Therapeutic value of stimulating the nitrate-nitrite-nitric oxide pathway to attenuate oxidative stress and restore nitric oxide bioavailability in cardiorenal disease.

Cardiovascular disorders including hypertension and associated renal disease are major health problems affecting more than 1.5 billion people worldwide. Apart from nonmodifiable factors such as ageing, family history and gender, both sedentary lifestyle and unhealthy dietary habits are considered as major risk factors. The disorders are interrelated suggesting common pathological pathways. Mechanistically, oxidative stress and compromised function of the nitric oxide synthase (NOS) system leading to endothelial dysfunction and reduction in nitric oxide (NO) bioavailability have been widely implicated and associated with development and progression of disease. New strategies that correct this redox imbalance and increase NO bioactivity may have major clinical implications. The inorganic anions, nitrate and nitrite, are endogenously formed by oxidization of NOS-derived NO, but there are also high amounts of nitrate in our daily diet. In this regard, accumulated evidence over the past two decades demonstrates that these anions can be recycled back to NO and other bioactive nitrogen oxides, thus offering an attractive alternative strategy for therapeutic exploitation. In this review, we describe how dietary stimulation of the nitrate-nitrite-NO pathway affects cardiovascular and renal functions in health and disease via modulation of oxidative stress and NO bioavailability. Clinical studies addressing potential effects on the renal system are still limited, but blood pressure-lowering effects of nitrate supplementation have been demonstrated in healthy and hypertensive subjects as well as in patients with chronic kidney disease. However, larger clinical studies are warranted to reveal whether chronic nitrate treatment can slow-down the progression of cardiorenal disease and associated complications.

Tumor suppressor SET9 guides the epigenetic plasticity of breast cancer cells and serves as an early-stage biomarker for predicting metastasis.

During the course of cancer progression, neoplastic cells undergo dynamic and reversible transitions between multiple phenotypic states, and this plasticity is enabled by underlying shifts in epigenetic regulation. Our results identified a negative feedback loop in which SET9 controls DNA methyltransferase-1 protein stability, which represses the transcriptional activity of the SET9 promoter in coordination with Snail. The modulation of SET9 expression in breast cancer cells revealed a connection with E2F1 and the silencing of SET9 was sufficient to complete an epigenetic program that favored epithelial-mesenchymal transition and the generation of cancer stem cells, indicating that SET9 plays a role in modulating breast cancer metastasis. SET9 expression levels were significantly higher in samples from patients with pathological complete remission than in samples from patients with disease recurrence, which indicates that SET9 acts as a tumor suppressor in breast cancer and that its expression may serve as a prognostic marker for malignancy.

Targeting the epigenetics of the DNA damage response in breast cancer.

Cancer is as much an epigenetic disease as it is a genetic disease, and epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Because the epigenetic factors involved in the DNA damage response are regulated by multiple elements, therapies to target specific components of the epigenetic machinery can be inefficient. In contrast, therapies aimed at inhibiting the methionine cycle can indirectly inhibit both DNA and protein methylation, and the wide variety of genes and pathways that are affected by these methylations make this global strategy very attractive. In the present study, we propose an adjuvant therapy that targets the epigenetics of the DNA damage response in breast cancer cells and that results in efficient apoptosis and a reduction in distant metastases in vivo. We observed that a combined therapy designed to uncouple adenosine metabolism using dipyridamole in the presence of a new synthetic antifolate, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin, simultaneously and efficiently blocked both the folic cycle and the methionine cycle in breast cancer cells and sensitized these cells to radiotherapy. The treatment impeded the recruitment of 53BP1 and BRCA1 to the chromatin regions flanking DNA double-strand breaks and thereby avoided the DNA damage responses in breast cancer cells that were exposed to ionizing radiation. In addition, this hypomethylating therapy was also efficient in reducing the self-renewal capability of breast cancer-initiating cells and induced reversion of mesenchymal phenotypes in breast cancer cells.

Cholinergic system and cell proliferation.

The cholinergic system, comprising acetylcholine, the proteins responsible for acetylcholine synthesis and release, acetylcholine receptors and cholinesterases, is expressed by most human cell types. Acetylcholine is a neurotransmitter, but also a local signalling molecule which regulates basic cell functions, and cholinergic responses are involved in cell proliferation and apoptosis. So, activation of nicotinic and muscarinic receptors has a proliferative and anti-apoptotic effect in many cells. The content of choline acetyltransferase, acetylcholine receptors and cholinesterases is altered in many tumours, and cholinesterase content correlates with patient survival in some cancers. During apoptosis, acetylcholinesterase is induced and appears in the nuclei. Acetylcholinesterase participates in the regulation of cell proliferation and apoptosis through hydrolysis of acetylcholine and by other catalytic and non catalytic mechanisms, in a variant-specific manner. This review gathers information on the role of cholinergic system and specially acetylcholinesterase in cell proliferation and apoptosis.

