Plaque stabilizing effects of apolipoprotein A-IV.

BACKGROUND AND AIMS: Apolipoprotein (apo) A-IV, the third most abundant HDL-associated protein, is atheroprotective and shares similar properties as apoA-I. We have reported previously that apoA-I, the most abundant apolipoprotein in HDL, inhibits plaque disruption in a mouse model. We aimed at examining the effects of apoA-IV on markers of plaque stability in vivo. METHODS: Plaques within brachiocephalic arteries of 16-week old apoE-knockout C57BL/6 mice were examined for changes in composition after 10 weeks on a high-fat diet (HFD). The animals received twice-weekly injections of human lipid-free apoA-IV (1 mg/kg, n = 31) or PBS (n = 32) during the 9th and 10th weeks of the HFD. RESULTS: In the apoA-IV treated mice, there were significantly fewer hemorrhagic plaque disruptions (9/31 vs. 18/32, p < 0.05), thicker fibrous caps, smaller lipid cores, a lower macrophage:SMC ratio, less MMP-9 protein, more collagen, and fewer proliferating cells. In the plaques of mice given apoA-IV, MCP-1, VCAM-1, and inducible NOS were also significantly lower. Based on the percentage of cleaved PARP-positive and TUNEL-positive plaque nuclei, apoA-IV reduced apoptosis. in HMDMs, apoA-IV reduced MMP-9 mRNA expression by half, doubled mRNA levels of TIMP1 and decreased MMP-9 activity. CONCLUSIONS: ApoA-IV treatment is associated with a more stable plaque phenotype and a reduced incidence of acute disruptions in this mouse model.

Assessment of cardiometabolic risk and prevalence of meeting treatment guidelines among patients with type 2 diabetes stratified according to their use of insulin and/or other diabetic medications: results from INSPIRE ME IAA.

AIM: Visceral adipose tissue (VAT) and liver fat (LF) are strongly associated with type 2 diabetes. It is not known, however, how diabetes treatment and/or risk factor management modulates the association between VAT, LF and diabetes. The aim was to determine the level of VAT and LF in patients with type 2 diabetes according to their treatment status and achievement of the American Diabetes Association's (ADA) diabetes management goals. METHODS: We performed a cross-sectional analysis of the baseline data of the International Study of the Prediction of Intra-Abdominal Adiposity and its Relationship with Cardiometabolic risk/Intra-Abdominal Adiposity (INSPIRE ME IAA), a 3-year prospective cardiometabolic imaging study conducted in 29 countries. Patients (n = 3991) were divided into four groups: (i) those without type 2 diabetes (noT2D n = 1003 men, n = 1027 women); (ii) those with type 2 diabetes but not treated with diabetes medications (T2Dnomeds n = 248 men, n = 198 women); (iii) those with type 2 diabetes and treated with diabetes medications but not yet using insulin (T2Dmeds-ins n = 591 men, n = 484 women) and (iv) those with type 2 diabetes and treated with insulin (T2Dmeds+ins n = 233 men, n = 207 women). Abdominal and liver adiposity were measured by computed tomography. RESULTS: Fewer patients with high VAT or LF achieved the ADA's goals for high-density lipoprotein cholesterol (HDL-C) or triglycerides compared to patients with low VAT or LF. Visceral adiposity (p = 0.02 men, p = 0.003 women) and LF (p = 0.0002 men, p = 0.0004 women) increased among patients who met fewer of the ADA treatment criteria, regardless of type 2 diabetes treatment. CONCLUSION: Residual cardiometabolic risk exists among patients with type 2 diabetes characterized by elevated VAT and LF.

Comparison of the effects of different statins and doses on lipid levels in patients with diabetes: results from VOYAGER.

