-standardized use of the plant leaves, studies have reported organ toxicity to the liver, kidney and the heart. As communities continue to use M. oleifera leaves for its medicinal and nutritional values, there is need to find an antidote for its hepatotoxicity. Aim: The study established the reversal effect of N-Acetyl Cysteine (NAC) on M. oleifera aqueous leaf extract-induced hepatotoxicity in Wistar albino rats. Methods: Twenty-four (24) rats received a toxic dose (8.05 g/kg bwt) of M. oleifera leaf extract for 28 days to cause sub-acute hepatotoxicity. They were divided into 4 groups of 6 rats each. Group I received 1 ml normal (control group), Group II received 1000 ng/kg NAC, Group III received 1200 mg/kg NAC and Group IV received 1500 mg/kg NAC. Another group of 6 rats (Group V) received 0.75 mg/kg Paracetamol to cause hepatotoxicity. Group V (a positive control) received the prescribed clinical dose of 1200 mg/kg NAC which reverses the hepatotoxicity. All the NAC doses were given once a day intragastric for 7 days. On days: 1, 3 and 7 of receiving NAC, liver serum enzymes and bilirubin were measured. On day 7 the animals were sacrificed and liver tissue harvested for histopathology analysis. Results: A dose of 8.05 g/kg of M. oleifera leaf extract and 0.75 mg/kg Paracetamol were able to induce hepatotoxicity in Wister albino rats in 28 days. The M. oleifera extract induced hepatotoxic rats treated with NAC at doses of 1000 mg/kg, 1200 mg/kg and 1500 mg/kg, had a reduction in mean serum liver enzymes, plus reduced mean serum bilirubin levels. The liver histopathological analysis showed reduced inflammation after treatment with NAC for 3 and 7 days in the M. oleifera and paracetamol induced hepatotoxic rats. Conclusion: NAC can reverse M. oleifera leaf aqueous extract-induced sub-acute hepatotoxicity in Wistar Albino rats.

Natural antibodies (NAbs) are important regulators of tissue homeostasis and inflammation and are thought to have diverse protective roles in a variety of pathological states. E06 is a T15 idiotype IgM NAb exclusively produced by B-1 cells, which recognizes the phosphocholine (PC) head group in oxidized phospholipids on the surface of apoptotic cells and in oxidized LDL (OxLDL), and the PC present on the cell wall of Streptococcus pneumoniae. Here we report that titers of the E06 NAb are selectively increased several-fold in Cd1d-deficient mice, whereas total IgM and IgM antibodies recognizing other oxidation specific epitopes such as in malondialdehyde-modified LDL (MDA-LDL) and OxLDL were not increased. The high titers of E06 in Cd1d-deficient mice are not due to a global increase in IgM-secreting B-1 cells, but they are specifically due to an expansion of E06-secreting splenic B-1 cells. Thus, CD1d-mediated regulation appeared to be suppressive in nature and specific for E06 IgM-secreting cells. The CD1d-mediated regulation of the E06 NAb generation is a novel mechanism that regulates the production of this specific oxidation epitope recognizing NAb.

Objective- To investigate the effect of gut microbiota and diet on atherogenesis. Approach and Results- Here, we investigated the interaction between the gut microbiota and diet on atherosclerosis by feeding germ-free or conventionally raised Apoe

Atherosclerosis is a chronic inflammation associated with increased expression of the acute phase isoforms of serum amyloid A (SAA) and in humans is a plasma biomarker for future cardiovascular events. However, whether SAA is only a biomarker or participates in the development of cardiovascular disease is not well characterized. The purpose of this review is to summarize putative functions of SAA relevant to atherogenesis and in-vivo murine studies that directly examine the effect of SAA on atherosclerosis.Modulation of the expression of SAA1 and/or SAA2 in murine models of atherosclerosis suggests that SAA promotes early atherogenesis. SAA secreted from bone-marrow-derived cells contributes to this antiatherogenic phenotype. SAA also promotes angiotensin-induced abdominal aneurysm in atherogenic mouse models. The reduction in atherosclerosis may be due, at least in part, to remodeling of the acute phase HDL to reduce its capacity to promote cholesterol efflux and reduce its anti-inflammatory ability.SAA is more than a marker of cardiovascular disease and is a participant in the early atherogenic process.

