Elevated leukocyte cell numbers (leukocytosis), and monocytes in particular, promote atherosclerosis; however, how they become increased is poorly understood. Mice deficient in the adenosine triphosphate-binding cassette (ABC) transporters ABCA1 and ABCG1, which promote cholesterol efflux from macrophages and suppress atherosclerosis in hypercholesterolemic mice, displayed leukocytosis, a transplantable myeloproliferative disorder, and a dramatic expansion of the stem and progenitor cell population containing Lin(-)Sca-1(+)Kit+ (LSK) in the bone marrow. Transplantation of Abca1(-/-) Abcg1(-/-) bone marrow into apolipoprotein A-1 transgenic mice with elevated levels of high-density lipoprotein (HDL) suppressed the LSK population, reduced leukocytosis, reversed the myeloproliferative disorder, and accelerated atherosclerosis. The findings indicate that ABCA1, ABCG1, and HDL inhibit the proliferation of hematopoietic stem and multipotential progenitor cells and connect expansion of these populations with leukocytosis and accelerated atherosclerosis.

The liver is an essential metabolic organ, and its metabolic function is controlled by insulin and other metabolic hormones. Glucose is converted into pyruvate through glycolysis in the cytoplasm, and pyruvate is subsequently oxidized in the mitochondria to generate ATP through the TCA cycle and oxidative phosphorylation. In the fed state, glycolytic products are used to synthesize fatty acids through de novo lipogenesis. Long-chain fatty acids are incorporated into triacylglycerol, phospholipids, and/or cholesterol esters in hepatocytes. These complex lipids are stored in lipid droplets and membrane structures, or secreted into the circulation as very low-density lipoprotein particles. In the fasted state, the liver secretes glucose through both glycogenolysis and gluconeogenesis. During pronged fasting, hepatic gluconeogenesis is the primary source for endogenous glucose production. Fasting also promotes lipolysis in adipose tissue, resulting in release of nonesterified fatty acids which are converted into ketone bodies in hepatic mitochondria though β-oxidation and ketogenesis. Ketone bodies provide a metabolic fuel for extrahepatic tissues. Liver energy metabolism is tightly regulated by neuronal and hormonal signals. The sympathetic system stimulates, whereas the parasympathetic system suppresses, hepatic gluconeogenesis. Insulin stimulates glycolysis and lipogenesis but suppresses gluconeogenesis, and glucagon counteracts insulin action. Numerous transcription factors and coactivators, including CREB, FOXO1, ChREBP, SREBP, PGC-1α, and CRTC2, control the expression of the enzymes which catalyze key steps of metabolic pathways, thus controlling liver energy metabolism. Aberrant energy metabolism in the liver promotes insulin resistance, diabetes, and nonalcoholic fatty liver diseases.

BACKGROUND

Multiple epidemiologic studies and some trials have linked diet with cardiovascular disease (CVD) prevention, but long-term intervention data are needed.

OBJECTIVE

To test the hypothesis that a dietary intervention, intended to be low in fat and high in vegetables, fruits, and grains to reduce cancer, would reduce CVD risk.

METHODS

Randomized controlled trial of 48,835 postmenopausal women aged 50 to 79 years, of diverse backgrounds and ethnicities, who participated in the Women's Health Initiative Dietary Modification Trial. Women were randomly assigned to an intervention (19,541 [40%]) or comparison group (29,294 [60%]) in a free-living setting. Study enrollment occurred between 1993 and 1998 in 40 US clinical centers; mean follow-up in this analysis was 8.1 years.

METHODS

Intensive behavior modification in group and individual sessions designed to reduce total fat intake to 20% of calories and increase intakes of vegetables/fruits to 5 servings/d and grains to at least 6 servings/d. The comparison group received diet-related education materials.

METHODS

Fatal and nonfatal coronary heart disease (CHD), fatal and nonfatal stroke, and CVD (composite of CHD and stroke).

