HIV infection and its treatment are associated with dyslipidemia, including hypoalphalipoproteinemia, and increased risk of cardiovascular disease. Parameters of HDL metabolism in HIV-positive patients were investigated in a cross-sectional study. The following groups of subjects were selected: i) 25 treatment-naïve HIV-infected patients or HIV-infected patients on long therapy break, ii) 28 HIV-infected patients currently treated with protease inhibitors, and iii) 33 HIV-negative subjects. Compared to the HIV-negative group, all groups of HIV-infected patients were characterized by significantly elevated triglyceride and apolipoprotein B levels, mass and activity of lecithin cholesterol acyl transferase and cholesteryl ester transfer protein (p<0.01). Total and LDL cholesterol was lower in treatment-naïve HIV-infected group only. HDL cholesterol and preβ 1 -HDL were significantly lower in all HIV-infected groups (p<0.05), while mean levels of apolipoprotein A-I (apoA-I) and ability of plasma to promote cholesterol efflux were similar in all groups. We found a positive correlation between apoA-I and levels of CD4+ cells (r 2 = 0.3, p<0.001). Plasma level of phospholipid transfer protein was reduced in the group on antiretroviral therapy. Taken together these results suggest that HIV infection is associated with modified HDL metabolism re-directing cholesterol to the apoB-containing lipoproteins and likely reducing the functionality of reverse cholesterol transport.

Both increased (mice study) and decreased (hamster study) CETP activity could result in enhanced RCT.

A cDNA encoding a rat intestinal Ca 2؉ -independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH 2 -terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A 2 , lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of >90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.Digestion of phospholipids and triacylglycerol in gastrointestinal tract involves several hydrolysis reactions. A variety of lipases, including acid lipase in lingual gland or stomach, and pancreatic lipase (which is of primary importance in luminal digestion of fats), hydrolyze triacylglycerol to produce monoacylglycerols and free fatty acids (see Refs. 1-3 for reviews)).Pancreatic phospholipases A 2 (PLA 2 ) 1 hydrolyze ester bonds at the sn-2 position of glycerophospholipids and produce fatty acids and lysophospholipids. These steps are prerequisites for lipid absorption by intestinal epithelium cells (4). Lysophospholipid can be directly absorbed, or hydrolyzed by pancreatic lysophospholipase and converted to glycerol 3-phosphate esters and fatty acids.Until recently, all those processes were believed to proceed in the lumen of alimentary tracts by the action of secretory enzymes mentioned above. However, recent studies suggest the presence of a lipid-hydrolyzing enzyme associated with intestinal brush border membranes (5, 6), named phospholipase B/lipase (PLB/LIP) because this enzyme displayed broad lipolytic activities (PLA 2 , lysophospholipase, and lipase activities) (7). PLB/LIP might participate in terminal digestion, or membrane digestion, of dietary lipids and biliary phospholipids, like well established glycosidases and pe...

Eight proteins potentially involved in cholesterol efflux [ABCA1, ABCG1, CYP27A1, phospholipid transfer protein (PLTP), scavenger receptor type BI (SR-BI), caveolin-1, cholesteryl ester transfer protein, and apolipoprotein A-I (apoA-I)] were overexpressed alone or in combination in RAW 264.7 macrophages. When apoA-I was used as an acceptor, overexpression of the combination of ABCA1, CYP27A1, PLTP, and SR-BI (Combination I) enhanced the efflux by 4.3-fold. It was established that the stimulation of efflux was due to increased abundance of ABCA1 and increased apoA-I binding to non-ABCA1 sites on macrophages. This combination caused only a small increase of the efflux to isolated HDL. When HDL was used as an acceptor, overexpression of caveolin-1 or a combination of caveolin-1 and SR-BI (Combination II) was the most active, doubling the efflux to HDL, without affecting the efflux to apoA-I. When tested in the in vivo mouse model of cholesterol efflux, overexpression of ABCA1 and Combination I elevated cholesterol export from macrophages to plasma, liver, and feces, whereas overexpression of caveolin-1 or Combination II did not have an effect. We conclude that pathways of cholesterol efflux using apoA-I as an acceptor make a predominant contribution to cholesterol export from macrophages in vivo.

