The anti inflammatory effects of high density lipoproteins.

It is well recognised that increased levels of high density lipoprotein (HDL) protect against atherosclerosis and correlate with improved prognosis for vascular disease associated events. While many of the atheroprotective effects of HDL are ascribed to the ability to remove cholesterol from the vasculature through the reverse cholesterol transport system, recent work has shown that HDL may be atheroprotective through its other functions, such as regulation of endothelial adhesion molecule expression, stimulation of endothelial nitric oxide synthase and inhibition of the damaging effects of oxidised low density lipoproteins. Recently, HDL has also been described to interact with circulating cells inhibiting both leukocyte and platelet activation, therefore having further systemic anti-inflammatory functions. This review summarises the studies and models used to examine the anti-inflammatory effects of HDL and details data describing the ability to inhibit leukocyte activation, contributing to the hypothesis that raised HDL is beneficial in the context of inflammation in atherosclerosis. Further, HDL modification in disease and current therapeutic strategies such as reconstituted HDL particles and apoA I mimetic peptides is discussed to provide insights to the potential applicability of raising HDL to regress cardiovascular disease.

L-arginine transporters in cardiovascular disease: a novel therapeutic target.

The amino acid l-arginine participates in a variety of key biochemical and physiological activities, including its well-recognized role as the key substrate for nitric oxide (NO) biosynthesis. The current review describes the cellular influences on arginine metabolism with particular focus on the transport of l-arginine in the endothelium. It details the processes by which intracellular and extracellular levels of l-arginine may influence nitric oxide production and further documents the imbalance that is evident in various cardiovascular disease states. In man, impairment of l-arginine transport has been observed in hypertension, heart failure, and renal disease, and it may thus be a relevant therapeutic target for rectification of nitric oxide pathogenesis in these conditions.

In vivo and in vitro evidence for impaired arginine transport in human heart failure.

BACKGROUND: The clinical features of congestive heart failure (CHF) result from a complex interaction between reduced ventricular function, neurohormonal activation, and impaired endothelial function. Although endothelial dysfunction has been well documented, the mechanisms that contribute to this abnormality remain unknown. Recent studies, however, indicate a potential therapeutic role for supplemental L-arginine, suggesting the presence of an underlying disorder of L-arginine metabolism. METHODS AND RESULTS: We used 2 complementary approaches to assess L-arginine transport in control subjects and patients with CHF. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), forearm clearance of [(3)H]L-arginine was significantly reduced in CHF patients compared with forearm kinetics in control subjects (64+/-2 versus 133+/-14 mL/min, P=0.002). In conjunction with this, [(3)H]L-arginine uptake by peripheral blood mononuclear cells (PBMCs) was also substantially reduced in heart failure patients compared with controls (V(max) 10. 1+/-1.3 versus 49.8+/-7.1 pmol/10(5) cells per 5 minutes, P<0.001). In association with this finding, we observed a 76% (P<0.01) reduction in mRNA expression for the cationic amino acid transporter CAT-1, as assessed by ribonuclease protection assay. CONCLUSIONS: These data document both in vivo and in vitro evidence for a marked depression of L-arginine transport in human CHF and therefore provide an explanation for the restorative actions of supplemental L-arginine on vascular function in CHF.

Diurnal variation in endothelium-dependent vasodilatation is not apparent in coronary artery disease.