RESULTS

By year 6, mean fat intake decreased by 8.2% of energy intake in the intervention vs the comparison group, with small decreases in saturated (2.9%), monounsaturated (3.3%), and polyunsaturated (1.5%) fat; increases occurred in intakes of vegetables/fruits (1.1 servings/d) and grains (0.5 serving/d). Low-density lipoprotein cholesterol levels, diastolic blood pressure, and factor VIIc levels were significantly reduced by 3.55 mg/dL, 0.31 mm Hg, and 4.29%, respectively; levels of high-density lipoprotein cholesterol, triglycerides, glucose, and insulin did not significantly differ in the intervention vs comparison groups. The numbers who developed CHD, stroke, and CVD (annualized incidence rates) were 1000 (0.63%), 434 (0.28%), and 1357 (0.86%) in the intervention and 1549 (0.65%), 642 (0.27%), and 2088 (0.88%) in the comparison group. The diet had no significant effects on incidence of CHD (hazard ratio [HR], 0.97; 95% confidence interval [CI], 0.90-1.06), stroke (HR, 1.02; 95% CI, 0.90-1.15), or CVD (HR, 0.98; 95% CI, 0.92-1.05). Excluding participants with baseline CVD (3.4%), the HRs (95% CIs) for CHD and stroke were 0.94 (0.86-1.02) and 1.02 (0.90-1.17), respectively. Trends toward greater reductions in CHD risk were observed in those with lower intakes of saturated fat or trans fat or higher intakes of vegetables/fruits.

CONCLUSIONS

Over a mean of 8.1 years, a dietary intervention that reduced total fat intake and increased intakes of vegetables, fruits, and grains did not significantly reduce the risk of CHD, stroke, or CVD in postmenopausal women and achieved only modest effects on CVD risk factors, suggesting that more focused diet and lifestyle interventions may be needed to improve risk factors and reduce CVD risk.

BACKGROUND

ClinicalTrials.gov Identifier: NCT00000611.

Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.

Here we demonstrate that the ABC transporter ABCG1 plays a critical role in lipid homeostasis by controlling both tissue lipid levels and the efflux of cellular cholesterol to HDL. Targeted disruption of Abcg1 in mice has no effect on plasma lipids but results in massive accumulation of both neutral lipids and phospholipids in hepatocytes and in macrophages within multiple tissues following administration of a high-fat and -cholesterol diet. In contrast, overexpression of human ABCG1 protects murine tissues from dietary fat-induced lipid accumulation. Finally, we show that cholesterol efflux to HDL specifically requires ABCG1, whereas efflux to apoA1 requires ABCA1. These studies identify Abcg1 as a key gene involved in both cholesterol efflux to HDL and in tissue lipid homeostasis.

BACKGROUND

Proteinuria or albuminuria is an established risk marker for progressive renal function loss. Albuminuria can be effectively lowered with antihypertensive drugs that interrupt the renin-angiotensin system (RAS). We investigated whether albuminuria could not only serve as a marker of renal disease, but also function as a monitor of the renoprotective efficacy of RAS intervention by the angiotensin II (Ang II) antagonist, losartan, in patients with diabetic nephropathy.

METHODS

The data from the RENAAL (Reduction in End Points in Noninsulin-Dependent Diabetes Mellitus with the Angiotensin II Antagonist Losartan) study, a double-blind, randomized trial, were used to examine the effects of losartan on the renal outcome [i.e., the primary composite end point of doubling of serum creatinine, end-stage renal disease (ESRD) or death] in 1513 type 2 diabetic patients with nephropathy. We examined the effect of the degree of albuminuria at baseline, initial antiproteinuric response to therapy, and the degree of remaining (residual) albuminuria on renal outcome (either the primary composite end point of RENAAL or ESRD). We also evaluated the contribution to renal protection of the antiproteinuric effect of losartan independently of changes in blood pressure.

RESULTS

Baseline albuminuria is almost linearly related to renal outcome, and is the strongest predictor among all measured well-known baseline risk parameters. After adjusting for baseline risk markers of age, gender, race, weight, smoking, sitting diastolic blood pressure, sitting systolic blood pressure, total cholesterol, serum creatinine, albuminuria, hemoglobin, and hemoglobin A(1c) (HbA(1c)) patients with high baseline albuminuria >> or =3.0 g/g creatinine) showed a 5.2-fold (95% CI 4.3-6.3) increased risk for reaching a renal end point, and a 8.1-fold (95% CI 6.1-10.8) increased risk for progressing to ESRD, compared to the low albuminuria group (<1.5 g/g). The changes in albuminuria in the first 6 months of therapy are roughly linearly related to the degree of long-term renal protection: every 50% reduction in albuminuria in the first 6 months was associated with a reduction in risk of 36% for renal end point and 45% for ESRD during later follow-up. Albuminuria at month 6, designated residual albuminuria, showed a linear relationship with renal outcome, almost identical to the relationship between baseline albuminuria and renal risk. Losartan reduced albuminuria by 28% (95% CI -25% to -36%), while placebo increased albuminuria by 4% (95% CI +8% to -1%) in the first 6 months of therapy. The specific (beyond blood pressure lowering) renoprotective effect of the Ang II antagonist, losartan, in this study is for the major part explained by its antialbuminuric effect (approximately 100% for the renal end point, and 50% for ESRD end point).

