Platelet inhibition by low-dose aspirin but not by clopidogrel reduces the axon-reflex current-induced vasodilation in humans

We previously showed a prolonged inhibition of current-induced vasodilation (CIV) after a single oral high dose of aspirin. In this study, we tested the hypothesis of platelet involvement in CIV. Nine healthy volunteers took 75 mg aspirin/day, 98 mg of clopidogrel bisulfate/day, or placebo for 4 days. CIV was induced by two consecutive 1-min anodal current applications (0.08 mA/cm2) through deionized water with a 10-min interval. CIV was measured with laser Doppler flowmetry and expressed as a percentage of baseline cutaneous vascular conductance: %Cb. In a second experiment in 10 volunteers, aspirin and placebo were given as in experiment 1, but a 26-h delay from the last aspirin intake elapsed before ACh iontophoresis and postocclusive hyperemia were studied in parallel to CIV. In experiment 1, the means ± SE amplitude of CIV was 822 ± 314, 313 ± 144, and 746 ± 397%Cb with placebo, aspirin (P < 0.05 from placebo and clopidogrel), and clopidogrel (NS from placebo), respectively. In experiment 2, CIV impairment with aspirin was confirmed: CIV amplitudes were 300 ± 99, and 916 ± 528%Cb under aspirin and placebo, respectively (P < 0.05), whereas vasodilation to ACh iontophoresis (322 ± 74 and 365 ± 104%Cb) and peak postocclusive hyperemia (491 ± 137 and 661 ± 248%Cb) were not different between aspirin and placebo, respectively. Low-dose aspirin, even 26 h after oral administration, impairs CIV, while ACh-mediated vasodilation and postocclusive hyperemia are preserved. If platelets are involved in the neurovascular mechanism triggered by galvanic current application in humans, it is likely to occur through the cyclooxygenase but not the ADP pathway. a significant increase in skin blood flow (SkBF) has been observed in response to non-noxious galvanic current application in humans. This current-induced vasodilation (CIV) has been described as the result of an axon reflex (3) and disappears in locally anesthetized or chronically capsaicin-treated skin. Then CIV depends on capsaicin-sensitive fibers and is an interesting model of the neurovascular interaction following non-noxious stimulation (11, 16). Prostaglandins are synthesized by cyclooxygenases (COX) and play a key role as mediators in the vascular response observed during CIV. COX are expressed in a large variety of human tissues, including endothelium, smooth muscles, nerves, and platelets (29). We recently reported a long-lasting inhibition of CIV (>5 days) following a single high (1,000 mg) oral dose of aspirin (11, 12, 40), which irreversibly blocks both isoforms of COX (COX-1 and COX-2). COX of neuronal origin does not seem responsible for this long-lasting effect (13). This long-lasting inhibition of CIV is consistent with the time required to resynthesize unblocked platelets after oral single-dose aspirin leading to the hypothesis that platelets participate in the vascular response to CIV. Indeed, a single oral high dose of aspirin irreversibly inactivates the platelet COX pathway for the duration of the life of the platelets (∼10 days) (1, 36). Indomethacin, a nonspecific COX inhibitor devoid of effect on vanilloid receptors and acid-sensing ion channels (41), abolished CIV, confirming that the inhibition of CIV by aspirin likely resulted from its effect on COX (and not on vanilloid receptors or on acid-sensing ion channels). Furthermore, the specific COX-2 inhibitor, celecoxib, failed to affect CIV, suggesting that CIV is mainly a COX-1-dependent phenomenon (39). Together, with the long-lasting effect of a single oral high dose of aspirin, the fact that COX-1 isoform participates in CIV raises the question of a possible platelet involvement in the axon-reflex CIV. Although there is, to date, no in vivo proof of a direct platelet-mediated vasodilation in humans, the hypothesis of a platelet involvement in axon-reflex vasodilation and other vasodilator mechanisms has previously been explored in vitro (15, 23, 31). Those authors provided evidence for a direct in vitro platelet-mediated endothelium-dependent vasodilation in preconstricted arteries but mainly by the ADP pathway. To investigate whether the platelet COX and ADP pathways are involved in vivo in CIV, we inhibited platelet function by aspirin (a platelet COX inhibitor) or clopidogrel (a platelet ADP-receptor inhibitor). Our hypothesis was that if platelets are involved, both clopidogrel and low-dose aspirin would impair the current-induced vasodilation

Low skin temperature impairs the cutaneous vasodilator response to local progressive pressure strain.