BACKGROUND: Acute coronary syndromes present with an increased incidence from 6:00 AM to 12:00 noon. Whether endothelial function follows a diurnal rhythm and whether this rhythm is impaired in coronary artery disease (CAD) has not previously been studied. METHODS AND RESULTS: Diurnal variation in endothelium-dependent vasodilatation was examined in 10 CAD patients and 10 control subjects. Forearm blood flow responses to acetylcholine, sodium nitroprusside, and N(G)-monomethyl-L-arginine were determined by plethysmography at 8:00 AM, 2:00 PM, and 8:00 PM. Heart rate, blood pressure, plasma cortisol, and inflammatory markers were also determined. Heart rate and the low-frequency component of heart rate variability were greatest in the morning in control subjects, suggesting a diurnal variation in sympathetic activity. Basal forearm blood flows were significantly reduced in control subjects at 8:00 PM compared with 8:00 AM and 2:00 PM (1.2+/-0.2 versus 2.1+/-0.2 [8:00 AM] and 2.1+/-0.3 [2:00 PM] mL. 100 mL(-1). min(-1); P<0.05) but unchanged in the CAD group. Acetylcholine (37 microgram/min) responses were greater at 8:00 AM than at 8:00 PM in control subjects (12.5+/-3.7 versus 19.6+/-2.9 mL. 100 mL(-1). min(-1), respectively; P<0.05), but these responses were not time dependent in the CAD group. Responses to sodium nitroprusside were similar at all time points and between those with and without CAD. CONCLUSIONS: Thus, normal volunteers have a diurnal variation in their endothelium-dependent vasodilatation that may counteract other, potentially adverse, diurnal variations in hemodynamic and other parameters. In contrast, CAD patients who had presented with acute coronary syndromes showed a loss of this protective mechanism.

Endothelium-dependent relaxation by acetylcholine is impaired in hypertriglyceridemic humans with normal levels of plasma LDL cholesterol.

OBJECTIVES: Patients with high triglyceride (of which very low density lipoproteins [VLDL] are the main carriers), but with normal low density lipoprotein (LDL) cholesterol levels, were examined for in vivo endothelium function status. BACKGROUND: Very low density lipoproteins inhibit endothelium-dependent, but not -independent, vasorelaxation in vitro. METHODS: Three groups were studied: 1) healthy volunteers (n = 10; triglyceride 1.24+/-0.14 mmol/liter, LDL cholesterol 2.99+/-0.24 mmol/liter); 2) hypertriglyceridemic (n = 11; triglyceride 6.97+/-1.19 mmol/liter, LDL cholesterol 2.17+/-0.2 mmol/liter, p < 0.05); and 3) hypercholesterolemic (n = 10; triglyceride 2.25+/-0.29 mmol/liter, LDL cholesterol 5.61+/-0.54 mmol/liter; p < 0.05) patients. Vasoactive responses to acetylcholine, sodium nitroprusside, noradrenaline, N(G)-monomethyl-L-arginine and postischemic hyperemia were determined using forearm venous occlusion plethysmography. RESULTS: Responses to acetylcholine (37 microg/min) were significantly dampened both in hypercholesterolemic (% increase in forearm blood flow: 268.2+/-62) and hypertriglyceridemic patients (232.6+/-45.2) when compared with controls (547.8+/-108.9; ANOVA p < 0.05). Responses to sodium nitroprusside (at 1.6 microg/min: controls vs. hypercholesterolemics vs. hypertriglyceridemic: 168.7+/- 25.1 vs. 140.6+/-38.9 vs. 178.5+/-54.5% increase), noradrenaline, N(G)-monomethyl-L-arginine and postischemic hyperemic responses were not different among the groups examined. CONCLUSIONS: Acetylcholine responses are impaired in patients with pathophysiologic levels of plasma triglycerides but normal plasma levels of LDL cholesterol. The impairment observed was comparable to that obtained in hypercholesterolemic patients. We conclude that impaired responses to acetylcholine normally associated with hypercholesterolemia also occur in hypertriglyceridemia. These findings identify a potential mechanism by which high plasma triglyceride levels may be atherogenic independent of LDL cholesterol levels.

Dietary supplementation with L-arginine fails to restore endothelial function in forearm resistance arteries of patients with severe heart failure.

