Adverse effects on macrophage lipid transport and survival by high density lipoprotein from patients with coronary heart disease.

High density lipoprotein (HDL)-macrophage interactions have the potential to modulate macrophage function in a beneficial way to prevent the development of lipid-loaded foam cell formation in atherosclerosis. Although HDL is atheroprotective, it can become dysfunctional in chronic inflammatory conditions and increase cardiovascular risk. Here, we examined the effect of dysfunctional-HDL from patients with coronary artery disease, on macrophage function in comparison to functional-HDL from controls. Exposure of macrophages to dysfunctional-HDL for 24 h resulted significant increase in cellular oxidative stress, cholesterol, and cytotoxicity. It also stimulated mitochondrial membrane depolarization, DNA damage, apoptosis, and cleavage of poly (ADP-ribose) polymerase, which are characteristics of proapoptotic pathways. In contrast, functional-HDL treatment maintained cholesterol homeostasis, essential membrane potential, DNA integrity, and cell survival. These results demonstrate that HDL from coronary artery disease (CAD) patient promotes proatherogenic effects that in turn trigger macrophage apoptosis, an important feature in atherogenesis and thereby providing new insight in our understanding of the atherogenic mechanisms.

Sleep promoting potential of low dose α-Asarone in rat model.

Commonly used hypnotics have undesirable side-effects, especially during continuous usage. On the other hand, some herbal products, which are used for prolonged periods, are suggested to have a sleep inducing property, though the claims have not been validated scientifically. The hypnotic potential of α-Asarone, an active principle of Acorus species, was tested in the present study by first identifying the optimal dose of α-Asarone for improving sleep, followed by studies that evaluated the effect of repeated administration of this optimal dose for five days on sleep deprived rats. Of all the doses tested (2, 10, 40, 80 and 120 mg/kg), 10 mg/kg α-Asarone improved the quality of sleep, as indicated by an increased NREM bout duration, reduced arousal index, and decreased bout frequencies of NREM sleep and wakefulness. A marginal decrease in the hypothalamic and body temperatures was also observed. Higher doses, on the other hand, not only reduced the quantity and quality of sleep, but also produced hypothermia. In sleep deprived rats, administration of 10 mg/kg α-Asarone for five consecutive days improved the quality of sleep in contrast to the vehicle and a known hypnotic midazolam. Improvement in NREM sleep quality was observed when the difference between the hypothalamic and the body temperature was minimum. An enhanced association between NREM sleep bout duration and hypothalamic temperature was also observed after administration of 10 mg/kg α-Asarone. This comprehensive study is the first report on the hypnotic property of α-Asarone, which validates its potential to be considered for treatment of insomnia.

High-density lipoprotein from subjects with coronary artery disease promotes macrophage foam cell formation: role of scavenger receptor CD36 and ERK/MAPK signaling.

Although high-density lipoprotein is atheroprotective, it can become dysfunctional in chronic inflammatory conditions and increase cardiovascular risk. We previously demonstrated that HDL from subjects with documented coronary artery disease is dysfunctional and is pro-oxidant/proinflammatory in macrophages. Here we examined the influence of dysfunctional/proinflammatory HDL (piHDL) on lipid accumulation in human macrophages, in comparison to functional HDL (nHDL). Exposure of macrophages to piHDL, in contrast to nHDL, resulted in oxidative stress and marked uptake of lipids from piHDL, leading to the formation of foam cell phenotype as noted by oil red O staining with concomitant increase in total cellular cholesterol content. Using western blotting, we identified that piHDL profoundly upregulated the expression of scavenger receptor CD36 and suppressed the expression of ABCG1 and SRB1 in macrophages, thereby facilitating cholesterol influx capacity of macrophages. We then identified that CD36 did not act alone, indeed it was activated in macrophages along with ERK/MAPK, in response to piHDL, which in turn led to lipid accumulation as well as proinflammatory response via activation of NFkB and subsequent release of proinflammatory markers-TNF-ά and MMP-9. These effects were confirmed using pharmacological inhibitors for either CD36 or ERK/MAPK. Furthermore, piHDL treatment moderately activated PPAR-γ and Nrf2, the known regulators of CD36 in macrophages, suggesting that the two forms of HDL differentially regulate CD36 expression. Taken together, the results demonstrate that a novel CD36-ERK/MAPK-dependent mechanism is involved in macrophage lipid accumulation by piHDL, there by revealing the importance of functional deficiency in HDL and its potential link to atherogenesis.

Evidence for an exclusive association of matrix metalloproteinase-9 with dysfunctional high-density lipoprotein: a novel finding.