Macrophages are core cellular elements of both early and advanced atherosclerosis. They take up modified lipoproteins and become lipid-loaded foam cells and secrete factors that influence other cell types in the artery wall involved in atherogenesis. Apoproteins E, AI, and SAA are all found on HDL which can enter the artery wall. In addition, apoE is synthesized by macrophages. These three apoproteins can promote cholesterol efflux from lipid-loaded macrophages and have other functions that modulate macrophage biology. Mimetic peptides based on the sequence or structure of these apoproteins replicate some of these properties and are potential therapeutic agents for the treatment of atherosclerosis to reduce cardiovascular diseases.

Murine models of atherosclerosis are useful for investigating the environmental and genetic influences on lesion formation and composition. Apoe −/− and Ldlr −/− mice are the 2 most extensively used models. The models differ in important ways with respect to the precise mechanism by which their absence enhances atherosclerosis, including differences in plasma lipoproteins. The majority of the gene function studies have utilized only 1 model, with the results being generalized to atherogenic mechanisms. In only a relatively few cases have studies been conducted in both atherogenic murine models. This review will discuss important differences between the 2 atherogenic models and will point out studies that have been performed in the 2 models where results are comparable and those where different results were obtained.

ApoE is a multifunctional protein that is expressed by many cell types that influences many aspects of cardiovascular physiology. In humans, there are three major allelic variants that differentially influence lipoprotein metabolism and risk for the development of atherosclerosis. Apoe-deficient mice and human apoE isoform knockin mice, as well as hypomorphic Apoe mice, have significantly contributed to our understanding of the role of apoE in lipoprotein metabolism, monocyte/macrophage biology, and atherosclerosis. This brief history of these mouse models will highlight their contribution to the understanding of the role of apoE in these processes. These Apoe(-/-) mice have also been extensively utilized as an atherosensitive platform upon which to assess the impact of modulator genes on the development and regression of atherosclerosis.

Purpose of review Macrophage foam cells are important cells in the vascular wall that contribute to the inflammation associated with atherosclerotic lesions. Recent studies have demonstrated the heterogeneity of macrophages in lesions. In this review, advances in our understanding of the formation of foam cells by macrophage subsets in atherosclerotic plaques will be discussed. Recent findings Macrophage subsets develop in response to the microenvironment in the arterial wall. The uptake of lipoproteins, particularly oxidized LDL, has been considered the major mechanism of foam cell formation. However, native and aggregated LDL can also be taken up by macrophages and M2 macrophages have been shown to be efficient in the uptake of apoptotic cells that can contribute lipids to the cells. The ability of the macrophage subsets to respond to bioactive lipids in the artery wall to either promote macrophage subset polarization and/or to promote foam cell formation is only beginning to be understood. Summary Although we are beginning to appreciate the heterogeneity of macrophages present in atherosclerotic plaques, further work is required to fully understand the molecular basis for the differential ability of macrophage subsets to form foam cells and to respond to bioactive lipids.

Background Hypercholesterolemia‐induced decreased availability of nitric oxide ( NO ) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K + (Kir) channels and that Kir2.1 is an upstream mediator of flow‐induced NO production. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia‐induced inhibition of flow‐induced NO production and flow‐induced vasodilation ( FIV ). We also tested the role of Kir2.1 in the development of atherosclerotic lesions. Methods and Results Kir2.1 currents are significantly suppressed in microvascular endothelial cells exposed to acetylated–low‐density lipoprotein or isolated from apolipoprotein E–deficient ( Apoe −/− ) mice and rescued by cholesterol depletion. Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe −/− mice, Kir2.1 +/− /Apoe −/− exhibit the same blunted FIV and flow‐induced NO response as Apoe −/− or Kir2.1 +/− alone, but while FIV in Apoe −/− mice can be rescued by cholesterol depletion, in Kir2.1 +/− /Apoe −/− mice cholesterol depletion has no effect on FIV . Endothelial‐specific overexpression of Kir2.1 in arteries from Apoe −/− and Kir2.1 +/− /Apoe −/− mice results in full rescue of FIV and NO production in Apoe −/− mice with and without the addition of a high‐fat diet. Conversely, endothelial‐specific expression of dominant‐negative Kir2.1 results in the opposite effect. Kir2.1 +/− /Apoe −/− mice also show increased lesion formation, particularly in the atheroresistant area of descending aorta. Conclusions We conclude that hypercholesterolemia‐induced reduction in FIV is largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow‐induced NO production, whereas the stages downstream of flow‐induced Kir2.1 activation appear to be mostly intact. Kir2.1 channels also have an atheroprotective role.

Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late-eluting peak was modestly reduced in APOA1-deficient mice but little affected in APOE- or APOA4-deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages.

PURPOSE OF REVIEW Previous epidemiological studies and studies in experimental animals have provided strong evidence for the atheroprotective effect of HDL and its major apoprotein, apolipoprotein A-I (apoA-I). Identification of genetic loci associating apoA-I/HDL with cardiovascular disease is needed to establish a causal relationship. RECENT FINDINGS Pharmacological interventions to increase apoA-I or HDL cholesterol levels in humans are not associated with reduction in atherosclerosis. Genome wide association study (GWAS) studies in humans and hybrid mouse diversity panel (HMDP) studies looking for genetic variants associated with apoA-I or HDL cholesterol levels with cardiovascular disease and atherosclerosis have not provided strong evidence for their atheroprotective function. SUMMARY These findings indicate that GWAS and HMDP studies identifying possible genetic determinants of HDL and apoA-I function are needed.

High-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) have been shown in epidemiological studies and in animal models to reduce the risk of atherosclerosis. Synthetic peptides modeling the repeating amphipathic a-helices in apoA-I, the major apoprotein of HDL, were originally designed to study the lipid binding and other physical properties of the helices. The interest in apoA-I mimetic peptides was greatly stimulated by the observation that some, but not all, of the peptides significantly reduce atherosclerosis in animal models. Both monohelical and tandem helical peptides have been studied in vitro and in vivo for properties that mimic the antiatherogenic functional properties of HDL and apoA-I, particularly their ability to promote cholesterol efflux and reduce inflammation and oxidation. Mimetic peptides related to apoE, another apoprotein with repeating amphipathic helices, have also been shown to be antiatherogenic. Both peptide families likely function via common mechanisms and unique mechanisms. The understanding of how the structural properties of the various mimetic peptides relate to their mechanism of action will facilitate the development of optimal antiatherogenic therapeutic mimetic peptides.

Chronic alcohol use is a common problem globally among the HIV-infected patients on ARV treatment regimens, leading to severe liver damage and increase in serum enzymes. The study determined effect of chronic alcohol intake on serum enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT)) in HIV-infected patients on d4T/3TC/NVP treatment regimen in Uganda using the WHO alcohol use disorders’ identification test (AUDIT) tool and chronic alcohol use biomarkers (ALT, AST, GGT, AST/ALT ≥ 2.0 and mean corpuscular volume (MCV)). A case control study using repeated measure design with serial measurements model was used. Alcohol use biomarkers were used to standardize the gender differences in alcohol use. A total of 41 patients (21 alcohol group and 20 control group) were followed up for 9 months with blood sampling done at 3 month intervals. The serum enzymes’ levels were determined by using the Cobas Intergra 400 Plus analyzer system. The mean GGT levels were higher in chronic alcohol use group as compared to control group in both groups. The levels were above reference ranges during 6 month and three times higher during 9-month follow-up period for both chronic alcohol use self reporting WHO AUDIT tool and biomarkers’ groups. Generally, the mean AST, ALT and AST/ALT levels were slightly higher in alcohol use group as compared to control group and were slightly higher in both groups as compared to reference ranges during the 9 month follow-up period. Chronic alcohol consumption by HIV-infected patients on d4T/3TC/NVP drug regimen increased GGT and AST/ALT serum enzyme levels and hence was used as chronic alcohol use biomarkers.