BACKGROUND AND AIMS: Diabetes mellitus is a well-known risk factor for cardiovascular disease, and brings an increased risk of vascular events and a higher mortality rate. Treatment guidelines recommend statins in patients with diabetes, with low-density lipoprotein cholesterol (LDL-C) targets of 100 mg dl(-1) (∼2.5 mmol l(-1)), and 80 (∼2.0 mmol l(-1)) or 70 mg dl(-1) (∼1.8 mmol l(-1)) in especially high-risk patients. The current study used the VOYAGER (an indiVidual patient data-meta-analysis Of statin therapY in At risk Groups: Effects of Rosuvastatin, atorvastatin, and simvastatin) database to characterise effects of rosuvastatin, atorvastatin and simvastatin in different doses on lipid levels in diabetes patients. METHODS AND RESULTS: The VOYAGER database included individual patient data from 37 studies involving comparisons of rosuvastatin with either atorvastatin or simvastatin. Of the 32 258 patients included, 8859 (27.5%) had diabetes. Rosuvastatin appeared to be the most efficacious of the three statins, both for lowering LDL-C and for reaching a target level of <70 mg dl(-1) for LDL-C. It was also more effective than atorvastatin at raising high-density lipoprotein cholesterol in the diabetes population. These results are consistent with the overall VOYAGER results. CONCLUSIONS: This meta-analysis of 8859 patients with diabetes mellitus shows favourable effects on lipids with the three statins studied, in line with results for the overall VOYAGER population. The importance of using an effective statin at an effective dose to reach treatment goals for such high-risk patients is evident.

Ability of traditional lipid ratios and apolipoprotein ratios to predict cardiovascular risk in people with type 2 diabetes.

AIMS/HYPOTHESIS: The apolipoprotein B (ApoB):apolipoprotein A (ApoA)-I ratio may be a better indicator of cardiovascular disease (CVD) risk in people with type 2 diabetes than traditional lipid risk markers (LDL-cholesterol, HDL-cholesterol and triacylglycerol), but whether the ApoB:ApoA-I ratio should be used to indicate lipid-lowering therapy is still debated. METHODS: The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study randomised 9,795 patients with type 2 diabetes to fenofibrate (200 mg daily) or placebo and followed them up for a median of 5 years. We compared ApoB, ApoA-I, ApoAII and the ApoB:ApoA-I ratio with traditional lipid variables as predictors of CVD risk. We estimated the HR of the effect of 1 SD difference in baseline concentrations of lipids, apolipoproteins and respective ratios on the risk of CVD events and also used receiver operating characteristic curve analysis. RESULTS: In the placebo group, the variables best predicting CVD events were non-HDL-cholesterol:HDL-cholesterol, total cholesterol:HDL-cholesterol (HR 1.21, p < 0.001 for both), ApoB:ApoA-I (HR 1.20, p < 0.001), LDL-cholesterol:HDL-cholesterol (HR 1.17, p < 0.001), HDL-cholesterol (HR 0.84, p < 0.001) and ApoA-I (HR 0.85, p < 0.001). In the fenofibrate group, the first four predictors were very similar (but ApoB:ApoA-I was fourth), followed by non-HDL-cholesterol and ApoB. Lipid ratios and ApoB:ApoA-I performed better than any single lipid or apolipoprotein in predicting CVD risk. CONCLUSIONS/INTERPRETATION: In patients with type 2 diabetes in the FIELD study, traditional lipid ratios were as strong as the ApoB:ApoA-I ratio in predicting CVD risk. The data provide little evidence for replacement of traditional lipids and their ratios with measures of ApoB, ApoA-I and their ratio.

Role of 3beta-hydroxysteroid-delta 24 reductase in mediating antiinflammatory effects of high-density lipoproteins in endothelial cells.