A pressure-induced vasodilation (PIV) was recently reported as a putative protective response in human skin. Therefore, we examined the influence of skin temperatures on cutaneous blood flow responses to local progressive pressure strain. Ten healthy volunteers were studied at different ambient temperatures leading to low (29.0 +/- 0.3 degrees C), intermediate (32.6 +/- 0.1 degrees C), high (33.9 +/- 0.1 degrees C) and very high (36.0 +/- 0.1 degrees C) skin temperatures. We measured cutaneous blood flow using laser Doppler flowmetry on the foot in response to a local progressive pressure increase of 5.0 mm Hg min(-1). Progressive pressure strain led to an almost linear cutaneous laser Doppler flow decrease at both low and intermediate skin temperatures (-40.1 +/- 6.6% and -31.2 +/- 6.5% from baseline at 30 +/- 1.25 mm Hg), whereas at both high and very high skin temperatures, subjects responded with a transient cutaneous vasodilation (+33.6 +/- 10.6% and +50.6 +/- 15.4% from baseline at 30 +/- 1.25 mm Hg). These findings suggest that high skin temperatures are required for the PIV to develop

Variability and short-term determinants of walking capacity in patients with intermittent claudication

OBJECTIVE: Global positioning system (GPS) recordings can provide valid information on walking capacity in patients with peripheral arterial disease (PAD) and intermittent claudication (IC) during community-based outdoor walking. This study used GPS to determine the variability of the free-living walking distance between two stops (WDBS), induced by lower-limb pain, which may exist within a single stroll in PAD patients with IC and the potential associated parameters obtained from GPS analysis.METHODS: This cross-sectional study of 57 PAD patients with IC was conducted in a university hospital. The intervention was a 1-hour free-living walking in a flat public park with GPS recording at 0.5 Hz. GPS-computed parameters for each patient were WDBS, previous stop duration (PSD), cumulated time from the beginning of the stroll, and average walking speed for each walking bout. The coefficient of variation of each parameter was calculated for patients with the number of walking bouts (N(WB)) >or=5 during their stroll. A multivariate analysis was performed to correlate WDBS with the other parameters. RESULTS: Mean (SD) maximal individual WDBS was 1905 (1189) vs 550 (621) meters for patients with N(WB) <5 vs N(WB) >or= 5, respectively (P < .001). In the 36 patients with N(WB) >or= 5, the coefficient of variation for individual WDBS was 43%. Only PSD and cumulated time were statistically associated with WDBS in 16 and 5 patients, respectively. CONCLUSIONS: A wide short-term variability of WDBS exists and likely contributes to the difficulties experienced by patients with IC to estimate their maximal walking distance at leisurely pace. Incomplete recovery from a preceding walk, as estimated through PSD, seems to dominantly account for the WDBS in patients with IC

Vascular uptake of rehydration fluids in hypohydrated men at rest and exercise

The purpose of this study was to formulate and to evaluate rehydration drinks, which would restore total body water and plasma volume (PV), for astronauts to consume before and during extravehicular activity, a few hours before reentry, and immediately after landing. In the first experiment (rest, sitting), five healthy men (23-41 yr), previously dehydrated for 24 hr., drank six (1a, 2, 4, 5, 6, 7) fluid formulations (one each at weekly intervals) and then sat for 70 min. Pre-test PV were measured with Evans blue dye and changes in PV were calculated with the hematocrit-hemoglobin transformation equation. This rest experiment simulated hypohydrated astronauts preparing for reentry. The second experiment (exercise, supine) followed the same protocol except four healthy men (30-46 yr) worked for 70 min. in the supine position on a cycle ergometer at a mean load of 71+/-1 percent of their peak aerobic work capacity. This exercise experiment simulated conditions for astronauts with reduced total body water engaging in extravehicular activity