Sustained heavy ethanol drinking is a common problem globally and ethanol is one of the most abused drugs among individuals of different socio-economic status including the HIV-infected patients on antiretroviral drugs. Ethanol is reward drug and a CNS depressant especially at high doses. The study determined the effect of sustained heavy ethanol drinking by HIV-infected patients on d4T/3TC/NVP regimen on CD4+ cell counts in Uganda using WHO AUDIT tool and chronic alcohol-use biomarkers. A case control study using repeated measures design with serial measurements model was used. The patients on stavudine (d4T) 30 mg, lamivudine (3TC) 150 mg and nevirapine (NVP) 200 mg and chronic alcohol use were recruited. A total of 41 patients (20 in alcohol group and 21 in control group) were screened for chronic alcohol use by WHO AUDIT tool and chronic alcohol use biomarkers. They were followed up for 9 months with blood sampling done at 3 months intervals. CD4+ cell count was determined using Facscalibur Flow Cytometer system. Results were then sorted by alcohol-use biomarkers (GGT, MCV and AST/ ALT ratio). Data were analysed using SAS 2003 version 9.1 statistical package with repeated measures fixed model and the means were compared using student t-test. The mean CD4+ cell counts in all the groups were lower than the reference ranges at baseline and gradually increased at 3, 6 and 9 months of follow-up. The mean CD4+ cell counts were higher in the control group as compared to the chronic alcohol use group in both WHO AUDIT tool group and chronic alcohol-use biomarkers group though there was no significant difference (p > 0.05). Chronic alcohol use slightly lowers CD4+ cell count in HIV-infected patients on d4T/3TC/NVP treatment regimen.

Atherosclerosis is a chronic inflammation initiated by the accumulation of low-density lipoproteins (LDL) in the subendothelial space of the artery wall in spatially selected regions of the artery. The LDL is derived from circulating plasma lipoproteins. The passage of macromolecules across the endothelial barrier may involve one of three mechanisms—diffusional, paracellular or transcellular.

The synthesis of apoE by adipocytes has profound effects on adipose tissue lipid flux and gene expression. Using adipose tissue transplantation from wild-type (WT) to apoE knockout (EKO) mice, we show that adipose tissue also contributes to circulating apoE. Different from circulating apoE produced by bone marrow transplantation (BMT), however, adipose tissue-derived apoE does not correct hyperlipidemia or suppress atherosclerosis. ApoE secreted by macrophages has a more acidic isoform distribution, and it increases binding of reconstituted VLDL particles to hepatocytes and fibroblasts more effectively than apoE secreted by adipocytes. The incremental binding can be entirely accounted for by binding to the LDL receptor. After BMT into EKO hosts, plasma cholesterol and macrophage-derived apoE are largely within IDL/LDL- and HDL-sized particles. After adipose tissue transplantation, most cholesterol and adipocyte apoE remain in VLDL. After BMT, circulating apoE no longer demonstrates predominance of acidic isoforms compared with that circulating after fat transplantation. In conclusion, fat transplantation provides circulating apoE levels similar to those provided by bone marrow transplantation, but it does not suppress hyperlipidemia or atherosclerosis. A potential mechanism contributing to this difference is differential binding to cell surface lipoprotein receptors.

Chronic consumption of aflatoxin-contaminated foods is a global problem in both developing and developed countries especially where there is poor regulation of their levels in foods. In the body, aflatoxins (AFBs) mainly AFB1 are biotransformed to various metabolites especially the active AFB1-exo-8,9-epoxide (AFBO). The AFB, AFBO and other metabolites interact with various biomolecules in the body including nucleic acids such as DNA and RNA and the various metabolic pathways such as protein synthesis, glycolytic pathway and electron transport chain involved in ATP production in body cells. The AFB interacts with DNA to form AFB-DNA adducts causing DNA breakages. The AFB and its metabolites induce the up regulation of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) through gene expression that regulates the metabolizing enzymes such as CYP450 involved in Phase I and Phase II metabolism of xenobiotics. AFB activates these nuclear receptors to produce the metabolizing enzymes. The AFB1 is metabolized in the body by cytochrome P450 (CYP450) enzyme isoforms such as CYP1A2, CYP1A2, CYP3A4/ CYP3A5, and CYP3A7 in fetus, glutathione S-transferase, aflatoxin B1-aldehyde reductase leading to reactive metabolites, some of which can be used as aflatoxin exposure biomarkers. These enzymes are involved in the Phase I and Phase II metabolic reactions of aflatoxins. The CYP1A2 is the principal metabolizer of aflatoxin at low concentrations while the reverse is true for CYP3A4. The accumulation of AFB and its metabolites in the body especially the AFB1-exo-8,9-epoxide depletes the glutathione (GSH) due to the formation of high amounts of epoxides and other reactive oxygen species (ROS). The AFB, AFB1-exo-8,9-epoxide and other metabolites also affect the epigenetic mechanisms including the DNA methylation, histone modifications, maturation of miRNAs as well as the daily formation of single nucleotide polymorphism (SNP) where AFB exposure may facilitate the process and induces G:C to T:A transversions at the third base in codon 249 of TP53 causing p53 mutations reported in hepatocellular carcinoma (HCC). The changes in epigenetic mechanisms lead to either epigenetic inactivation or epigenetic derepression and all these affect the gene expression, cellular differentiation and growth. AFB also through epigenetic mechanisms promotes tumorigenesis, angiogenesis, invasion and metastasis in hepatocellular carcinoma. However, the formation of the small amounts of AFB1 from AFB2 is suspected to cause the carcinogenicity of AFB2 in humans and animals. Chronic aflatoxins exposure leads to formation of reactive AFBO metabolites in the body that could activate and de-activates the various epigenetic mechanisms leading to development of various cancers.

