Myocardial glutathione metabolic status in fat-fed rabbits.

Short-term fat feeding could exert adverse cardiac effects by altering myocardial glutathione-related antioxidant defenses. We have here assessed total glutathione (TG), the activities of glutathione reductase (GSSG-Red), γ-glutamylcysteine synthetase (γ-GCS), γ-glutamyl transpeptidase (γ-GT) and glutathione peroxidase (GSH-Px), fluorescent damage products of lipid peroxidation (FDPL), thiobarbituric acid-reactive substances (TBARS), H2O2, and ATP in the aerobically perfused hearts of control rabbits and of rabbits fed a fat-enriched diet for 18 days. Such biochemical parameters, myocardial hemodynamics and infarct size were assessed in the perfused hearts of other control and fat-fed rabbits subjected to 60 min global ischemia plus 30 min reperfusion. Compared to controls, a reduced activity of GSSG-Red and γ-GT associated with decreased TG content was detected in the aerobically perfused hearts of fat-fed rabbits, which also showed insignificant γ-GCS activation, GSH-Px depressed activity, FDPL, TBARS and H2O2 burden, and unaltered ATP content. Ischemia-reperfusion decreased the myocardial levels of TG, ATP, and γ-GCS activity and augmented those of FDPL, TBARS, and H2O2 especially in the fat-fed rabbits, without significant changes in myocardial GSSG-Red, γ-GT, and GSH-Px activities. Ischemia-reperfusion induced greater hemodynamic dysfunction and infarct size in the hearts of fat-fed rabbits than in those of controls. Thus, short-term fat feeding and hyperlipidemia alter glutathione metabolic status of the rabbit myocardium, inducing a GSSG-Red- and γ-GT-related decrement of myocardial glutathione content, which, together with GSH-Px dysfunction, may favor tissue oxidative stress and render the myocardium more susceptible to ischemia-reperfusion injury.

Thrombophilia and outcomes of assisted reproduction technologies: a systematic review and meta-analysis.

Thrombophilia has been associated with pregnancy complications and recurrent miscarriage. The aim of this systematic review was to evaluate the controversial association between thrombophilia and failures of assisted reproduction technology (ART). A systematic search of the literature for studies reporting on thrombophilia in women undergoing ART up to April 2011 yielded 33 studies (23 evaluating anti-phospholipid antibodies, 5 inherited thrombophilia, and 5 both) involving 6092 patients. Overall, methodologic quality of the studies was poor. Combined results from case-control studies showed that factor V Leiden was significantly more prevalent among women with ART failure compared with fertile parous women or those achieving pregnancy after ART (odds ratio = 3.08; 95% confidence interval, 1.77-5.36). The prothrombin mutation, methylenetetrahydrofolate reductase mutation, deficiency of protein S, protein C, or anti-thrombin were all not associated with ART failure. Women with ART failure tested more frequently positive for anti-phospholipids antibodies (odds ratio = 3.33; 95% confidence interval, 1.77-6.26) with evidence of high degree of between-study heterogeneity (I(2) = 75%; P < .00001). Prospective cohort studies did not show significant associations between thrombophilia and ART outcomes. Although case-control studies suggest that women experiencing ART failures are more frequently positive for factor V Leiden and anti-phospholipid antibodies, the evidence is inconclusive and not supported by cohort studies.

Prediction of carotid plaques in hypertensive patients by risk factors, left ventricular hypertrophy, and epicardial adipose tissue thickness.

Hypertension and other risk factors (RFs) predispose to carotid plaques (CPs). An association between left ventricular hypertrophy (LVH) or epicardial adipose tissue (EAT) and CPs has also been reported. The aim of the study was to evaluate whether the assessment of LVH and EAT thickness, beyond RFs, would be of additive value in predicting CPs in hypertensive subjects. We studied 548 hypertensive patients aged ≥ 50 years without carotid bruit. LVH and CPs were evaluated and defined according to standard criteria. EAT was measured by echocardiography above the free wall of the right ventricle at end diastole. The presence of LVH and EAT thickness above the median value (3.9 mm) together significantly increased prevalence of CPs in subjects with 0-1 risk factor, but not in those with ≥ 2 RFs who showed high prevalence of CPs independently of LVH and/or EAT. Receiver operating characteristic curve analysis showed that the addition of LVH and higher EAT thickness together significantly improved prediction of CPs in patients with 0-1 risk factor. Indeed, the area under the curve improved from 0.63 (0.56-0.69) to 0.73 (0.67-0.79), which was significantly higher (p < 0.05). In patients with ≥ 2 RFs, the addition of LVH and EAT did not significantly improve prediction of CPs. This study shows that the presence of LVH and higher EAT thickness together improves prediction of CPs in hypertensive patients with 0-1 risk factor and that those with ≥ 2 RFs show high prevalence of CPs independently of LVH and/or EAT.

