Direct determination of trace elements in powdered samples by in-cell isotope dilution femtosecond laser ablation ICPMS

A method has been developed for the direct and simultaneous multielement determination of Cu, Zn, Sn, and Pb in soil and sediment samples using femtosecond laser ablation inductively coupled plasma mass spectrometry (fs-LA-ICPMS) in combination with isotope dilution mass spectrometry (IDMS). The in-cell isotope dilution fs-LA-ICPMS method proposed in this work was based on the quasi-simultaneous ablation of the natural abundance sample and the isotopically enriched solid spike, which was performed using a high repetition rate laser and a fast scanning beam device in a combined manner. Both the sample preparation procedure and the total analysis time have been drastically reduced, in comparison with previous approaches, since a unique multielement isotopically enriched solid spike was employed to analyze different powdered samples. Numerous experimental parameters were carefully selected (e.g., carrier gas flow rate, inlet diameter of the ablation cell, sample translation speed, scanner speed, etc.) in order to ensure the complete mixing between the sample and the solid spike aerosols. The proposed in-cell fs-LA-ICP-IDMS method was tested for the analysis of two soil (CRM 142R, GBW-07405) and two sediment (PACS-2, IAEA-405) reference materials, and the analysis of Cu, Zn, Sn, and Pb yielded good agreement of usually not more than 10% deviation from the certified values and precisions of less than 15% relative standard deviation. Furthermore, the concentrations were in agreement not only with the certified values but also with those obtained by ICP-IDMS after the microwave-assisted digestion of the solid samples, demonstrating therefore that in-cell fs-LA-ICP-IDMS opens the possibility for accurate and precise determinations of trace elements in powdered samples reducing the total sample preparation time to less than 5 min. Additionally, scanning electron microscope measurements showed that the aerosol generated by in-cell fs-LA-ICP-IDMS predominantly consisted of linear agglomerates of small particles (in the order of few tens of nanometers) and a few large spherical particles with diameters below 225 nm

Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseases

Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and gamma-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein gamma-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNF alpha, IL-1 beta and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.Portuguese Science and Technology Foundation (FCT) [PTDC/SAU-ORG/117266/2010, PTDC/BIM-MEC/1168/2012, UID/Multi/ 04326/2013]; FCT fellowships [SFRH/BPD/70277/2010, SFRH/BD/111824/2015

Receptores tipo Toll y de Esfingosina1 fosfato y su implicación en la fisiopatología cardiovascular

Dado que los receptores tipo Toll (TLR) y esfingosina-1-fosfato (S1P) juegan un papel muy importante en inflamación, en esta tesis se estudió la potencial interacción entre los receptores de S1P y TLRs en las vías de adhesión/ inflamación en células endoteliales de origen humano. Determinamos por Western Blot y por citometría de Flujo, que las células tratadas con LPS (ligando de TLR4) y S1P, expresaban una mayor cantidad de ICAM-1 y E-selectina comparado con las células estimuladas con cada ligando por separado, observando diferencias entre las endoteliales de origen venoso y aórtico en cuanto a la expresión de E-selectina. Sin embargo, no observamos ningún efecto cooperativo cuando estimulamos las células con un ligando de TLR2/TLR1. En consecuencia con el aumento de la expresión de las moléculas de adhesión, los experimentos de adhesión bajo flujo laminar mostraron que LPS y S1P cooperan para aumentar el número de leucocitos adheridos a la monocapa endotelial y la fuerza de adhesión en comparación con las células estimuladas con los ligandos por separado. Además la fuerza de adhesión resultó ser similar a la inducida por TNF-¿. Además, la coestimulación con LPS y S1P coopera en el aumento de la expresión de moléculas proinflamatorias como IL-6, COX-2 y prostaciclina. El análisis de las rutas de señalización implicadas reveló una fosforilación sinergística de la MAPK ERK tras el tratamiento con LPS y S1P y la implicación del factor de tanscripción NF-kB, además en el caso de las endoteliales de origen venoso, también participa la MAPK p38. Además, dilucidamos los receptores de S1P implicados en el efecto cooperativo, así como la posible implicación de la enzima esfingosina Kinasa en este efecto. Por otro lado, debido a la prominente acumulación lipídica y los cambios inflamatorios en la estenosis aórtica, y la implicación de S1P en la patofisiología cardiovascular, estudiamos el papel de S1P en las vías proinflamatorias/proosteogénicas en células intersticiales procedentes de válvulas aórticas (AVIC) tanto sanas como estenóticas y de válvulas pulmonares (PVIC). Los resultados fueron que la coestimulación con LPS y S1P tenía un efecto sinérgico en el aumento de la expresión de ICAM-1, COX-2 y cooperan en la expresión de ALP (un marcador inicial de calcificación), en la de VEGF (un factor angiogénico) y en la secreción de sICAM-1 (un marcador cardiovascular) y prostaglandinas (un mediador inflamatorio derivado de la expresión de COX-2). Además, estos efectos, son mayores en las células AVIC provenientes de válvulas estenóticas que de las sanas, y más aún que en las PVIC.Premio extraordinario de Doctorado.Peer Reviewe

