Ada Popolo

Oxidative stress in patients with cardiovascular disease and chronic renal failure

Abstract Oxidative response regulates many physiological response in human health, but if not properly regulated it could also lead to a number of deleterious effects. The importance of oxidative stress injury depends on the molecular target, the severity of the stress, and the mechanism by which the oxidative stress is imposed: it has been implicated in several diseases including cancer, neurodegenerative diseases, malaria, rheumatoid arthritis and cardiovascular and kidney disease. Most of the common diseases, such as hypertension, atherosclerosis, heart failure, and renal dysfunction, are associated with vascular functional and structural alterations including endothelial dysfunction, altered contractility, and vascular remodeling. Common to these processes is increased bioavailability of reactive oxygen species (ROS), decreased nitric oxide (NO) levels, and reduced antioxidant capacity. Oxidative processes are up-regulated also in patients with chronic renal failure (CRF) and seem to be a cause of elevated risk of morbidity and mortality in these patients. In this review, we highlight the role of oxidative stress in cardiovascular and renal disease.

Involvement of β3-adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function

The β3-adrenoreceptor plays a major role in lipolysis but the role and distribution of β3-receptors in other specific sites have not been extensively studied. β3-adrenergic receptors are present not only in adipose tissue but also in human gall bladder, colon, prostate, and skeletal muscle. Recently, β3-adrenergic receptor stimulation was shown to elicit vasorelaxation of rat aorta through the NO–cGMP signal transduction pathway. Here we show that β3-receptors are present in human corpus cavernosum and are localized mainly in smooth muscle cells. After activation by a selective β3-adrenergic receptor agonist, BRL 37344, there was a cGMP-dependent but NO-independent vasorelaxation that was selectively blocked by a specific β3-receptor antagonist. In addition, we report that the human corpus cavernosum exhibits basal β3-receptor-mediated vasorelaxant tone and that β3-receptor activity is linked to inhibition of the RhoA/Rho-kinase pathway. These observations indicate that β3-receptors may play a physiological role in mediating penile erection and, therefore, could represent a therapeutic target for treatment of erectile dysfunction.

The Uremic Toxin Indoxyl Sulphate Enhances Macrophage Response to LPS

Indoxyl sulphate (IS) is a protein-bound uremic toxin that results from the metabolism of dietary tryptophan normally excreted by kidney through the proximal tubules. Thus the toxin accumulates in the blood of patients with impaired renal function such as in chronic kidney disease (CKD). High IS serum levels in patients with CKD suggest its involvement in CKD progression and in the onset of complications. Its presence in plasma is also a powerful predictor of overall and cardiovascular morbidity/mortality. IS is a well known nephrovascular toxin but very little is known regarding its effects on the immune system and in particular during inflammation. In this study we examined the effect of IS on macrophage activation in response to lipopolysaccharide from E. coli (LPS), a gram negative bacterial endotoxin associated with inflammation and septic shock. To simulate the uremic condition, J774A.1 macrophages were incubated with IS at concentrations observed in uremic patients (1000–62.5 µM) both alone and during LPS challenge. IS alone induced release of reactive oxygen species (ROS), through a mechanism involving pro- and anti-oxidant systems, and alteration in intracellular calcium homeostasis. When added to J774A.1 macrophages in presence of LPS, IS significantly increased the nitric oxide (NO) release, inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2) expression. IS pre-treatment was also associated with an increase in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production by macrophages stimulated with LPS. Mechanistic studies revealed that IS increased LPS-induced NF-kB nuclear translocation, ROS release and altered calcium concentrations, mainly because of mitochondrial calcium overloading. Moreover also in primary mouse peritoneal macrophages IS enhances the inflammatory response to LPS increasing ROS, NO, iNOS, COX-2, TNF-α, IL-6 and NF-kB levels. This study provides evidences that IS stimulates macrophage function and enhances inflammatory reasponse associated with LPS, thus contributing to altered immune response dysfunctions observed in CKD.