Primary prophylaxis for venous thromboembolism in ambulatory cancer patients receiving chemotherapy.

BACKGROUND: Venous thromboembolism (VTE) often complicates the clinical course of cancer disease. The risk is further increased by chemotherapy but the safety and efficacy of primary thromboprophylaxis in cancer patients treated with chemotherapy is uncertain. OBJECTIVES: To assess the efficacy and safety of primary thromboprophylaxis in ambulatory cancer patients receiving chemotherapy. SEARCH METHODS: The Cochrane Peripheral Vascular Diseases Group searched their Specialised Register (last searched 3 May 2011) and CENTRAL (2011, Issue 2). The authors searched clinical trials registries and reference lists of relevant studies. SELECTION CRITERIA: Randomised controlled trials (RCTs) comparing unfractionated heparin (UFH), low molecular weight heparin (LMWH), vitamin K antagonists (VKA), direct thrombin inhibitors, direct factor Xa inhibitors or mechanical intervention to no intervention or placebo; or comparing two different anticoagulants. DATA COLLECTION AND ANALYSIS: Data were extracted on methodological quality, patients, interventions and outcomes including symptomatic VTE and major bleeding as the primary effectiveness and safety outcomes, respectively. MAIN RESULTS: Nine RCTs with a total of 3538 patients were considered. None of the RCTs tested UFH, fondaparinux, direct factor Xa inhibitors or mechanical interventions. Overall, the risk of bias was low in most of the studies. LMWH, when compared with inactive control, significantly reduced the incidence of symptomatic VTE (risk ratio (RR) 0.62, 95% confidence interval (CI) 0.41 to 0.93) with no evidence of heterogeneity (I(2) = 0%). The number needed to treat to prevent a symptomatic VTE was 60. LMWH was associated with a 60% increase in major bleeding when compared with inactive control, although this was not statistically significant (RR 1.57, 95% CI 0.69 to 3.60; I(2) = 10%). There was a 45% reduction in overall VTE (RR 0.55, 95% CI 0.34 to 0.88; I(2) = 0%) while for symptomatic pulmonary embolism, asymptomatic VTE, minor bleeding and one-year mortality the differences between the LMWH and control groups were not statistically significant. The effect of the vitamin K antagonist warfarin on preventing symptomatic VTE, measured in only one study, was not statistically significant (RR 0.15, 95% CI 0.02 to 1.20). In one RCT of patients with myeloma, LMWH was associated with a 67% reduction in symptomatic VTE (RR 0.33, 95% CI 0.14 to 0.83) compared with warfarin, with no differences in major bleeding. Antithrombin, evaluated in one study on paediatric patients, had no significant effect on VTE nor major bleeding when compared with inactive control. AUTHORS' CONCLUSIONS: Primary thromboprophylaxis with LMWH significantly reduced the incidence of symptomatic VTE in ambulatory cancer patients treated with chemotherapy. However, the lack of power hampers definite conclusions on the effects on major safety outcomes, which mandates additional studies to determine the risk to benefit ratio of LMWH in this setting.

A prospective evaluation of iron deficiency anemia in the GI endoscopy setting: role of standard endoscopy, videocapsule endoscopy, and CT-enteroclysis.

BACKGROUND: Iron deficiency anemia (IDA) is a frequently encountered condition in clinical practice. After conventional endoscopy, the cause of anemia remains unknown in up to 40% of patients. OBJECTIVE: To evaluate prospectively the diagnostic efficacy of a systematic endoscopic approach to IDA and to compare the diagnostic yield of videocapsule endoscopy (VCE) and CT-enteroclysis in endoscopy-negative patients. DESIGN: Consecutive patients with IDA were enrolled prospectively. SETTING: Open-access endoscopy within an academic hospital. PATIENTS: This study involved 189 patients with IDA, including 98 women and 91 men; mean (±standard deviation) age 68 years±16.6 years. INTERVENTION: Patients with IDA underwent gastroscopy and colonoscopy plus ileoscopy. Endoscopy-negative patients were further blindly evaluated by both CT-enteroclysis and VCE. MAIN OUTCOME MEASUREMENTS: Diagnostic yield of conventional endoscopy; diagnostic yield of VCE versus CT-enteroclysis. RESULTS: Endoscopy results were positive in 144 of 189 patients (76.2%). CT-enteroclysis and VCE allowed a diagnosis in 37 of 45 endoscopy-negative patients (82.2%). Overall, VCE was superior to CT-enteroclysis (77.8% vs 22.2%; P<.001), in particular when flat lesions were found. LIMITATIONS: Single-center study. CONCLUSION: A systematic approach to IDA, which includes standard endoscopy, VCE, and CT-enteroclysis allows an overall diagnostic rate of 95.7%; however, CT-enteroclysis should be limited to cases of nondiagnostic VCE.

