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Attività pro-angiogenica di urotensina-II su cellule endoteliali vascolari umane
Full text linkUrotensin-II (U-II) is a cyclic peptide, originally isolated from the urophysis of the goby Gillichthys mirabilis. The human form of U-II (hU-II) is a cyclic undecapeptide. Mature hU-II is synthesized from a large precursor molecule, the prepro-U-II, whose mRNA has been found in many tissues. U-II has been identified as the endogenous ligand of a specific high-affinity receptor, recently identified as the orphan receptor GPR14 which has been renamed urotensin receptor (UT). The human UT isoform belongs to the class A superfamily of G-protein-coupled receptors. The principal physiological role of U-II in mammals is in the cardiovascular system, where it exerts a potent systemic vasoconstrictor and hypertensive effect and several lines of evidence suggested that U-II might be involved in the pathophysiology of the cardiovascular system. On endothelial cells (EC) of animal origin U-II has also been shown to exert a clearcut pro-angiogenic effect, but few experimental data are presently available clarifying the effect of U-II in human endothelium.
Thus, in the present study in vitro models based on human vascular endothelial cells directly isolated from different normal human vessels, including saphenous vein (HSVEC), jugular vein (HJVEC), umbilical vein (HUVEC) and aorta (HAEC) have been used to characterize different aspects of the pro-angiogenic profile exhibited by U-II.
RT-PCR and ICC analyses indicated that UT was expressed by all the human EC considered, while the expression of U-II resulted heterogeneous, in fact the peptide was detectable in HAEC and HUVEC only. When tested in the Matrigel assay all the investigated EC exhibited a strong angiogenic response to the peptide, with the formation of a meshwork of capillary-like structures of increased density and complexity when compared to the unstimulated condition. The effect was comparable to that of FGF-2 and was counteracted by Palosuran, a specific antagonist of UT, indicating that they were triggered by the binding of U-II to its receptor. Interestingly, in EC derived from adult vessels this activity was not associated with a proliferogenic effect. On the contrary, U-II induced a moderate but significant increase of cell proliferation in HUVEC.
Experiments performed in the presence of specific inhibitors of various steps of the signaling cascade showed that the U-II induced self organization of the cells in capillary-like structures is PKC dependent and involves the activation of the ERK1/2 transduction pathway. Western blot analyses on the phosphorilated forms of these kinases provided further support to this finding. Interestingly, the pharmacological inhibition of PI3K, hindered the capacity of U-II to induce a pro-angiogenic effect on HUVEC, indicating that the PI3K/Akt pathway is also involved in regulating the process.
It has also to be observed that some of the signalling pathways activated as a consequence of the binding of U-II to UT can, in principle, be started in several ways. In fact GPCR can also indirectly activate them by inducing the synthesis and release of growth factors. In this respect, the stimulation of HUVEC with U-II for 24 hours, induced AM, ET-1 and VEGF expression, as mRNA and proteins. All these factors are characterized by well known pro-angiogenic properties. Therefore U-II can also act indirectly by stimulating in HUVEC the expression of other pro-angiogenic factors.
Altogether, the results of the present study suggested that U-II, in addition to regulating cardiovascular function, also exerts a direct action on the development and remodelling of the vascular network.Urotensina-II (U-II) è un peptide ciclico, originariamente isolato dall’urofisi del pesce Gillichthys mirabilis, la cui isoforma umana (hU-II) è costituita di 11 aminoacidi. hU-II matura è sintetizzata da un precursore più grande, la prepro-U-II, il cui mRNA è stato individuato in vari tessuti. U-II è stata identificata quale ligando endogeno di uno specifico recettore ad alta affinità, il recettore orfano GPR14 che è stato successivamente rinominato UT. L’isoforma umana di UT appartiene alla classe A della superfamiglia di recettori accoppiati alle proteine G (GPCR). Il principale ruolo fisiologico di U-II nei mammiferi si esplica a livello del sistema cardiovascolare, dove il peptide esercita un notevole effetto ipertensivo e vasocostrittore e proprio per questo molti dati suggeriscono che U-II possa essere coinvolta nella regolazione del sistema cardiovascolare in condizioni sia fisiologiche che patologiche. In cellule endoteliali (EC) di origine animale sembra esercitare un chiaro effetto pro-angiogenico, ma pochi dati sperimentali sono attualmente disponibili per chiarire le azioni del peptide sull’endotelio vascolare umano.
In questo lavoro modelli in vitro basati sull’uso di cellule endoteliali vascolari umane isolate direttamente da vasi diversi, quali vena safena (HSVEC), vena giugulare (HJVEC), vena ombelicale (HUVEC) e aorta (HAEC), sono stai utilizzati per caratterizzare vari aspetti del profilo pro-angiogenico di U-II.
Analisi di RT-PCR ed immunocitochimica indicano che UT è espresso da tutte le EC umane considerate, mentre l’espressione di U-II risulta eterogenea, infatti il peptide si ritrova solo nelle HUVEC e nelle HAEC. Sottoposte a coltura su Matrigel, tutte le EC analizzate presentano una forte risposta angiogenica al peptide, con conseguente formazione di una rete di strutture capillaro-simili di maggiore densità e complessità rispetto alla situazione di controllo. L’effetto è comparabile a quello indotto da FGF-2 ed è antagonizzato da Palosuran, antagonista specifico di UT, ad indicare che l’attività del peptide è innescata dal legame con il suo recettore funzionale. E’ stato interessante notare che nelle EC derivate da vasi adulti questa azione non è associata ad un effetto proliferativo. Al contrario, U-II induce un moderato ma significativo aumento della proliferazione cellulare nelle HUVEC.
