Judith Alonso

CCL20 Is Increased in Hypercholesterolemic Subjects and Is Upregulated By LDL in Vascular Smooth Muscle Cells Role of NF-κB

Objective—Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC). Methods and Results—In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5±3.2 versus 9.1±1.3 pg/mL; P<0.01). LDL induced the expression of CCL20 in VSMC in a dose- and time-dependent manner. Increased levels of CCL20 secreted by LDL-treated VSMC significantly induced human lymphocyte migration, an effect reduced by CCL20 silencing. The upregulation of CCL20 by LDL was dependent on the activation of kinase signaling pathways and NF-&kgr;B. By site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified a NF-&kgr;B site (−80/−71) in CCL20 promoter critical for LDL responsiveness. Lysophosphatidic acid mimicked the upregulation of CCL20 induced by LDL, and minimal oxidation of LDL increased the ability of LDL to induce CCL20 through a mechanism that involves lysophosphatidic acid receptors. CCL20 was overexpressed in atherosclerotic lesions from coronary artery patients, colocalizing with VSMC. CCL20 was detected in conditioned media from healthy human aorta and its levels were significantly higher in secretomes from carotid endarterectomy specimens. Conclusion—This study identifies CCL20 in atherosclerotic lesions and recognizes this chemokine as a mediator highly sensitive to the inflammatory response elicited by LDL.

Over-expression of Neuron-derived Orphan Receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury

We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

Giant Seebeck effect in Ge-doped SnSe.

Thermoelectric materials may contribute in the near future as new alternative sources of sustainable energy. Unprecedented thermoelectric properties in p-type SnSe single crystals have been recently reported, accompanied by extremely low thermal conductivity in polycrystalline samples. In order to enhance thermoelectric efficiency through proper tuning of this material we report a full structural characterization and evaluation of the thermoelectric properties of novel Ge-doped SnSe prepared by a straightforward arc-melting method, which yields nanostructured polycrystalline samples. Ge does not dope the system in the sense of donating carriers, yet the electrical properties show a semiconductor behavior with resistivity values higher than that of the parent compound, as a consequence of nanostructuration, whereas the Seebeck coefficient is higher and thermal conductivity lower, favorable to a better ZT figure of merit.

NOR-1 modulates the inflammatory response of vascular smooth muscle cells by preventing NFκB activation

Recent work has highlighted the role of NR4A receptors in atherosclerosis and inflammation. In vascular smooth muscle cell (VSMC) proliferation, however, NOR-1 (neuron-derived orphan receptor-1) exerts antagonistic effects to Nur77 and Nurr1. The aim of this study was to analyse the effect of NOR-1 in VSMC inflammatory response. We assessed the consequence of a gain-of-function of this receptor on the response of VSMC to inflammatory stimuli. In human VSMC, lentiviral over-expression of NOR-1 reduced lipopolysaccharide (LPS)-induced up-regulation of cytokines (IL-1β, IL-6 and IL-8) and chemokines (MCP-1 and CCL20). Similar effects were obtained in cells stimulated with TNFα or oxLDL. Conversely, siRNA-mediated NOR-1 inhibition significantly increased the expression of pro-inflammatory mediators. Interestingly, in the aortas from transgenic mice that over-express human NOR-1 in VSMC (TgNOR-1), the up-regulation of cytokine/chemokine by LPS was lower compared to wild-type littermates. Similar results were obtained in VSMC from transgenic animals. NOR-1 reduced the transcriptional activity of NFκB sensitive promoters (in transient transfections), and the binding of NFκB to its responsive element (in electrophoretic mobility shift assays). Furthermore, NOR-1 prevented the activation of NFκB pathway by decreasing IκBα phosphorylation/degradation and inhibiting the phosphorylation and subsequent translocation of p65 to the nucleus (assessed by Western blot and immunocytochemistry). These effects were associated with an attenuated phosphorylation of ERK1/2, p38 MAPK and Jun N-terminal kinase, pathways involved in the activation of NFκB. In mouse challenged with LPS, the activation of the NFκB signalling was also attenuated in the aorta from TgNOR-1. Our data support a role for NOR-1 as a negative modulator of the acute response elicited by pro-inflammatory stimuli in the vasculature.

