1,720,987 research outputs found
Chronic nitric oxide deprivation induces hypoxia inducible factor-1α stabilization in human endothelial cells
No full textThe endothelium explicates its physiological functions by producing active molecules, among which nitric oxide (NO) is particularly important. By diffusing into neighbouring smooth muscle cells, endothelial-produced NO induces vasorelaxation, thereby controlling blood pressure levels. NO generated in the endothelium also has antiaggregant activity that protects the cardiovascular system from thrombosis and acute events. Consistent with the key role of this gaseous messenger in cardiovascular physiology, NO loss is a dangerous event which is associated with endothelial dysfunction typical of diffuse pathological conditions like atherosclerosis, pulmonary hypertension and senescence.
In the present study, the behavioural and molecular consequences deriving from chronic NO deprivation in human primary endothelial cells (human umbilical vein endothelial cells, HUVECs) were investigated. To inhibit NO formation, endothelial nitric oxide synthase (eNOS) was chronically inhibited by treatment with NG-Nitro-L-arginine methyl ester (L-NAME), a structural analogue of L-arginine that competitively block the active site of the enzyme. After 48 h of L-NAME treatment, HUVECs displayed a higher migratory capability as evaluated by chemotaxis assays in Boyden’s chamber. The possible involvement of the cyclic GMP/protein kinase G (cGMP/PKG) pathway in the observed effect was excluded, since the increased HUVEC migratory behaviour was not induced by a long term treatment with the guanylate cyclase inhibitor ODQ, indicating that cGMP and/or PKG are not involved in the regulation of cell motility.
In an attempt to explain the mechanism through which NO deprivation enhances migration, we investigated by quantitative real time PCR (RT-qPCR) how chronic L-NAME treatment affects the expression level of the Vascular Endothelial Growth Factor (VEGF) receptor-2 (kinase insert domain receptor, KDR). We also analyzed VEGF itself, as endogeous production of the growth factor could potentiate migration by an autocrine loop. RT-qPCR analyses demonstrated that both VEGF and KDR mRNA levels were significantly augmented in L-NAME treated cells.
Increased VEGF production and cell motility are typical events occurring in hypoxic cancer cells, due to the accumulation of hypoxia-inducible factor-1 (HIF-1), which plays a major role in the transcriptional activation of genes encoding angiogenic factors. Similarly, induction of VEGF expression during hypoxia has been described also in endothelial cells. We therefore analysed the effect of long term L-NAME treatment on HIF-1 levels in HUVECs. Most interestingly, we observed that, after 48 h of treatment, L-NAME induced in HUVECs nuclear accumulation of HIF-1α. RT-qPCR analysis revealed no significant change in HIF-1α mRNA levels, suggesting that HIF-1 accumulation in L-NAME-treated cells was mainly due to its stabilization, as occurs under hypoxic conditions.
Taken together, these data suggest that prolonged L-NAME treatment induces in endothelial cells a pseudohypoxic state, with the consequent stabilization of HIF-1α under normoxic conditions. These events may very well occur in all of those pathological conditions where eNOS expression and/or activity are impaired thus contributing to the endothelial dysfunction typical of such diseases like hypertension and diabetes
Silencing of Eps8 blocks migration and invasion in human glioblastoma cell lines
No full textGlioblastoma multiforme (GBM) is the most malignant human primary brain tumor, and its infiltrative nature represents the leading cause for the failure of therapies and tumor recurrences. It is therefore crucial the knowledge of the molecular mechanisms underlying GBM invasion to identify novel therapeutic targets to limit motility. In this study, we evaluated the role of Epidermal growth factor receptor Pathway Substrate 8 (Eps8), a crucial regulator of the actin cytoskeleton dynamics accompanying cell motility and invasion, in GBM migration and invasiveness. We found that silencing of the protein by small interfering RNAs (siRNAs) abrogated the migratory and invasive capacity of three different human GBM cell lines both in 2-dimensional (2-D) and 3-dimensional (3-D) in vitro assays. The inhibitory effect on invasion was maintained independently by the migration mode utilized by the cells in our 3-D model, and was accompanied by an impaired formation of actin-based cytoskeletal protrusive structures. Our data propose Eps8 as a key molecule involved in the control of the intrinsic invasive behavior of GBM cells, and suggest that this protein might represent a useful target for the design of new drugs for the treatment of these tumors
EFFECTS OF CHRONIC NITRIC OXIDE DEPRIVATION ON ENDOTHELIAL CELL BEHAVIOUR
Full text linkThe endothelium explicates its physiological functions by producing active molecules, among which nitric oxide (NO) is particularly important. It is well known that endothelial dysfunction (ED), i.e. an impaired function of the endothelium coupled with a reduced release of NO, is a risk factor for atherosclerosis together with a list of conditions such as hypertension, hypercholesterolemia, smoking, diabetes, and the aging process itself. These conditions are also associated with a significant increase in Reactive Oxygen Species (ROS) in the vascular wall that may contribute to the establishment of ED and to the development of its late effect on cardiovascular system.