Background: The cholesterol efflux capacity (CEC) of serum HDL, measured using cultured macrophages predicts incident and prevalent CVD risk in humans. The ABCA1 pathway is a key regulator of macrophage cholesterol homeostasis in vivo. Methods: We used genetic and biochemical approaches in mice to identify important mediators of CEC. Results: On high-resolution size-exclusion chromatography of mouse plasma, macrophage CEC and HDL co-eluted as a single major peak, suggesting that HDL mediates cholesterol efflux. In contrast, size-exclusion chromatography revealed two major peaks of material that promoted ABCA1-specific CEC, one of which was distinct from HDL. HDL particle concentration was reduced by 75% in Apoa1 -/- mice; this resulted in a 50% decrease in macrophage CEC but, surprisingly, had no impact on ABCA1-specific CEC. Orthogonal chromatography-mass spectrometric analysis of the non-HDL-associated efflux inducing material isolated from wild-type and APOA1 deficient plasma showed that plasminogen strongly correlated with ABCA1-specific CEC. Moreover, isolated plasminogen promoted cholesterol efflux by the ABCA1 pathway, and the specific activity of ABCA1-specific CEC of non-HDL-associated material was reduced by 50% in plasminogen deficient plasma. Imaging of cells treated with fluorescently-labeled antibodies demonstrated that ABCA1 and plasminogen co-localized on the plasma membrane. Conclusions: HDL particle concentration is an important contributor to macrophage CEC. However, other pathways contribute to ABCA1-specific CEC; our studies identify plasminogen as one potential mediator. Plasminogen associates with CVD risk in human genetic studies, raising the possibility that it plays a role in atherosclerosis by modulating ABCA1-mediated sterol efflux from macrophages.

Although vitamin D has been implicated in cardiovascular protection, few studies have addressed the role of vitamin D receptor (VDR) in atherosclerosis. Here we investigate the effect of inactivation of the VDR signaling on atherogenesis and the antiatherosclerotic mechanism of vitamin D. Low density lipoprotein receptor (LDLR)(-/-)/VDR(-/-) mice exhibited site-specific accelerated atherogenesis, accompanied by increases in adhesion molecules and proinflammatory cytokines in the aorta and cholesterol influx in macrophages. Macrophages showed marked renin up-regulation in the absence of VDR, and inhibition of renin by aliskiren reduced atherosclerosis in LDLR(-/-)/VDR(-/-) mice, suggesting that the renin-angiotensin system (RAS) promotes atherosclerosis in the absence of VDR. LDLR(-/-) mice receiving LDLR(-/-)/VDR(-/-) BMT developed larger lesions than LDLR(-/-) BMT controls. Moreover, LDLR(-/-) mice receiving Rag-1(-/-)/VDR(-/-) BMT, which were unable to generate functional T and B lymphocytes, still had more severe atherosclerosis than Rag-1(-/-) BMT controls, suggesting a critical role of macrophage VDR signaling in atherosclerotic suppression. Aliskiren treatment eliminated the difference in lesions between Rag-1(-/-)/VDR(-/-) BMT and Rag-1(-/-) BMT recipients, indicating that local RAS activation in macrophages contributes to the enhanced atherogenesis seen in Rag-1(-/-)/VDR(-/-) BMT mice. Taken together, these observations provide evidence that macrophage VDR signaling, in part by suppressing the local RAS, inhibits atherosclerosis in mice.