Targeting the epigenetic machinery of cancer cells.

Cancer is characterized by uncontrolled cell growth and the acquisition of metastatic properties. In most cases, the activation of oncogenes and/or deactivation of tumour suppressor genes lead to uncontrolled cell cycle progression and inactivation of apoptotic mechanisms. Although the underlying mechanisms of carcinogenesis remain unknown, increasing evidence links aberrant regulation of methylation to tumourigenesis. In addition to the methylation of DNA and histones, methylation of nonhistone proteins, such as transcription factors, is also implicated in the biology and development of cancer. Because the metabolic cycling of methionine is a key pathway for many of these methylating reactions, strategies to target the epigenetic machinery of cancer cells could result in novel and efficient anticancer therapies. The application of these new epigenetic therapies could be of utility in the promotion of E2F1-dependent apoptosis in cancer cells, in avoiding metastatic pathways and/or in sensitizing tumour cells to radiotherapy.

Assessment of oxidative status markers and NO bioavailability in hypertensive disorders of pregnancy.

The systemic oxidative status in hypertensives disorders of pregnancy (HDP) and its association with endothelial dysfunction is controversial. In the present study, we evaluated systemic plasma levels of oxidative stress markers (TBARS (thiobarbituric acid-reactive substances) and carbonyl) and total antioxidant status (FRAP (ferric reducing ability of plasma (ferric reducing/antioxidant power) and reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide))), as well as assessed the impact these markers have on nitric oxide (NO) status in healthy pregnant (HP, n=38), gestational hypertensive (GH, n=33) and preeclamptic pregnant women (PE, n=28). We found similar values of TBARS among all groups, and reduced carbonyl levels in HDP between the PE and GH. Conversely, significant increases in plasma activity of antioxidant status were observed in the GH and PE groups compared to the HP group (using both MTT or FRAP method). Importantly, HDP present significantly lower nitrite levels compared to HP women. In Conclusion, our findings show a compensatory antioxidant mechanism against reactive oxygen species (ROS) generation in HDP, which is not associated with nitrite levels restoration.

The AChE membrane-binding tail PRiMA is down-regulated in muscle and nerve of mice with muscular dystrophy by merosin deficiency.

Since Duchenne muscular dystrophy was attributed to mutations in the dystrophin gene, more than 30 genes have been found to be causally related with muscular dystrophies, about half of them encoding proteins of the dystrophin-glycoprotein complex (DGC). Through laminin-2, the DGC bridges the muscle cytoskeleton and the extracellular matrix. Decreased levels of PRiMA-linked acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) have been observed in dystrophic muscle and nerve of dystrophin-deficient (mdx) and laminin-2 deficient (Lama2dy) mice. To help explain these observations, the relative content of AChE, BuChE and PRiMA mRNAs were compared in normal and Lama2dy mouse muscle and sciatic nerve. The 17-fold lower level of PRiMA mRNA in Lama2dy muscle explained the deficit in PRiMA-linked ChEs. This would increase acetylcholine availability and, eventually, the desensitization of nicotinic receptors. Abnormal development of the Schwann cells led to peripheral neuropathy in the Lama2dy mouse. Compared with normal nerve, dystrophic nerve displayed 4-fold less AChE-T mRNA, 3-fold more BuChE mRNA and 2.5-fold less PRiMA mRNA, which agreed with the lower AChE activity in dystrophic nerve, its increased BuChE activity and the specific drop in PRiMA-linked BuChE. The widely accepted role of glial cells as the source of BuChE, the observed dysmyelination of Lama2dy nerve and its increased BuChE activity support the idea that BuChE up-regulation is related with the aberrant differentiation of the Schwann cells.

Spironolactone and hydrochlorothiazide exert antioxidant effects and reduce vascular matrix metalloproteinase-2 activity and expression in a model of renovascular hypertension.