OBJECTIVE: The purpose of this study was to investigate the ability of high-density lipoproteins (HDLs) to upregulate genes with the potential to protect against inflammation in endothelial cells. METHODS AND RESULTS: Human coronary artery endothelial cells (HCAECs) were exposed to reconstituted HDLs (rHDLs) for 16 hours before being activated with tumor necrosis factor-alpha (TNF-alpha) for 5 hours. rHDLs decreased vascular cell adhesion molecule-1 (VCAM-1) promoter activity by 75% (P<0.05), via the nuclear factor-kappa B (NF-kappaB) binding site. rHDLs suppressed the canonical NF-kappaB pathway and decreased many NF-kappaB target genes. Suppression of NF-kappaB and VCAM-1 expression by rHDLs or native HDLs was dependent on an increase in 3beta-hydroxysteroid-Delta 24 reductase (DHCR24) levels (P<0.05). The effect of HDLs on DHCR24 is dependent on SR-BI but not ABCAI or ABCGI. Silencing DHCR24 expression increased NF-kappaB (1.2-fold, P<0.05), VCAM-1 (30-fold, P<0.05), and NF-kappaB p50 (4-fold, P<0.05) and p65 subunits (150-fold, P<0.05). TNF-alpha activation of siDHCR24-treated cells increased expression of VCAM-1 (550-fold, P<0.001) and NF-kappaB (9-fold, P<0.001) that could no longer be suppressed by rHDLs. CONCLUSIONS: Results suggest that antiinflammatory effects of rHDLs are mediated partly through an upregulation of DHCR24. These findings raise the possibility of considering DHCR24 as a target for therapeutic modulation.

Effects of cross-link breakers, glycation inhibitors and insulin sensitisers on HDL function and the non-enzymatic glycation of apolipoprotein A-I.

AIMS/HYPOTHESIS: Hyperglycaemia, a key feature of diabetes, is associated with non-enzymatic glycation of plasma proteins. We have shown previously that the reactive alpha-oxoaldehyde, methylglyoxal, non-enzymatically glycates apolipoprotein (Apo)A-I, the main apolipoprotein of HDL, and prevents it from activating lecithin:cholesterol acyltransferase (LCAT), the enzyme that generates almost all of the cholesteryl esters in plasma. This study investigates whether the glycation inhibitors aminoguanidine and pyridoxamine, the insulin sensitiser metformin and the cross-link breaker alagebrium can inhibit and/or reverse the methylglyoxal-mediated glycation of ApoA-I and whether these changes can preserve or restore the ability of ApoA-I to activate LCAT. METHODS: Inhibition of ApoA-I glycation was assessed by incubating aminoguanidine, pyridoxamine, metformin and alagebrium with mixtures of methylglyoxal and discoidal reconstituted HDL (rHDL) containing phosphatidylcholine and ApoA-I, ([A-I]rHDL). Glycation was assessed as the modification of ApoA-I arginine, lysine and tryptophan residues, and by the extent of ApoA-I cross-linking. The reversal of ApoA-I glycation was investigated by pre-incubating discoidal (A-I)rHDL with methylglyoxal, then incubating the modified rHDL with aminoguanidine, pyridoxamine or alagebrium. RESULTS: Aminoguanidine, pyridoxamine, metformin and alagebrium all decreased the methylglyoxal-mediated glycation of the ApoA-I in discoidal rHDL and conserved the ability of the particles to act as substrates for LCAT. However, neither aminoguanidine, pyridoxamine nor alagebrium could reverse the glycation of ApoA-I or restore its ability to activate LCAT. CONCLUSIONS/INTERPRETATION: Glycation inhibitors, insulin sensitisers and cross-link breakers are important for preserving normal HDL function in diabetes.

Aggressive management of obesity in children and young adults: the known challenges and potential opportunities.

The world is confronted with an impending epidemic of premature cardiovascular disease (CVD) that is being fueled by an increase in the prevalence of central obesity.

The impact of glycation on apolipoprotein A-I structure and its ability to activate lecithin:cholesterol acyltransferase.