Inhibitory activity of salicylic acid on lipoxygenase-dependent lipid peroxidation.

BACKGROUND: Since iron is essential for lipoxygenase activity and salicylic acid (SA) can interact with the metal, possible lipoxygenase inhibition by SA was investigated. METHODS: Kinetic spectrophotometric evaluation of enzymatic lipid peroxidation catalyzed by soybean lipoxygenase (SLO), rabbit reticulocyte 15-lipoxygenase (RR15-LOX), porcine leukocyte 12-lipoxygenase (PL12-LOX) and human recombinant 5-lipoxygenase (HR5-LOX) with and without SA. RESULTS: SA inhibited linoleic, arachidonic and docosahexaenoic acid or human lipoprotein peroxidation catalyzed by SLO with IC50 of, respectively, 107, 153, 47 and 108 microM. Using the same substrates, SA inhibited RR15-LOX with IC50 of, respectively, 49, 63, 27 and 51 microM. Further, arachidonic acid peroxidation catalyzed by PL12-LOX and HR5-LOX was inhibited by SA with IC50 of 101 and 168 microM, respectively. Enzymatic inhibition was complete, reversible and non-competitive. Conceivably due to its lower hydrophobicity, aspirin was less effective, indicating acetylation-independent enzyme inhibition. SA and aspirin were ineffective peroxyl radical scavengers but readily reduced Fe3+, i.e. FeCl3, to Fe2+, suggesting their capacity to reduce Fe3+ at the enzyme active site. Indeed, similar to the catecholic redox inhibitor nordihydroguaiaretic acid, SA inhibited with the same efficiency both ferric and the native ferrous SLO form, indicating that these compounds reduce the active ferric enzyme leading to its inactivation. GENERAL SIGNIFICANCE: SA can inhibit lipoxygenase-catalyzed lipid peroxidation at therapeutic concentrations, suggesting its possible inhibitory activity against enzymatic lipid peroxidation in the clinical setting.

Regression of echocardiographic left ventricular hypertrophy after 2 years of therapy reduces cardiovascular risk in patients with essential hypertension.

BACKGROUND: There is still ambiguity about the prognostic relevance of regression of left ventricular hypertrophy (LVH) (as revealed by echocardiography) in a large population of subjects with hypertension, with and without evidence of LVH in their electrocardiograms (ECGs). This holds true even after adjusting for various confounders including in treatment ambulatory blood pressure (BP). The most suitable time point for a follow-up echocardiography also remains a matter for debate. In this study, we investigated the prognostic relevance of regression of LVH after 2 years of therapy, in a large population of subjects with hypertension, and possessing the aforesaid characteristics. METHODS: The occurrence of adverse cardiovascular events was evaluated in 387 patients with LVH shown by echocardiography at baseline, and these patients were studied again after 2 years of therapy. At the second examination, 245 subjects showed regression of LVH, whereas 142 did not. RESULTS: During the time period before the subsequent follow up (6.2 +/- 3 years, range 1.9-12.9 years), 59 first adverse events (26 cardiac and 33 cerebrovascular) had occurred among these subjects. The event rates per 100 patient-years in patients with and without LVH regression were 1.06 and 4.4, respectively. After adjusting for several covariates at the 2-year visit, including in treatment ambulatory BP, Cox regression analysis showed that cardiovascular risk was significantly lower in patients with LVH regression than in those without (RR 0.36, 95% CI 0.19-0.68, P = 0.002). When left ventricular (LV) mass index reduction was analyzed instead of LVH status, it was found to be significantly associated with reduced risk (RR 0.62 per 1-s.d. decrease, 95% CI 0.44-0.88, P = 0.01). CONCLUSIONS: Regression of LVH, as revealed by echocardiography after 2 years of therapy, is associated with reduced cardiovascular risk in patients with hypertension, whether or not LVH was revealed in their ECGs. This holds true even after adjusting for various confounders including in treatment ambulatory BP.

Prognostic relevance of masked hypertension in subjects with prehypertension.

