Ramón Deulofeu

A Walnut Diet Improves Endothelial Function in Hypercholesterolemic Subjects A Randomized Crossover Trial

Background—Epidemiological studies suggest that nut intake decreases coronary artery disease (CAD) risk. Nuts have a cholesterol-lowering effect that partly explains this benefit. Endothelial dysfunction is associated with CAD and its risk factors and is reversed by antioxidants and marine n-3 fatty acids. Walnuts are a rich source of both antioxidants and &agr;-linolenic acid, a plant n-3 fatty acid. Methods and Results—To test the hypothesis that walnut intake will reverse endothelial dysfunction, we randomized in a crossover design 21 hypercholesterolemic men and women to a cholesterol-lowering Mediterranean diet and a diet of similar energy and fat content in which walnuts replaced ≈32% of the energy from monounsaturated fat. Participants followed each diet for 4 weeks. After each intervention, we obtained fasting blood and performed ultrasound measurements of brachial artery vasomotor function. Eighteen subjects completing the protocol had suitable ultrasound studies. Compared with the Mediterranean diet, the walnut diet improved endothelium-dependent vasodilation and reduced levels of vascular cell adhesion molecule-1 (P <0.05 for both). Endothelium-independent vasodilation and levels of intercellular adhesion molecule-1, C-reactive protein, homocysteine, and oxidation biomarkers were similar after each diet. The walnut diet significantly reduced total cholesterol (−4.4±7.4%) and LDL cholesterol (−6.4±10.0%) (P <0.05 for both). Cholesterol reductions correlated with increases of both dietary &agr;-linolenic acid and LDL &ggr;-tocopherol content, and changes of endothelium-dependent vasodilation correlated with those of cholesterol-to-HDL ratios (P <0.05 for all). Conclusions—Substituting walnuts for monounsaturated fat in a Mediterranean diet improves endothelium-dependent vasodilation in hypercholesterolemic subjects. This finding might explain the cardioprotective effect of nut intake beyond cholesterol lowering.

Sympathetic nervous activity, renin-angiotensin system and renal excretion of prostaglandin E2 in cirrhosis. Relationship to functional renal failure and sodium and water excretion

Abstract. To investigate if functional renal failure in cirrhosis could be related to disturbances of vasoactive systems, plasma renin activity, plasma catecholamines and urinary prostaglandin E2 (PGE2) were determined in twenty‐two normal subjects and sixty‐five cirrhotics. Furthermore, in thirty‐three of these subjects, the effect of lysine‐acetylsalicylate (450 mg i.v.) on renal function was studied.

Neutrophil infiltration increases matrix metalloproteinase-9 in the ischemic brain after occlusion/reperfusion of the middle cerebral artery in rats.

Matrix metalloproteinase-9 (MMP-9) activity increases in the brain during the first day after focal ischemia and might be involved in the pathogenesis of tissue damage. We previously showed MMP-9 in the extracellular space of brain parenchyma along with neutrophil recruitment after ischemia. In the present study, we tested whether neutrophils were a direct source of enhanced MMP-9 in the ischemic brain. Neutrophil infiltration was prevented either by injecting an antibody against ICAM-1, which abrogates neutrophil adhesion to the endothelial vessel wall, or by inducing neutropenia. One-hour intraluminal middle cerebral artery occlusion with reperfusion was induced, and studies were performed at 24 hours. Circulating neutrophils expressed 95-kDa MMP-9 and dimers, and infiltrated neutrophils stained positive for MMP-9. The expression of MMP-9 (mainly 95-kDa proform and dimers and, to a lesser extent, 88-kDa form) increased in brain after ischemia/reperfusion. Treatments preventing neutrophil infiltration failed to preclude the ischemia-induced increase in 88-kDa MMP-9 form and gelatinase activity in neurons and blood vessels. However, these treatments prevented the major increase in 95-kDa MMP-9 form and dimers. We conclude that neutrophil infiltration highly contributes to enhanced MMP-9 in the ischemic brain by releasing MMP-9 proform, which might participate in the tissular inflammatory reaction.

Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA

Docosahexaenoic acid (DHA) is a ω‐3 essential fatty acid that reduces the incidence and severity of a number of diseases. Recently, a novel series of DHA‐derived lipid mediators with potent protective actions has been identified. In this study we demonstrate that dietary amplification of these DHA‐derived products protects the liver from necroinflammatory injury. In vitro, supplementation of hepatocytes with DHA significantly reduced hydrogen peroxide‐induced DNA damage, evaluated by the “comet assay,” and oxidative stress, determined by measurement of malondialdehyde levels. In vivo, dietary supplementation of mice with DHA ameliorated carbon tetrachloride‐induced necroinflammatory damage. In addition, hepatic cyclooxygenase‐2 expression and PGE2 levels were significantly reduced in mice fed DHA‐enriched diets. In these animals, increased hepatic formation of DHA‐derived lipid mediators (i.e., 17S‐hydroxy‐DHA (17S‐HDHA) and protectin D1) was detected by HPLC‐gas chromatography/mass spectrometry analysis. Consistent with these findings, synthetic 17‐HDHA abrogated genotoxic and oxidative damage in hepatocytes and decreased TNF‐α release and 5‐lipoxygenase expression in macrophages. In a transactivation assay, 17‐ HDHA acted in a concentration‐dependent manner as a PPARγ agonist. Taken together, these findings identify a potential role for DHA‐derived products, specifically 17S‐HDHA and protectin D1, in mediating the protective effects of dietary DHA in necroinflammatory liver injury.—González‐Périz, A., Planagumà, A., Gronert, K., Miquel, R., López‐Parra, M., Titos, E., Horrillo, R., Ferré, N., Deulofeu, R., Arroyo, V., Rodés, J., Clària, J. Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S‐hydroxy‐DHA. FASEB J. 20, E1844–E1855 (2006)

Safety and efficacy of uric acid in patients with acute stroke (URICO-ICTUS): a randomised, double-blind phase 2b/3 trial

INTRODUCTION Uric acid is an antioxidant with neuroprotective effects in experimental models of stroke. We assessed whether uric acid therapy would improve functional outcomes at 90 days in patients with acute ischaemic stroke. METHODS URICO-ICTUS was a randomised, double-blind, placebo-controlled, phase 2b/3 trial that recruited patients with acute ischaemic stroke admitted to ten Spanish stroke centres. Patients were included if they were aged 18 years or older, had received alteplase within 4·5 h of symptom onset, and had an eligible National Institutes of Health Stroke Scale (NIHSS) score (>6 and ≤25) and premorbid (assessed by anamnesis) modified Rankin Scale (mRS) score (≤2). Patients were randomly allocated (1:1) to receive uric acid 1000 mg or placebo (both infused intravenously in 90 min during the infusion of alteplase), stratified by centre and baseline stroke severity. The primary outcome was the proportion of patients with excellent outcome (ie, an mRS score of 0-1, or 2 if premorbid score was 2) at 90 days, analysed in the target population (all randomly assigned patients who had been correctly diagnosed with ischaemic stroke and had begun study medication). The study is registered with ClinicalTrials.gov, number NCT00860366. FINDINGS Between July 1, 2011, and April 30, 2013, we randomly assigned 421 patients, of whom 411 (98%) were included in the target population (211 received uric acid and 200 received placebo). 83 (39%) patients who received uric acid and 66 (33%) patients who received placebo had an excellent outcome (adjusted risk ratio 1·23 [95% CI 0·96-1·56]; p=0·099). No clinically relevant or statistically significant differences were reported between groups with respect to death (28 [13%] patients who received uric acid vs 31 [16%] who received placebo), symptomatic intracerebral haemorrhage (nine [4%] vs six [3%]), and gouty arthritis (one [<1%] vs four [2%]). 516 adverse events occurred in the uric acid group and 532 in the placebo group, of which 61 (12%) and 67 (13%), respectively, were serious adverse events (p=0·703). INTERPRETATION The addition of uric acid to thrombolytic therapy did not increase the proportion of patients who achieved excellent outcome after stroke compared with placebo, but it did not lead to any safety concerns. FUNDING Institute of Health Carlos III of the Spanish Ministry of Health and Fundación Doctor Melchor Colet.