Cl-IB-MECA inhibits human thyroid cancer cell proliferation independently of A3 adenosine receptor activation

A3 adenosine receptor (A3AR) agonists have been reported to modulate cellular proliferation. This work was aimed to investigate the expression and the possible action of A3 receptor in the human thyroid carcinomas. Normal thyroid tissue samples did not express A3 adenosine receptor, while primary thyroid cancer tissues expressed high level of A3AR, as determined by immunohistochemistry analysis. In human papillary thyroid carcinoma cell line, NPA, at concentrations ≥10µM, the A3AR-selective agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5’-N-methylcarboxamide (Cl-IB-MECA) produced inhibition of cell growth, by blocking the G1 cell cycle phase in a concentration- and time-dependent manner. This effect was well correlated with a reduction of protein expression of cyclins D1 and E2 after 24 hours of Cl-IB-MECA treatment. Moreover Cl-IB-MECA induced dephosphorylation of ERK1/2 in a time- and concentration-dependent manner, which in turn inhibits cell proliferation. The effect of Cl-IB-MECA was not prevented by A3AR antagonists, MRS1191 or MRS1523 or FA385. Furthermore, neither nucleoside transporter inhibitors, Dypiridamole and NBTI, nor the A1, A2A and A2B receptors antagonists were able to block the response to Cl-IB-MECA. Although Cl-IB-MECA has been shown to influence cell death and survival in other systems through an A3AR-mediated mechanism, in NPA cells the growth inhibition induced by micromolar concentrations of Cl-IB-MECA appeared not mediated through activation of this receptor and hence that its effects on human papillary carcinoma cell line seem to be independent of the presence of this receptor subtype.

Cl‐IB‐MECA enhances TRAIL‐induced apoptosis via the modulation of NF‐κB signalling pathway in thyroid cancer cells

Apoptosis is an endogenous process that can be a useful anti‐cancer tool. This study aimed to investigate the effect of Cl‐IB‐MECA, adenosine receptor A3 agonist, on TRAIL‐induced apoptosis of thyroid carcinoma cells. Cl‐IB‐MECA enhanced TRAIL‐mediated apoptosis in FRO but not in ARO cells. This effect was correlated to higher expression levels of DR5 on FRO than ARO cells, that instead presented higher levels of decoy receptors, DcR1 and DcR2. To understand the cross‐talk between the effect of Cl‐IB‐MECA and TRAIL, we evaluated the nuclear translocation of p65 and c‐Rel. Since the dependency by NF‐κB, TRAIL promoted the nuclear translocation of both p65 and c‐Rel subunits. However, the addition of Cl‐IB‐MECA led to the predominant translocation of c‐Rel after TRAIL addition. Furthermore, Bcl‐2, cFLIP and pAkt were lower induced than caspase‐3 and ‐9 in FRO cells. To discriminate a specific effect of TRAIL, we used tumour necrosis factor‐alpha (TNF‐α) with Cl‐IB‐MECA. In this case, no synergism was observed. In addition, the effect of Cl‐IB‐MECA was not A3 receptor‐dependent since its antagonists, MRS1191 and FA385, failed to block Cl‐IB‐MECA activity on TRAIL‐treated FRO cells. In conclusion, Cl‐IB‐MECA enhanced TRAIL‐mediated apoptosis via NF‐κB/c‐Rel activation and DR5‐dependent manner. This study may shed light on a potential drug cocktail that may prove useful as anti‐cancer in an in vivo animal model. J. Cell. Physiol. 221: 378–386, 2009.

Acetamide Derivatives with Antioxidant Activity and Potential Anti-Inflammatory Activity

This study reports the synthesis and antioxidant activity of some new acetamide derivatives. The compounds’ structures were elucidated by NMR analysis and their melting points were measured. The in vitro antioxidant activity of these compounds was tested by evaluating the amount of scavenged ABTS radical and estimating ROS and NO production in tBOH- or LPS-stimulated J774.A1 macrophages. All compounds were tested for their effect on cell viability by an MTT assay and by a Brine Shrimp Test.