CONCLUSIONS

Albuminuria is the predominant renal risk marker in patients with type 2 diabetic nephropathy on conventional treatment; the higher the albuminuria, the greater the renal risk. Reduction in albuminuria is associated with a proportional effect on renal protection, the greater the reduction the greater the renal protection. The residual albuminuria on therapy (month 6) is as strong a marker of renal outcome as is baseline albuminuria. The antiproteinuric effect of losartan explains a major component of its specific renoprotective effect. In conclusion, albuminuria should be considered a risk marker for progressive loss of renal function in type 2 diabetes with nephropathy, as well as a target for therapy. Reduction of residual albuminuria to the lowest achievable level should be viewed as a goal for future renoprotective treatments.

Our review focuses on all articles in the English language that provide sufficient data to calculate a relative risk or odds ratio for CHD at different levels of physical activity. The inverse association between physical activity and incidence of CHD is consistently observed, especially in the better designed studies; this association is appropriately sequenced, biologically graded, plausible, and coherent with existing knowledge. Therefore, the observations reported in the literature support the inference that physical activity is inversely and causally related to the incidence of CHD. The two most important observations in this review are, first, better studies have been more likely than poorer studies to report an inverse association between physical activity and the incidence of CHD and, second, the relative risk of inactivity appears to be similar in magnitude to that of hypertension, hypercholesterolemia, and smoking. These observations suggest that in CHD prevention programs, regular physical activity should be promoted as vigorously as blood pressure control, dietary modification to lower serum cholesterol, and smoking cessation. Given the large proportion of sedentary persons in the United States (91), the incidence of CHD attributable to insufficient physical activity is likely to be surprisingly large. Therefore, public policy that encourages regular physical activity should be pursued.

BACKGROUND

Exenatide, an incretin mimetic for adjunctive treatment of type 2 diabetes (T2DM), reduced hemoglobin A(1c) (A1C) and weight in clinical trials. The objective of this study was to evaluate the effects of>> or = 3 years exenatide therapy on glycemic control, body weight, cardiometabolic markers, and safety.

METHODS

Patients from three placebo-controlled trials and their open-label extensions were enrolled into one open-ended, open-label clinical trial. Patients were randomized to twice daily (BID) placebo, 5 mug exenatide, or 10 mug exenatide for 30 weeks, followed by 5 mug exenatide BID for 4 weeks, then 10 mug exenatide BID for>> or = 3 years of exenatide exposure. Patients continued metformin and/or sulfonylureas.

RESULTS

217 patients (64% male, age 58 +/- 10 years, weight 99 +/- 18 kg, BMI 34 +/- 5 kg/m(2), A1C 8.2 +/- 1.0% [mean +/- SD]) completed 3 years of exenatide exposure. Reductions in A1C from baseline to week 12 (-1.1 +/- 0.1% [mean +/- SEM]) were sustained to 3 years (-1.0 +/- 0.1%; p < 0.0001), with 46% achieving A1C < or = 7%. Exenatide progressively reduced body weight from baseline (-5.3 +/- 0.4 kg at 3 years; p < 0.0001). Patients with elevated serum alanine aminotransferase (ALT) at baseline (n = 116) had reduced ALT (-10.4 +/- 1.5 IU/L; p < 0.0001) and 41% achieved normal ALT. Patients with elevated ALT at baseline tended to lose more weight than patients with normal ALT at baseline (-6.1 +/- 0.6 kg vs. -4.4 +/- 0.5 kg; p = 0.03), however weight change was minimally correlated with baseline ALT (r = -0.01) or ALT change (r = 0.31). Homeostasis Model Assessment B (HOMA-B), blood pressure, and aspartate aminotransferase (AST) all improved. A subset achieved 3.5 years of exenatide exposure and had serum lipids available for analysis (n = 151). Triglycerides decreased 12% (p = 0.0003), total cholesterol decreased 5% (p = 0.0007), LDL-C decreased 6% (p < 0.0001), and HDL-C increased 24% (p < 0.0001). Exenatide was generally well tolerated. The most frequent adverse event was mild-to-moderate nausea. The main limitation of this study is the open-label, uncontrolled nature of the study design which does not provide a placebo group for comparison.