OBJECTIVES: We sought to examine the efficacy of dietary supplementation of L-arginine on endothelium-dependent vasodilation in patients with congestive heart failure. BACKGROUND: Endothelial dysfunction, as evidenced by a diminished response to such vasodilators as acetylcholine, is well defined in patients with heart failure. These responses are improved by intraarterial infusion with L-arginine. Because L-arginine is a semi-essential amino acid, we investigated the effects of dietary L-arginine on endothelium-dependent vasodilation in these patients. METHODS: Twenty patients with heart failure (New York Heart Association functional class III/IV, mean [+/- SE] age 51.3 +/- 1.7 years) and seven healthy control subjects (mean age 52.6 +/- 3.3 years) were studied. All patients continued taking their usual treatment. Responses to acetylcholine and sodium nitroprusside were determined using forearm plethysmography. Patients with heart failure received either L-arginine (20 g/day every day for 28 days) or placebo (vehicle syrup in equal amounts) in a double-blind protocol. The calculated power of the study was between 62% and 80% to detect a 30% to 40% change in area under the dose-response (forearm vascular resistance) curve. RESULTS: Responses to acetylcholine, but not to sodium nitroprusside, were significantly attenuated in patients with heart failure compared with control subjects (mean area under curve [AUC], control subjects vs. patients with heart failure: 1,125.4 +/- 164.5 vs. 617.3 +/- 116.6 U, p < 0.05, by Student t test). A significant increase in urea and aspartate transaminase levels in patients receiving active L-arginine treatment was observed. Responses to acetylcholine (AUC; before vs. after L-arginine: 641.5 +/- 126.7 vs. 695.9 +/- 151.9 U) and sodium nitroprusside were not affected by either L-arginine or placebo. CONCLUSIONS: Endothelial dysfunction was apparent in patients with heart failure despite rigorous vasoactive treatment. Oral administration with L-arginine was ineffective in influencing endothelial function in these patients.

Lipids and the endothelium.

The normal endothelium is characterised by the production of a number of molecules which affect the contractile state of adjacent myocytes and the behavior of formed elements within the blood stream, and by the absence of cell surface adhesion molecules. In addition, endothelial cells are important modulators of coagulation and fibrinolysis. Whilst effects of lipids have been documented on many of these endothelial processes, there is particularly strong evidence for effects on the vasodilatation mediated by endothelium derived nitric oxide and on the interaction between leukocytes and the endothelial surface. Both LDL cholesterol and triglyceride rich lipoproteins impair endothelium dependent vasodilatation. The effects of LDL cholesterol are primarily evident for lipoprotein particles that have been oxidised with evidence for effects of specific constituents of oxidised LDL, such as lysophosphatidylcholine (LPC). LDL effects have been demonstrated at numerous sites of the nitric oxide signaling pathway including receptor-G protein coupling, nitric oxide synthase and NO bioactivity, with evidence for enhanced superoxide formation and the consequent production of the less potent dilator peroxynitrite. The effects of lipids on endothelium dependent vasodilatation can be reversed not only by reducing the level of elevated lipids levels but also by provision of the NOS substrate, L-arginine and by the provision of antioxidants, although the mechanism for these effects are not fully elucidated. The adhesion of leukocytes to the endothelial surface is stimulated by low density and triglyceride rich lipoproteins. As with endothelium dependent vasodilatation, the effects of LDL cholesterol are primarily evident for low-density lipoprotein particles that have been oxidised, and many of the effects of oxidised LDL can be mimicked by LPC. HDL can overcome pro-adhesive effects of oxidised LDL. The effects of LDL on leukocyte adhesion are secondary to the expression of adhesion molecules on the luminal surfaces of endothelial cells. In addition to the likely deleterious effects of lipids on endothelium-mediated vasodilatation and leukocyte-endothelial cell interaction, lipids have been shown to affect a number of other endothelial processes and function. Thus, oxidised LDL affects endothelial ET1 and PGI2 release. Although effects have been shown on endothelial cell growth and apoptosis and on endothelial processes related to thrombosis and fibrinolysis, these effects have been less extensively studied than endothelial dependent vasodilatation and leukocyte-endothelial cell interaction. Many of the effects of elevated or modified low density and TG rich lipoproteins on endothelial cells and endothelial cell processes could be expected to contribute to the development of atherosclerosis and therefore, to the association between lipids and atherosclerotic, particularly coronary, vascular disease. However, the extent to which "endothelial dysfunction" accounts for the known relationships between serum lipid concentrations and CHD is yet to be established.

