Advanced glycation end products, oxidative stress and metalloproteinases are altered in the cerebral microvasculature during aging

Biological aging is associated with an increased incidence of cerebrovascular disease. Recent findings indicate that oxidative stress promoting age-related changes of cerebral circulation are involved in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease. The aim of this study was to evaluate the contribution of cerebral microvessels to the oxidative stress during brain aging, by: (i) assessment of precursors for advanced glycation end products (AGE) formation, (ii) activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR), and (iii) the activities of metalloproteinases (MMPs), MMP-2 and MMP-9, involved in synaptogenesis and memory consolidation. The experiments were performed on two groups of male Wistar rats: 15 young (3-6 months old) and 15 aged (18-24 months old) animals. The cerebral microvessels were isolated by mechanical homogenization, the concentration of protein carbonyls and the activity of antioxidant enzymes were evaluated by spectrophotometry, and gelatin SDS-PAGE zymography was employed to evaluate MMP-2 and MMP-9 activities. The results showed that, by comparison with young rats, aged brain microvessels contain: (i) approximately 106 % increase of protein carbonyls production; (ii) approximately 68% higher GPx activity, unmodified activities of SOD and GR; (iii) approximately 30% diminishment in MMP-2 activity, and the specific occurrence of MMP-9 enzyme. The data suggest that the age-related changes of microvessels could increase the propensity for cerebral diseases and might represent, at least in part, a prerequisite for the deterioration of mental and physical status in the elderly.     

In vitro evaluation of (S)-ibuprofen toxicity on joint cells and explants of cartilage and synovial membrane

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the toxicity of (S)-ibuprofen on chondrocytes and synoviocytes isolated from sheep shoulder joint and cultured in monolayers during 72 h, and on joint explants (cartilage and capsule) cultured in mono- or in co-culture for 13 days. (S)-ibuprofen (5 μM up to 1 mM) did not reduce the cell viability and protein content when added on chondrocyte monolayers, while at 1 mM (S)-ibuprofen reduced (by 8%, p = 0.01) the synoviocytes viability compared to untreated cells. During co-culture of joint explants, (S)-ibuprofen at 50 μM significantly reduced by 35% the spontaneous release of glycosaminoglycans (GAGs) from cartilage (p = 0.0065) whereas in monoculture, (S)-ibuprofen was inactive on GAG metabolism. (S)-ibuprofen at 1 mM significantly reduced cell lysis (lactate dehydrogenase leakage) by 74% during monoculture of capsule explants (p = 0.0136) and by 35% during co-culture of explants (p = 0.0013). Our findings demonstrate that the active isomer of ibuprofen at micro- and millimolar levels was not toxic for chondrocytes and synoviocytes and may reduce at 1 mM the cell lysis during culture of joint explants. The limited toxicity of (S)-ibuprofen at low and high concentration in sheep joint shoulder makes this enantiomer a promising drug candidate for the loading of intra-articular DDS.     

Silencing of epidermal growth factor,latrophilin and seven transmembrane domain-containing protein 1 (ELTD1) via siRNA-induced cell death in glioblastoma

The failure of therapies targeting tumor angiogenesis may be caused by anti-angiogenic resistance mechanisms induced by VEGF and non-VEGF pathways alterations. Anti-angiogenic therapy failure is also attributed to immune system, acting by tumor-associated macrophages that release pro-angiogenic factors and a consequent increase of blood vessels. Recently, in a study by Rheal et al., a new angiogenic receptor, epidermal growth factor, latrophilin, and 7 trans-membrane domain-containing protein 1 on chromosome 1(ELTD1) has been identified as a promising glioma biomarker. In this study we aim to analyse whether this receptor may be used as a target molecule in glioblastoma therapy. Our results showed that small interfering RNA silencing ELTD1 caused cytotoxicity in glioblastoma cells. We also found that PDGFR, VEGFR, and their common PI3K/mTOR intracellular pathway inactivation-induced cytotoxicity in glioblastoma cells. Further, we found high percent of cytotoxicity in a low passage glioblastoma cell line after BEZ235 (a dual inhibitor of PI3K/mTOR pathway) treatment at nanomolar concentrations, compared to AG1433 (a PDGFR inhibitor) and SU1498 (a VEGFR inhibitor) that were only cytotoxic at micromolar ranges. In the future, these could prove as attractive therapeutic targets in single therapy or coupled with classic therapeutic approaches such as chemotherapy of radiotherapy.     

Macrophage stimulating protein variation enhances the risk of sporadic extrahepatic cholangiocarcinoma

BackgroundPrimary sclerosing cholangitis confers risk of cholangiocarcinoma. Here, we assessed the primary sclerosing cholangitis-associated variant rs3197999 in the MST1 gene, coding for RON receptor tyrosine kinase ligand macrophage stimulating protein, in a large European cholangiocarcinoma cohort.Materials and methods223 cholangiocarcinoma patients including three primary sclerosing cholangitis individuals and 355 cancer- and primary sclerosing cholangitis-free controls were genotyped for MST1 rs3197999.ResultsThe cancer group departed from Hardy–Weinberg equilibrium (p = 0.022) and exhibited a trend for rs3197999 [A] overrepresentation (31% vs. 26%: p = 0.10). Homozygous rs3197999 [AA] carrier status significantly increased overall (OR = 1.97; p = 0.023) and primary sclerosing cholangitis-unrelated biliary tract cancer risk (OR = 1.84; p = 0.044), relative to homozygous common allele carriers. The association was most pronounced in patients with extrahepatic tumours. This finding was robust to multivariate analysis (p < 0.05), validating the [AA] genotype as an independent cholangiocarcinoma risk factor.ConclusionsThese results suggest that the [AA] genotype of the common MST1 variant rs3197999 enhances genetic risk of sporadic extrahepatic cholangiocarcinoma irrespective of primary sclerosing cholangitis status, presumably by modulating inflammatory responses and/or altered MSP/RON signalling.     