In the present study, the behavioural and molecular consequences deriving from chronic NO deprivation were investigated in human primary endothelial cells (human umbilical vein endothelial cells, HUVECs). To inhibit NO formation, endothelial nitric oxide synthase (eNOS) was chronically inhibited by treatment with L-NG-Nitroarginine methyl ester (L-NAME), a structural analogue of L-arginine that competitively block the active site of the enzyme, or by transfection with a siRNA specific for eNOS.
We observed that a 48-h L-NAME treatment induced in HUVECs a higher migratory capability (evaluated by chemotaxis assays in Boyden’s chamber) which was independent of the reduced activity of the cyclic GMP/protein kinase G pathway present in chronically NO deprived HUVECs. In the attempt to explain the mechanism(s) through which NO deficiency enhances migration, we investigated if chronic L-NAME treatment affected the expression and production of Vascular Endothelial Growth Factor (VEGF) and of its receptor KDR. RT-qPCR analyses, accompanied by ELISA assays and western blot analyses, demonstrated that both VEGF and KDR mRNAs and proteins were significantly augmented in L-NAME treated cells, thus suggesting the establishment of an autocrine loop responsible for the increased migration.
Increased VEGF production and cell motility are typical events occurring in hypoxic cancer cells, due to the accumulation of hypoxia-inducible factor-1α (HIF-1α), which plays a major role in the transcriptional activation of genes encoding angiogenic factors. Similarly, induction of VEGF expression during hypoxia has been described in endothelial cells (ECs). Interestingly, we observed a significant nuclear accumulation of HIF-1α in HUVECs chronically treated with L-NAME. Moreover, the transcriptional activity of HIF-1α was responsible for the increases in VEGF/KDR expression and migration since the transfection with ΔARNT (a dominant negative form of the HIF-1β subunit that maintains the capacity of forming an heterodimer but cannot bind DNA) is able to totally blunt both the effects in L-NAME treated HUVECs, thus confirming the involvement of an autocrine loop in the pro-migratory effect induced by NO deprivation. The dependence of HIF-1α stabilization from NO deficiency was confirmed by using the NO donor DETA/NO. Very low doses of DETA/NO reverted both the HIF-1α accumulation and the consequent increases in VEGF expression and cell motility induced by L-NAME treatment. Furthermore, to investigate whether the observed effects were due to the specific inhibitory effect of L-NAME on eNOS activity, we knocked-down the enzyme by using RNA interference methodology. In eNOS silenced cells, HIF-1α accumulated in the nucleus and VEGF production was enhanced thus confirming the dependence of the observed effects on eNOS inhibition. All these results suggest that basal release of NO may act as a negative controller of HIF-1α levels and cell motility in HUVECs with important consequences on ECs physiology.
In the attempt to unravel the pathway(s) linking NO deficiency to HIF-1α accumulation and activity, we focus our attention on ROS since their formation has been involved in HIF-1α stabilization in normoxia. We found that acute treatment with L-NAME induced in HUVECs an early and transient burst in ROS formation that was fully prevented by the presence of the antioxidant N-acetylcysteine (NAC). HIF-1α accumulation was reduced by 45% in the presence of NAC indicating that the peak of ROS was only partially involved in its stabilization. On the contrary, NAC did not affect the increase in cell migration in ECs chronically deprived of NO. At variance with acute treatment, chronic L-NAME exposure gave rise to an antioxidant environment characterized by a reduction in cellular ROS content accompanied by an increase in superoxide dismutase-2 (SOD-2) expression and activity. Importantly, this protective response was accompanied by the nuclear accumulation of the transcription factor NF-E2-related factor-2 (Nrf2) that was fully prevented in the presence of NAC. These results suggest the establishment of an antioxidant status in HUVECs chronically deprived of NO in the attempt to neutralize any further cell damage induced by loss of NO.