Non-specific inhibition by human lipoproteins of endothelium dependent relaxation in rat aorta may be attributed to lipoprotein phospholipids.

OBJECTIVE: In vitro incubation of low-density lipoprotein (LDL) is reported to attenuate endothelium dependent relaxation mediated by acetylcholine (ACh) while not affecting endothelium-independent relaxation. This study was designed to examine the effects of other lipid-carrying lipoproteins as well as to study their effects on responses mediated by endothelium dependent agonists other than ACh. METHODS: The effects of human LDL, very-low-density lipoprotein (VLDL) and high density lipoprotein (HDL) on endothelium-dependent relaxation by ACh, histamine and the calcium ionophore, A23187, and endothelium-independent relaxation by sodium nitroprusside (SNP) were investigated is rat isolated aortic rings. The effects of combined LDL and HDL incubation on responses mediated by ACh were also examined. Control experiments included experiments examining the effects of bovine serum albumin on responses mediated by ACh. Thiobarbituric-acid-reactive substances (TBARS) measured before and after organ bath incubation indicated little oxidation of the lipoproteins used. RESULTS: Maximal responses to ACh were inhibited by LDL, VLDL and HDL (0.02 and 0.2 mg protein/ml), to histamine by LDL (0.2 mg protein/ml), VLDL (0.02 and 0.2 mg protein/ml) and HDL (0.02 mg protein/ml) and to A23187 by LDL (0.2 mg protein/ml). VLDL (0.2 mg protein/ml) and HDL (0.02 and 0.2 mg protein/ml). A small but significant correlation (r = 0.54, P = 0.01) was observed between the level of inhibition of the endothelium-dependent responses and lipoprotein phospholipid concentration in the organ bath but not between the level of inhibition and cholesterol (free and esterified) or triglyceride concentrations. Responses to SNP were unaffected by LDL, VLDL and HDL. Combined incubation of tissues with LDL (0.2 mg protein/ml) and HDL (0.2 mg protein/ml) significantly increased maximal responses to ACh (pre-lipoproteins 81.8 +/- 5.7 vs plus-LDL/HDL 100 +/- 0.0; P < 0.05). Bovine serum albumin had no effect on the maximal responses to ACh. CONCLUSIONS: We conclude that inhibition by human lipoproteins of endothelium-dependent agonists occurs with LDL, HDL and VLDL and suggest that this may be due to the phospholipid content of each lipoprotein. However, combined incubation of HDL with LDL negates this effect and an increased maximal response to ACh is reported.

The isoflavone metabolite dehydroequol produces vasodilatation in human resistance arteries via a nitric oxide-dependent mechanism.

BACKGROUND: Isoflavones (phytoestrogens) offer potential cardioprotective benefits. We recently reported on the vasodilatory activity of the isoflavone metabolite, dehydroequol, in rat isolated aortic ring preparations. In the current study, we examine the effect of this metabolite on the vascular haemodynamic profile in human forearm resistance arteries. METHODS AND RESULTS: Responses to brachial artery infusion of dehydroequol (0.1, 0.3, 1 and 3 micromol/min) in forearm resistance arteries were obtained in six healthy males. These were done, on two separate occasions, in the absence and presence of endogenous nitric oxide synthase inhibition using NG-monomethyl-L-arginine, with sufficient sodium nitroprusside to maintain vascular tone. Dehydroequol produced a dose-dependent increase in forearm blood flow from 2.44 +/- 0.37 (basal) to 5.25 +/- 1.07 mL/100 mL/min (P < 0.05) at dehydroequol 3 micromol/min. Responses to dehydroequol were significantly dampened with inhibition of endogenous nitric oxide synthase (at 3 micromol/min: % increase in forearm blood flow fell from 114.3 +/- 22.81 to 19.45 +/- 9.19; P < 0.01). CONCLUSION: This is the first report of dehydroequol, a metabolite derived from the isoflavone diadzein, demonstrating potent vasodilatory properties in human resistance arteries via a nitric oxide-dependent mechanism.

