1,720,972 research outputs found
Downregulation of HD-PTP by high magnesium concentration : novel insights into magnesium-induced endothelial migration
No full textMagnesium promotes endothelial migration, an event which is orchestrated by a complex interplay between protein tyrosine kinases and phosphatases. We found that high extracellular concentrations of magnesium do not modulate the levels and the activation of FAK and Src, two tyrosine kinases involved in driving cell migration. Interestingly, we show that magnesium induced-endothelial motility correlates with the downregulation of HD-PTP, a potential tyrosine phopshatase previously shown to be involved in modulating cell migration. The decreased amounts of HD-PTP are not dependent upon transcriptional mechanisms. In contrast to Fibroblast Growth Factor-induced HD-PTP downregulation, the proteasome seems not to be involved in regulating HD-PTP levels in endothelial cells cultured in high magnesium containing medium. Our results indicate that, in the presence of high magnesium concentrations, endothelial cells are stimulated to migrate through complex mechanisms involving also HD-PTP
The effects of silencing EDF-1 in human endothelial cells
No full textOBJECTIVE:
EDF-1, a 16 kDa highly conserved intracellular protein, serves as a calmodulin binding protein and, upon nuclear translocation, functions as a coactivator of several transcription factors. To understand whether EDF-1 is implicated in regulating endothelial function, we silenced EDF-1 expression using small hairpin (sh) RNA.
METHODS:
Human umbilical vein endothelial cells (HUVEC) were utilized and EDF-1 levels were detected by western blot. Cell proliferation, cell organization in fibrin gel and nitric oxide release were evaluated in cells silencing EDF-1 after transfection with shRNA.
RESULTS:
EDF-1 was downregulated in quiescent and senescent HUVEC, whereas it was upregulated in proliferating cells. Knocking down EDF-1 promoted the acquisition of a spindle phenotype, inhibited cell proliferation, accelerated the organization into capillary-like networks on fibrin gels and induced the production of nitric oxide (NO). While the total amounts and the degree of phosphorylation of endothelial NO synthase are not altered in cells silencing EDF-1, we found an increased interaction between calmodulin and endothelial NO synthase. Accordingly, the calmodulin inhibitor calmidazolium significantly decreased NO release in cells silencing EDF-1. These results suggest that knocking down EDF-1 might increase free calmodulin which ultimately activates endothelial NO synthase.
CONCLUSIONS:
Since EDF-1 (i) is involved in the control of endothelial proliferation and organization, events which are crucial to repair damages to the vessel wall, and (ii) increases NO, which exerts anti-atherogenic and anti-thrombotic effects, we conclude that EDF-1 is implicated in molecular events that are pivotal to endothelial function and, therefore, to vascular integrity
Extracellular magnesium and in vitro cell differentiation : different behaviour of different cells
No full textThe contribution of magnesium to cell differentiation is not clear. Some studies indicate that low extracellular magnesium promotes cell differentiation, while others reach opposite conclusions. We evaluated the effects of different concentrations of extracellular magnesium on the differentiation of three in vitro experimental models: human endothelial cells seeded onto Matrigel, phorbol ester-treated myeloid leukemia U937 cells, and 3T3-L1 pre-adipocytes exposed to a hormonal cocktail containing dexamethasone and insulin. The differentiation of endothelial cells and pre-adipocytes seems to be independent of extracellular Mg concentration. Conversely, magnesium deficiency retards, while high extracellular magnesium accelerates phorbol ester-induced U937 cell differentiation, probably by interfering with calcium homeostasis or with the activity of kinases. We conclude that the extracellular magnesium concentration affects the differentiation of various cell types
High magnesium inhibits human osteoblast differentiation in vitro
No full textSeveral studies in humans indicate that both high and low concentrations of magnesium have harmful effects on bone metabolism and homeostasis. However, little is known about the effects of different concentrations of magnesium on bone cells. Considering that 1mM is the physiological concentration of extracellular magnesium for cultured cells, in our experimental model we exposed osteoblast like SaOS-2 cells and normal human osteoblasts to low (0.1mM) and high (5.0mM) concentrations of magnesium. We found that high concentrations of magnesium markedly inhibited the deposition of mineral matrix by SaOS-2 as well as the activity of alkaline phosphatase, a marker of osteoblast differentiation. We then evaluated the differentiation of normal human osteoblasts by measuring alkaline phosphatase activity and again found a marked inhibition by high concentrations of magnesium. Nitric oxide, which is known to play a role in bone formation, does not seem to be involved. We hypothesize that high levels of magnesium might alter the intracellular concentration of various cations - among which calcium - by competing for the same transporters. We conclude that high magnesium levels impair osteoblast activity and might therefore contribute to bone disease
EDF-1 contributes to the regulation of nitric oxide release in VEGF treated human endothelial cells
No full textVascular endothelial growth factor (VEGF) induces nitric oxide (NO) release by triggering multiple intracellular signals, among others the calcium/calmodulin pathway and the activation of Akt, events which induce endothelial NO synthase (eNOS) activity. Because Endothelial Differentiation-related Factor (EDF)-1 is a calmodulin binding protein and plays a role in modulating endothelial functions, we evaluated whether EDF-1 is implicated in the regulation of eNOS activity in VEGF-treated human endothelial cells. While VEGF does not modulate the total amounts of EDF-1, it promotes the dissociation of calmodulin from EDF-1 which correlates with the increase of calmodulin bound to eNOS and the induction of NO release. To better characterize the contribution of EDF-1 to the regulation of VEGF-induced NO release, we stably silenced EDF-1 in endothelial cells. We here show that endothelial cells silencing EDF-1 produce more NO than controls and do not increase NO release in response to VEGF. The insensitivity to VEGF results from the incapability of cells silencing EDF-1 to phosphorylate eNOS Ser(1177), even though Akt is activated. Interestingly, okadaic acid, a pharmacologic inhibitor of the serine/threonine phosphatase PP2A, which preferentially dephosphorylates eNOS Ser(1177), restores NO release and eNOS Ser(1177) phosphorylation in cells silencing EDF-1. Our results suggest EDF-1 as a novel contributor to the complex regulation of eNOS activity in human endothelial cells
Transcriptional coactivator EDF-1 is required for PPARγ-stimulated adipogenesis
No full textPeroxisome proliferator-activated receptor-γ (PPARγ) is essential for adipogenesis. Since EDF-1 is a cofactor of PPARγ, we investigated the molecular cross-talk between EDF-1 and PPARγ in adipogenesis. While EDF-1 was not modulated during differentiation of 3T3-L1 cells, it co-immunoprecipitated with PPARγ. Silencing EDF-1 by shRNAs inhibited the differentiation in adipocytes of 3T3-L1 cells, as detected by the staining of intracellular triglycerides and the expression of the PPARγ target gene aP2. Accordingly, we found that anti-EDF-1 shRNAs decreased ligand dependent activation of PPARγ in 3T3-L1 transiently transfected with a vector expressing luciferase under the control of a PPARγ responsive consensus. To rule out that this inhibition is due to the concomitant downregulation of PPARγ levels, we overexpressed PPARγ in 3T3-L1 silencing EDF-1 and found a decrease of ligand dependent activation of PPARγ, in spite of the high amounts of PPARγ. These results demonstrate that EDF-1 is required for PPARγ transcriptional activation during 3T3-L1 differentiation
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
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