Dexamethasone improves vascular hyporeactivity induced by LPS in vivo by modulating ATP-sensitive potassium channels activity

Septic shock represents an important risk factor for patients critically ill. This pathology has been largely demonstrated to be a result of a myriad of events. Glucocorticoids represent the main pharmacological therapy used in this pathology. Previously we showed that ATP‐sensitive potassium (KATP) channels are involved in delayed vascular hyporeactivity in rats (24 h after Escherichia coli lipopolysaccharide (LPS) injection). In LPS‐treated rats, we observed a significant hyporeactivity to phenylephrine (PE) that was reverted by glybenclamide (GLB), and a significant increase in cromakalim (CRK)‐induced hypotension. We evaluated the effect of dexamethasone (DEX 8 mg kg−1 i.p.) whether on hyporeactivity to PE or on hyperreactivity to CRK administration, in vivo, in a model of LPS (8 × 106 U kg−1 i.p.)‐induced endotoxemia in urethane‐anaesthetised rats. DEX treatment significantly reduced, in a time‐dependent manner, the increased hypotensive effect induced by CRK in LPS‐treated rats. This effect was significantly (P<0.05) reverted by the glucocorticoid receptor antagonist RU38486 (6.6 mg kg−1 i.p.). GLB‐induced hypertension (40 mg kg−1 i.p.), in LPS‐treated rats, was significantly inhibited by DEX if administered at the same time of LPS. Simultaneous administration of DEX and LPS to rats completely abolished the hyporeactivity to PE observed after 24 h from LPS injection. In conclusion, our results suggest that the beneficial effect of DEX in endotoxemia could be ascribed, at least in part, to its ability to interfere with KATP channel activation induced by LPS. This interaction may explain the improvement of vascular reactivity to PE, mediated by DEX, in LPS‐treated rats, highlighting a new pharmacological activity to the well‐known anti‐inflammatory properties of glucocorticoids.

Inflammatory mediators in a short-time mouse model of doxorubicin-induced cardiotoxicity.

Doxorubicin (DOXO) is commonly used to treat a wide range of malignant tumors, but its clinical use is limited by acute and chronic cardiotoxicity. The precise mechanism underlying DOXO-induced cardiotoxicity is still not completely elucidated, but cardiac inflammation seems to be involved. Effects of DOXO on proinflammatory cytokines, inflammatory cell infiltration, and necrosis have been proven only when a functional impairment has already occurred, so this study aimed to investigate the acute effect of DOXO administration in mouse heart. The results of our study demonstrated alterations in cardiac function parameters assessed by ultrasound within 24h after a single injection of DOXO, with a cumulative effect along the increase of the dose and the number of DOXO administrations. At the same time, DOXO causes a significant production of proinflammatory cytokines (such as TNF-α and IL-6) with a concomitant reduction of IL-10, a well-known antiinflammatory cytokine. Furthermore, overexpression of inducible nitric oxide synthase (iNOS) in heart tissue and increased levels of serum nitrite in DOXO-treated mice were detected. Notably, DOXO administration significantly increased nitrotyrosine expression in mouse heart. Our data support the hypothesis that these early events, could be responsible for the later onset of more severe deleterious remodeling leading to DOXO induced cardiomyopathy.

Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells.

Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern.

l-Arginine and its metabolites in kidney and cardiovascular disease

Abstractl-Arginine is a semi essential amino acid synthesised from glutamine, glutamate and proline via the intestinal-renal axis in humans and most mammals. l-Arginine degradation occurs via multiple pathways initiated by arginase, nitric-oxide synthase, Arg: glycine amidinotransferase, and Arg decarboxylase. These pathways produce nitric oxide, polyamines, proline, glutamate, creatine and agmatine with each having enormous biological importance. Several disease are associated to an l-arginine impaired levels and/or to its metabolites: in particular various l-arginine metabolites may participate in pathogenesis of kidney and cardiovascular disease. l-Arginine and its metabolites may constitute both a marker of pathology progression both the rationale for manipulating l-arginine metabolism as a strategy to ameliorate these disease. A large number of studies have been performed in experimental models of kidney disease with sometimes conflicting results, which underlie the complexity of Arg metabolism and our incomplete knowledge of all the mechanisms involved. Moreover several lines of evidence demonstrate the role of l-arg metabolites in cardiovascular disease and that l-arg administration role in reversing endothelial dysfunction, which is the leading cause of cardiovascular diseases, such as hypertension and atherosclerosis. This review will discuss the implication of the mains l-arginine metabolites and l-arginine-derived guanidine compounds in kidney and cardiovascular disease considering the more recent literature in the field.