Responses to endothelium-dependent agonists in subcutaneous arteries excised from hypercholesterolaemic men.

1. Vasomotor function of the vascular endothelium was examined in human subcutaneous arteries excised from 8 hypercholesterolaemic and 7 normolipidaemic subjects. 2. Left gluteal skin biopsies were performed under local anaesthesia. Subcutaneous arteries were isolated and two vessels from each subject mounted in separate myographs. A 20 ml fasting blood sample was taken at the time of the biopsy. 3. Hypercholesterolaemic subjects had either never been treated with lipid lowering therapy or therapy had been stopped at least two weeks before the study (n = 2). At the time of the study total plasma cholesterol levels (control: 4.6+/-0.3 vs hypercholesterolaemic: 8.3+/-0.6 mmol l(-1): P < 0.01) were significantly elevated in hypercholesterolaemic subjects when compared with controls. 4. Full concentration-response curves to the vasoconstrictor noradrenaline and the vasodilators acetylcholine and substance P were constructed. A single point concentration-response to sodium nitroprusside (10 microM) was also obtained. Dilator responses were obtained in vessels pre-constricted with a submaximal concentration of noradrenaline. Vessels were then incubated for 30 min with either L- or D-arginine (10 microM) and the concentration-response curves to the three dilator agonists repeated in the presence of the amino acid. 5. Maximum relaxation responses to acetylcholine (control vs hypercholesterolaemic: 83.3+/-6.1% vs 47.4+/-13.5%; P < 0.05), but not to substance P or sodium nitroprusside, were dampened in the hypercholesterolaemic group when compared with controls. 6. Neither incubation with L-arginine nor D-arginine had any effect on maximum relaxation responses to acetylcholine in either the control group (pre L-arginine vs plus L-arginine: 83.3+/-6.1 vs 82.3+/-5.5%, pre D-arginine vs plus D-arginine: 98.9+/-1.2 vs 98.2+/-1.1%) or the hypercholesterolaemic group (pre L-arginine vs plus L-arginine: 47.4+/-13.5 vs 55.3+/-14.3%, pre D-arginine vs plus D-arginine: 43.3+/-13.6 vs 65.4+/-12.3%). 7. When results from the two study groups were pooled, the strongest predictor of maximum relaxation obtained to acetylcholine was apolipoprotein A1 (r = 0.67; P = 0.001). 8. In conclusion, relaxation responses mediated by the endothelium-dependent agonist acetylcholine, but not by substance P, are impaired in hypercholesterolaemic patients. L-Arginine did not improve the impaired relaxation responses to acetylcholine. We suggest that impaired endothelium-dependent relaxation is specific to acetylcholine and not to an abnormal L-arginine-nitric oxide pathway in subcutaneous arteries excised from this study group.

The vascular activity of some isoflavone metabolites: implications for a cardioprotective role.

Legume-derived isoflavones such as genistein, diadzein and equol have been associated with a reduction in risk of cardiovascular disease. In the current study, we explore the vascular activity of several isoflavone metabolites namely dihydrodaidzein, cis and trans-tetrahydrodaidzein and dehydroequol for potential cardioprotective properties. Rat isolated aortic rings were used. 17beta-oestradiol, equol, and all four of the metabolites studied significantly antagonized contractile responses to noradrenaline. The direct vasodilatory action of these compounds were examined and in contrast to 17beta-oestradiol, the vasodilatory effect of which was demonstrated to be endothelium independent, the dilatory action of all four compounds could be inhibited by endothelium denudation. Further, the dilatory action of both dihydrodaidzein and cis-tetrahydrodaidzein were inhibited by the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (NOLA), by the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by 40 mM KCl. Dilatory responses to dehydroequol and trans-tetrahydrodaidzein, on the other hand, were inhibited by 40 mM KCL but not by NOLA nor ODQ. Finally, we examined the protective potential of these compounds in inhibiting endothelium damage by oxidized low density lipoprotein (ox-LDL). Trans-tetrahydrodaidzein was at least 10 fold more potent than 17beta-oestradiol in protecting against ox-LDL induced damage. We conclude that the isoflavone metabolites, dihydrodaidzein, cis- and trans-tetrahydrodaidzein and dehydroequol, may potentially represent a novel series of cardioprotective therapeutics.