AIMS/HYPOTHESIS: Hyperglycaemia, one of the main features of diabetes, results in non-enzymatic glycation of plasma proteins, including apolipoprotein A-I (apoA-I), the most abundant apolipoprotein in HDL. The aim of this study was to determine how glycation affects the structure of apoA-I and its ability to activate lecithin:cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport. MATERIALS AND METHODS: Discoidal reconstituted HDL (rHDL) containing phosphatidylcholine and apoA-I ([A-I]rHDL) were prepared by the cholate dialysis method and glycated by incubation with methylglyoxal. Glycation of apoA-I was quantified as the reduction in detectable arginine, lysine and tryptophan residues. Methylglyoxal-AGE adduct formation in apoA-I was assessed by immunoblotting. (A-I)rHDL size and surface charge were determined by non-denaturing gradient gel electrophoresis and agarose gel electrophoresis, respectively. The kinetics of the LCAT reaction was investigated by incubating varying concentrations of discoidal (A-I)rHDL with a constant amount of purified enzyme. The conformation of apoA-I was assessed by surface plasmon resonance. RESULTS: Methylglyoxal-mediated modifications of the arginine, lysine and tryptophan residues in lipid-free and lipid-associated apoA-I were time- and concentration-dependent. These modifications altered the conformation of apoA-I in regions critical for LCAT activation and lipid binding. They also decreased (A-I)rHDL size and surface charge. The rate of LCAT-mediated cholesterol esterification in (A-I)rHDL varied according to the level of apoA-I glycation and progressively decreased as the extent of apoA-I glycation increased. CONCLUSIONS/INTERPRETATION: It is concluded that glycation of apoA-I may adversely affect reverse cholesterol transport in subjects with diabetes.

The rationale for using apoA-I as a clinical marker of cardiovascular risk.

An inverse relationship between the concentration of high-density lipoprotein (HDL) cholesterol and the risk of developing cardiovascular is well established. There are several documented functions of HDLs that may contribute to a protective role of these lipoproteins. These include the ability of HDLs to promote the efflux of cholesterol from macrophages and foam cells in the artery wall and to anti-inflammatory/antioxidant properties of these lipoproteins. The fact that the main apolipoprotein of HDLs, apoA-I, plays a prominent role in each of these functions adds support to the view that apoA-I should be measured as a component of the assessment of cardiovascular risk in humans.

Apo B versus cholesterol in estimating cardiovascular risk and in guiding therapy: report of the thirty-person/ten-country panel.

There is abundant evidence that the risk of atherosclerotic vascular disease is directly related to plasma cholesterol levels. Accordingly, all of the national and transnational screening and therapeutic guidelines are based on total or LDL cholesterol. This presumes that cholesterol is the most important lipoprotein-related proatherogenic risk variable. On the contrary, risk appears to be more directly related to the number of circulating atherogenic particles that contact and enter the arterial wall than to the measured concentration of cholesterol in these lipoprotein fractions. Each of the atherogenic lipoprotein particles contains a single molecule of apolipoprotein (apo) B and therefore the concentration of apo B provides a direct measure of the number of circulating atherogenic lipoproteins. Evidence from fundamental, epidemiological and clinical trial studies indicates that apo B is superior to any of the cholesterol indices to recognize those at increased risk of vascular disease and to judge the adequacy of lipid-lowering therapy. On the basis of this evidence, we believe that apo B should be included in all guidelines as an indicator of cardiovascular risk. In addition, the present target adopted by the Canadian guideline groups of an apo B <90 mg dL(-1) in high-risk patients should be reassessed in the light of the new clinical trial results and a new ultra-low target of <80 mg dL(-1) be considered. The evidence also indicates that the apo B/apo A-I ratio is superior to any of the conventional cholesterol ratios in patients without symptomatic vascular disease or diabetes to evaluate the lipoprotein-related risk of vascular disease.

Effect of inhibiting cholesteryl ester transfer protein on the kinetics of high-density lipoprotein cholesteryl ester transport in plasma: in vivo studies in rabbits.

OBJECTIVE: Inhibitors of cholesteryl ester transfer protein (CETP) have been developed as potential anti-atherogenic agents. Theoretically, however, they may be pro-atherogenic by blocking one of the pathways for removing high-density lipoprotein (HDL) cholesteryl esters (CE) from plasma in the final step of reverse cholesterol transport. Here we describe how CETP inhibition in rabbits impacts on the kinetics of HDL CE transport in plasma. METHODS AND RESULTS: Administration of a CETP inhibitor reduced CETP activity by 80% to 90% and doubled the HDL cholesteryl ester concentration. Multi-compartmental analysis was used to determine HDL CE kinetics in CETP-inhibited and control rabbits after injection of tracer amounts of both native and reconstituted HDL labeled with 3H in the CE moiety. In control rabbits, HDL CE was removed from plasma by both a direct pathway and an indirect pathway after transfer of HDL CE to the very-low-density lipoprotein/low-density lipoprotein fraction. In CETP-inhibited rabbits there was an almost complete block in removal via the indirect pathway. This did not compromise the overall removal of HDL CE from plasma, which was not different in control and inhibited animals. CONCLUSIONS: Inhibiting CETP in rabbits does not compromise the removal of HDL CE from plasma.

