Variation in the SLC23A1 gene does not influence cardiometabolic outcomes to the extent expected given its association with L-ascorbic acid.

BACKGROUND: Observational studies showed that circulating L-ascorbic acid (vitamin C) is inversely associated with cardiometabolic traits. However, these studies were susceptible to confounding and reverse causation. OBJECTIVES: We assessed the relation between L-ascorbic acid and 10 cardiometabolic traits by using a single nucleotide polymorphism in the solute carrier family 23 member 1 (SLC23A1) gene (rs33972313) associated with circulating L-ascorbic acid concentrations. The observed association between rs33972313 and cardiometabolic outcomes was compared with that expected given the rs33972313-L-ascorbic acid and L-ascorbic acid-outcome associations. DESIGN: A meta-analysis was performed in the following 5 independent studies: the British Women's Heart and Health Study (n = 1833), the MIDSPAN study (n = 1138), the Ten Towns study (n = 1324), the British Regional Heart Study (n = 2521), and the European Prospective Investigation into Cancer (n = 3737). RESULTS: With the use of a meta-analysis of observational estimates, inverse associations were shown between L-ascorbic acid and systolic blood pressure, triglycerides, and the waist-hip ratio [the strongest of which was the waist-hip ratio (-0.13-SD change; 95% CI: -0.20-, -0.07-SD change; P = 0.0001) per SD increase in L-ascorbic acid], and a positive association was shown with high-density lipoprotein (HDL) cholesterol. The variation at rs33972313 was associated with a 0.18-SD (95% CI: 0.10-, 0.25-SD; P = 3.34 × 10⁻6) increase in L-ascorbic acid per effect allele. There was no evidence of a relation between the variation at rs33972313 and any cardiometabolic outcome. Although observed estimates were not statistically different from expected associations between rs33972313 and cardiometabolic outcomes, estimates for low-density lipoprotein cholesterol, HDL cholesterol, triglycerides, glucose, and body mass index were in the opposite direction to those expected. CONCLUSIONS: The nature of the genetic association exploited in this study led to limited statistical application, but despite this, when all cardiometabolic traits were assessed, there was no evidence of any trend supporting a protective role of L-ascorbic acid. In the context of existing work, these results add to the suggestion that observational relations between L-ascorbic acid and cardiometabolic health may be attributable to confounding and reverse causation.

Reduced levels of S-nitrosothiols in plasma of patients with systemic sclerosis and Raynaud's phenomenon.

OBJECTIVE: S-Nitrosothiols (RSNOs) are bioactive forms of nitric oxide which are involved in cell signalling and redox regulation of vascular function. Circulating S-nitrosothiols are predominantly in the form of S-nitrosoalbumin. In this study plasma concentrations of S-nitrosothiols were measured in patients with systemic sclerosis (SSc) where NO metabolism is known to be abnormal. PATIENTS AND METHODS: Venous blood was collected from 16 patients with Raynaud's phenomenon (RP), 45 with systemic sclerosis (SSc) (34 patients had limited SSc (IcSSc) and 11 diffuse cutaneous disease (dcSSc)). Twenty six healthy subjects were used as controls. Plasma S-nitrosothiol concentrations were measured by chemiluminescence. The measurements were related to the extent of biological age, capillary/skin scores and disease duration. RESULTS: Plasma RSNO levels in patients with Raynaud's phenomenon (RP) and in those with SSc was significantly lower compared to the concentrations in control subjects. In SSc, plasma S-nitrosothiols were often below the level of detection (1nM). CONCLUSIONS: Low S-nitrosothiol concentrations were observed in the blood of patients with SSc and patients with RP indicating a profound disturbance of nitric oxide metabolism.

Modulation of fibrosis in systemic sclerosis by nitric oxide and antioxidants.

SYSTEMIC SCLEROSIS (SCLERODERMA: SSc) is a multisystem, connective tissue disease of unknown aetiology characterized by vascular dysfunction, autoimmunity, and enhanced fibroblast activity resulting in fibrosis of the skin, heart, and lungs, and ultimately internal organ failure, and death. One of the most important and early modulators of disease activity is thought to be oxidative stress. Evidence suggests that the free radical nitric oxide (NO), a key mediator of oxidative stress, can profoundly influence the early microvasculopathy, and possibly the ensuing fibrogenic response. Animal models and human studies have also identified dietary antioxidants, such as epigallocatechin-3-gallate (EGCG), to function as a protective system against oxidative stress and fibrosis. Hence, targeting EGCG may prove a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc.