5-Lipoxygenase Activating Protein Signals Adipose Tissue Inflammation and Lipid Dysfunction in Experimental Obesity

The presence of the so-called low-grade inflammatory state is recognized as a critical event in adipose tissue dysfunction, leading to altered secretion of adipokines and free fatty acids (FFAs), insulin resistance, and development of hepatic complications associated with obesity. This study was designed to investigate the potential contribution of the proinflammatory 5-lipoxygenase (5-LO) pathway to adipose tissue inflammation and lipid dysfunction in experimental obesity. Constitutive expression of key components of the 5-LO pathway, as well as leukotriene (LT) receptors, was detected in adipose tissue as well as in adipocyte and stromal vascular fractions. Adipose tissue from obese mice, compared with that from lean mice, exhibited increased 5-LO activating protein (FLAP) expression and LTB4 levels. Incubation of adipose tissue with 5-LO products resulted in NF-κB activation and augmented secretion of proinflammatory adipokines such as MCP-1, IL-6, and TNF-α. In addition, LTB4, but not LTD4, reduced FFA uptake in primary adipocytes, whereas 5-LO inhibition suppressed isoproterenol-induced adipose tissue lipolysis. In mice with dietary obesity, elevated FLAP expression in adipose tissue was paralleled with macrophage infiltration, increased circulating FFA levels, and hepatic steatosis, phenomena that were reversed by FLAP inhibition with Bay-X-1005. Interestingly, FLAP inhibition induced AMP-activated protein kinase phosphorylation in parallel with decreases in hormone-sensitive lipase activity and the expression and secretion of TNF-α and IL-6. Similar effects were observed in differentiated 3T3-L1 adipocytes incubated with either Bay-X-1005 or the selective LTB4 receptor antagonist U-75302. Taken together, these findings indicate that the 5-LO pathway signals the adipose tissue low-grade inflammatory state and steatogenic potential in experimental obesity.

Ischemic pre-conditioning in deceased donor liver transplantation: a prospective randomized clinical trial.

To assess the immediate and long‐term effects of ischemic preconditioning (IPC) in deceased donor. liver transplantation (LT), we designed a prospective, randomized controlled trial involving 60 donors: control group (CTL, n = 30) or study group (IPC, n = 30). IPC was induced by 10‐min hiliar clamping immediately before recovery of organs. Clinical data and blood and liver samples were obtained in the donor and in the recipient for measurements. IPC significantly improved biochemical markers of liver cell function such as uric acid, hyaluronic acid and Hypoxia‐Induced Factor‐1alpha (HIF‐1α) levels. Moreover, the degree of apoptosis was significantly lower in the IPC group. On clinical basis, IPC significantly improved the serum aspartate aminotransferase (AST) levels and reduced the need for reoperation in the postoperative period. Moreover, the incidence of primary nonfunction (PNF) was lower in the IPC group, but did not achieve statistical significance. We conclude that 10‐min IPC protects against I/R injury in deceased donor LT.

Ascorbic acid improves the intrahepatic endothelial dysfunction of patients with cirrhosis and portal hypertension

Patients with cirrhosis show intrahepatic endothelial dysfunction, characterized by an impaired flow‐dependent vasorelaxation. This alteration is responsible for the marked postprandial increase in portal pressure and is attributed to an insufficient release of nitric oxide (NO). Ascorbic acid reverts endothelial dysfunction in other vascular disorders, via the increase of NO bioavailability through the neutralization of superoxide anions, thus preventing the scavenging of NO by superoxide. This study examined whether acute ascorbic acid administration might improve endothelial dysfunction in cirrhosis. Thirty‐seven portal hypertensive patients with cirrhosis had measurements of hepatic and systemic hemodynamics, ascorbic acid, and malondialdehyde (MDA). Patients were randomly allocated to receive ascorbic acid (3 g, intravenously, n = 15) or placebo (n = 12) followed by a liquid meal. A third group received ascorbic acid followed by a sham meal (n = 10). Measurements were repeated after 30 minutes (hepatic venous pressure gradient at 15 and 30 minutes). Patients with cirrhosis had significantly lower ascorbic acid levels and higher MDA than healthy controls. Ascorbic acid significantly reduced MDA levels and markedly attenuated the postprandial increase in the hepatic venous pressure gradient (4% ± 7% vs. 18% ± 10% in placebo at 30 minutes, P < .001). Ascorbic acid followed by sham meal did not modify hepatic or systemic hemodynamics. In conclusion, patients with cirrhosis exhibited intrahepatic endothelial dysfunction, associated with decreased levels of ascorbic acid and increased levels of MDA. Ascorbic acid improved intrahepatic endothelial dysfunction, blunting the postprandial increase in portal pressure. These results encourage the performance of further studies testing antioxidants as adjunctive therapy in the treatment of portal hypertension. (HEPATOLOGY 2006;43:485–491.)