Investigation of pond velocities using dye and small drogues: a case study of the Nelson City waste stabilisation pond.

The city of Nelson, New Zealand, has a 27 hectare oxidation pond as its primary wastewater treatment facility. Recent changes in the configuration of the pond and installation of a mixer/aerator raised concerns that pond treatment rates and effluent quality may be affected by high internal pond velocities and short retention times. This paper presents the findings of an investigation into wastewater velocity and movement within the pond using qualitative dye dispersion and tracking of small-scale "holey-sock" drogues. Simultaneous deployment of drogues and dye allowed methods to be compared, since small-scale drogues have not commonly been used in wastewater ponds. Dye dispersion was assessed using low-level aerial photography from a tethered helium blimp to track short term movement and mixing, while a datalogger and fluorometer were used to measure pond retention time. Drogue movement was tracked in conjunction with the dye study from a small boat using hand-held GPS. The dye study found that: (i) the first portion of pond influent discharged from the pond after 37.5 hours, substantially quicker than the theoretical pond retention time of 27 days. However, the measured retention time was with a mixer in place and the theoretical time was without a mixer; (ii) the position of the paddle wheel mixer/aerator was not optimally placed for mixing the influent and a quiescent region existed adjacent to the influent point; and (iii) the low-level aerial photography was an effective method of evaluating larger pond systems. The "holey-sock" drogue studies showed that: (i) the drogues accurately followed the movement and velocity of dyed influent within the pond; (ii) wastewater velocity and movement was dominated by the paddle wheel mixer/aerator; and (iii) wind direction had a minor influence on wastewater velocity and movement in areas not directly affected by the paddle wheel mixer/aerator. The study demonstrated that the combined use of dye and drogues was a relatively low-cost and effective means of determining internal pond velocities and movement. Future studies using similar methods will be useful in helping validate computer-modelled movement and velocity.

Hugh sinclair lecture: the regulation and remodelling of HDL by plasma factors.

High density lipoproteins (HDLs) originate as lipid-free or lipid-poor apolipoproteins that acquire most of their lipid in the extracellular space. They accept phospholipids from cells in a process promoted by the ATP binding cassette A1 transporter to form prebeta-migrating discoidal HDL that are efficient acceptors of cholesterol released from cell membranes. The cholesterol in discoidal HDL is esterified by lecithin:cholesterol acyltransferase (LCAT) in a process that converts the prebeta-migrating disc into an alpha-migrating, spherical HDL. Spherical HDL are further remodelled by cholesteryl ester transfer protein (CETP) that transfers cholesteryl esters from HDL to other lipoproteins and by hepatic lipase that hydrolyses HDL triglyceride in processes that reduce HDL size and lead to the dissociation of prebeta-migrating, lipid-poor apolipoprotein (apo)A-I from the particle. Prebeta-migrating, lipid-poor apoA-I is also generated as a product of the remodelling of HDL by phospholipid transfer protein. Thus, apoA-I cycles between lipid-poor and lipid associated forms as part of a highly dynamic metabolism of HDL. The other main HDL apolipoprotein, apoA-II is incorporated into apoA-I-containing particles in a process of particle fusion mediated by LCAT. Extracellular assembly and remodelling of HDL not only plays a major role in HDL regulation but also provides potential targets for therapeutic intervention. One example of this is the development of inhibitors of CETP.

Cholesteryl ester transfer protein, high density lipoprotein and arterial disease.

The reported relationships between cholesteryl ester transfer protein, high density lipoproteins and arterial disease are confusing and conflicting. Several papers published during the review period add substantially to the evidence base regarding the atherogenicity (or anti-atherogenicity) of cholesteryl ester transfer protein, although none clearly resolves the continuing conflict. These new papers are presented against the backdrop of the previous state of knowledge.

Threshold level or not for low-density lipoprotein cholesterol.

