Cardiac Explant-Derived Cells Are Regulated by Notch-Modulated Mesenchymal Transition

Progenitor cell therapy is emerging as a novel treatment for heart failure. However the molecular mechanisms regulating the generation of cardiac progenitor cells is not fully understood. We hypothesized that cardiac progenitor cells are generated from cardiac explant via a process similar to epithelial to mesenchymal transition (EMT).Explant-derived cells were generated from partially digested atrial tissue. After 21 days in culture, c-Kit+ cells were isolated from cell outgrowth. The majority of explant-originated c-Kit+ cells expressed the epicardial marker Wt1. Cardiac cell outgrowth exhibits a temporal up-regulation of EMT-markers. Notch stimulation augmented, while Notch inhibition suppressed, mesenchymal transition in both c-Kit+ and c-Kit- cells. In c-Kit+ cells, Notch stimulation reduced, while Notch inhibition up-regulated pluripotency marker expressions such as Nanog and Sox2. Notch induction was associated with degradation of β-catenin in c-Kit- cells. In contrast, Notch inhibition resulted in β-catenin accumulation, acquisition of epitheloid morphology, and up-regulation of Wnt target genes in c-Kit- cells.Our study suggests that Notch-mediated reversible EMT process is a mechanism that regulates explant-derived c-Kit+ and c-Kit- cells

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Mab21l2 Is Essential for Embryonic Heart and Liver Development

During mouse embryogenesis, proper formation of the heart and liver is especially important and is crucial for embryonic viability. In this study, we showed that Mab21l2 was expressed in the trabecular and compact myocardium, and that deletion of Mab21l2 resulted in a reduction of the trabecular myocardium and thinning of the compact myocardium. Mab21l2-deficient embryonic hearts had decreased expression of genes that regulate cell proliferation and apoptosis of cardiomyocytes. These results show that Mab21l2 functions during heart development by regulating the expression of such genes. Mab21l2 was also expressed in the septum transversum mesenchyme (STM). Epicardial progenitor cells are localized to the anterior surface of the STM (proepicardium), and proepicardial cells migrate onto the surface of the heart and form the epicardium, which plays an important role in heart development. The rest of the STM is essential for the growth and survival of hepatoblasts, which are bipotential progenitors for hepatocytes and cholangiocytes. Proepicardial cells in Mab21l2-deficient embryos had defects in cell proliferation, which led to a small proepicardium, in which α4 integrin expression, which is essential for the migration of proepicardial cells, was down-regulated, suggesting that defects occurred in its migration. In Mab21l2-deficient embryos, epicardial formation was defective, suggesting that Mab21l2 plays important roles in epicardial formation through the regulation of the cell proliferation of proepicardial cells and the migratory process of proepicardial cells. Mab21l2-deficient embryos also exhibited hypoplasia of the STM surrounding hepatoblasts and decreased hepatoblast proliferation with a resultant loss of defective morphogenesis of the liver. These findings demonstrate that Mab21l2 plays a crucial role in both heart and liver development through STM formation

In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFβ-signaling and WT1

Adult epicardial cells are required for endogenous cardiac repair. After myocardial injury, they are reactivated, undergo epithelial-to-mesenchymal transformation (EMT) and migrate into the injured myocardium where they generate various cell types, including coronary smooth muscle cells and cardiac interstitial fibroblasts, which contribute to cardiac repair. To understand what drives epicardial EMT, we used an in vitro model for human adult epicardial cells. These cells have an epithelium-like morphology and markedly express the cell surface marker vascular cell adhesion marker (VCAM-1). In culture, epicardial cells spontaneously undergo EMT after which the spindle-shaped cells now express endoglin. Both epicardial cells before and after EMT express the epicardial marker, Wilms tumor 1 (WT1). Adding transforming growth factor beta (TGFβ) induces loss of epithelial character and initiates the onset of mesenchymal differentiation in human adult epicardial cells. In this study, we show that TGFβ-induced EMT is dependent on type-1 TGFβ receptor activity and can be inhibited by soluble VCAM-1. We also show that epicardial-specific knockdown of Wilms tumor-1 (WT1) induces the process of EMT in human adult epicardial cells, through transcriptional regulation of platelet-derived growth factor receptor alpha (Pdgfrα), Snai1 and VCAM-1. These data provide new insights into the process of EMT in human adult epicardial cells, which might provide opportunities to develop new strategies for endogenous cell-based cardiac repair

Acute Multiple Organ Failure in Adult Mice Deleted for the Developmental Regulator Wt1

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal–epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source