Hypercholesterolemia and endothelium dysfunction: role of dietary supplementation as vascular protective agents.

There is increasing evidence that dietary supplementation, such as L-arginine, anti-oxidant vitamins, soy phytoestrogens, flavonoids and omega-3 fatty acids exert vascular protective benefits particularly in terms of restoring endothelial function in cardiovascular disease states. The endothelium has been a major focus over the past 20 years as being a primary site at which dysfunction occurs in association with, and contributing to, vascular pathologies. Such states include mild compromise of the cardiovascular system as observed in smokers, hypercholesterolemics and hypertensives, through to end-point heart failure. This review will focus on the experimental and clinical evidence examining the effect of nutriceuticals on vascular function, in particular endothelium-derived factors, and argues that there is a role for nutriceuticals in the clinical management of the cardiovascular compromised individual.

Effect of fish oil supplementation on aortic compliance in rats: role of the endothelium.

Arterial compliance improves with dietary fish oils in patients with high cardiovascular risk. Since fish oils alter prostaglandin metabolism and the L-arginine-nitric oxide pathway, and since compliance may be modified by vasoactive substances, the effect of the endothelium and some of its derivatives on aortic complaince were examined. Rats were randomly allocated to four groups, the first of which fed only the regular chow. The remaining three groups were fed the chow supplemented by daily gavage with either coconut, fish or safflower oil for 8 weeks. The thoracic aorta was removed and six 2 mm rings obtained. Rings were paired and one from each pair treated with either N(W)-nitro-L-arginine, indomethacin or de-endothelialized. A diameter-tension curve was initiated from wire touch position using incremental increases in wire distance until no further response observed. The data was transformed to a diameter-pressure relationship and fitted with a linear equation, the slope of which related directly to compliance. De-endothelialization (slopes: control vs de-endothelialized: 9.05+/-0.15 vs 8.31+/-0.24; P< 0.05) and indomethacin (slopes: control vs indomethacin: 9.11+/-0.15 vs 7.76+/-0.37; P< 0.05) significantly decreased arterial compliance as did dietary fish oils (slopes: control vs n-3: 9.16+/-0.11 vs 7.84+/-0.39; P< 0.05). No further effect was seen with indomethacin in the fish oil treated group. It is concluded that the endothelium and in particular, endothelium derived prostanoids, contribute to vessel compliance. We also conclude that fish oils have a similar action to indomethacin, leading to the increase in aortic stiffness observed.

Impaired endothelium-dependent relaxation in large, but not small arteries in rats after coronary ligation.

Vascular responses were studied in both large and small arteries of rats following 8 weeks of heart failure produced by coronary ligation. Responses to noradrenaline, acetylcholine and sodium nitroprusside were studied in isolated thoracic aorta and mesenteric arteries. In the aorta, concentration-response curves for noradrenaline were similar between heart failure and sham animals and unaffected by the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG). Relaxation by acetylcholine was impaired in heart failure rats (EC50-6.79 log M heart failure vs. -7.15 log M sham). In the presence of L-NOARG, relaxation by acetylcholine was completely abolished in rings from sham rats, whereas constriction was observed in rings from heart failure rats. Relaxation by sodium nitroprusside was not different between sham and heart failure rats. In mesenteric arteries, responses to noradrenaline, acetylcholine and sodium nitroprusside were not different between heart failure and sham rats. L-NOARG reduced the maximum response to acetylcholine in both heart failure (82% to 50%) and shams (89% to 49%) by a similar magnitude, with no effect on relaxation to sodium nitroprusside. These data suggest that acetylcholine-induced relaxation is impaired in the aorta, but not mesenteric arteries in rats with heart failure. The mechanism is not solely due to impaired nitric oxide release and may be due to acetylcholine-induced contraction.

