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The structure and composition of the endothelial cell (EC) glycocalyx reflect a balance of the biosynthesis of glycans and their shear dependent removal. Shedding of glycans from the EC surface has been shown to occur in response to reactive oxygen species (ROS) and inflammatory mediators. Using sub-antimicrobial doses of doxycycline, a broad spectrum matrix metalloprotease (MMP) inhibitor, inhibition of chemoattractant induced glycan shedding has suggested that MMPs may be a major effector of the loss of glycans. However, it has also been reported that doxycycline is a scavenger of ROS that may also activate MMPs. To clarify the basis for doxycycline as an inhibitor of glycan shedding, the present studies were undertaken to determine its effect on ROS induced shedding in post-capillary venules of the exteriorized mesentery of the rat. To this end, hypoxanthine (HX) and xanthine oxidase (XO) were rapidly mixed on the mesenteric surface for a 2min period to generate superoxide anion (O2(-)·) and the time course of glycan shedding was monitored in post-capillary venules over a 30min period. Glycan shedding was quantitated by loss of adherent fluorescently labeled lectin coated microspheres (FLMs, 0.1μm diameter) that were systemically infused. It was found that HX/XO caused FLM adhesion to decrease 45% within 30min. This effect could be inhibited in a dose dependent manner by the addition of superoxide dismutase to the superfusion solution, thus confirming the role of O2(-)·. In contrast, 0.5μM doxycycline had no effect on FLM shedding in response to HX/XO, contrary to its ability to attenuate shedding in response to the chemoattractant fMLP. Thus it is suggested that the efficacy of doxycycline as an inhibitor of glycan shedding during inflammation arises from its ability to inhibit MMP activation., (© 2013.)

Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKCα and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis., (Copyright © 2011. Published by Elsevier Inc.)

We report peripheral actions in rats of Neotrofin, a purine derivative of therapeutic interest. Systemic injections mimicked NGF in eliciting sprouting of nociceptive nerves without affecting their regeneration. The sprouting was prevented by anti-NGF treatment, implicating endogenous NGF. We detected no Neotrofin-induced increases in cutaneous NGF levels or in retrograde NGF transport. In contrast, both NGF and phosphorylation of trkA increased significantly in DRGs, with a marginal appearance of phosphorylated trkA in axons. We conclude that the DRG effects of Neotrofin are responsible for its induction of sprouting. Neotrofin also induced a striking phosphorylation of axonal erk 1 and 2, which was, however, unaffected by anti-NGF treatment. We suggest that this NGF-independent MAP kinase activation is involved in nonsprouting functions of Neotrofin such as neuroprotection. Unlike injected NGF, Neotrofin did not induce hyperalgesia, supporting its candidacy as a treatment for peripheral neuropathies like those induced by diabetes and anticancer chemotherapy.

The formation of reactive oxygen species (ROS) may be important in the pathogenesis of microvascular dysfunction and injury in ischemic retinopathies. The authors hypothesized that retinal endothelial cells can generate injurious levels of superoxide radical in response to ischemia/reperfusion, that endothelial xanthine oxidase and cyclooxygenase are important enzymatic sources of superoxide radical under these conditions, and that superoxide scavengers and inhibitors of these enzymes can protect endothelium from ischemic injury. The authors used confluent cultures of mouse retinal endothelial cells (MREC) subjected to exogenously generated superoxide or simulated ischemia-reperfusion to test these hypotheses. Cell injury was assessed biochemically by lactate dehydrogenase release into the culture medium. MREC were injured in a duration-dependent fashion by exposure to the superoxide-generating mix of hypoxanthine and xanthine oxidase. Increasing periods of oxygen and glucose deprivation (OGD) for 5-9 hr followed by replenishment of substrates for 2 hr led to progressive increases in endothelial cell injury; a significant proportion of the injury occurred during the period of substrate replenishment. Significant MREC protection was achieved by the superoxide scavengers SOD (1000 U ml(-1)) and a carboxylic acid derivative of carboxyfullerene (10 microM), the xanthine oxidase inhibitors oxypurinol (100 microM) and diphenyleneiodonium (DPI) (100 n M), and the cyclooxygenase inhibitors indomethacin (300 microM) and ibuprofen (300 microM). It is concluded that MREC are vulnerable to auto-oxidative injury by superoxide radical generated following a period of OGD. Both xanthine oxidase- and cyclooxygenase-dependent pathways are important enzymatic sources of superoxide formation in this setting. These enzymes and the ROS produced from their activity may be viable therapeutic targets to reduce microvascular dysfunction and injury in ischemic retinopathies., (Copyright 2002 Elsevier Science Ltd.)