CONCLUSIONS

Adjunctive exenatide treatment for>> or = 3 years in T2DM patients resulted in sustained improvements in glycemic control, cardiovascular risk factors, and hepatic biomarkers, coupled with progressive weight reduction.

BACKGROUND

Elevated plasma concentrations of C-reactive protein (CRP) are associated with increased cardiovascular risk. We evaluated whether long-term therapy with pravastatin, an agent that reduces cardiovascular risk, might alter levels of this inflammatory parameter.

RESULTS

CRP levels were measured at baseline and at 5 years in 472 randomly selected participants in the Cholesterol and Recurrent Events (CARE) trial who remained free of recurrent coronary events during follow-up. Overall, CRP levels at baseline and at 5 years were highly correlated (r=0.60, P<0.001). However, among those allocated to placebo, median CRP levels and the mean change in CRP tended to increase over time (median change, +4. 2%; P=0.2 and mean change, +0.07 mg/dL; P=0.04). By contrast, median CRP levels and the mean change in CRP decreased over time among those allocated to pravastatin (median change, -17.4%; P=0.004 and mean change, -0.07 mg/dL; P=0.002). Thus, statistically significant differences were observed at 5 years between the pravastatin and placebo groups in terms of median CRP levels (difference, -21.6%; P=0.007), mean CRP levels (difference, -37.8%; P=0.002), and absolute mean change in CRP (difference, -0.137 mg/dL; P=0.003). These effects persisted in analyses stratified by age, body mass index, smoking status, blood pressure, and baseline lipid levels. Attempts to relate the magnitude of change in CRP to the magnitude of change in lipids in both the pravastatin and placebo groups did not reveal any obvious relationships.

CONCLUSIONS

Among survivors of myocardial infarction on standard therapy plus placebo, CRP levels tended to increase over 5 years of follow-up. In contrast, randomization to pravastatin resulted in significant reductions in this inflammatory marker that were not related to the magnitude of lipid alterations observed. Thus, these data further support the potential for nonlipid effects of this agent.

Lipid peroxidation is a well known example of oxidative damage in cell membranes, lipoproteins, and other lipid-containing structures. Peroxidative modification of unsaturated phospholipids, glycolipids, and cholesterol can occur in reactions triggered by i) free radical species such as oxyl radicals, peroxyl radicals, and hydroxyl radicals derived from iron-mediated reduction of hydrogen peroxide or ii) non-radical species such as singlet oxygen, ozone, and peroxynitrite generated by the reaction of superoxide with nitric oxide. Lipid hydroperoxides (LOOHs) are prominent non-radical intermediates of lipid peroxidation whose identification can often provide valuable mechanistic information, e.g., whether a primary reaction is mediated by singlet oxygen or oxyradicals. Certain cholesterol-derived hydroperoxides (ChOOHs) have been used very effectively in this regard, both in model systems and cells. Being more polar than parent lipids, LOOHs perturb membrane structure/function and can be deleterious to cells on this basis alone. However, LOOHs can also participate in redox reactions, the nature and magnitude of which often determines whether peroxidative injury is exacerbated or prevented. Exacerbation may reflect iron-catalyzed one-electron reduction of LOOHs, resulting in free radical-mediated chain peroxidation, whereas prevention may reflect selenoperoxidase-catalyzed two-electron reduction of LOOHs to relatively non-toxic alcohols. LOOH partitioning between these two pathways in an oxidatively stressed cell is still poorly understood, but recent cell studies involving various ChOOHs have begun to shed light on this important question. An aspect of related interest that is under intensive investigation is lipid peroxidation/LOOH-mediated stress signaling, which may evoke a variety of cellular responses, ranging from induction of antioxidant enzymes to apoptotic death. Ongoing exploration of these processes will have important bearing on our understanding of disease states associated with peroxidative stress.

Laboratory measurements of plasma lipids (principally cholesterol and triglycerides) and lipoprotein lipids (principally low-density lipoprotein [LDL] and low-density lipoprotein [HDL] cholesterol) are the cornerstone of the clinical assessment and management of atherosclerotic cardiovascular disease (CVD) risk. LDL particles, and to a lesser extent very-low-density lipoprotein [VLDL] particles, cause atherosclerosis, whereas HDL particles prevent or reverse this process through reverse cholesterol transport. The overall risk for CVD depends on the balance between the "bad" LDL (and VLDL) and "good" HDL particles. Direct assessment of lipoprotein particle numbers us now possible through nuclear magnetic resonance spectroscopic analysis.

The PGC-1 family of coactivators stimulates the activity of certain transcription factors and nuclear receptors. Transcription factors in the sterol responsive element binding protein (SREBP) family are key regulators of the lipogenic genes in the liver. We show here that high-fat feeding, which induces hyperlipidemia and atherogenesis, stimulates the expression of both PGC-1beta and SREBP1c and 1a in liver. PGC-1beta coactivates the SREBP transcription factor family and stimulates lipogenic gene expression. Further, PGC-1beta is required for SREBP-mediated lipogenic gene expression. However, unlike SREBP itself, PGC-1beta reduces fat accumulation in the liver while greatly increasing circulating triglycerides and cholesterol in VLDL particles. The stimulation of lipoprotein transport upon PGC-1beta expression is likely due to the simultaneous coactivation of the liver X receptor, LXRalpha, a nuclear hormone receptor with known roles in hepatic lipid transport. These data suggest a mechanism through which dietary saturated fats can stimulate hyperlipidemia and atherogenesis.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease. To elucidate the molecular basis of NAFLD, we performed an exome-wide association study of liver fat content. Three variants were associated with higher liver fat levels at the exome-wide significance level of 3.6 × 10(-7): two in PNPLA3, an established locus for NAFLD, and one (encoding p.Glu167Lys) in TM6SF2, a gene of unknown function. The TM6SF2 variant encoding p.Glu167Lys was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and with lower levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides and alkaline phosphatase in 3 independent populations (n>> 80,000). When recombinant protein was expressed in cultured hepatocytes, 50% less Glu167Lys TM6SF2 protein was produced relative to wild-type TM6SF2. Adeno-associated virus-mediated short hairpin RNA knockdown of Tm6sf2 in mice increased liver triglyceride content by threefold and decreased very-low-density lipoprotein (VLDL) secretion by 50%. Taken together, these data indicate that TM6SF2 activity is required for normal VLDL secretion and that impaired TM6SF2 function causally contributes to NAFLD.

Enzyme inducers such as 3H-1,2-dithiole-3-thione (D3T) enhance the detoxication of environmental carcinogens and protect against neoplasia. The putative molecular sensor for inducers is Keap1, a sulfhydryl-rich protein that sequesters the transcription factor Nrf2 in the cytoplasm. Expression of these detoxication enzymes is blunted in nrf2-deficient mice; moreover, these mice are more sensitive to carcinogenesis, and the protective actions of dithiolethiones are lost with nrf2 disruption. Hepatic gene expression profiles were examined by oligonucleotide microarray analysis in vehicle- or D3T-treated wild-type mice as well as in nrf2 single and keap1-nrf2 double knockout mice to identify those genes regulated by the Keap1-Nrf2 pathway. Transcript levels of 292 genes were elevated in wild-type mice 24 h after treatment with D3T; 79% of these genes were induced in wild-type, but not nrf2-deficient mice. These nrf2-dependent, D3T-inducible genes included known detoxication and antioxidative enzymes. Unexpected clusters included genes for chaperones, protein trafficking, ubiquitin/26 S proteasome subunits, and signaling molecules. Gene expression patterns in keap1-nrf2 double knockout mice were similar to those in nrf2-single knockout mice. D3T also led to nrf2-dependent repression of 31 genes at 24 h; principally genes related to cholesterol/lipid biosynthesis. Collectively, D3T increases the expression of genes through the Keap1-Nrf2 signaling pathway that directly detoxify toxins and generate essential cofactors such as glutathione and reducing equivalents. Induction of nrf2-dependent genes involved in the recognition and repair/removal of damaged proteins expands the role of this pathway beyond primary control of electrophilic and oxidative stresses into secondary protective actions that enhance cell survival.

BACKGROUND

Cardiovascular disease causes severe morbidity and mortality in type 1 diabetes, although the specific risk factors and whether chronic hyperglycemia has a role are unknown. We examined the progression of carotid intima-media thickness, a measure of atherosclerosis, in a population with type 1 diabetes.

METHODS

As part of the Epidemiology of Diabetes Interventions and Complications (EDIC) study, the long-term follow-up of the Diabetes Control and Complications Trial (DCCT), 1229 patients with type 1 diabetes underwent B-mode ultrasonography of the internal and common carotid arteries in 1994-1996 and again in 1998-2000. We assessed the intima-media thickness in 611 subjects who had been randomly assigned to receive conventional diabetes treatment during the DCCT and in 618 who had been assigned to receive intensive diabetes treatment.

RESULTS

At year 1 of the EDIC study, the carotid intima-media thickness was similar to that in an age- and sex-matched nondiabetic population. After six years, the intima-media thickness was significantly greater in the diabetic patients than in the controls. The mean progression of the intima-media thickness was significantly less in the group that had received intensive therapy during the DCCT than in the group that had received conventional therapy (progression of the intima-media thickness of the common carotid artery, 0.032 vs. 0.046 mm; P=0.01; and progression of the combined intima-media thickness of the common and internal carotid arteries, -0.155 vs. 0.007; P=0.02) after adjustment for other risk factors. Progression of carotid intima-media thickness was associated with age, and the EDIC base-line systolic blood pressure, smoking, the ratio of low-density lipoprotein to high-density lipoprotein cholesterol, and urinary albumin excretion rate and with the mean glycosylated hemoglobin value during the mean duration (6.5 years) of the DCCT.

CONCLUSIONS

Intensive therapy during the DCCT resulted in decreased progression of intima-media thickness six years after the end of the trial.

To assess the genetics of hyperlipidemia in coronary heart disease, family studies were carried out in 2520 relatives and spouses of 176 survivors of myocardial infarction, including 149 hyperlipidemic and 27 normolipidemic individuals. The distribution of fasting plasma cholesterol and triglyceride values in relatives, together with segregation analyses, suggested the presence of five distinct lipid disorders. Three of these-familial hypercholesterolemia, familial hypertriglyceridemia, and familial combined hyperlipidemia-appeared to represent dominant expression of three different autosomal genes, occurring in about 20% of survivors below 60 yr of age and 7% of all older survivors. Two other disorders-polygenic hypercholesterolemia and sporadic hypertriglyceridemia-each affected about 6% of survivors in both age groups. The most common genetic form of hyperlipidemia identified in this study has hitherto been poorly defined and has been designated as familial combined hyperlipidemia. Affected family members characteristically had elevated levels of both cholesterol and triglyceride. However, increased cholesterol or increased triglyceride levels alone were also frequently observed. The combined disorder was shown to be genetically distinct from familial hypercholesterolemia and familial hypertriglyceridemia for the following reasons: (a) the distribution pattern of cholesterol and triglyceride levels in relatives of probands was unique; (b) children of individuals with combined hyperlipidemia did not express hypercholesterolemia in contrast to the finding of hypercholesterolemic children from families with familial hypercholesterolemia; and (c) analysis of informative matings suggested that the different lipid phenotypes owed their origin to variable expression of a single autosomal dominant gene and not to segregation of two separate genes, such as one elevating the level of cholesterol and the other elevating the level of triglyceride. Heterozygosity for one of the three lipid-elevating genes identified in this study may have a frequency in the general population of about 1%, constituting a major problem in early diagnosis and preventive therapy.

A growing amount of evidence suggests that regional fat distribution plays an important part in the development of an unfavorable metabolic and cardiovascular risk profile. Epicardial fat is a metabolically active organ that generates various bioactive molecules, which might significantly affect cardiac function. This small, visceral fat depot is now recognized as a rich source of free fatty acids and a number of bioactive molecules, such as adiponectin, resistin and inflammatory cytokines, which could affect the coronary artery response. The observed increases in concentrations of inflammatory factors in patients who have undergone coronary artery bypass grafting remain to be confirmed in healthy individuals. Furthermore, epicardial adipose mass might reflect intra-abdominal visceral fat. Therefore, we propose that echocardiographic assessment of this tissue could serve as a reliable marker of visceral adiposity. Epicardial adipose tissue is also clinically related to left ventricular mass and other features of the metabolic syndrome, such as concentrations of LDL cholesterol, fasting insulin and adiponectin, and arterial blood pressure. Echocardiographic assessment of epicardial fat could be a simple and practical tool for cardiovascular risk stratification in clinical practice and research. In this paper, we briefly review the rapidly emerging evidence pointing to a specific role of epicardial adipose tissue both as a cardiac risk marker and as a potentially active player in the development of cardiac pathology.

Particulate ligands, including cholesterol crystals and amyloid fibrils, induce production of interleukin 1β (IL-1β) dependent on the cytoplasmic sensor NLRP3 in atherosclerosis, Alzheimer's disease and diabetes. Soluble endogenous ligands, including oxidized low-density lipoprotein (LDL), amyloid-β and amylin peptides, accumulate in such diseases. Here we identify an endocytic pathway mediated by the pattern-recognition receptor CD36 that coordinated the intracellular conversion of those soluble ligands into crystals or fibrils, which resulted in lysosomal disruption and activation of the NLRP3 inflammasome. Consequently, macrophages that lacked CD36 failed to elicit IL-1β production in response to those ligands, and targeting CD36 in atherosclerotic mice resulted in lower serum concentrations of IL-1β and accumulation of cholesterol crystals in plaques. Collectively, our findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation.