In addition, since NO plays an important role in promoting mitochondrial biogenesis in different cell types and tissues, we analyzed the mitochondrial mass and function in HUVECs after NO deprivation. Long term L-NAME treatment induced a significant reduction in mitochondrial DNA (mtDNA) accompanied by decreases in the incorporation of the metabolic indicator MTS, in cellular ATP content, and in oxygen consumption. In agreement, the silencing of eNOS was able to decrease mtDNA and total cellular ATP levels thus confirming that loss of NO sustained the onset of mitochondrial dysfunction in HUVECs. Importantly, metabolic effects observed in chronically NO deprived ECs was independent of both HIF-1α activity and ROS generation.
In conclusion, we demonstrated that an endothelial deficit of NO, by mimicking the in vivo early phase of ED, induces important physiological modifications in human ECs. In particular, loss of NO leads to the accumulation and transcriptional activation of HIF-1α responsible for the enhanced VEGF/KDR expression and cell motility, and to the establishment of mitochondrial dysfunction. Importantly, most of the peculiar features shown by long term NO deprived HUVECs are independent of acute ROS generation, and must therefore depend on other pathways triggered by NO loss. On the contrary, ROS formation appears to be totally responsible for the Nrf2 accumulation that might account for the establishment of an adaptive antioxidant status in response to oxidative stress. Further experiments will be necessary to fully characterize our in vitro model of ED and to elucidate the molecular mechanism(s) involved in HIF-1α stabilization. Our model should however represents an useful system for the study and the identification of innovative pharmacological targets and markers for ED, thus contributing to a better knowledge of the endothelium behavior in the absence of NO and to an improved comprehension of the molecular mechanisms involved in the onset of cardiovascular pathologies
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Charcoal-stripped serum impairs growth and sprouting of male and female human umbilical vein endothelial cells (HUVECs) regardless of estrogens
No full textLoss of a normal endothelial function crucially contributes to the development and clinical expression of atherosclerosis and cardiovascular disease (CVD). Despite substantial sex/gender differences have been described in the occurrence of CVD, very little is known about innate sex properties of male and female endothelial cells (ECs).
To characterize putative sex-dependent differences, we compared in vitro properties of male and female human umbilical vein ECs (HUVECs) showing that female HUVECs express an higher amount of eNOS mRNA and protein, and possess greater migratory capabilities in comparison to male cells. Male and female HUVECs did not show however any significant difference in metabolic activity.
Since a crucial issue in the sex-related incidence of CVD concerns estrogens, we compared male and female HUVECs grown in standard medium or in a nominally estrogen-free medium containing charcoal-stripped FBS. We found that lack of hormones induced in both sexes: i) a decrease in cell number, and as a consequence in metabolic activity (evaluated by MTT and ATP); ii) an impaired in vitro angiogenesis. All the effects were not reverted by 17-beta-estradiol (E2). These results suggest that estrogens did not represent a critical factor for the maintenance of EC behavior regardless of cell sex. Experiments are ongoing to identify substance(s) responsible for EC rescue from charcoal-stripped FBS-induced impairment in growth and angiogenesis
Silencing of Eps8 inhibits in vitro angiogenesis
No full textAims: Eps8 is an actin-binding protein which has been proposed as a regulator of cancer cell motility and invasion. However, nothing much is known about its contribution to the invasive properties of endothelial cells (ECs), and more generally to angiogenesis. Main methods: Expression and silencing of Eps8 were evaluated by western blot analysis. The effect of Eps8 silencing on cell number and VEGF-induced signaling was tested with standard methods. Migration was evaluated by scratch wound assay and morphogenesis with 2-dimensional (2-D) tube formation and 3-dimensional (3-D) sprouting assays. Actin cytoskeleton was visualized by immunofluorescence. Key findings: We found that silencing of Eps8 profoundly affected the ability of human ECs to migrate and to undergo tube formation and sprouting in 2-D and 3-D in vitro assays, respectively. Notably, capillary-like outgrowth was strictly depending on Eps8 expression also in human tumor-derived ECs. Significance: Our data demonstrate for the first time the involvement of Eps8 in the morphological processes required for in vitro angiogenesis, and suggest that this protein might represent a common target for the design of new anticancer drugs, acting at the same time on both tumor and endothelial cells
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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