As drugs, such as the statins, and other therapies demonstrate the ability to significantly lower levels of low-density lipoprotein cholesterol (LDL-C), one issue is whether there is a lower threshold below which no further decline in coronary heart disease occurs. Those who evaluate the data from multiple trials and conclude that no significant decrease in coronary event rates occurs at or below 125 mg/dL suggest using this level as a guideline for clinical application of cholesterol-lowering therapy. On the other hand, analysis of the results of the same population and primary prevention studies concludes that no such threshold exists. The issues affected by the decision of whether to use a threshold include costs to the healthcare system for additional physician time, tests, and medication; unknown clinical events and safety related to very low LDL-C; and resource prioritization to an unestablished therapeutic approach.

Time sequence of the inhibition of endothelial adhesion molecule expression by reconstituted high density lipoproteins.

We have used discoidal reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I and dimyristoyl phosphatidylcholine (DMPC) as a tool to investigate the time sequence of the HDL-mediated inhibition of vascular cell adhesion molecule (VCAM)-1 and E-selectin expression in cytokine-activated human umbilical vein endothelial cells (HUVECs). Specifically, we have asked a few questions - (i) how long do the cells need to be exposed to the rHDL before adhesion molecule expression is inhibited and (ii) how long does the inhibition persist after removing the rHDL from the cells. When the cells were not pre-incubated with the rHDL, there was no inhibition. The magnitude of the inhibition increased progressively with increasing duration of pre-incubation up to 16 h. Inhibition did not require the rHDL to be physically present during the activation of adhesion molecule expression by tumour necrosis factor(TNF)-alpha, excluding the possibility that the rHDL was merely interfering with the interaction between TNF-alpha and the cells. When HUVECs were pre-incubated for 16 h with rHDL, the inhibition remained substantial even if the rHDL were removed from the medium up to 8 h prior to addition of TNF-alpha. The HDL-mediated inhibition of VCAM-1 in HUVECs was unaffected by the presence of puromycin, an inhibitor of protein synthesis, excluding the possibility that HDL may have acted by stimulating the synthesis of a cell protein that itself inhibits adhesion molecule expression. These results have important implications in terms of understanding the mechanism(s) of the HDL-mediated inhibition of endothelial adhesion molecule expression.

Evidence that apolipoprotein A-I facilitates hepatic lipase-mediated phospholipid hydrolysis in reconstituted HDL containing apolipoprotein A-II.

This study examines hepatic lipase (HL) mediated phospholipid hydrolysis in mixtures of apolipoprotein-specific, spherical reconstituted high-density lipoproteins (rHDL). We have shown previously that apolipoprotein A-I (apoA-I) and apoA-II have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in well-characterized, homogeneous preparations of spherical rHDL [Hime, N. J., Barter, P. J., and Rye, K.-A. (1998) J. Biol. Chem. 273, 27191-27198]. In the present study, phospholipid hydrolysis was assessed in mixtures of rHDL containing either apoA-I only, (A-I)rHDL, apoA-II only, (A-II)rHDL, or both apoA-I and apoA-II, (A-I/A-II)rHDL. The rHDL contained trace amounts of radiolabeled phospholipid, and hydrolysis was measured as the formation of radiolabeled nonesterified fatty acids (NEFA). As predicted from our previous kinetic studies, the (A-II)rHDL acted as competitive inhibitors of HL-mediated phospholipid hydrolysis in (A-I)rHDL. Less expected was the observation that the rate of phospholipid hydrolysis in (A-II)rHDL was enhanced when (A-I)rHDL were also present in the incubation mixture. The rate of phospholipid hydrolysis in (A-I/A-II)rHDL was also greater than in (A-II)rHDL, indicating that apoA-I enhances phospholipid hydrolysis when it is present as a component of (A-I/A-II)rHDL. It is concluded that apoA-I enhances HL-mediated phospholipid hydrolysis in apoA-II containing rHDL, irrespective of whether the apoA-I is present in the same particle as the apoA-II [as in (A-I/A-II)rHDL] or whether it is present as a component of a different particle, such as when (A-I)rHDL are added to incubations of (A-II)rHDL.