Lipids and atherosclerosis: clinical management of hypercholesterolaemia.

Hypercholesterolaemia is a major risk factor for the development of atherosclerosis which, in turn, underlies most ischaemic heart disease (IHD). This review deals briefly with the pathophysiology of lipids in humans and follows with a discussion of current lipid-lowering therapies. In those patients with a history of myocardial infarction (MI) or unstable angina, appropriate lipid-lowering therapy has been convincingly shown to reduce not only cardiac events but also overall mortality. The advent of the HMG CoA reductase inhibitors in the late 1980s has had a revolutionary impact in the clinical management of hypercholesterolaemia, not only because of their efficacy but especially because they are well-tolerated. The use of other treatments such as the fibrates and bile acid resins are also discussed. Given the successful use of the statins, it is felt that an emergence of a different class of LDL-cholesterol lowering compound is unlikely in the near future and rather that compounds which can increase HDL-cholesterol while lowering LDL will be of greater impact. There may also be a shifting trend towards such naturally occurring compounds as plant stanols and phytoestrogens.

Arginase II inhibition prevents nitrate tolerance.

BACKGROUND AND PURPOSE: Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance. EXPERIMENTAL APPROACH: Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII -/-) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence. KEY RESULTS: Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration-response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII -/- mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII -/- mice did not. CONCLUSION AND IMPLICATIONS: Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.

Advanced glycation of apolipoprotein A-I impairs its anti-atherogenic properties.

AIMS/HYPOTHESIS: AGE contribute to the pathogenesis of diabetic complications, including dyslipidaemia and atherosclerosis. However, the precise mechanisms remain to be established. In the present study, we examined whether AGE modification of apolipoprotein A-I (apoA-I) affects its functionality, thus altering its cardioprotective profile. MATERIALS AND METHODS: The ability of AGE-modified apoA-I to facilitate cholesterol and phospholipid efflux, stabilise ATP-binding cassette transporter A1 (ABCA1) and inhibit expression of adhesion molecules in human macrophages and monocytes was studied. RESULTS: The ability of AGE-modified apoA-I to promote cholesterol efflux from THP-1 macrophages, isolated human monocytes and from ABCA1-transfected HeLa cells was significantly reduced (>70%) compared with unmodified apoA-I. This effect was reversed by preventing AGE formation with aminoguanidine or reversing AGE modification using the cross-link breaker alagebrium chloride. AGE-modification of HDL also reduced its capacity to promote cholesterol efflux. AGE-apoA-I was also less effective than apoA-I in stabilising ABCA1 in THP-1 cells as well as in inhibiting expression of CD11b in human monocytes. CONCLUSIONS/INTERPRETATION: AGE modification of apoA-I considerably impairs its cardioprotective, antiatherogenic properties, including the ability to promote cholesterol efflux, stabilise ABCA1 and inhibit the expression of adhesion molecules. These findings provide a rationale for targeting AGE in the management of diabetic dyslipidaemia.

Transient improvement of acetylcholine responses after short-term oral L-arginine in forearms of human heart failure.