BACKGROUND: The prognostic impact of masked hypertension is not yet completely clear. The aim of this study was to evaluate the prognostic relevance of masked hypertension in subjects with prehypertension. METHODS: The occurrence of fatal and nonfatal cardiovascular events was evaluated in 591 subjects with prehypertension defined as clinic blood pressure (BP) in the range of 120-139 mm Hg for systolic BP and 80-89 mm Hg for diastolic BP. Among them, 471 were classified as having true prehypertension (clinic BP <140/90 mm Hg and daytime BP <135/85 mm Hg) and 120 as having masked hypertension (clinic BP <140/90 mm Hg and daytime BP > or =135 or 85 mm Hg). RESULTS: During the follow-up (6.6 +/- 4.3 years, range 0.5-15.5 years), 29 cardiovascular events occurred. In subjects with true prehypertension and masked hypertension the event-rates per 100 patient-years were 0.57 and 1.51, respectively. Event-free survival was significantly different between the groups (P < 0.005). After adjustment for other covariates, including clinic BP (forced into the model), Cox regression analysis showed that cardiovascular risk was significantly higher in masked hypertension than in true prehypertension (masked vs. true prehypertension, relative risk 2.65, 95% confidence interval 1.18-5.98, P = 0.018). CONCLUSIONS: Among subjects with prehypertension, those with masked hypertension are at higher cardiovascular risk than those with true prehypertension. Out-of-office BP should be known in individuals with prehypertension, preferably by ambulatory BP monitoring or alternatively by home BP measurement, to obtain a better prognostic stratification.

Reversal of P-glycoprotein-mediated multidrug resistance in human sarcoma MES-SA/Dx-5 cells by nonsteroidal anti-inflammatory drugs.

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is one of the major reasons for the failure of cancer therapy. Several chemosensitizers are able to reverse in vitro MDR by inhibiting P-gp, although high toxicity limits their clinical application. In this study, we aimed to investigate the in vitro effectiveness of four common non-steroidal anti-inflammatory drugs (NSAIDs) such as Curcumin (Cur), Sulindac (Sul), Ibuprofen (Ibu) and NS-398 (NS) to inhibit P-gp activity at clinically achievable doses and to evaluate their potential use as sensitizers in anti-cancer chemotherapy. The human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx-5) expressing high levels of P-gp, were treated with different doxo concentrations in the presence or absence of NSAIDs. Cellular accumulation of doxo, cytotoxicity and apoptosis induction were measured in comparison with Verapamil, a specific P-gp inhibitor, used as a reference molecule. We found that Ibu, Cur and NS-398 enhanced significantly doxo retention, cytotoxicity and apoptosis on resistant MES-SA/Doxo-5 cells when compared with doxo alone. In contrast, no significant changes were found in resistant cells treated with Sul-doxo combinations. Our results demonstrate that Ibu, Cur and NS-398 below their therapeutic plasma concentrations were able to overcome P-gp-mediated MDR in MES-SA/Dx-5 cells. These findings provide the rationale for clinical studies of NSAIDs and/or derivatives as a new potential generation of chemosensitizers to improve effectiveness of the anti-cancer drugs in the treatment of human cancer.

Risk of atrial fibrillation in dipper and nondipper sustained hypertensive patients.

OBJECTIVE: The risk of atrial fibrillation (AF) in sustained hypertensive patients with different circadian blood pressure (BP) patterns is unknown. We investigated the risk of new onset AF in dipper and nondipper sustained hypertensive patients. METHODS: The occurrence of AF was evaluated in 1141 patients aged > or = 40 years with sustained hypertension (clinic BP > or = 140 and/or 90 mmHg and daytime BP > or = 135 and/or 85 mmHg). Among these patients, 783 had night-time systolic BP fall > or = 10% (dippers) and 358 had night-time BP decline <10% (nondippers). RESULTS: During the follow-up (6.1+/-3.2, range 0.5-12.9 years), AF occurred in 43 patients. The AF rate per 100 patient-years in dippers and nondippers was 0.38 and 1.13, respectively. AF free survival was significantly different between the groups (P=0.0002). After adjustment for other covariates, including left atrial enlargement or left ventricular hypertrophy (these variables were analyzed in separate models because of a strong association between them) and 24-h BP, Cox regression analysis showed that the risk of AF was significantly higher in nondippers than in dippers [nondippers vs. dippers, relative risk (RR) 2.02, 95% confidence interval (CI) 1.08-3.79, P=0.028 in the model including left atrial enlargement, and RR 1.97, 95% CI: 1.05-3.69, P=0.035 in the model including left ventricular hypertrophy]. CONCLUSION: This study shows that nondipper sustained hypertensive patients have a two-fold greater risk of developing AF than dipper ones. This aspect could partly contribute to explain the higher cardiovascular risk previously observed in nondipper hypertensive patients.

Association of body iron stores with low molecular weight iron and oxidant damage of human atherosclerotic plaques.