BACKGROUND AND PURPOSE: Increased oxidative stress and up-regulation of matrix metalloproteinases (MMPs) may cause structural and functional vascular changes in renovascular hypertension. We examined whether treatment with spironolactone (SPRL), hydrochlorothiazide (HCTZ) or both drugs together modified hypertension-induced changes in arterial blood pressure, aortic remodelling, vascular reactivity, oxidative stress and MMP levels and activity, in a model of renovascular hypertension. EXPERIMENTAL APPROACH: We used the two-kidney,one-clip (2K1C) model of hypertension in Wistar rats. Sham-operated or hypertensive rats were treated with vehicle, SPRL (25 mg.kg(-1).day(-1)), HCTZ (20 mg.kg(-1).day(-1)) or a combination for 8 weeks. Systolic blood pressure was monitored weekly. Aortic rings were isolated to assess endothelium-dependent and -independent relaxations. Morphometry of the vascular wall was carried out in sections of aorta. Aortic NADPH oxidase activity and superoxide production were evaluated. Formation of reactive oxygen species was measured in plasma as thiobarbituric acid-reactive substances. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry and immunohistochemistry. KEY RESULTS: Treatment with SPRL, HCTZ or the combination attenuated 2K1C-induced hypertension, and reversed the endothelial dysfunction in 2K1C rats. Both drugs or the combination reversed vascular aortic remodelling induced by hypertension, attenuated hypertension-induced increases in oxidative stress and reduced MMP-2 levels and activity. CONCLUSIONS AND IMPLICATIONS: SPRL or HCTZ, alone or combined, exerted antioxidant effects, and decreased renovascular hypertension-induced MMP-2 up-regulation, thus improving the vascular dysfunction and remodelling found in this model of hypertension.

Targeting of acetylcholinesterase to lipid rafts of muscle.

Despite the great progress made in setting the basis for the molecular diversity of acetylcholinesterase (AChE), an explanation for the existence of two types of amphiphilic subunits, with and without glicosylphosphatidylinositol (GPI) (Types I and II), has not been provided yet. In searching whether, as for the deficiency of dystrophin, that of merosin (laminin-alpha2 chain) alters the number of caveolae in muscle, a high increase in caveolin-3 (Cav3) was observed in the Triton X-100-resistant membranes (TRM) isolated from muscle of merosin-deficient dystrophic mice (Lama2dy). The rise in Cav3 was accompanied by that of non-caveolar lipid rafts, as showed by the greater ecto-5'-nucleotidase (eNT) activity, a marker of non-caveolar rafts, in TRM of dystrophic muscle. The observation of AChE activity in TRM, the increased levels of rafts and raft-bound AChE activity in merosin-deficient muscle and the presence of phospholipase C-sensitive AChE dimers in TRM supported targeting of glypiated AChE to rafts. This issue and the involvement of TRM in conveying nicotinic receptors to the neuromuscular junction and particular muscarinic receptors to cardiac sarcolemma strongly support a role for lipid rafts in targeting ACh receptors and glypiated AChE. Their nearby location in the surface membrane may provide cells with a fine tuning for regulating cholinergic responses.

The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution.

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.

Cholinesterases are down-expressed in human colorectal carcinoma.

The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G2(A)) and monomers (G1(A)) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A12, G4(H) and PRiMA-containing G4(A) AChE forms, besides G4(H), G4(A) and G1(H) BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and over-stimulating muscarinic receptors.

Colostoma - tecnica do suporte modificado. / Colostomy-modified support technic

Com o objetivo de facilitar o manuseio imediato do colostoma, os autores apresentam a tecnica do suporte subcutaneo da alca intestinal, utilizada num total de trinta pacientes.Apresenta as complicacoes precoces e tardias da tecnica, observando-se uma incidencia de oito precoces e duas tardias

Quando lesoes da boca significam cancer. / When mouth lesions mean cancer

A casuistica levantada, consta de 904 casos de lesoes de cavidade bucal e orofaringe, sendo 714 casos de lesoes ulceradas e, destes 91% tem como laudo anatomo-patologico CEC (Carcinoma Espinocelular). Outrossim, e dado importancia para comentarios sobre PAR (Pacientes de Alto Risco) e AAR (Areas de Alto Risco), confirmando certos dados da literatura com os nossos.Por fim, menciona-se a importancia da caracterizacao dos tres tipos de lesoes mais comumente encontradas neste sitio anatomico e a referida conduta diagnostica a ser tomada em consideracao

A histologia do linfonodo axilar como elemento prognostico nos tumores malignos da mama. / Histology of axillary lymph nodes as a prognostic element in carcinoma of the breast

E analisada a relacao existente entre o aspecto histologico reacional de linfonodos axilares em 83 casos de pacientes portadores de tumores malignos de mama, com o grau de imunidade sistemica determinada pelo teste cutaneo do dinitroclorobenzeno (DNCB). Observou-se que nao existe correlacao entre a histologia do linfodono e do prognostico. Concluiu-se que o numero de linfonodos axiliares acometidos por metastases e ainda o melhor parametro para o prognostico e a selecao de pacientes que devem receber tratamento adjuvante