OBJECTIVE: High-density lipoprotein is a heterogeneous class of lipoprotein with diverse antiatherogenic functions. However, these antiatherogenic properties of HDL can be compromised in atherosclerotic conditions. We have recently identified dysfunctionality in HDL even among healthy subjects, during systemic inflammation. This study was carried out with the objective of examining whether dysfunctional HDL is associated with pro-inflammatory proteins other than the acute phase proteins as reported earlier. METHODS: Serum HDL was isolated by three different methods-density gradient ultracentrifugation, PEG precipitation and electroelution. The antioxidant property of HDL was assessed as change in oxidation of LDL based on Dichloro-dihydro-fluorescein diacetate assay. HDL was subjected to gelatin zymography and western blot for assessment of MMP 9 activity. RESULTS: Dysfunctional HDL did not prevent the auto-oxidation of LDL. On the contrary the oxidation was enhanced. The zymogram data indicated enhanced MMP-9 activity selectively in dysfunctional HDL, irrespective of HDL isolation methods. This was confirmed by western blot of HDL probed with antibody specific to MMP 9. We also observed that dysfunctional HDL induced inflammatory response in monocyte/macrophages as evidenced by enhanced TNF-α and decreased IL-10 production. Further, invitro incubation of functional HDL with MMP-9 provided direct evidence for the association of MMP-9 with HDL and the role of MMP-9 in HDL dysfunction. CONCLUSION: A remarkable finding in the present study is the previously unrecognized association of MMP-9 with dysfunctional HDL and its proinflammatory property, indicating a novel molecular connection that can enhance the risk of cardiovascular disease in subjects with dysfunctional HDL.

Oxidatively modified high density lipoprotein promotes inflammatory response in human monocytes-macrophages by enhanced production of ROS, TNF-α, MMP-9, and MMP-2.

It has been proposed that high-density lipoprotein (HDL) loses its cardioprotective ability through oxidative modifications by reactive oxygen species (ROS) and promote atherogenesis. However, the pro-atherogenic pathways undergone by oxidized HDL remain poorly understood. Since monocytes play a crucial role in atherogenesis, this study was aimed to investigate the influence of both native and oxidized HDL (oxHDL) on monocytes-macrophages functions relevant to atherogenesis. HDL particles were isolated from human blood samples by ultracentrifugation and subjected to in vitro oxidation with CuSO(4). The extent of oxidation was quantitated by measurement of lipid peroxides. Human peripheral blood mononuclear cells were isolated and cultured under standard conditions. Cells were treated with native and oxHDL at varying concentrations for different time intervals and used for several analyses. Intracellular ROS production was assessed based on ROS-mediated DCFH fluorescence of the cells. The release of TNF-α and matrix metalloproteinases (MMPs) was quantitated using ELISA kit and gelatine zymography, respectively. Treatment of cells with oxidized HDL enhanced the production of ROS in a concentration-dependent way, while native HDL had no such effect. Further, the release of TNF-α, MMP-9, and MMP-2 was found to be remarkably higher in cells incubated with oxHDL than that of native HDL. Results demonstrate that oxidative modification of HDL induces pro-inflammatory response and oxidative stress in human monocytes-macrophages.

A Simple Economical Method for Assay of Atherogenic Small Dense Low-Density Lipoprotein-Cholesterol (sdLDL-C).

In the present study, we report a simple and economical precipitation method for the quantitative determination of small, dense LDL-cholesterol (sdLDL-C) in serum that is considered to be an emerging risk factor for cardiovascular disease. This method consisted of precipitation of lipoproteins of density <1.044 g/ml using heparin-MnCl(2) and quantification of cholesterol existed in the supernatant using reagents for routine cholesterol assay instead of the costly direct low density lipoprotein-cholesterol assay kit. The supernatant contained sdLDL and high-density lipoprotein (HDL) that was confirmed by polyacrylamide gel electrophoresis. sdLDL-C concentration can be calculated by subtracting the HDL-C value from the total cholesterol concentration of the supernatant. sdLDL-C values obtained by this modified method were similar to those obtained by direct assay of sdLDL-C and there was significant correlation between the two methods. In conclusion, this method is highly economical, do not require special equipments and is useful to evaluate atherogenic risk.

IgA1 desialylated by microbial neuraminidase forms immune complex with naturally occurring anti-T antibody in human serum.