The association between iron, an oxidant catalyst, and atherosclerosis is controversial. In particular, it is unknown whether: (1) stored iron, namely serum ferritin, is correlated with catalytic iron and oxidant damage of human atherosclerotic plaques; (2) catalytic iron is related to oxidative injury within such plaques; (3) plaque oxidant burden is associated with the severity of atherosclerosis. Thus, we assessed low molecular weight iron (LMWI), which represents the metal catalytically active form, together with fluorescent damage products of lipid peroxidation (FDPL) and lipid hydroperoxides (LOOH), in 38 atherosclerotic plaques surgically removed from 38 patients who had undergone selective carotid endarterectomy. In each patient, the levels of serum ferritin were measured and correlated with those of plaque LMWI and lipoperoxides by the Spearman rank correlation test with Spearman rank correlation coefficient (r(S)) calculation. Moreover, in patients selected from the same study population, we compared plaque analyte levels between two groups with different severity of atherosclerotic carotid stenosis, i.e., <90% (group A, n = 25) or > or =90% (group B, n = 13), and between another two groups without (group C, n = 27) and with (group D, n = 11) associated contralateral carotid stenosis > or =50%, indicative of "extensive" and more severe atherosclerotic disease. In group A patients, serum ferritin was directly and significantly correlated with plaque LMWI (r(S) = 0.46, P < 0.025) and FDPL (r(S) = 0.58, P < 0.005), while its correlation with plaque LOOH, albeit direct, did not attain statistical significance. Moreover, a direct and significant relationship was evident between the plaque content of LMWI and that of both FDPL (r(S) = 0.61, P < 0.0025) and LOOH (r(S) = 0.51, P < 0.025), suggesting a prooxidant role of catalytic iron within human atherosclerotic plaques. Considering the 13 patients of group B, a positive and significant correlation was observed between the levels of serum ferritin and those of plaque LMWI (r(S) = 0.83, P < 0.0001); on the other hand, serum ferritin, as well as plaque LMWI, showed no significant correlation with either plaque FDPL or LOOH, conceivably reflecting the small number of patients belonging to group B. Finally, plaque LMWI, FDPL, and LOOH content was significantly higher in group B than in group A, and in group D than in group C. These data suggest a role for catalytic iron in atherosclerotic plaque oxidation and in the severity of atherosclerosis, which appears indeed associated with plaque oxidant burden.

Effects of toll-like receptor-4 gene polymorphisms on soluble P-selectin and von Willebrand factor levels in hypercholesterolemic patients.

Toll-like receptor-4 (TLR-4) gene polymorphisms have been associated with a lower risk of atherosclerosis. High levels of soluble P-selectin (sP-selectin) and von Willebrand factor predict an increased risk for cardiovascular events and correlate to atherosclerotic risk factors. The relationship between these markers and TLR-4 gene polymorphisms was evaluated in a cohort of consecutive hypercholesterolemic outpatients. TLR-4 gene polymorphisms were detected in 48 out of 330 (14%) patients with hypercholesterolemia. Lipid and inflammatory markers, sP-selectin and von Willebrand were evaluated in carriers and in 96 (ratio 2:1 to cases) age- and sex-matched TLR-4 wild-type patients randomly selected from the same population. A cohort of normocholesterolemic outpatients (n = 262) served as the control group. sP-selectin was sensibly lower in carriers of TLR-4 variants as compared to wild-types and controls (89 ng/ml vs. 162 ng/ml and 163 ng/dl, respectively, p = 0.0001). Similarly, carriers showed lower von Willebrand factor values (683 mU/ml) than wild-types (910 mU/ml; p = 0.001). In multivariate analysis, TLR-4 gene polymorphisms were positively associated with sP-selectin, whereas the relationship with von Willebrand factor was no longer significant. HMG-CoA reductase inhibitors reduced sP-selectin and von Willebrand factor levels independently of TLR-4 gene variants. Plasma concentrations of these markers, however, remained lower in carriers of TLR-4 gene polymorphisms even after cholesterol lowering. In conclusion, carriership of Asp299 and Thr399Ile TLR-4 gene polymorphisms is associated with lower levels of sP-selectin and von Willebrand factor among hypercholesterolemic patients. While the underlying mechanisms remain to be investigated, such an association may indicate a protective effect of TLR-4 variants for atherosclerosis.

Amiodarone inhibits tocopherol-mediated human lipoprotein peroxidation.