Uncompetitive antagonism of AMPA receptors: Mechanistic insights from studies of polyamine toxin derivatives

Philanthotoxins are uncompetitive antagonists of Ca2+-permeable AMPA receptors presumed to bind to the pore-forming region, but a detailed molecular mechanism for this interaction is missing. Here a small library of novel philanthotoxins was designed and synthesized using a solid-phase strategy. The biological activities were investigated at cloned and "native" AMPA receptors using electrophysiological techniques. A distinct relationship between length of the polyamine moiety and the location of a secondary amino group was observed. Fitting the data to the Woodhull equation allowed the first experimental demonstration of the relative location and orientation of the philanthotoxin molecule in the receptor. These results were corroborated by in silico studies using a homology model of the AMPA receptor ion channel. Together these studies provide strong evidence for a molecular mechanism by which polyamine toxins antagonize the AMPA receptor ion channel and provide the basis for rational development of uncompetitive antagonists of AMPA receptors.     

Uveal melanoma

The paper present a clinic and histological study, including all cases of uveal malignant tumours, hospitalized in Ophthalmological Clinic of Craiova in the last 15 years. The follow-up period of cases were 4 years. The diagnosis was established by clinic and paraclinic examination (imaging methods-- MRI, CT, Echo) and histologic examination. The histologic examination has been done for all cases. There were investigated several parametres: tumour cytology, the level of pigmentation, intravascular embolus, sclera infiltration, vascular invasion, vascular pattern, tumoral necrosis, mitotic activity. The histologic aspects of tumoral progression were: the high level mitotic activity, high surface area of intratumoral necrosis, the infiltrative process to the vessels, sclera, and ciliary body. The arcuat and network vascular patterns were involved in the systemic metastasis and local invasion. The most predominant type of cellularity related with a severe evolution of cases were the epithelioid cells and the mixed type cells.     

Sex-related differences in the immune response of weanling piglets exposed to low doses of fumonisin extract

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides, a fungus that commonly contaminates maize. Sex-related effects of FB1 have been observed with respect to carcinogenicity in rodents, to performances in pigs and immunosuppression in mice. In the present study the sex-related effect of FB1 on the pig immune response was determined. Female and castrated male piglets received for 28 d either control feed or feed contaminated with 8 mg FB1/kg feed in the form of F. verticillioides culture material. At day 7 and day 21, animals were immunised subcutaneously with a Mycoplasma agalactiae vaccine. Ingestion of FB1-contaminated feed significantly decreased weight gain in males but had no effect in females. No sex-related difference was observed in biochemical parameters, but a higher level of creatinine was noted in toxin-treated animals. FB1 also altered the pig immune response in a sex-specific manner. In males, ingestion of FB1-contaminated feed significantly decreased specific antibody levels after vaccination as well as the mRNA expression level of IL-10. In females, the toxin has no effect on specific antibodies or on cytokine mRNA levels. The results of the present study indicate that FB1 is immunosuppressive in pigs. The magnitude of this FB1-induced immunosuppression is highly dependent on sex, with males being more susceptible than females.     

The cellular mechanisms involved in the vasodilator effect of nebivolol on the renal artery

Nebivolol is known as a highly selective beta1-adrenoceptor antagonist. Based on the reported vasodilator effect of nebivolol, we examined the cellular mechanisms by which the drug induces renal artery vasodilation, an issue of potential relevance for condition associated with high blood pressure. To this purpose, myograph and patch-clamp techniques were used. Small mouse renal arteries were placed in the myograph chamber, and after the optimal concentration for the vasodilator effect of nebivolol was established (50 microM), the arteries were further investigated to assess the potential contribution of nitric oxide (NO) and of Ca2+ ions to the nebivolol-induced effect, by exposing the arteries to the specific inhibitors such as N(G)-nitro-L-arginine methylester (L-NAME, 100 microM), ethylenglycol-bis-(beta-amino-ethylen ester) N,N'-tetraacetic acid (EGTA, 4 microM) and thapsigargin (1 microM). The expression of NO synthase was evaluated by the Western-blot technique. Using myograph and patch-clamp techniques applied on intact renal artery, we investigated the role of beta2-adrenoceptors, of myoendothelial junctions and of Ca(2+)-activated K+ channels in the vasodilatory effects of nebivolol, using 100 microM butoxamine, 40 microM 18 beta-glycyrrhetinic acid, 1 mM tetraethylammonium, and 100 nM iberiotoxin, respectively. The results showed that the cellular mechanisms of the vasodilator effect of nebivolol on the renal artery entail (i) activation of the endothelial beta2-adrenoceptor, (ii) participation of [Ca2+]i, (iii) increase in NO and eNOS, and (iv) activation of Ca(2+)-activated K+ channels. The cellular mechanisms underlying vasodilator effect of nebivolol on the artery explain the favorable effect of this drug in hypertension.