Synergy between sphingosine 1-phosphate and lipopolysaccharide signaling promotes an inflammatory, angiogenic and osteogenic response in human aortic valve interstitial cells

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Given that the bioactive lipid sphingosine 1-phosphate is involved in cardiovascular pathophysiology, and since lipid accumulation and inflammation are hallmarks of calcific aortic stenosis, the role of sphingosine 1-phosphate on the pro-inflammatory/pro-osteogenic pathways in human interstitial cells from aortic and pulmonary valves was investigated. Realtime PCR showed sphingosine 1-phosphate receptor expression in aortic valve interstitial cells. Exposure of cells to sphingosine 1-phosphate induced pro-inflammatory responses characterized by interleukin-6, interleukin-8, and cyclooxygenase-2 up-regulations, as observed by ELISA and Western blot. Strikingly, cell treatment with sphingosine 1-phosphate plus lipopolysaccharide resulted in the synergistic induction of cyclooxygenase-2, and intercellular adhesion molecule 1, as well as the secretion of prostaglandin E2, the soluble form of the intercellular adhesion molecule 1, and the pro-angiogenic factor vascular endothelial growth factor-A. Remarkably, the synergistic effect was significantly higher in aortic valve interstitial cells from stenotic than control valves, and was drastically lower in cells from pulmonary valves, which rarely undergo stenosis. siRNA and pharmacological analysis revealed the involvement of sphingosine 1-phosphate receptors 1/3 and Toll-like receptor-4, and downstream signaling through p38/MAPK, protein kinase C, and NF-kB. As regards pro-osteogenic pathways, sphingosine 1-phosphate induced calcium deposition and the expression of the calcification markers bone morphogenetic protein-2 and alkaline phosphatase, and enhanced the effect of lipopolysaccharide, an effect that was partially blocked by inhibition of sphingosine 1-phosphate receptors 3/2 signaling. In conclusion, the interplay between sphingosine 1-phosphate receptors and Toll-like receptor 4 signaling leads to a cooperative up-regulation of inflammatory, angiogenic, and osteogenic pathways in aortic valve interstitial cells that seems relevant to the pathogenesis of aortic stenosis and may allow the inception of new therapeutic approaches.This study was supported by grants from Spanish Ministry of Science, Plan Nacional de Salud y Farmacia SAF2006/08031 (co-funded by European FEDER-FSE2000/2006), and SAF2009/08692; Junta de Castilla y León (Regional Funding) BIO39/VA28/10 and GR230; Fundación Caja Burgos 2010; and the Instituto de Salud Carlos III (Spanish Ministry of Health): FIS PI11/00264, RECAVA network. IFP was a fellow from the FPI Program (Ministry of Science). AID was a Miguel Servet researcher (ISCIII).Peer Reviewe

Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells

[Background]: Aortic stenosis shares some ethiopathological features with atherosclerosis and increasing evidence links Toll-like receptors (TLRs) to atherogenesis. [Methods]: TLR-mediated inflammation and osteogenesis were investigated in human interstitial cells isolated from stenotic and non-stenotic aortic valves. TLR expression and signalling were evaluated by quantitative RT-PCR, flow cytometry, Western blot analysis, ELISA, and cytokine arrays. Osteogenesis was evaluated by measuring alkaline phosphatase activity. [Results]: Interstitial cells from control valves express most TLRs, being TLR4 the most abundant, whereas cells from stenotic valves express higher TLR4 and TLR2 and lower TLR5 and TLR9 transcript levels. When pro-inflammatory pathways were analyzed, we observed that TLR4, TLR2 and TLR3 ligands induced an early activation of NF-κB and p38 MAPK activation in cells from control and stenotic valves. Strikingly, when TLRs sensing viral patterns were studied, a sustained TLR3-mediated activation of NF-κB, a κB-independent induction of catalytically active cyclooxigenase (COX)-2 and ICAM-1 expression, and induction of expression of several chemokines were observed. TLR4, but not TLR2, engagement produced a similar but NF-κB-dependent effect. Moreover, TLR3 and TLR4 agonists induced alkaline phosphatase expression and activity. [Conclusions]: Exposure of aortic valve interstitial cells to viral and Gram-negative bacteria molecular patterns induces distinct and long-term TLR-mediated pro-inflammatory and pro-osteogenic responses that might be relevant to the pathogenesis of degenerative aortic stenosis.This study was supported by Red Recava from Instituto de Salud Carlos III, and grants from CNIC-Translational CNIC-09-2007, Junta de Castilla y León HUV03A08, BIO39/VA28/10 and GR230; grants from Plan Nacional de Salud y Farmacia SAF2006/08031 (co-funded by FEDER-FSE2000/ 2006), and SAF2009/08692. MIFP is a fellow from the FPI Program, Ministerio de Ciencia y Tecnología. A.I.D. is a researcher from the Miguel Servet Program, Instituto de Salud Carlos III.Peer Reviewe

Lipopolysaccharide and sphingosine-1-phosphate cooperate to induce inflammatory molecules and leukocyte adhesion in endothelial cells

Given that TLRs and sphingosine-1-phosphate (S1P) are key players in inflammation, we explored the potential interplay between TLRs and S1P in the adhesion/inflammatory pathways in primary human endothelial cells. As determined byWestern blot and flow cytometry, cells treated with LPS (a TLR4 ligand) and S1P showed significantly enhanced expression of adhesion molecules such as ICAM-1 and E-selectin compared with the effect of either ligand alone. Cell-type differences on E-selectin upregulation were observed. In contrast, no cooperation effect on ICAM-1 or E-selectin was observed with a TLR2/TLR1 ligand. Consistent with an increase in adhesion molecule expression, endothelial cell treatment with LPS plus S1P significantly enhanced adhesion of PBMCs under shear stress conditions compared with the effect of either ligand alone and exhibited comparable levels of cell adhesion strength as those after TNF-α treatment. Moreover, LPS and S1P cooperated to increase the expression of proinflammatory molecules such as IL-6, cyclooxygenase-2, and prostacyclin, as determined by ELISA and Western blot. The analysis of signaling pathways revealed the synergistic phosphorylation of ERK upon LPS plus S1P treatment of HUVEC and human aortic endothelial cells and cell-type differences on p38 and NF-κB activation. Moreover, pharmacological and small interfering RNA experiments disclosed the involvement of S1P1/3 and NF-κB in the cooperation effect and that cell origin determines the S1P receptors and signaling routes involved. Sphingosine kinase activity induction upon LPS plus S1P treatment suggests S1P- Sphingosine kinase axis involvement. In summary, LPS and S1P cooperate to increase proinflammatory molecules in endothelial cells and, in turn, to augment leukocyte adhesion, thus exacerbating S1P-mediated proadhesive/proinflammatory properties. Copyright © 2012 by The American Association of Immunologists, Inc.This work was supported by Plan Nacional de Salud y Farmacia Grants SAF2006/08031 (cofunded by the European Fund for Economic and Regional Development FEDER-FSE2000/2006) and SAF2009/08692; Junta de Castilla y León Grants BIO39/VA28/10 and GR230; and by Consejo Superior de Investigaciones Científicas (Proyecto Intramural 2007-20I015). I.F.-P. was a fellow from the “Formación del Personal Investigador” Program (Ministerio de Ciencia y Tecnología). A.I.D. was a researcher from the Miguel Servet Program, Instituto de Salud Carlos III.Peer Reviewe