Macrophages play an important role in host defense pathways and are also involved in a variety of diseases, including atherosclerosis (1, 2). The key role of macrophages in the development of atherosclerosis has made this cell type a versatile in vitro model of this disease (3).Transfection of macrophages is a powerful tool to study their function, and a number of methods have been described to achieve high levels of expression of different genes through transient transfection (4-6). These levels of expression are sufficiently high to study synthetic processes, when proteins are tagged or otherwise distinguished from host proteins. However, studying cell functions such as growth, lipoprotein binding, lipid uptake, and efflux requires not only high levels of gene expression but also for the gene to be expressed in a majority of cells, a highefficiency transfection. High efficiency of transfection is also critical for a multiple gene transfection, as it requires that all transfected genes be expressed in the same population of cells. Viral and stable transfections offer adequate efficiency of DNA transfer; however, they are often laborintensive and time-consuming. High-efficiency transient transfection of macrophages has proved to be difficult.Here, we describe a method for high-efficiency transient transfection of RAW 264.7 mouse macrophages. We fortuitously found that the low efficiency of expression of transfected genes in macrophages is a consequence of methylation-mediated silencing of transfected genes rather than of low uptake of DNA into cells. To maximize the efficiency of macrophage transfection, we evaluated the DNA methylation inhibitor, 5-azacytidine, an epigenetic modifier often used to reactivate methylation-dependent transcriptionally silent genes (7). We demonstrated by methylationspecific PCR that 5-azacytidine prevents methylation of the promoter of transfected genes, and for the first time we achieved transient expression of a reporter protein in 80-100% of macrophage cells. The method was then used for the high-efficiency transient transfection of RAW 264.7 macrophages with sterol 27-hydroxylase (CYP27A1), which led to the stimulation of cholesterol efflux from these cells.Abbreviations: apoA-I, apolipoprotein A-I; CMV, cytomegalovirus; CYP27A1, sterol 27-hydroxylase; GFP, green fluorescent protein; LXR, liver X receptor.

HDL-phospholipids (HDL-PL) play an important role in reverse cholesterol transport (RCT). Phosphatidylcholine (PC) is the most important phospholipid in RCT because it is the essential cholesterol-binding component of lipoproteins and is the acyl donor in the esterification of FC by lecithin:cholesterol acyltransferase (LCAT). FC efflux to sera is a positive anti-atherogenic function of HDL-PL. Although PC has long been recognized as an anti-atherogenic agent, developement of new HDL therapies based on PC have been fraught with issues of efficacy, cost, and safety. Moreover, some methods to increase HDL-PC perturb HDL and release lipid-free apolipoproteins (apo) A-I. We developed a new method, HDL SPLn (SPLn) using a modified detergent removal method that obviates these concerns. SPLn can incorporate PC into HDL and increase HDL-PC >10 fold. This is achieved with no loss of apo A-I. According to size exclusion chromatography and native gradient gel electrophoresis, SPLn raises the HDL particle weight in a dose-dependent way, from ~120 kDa to ~350 kDa. Kinetic analysis of FC efflux to the resulting SPLn particles shows that K m and V max for SPLn HDL are lower and higher respectively than for native HDL. As a consequence, the catalytic efficiency, Vmax/Km, increases by more than 400%. Clinically, small increases in serum HDL-PL are associated with significant and profound increases in FC efflux to serum. Treatment of relatively small amounts of plasma by SPLn is a potential method of improving at least one step in RCT.

Phospholipase A 2 (PLA 2 ) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca 2؉ ions for activity and exhibited both phosphatidic acid-preferring PLA 2 and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA 2 and lipase activities. The sequence of NH 2 -terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA 2 activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA 2 -resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquinetreated rats, treated with excess of pancreatic PLA 2 , and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.

BACKGROUND Obesity-linked metabolic syndrome (MetS) is associated with a dyslipidemic profile that includes hypertriglyceridemia and low plasma high-density lipoprotein cholesterol (HDL-C). HDL initiates reverse cholesterol transport (RCT) via macrophage cholesterol efflux (MCE). Some hypothesize that dyslipidemic patients have impaired RCT. MCE to patient plasma, a metric of HDL function, inversely correlates with atherosclerotic burden. Paradoxically, MCE to plasma of hypertriglyceridemic subjects is higher than that to normolipidemic (NL) plasma. OBJECTIVE Although weight-loss reduces dyslipidemia, its effect on MCE to the plasma of obese, MetS patients is unknown. Thus, we tested the hypothesis that reducing dyslipidemia with weight-loss reduces the MCE capacity of MetS plasma to that of NL plasma. METHODS Cholesterol efflux (MCE) from THP-1 macrophages to plasma from NL controls and to obese, MetS patients before and after weight-loss was measured. RESULTS MCE to plasma of obese, MetS patients was higher than that to control plasma (p=0.006). Weight-loss in MetS patients (mean = −9.77 kg) reduced dyslipidemia, insulin resistance and systolic blood pressure. HDL-C was unchanged and apolipoprotein A-I decreased with weight-loss. Weight-loss in MetS patients normalized MCE (p<0.001) to that of NL subjects. MCE correlated with apolipoprotein B levels (r2 = 0.13 – 0.38). Chromatography showed that macrophage cholesterol initially associates with HDL but accumulates in apolipoprotein B-containing lipoproteins at later times. CONCLUSIONS While the initial acceptor of macrophage cholesterol efflux is HDL, the elevated apo B lipoproteins are a cholesterol sink that increase MCE in MetS patients. Weight loss results in decreased apo B-lipoproteins and decreased MCE to plasma of MetS patients.