Transient self-potential anomalies associated with recent lava flows at Piton de la Fournaise volcano (Réunion Island, Indian Ocean)

International audienceSelf-potential signals are sensitive to various phenomena including ground water flow (streaming potential), thermal gradients (thermoelectric potential), and potentially rapid fluid disruption associated with vaporization of water. We describe transient self-potential anomalies observed over recent (< 9 years) lava flows at Piton de la Fournaise volcano (Reunion Island, Indian Ocean). Repeated self-potential measurements are used to determine the decay of the self-potential signals with time since the emplacement of a set of lava flow. We performed a 9 km-long self-potential profile in February 2004 in the Grand Brûlé area. This profile was repeated in July–August 2006. The second repetition of this profile crossed eight lava flows emplaced between 1998 and 2005 during seven eruptions of Piton de la Fournaise volcano. The self-potential data show clear positive anomalies (up to 330 mV) and spatially correlated with the presence of recent lava flows. The amplitude of the self-potential anomalies decreases exponentially with the age of the lava flows with a relaxation time of not, vert, similar 44 months. We explain these anomalies by the shallow convection of meteoric water and the associated streaming potential distribution but we cannot exclude possible contributions from the thermoelectric effect and the rapid fluid disruption mechanism. This field case evidences for the first time transient self-potential signals associated with recent volcanic deposits. It can be also a shallow analogue to understand the variation of self-potential signals in active geothermal areas and transient self-potential signals associated with dike intrusion at larger depths. The empirical equation we proposed can also be used to diagnose the cooling of recent lava flow on shield volcanoes

Small RNA analysis in Sindbis virus infected human HEK293 cells

In contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. Since RNAi has a well-established role in controlling infection of the alphavirus Sindbis virus (SINV) in insects, we have used this virus to investigate the role of RNAi in SINV infection of human cells

The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment

A Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition

BACKGROUND: The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity. RESULTS: By a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity. CONCLUSION: By combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression

Improving model predictions for RNA interference activities that use support vector machine regression by combining and filtering features

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is a naturally occurring phenomenon that results in the suppression of a target RNA sequence utilizing a variety of possible methods and pathways. To dissect the factors that result in effective siRNA sequences a regression kernel Support Vector Machine (SVM) approach was used to quantitatively model RNA interference activities.</p> <p>Results</p> <p>Eight overall feature mapping methods were compared in their abilities to build SVM regression models that predict published siRNA activities. The primary factors in predictive SVM models are position specific nucleotide compositions. The secondary factors are position independent sequence motifs (<it>N</it>-grams) and guide strand to passenger strand sequence thermodynamics. Finally, the factors that are least contributory but are still predictive of efficacy are measures of intramolecular guide strand secondary structure and target strand secondary structure. Of these, the site of the 5' most base of the guide strand is the most informative.</p> <p>Conclusion</p> <p>The capacity of specific feature mapping methods and their ability to build predictive models of RNAi activity suggests a relative biological importance of these features. Some feature mapping methods are more informative in building predictive models and overall <it>t</it>-test filtering provides a method to remove some noisy features or make comparisons among datasets. Together, these features can yield predictive SVM regression models with increased predictive accuracy between predicted and observed activities both within datasets by cross validation, and between independently collected RNAi activity datasets. Feature filtering to remove features should be approached carefully in that it is possible to reduce feature set size without substantially reducing predictive models, but the features retained in the candidate models become increasingly distinct. Software to perform feature prediction and SVM training and testing on nucleic acid sequences can be found at the following site: <url>ftp://scitoolsftp.idtdna.com/SEQ2SVM/</url>.</p

Identification of Keratinocyte Growth Factor as a Target of microRNA-155 in Lung Fibroblasts: Implication in Epithelial-Mesenchymal Interactions

International audienceBACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury