Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17β-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17β-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity

Regionalization of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder.

BACKGROUND: Reciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue. RESULTS: As a first step, we performed morphological observations to understand how the extra-embryonic region of the egg cylinder forms from the blastocyst. Our analysis suggests a new model for the formation of this region involving cell rearrangements such as folding of the extra-embryonic ectoderm at the early egg cylinder stage. To trace visceral endoderm cells, we microinjected mRNAs encoding fluorescent proteins into single surface cells of the inner cell mass of the blastocyst and analysed the distribution of labelled cells at E5.0, E5.5 and E6.5. We found that at E5.0 the embryonic and extra-embryonic regions of the visceral endoderm do not correspond to distinct cellular compartments. Clusters of labelled cells may span the junction between the two regions even after the appearance of histological and molecular differences at E5.5. We show that in the embryonic region cell dispersion increases after the migration of the AVE. At this time, visceral endoderm cell clusters tend to become oriented parallel to the junction between the embryonic and extra-embryonic regions. Finally we investigated the origin of the AVE and demonstrated that this anterior signalling centre arises from more than a single precursor between E3.5 and E5.5. CONCLUSION: We propose a new model for the formation of the extra-embryonic region of the egg cylinder involving a folding of the extra-embryonic ectoderm. Our analyses of the pattern of labelled visceral endoderm cells indicate that distinct cell behaviour in the embryonic and extra-embryonic regions is most apparent upon AVE migration. We also demonstrate the polyclonal origin of the AVE. Taken together, these studies lead to further insights into the formation of the extra-embryonic tissues as they first develop after implantation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

A009 Importance of tweak-CD163 system in peripheral artery disease

IntroductionCD163 is a macrophage receptor of haptoglogin/ haemoglobin complexes responsible for clearance of hemogloin. It has been recently suggested to be a potential scavenger receptor for TWEAK (Tumor necrosis factor-like weak inducer of apoptosis). TWEAK levels were reported to be decreased in carotid atherosclerosis. Our hypothesis is that decreased circulating TWEAK could be paralleled by an increased presence of CD163-expressing macrophage in atherosclerotic plaques. Since peripheral artery disease (PAD) is an important manifestation of systemic atherosclerosis, we have assessed the levels of circulating TWEAK-CD163 in PAD.Methods and ResultsPatients with PAD (n=184) had lower TWEAK (169.2±8.3vs 211.9±15.4pg/mL; p<0.05) and higher sCD163 (408.1±14.5vs 317.4±8.4ng/mL; p<0.05) plasma concentration than age-matched controls (n=330). After stratification according to the severity of disease, we observed that TWEAK/sCD163 ratio was significantly decreased in those patients with higher degree of disease (0.39±0.06vs 0.66±0.08, p<0.05) relative to the other groups. Analysis of conditioned medium obtained from cultured human atherosclerotic femoral plaque samples (n=38) and healthy aortas (n=14) revealed that higher amount of sCD163 was released by the atherosclerotic tissue, whereas TWEAK presented the opposite trend.ConclusionsOur results suggest that CD163/TWEAK plasma ratio could be a potential biomarker of clinical peripheral artery disease. We can hypothesized that decreased levels of circulating TWEAK observed in atherosclerosis may be the result of a trapping by plaque macrophages through their CD163

Searching for biomarkers of aneurysmal disease

Comunicaciones a congreso

Simulating the Mammalian Blastocyst - Molecular and Mechanical Interactions Pattern the Embryo

Mammalian embryogenesis is a dynamic process involving gene expression and mechanical forces between proliferating cells. The exact nature of these interactions, which determine the lineage patterning of the trophectoderm and endoderm tissues occurring in a highly regulated manner at precise periods during the embryonic development, is an area of debate. We have developed a computational modeling framework for studying this process, by which the combined effects of mechanical and genetic interactions are analyzed within the context of proliferating cells. At a purely mechanical level, we demonstrate that the perpendicular alignment of the animal-vegetal (a-v) and embryonic-abembryonic (eb-ab) axes is a result of minimizing the total elastic conformational energy of the entire collection of cells, which are constrained by the zona pellucida. The coupling of gene expression with the mechanics of cell movement is important for formation of both the trophectoderm and the endoderm. In studying the formation of the trophectoderm, we contrast and compare quantitatively two hypotheses: (1) The position determines gene expression, and (2) the gene expression determines the position. Our model, which couples gene expression with mechanics, suggests that differential adhesion between different cell types is a critical determinant in the robust endoderm formation. In addition to differential adhesion, two different testable hypotheses emerge when considering endoderm formation: (1) A directional force acts on certain cells and moves them into forming the endoderm layer, which separates the blastocoel and the cells of the inner cell mass (ICM). In this case the blastocoel simply acts as a static boundary. (2) The blastocoel dynamically applies pressure upon the cells in contact with it, such that cell segregation in the presence of differential adhesion leads to the endoderm formation. To our knowledge, this is the first attempt to combine cell-based spatial mechanical simulations with genetic networks to explain mammalian embryogenesis. Such a framework provides the means to test hypotheses in a controlled in silico environment

Identification of novel biomarkers of abdominal aortic aneurysms by 2D-DIGE and MALDI-MS from AAA-thrombus-conditioned media

In the search for novel biomarkers, noncandidate-based proteomic strategies open up new opportunities to gain a deeper insight into disease processes regarding their molecular mechanisms, the risk factors involved, and the monitoring of disease progression. To carry out these complex analyses, the combined use of gel electrophoresis with mass spectrometry (MS) represents a powerful choice. In addition, the introduction of protein dye labeling has notably improved the reliability of differential expression studies by increasing the statistical significance of the protein candidates. Here, we describe a strategy where different layers (luminal/abluminal) from the intraluminal thrombus (ILT) of human abdominal aortic aneurysm (AAA) patients were incubated in protein-free medium. Then, the levels of the proteins released were compared by two-dimensional differential in-gel electrophoresis (2D-DIGE) and the proteins of interest identified by MS. We consider that the use of tissue-conditioned media could offer a substantial advantage in the analytical study of biological fluids, as they provide a source of proteins to be released to the bloodstream, which could serve as potential circulating biomarkers.This chapter has been supported by the EC, FAD project (FP-7, HEALTH F2-2008-200647), the Spanish MICIN (SAF2010/21852), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Redes RECAVA (RD06/0014/0035), EUS2008-03565, and Fundacion Pro CNIC.S

Identification of soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) as a possible biomarker of subclinical atherosclerosis

OBJECTIVES: Assessment of vascular risk in asymptomatic patients and the response to medical therapy is a major challenge for prevention of cardiovascular events. Our aim was to identify proteins differentially released by healthy versus atherosclerotic arterial walls, which could be found in plasma and serve as markers of atherosclerosis. METHODS AND RESULTS: We have analyzed supernatants obtained from cultured human carotid plaques and healthy arteries by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry ProteinChip System. Surface-enhanced laser-desorption/ionization analysis unveiled an 18.4-kDa peak released in lower amount by carotid plaques than normal endarteries. This protein was identified as soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK). To confirm that sTWEAK was the protein of interest, Western blot and enzyme-linked immunosorbent assay were performed. Both techniques confirmed that sTWEAK levels were decreased in carotid plaque supernatants. Subsequent measurement of sTWEAK in plasma showed a reduced concentration in subjects with carotid stenosis (N=30) compared with healthy subjects matched by sex and age (N=28) (P<0.001). Furthermore, in a test population of 106 asymptomatic subjects, we showed that sTWEAK concentrations negatively correlated with the carotid intima-media thickness (r=-0.4; P<0.001), an index of subclinical atherosclerosis. CONCLUSIONS: These results suggest that sTWEAK could be a potential biomarker of atherosclerosis

Origin of congenital coronary arterio-ventricular fistulae from anomalous epicardial and myocardial development.