Esperimenti condotti in presenza di specifici inibitori di vari step della cascata di segnalazione intracellulare dimostrano che l’auto-organizzazione in strutture capillaro-simili delle EC indotta da U-II è PKC-dipendente e coinvolge l’attivazione del pathway di trasduzione di ERK1/2. Analisi di western blot sulle forme fosforilate di queste chinasi hanno confermato i dati ottenuti con il Matrigel. Inoltre, l’inibizione farmacologica di PI3K, ostacola la capacità di U-II di indurre un effetto pro-angiogenico nelle HUVEC, indicando che anche il pathway PI3K/Akt potrebbe essere coinvolto nella regolazione del processo.
E’ da considerare che alcuni pathway attivati come conseguenza del legame di U-II ad UT, potrebbero essere innescati in diversi modi. Infatti, i GPCR potrebbero anche attivarli indirettamente inducendo la sintesi ed il rilascio di fattori di crescita. A questo proposito, si è messo in evidenza come, dopo 24 ore di stimolazione, U-II induca nelle HUVEC l’espressione di AM, ET-1 e VEGF, tutti fattori con proprietà pro-angiogeniche note. A tempi più lunghi, quindi, U-II potrebbe anche agire indirettamente stimolando nelle HUVEC l’espressione di altri fattori pro-angiogenici.
I risultati di questo lavoro suggeriscono quindi che U-II, oltre a regolare la funzionalità cardiovascolare, potrebbe anche esercitare un’azione diretta sullo sviluppo e il rimodellamento vascolare
Involvement of vascular endothelial growth factor signaling inCLR/RAMP1 and CLR/RAMP2-mediated pro-angiogenic effect of intermedin on human vascular endothelial cells.
No full textIntermedin (IMD) is a recently discovered peptide closely related to
adrenomedullin. Its principal physiological activity is its role in the
regulation of the cardiovascular system, where it exerts a potent hypotensive
effect. In addition, data were recently provided showing that this peptide is
able to exert a clearcut pro-angiogenic effect both in vitro and in vivo. IMD
acts through the non-selective interaction with receptor complexes formed by the
dimerization of calcitonin-like receptor (CLR) with the receptor
activity-modifying proteins RAMP1, 2 or 3. Thus, in the present study, the role
of CLR/RAMP complexes in mediating the pro-angiogenic effect induced by IMD on
human umbilical vein endothelial cells (HUVECs) cultured on Matrigel was
examined. Real-time PCR demonstrated the expression of IMD, CLR/RAMP1 and
CLR/RAMP2 (but not CLR/RAMP3) mRNA in HUVECs. IMD exerted a significant in vitro
angiogenic action, specifically triggered by the binding of the peptide to
CLR/RAMP complexes. Both CLR/RAMP1 and CLR/RAMP2 appeared to mediate the
pro-angiogenic effect, which was associated with a significant increase of
vascular endothelial growth factor (VEGF) mRNA expression 18 h following IMD
administration, indicating that the observed pro-angiogenic effects are related,
at least in part, to an increased synthesis of this growth factor promoted by the
peptide. Western blot analysis, however, showed a significant increase of VEGF
receptor-2 phosphorylation as early as 5 min following IMD administration,
indicating that IMD induces a pro-angiogenic response in human vascular
endothelial cells not only via CLR/RAMP-induced release of VEGF, but also during
signal initiation and propagation by transactivating the VEGF receptor-2
machinery
Urotensin-II-stimulated expression of pro-angiogenic factors in human vascular endothelial cells
No full textUrotensin-II (U-II) is an endogenous peptide recently characterized as a "nonclassic" pro-angiogenic cytokine. In fact, human vascular endothelial cells express the U-II receptor and exhibit a strong in vitro angiogenic response to the peptide, which was specifically triggered by the binding of U-II to its receptor and involved the activation of ERK1/2 and PI3K/Akt signaling pathways. Moreover, available studies, designed to investigate the pro-angiogenic effect quite shortly following U-II stimulation, suggested that the angiogenic action of the peptide was direct and not associated with an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. In the present study, the expression of three pro-angiogenic factors, namely VEGF, endothelin-1, and adrenomedullin, was studied in human umbilical vein endothelial cells (HUVEC) for longer times of U-II stimulation. RT-PCR and Western blot indicated that in HUVEC, exposed for at least 24h to U-II, the expression of the three angiogenic molecules was significantly increased at both mRNA and protein level, opening the possibility that U-II, not only could exert a direct stimulation of an angiogenic phenotype in endothelial cells quite shortly following exposure to the peptide, but could also further enhance the process indirectly by inducing in the cells a delayed production of other pro-angiogenic factors. Interestingly, a preliminary analysis of the time course of the in vitro capillary-like pattern formation was consistent with this view, suggesting a two phase temporal dynamics of the process
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
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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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