Plasma HDL levels are inversely associated with atherosclerosis. Inbred mouse strains differ in plasma HDL levels and susceptibility to atherosclerosis. Atherosclerosis-susceptible C57BL/6J mice possess plasma HDL levels 2-fold lower than atherosclerosis-resistant FVB/NJ mice. Polymorphisms have been previously identified between the two mouse strains in the major HDL apolipoproteins, ApoA-I and ApoA-II, which may affect their function on HDL. To begin to understand the HDL differences, we here report on a detailed comparison of the lipid-associated functions of the two mouse ApoA-I proteins. We demonstrate that these polymorphisms significantly alter the protein self-association properties, the ability of the proteins to clear lipid micelles from solution, and their binding affinity for mature mouse HDL. The changes in lipid binding do not appear to alter the ability of the protein to promote cholesterol efflux from cells or the formation of nascent HDL from primary hepatocytes. These apolipoprotein polymorphisms do not change the rate at which HDL protein or cholesterol are catabolized in vivo. Although the presence of the polymorphisms in ApoA-I alters important factors in HDL formation, the basis for the differences in the HDL plasma levels observed in the various mouse strains is more complex and requires additional investigation.

The association between apolipoprotein E (apoE) and amyloid-β peptide (Aβ) may significantly impact the function of both proteins, thus affecting the etiology of Alzheimer’s disease (AD). However, apoE/Aβ interactions remain fundamentally defined by the stringency of the detection method. Here we use size exclusion chromatography (SEC) as a non-stringent approach to the detection of apoE/Aβ interactions in solution, specifically apoE and both endogenous and exogenous Aβ from plasma, CSF and astrocyte conditioned media. By SEC analysis, Aβ association with plasma and CNS lipoproteins is apoE-dependent. While endogenous Aβ elutes to specific human plasma lipoproteins distinct from those containing apoE, it is the apoE-containing lipoproteins that absorb excess amounts of exogenous Aβ40. In human CSF, apoE, endogenous Aβ and phospholipid elute in an almost identical profile, as do apoE, exogenous Aβ and phospholipid from astrocyte conditioned media. Combining SEC fractionation with subsequent analysis for SDS-stable apoE/Aβ complex reveals that apoE-containing astrocyte lipoproteins exhibit the most robust interactions with Aβ. Thus, standardization of the methods for detecting apoE/Aβ complex is necessary to determine its functional significance in the neuropathology characteristic of AD. Importantly, a systematic understanding of the role of apoE-containing plasma and CNS lipoproteins in Aβ homeostasis could potentially contribute to identifying a plasma biomarker currently over-looked because it has multiple components.

Objective— Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC). Approach and Results— Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft–mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE −/− SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE −/− SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE −/− SMC, but only partially in wild-type SMC. Conclusions— ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL.

BACKGROUND AND PURPOSE Potent synthetic nonsteroidal liver X receptor (LXR) agonists like T0901317 induce triglyceridaemia and fatty liver, effects not observed with some natural and synthetic steroidal, relatively weak agonists of LXR. To determine if potency is responsible for the lack of side effects with some steroidal agonists, we investigated the in vivo effects of a novel steroidal LXR agonist, ATI-111, that is more potent than T0901317. EXPERIMENTAL APPROACH Eight week old male LDLR–/– mice fed an atherogenic diet were orally treated with vehicle or ATI-111 at 3 and 5 mg·kg−1·day−1 for 8 weeks, and effects on plasma and liver lipid levels, expression of genes involved in lipid metabolism and on atherogenesis were analysed. KEY RESULTS ATI-111 increased the expression of genes involved in lipid transport, such as ABCA1, ABCG1 and ABCG5/G8, in intestine and macrophages; decreased ABCG1, apoE; and slightly increased ABCA1 and ABCG5/G8 expression in liver. ATI-111 markedly increased sterol regulatory element-binding protein (SREBP)-1c mRNA in some tissues, whereas acetyl-coenzyme A carboxylase and fatty acid synthase expression was unaffected or only slightly increased in intestine and liver. ATI-111 inhibited the conversion of SREBP-1c precursor form to its active form. Compared with vehicle-treated mice, the levels of hepatic lipids and liver-secreted nascent lipoproteins were not altered, while a significant decrease in plasma cholesterol and triglyceride levels was observed in ATI-111-treated mice. ATI-111 significantly inhibited atherogenesis in three separate vascular sites. CONCLUSIONS AND IMPLICATIONS ATI-111 is a promising candidate for further development as a treatment of certain vascular diseases as it lacks the significant side effects associated with nonsteroidal LXR agonists, the induction of fatty liver and hypertriglyceridaemia.

