Nitric oxide and atherosclerosis: an update

Nitric oxide (NO) is a molecule that has gained recognition as a crucial modulator of vascular disease. NO has a number of intracellular effects that lead to vasorelaxation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet adhesion. Endothelium damage induced by atherosclerosis leads to the reduction in bioactivity of endothelial NO synthase (eNOS) with subsequent impaired release of NO together with a local enhanced degradation of NO by increased generation of reactive oxygen species with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Many commonly used vasculoprotective agents have their therapeutic actions through the production of NO. L-Arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. Finally, eNOS gene polymorphism might be an additional risk factor that may contribute to predict cardiovascular events. However, further studies are needed to understand the possible clinical implications of these correlations

Pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells

We examined the hypothesis that pomegranate juice (PJ) can revert the potent downregulation of the expression of endothelial nitric-oxide synthase (NOSIII) induced by oxidized low-density liporotein (oxLDL) in human coronary endothelial cells. Western blot and Northern blot analyses showed a significant decrease of NOSIII expression after a 24-h treatment with oxLDL. Accordingly, we observed a significant dose-dependent reduction in nitric oxide bioactivity represented by both basal and bradykinin-stimulated cellular cGMP accumulation. These phenomena were corrected significantly by the concomitant treatment with PJ. Our data suggest that PJ can exert beneficial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the NOSIII bioactivity

The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols

Study of temozolomide and sulfasalazine action on human glioblastoma and rat glioma cells

Orientadores: Fabio Rogerio, Roger Frigério CastilhoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Gliomas são os tumores cerebrais mais comuns em adultos. Astrocitomas são gliomas derivados de astrócitos. Temozolamida (TMZ) é um agente alquilante do DNA usado para tratamento de astrocitomas de comportamento biológico agressivo. Apesar da melhora do prognóstico, em comparação com os pacientes submetidos apenas à radioterapia, o tratamento com TMZ apresenta eficácia limitada, visto que alguns tumores apresentam resistência a esta droga. Os astrócitos neoplásicos possuem um transportador (sistema Xc-) na membrana citoplasmática, o qual importa cistina para a síntese do antioxidante glutationa. O sistema Xc- pode ser inibido farmacologicamente pela sulfasalazina (SAS). No presente estudo, avaliamos abordagem terapêutica que consiste na administração in vitro de TMZ associada com SAS em três linhagens de astrocitoma humano (U87MG, A172 e T98G) e uma linhagem de glioma de rato (C6). Os parâmetros avaliados foram viabilidade celular, morte por apoptose, capacidade de proliferação e invasão, expressão gênica, níveis de glutationa total e peroxidação lipídica. As metodologias adotadas foram, respectivamente, ensaio colorimétrico (MTT), reação de TUNEL quantificada por citometria de fluxo, Western Blotting para a proteína PCNA, atividade enzimática de metaloproteinases 2 e 9 (MMP2 e MMP9), PCR em tempo real e transcriptoma, método de reciclagem da glutationa e TBARS. Verificamos que as quatro linhagens não apresentaram alterações significativas de viabilidade após tratamento com TMZ 25 µM ou 50 µM por 1, 3 ou 5 dias. Por outro lado, a associação de TMZ 25 µM com SAS 0.5 mM após 5 dias levou à queda significativa de viabilidade tanto na linhagem U87MG, quanto nas células A172....Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: Gliomas are the most common brain tumors in adults. Astrocytomas are gliomas derived from astrocytes. Temozolomide (TMZ) is an alkylating agent of the DNA used for treatment of astrocytomas with aggressive behavior. Despite the improvement of prognosis compared to patients subjected to radiotherapy alone, treatment with TMZ has limited effectiveness, since some tumors are resistant to this drug. Neoplastic astrocytes have a transporter in the cytoplasmic membrane (Xc- system), which imports cystine for the synthesis of the antioxidant glutathione. The Xc- system can be inhibited pharmacologically by sulfasalazine (SAS). In the present study, we evaluated an therapeutic approach in vitro that consists in TMZ administration associated with SAS in three lineages of human astrocytoma (U87MG, T98G and A172) and a rat glioma cell line (C6). We assessed cell viability, death by apoptosis, proliferation and invasion capacity, differential gene expression, the total glutathione levels and lipid peroxidation.The adopted methodologies were, respectively, colorimetric assay (MTT), TUNEL reaction quantified by flow cytometry, Western blot for PCNA protein, enzymatic activity of metalloproteinases 2 and 9 (MMP2 and MMP9), real time PCR and transcriptome, recycling method of glutathione and TBARS. We found that the four cell lines showed no significant changes in viability after treatment with TMZ 25 µM or 50 µM for 1, 3 or 5 days. On the other hand, the combination of 25 µM TMZ with SAS 0.5 mM after 5 days led to a significant decrease in viability in both U87MG and A172 lineages...Note: The complete abstract is available with the full electronic documentDoutoradoFisiologiaDoutora em Biologia Funcional e Molecular2013/02618-1, 2011/50400-0FAPESPCAPE