Coronary Artery Fistulae (CAFs) are cardiac congenital anomalies consisting of an abnormal communication of a coronary artery with either a cardiac chamber or another cardiac vessel. In humans, these congenital anomalies can lead to complications such as myocardial hypertrophy, endocarditis, heart dilatation, and failure. Unfortunately, despite their clinical relevance, the aetiology of CAFs remains unknown. In this work, we have used two different species (mouse and avian embryos) to experimentally model CAFs morphogenesis. Both conditional Itga4 (alpha 4 integrin) epicardial deletion in mice and cryocauterisation of chick embryonic hearts disrupted epicardial development and ventricular wall growth, two essential events in coronary embryogenesis. Our results suggest that myocardial discontinuities in the embryonic ventricular wall promote the early contact of the endocardium with epicardial-derived coronary progenitors at the cardiac surface, leading to ventricular endocardial extrusion, precocious differentiation of coronary smooth muscle cells, and the formation of pouch-like aberrant coronary-like structures in direct connection with the ventricular lumen. The structure of these CAF-like anomalies was compared with histopathological data from a human CAF. Our results provide relevant information for the early diagnosis of these congenital anomalies and the molecular mechanisms that regulate their embryogenesis.The authors thank Dr. A. Rojas (CABIMER, Sevilla, Spain) and Prof. Thalia Papayannopoulou (University of Washington, WA, USA) for sharing with us the G2- Gata4-Cre and Itga4-floxed mouse lines, respectively. We also thank Vanessa Benhamo (Institut Imagine) for her expert support with HREM. Finally, we thank all members of “DeCA” laboratory (University of Málaga, Málaga, Spain), and the “Heart Morphogenesis” laboratory (Institut Imagine and Institut Pasteur, Paris, France) for their help and fruitful discussions on this paper. This work was supported by the Spanish Ministry of Science, R+D+i National Programme [grants RTI2018-095410-RBI00 and PID2021-122626-OB-I00], Spanish Ministry of Science-ISCIII [grant number RD16/0011/0030], and University of Málaga [grant number UMA18-FEDERJA-146] to [JMPP]; Consejería de Salud y Familias, Junta de Andalucía [grant number PIER-0084- 2019] to [JAGD]; University of Málaga [grant number I Plan Propio-UMA-A.4] to [ARV]; Spanish Ministry of Science, Innovation, and Universities (MCIU) (CIBER CV) [grant numbers PID2019-104776RB-I00 and CB16/11/00399] to [JLDLP].S

The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation

Initiation of the development of the anterior–posterior axis in the mouse embryo has been thought to take place only when the anterior visceral endoderm (AVE) emerges and starts its asymmetric migration. However, expression of Lefty1, a marker of the AVE, was recently found to initiate before embryo implantation. This finding has raised two important questions: are the cells that show such early, preimplantation expression of this AVE marker the real precursors of the AVE and, if so, how does this contribute to the establishment of the AVE? Here, we address both of these questions. First, we show that the expression of another AVE marker, Cer1, also commences before implantation and its expression becomes consolidated in the subset of ICM cells that comprise the primitive endoderm. Second, to determine whether the cells showing this early Cer1 expression are true precursors of the AVE, we set up conditions to trace these cells in time-lapse studies from early periimplantation stages until the AVE emerges and becomes asymmetrically displaced. We found that Cer1-expressing cells are asymmetrically located after implantation and, as the embryo grows, they become dispersed into two or three clusters. The expression of Cer1 in the proximal domain is progressively diminished, whilst it is reinforced in the distal–lateral domain. Our time-lapse studies demonstrate that this distal–lateral domain is incorporated into the AVE together with cells in which Cer1 expression begins only after implantation. Thus, the AVE is formed from both part of an ancestral population of Cerl-expressing cells and cells that acquire Cer1 expression later. Finally, we demonstrate that when the AVE shifts asymmetrically to establish the anterior pole, this occurs towards the region where the earlier postimplantation expression of Cer1 was strongest. Together, these results suggest that the orientation of the anterior–posterior axis is already anticipated before AVE migration

APM_GUI: analyzing particle movement on the cell membrane and determining confinement

<p>Abstract</p> <p>Background</p> <p>Single-particle tracking is a powerful tool for tracking individual particles with high precision. It provides useful information that allows the study of diffusion properties as well as the dynamics of movement. Changes in particle movement behavior, such as transitions between Brownian motion and temporary confinement, can reveal interesting biophysical interactions. Although useful applications exist to determine the paths of individual particles, only a few software implementations are available to analyze these data, and these implementations are generally not user-friendly and do not have a graphical interface,.</p> <p>Results</p> <p>Here, we present APM_GUI (Analyzing Particle Movement), which is a MatLab-implemented application with a Graphical User Interface. This user-friendly application detects confined movement considering non-random confinement when a particle remains in a region longer than a Brownian diffusant would remain. In addition, APM_GUI exports the results, which allows users to analyze this information using software that they are familiar with.</p> <p>Conclusions</p> <p>APM_GUI provides an open-source tool that quantifies diffusion coefficients and determines whether trajectories have non-random confinements. It also offers a simple and user-friendly tool that can be used by individuals without programming skills.</p