IgA1 was identified as the most prominent O-glycosylated protein of human serum. Desialylation by bacterial (Clostridium perfringens) neuraminidase rendered dot-blotted IgA1 recognizable by the naturally occurring serum antibody (anti-T) directed against Thomsen-Friedenreich antigen, Galbeta1-->3GalNAc-alpha-. On Western blot of serum O-glycosylated proteins anti-T recognized nearly all the bands including IgA1 as did the T antigen-specific animal lectin galectin-1 but only after their desialylation. Agglutination of desialylated human erythrocytes by anti-T was effectively inhibited by desialylated IgA1, but not by native IgA1 or other immunoglobulins. Desialylation of serum by neuraminidase led to significantly increased formation of immune complexes containing IgM, the major immunoglobulin type in anti-T on one hand and O-glycosylated proteins/IgA1 on the other. In further evidence for anti-T-desialylated IgA1 immune complex formation, purified anti-T added to desialylated, but not native serum led to formation of additional IgA-IgM immune complexes. Also neuraminidase treatment significantly reduced the titre of free (non-immune complexed) anti-T in serum, while selective removal of anti-T by affinity absorption resulted in considerable decrease in the amount of IgA1 that got converted to immune complexes following enzymatic desialylation of serum. Formation of immune complex between anti-T and neuraminidase-treated IgA1 in serum may be significant since many disease pathogens release neuraminidase and since IgA1 is a powerful ligand for tissue galectin-1 more so after desialylation. Diabetes also raises serum IgA and neuraminidase levels.

Electroelution of lipoprotein(a) [Lp(a)] from native polyacrylamide gels: a new, simple method to purify Lp(a).

Lipoprotein(a), Lp(a), is an atherogenic lipoprotein consisting of an LDL like core particle and a covalently linked glycoprotein of variable size. Lp(a), isolated from serum always contains LDL and HDL(2) as contaminants since Lp(a) floats in the density range 1.05-1.12 g/ml which overlaps that of LDL and HDL(2). Purified Lp(a) is increasingly needed as a standard to overcome various problems in the standardization of Lp(a) measurements and for in vitro biological studies. Problems inherent to the purification of Lp(a) include the aggregation of Lp(a) with LDL, overlapping size distribution and the inability of some fractions to bind to affinity columns. Here, we describe the development of a new method to purify Lp(a) from contaminating LDL and HDL(2) particles. Lp(a) was isolated from serum by sequential ultracentrifugation, resolved by native polyacrylamide gel electrophoresis and the gel segments were electroeluted to obtain pure Lp(a). l-Proline was added to the sample to a final concentration of 0.1 M to prevent the aggregation of Lp(a) with LDL.

Enhanced lipid peroxidation in patients during coronary artery bypass grafting.

Lipid peroxidation products were measured at various time intervals in 20 patients with coronary artery disease, who underwent coronary artery bypass graft (CABG) surgery. Post-operative blood lipid peroxides were found to be significantly higher (p < 0.001) than the preoperative value. Lipid peroxides raised to a peak value of 46.42 +/- 12.86 n mol/g Alb at 5 min of reperfusion when compared to the basal value and afterwards the level declined to 41.02 +/- 7.09 at 2 hrs and remained in that level even at 24 hrs of reperfusion. This increase implies an enhancement in free radical mediated oxidation of membrane lipids during bypass surgery and thus provides evidence for free radical generation during myocardial reperfusion.

Distribution of cholesterol in HDL and its subfractions in patients with coronary atherosclerotic heart disease.

The distribution of HDL-C and its subclasses HDL3-C and HDL2-C and other serum lipids was studied in patients with coronary atherosclerotic heart disease grouped as young males (group 1) and older males (group 2) along with age matched controls. All the patients had significantly higher levels of serum cholesterol, triglycerides, LDL-C and VLDL-C and lower levels of HDL-C. The analysis of HDL - subclasses clearly demonstrated that the low levels of HDL-C was due to the significant decrease of cholesterol in both HDL3 (group 1: 32%; group 2: 30%) and HDL2 subclasses (group 1: 55%; group 2: 48%) compared to the respective control values. Further it has also been observed that this low level of HDL-C is a characteristic feature of patients irrespective of whether the levels of serum cholesterol and triglycerides are high or normal. Although both the HDL subclasses were decreased, the percentage of reduction of cholesterol was greater in the HDL2 than in HDL3 subclass. In addition, the levels of cholesterol either in HDL or any of its subclasses, HDL3 and HDL2 did not show any change in relation to the extent of coronary disease which was assessed by coronary angiography. This study confirms the inverse relation of HDL-C with coronary atherosclerosis and also indicates that, of all the lipid parameters examined, only HDL-C, particularly its subclass HDL2-C, shows independent inverse relation to the incidence of coronary atherosclerotic artery disease in men.

Antioxidant status in relation to free radical production during stable and unstable anginal syndromes.