The effects of liver X receptor (LXR) agonists on plasma lipid homeostasis, especially triglyceride metabolism are controversial. Here we examined the effect of long-term activation of LXR on plasma lipid homeostasis in wild-type C57BL/6 and LDL receptor deficient (LDLR−/−) mice given the LXR agonist T0901317 for 4 weeks. LXR agonist treatment of wild-type mice decreased plasma total triglycerides by 35% due to a significant reduction of plasma VLDL triglycerides. In contrast, in LDLR−/− mice T0901317 treatment increased plasma total cholesterol and triglycerides. An increase in the level of smaller VLDL particles was also observed in T0901317-treated LDLR−/− mice. The changes in circulating lipoprotein profiles in response to T0901317 treatment in these two animal models reflect the balance between synthesis and secretion on the one hand and lipolysis and clearance on the other. In both models there was both an increase in VLDL production and secretion and in an increase in LPL production and activity in T0901317-treated animals. In wild-type mice lipolysis and clearance predominates, while in the absence of the LDLR, which plays a major role in the clearance of apoB-containing lipoproteins, the increased output predominates. The generation of elevated levels of small VLDL particles due to increased lipolysis may represent an additional risk factor for atherosclerosis.

Modifying apolipoprotein (apo) A-I mimetic peptides to include a proline-punctuated alpha-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function. This study compares the following mimetics, either acetylated or biotinylated (b): 4F (18mer) and 4F-proline-4F (37mer, Pro). b4F interacts with both mouse HDL (moHDL) and LDL in vitro. b4F in vivo plasma clearance kinetics are not affected by mouse HDL level. Administration of biotinylated peptides to mice demonstrates that b4F does not associate with lipoproteins smaller than LDL in vivo, though it does associate with fractions containing free hemoglobin (Hb). In contrast, bPro specifically interacts with HDL. b4F and bPro show opposite binding responses to HDL by surface plasmon resonance. Administration of acetylated Pro to apoE(-/-) mice significantly decreases plasma serum amyloid A levels, while acetylated 4F does not have this ability. In contrast to previous reports that inferred that 4F associates with HDL in vivo, we systematically examined this potential interaction and demonstrated that b4F does not interact with HDL in vivo but rather elutes with Hb-containing plasma fractions. bPro, however, specifically binds to moHDL in vivo. In addition, the number of amphipathic alpha-helices and their linker influences the anti-inflammatory effects of apoA-I mimetic peptides in vivo.

Hyperlipidemia is a major risk factor for developing atherosclerosis in humans, and epidemiological studies have correlated specific lipoprotein levels with cardiovascular disease risk. Murine models of atherosclerosis rely on the induction of hyperlipidemia for vascular lesions to form, but the pathogenic contributions attributed to different lipoprotein populations are not well defined. To address this issue, we analyzed over 300 LDL receptor (LDLR) deficient mice that have been fed a high-fat diet and for which a full lipoprotein profile and aortic root atherosclerosis values were assessed. Overall, aortic root atherosclerosis is best predicted by plasma VLDL cholesterol levels with less predictive value derived from either LDL or HDL cholesterol. Triglyceride levels are more atherogenic in female mice, especially immune competent females, and depletion of the adaptive immune system leads to a global reduction in plasma lipid levels and aortic root lesion size yet does not appear to alter the atherogenic potential of individual lipoprotein subspecies. In contrast, HDL-cholesterol is a better predictor of aortic root atherosclerosis in apoE-deficient mice. In summary, this large scale analysis of high-fat diet fed LDLR deficient mice highlight the relationship between different plasma lipid components, especially VLDL-cholesterol, and aortic root atherosclerosis.