NR4A receptors up-regulate the antiproteinase alpha-2 macroglobulin (A2M) and modulate MMP-2 and MMP-9 in vascular smooth muscle cells

Matrix metalloproteinases (MMPs) are associated with tissue remodelling and repair. In non-vascular tissues, NR4A receptors have been involved in the regulation of MMPs by transcriptional repression mechanisms. Here, we analyse alternative mechanisms involving NR4A receptors in the modulation of MMP activity in vascular smooth muscle cells (VSMC). Lentiviral overexpression of NR4A receptors (NOR-1, Nurr1 and Nur77) in human VSMC strongly decreased MMP-2 and MMP-9 activities (analysed by zymography and DQ-gelatin assays) and protein levels. NR4A receptors also down-regulated MMP-2 mRNA levels. Real-time PCR analysis evidenced that alpha-2-macroglobulin (A2M), but not other MMP inhibitors (TIMP-1 and TIMP-2) were up-regulated in NR4A-transduced cells. Interestingly, A2M was expressed in human vascular tissues including the smooth muscle media layer. While NR4A receptors increased A2M expression and secretion in VSMC, NR4A knockdown significantly reduced basal A2M expression in these cells. The direct transcriptional regulation of the human A2M promoter by NR4A receptors was characterised in luciferase reporter assays, electrophoretic mobility shift assays and by chromatin immunoprecipitation, identifying a NGFI-B response element (NBRE-71/-64) essential for the NR4A-mediated induction. The blockade of A2M partially prevented the reduction of MMPs activity observed in NR4A-transduced cells. Although mouse A2M promoter was unresponsive to NR4A receptors, vascular MMP expression was attenuated in transgenic mice over-expressing human NOR-1 in VSMC challenged with lipopolysaccharide. Our results show that the pan-proteinase inhibitor A2M is expressed in the vasculature and that NR4A receptors modulate VSMC MMP activity by several mechanisms including the up-regulation of A2M.

NOR-1/NR4A3 regulates the cellular inhibitor of apoptosis 2 (cIAP2) in vascular cells: role in the survival response to hypoxic stress.

Vascular cell survival is compromised under pathological conditions such as abdominal aortic aneurysm (AAA). We have previously shown that the nuclear receptor NOR-1 is involved in the survival response of vascular cells to hypoxia. Here, we identify the anti-apoptotic protein cIAP2 as a downstream effector of NOR-1. NOR-1 and cIAP2 were up-regulated in human AAA samples, colocalizing in vascular smooth muscle cells (VSMC). While NOR-1 silencing reduced cIAP2 expression in vascular cells, lentiviral over-expression of this receptor increased cIAP2 mRNA and protein levels. The transcriptional regulation of the human cIAP2 promoter was analyzed in cells over-expressing NOR-1 by luciferase reporter assays, electrophoretic mobility shift analysis and chromatin immunoprecipitation, identifying a NGFI-B site (NBRE-358/-351) essential for NOR-1 responsiveness. NOR-1 and cIAP2 were up-regulated by hypoxia and by a hypoxia mimetic showing a similar time-dependent pattern. Deletion and site-directed mutagenesis studies show that NOR-1 mediates the hypoxia-induced cIAP2 expression. While NOR-1 over-expression up-regulated cIAP2 and limited VSMC apoptosis induced by hypoxic stress, cIAP2 silencing partially prevented this NOR-1 pro-survival effect. These results indicate that cIAP2 is a target of NOR-1, and suggest that this anti-apoptotic protein is involved in the survival response to hypoxic stress mediated by NOR-1 in vascular cells.

The nuclear receptor NOR-1 regulates the small muscle protein, X-linked (SMPX) and myotube differentiation.