Selective attenuation of Toll-like receptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate

[Aims]: Vascular inflammation is a major atherogenic factor and Toll-like receptor (TLR) 2 ligands, including bacterial and serum lipoproteins, seem to be involved in atherogenesis. On this basis, we analysed the effect of lipoproteins and different lipid components on TLR2-dependent signalling. [Methods and results]: In TLR2-transfected human embryonic kidney 293 cells and human monocytes, oxidized low-density lipoproteins inhibited nuclear factor (NF)-κB-driven transcriptional activity and chemokine gene expression in response to TLR2 ligands. Sphingosine 1-phosphate (S1P) and oxidized palmitoyl-arachidonoyl-phosphatidylcholine, but not lipoprotein-carried lysophospholipids, inhibited TLR2 activation. Silencing experiments in TLR2-transfected 293 cells showed that the S1P-mediated attenuation effect is mediated by S1P receptors type 1 and type 2. To address the physiological significance of these findings, additional experiments were performed in human peripheral blood monocytes and monocyte-derived macrophages. In both cell types, S1P selectively attenuated TLR2 signalling, as NF-κB and extracellular signal-regulated kinase activation, but not c-Jun amino terminal kinase phosphorylation, were inhibited by physiologically relevant concentrations of S1P. Moreover, the attenuation of TLR2 signalling was partially reverted by pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and Ras pathways. In addition, S1P inhibited the chemokine gene expression elicited by TLR2, but not by TLR4 ligands. [Conclusion]: These findings disclose a cross-talk mechanism between lipoprotein components and TLR in which engagement of S1P receptors exert selective attenuation of TLR2-dependent activation via PI3K and Ras signalling. A corollary to these data is that the negative cross-talk of S1P receptors and TLR2 signalling might be involved in the atheroprotective effects of S1P.Co-funded by FEDER-FSE 2000/2006, from Plan Nacional de Salud y Farmacia (SAF2004-01232, SAF2006/08031, Ministerio de Sanidad (FIS 03/1489), Red Recava and Red Brucelosis from Instituto de Salud Carlos III, and Fundación de Investigación Médica Mutua Madrileña (FMM). Instituto de Salud Carlos III supported post-doctoral contract fund to A.I.D.Peer Reviewe

Sphingosine 1-phosphate induces inflammation and osteogenesis and increases the activity of the LPS/TLR4 route in human aortic valve interstitial cells

Resumen del póster presentado al 22nd IUBMB & 37th FEBS Congress: "From Single Molecules to Systems Biology" celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012.-- et al.[Aims]: Lipid accumulation in the aortic valve is a characteristic of aortic stenosis and sphingosine 1-phosphate (S1P) plays a relevant functional role in the cardiovascular system. On this basis, we investigated the possible role of S1P on the induction of proinflammatory and pro-osteogenic changes in human interstitial cells from aortic valve (AVIC) and pulmonary valves (PVIC). [Methods and Results]: As regards pro-inflammatory routes, S1P up-regulated IL-6, IL-8, and cyclooxygenase (COX)-2 in AVICs, as determined by Western blot and ELISA experiments. AVIC exposure to a combination of S1P and bacterial lipopolysaccharide (LPS) a Toll-like receptor (TLR)-4 ligand known to promote pro-inflammatory and pro-osteogenic phenotypes in AVICs, resulted in the synergistic induction of COX-2, PGE2, and intercellular adhesion molecule (ICAM)-1. Strikingly, the cooperative effect was stronger in stenotic than in control cells, and more prominent in cells from the aortic valves than from the pulmonary valves, which rarely undergo stenosis. Pharmacological and gene silencing experiments revealed the involvement of several S1P receptors in the synergistic effect. As regards proosteogenic processes, S1P induced the expression/activity of the calcification marker alkaline phosphatase (ALP) in in vitro calcification experiments. In addition, S1P cooperated with LPS to enhance significantly ALP activity and the cooperative effect was partially blocked by S1P receptor antagonists. [Conclusions]: S1P induces pro-inflammatory and pro-osteogenic changes and increases the effect of TLR4 ligands in AVICs, what might be relevant for the pathogenesis of aortic stenosis and could open the way for new therapeutic approaches for this disease.Peer Reviewe