Genetic variation at the SLC23A1 locus is associated with circulating concentrations of L-ascorbic acid (vitamin C): evidence from 5 independent studies with >15,000 participants.

BACKGROUND: L-ascorbic acid is an essential part of the human diet and has been associated with a wide range of chronic complex diseases, including cardiovascular outcomes. To date, there are no confirmed genetic correlates of circulating concentrations of L-ascorbic acid. OBJECTIVE: We aimed to confirm the existence of an association between common variation at the SLC23A1 gene locus and circulating concentrations of L-ascorbic acid. DESIGN: We used a 2-stage design, which included a discovery cohort (the British Women's Heart and Health Study), a series of follow-up cohorts, and meta-analysis (totaling 15,087 participants) to assess the relation between variation at SLC23A1 and circulating concentrations of L-ascorbic acid. RESULTS: In the discovery cohort, variation at rs33972313 was associated with a reduction in circulating concentrations of L-ascorbic acid (-4.15 micromol/L; 95% CI: -0.49, -7.81 micromol/L; P = 0.03 reduction per minor allele). Pooled analysis of the relation between rs33972313 and circulating L-ascorbic acid across all studies confirmed this and showed that each additional rare allele was associated with a reduction in circulating concentrations of L-ascorbic acid of -5.98 micromol/L (95% CI: -8.23, -3.73 micromol/L; P = 2.0 x 10(-7) per minor allele). CONCLUSIONS: A genetic variant (rs33972313) in the SLC23A1 vitamin C active transporter locus was identified that is reliably associated with circulating concentrations of L-ascorbic acid in the general population. This finding has implications more generally for the epidemiologic investigation of relations between circulating L-ascorbic acid and health outcomes.

Antioxidants and CVD.

The involvement of free radicals and reactive oxygen and nitrogen species in the pathology of inflammatory and degenerative disease has been widely accepted, although the centrality of these species to the outcome of these diseases is still a matter for debate. In the case of CVD, and particularly the development of the atherosclerotic plaque, the oxidation of LDL is of particular importance and appears to explain many of the events that occur during the life history of the plaque. The corollary of this situation is that antioxidants must be a benign force to protect the population from the modern scourge of heart disease. In fact, recent evidence from intervention studies with large doses of the antioxidant vitamins and other antioxidants in foods has been very disappointing. Here, the background for the belief that antioxidants ought to be beneficial is examined and an attempt made to explain why the results of these intervention studies have been unsuccessful. It is agreed that a diet rich in fruit and vegetables is protective for both CVD and cancer, but the explanation for this effect may not necessarily lie with the presence of antioxidants.

Effect of nitric oxide and peroxynitrite on type I collagen synthesis in normal and scleroderma dermal fibroblasts.

Nitric oxide ((.-)NO) is an important physiological signaling molecule and potent vasodilator. Recently, we have shown abnormal (.-)NO metabolism in the plasma of patients with systemic sclerosis (SSc), a disease that features excessive collagen overproduction as well as vascular dysfunction. The current study investigates the effects of (.-)NO and peroxynitrite (ONOO(-)) on secretion of type I collagen by SSc dermal fibroblasts, compared with those from normal dermal fibroblasts (CON) and a dermal fibroblast cell line (AG). Dermal fibroblasts were incubated with (.-)NO donors (SNP, DETA-NONOate) with or without the antioxidant ascorbic acid, or ONOO(-) for 24-72 h. In CON and AG fibroblasts, type I collagen was dose dependently decreased by SNP or DETA-NONOate. However, (.-)NO had no effect in SSc fibroblasts. Furthermore, the inhibition of collagen synthesis by (.-)NO was reversed by ascorbic acid and was not affected by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanyl cyclase, or 8-bromoguanosine cyclic 3',5'-monophosphate, a cGMP agonist. SNP also showed a significant up-regulation of matrix metalloproteinase-1 (MMP-1) protein and activity levels, an essential collagenase involved in collagen degradation, in the AG fibroblasts. Additionally, (.-)NO-treated fibroblasts had lower prolyl hydroxylase activity, an enzyme important in the post-translational processing of collagen, while there was no effect on total protein levels. There were no significant effects on type I collagen levels when dermal fibroblasts were treated with ONOO(-). Taken together, ()NO inhibits collagen secretion in normal dermal fibroblasts but regulation is lost in SSc fibroblasts, while ONOO(-) itself is ineffective. (.-)NO inhibition of collagen was by cGMP-independent regulatory mechanisms and in part may be due to up-regulation of MMP-1 and/or inhibition of prolyl hydroxylase. These differences may contribute to the observed pathology of SSc.

Differential functions of tissue factor in the trans-activation of cellular signalling pathways.