Patients with heart failure exhibit impaired endothelium-dependent vasodilation. Although brief intraarterial administration of L-arginine improves endothelium-dependent vasodilatation in these patients, long-term oral supplementation is ineffective. To resolve these conflicting findings, we examined the effect of a single, short-term oral dose of L-arginine on serial, hourly forearm vascular responses to acetylcholine, sodium nitroprusside, and norepinephrine. Eighteen patients with heart failure were randomly allocated in a double-blinded, crossover study to receive either a single 20-g oral dose of L-arginine or placebo. Vascular responses were measured by forearm venous occlusion plethysmography before and at 60, 120, and 180 min after dosage. Blood was obtained for measurement of L-arginine and nitric oxide metabolite levels. Oral L-arginine increased plasma levels by fourfold at 60, 120, and 180 min. Vasodilatation to acetylcholine, 37 microg/min, was significantly enhanced at 60 min (percentage increase in forearm blood flow: placebo, 413 +/- 64%; L-arginine, 587 +/- 94%; p < 0.05), discernible at 120 min (p = 0.058) but no longer apparent at 180 min. Neither basal forearm blood flow, sodium nitroprusside, nor norepinephrine responses nor plasma levels of nitrite and nitrate were altered. We conclude that although short-term oral supplementation with L-arginine produced marked sustained elevation of plasma levels of L-arginine in patients with heart failure, responses to acetylcholine were only transiently improved.

Restoration of nitric oxide function in human hyperlipidaemia, congestive heart failure and liver cirrhosis.

1. There is accumulating evidence for a range of abnormalities in the nitric oxide (NO) signalling cascade in human cardiovascular disorders. 2. In the present review we assess the literature detailing such evidence in early (hyperlipidaemia) and end-stage (heart failure) disease, with emphasis on the mechanisms by which the disturbances are thought to occur. 3. Strategies for the correction of disturbed NO signalling in these states are reviewed and include both prescribed pharmacological interventions, such as lipid-lowering therapy and novel uses of angiotensin-converting enzyme inhibitors, as well as non-pharmacological interventions, such as exercise and dietary supplementation with L-arginine and n-3 polyunsaturated fatty acids. 4. In addition to a decreased production/function of NO, the possible detrimental effects of a chronic elevation in NO production in patients with liver cirrhosis, together with a novel use of antibiotics to correct this perturbation, is outlined.

Evidence for altered vascular responses to exogenous endothelin-1 in patients with advanced cirrhosis with restoration of the normal vasoconstrictor response following successful liver transplantation.

BACKGROUND AND AIMS: There is evidence that dampened responses to endogenous vasoconstrictors contribute to the hyperdynamic circulation that is characteristic of advanced cirrhosis. The aim of this study was to determine whether there is an altered vascular responsiveness to the endothelium derived constricting factor endothelin-1 (ET-1) in patients with decompensated chronic liver disease which might contribute to this abnormal circulatory state, and whether normal endothelin responses are restored following liver transplantation. METHODS: Using forearm plethysmography, we studied the vascular response to an intra-arterial ET-1 infusion in six patients with end stage cirrhosis, before and after liver transplantation, compared with six normal control subjects. Responses to the selective endothelin A (ET(A)) receptor subtype antagonist, BQ123, were also examined. RESULTS: The forearm vessels of patients with cirrhosis vasodilated in response to ET-1 infusion while in healthy controls a marked vasoconstriction response was observed (p<0.0001, area under the curve time-blood flow was normal compared with the cirrhosis groups, ANOVA). Prior to commencement of liver transplant surgery, cirrhotic patients were confirmed to have a hyperdynamic circulation with a high cardiac index (4.07 (0.23) l/min/m(2) (normal range 2.8-3.6 l/min/m(2))) and low systemic vascular resistance index (1284 (115) dynxs/cm(5)/m(2) (normal range 1760-2600 dynxs/cm(5)/m(2))). Following transplantation, normal vasoconstrictor responses to ET-1 were restored. Responses to BQ123 were not different in patients with advanced cirrhosis compared with controls. CONCLUSION: In patients with end stage cirrhosis, ET-1 produces vasodilatation at a dose that causes marked vasoconstriction in normal control subjects. This effect is not attributable to impairment of ET(A) receptor responses. Our findings suggest that altered endothelin responses may contribute to the generalised dilatation of the circulation that occurs in patients with advanced liver disease.