It is unknown whether lipoprotein tocopherol-mediated peroxidation (TMP) is influenced by peculiar drug physicochemical properties such as hydrophobicity. Thus, we studied the effect of the extremely hydrophobic agent amiodarone on human non-HDL TMP. The drug, albeit devoid of specific radical-scavenging effects, inhibited TMP at therapeutic concentrations and with an efficiency similar to that of the physiological co-antioxidant ascorbic acid, showing indeed an IC(50) of 5microM. A comparable efficiency was observed with human LDL, and with a pure LDL-VLDL mixture. TMP was also inhibited by other hydrophobic cationic amphiphiles without radical-scavenging activity, namely desethylamiodarone, chlorpromazine, clomipramine, promethazine, promazine, verapamil, bromhexine, propranolol, mepivacaine, metoprolol, tramadol and ranitidine, whose anti-TMP potency was far lower than that of amiodarone and related to drug hydrophobicity degree. Further, TMP was strongly inhibited by butylhydroxytoluene, a lipophilic radical scavenger. Hydrophobic acidic (diclofenac, indomethacin, ibuprofen and ketoprofen) or neutral (n-hexane, anthracene, o-xylene and toluene) compounds could not instead inhibit TMP, indicating a stringent requirement for drug basicity in the pharmacological inhibition of TMP. Amiodarone effectiveness was lowered by lipoprotein alpha-tocopherol enrichment, suggesting some drug-alpha-tocopherol interaction and less lipid pharmacological protection at higher alpha-tocopheroxyl radical generation. Drug anti-TMP activity may so be related to electrostatic and hydrophobic interactions with lipoprotein alpha-tocopherol and lipid moiety, resulting in decreased radical phase transfer and lipid propensity to undergo radical-driven peroxidation. In conclusions, primarily drug basicity and then hydrophobicity are solely relevant to TMP inhibition. Amiodarone, at therapeutic concentrations, has anti-TMP properties, which could occur in the clinical setting.

Prognostic relevance of metabolic syndrome in hypertensive patients at low-to-medium risk.

BACKGROUND: The prognostic impact of metabolic syndrome (MetS) in the hypertensive population at low-medium risk is unknown. In this study, we evaluated the prognostic relevance of MetS in hypertensive patients at low-medium risk. METHODS: The occurrence of nonfatal and fatal cardiac and cerebrovascular events was evaluated in 802 patients with mild to moderate essential hypertension at low-medium risk according to the 2003 World Health Organization/International Society of Hypertension statement on the management of hypertension. Among these patients, 218 (27.2%) had MetS according to a modified National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) definition (body mass index in place of waist circumference). RESULTS: During follow-up (6.9 +/- 3.1 years; range, 0.5 to 13.1 years, mean +/- SD), 58 first cardiovascular events occurred. The event rates per 100 patient-years in patients without and with MetS were 0.87 and 1.51, respectively. Event-free survival was significantly different between groups (P = .03). After adjustment for several covariates, Cox regression analysis showed that cardiovascular risk was significantly higher in patients with than in patients without MetS (relative risk, 2.64; 95% confidence interval, 1.52 to 4.58; P = .001). Other independent predictors of outcome were age, smoking habit, 24-h systolic BP, and LDL cholesterol. CONCLUSIONS: Hypertensive patients at low-medium risk with MetS are at higher cardiovascular risk than those without MetS. Metabolic syndrome may be a useful tool for clinicians to identify subjects who are at increased risk when traditional assessment may indicate low-medium risk.

Suppression of RAGE as a basis of simvastatin-dependent plaque stabilization in type 2 diabetes.

OBJECTIVE: Receptor for advanced glycation end products (AGEs) (RAGE) plays a central role in the process of plaque rupture in diabetic patients. Recently, it has been reported that RAGE may be downregulated by improving glycemic control. In contrast, despite being well known that RAGE may be induced in human vessels in a glucose-independent fashion, also by myeloperoxidase (MPO)-dependent AGE generation, no data exist regarding the possibility of a pharmacological modulation of glucose-independent RAGE generation. Thus, the aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques. METHODS AND RESULTS: Seventy type 2 diabetic patients with asymptomatic carotid artery stenosis (>70%) were randomized to American Heart Association (AHA) step 1 diet plus simvastatin (40 mg/d) or AHA step 1 diet alone for 4 months before endarterectomy. Plaque expression of MPO, AGEs, RAGE, NF-kappaB, COX-2, mPGES-1, matrix metalloproteinase (MMP)-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, procollagen 1, and interstitial collagen was analyzed by immunohistochemistry and Western blot; zymography was used to detect MMP activity. Plaques from the simvastatin group had less (P<0.0001) immunoreactivity for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9, lipids and oxLDL; reduced (P<0.0001) gelatinolytic activity; increased (P<0.0001) procollagen 1 and collagen content; and fewer (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells. Of interest, RAGE inhibition by simvastatin, observed not only in plaque sections but also in plaque-derived macrophages, was reverted by addition of AGEs in vitro. CONCLUSIONS: This study supports the hypothesis that simvastatin inhibits plaque RAGE expression by decreasing MPO-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the biosynthesis of PGE2-dependent MMPs, responsible for plaque rupture.