In this study we examined the ability of tissue factor (TF) alone, or in conjunction with factor VIIa, factor Xa and TFPI in activating a number of key signalling pathways associated with cellular growth, stress and differentiation responses in human endothelial cells. We used luciferase reporter systems to demonstrate the activation of p42/44 MAPK by the TF-FVIIa complex, mediated via the PAR1 receptor. TF alone was capable of interacting with the cell surface and was sufficient to activate the JNK-SAPK pathway and subsequently AP-1, but the level of activation was enhanced by the activity of FXa on PAR1 and 2. Furthermore, the phosphorylated form of the transmembrane-cytoplasmic domain of TF was directly responsible for activation of these pathways. CREB activation occurred in response to TF-FVIIa in a non-protease dependent manner but was lowered on addition of FXa. Finally, NFkappaB activation occurred in response to FVIIa or FXa, with the latter exhibiting higher levels of activation. In conclusion, we have shown that TF is capable of activating differing signalling pathways, via more than one mechanism. The differential influence of TF is modified depending on the presence of other coagulation factors and ultimately acts as a deciding factor in the determination of cellular fate.

Associations of vitamin C status, fruit and vegetable intakes, and markers of inflammation and hemostasis.

BACKGROUND: It has been suggested that a high dietary intake and high circulating concentrations of vitamin C may protect against ischemic heart disease. OBJECTIVES: The objective was to examine the associations between dietary and plasma vitamin C concentrations, fruit and vegetable intakes, and markers of inflammation and hemostasis associated with cardiovascular disease in older men free of cardiovascular disease. DESIGN: This cross-sectional study examined 3258 men aged 60-79 y with no physician diagnosis of myocardial infarction, stroke, or diabetes and who were drawn from general practices in 24 British towns. Fruit and vegetable intakes and dietary vitamin C were assessed by using a food-frequency questionnaire. RESULTS: Plasma vitamin C, fruit intake, and dietary vitamin C intake were significantly and inversely associated with mean concentrations of C-reactive protein, an acute phase reactant, and tissue plasminogen activator (t-PA) antigen, a marker of endothelial dysfunction, even after adjustment for confounders. Vegetable intake was correlated significantly (inversely) only with t-PA. For plasma vitamin C (highest versus lowest quartile), the adjusted odds of elevated C-reactive protein and t-PA (highest tertile versus lowest tertile) were 0.56 (95% CI: 0.44, 0.71) and 0.79 (0.62, 1.00); for fruit intake, the corresponding odds ratios were 0.76 (0.60, 0.95) and 0.76 (0.61, 0.95). Plasma (but not dietary) vitamin C also showed inverse associations with both fibrinogen concentrations and blood viscosity. No associations were seen with von Willebrand factor or factor VIII. CONCLUSION: The findings suggest that vitamin C has antiinflammatory effects and is associated with lower endothelial dysfunction in men with no history of cardiovascular disease or diabetes.

Roles for prostaglandins in the steroidogenic response of human granulosa cells to high-density lipoproteins.

In human granulosa-lutein cells, high-density lipoproteins (HDL) can stimulate progesterone synthesis. The objective of the present study was to establish whether prostaglandins (PGs) participate in the steroidogenic response to HDL. Both HDL and apolipoprotein AI (ApoAI) stimulated concentration-dependent increases in PGE2, cAMP and progesterone accumulation. The minimum concentrations of HDL and ApoAI required to elevate PGE2 production were the same as those required to stimulate cAMP accumulation and progesterone synthesis. Concentrations of PGE2 were elevated within 10 min in cells exposed to HDL and rose progressively over 24 h, whereas cAMP and progesterone were only increased significantly after 24 h of treatment with HDL. Co-treatment with prostaglandin H synthase inhibitors (meclofenamic acid and indomethacin) abolished the cAMP and progesterone responses to both HDL and ApoAI. Hence, the ability of HDL to stimulate progesterone synthesis can be mimicked by ApoAI and appears to involve increased generation of one or more luteotrophic PGs, possibly acting via cAMP.

Anticoagulant and antiplatelet agents: their clinical and device application(s) together with usages to engineer surfaces.

An essential aspect of the treatment of patients with cardiovascular disease is the use of anticoagulant and antiplatelet agents for the prevention of further ischaemic events and vascular death resulting from thrombosis. Aspirin and heparin have been the standard therapy for the management of such conditions to date. Recently, numerous more potent platelet inhibitors together with anticoagulant agents have been developed and tested in randomized clinical trials. This article reviews the current state of the art of antiplatelet and anticoagulant therapy in light of its potential clinical efficacy. It then focuses on the usages of these agents in order to improve the performance of clinical devices such as balloon catheters, coronary stents, and femoropopliteal bypass grafting and extra corporeal circuits for cardiopulmonary bypass. The article then goes on to look at the usage of these agents more specifically heparin, heparan, hirudin, and coumarin in the development of more biocompatible scaffolds for tissue engineering.