Lipid peroxidation and the antioxidant status were studied in male patients having stable angina (SA) and unstable angina (UA) pectoris and the results were compared with that of controls. Lipid peroxides (LPx) and conjugated dienes (CD) were found to be elevated in patients with both SA (LPx: 3.96 +/- 1.07, P less than 0.001; CD: 357.09 +/- 66.23, P less than 0.01) and UA (LPx: 4.66 +/- 1.33, CD: 373.33 +/- 49.82, P less than 0.001) than in controls (LPx: 3.22 +/- 0.86, CD: 335.15 +/- 60.27). In SA, the erythrocytes expressed a diminished activity of superoxide dismutase (SOD) (SA: 435.59 +/- 76.02, control: 651.69 +/- 145.90, P less than 0.001) and normal activities of catalase and glutathione peroxidase, whereas in UA it showed enhanced activities of both SOD (UA: 735.72 +/- 145.67, P less than 0.01) and catalase (UA: 21.94 +/- 6.26, control: 18.69 +/- 6.37, P less than 0.01). A significant increase was also noticed in the levels of ceruloplasmin and vitamin E during both types of angina, but not alteration was observed in the levels of transferrin. Further, the patients with diabetes showed maximum levels of lipid peroxides compared to smokers and hypertensives. The level of lipid peroxides was also observed to increase with the severity of disease. This study indicates that free radicals are involved in the pathogenesis and progression of atherosclerotic heart disease.

Interaction of human serum lipoproteins with biomaterials.

Human serum was incubated with representative portions of polyvinyl chloride (PVC) blood storage bags and vascular prostheses. The in vitro interaction process with lipoprotein was followed by polyacrylamide gel electrophoresis (PAGE) using sudan black and nitroblue tetrazolium (NBT) in the prestaining procedure. Densitometric scan of all the lipoprotein bands in serum after incubation with PVC bag material when prestained with sudan black showed remarkable increase in intensity. However, in the same experiment when NBT was used for prestaining no increase in the intensity of any of the lipoprotein bands could be observed. Since sudan black is known to bind cholesterol specifically we suggest that a molecular unfolding occurs when lipoprotein interacts with PVC bag material. When similar experiments were carried out with vascular prosthesis there was conspicuous decrease in the intensity of the high-density lipoprotein (HDL) band especially when stained with NBT. This indicates preferential adhesion of HDL during interaction with vascular prosthesis.

Diet, nutrition intake, and metabolism in populations at high and low risk for colon cancer. Composition, intake, and excretion of fiber constituents.

The role of fiber in human diet in preventing a number of chronic diseases has been a widely debated topic in recent years. The claim that populations at low risk for colon cancer generally consume a more fiber-rich diet than those at high risk, has been used to postulate a protective role for this group of substances. In this study we asked the question whether populations leading different dietary lifestyles and who are at varying risks for colon cancer show marked differences in their dietary and fecal profiles of various fiber components. Four study groups consisting of Seventh-day Adventist (SDA) pure vegetarians, SDA lacto-ovo vegetarians, SDA nonvegetarians, and a group of general population nonvegetarians were selected from the greater Los Angeles Basin area. Three-day composite diets, and stools were analyzed for neutral detergent fiber (NDF), hemi-cellulose, lignin, cellulose, cutin + silica, and pectin. The percentage composition and the daily intake and output of each of these components were computed for each population group. The dietary profile revealed a trend (not statistically significant) toward generally higher daily intake values among the vegetarian subgroups, neutral detergent fiber values in g/day: SDA pure vegetarians, 63.0 +/- 7.9; SDA-lacto-ovo vegetarians, 55.8 +/- 3.5; SDA nonvegetarians, 57.2 +/- 3.5; general population nonvegetarians, 52.5 +/- 4.9), lignin, cellulose, and pectin being the major contributors to this difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Diet, nutrition intake, and metabolism in populations at high and low risk for colon cancer. Determination of fiber and fiber constituents in food and stools.

A procedure is described for the determination of total neutral detergent fiber in composite food and stool samples. This procedure also determines hemicellulose, cellulose, lignin, and cutin + silica in these samples. The quantitation of pectins which involves a separate independent procedure is also described. The present modified neutral detergent fiber procedure yielded values that were significantly higher than the crude fiber method described in previous literature.

Dietary fiber and cholesterol metabolism in rats fed a high cholesterol diet.

The effect of administering blackgram (Phaseolus mungo) fiber (isolated as neutral detergent residue) at the 30% dietary level has been studied with regard to lipid concentration in the tissues and that of biliary and fecal bile acids and sterols. Rats were fed a high fat-cholesterol diet and compared with those fed a cellulose diet. The results indicate that blackgram fiber significantly lowers cholesterol in both serum and aorta [11]. There was an increased concentration of biliary sterols and bile acids and increased fecal excretion of sterols and bile acids, each of these effects being significantly greater than those observed in the rats fed cellulose.