Modulatory effects of heparin on cellular accumulation and cytotoxicity of doxorubicin in MRP1-overexpressing HL60/doxo cells.

BACKGROUND: The overexpression of multidrug resistance protein (MRP1), associated with high levels of intracellular glutathione (GSH), is a well characterized mechanism of multidrug resistance (MDR) in several malignancies. Various chemosensitizers have been used in vitro to modulate the MRP1 activity, but the high toxicity limits their clinical application. Unfractionated heparin (UFH), is frequently used to prevent thrombo-embolic complications in cancer patients. This in vitro study aimed to elucidate the potential role of UFH as a sensitizer in anticancer clinical chemotherapy. MATERIALS AND METHODS: The human leukemic doxorubicin-resistant cell line (HL60/doxo), which overexpresses the MRP1 protein was treated with UFH alone or in combination with three different concentrations of doxo. The intracellular accumulation and cytotoxicity of doxo and the cellular GSH content were measured in comparison with the leukotriene LTD4 receptor antagonist, MK571, a specific MRP1 inhibitor. RESULTS: UFH increased doxo accumulation and cytotoxicity in the HL60/doxo cell line with respect to cells treated with doxo alone. UFH also decreased the cellular GSH content in the HL60/doxo cells with respect to the control, suggesting a potential involvement of UFH in doxo co-transport with GSH. CONCLUSION: Our results demonstrate that UFH modulates MRP1-mediated MDR in HL60/doxo cells expressing high MRP1 levels. These findings suggest a potential clinical application of heparin as an adjuvant to overcome MRP1-mediated drug resistance in cancer patients.

EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment.

PURPOSE: It has been reported that EGFR mutations in lung carcinomas make the disease more responsive to treatment with tyrosine kinase inhibitors. We decided to evaluate the prevalence of EGFR mutations in a large series of non-small-cell lung carcinomas (NSCLCs) and to develop a rapid and sensitive screening method. PATIENTS AND METHODS We examined 860 consecutive NSCLC patients for EGFR mutations in exons 18, 19, and 21 using a dual technical approach--direct sequencing of polymerase chain reaction (PCR) products and PCR single-strand conformation polymorphism (SSCP) analysis. Moreover, all lung adenocarcinomas were analyzed for K-ras mutations at codon 12 by allele-specific oligoprobe hybriditations. RESULTS: There were no EGFR mutations in 454 squamous carcinomas and 31 large cell carcinomas investigated. Thirty-nine mutations were found in the series of 375 adenocarcinomas (10%). Mutations were present in 26% of 86 bronchioloalveolar carcinomas (BACs) and in 6% of 289 conventional lung adenocarcinomas; P = .000002. EGFR mutations and K-ras mutations were mutually exclusive. A multivariable analysis revealed that BAC histotype, being a never smoker, and female sex were independently associated with EGFR mutations (odds ratios: 4.542, 3.632, and 2.895, respectively). The SSCP analysis was accurate and sensitive, allowing identification of mutations that were undetectable (21% of cases) by direct sequencing. CONCLUSION: Mutations in the EGFR tyrosine kinase domain define a new molecular type of lung carcinoma, more frequent in particular subsets of patients. The SSCP assay is a rapid and reliable method for the detection of EGFR kinase domain mutations in lung cancer.

Blood pressure variability and cardiovascular risk in treated hypertensive patients.

BACKGROUND: The independent prognostic value of blood pressure (BP) variability in treated hypertension is not yet clear. We investigated the relationship between BP variability, evaluated by noninvasive monitoring, and cardiovascular outcome in treated hypertensive patients. METHODS: The occurrence of fatal and nonfatal cardiovascular events was evaluated in 1472 treated patients. Subjects with the standard deviation of daytime or night-time systolic BP below or above the median of the population were classified as having low or high BP variability. Specifically, 738 and 734 patients had low and high daytime BP variability, respectively, and 739 and 733 subjects had low and high night-time BP variability, respectively. RESULTS: During follow-up (4.88 +/- 2.9 years, range 0.2-11.6 years) there were 119 events. The event rates per 100 patient-years in subjects with low and high BP variability according to daytime BP were 1.18 and 2.01, respectively, and in those with low and high BP variability according to night-time BP were 1.2 and 2.05, respectively. Event-free survival was significantly different between the low and high BP variability groups (P = .006 for both daytime and night-time BP). However, after adjustment for other covariates in a Cox multivariate analysis, the adverse prognostic relevance of high BP variability was no longer detectable, whereas age, smoking habit, LDL cholesterol, diabetes, previous events, LV hypertrophy, and daytime or night-time systolic BP resulted independent predictors of risk. CONCLUSIONS: Increased BP variability is associated with higher incidence of cardiovascular events, but also with other relevant prognostic factors. Indeed, in multivariate analysis the possible adverse prognostic impact of BP variability is no longer evident. Thus, in treated hypertension, BP variability evaluated by noninvasive monitoring is not an independent predictor of outcome.