OBJECTIVE

Pulse pressure is a stronger predictor of cardiovascular events than systolic or diastolic blood pressure in large cohorts of French and North American patients. However, its influence on stroke is controversial. Large-artery stiffness is the main determinant of pulse pressure. The influence of arterial stiffness on the occurrence of stroke has never been demonstrated. Our aim was to establish the relationship between aortic stiffness and stroke death in hypertensive patients.

METHODS

We included, in a longitudinal study, 1715 essential hypertensive patients who had a measurement of arterial stiffness at entry (ie, between 1980 and 2001) and no overt cardiovascular disease or symptoms. Mean follow-up was 7.9 years. At entry, aortic stiffness was assessed from the carotid-femoral pulse wave velocity. A Cox proportional hazard regression model was used to estimate the relative risk (RR) of stroke and coronary deaths.

RESULTS

Mean+/-SD age at entry was 51+/-13 years. Twenty-five fatal strokes and 35 fatal coronary events occurred. Pulse wave velocity significantly predicted the occurrence of stroke death in the whole population. There was a RR increase of 1.72 (95% CI, 1.48 to 1.96; P<0.0001) for each SD increase in pulse wave velocity (4 m/s). The predictive value of pulse wave velocity remained significant (RR=1.39 [95% CI, 1.08 to 1.72]; P=0.02) after full adjustment for classic cardiovascular risk factors, including age, cholesterol, diabetes, smoking, mean blood pressure, and pulse pressure. In this population, pulse pressure significantly predicted stroke in univariate analysis, with a RR increase of 1.33 (95% CI, 1.16 to 1.51) for each 10 mm Hg of pulse pressure (P<0.0001) but not after adjustment for age (RR=1.19 [95% CI, 0.96 to 1.47]; P=0.10).

CONCLUSIONS

This study provides the first evidence, in a longitudinal study, that aortic stiffness is an independent predictor of fatal stroke in patients with essential hypertension.

Hepatic fibrosis is a dynamic process characterized by the net accumulation of extracellular matrix resulting from chronic liver injury of any aetiology, including viral infection, alcoholic liver disease and NASH. Activation of hepatic stellate cells (HSCs) - transdifferentiation of quiescent, vitamin-A-storing cells into proliferative, fibrogenic myofibroblasts - is now well established as a central driver of fibrosis in experimental and human liver injury. Yet, the continued discovery of novel pathways and mediators, including autophagy, endoplasmic reticulum stress, oxidative stress, retinol and cholesterol metabolism, epigenetics and receptor-mediated signals, reveals the complexity of HSC activation. Extracellular signals from resident and inflammatory cells including macrophages, hepatocytes, liver sinusoidal endothelial cells, natural killer cells, natural killer T cells, platelets and B cells further modulate HSC activation. Finally, pathways of HSC clearance have been greatly clarified, and include apoptosis, senescence and reversion to an inactivated state. Collectively, these findings reinforce the remarkable complexity and plasticity of HSC activation, and underscore the value of clarifying its regulation in hopes of advancing the development of novel diagnostics and therapies for liver disease.

We have produced transgenic mice whose livers express a dominant positive NH2-terminal fragment of sterol regulatory element binding protein-1c (SREBP-1c). Unlike full-length SREBP-1c, the NH2-terminal fragment enters the nucleus without a requirement for proteolytic release from cell membranes, and hence it is immune to downregulation by sterols. We compared SREBP-1c transgenic mice with a line of transgenic mice that produces an equal amount of the NH2-terminal fragment of SREBP-1a. SREBP-1a and -1c are alternate transcripts from a single gene that differ in the first exon, which encodes part of an acidic activation domain. The 1a protein contains a long activation domain with 12 negatively charged amino acids, whereas the 1c protein contains a short activation domain with only 6 such amino acids. As previously reported, livers of the SREBP-1a transgenic mice were massively enlarged, owing to accumulation of triglycerides and cholesterol. SREBP-1c transgenic livers were only slightly enlarged with only a moderate increase in triglycerides, but not cholesterol. The mRNAs for the LDL receptor and several cholesterol biosynthetic enzymes were elevated in SREBP-la transgenic mice, but not in 1c transgenic mice. The mRNAs for fatty acid synthase and acetyl CoA carboxylase were elevated 9- and 16-fold in la animals, but only 2- and 4-fold in 1c animals. Experiments with transfected cells confirmed that SREBP-1c is a much weaker activator of transcription than SREBP-1a when both are expressed at levels approximating those found in nontransfected cells. SREBP-1c became a strong activator only when expressed at supraphysiologic levels. We conclude that SREBP-1a is the most active form of SREBP-1 and that SREBP-1c may be produced when cells require a lower rate of transcription of genes regulating cholesterol and fatty acid metabolism.