International audience: Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes

ERYTHROPOIETIN FOR THE TREATMENT OF SUBARACHNOID HEMORRAGE: A FEASIBLE INGREDIENT FOR A SUCCESS MEDICAL RECIPE

Subaracnhoid hemorrage (SAH) following aneurysm bleeding accounts for 6% to 8% of all cerebrovascular accidents. Althoug an aneurysm can be effectively managed by surgery or endovascular therapy, delayed cerebral ischemia is diagnosed in a high percentage of patients resulting in significant morbility and mortality. Cerebral vasospasm occurs in more than half of all patients after aneurysm rupture and is recognized as the leading cause of delayed cerebral ischemia after SAH. Hemodynamic strategies and endovascular procedures may be considered fo the treatment of cerebral vasospasm. In recent years, the mechanism contributing to the development of vasospasm, abnormal reactivity of cerebral arteries and cerebral ischemia following SAH, have been intensively investigated. A number of pathological processes have been identified in the pathogenesis of vasospasm including endothelial injury, smooth muscle cell contraction from spasmogenic substances produced by the subarachnoid blood clots, changes in vascular responsiveness and inflammatory response of the vascular endothelium. to date, the current therapeutic interventions remain ineffective being limited to the manipulation os systemic blood pressure, variation of blood volume and viscosity, and control of arterial carbon dioxide tension. In this scenario, the hormone erythropoietin (EPO), has been found to exert neuroprotective action during experimental SAH when its recombinant form (rHuEPO) is systematically administered. However, recent translation of experimental data into clinical trials has suggested an unclear role of recombinant human EPO in the setting of SAH. In this context, the aim of the recurrent review is to present current evidence on the potential role of EPO in cerebrovascular dysfunction following aneurysmal subarachnoid hemorrage

Linear domain interactome and biological function of anterior gradient 2

The Anterior Gradient 2 (AGR2) protein has been implicated in a variety of biological systems linked to cancer and metastasis, tamoxifen-induced drug resistance, pro-inflammatory diseases like IBD and asthma, and limb regeneration. The molecular mechanisms by which AGR2 mediates these various phenotypes in disease progression in both cancer and IBD are poorly understood, as is the biological function(s) of AGR2 under non-disease conditions. Here, we use a combination of biochemical techniques, organ culture, cell biology and mouse genetics to investigate the biological significance of AGR2 both in cell lines and in vivo. We present data based on phage-peptide inter-actomics screens suggesting a role for AGR2 in mediating the maturation and trafficking of a class of membrane and secretory proteins, and investigate a putative interaction between AGR2 and one member of this class of proteins. We also describe the construction of a universal vector for use in making a variety of transgenic animals, and then present data showing its use as a promoter reporter, and attempt to investigate the temporal and spatial expression of AGR2 in the developing and adult mouse. Further, we present data describing the localisation pattern of AGR2 in the developing murine kidney using a combination of organ culture and antibody staining, and suggest a role for AGR2 in the developing kidney based on this data that is in agreement with a chaperone function for membrane and secretory proteins. Together, these data suggest that AGR2 has an intrinsic consensus docking site for a subset of its client proteins, that AGR2 plays a role in protein maturation in ciliated cell types, and provides a novel biological model to dissect the role of AGR2 in ER-trafficking

PAI-1 and functional blockade of SNAI1 in breast cancer cell migration

12 pages, 5 figures.-- PMID: 19055748 [PubMed].-- et al.[Introduction]: Snail, a family of transcriptional repressors implicated in cell movement, has been correlated with tumour invasion. The Plasminogen Activation (PA) system, including urokinase plasminogen activator (uPA), its receptor and its inhibitor, plasminogen activator inhibitor type 1(PAI-1), also plays a key role in cancer invasion and metastasis, either through proteolytic degradation or by non-proteolytic modulation of cell adhesion and migration. Thus, Snail and the PA system are both over-expressed in cancer and influence this process. In this study we aimed to determine if the activity of SNAI1 (a member of the Snail family) is correlated with expression of the PA system components and how this correlation can influence tumoural cell migration.[Methods]: We compared the invasive breast cancer cell-line MDA-MB-231 expressing SNAI1 (MDA-mock) with its derived clone expressing a dominant-negative form of SNAI1 (SNAI1-DN). Expression of PA system mRNAs was analysed by cDNA microarrays and real-time quantitative RT-PCR. Wound healing assays were used to determine cell migration. PAI-1 distribution was assessed by immunostaining.[Results]: We demonstrated by both cDNA microarrays and realtime quantitative RT-PCR that the functional blockade of SNAI1 induces a significant decrease of PAI-1 and uPA transcripts. After performing an in vitro wound-healing assay, we observed that SNAI1-DN cells migrate more slowly than MDA-mock cells and in a more collective manner. The blockade of SNAI1 activity resulted in the redistribution of PAI-1 in SNAI1-DN cells decorating large lamellipodia, which are commonly found structures in these cells.[Conclusions]: In the absence of functional SNAI1, the expression of PAI-1 transcripts is decreased, although the protein is redistributed at the leading edge of migrating cells in a manner comparable with that seen in normal epithelial cells.This work was supported by the CNRS ACI Program "Complexité du vivant" (grant # 050009DR11) and by the Evry Genopole grant "Aide à l'acquisition d'équipement semi-lourd" 2007 and 2008.Peer reviewe