Endogenous nitric oxide and exogenous nitric oxide supplementation in hepatic ischemia-reperfusion injury in the rat.

BACKGROUND Although nitric oxide (NO) is thought to be beneficial in hepatic ischemia-reperfusion (I/R), the mechanisms for this effect are not well established. METHODS To investigate the effects of endogenous NO and exogenous NO supplementation on hepatic I/R injury and their pathogenic mechanisms, serum ALT and hyaluronic acid (endothelial cell damage), and hepatic malondialdehyde and H2O2 (oxidative stress), myeloperoxidase activity (leukocyte accumulation), and endothelin (vasoconstrictor peptide opposite to NO) were determined at different reperfusion periods in untreated rats and rats receiving L-NAME, L-NAME+L-arginine, and spermine NONOate (exogenous NO donor). RESULTS After reperfusion every parameter increased in untreated animals. Endogenous NO synthesis inhibition by L-NAME increased hepatocyte and endothelial damage as compared to untreated rats, which was reverted and even improved by the addition of L-arginine. Spermine NONOate also improved this damage. However, different mechanisms account for the beneficial effect of endogenous and exogenous NO. Oxidative stress decreased by both L-NAME and L-NAME+L-arginine, but remained unmodified by spermine NONOate. Myeloperoxidase increased by L-NAME and this effect was reverted by the addition of L-arginine, whereas no change was observed with spermine NONOate. Endothelin levels were not modified by L-NAME and L-NAME+L-arginine, but decreased with spermine NONOate. CONCLUSIONS These results suggest that, although both endogenous and exogenous NO exert a protective role in experimental hepatic I/R injury, the mechanisms of the beneficial effect of the two sources of NO are different.

The eNOS cofactor tetrahydrobiopterin improves endothelial dysfunction in livers of rats with CCl4 cirrhosis

In cirrhosis, intrahepatic endothelial dysfunction is one of the mechanisms involved in the increased resistance to portal blood flow and therefore in the development of portal hypertension. Endothelial nitric oxide synthase (eNOS) uncoupling due to deficiency of tetrahydrobiopterin (BH4) results in decreased production of NO and plays a major role in endothelial dysfunction in other conditions. We examined whether eNOS uncoupling is involved in the pathogenesis of endothelial dysfunction of livers with cirrhosis. Basal levels of tetrahydrobiopterin and guanosine triphosphate (GTP)‐cyclohydrolase (BH4 rate‐limiting enzyme) expression and activity were determined in liver homogenates of control and rats with CCl4 cirrhosis. Thereafter, rats were treated with tetrahydrobiopterin, and eNOS activity, NO bioavailability, assessed with a functional assay, and the vasodilator response to acetylcholine (endothelial function) were evaluated. Livers with cirrhosis showed reduced BH4 levels and decreased GTP‐cyclohydrolase activity and expression, which were associated with impaired vasorelaxation to acetylcholine. Tetrahydrobiopterin supplementation increased BH4hepatic levels and eNOS activity and significantly improved the vasodilator response to acetylcholine in rats with cirrhosis. In conclusion, the impaired response to acetylcholine of livers with cirrhosis is modulated by a reduced availability of the eNOS cofactor, tetrahydrobiopterin. Tetrahydrobiopterin supplementation improved the endothelial dysfunction of cirrhotic livers. (HEPATOLOGY 2006;44:44–52.)