The state of macrophage differentiation determines the TNF alpha response to nitrated lipoprotein uptake.

Inflammatory cytokine synthesis by monocyte-macrophages in the developing plaque represents an important amplification point in atherosclerotic disease progression. Here we have investigated whether the state of monocyte-macrophage differentiation can influence TNF alpha synthesis in response to scavenged modified low-density lipoprotein (LDL). We show that LDL modified by nitration induces TNF alpha synthesis when added to undifferentiated human monocytes or a mouse cell line (RAW264.7) bearing an incompletely differentiated phenotype. However, significantly reduced levels of TNF alpha were released from in vitro differentiated human macrophages (P=0.006) or a mouse cell line (IC-21) bearing a well-differentiated macrophage phenotype (P<0.001). A possible scavenging insufficiency in macrophagic cell types was ruled out by lipoprotein-uptake studies and competency to synthesise TNF alpha was confirmed using lipopolysaccharide (LPS) as a stimulus. However, LPS-induced TNF alpha secretion in IC-21 cells was partially suppressed by pre-treatment with nitrated LDL (46%, P=0.0076), with no equivalent effect seen in RAW264.7 cells. Based on these data, we hypothesise that the state of differentiation of intimal monocyte-macrophages may play an important role in their inflammatory response to scavenged modified lipoproteins and that the fully differentiated macrophage end-point may be associated with a non-inflammatory and therefore, atheroprotective, phenotype.

Temporal relationships between circulating levels of CC and CXC chemokines and developing atherosclerosis in apolipoprotein E*3 Leiden mice.

OBJECTIVE: CC and CXC chemokines are implicated in leukocyte recruitment during development of atherosclerotic lesions, suggesting circulating levels of chemokines may be useful serum markers of atherogenesis. Serum chemokine concentrations were measured in apolipoprotein (apo) E*3 Leiden mice and their nontransgenic littermates and related to the differing rates of atherogenesis in these animals. METHODS AND RESULTS: Mice were fed a high-fat, high-cholesterol/cholate (HFC/C) diet for 18 weeks. Circulating levels of JE/monocyte chemotactic protein-1 increased (P<0.05) after 2 to 4 weeks, coincident with development of diet-induced hypercholesterolemia, and remained elevated throughout the study. Circulating KC concentrations increased (P<0.05) after consumption of HFC/C diet; however, unlike JE, serum KC concentrations increased more rapidly in apoE*3 Leiden mice than their nontransgenic littermates. Hepatic expression of JE and KC mRNA were detected by in situ hybridization in all mice fed HFC/C diet. Aortic expression of JE mRNA was seen only in apoE*3 Leiden mice within macrophage-rich atherosclerotic lesions. By contrast, no aortic expression of KC mRNA was detected by in situ hybridization. CONCLUSIONS: Increases in serum chemokine concentrations did not reflect temporal aortic production of these molecules and proved less predictive than serum cholesterol of the markedly different extent of atheroma in apoE*3 Leiden and nontransgenic mice.

The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans.

Dietary nitrate is metabolized to nitrite by bacterial flora on the posterior surface of the tongue leading to increased salivary nitrite concentrations. In the acidic environment of the stomach, nitrite forms nitrous acid, a potent nitrating/nitrosating agent. The aim of this study was to examine the pharmacokinetics of dietary nitrate in relation to the formation of salivary, plasma, and urinary nitrite and nitrate in healthy subjects. A secondary aim was to determine whether dietary nitrate increases the formation of protein-bound 3-nitrotyrosine in plasma, and if dietary nitrate improves platelet function. The pharmacokinetic profile of urinary nitrate excretion indicates total clearance of consumed nitrate in a 24 h period. While urinary, salivary, and plasma nitrate concentrations increased between 4- and 7-fold, a significant increase in nitrite was only detected in saliva (7-fold). High dietary nitrate consumption does not cause a significant acute change in plasma concentrations of 3-nitrotyrosine or in platelet function.

Ghrelin can bind to a species of high density lipoprotein associated with paraoxonase.