OBJECTIVE

To determine the effects of longer term modest salt reduction on blood pressure, hormones, and lipids.

METHODS

Systematic review and meta-analysis.

METHODS

Medline, Embase, Cochrane Hypertension Group Specialised Register, Cochrane Central Register of Controlled Trials, and reference list of relevant articles.

METHODS

Randomised trials with a modest reduction in salt intake and duration of at least four weeks.

METHODS

Data were extracted independently by two reviewers. Random effects meta-analyses, subgroup analyses, and meta-regression were performed.

RESULTS

Thirty four trials (3230 participants) were included. Meta-analysis showed that the mean change in urinary sodium (reduced salt v usual salt) was -75 mmol/24 h (equivalent to a reduction of 4.4 g/day salt), and with this reduction in salt intake, the mean change in blood pressure was -4.18 mm Hg (95% confidence interval -5.18 to -3.18, I(2)=75%) for systolic blood pressure and -2.06 mm Hg (-2.67 to -1.45, I(2)=68%) for diastolic blood pressure. Meta-regression showed that age, ethnic group, blood pressure status (hypertensive or normotensive), and the change in 24 hour urinary sodium were all significantly associated with the fall in systolic blood pressure, explaining 68% of the variance between studies. A 100 mmol reduction in 24 hour urinary sodium (6 g/day salt) was associated with a fall in systolic blood pressure of 5.8 mm Hg (2.5 to 9.2, P=0.001) after adjustment for age, ethnic group, and blood pressure status. For diastolic blood pressure, age, ethnic group, blood pressure status, and the change in 24 hour urinary sodium explained 41% of the variance between studies. Meta-analysis by subgroup showed that in people with hypertension the mean effect was -5.39 mm Hg (-6.62 to -4.15, I(2)=61%) for systolic blood pressure and -2.82 mm Hg (-3.54 to -2.11, I(2)=52%) for diastolic blood pressure. In normotensive people, the figures were -2.42 mm Hg (-3.56 to -1.29, I(2)=66%) and -1.00 mm Hg (-1.85 to -0.15, I(2)=66%), respectively. Further subgroup analysis showed that the decrease in systolic blood pressure was significant in both white and black people and in men and women. Meta-analysis of data on hormones and lipids showed that the mean change was 0.26 ng/mL/h (0.17 to 0.36, I(2)=70%) for plasma renin activity, 73.20 pmol/L (44.92 to 101.48, I(2)=62%) for aldosterone, 187 pmol/L (39 to 336, I(2)=5%) for noradrenaline (norepinephrine), 37 pmol/L (-1 to 74, I(2)=12%) for adrenaline (epinephrine), 0.05 mmol/L (-0.02 to 0.11, I(2)=0%) for total cholesterol, 0.05 mmol/L (-0.01 to 0.12, I(2)=0%) for low density lipoprotein cholesterol, -0.02 mmol/L (-0.06 to 0.01, I(2)=16%) for high density lipoprotein cholesterol, and 0.04 mmol/L (-0.02 to 0.09, I(2)=0%) for triglycerides.

CONCLUSIONS

A modest reduction in salt intake for four or more weeks causes significant and, from a population viewpoint, important falls in blood pressure in both hypertensive and normotensive individuals, irrespective of sex and ethnic group. Salt reduction is associated with a small physiological increase in plasma renin activity, aldosterone, and noradrenaline and no significant change in lipid concentrations. These results support a reduction in population salt intake, which will lower population blood pressure and thereby reduce cardiovascular disease. The observed significant association between the reduction in 24 hour urinary sodium and the fall in systolic blood pressure, indicates that larger reductions in salt intake will lead to larger falls in systolic blood pressure. The current recommendations to reduce salt intake from 9-12 to 5-6 g/day will have a major effect on blood pressure, but a further reduction to 3 g/day will have a greater effect and should become the long term target for population salt intake.

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.