Recent works have highlighted the role of NOR-1 in both smooth and skeletal muscle, and have proposed this nuclear receptor as a nexus that coordinates muscle performance and metabolic capacity. However, no muscle specific genes regulated by NOR-1 have been identified so far. To identify NOR-1 target genes, we over-expressed NOR-1 in human vascular smooth muscle cells (VSMC). These cells subjected to sustained over-expression of supraphysiological levels of NOR-1 experienced marked phenotypic changes and up-regulated the skeletal muscle protein X-linked (SMPX), a protein typically expressed in striated muscle and associated to cell shape. By transcriptional studies and DNA-protein binding assays, we identified a non-consensus NBRE site in human SMPX promoter, critical for NOR-1 responsiveness. The expression of SMPX was higher in human skeletal muscle myoblasts (HSMM) than in human VSMC, and further increased in HSMM differentiated to myotubes. NOR-1 silencing prevented SMPX expression in HSMM, as well as their differentiation to myotubes, but the up-regulation of SMPX was dispensable for HSMM differentiation. Our results indicate that NOR-1 regulate SMPX in human muscle cells and acts as a muscle regulatory factor, but further studies are required to unravel its role in muscle differentiation and hypertrophy.

Enhanced figure of merit in nanostructured (Bi,Sb) 2 Te 3 with optimized composition, prepared by a straightforward arc-melting procedure

Sb-doped Bi2Te3 is known since the 1950s as the best thermoelectric material for near-room temperature operation. Improvements in material performance are expected from nanostructuring procedures. We present a straightforward and fast method to synthesize already nanostructured pellets that show an enhanced ZT due to a remarkably low thermal conductivity and unusually high Seebeck coefficient for a nominal composition optimized for arc-melting: Bi0.35Sb1.65Te3. We provide a detailed structural analysis of the Bi2−xSbxTe3 series (0 ≤ x ≤ 2) based on neutron powder diffraction as a function of composition and temperature that reveals the important role played by atomic vibrations. Arc-melting produces layered platelets with less than 50 nm-thick sheets. The low thermal conductivity is attributed to the phonon scattering at the grain boundaries of the nanosheets. This is a fast and cost-effective production method of highly efficient thermoelectric materials.

Extra-low thermal conductivity in unfilled CoSb3-δ skutterudite synthesized under high-pressure conditions

Thermoelectric CoSb3-δ skutterudite was synthesized and sintered in one step under high-pressure conditions at 3.5 GPa in a piston-cylinder hydrostatic press. Structural analysis carried out from synchrotron x-ray powder diffraction data reveals a significant Sb deficiency in this material. The introduction of point defects in the form of Sb vacancies distributed at random in the structure leads to an impressive reduction (>50%) of the total thermal conductivity, κ, which is one of the main ingredients of good thermoelectric materials. This suggests phonon scattering effects originated in the Sb defects, which drives to a better improvement in κ than that achieved by the conventional strategy of filling the cages of the skutterudite structure with rare earths or other heavy cations. In parallel, changes in the electronic band structure caused by point variation of the stoichiometry produce an undesired increment in the electrical resistivity. Nevertheless, the low thermal conductivity combined with a high...

El receptor nuclear NOR-1 regula la activación de las células vasculares y el remodelado vascular en respuesta a estrés hemodinámico

INTRODUCTION Previous studies have shown that the loss of NOR-1 function modulates the activation of vascular smooth muscle cells (VSMC). In this study we use a mouse that over-expresses human NOR-1 in VSMC to analyze the effect of a gain of NOR-1 function on the activation of VSMC and in the hyperplasia of the intima induced by hemodynamic stress. METHODS To generate the transgenic animal the human NOR-1 cDNA was placed under the control of the SM22α promoter. The expression of NOR-1 was analyzed by real time PCR, Western blot, immunohistochemistry and immunocitochemistry, and NOR-1 functionality was evaluated by luciferase activity assays. The incorporation of tritiated thymidine was determined as a cell proliferation index. The left carotid artery was ligated, and cross-sections were subjected to morphometric and immunostaining analysis. RESULTS The transgenic mouse exhibited significant levels of human NOR-1 in aorta and carotid arteries. In aortic VSMC from transgenic mice an increase in the transcriptional activity of ciclin D2 was detected, as well as higher proliferative rates and increased levels of the marker Myh10. In these animals, carotid artery ligation induced a greater neointimal formation and a higher stenotic grade than in wild-type animals, in accordance with the labelling detected for Myh10 and phosphorylated Histone H3. CONCLUSIONS These results reinforce the role of NOR-1 in VSMC proliferation and in vascular remodelling, and allow us to propose this model as a useful tool to study the involvement of NOR-1 in vascular function and in vascular diseases such as atherosclerosis and restenosis.