Ghrelin is a 28-residue peptide hormone that is principally released from the stomach during fasting and prior to eating. Two forms are present in human plasma: the unmodified peptide and a less abundant acylated version, in which octanoic acid is attached to the third residue, a serine, via an ester linkage. The acylated form of ghrelin acts as a ligand for the growth hormone secretagogue receptor and can stimulate the release of growth hormone from the pituitary gland. It also initiates behavioral and metabolic adaptations to fasting. Here we show that an immobilized form of ghrelin specifically binds a species of high density lipoprotein associated with the plasma esterase, paraoxonase, and clusterin. Both free ghrelin and paraoxon, a substrate for paraoxonase, can inhibit this interaction. An endogenous species of ghrelin is found to co-purify with high density lipoprotein during density gradient centrifugation and subsequent gel filtration. This interaction links the orexigenic peptide hormone ghrelin to lipid transport and metabolism. Furthermore, the interaction of the esterified hormone ghrelin with a species of HDL containing an esterase suggests a possible mechanism for the conversion of ghrelin to des-acyl ghrelin.

Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis.

Activation of the phosphatidylinositol 3- kinase/AKT pathway antagonizes apoptosis in diverse cellular systems. We previously showed that human plasma activated AKT and potently blocked the ability of chlorambucil or gamma radiation to induce apoptosis of B-chronic lymphocytic leukemia (CLL) cells. Here we report experiments that identify albumin as the major component of plasma that blocks CLL cell killing by chlorambucil or radiation. Intact plasma depleted of albumin by chromatography on Cibacron blue-Sepharose or plasma from a subject with analbuminemia failed either to activate AKT or to protect CLL cells from chlorambucil-induced apoptosis. Both functions were restored by re-addition of albumin. The protective action of albumin as well as AKT activation was compromised by the binding of lipids. Fluorescence-activated cell sorter (FACScan) analysis demonstrated the uptake of fluoresceinated albumin by CLL cells. Accumulation of albumin in intracellular vesicles was also shown by confocal microscopy. Indirect inhibition of AKT activation by the phosphatidylinositol 3-kinase inhibitor LY294002 reversed the blockade of chlorambucil-induced killing by plasma albumin. The data suggest that activation of AKT consequent to binding of albumin by CLL cells blocks chlorambucil- and radiation-induced apoptosis. Strategies designed to block albumin-induced antiapoptotic signaling may, therefore, be of value in enhancing cytotoxic drug action on CLL cells.

The nitration of proteins in platelets: significance in platelet function.

Exogenous peroxynitrite has been shown to inhibit or activate platelets according to the concentration added and, at the same time, nitrate platelet proteins. Here, recent evidence is discussed which indicates that nitration of proteins may also occur during normal platelet activation by collagen, by mechanical stimulation during isolation and by exposure to low levels of hydrogen peroxide. Furthermore, this nitration appears to be transient. The implications of these findings are discussed in terms of platelet biology and cell signaling processes.

Oral L-arginine does not improve endothelial dysfunction in children with chronic renal failure.

BACKGROUND: Cardiovascular disease is a major cause of mortality amongst patients with chronic renal failure (CRF). L-arginine has been used to improve endothelial function by increasing nitric oxide (NO) bioavailability and in animal models this in turn has attenuated the progression of atherosclerosis. We examined whether dietary L-arginine supplementation improved endothelial function in children with CRF. METHODS: A randomized, double-blind, placebo-controlled, crossover trial of L-arginine was conducted in 21 normotensive children aged 11.5 +/- 3 (7 to 17) years with CRF (GFR 27.4 +/- 13.2 mL/min/1.73 m(2)) in whom endothelial dysfunction had previously been demonstrated. We examined the effect of L-arginineon the endothelial response to shear stress (NO-dependent) using a non-invasive technique of high-resolution ultrasound. Each subject was studied before and after 4 weeks of L-arginine (2.5 g/m(2) or 5 g/m(2) x 3/day) or placebo, separated by a rest period of 4 weeks. Brachial artery diameter was measured at rest, during increased flow (endothelial-dependent dilation) and after 25 microg of glyceryl trinitrate (endothelial-independent dilation) at each visit. RESULTS: After oral L-arginine, plasma L-arginine levels rose from 82 +/- 20 to 179 +/- 110 micromol/L (P < 0.001). No significant change in endothelial-dependent dilation during L-arginine (7.96 +/- 2.35 to 7.71 +/- 3.22%; P> 0.05) or placebo (8.2 +/- 2.89 to 8.3 +/- 3.14%; P> 0.05) was noted. There was no change in endothelial-independent dilation. CONCLUSION: Endothelial function was not improved with L-arginine, suggesting that dietary supplementation is not a useful clinical approach in children with CRF.