Association between 5-lipoxygenase expression and plaque instability in humans.

OBJECTIVE: The participation of 5-lipoxygenase (5-LO) in the development of atherosclerosis has been suggested by recent studies. However, a role for 5-LO as a modulator of atherosclerotic plaque instability has not been previously reported in humans. Thus, the aims of this study was to analyze the expression of 5-LO in human carotid plaques and to investigate the mechanism by which this enzyme could lead to plaque instability and rupture. METHODS AND RESULTS: We obtained atherosclerotic plaques from 60 patients undergoing carotid endarterectomy. We divided the plaques into symptomatic and symptomatic according to clinical evidence of plaque instability. Clinical evidence of plaque instability was provided by the assessment of recent ischemic symptoms attributable to the stenosis and by the presence of ipsilateral cerebral lesion(s) determined by computed tomography. Plaques were analyzed for CD68+ macrophages, CD3+ T cells, alpha-actin+ smooth muscle cells, 5-LO, cyclooxygenase 2, matrix metalloproteinase (MMP)-2, and MMP-9 by immunohistochemical, immunoblotting, and densitometric analyses. MMP activity was assessed by zymography. Leukotriene (LT) B4 and collagen were quantified by ELISA and Sirius red polarization, respectively. The percentage of macrophage-rich and T-cell-rich areas was larger in symptomatic compared with asymptomatic patients (25+/-6% versus 8+/-4%, P<0.0001, and 74+/-17 versus 18+/-4 cell/mm2, P<0.003). 5-LO expression was higher in symptomatic compared with asymptomatic plaques (24+/-4% versus 6+/-3%, P<0.0001) and was associated with increased MMP-2 and MMP-9 expression (27+/-4% versus 7+/-3%, P<0.0001, and 29+/-5% versus 8+/-2%, P<0.0001) and activity and with decreased collagen content (6.9+/-2.4% versus 17.8+/-3.1%, P<0.01). Immunofluorescence showed that 5-LO and MMPs colocalize in activated macrophages. Notably, higher 5-LO in symptomatic plaques correlated with increased LTB4 production (18.15+/-3.56 versus 11.27+/-3.04 ng/g tissue, P<0.0001). CONCLUSIONS: The expression of 5-LO is elevated in symptomatic compared with asymptomatic plaques and is associated with acute ischemic syndromes, possibly through the generation of LTB4, subsequent MMP biosynthesis, and plaque rupture.

Helicobacter pylori infection causes persistent platelet activation in vivo through enhanced lipid peroxidation.

OBJECTIVE: We aimed at investigating the relationship between Helicobacter pylori infection and in vivo lipid peroxidation and platelet activation, as reflected by urinary 8-iso-prostaglandin (PG)F(2alpha) and 11-dehydro-thromboxane (TX)B2, respectively, in otherwise healthy dyspeptic subjects. METHODS AND RESULTS: We measured urinary 8-iso-PGF2alpha and 11-dehydro-TXB2 excretion in 40 dyspeptic subjects with a positive 13C-urea breath test and 38 dyspeptic individuals with a negative test. Moreover, we investigated the effects of H pylori eradication on prostanoid metabolite excretion in 23 H pylori-positive subjects. We also measured prostanoid metabolite excretion before and after selective cyclooxygenase-2 inhibition with rofecoxib in 4 H pylori-positive subjects. Urinary 8-iso-PGF2alpha and 11-dehydro-TXB2 excretion was significantly higher in the H pylori-positive individuals than in controls. A significant direct correlation was found between the degree of positivity to the 13C-urea breath test and urinary 8-iso-PGF2alpha excretion. The latter was linearly correlated with urinary 11-dehydro-TXB2. Successful eradication of H pylori infection led to a significant reduction in both 8-iso-PGF(2alpha) and 11-dehydro-TXB2. Furthermore, their levels were unaffected after treatment with rofecoxib. CONCLUSIONS: Our study provides evidence of enhanced in vivo lipid peroxidation and platelet activation in association with H pylori infection and suggests a novel mechanism by which an infectious agent could contribute to atherothrombosis.