This study investigates whether the neurohormone melatonin can prevent the retinal neuronal injury caused by reactive oxygen species (ROS) in cultured human retinal neuronal cells. Cultures of human retinal neuronal cells established from a variety of donors were grown to 14 days and then subjected to experimental hypoxanthine/xanthine oxidase (HX/XO)-induced injury. Intracellular production of ROS by administration of HX/XO was confirmed by flow cytometry; the ROS resulted in both apoptotic and necrotic pattern of cell death in the retinal neuron cultures. The efficacy of melatonin against ROS injury was quantitated by MTT assay, enzyme immunoassay, and immunocytochemistry for neurofilament protein. The antioxidative effect of melatonin was compared with that of alpha-tocopherol. Retinal neuronal injury significantly reduced in a dose-response manner by a treatment of 1.0-8.0 mM alpha-tocopherol. Melatonin, in concentrations of more than 2.0 mM, also significantly reduced the injury. About 70% of cells are rescued by pretreatment with 1.0 mM alpha-tocopherol and 8.0 mM melatonin in the MTT assay. Our observations suggest that melatonin can rescue retinal neurons from ROS injury in human retinal cell cultures., ((c)2001 Elsevier Science.)

Background: Recently we reported that bacterial lipopolysaccharide (LPS) stimulates release of tumor necrosis factor alpha (TNF-alpha) from porcine coronary arteries and smooth muscle cells cultured from those vessels. It has also been reported that plasma levels of TNF-alpha are elevated after myocardial infarction. Since it is known that the production of reactive oxygen intermediates (ROI) occurs during ischemia and ROI are suggested activators of the nuclear regulatory factor kappaB (NF-kappaB), we tested the hypothesis that release of TNF-alpha from smooth muscle cells could also be stimulated with a ROI-generating system., Materials and Methods: Smooth muscle cells were isolated from porcine coronary arteries. Confluent cells in 48-well culture dishes were treated for 30 min with 0.003 units/ml xanthine oxidase (XO) and 2 mM hypoxanthine (HX) added to the culture medium. The medium was then removed and the cells were washed three times and fresh medium without HX-XO was added. Then, at 1, 3, and 6 h the medium was removed and analyzed for biologically active TNF-alpha. In other experiments, smooth muscle cells were treated with 20 micrograms/ml LPS for 6 h and aliquots of medium analyzed for TNF-alpha. Untreated cells served as controls. Data were analyzed by two-way ANOVA with repeated measures. Extracts of total cell protein were prepared and activation of NF-kappaB was determined by electrophoretic mobility shift assay., Results: Treatment of cells with HX-XO stimulated release of TNF-alpha, which rose to a maximum of 17.5 +/- 1.7 units/mg cell protein at 6 h. This was significantly higher (P < 0. 05) than release stimulated by LPS (10.2 +/- 1.0 units/mg at 6 h) or TNF-alpha detected in the culture medium from untreated control cells (4.2 +/- 0.9 units/mg protein at 6 h). Both HX/XO and LPS activated NF-kappaB., Conclusions: These results support the conclusion that coronary smooth muscle cells are a potential source of TNF-alpha during events that are associated with formation of ROI such as myocardial ischemia., (Copyright 1999 Academic Press.)