Humans have two major high density lipoprotein (HDL) sub-fractions, HDL2 and HDL3, whereas mice have a monodisperse HDL profile. Epidemiological evidence has suggested that HDL2 is more atheroprotective; however, currently there is no direct experimental evidence to support this postulate. The amino acid sequence of apoA-I is a primary determinant of HDL subclass formation. The majority of the α-helical repeats in human apoA-I are proline-punctuated. A notable exception is the boundary between helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain and the C-terminal hydrophobic domain. In this study we ask whether the substitution of a proline-containing sequence (PCS) separating other helices in human apoA-I for the non-proline-containing sequence (NPCS) between helices 7 and 8 (residues 184–190) influences HDL subclass association. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL2. In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL3. Thus, the specific amino acid sequence between helices 7 and 8 influences HDL subclass association. The wild-type and mutant proteins exhibited similar physicochemical properties except that the two mutants displayed greater lipid-associated stability versus wild-type human apoA-I. These results focus new attention on the influence of the boundary between helices 7 and 8 on the properties of apoA-I. The expression of these mutants in mice may result in the preferential generation of HDL2 or HDL3 and allow us to examine experimentally the anti-atherogenicity of the HDL subclasses.

Leaf extracts of Mangifera indica (L.), a medicinal and horticultural plant were investigated for antibacterial activity against Staphylococcus aureus, Esherichia coli and Pseudomonas aeruginosa. Using the agar-well diffusion and the gradient serial dilution methods the extracts showed weak antibacterial activity against the study organisms compared with the positive control (gentamycin). The ethanolic extract was most active with minimum inhibitory concentration ranging from 5481.0 to 43750.0 l gm l )1 . Chemical tests showed the presence of saponins, steroids and triterpenoids in the ether fraction, alkaloids, anthracenocides, coumarins, flavonones, reducing sugars, catechol and gallic tannins, saponins, steroids and triterpenoids in the ethanolic portion and anthracenocides, flavonones, reducing sugars in the aqueous fraction of the plant extract. These results shows that leaf extracts of M. indica possess some antibacterial activity that could be the basis for their medicinal use in Uganda.

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL's APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.

A new method of performing pump–probe experiments is proposed and experimentally demonstrated by a proof of concept on the millisecond scale. The idea behind this method is to measure the total probe intensity arising from several time points as a group, instead of measuring each time separately. These measurements are multiplexes that are then transformed into the true signal via multiplication with a binary Hadamard S matrix. Each group of probe pulses is determined by using the pattern of a row of the Hadamard S matrix and the experiment is completed by rotating this pattern by one step for each sample excitation until the original pattern is again produced. Thus to measure n time points, n excitation events are needed and n probe patterns each taken from the n × n S matrix. The time resolution is determined by the shortest time between the probe pulses. In principle, this method could be used over all timescales, instead of the conventional pump–probe method which uses delay lines for picosecond and faster time resolution, or fast detectors and oscilloscopes on longer timescales. This new method is particularly suitable for situations where the probe intensity is weak and/or the detector is noisy. When the detector is noisy, there is in principle a signal to noise advantage over conventional pump–probe methods.

Lipid-enriched diets are often used to induce or accelerate the rate of atherosclerotic lesion development in murine models of atherosclerosis. It appears that the induction of persistent hypercholesterolemia to levels >&300 mg/dL is required for the development of experimental atherosclerosis in the mouse. A variety of different diets have been used that vary in the level of cholesterol, the level and type of fatty acid, and the absence or presence of cholate. Each of these components as well as the protein source has been shown to influence lipoprotein level and/or atherosclerosis, with dietary cholesterol being the major proatherogenic component. In some instances the effects of these components on the expression of hepatic genes relevant to lipid homeostasis has been observed. An appreciation of the effect of the differences in diet composition on these processes is important to compare results from different atherosclerosis studies, so the composition of the diets used should always be reported or referenced. Cholate should not be used unless its effects are being specifically investigated.

Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis.