Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1^Cre) mouse. Embryonic Wt1^Cre;Alk3^fl/fl specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1^Cre;Alk3^fl/fl mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1^Cre;Alk3^fl/fl specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.     

Electroacupuncture inhibits apoptosis in annulus fibrosis cells through suppression of the mitochondria-dependent pathway in a rat model of cervical intervertebral disc degradation

The purpose of this study was to investigate whether treatment with electroacupuncture (EA) inhibited mitochondria-dependent apoptosis in annulus fibrosis (AF) cells in a rat model of cervical intervertebral disc degradation induced by unbalanced dynamic and static forces. Forty Sprague-Dawley rats were used in this study, of which 30 underwent surgery to induce cervical intervertebral disc degradation, 10 rats received EA at acupoints Dazhui (DU 14) and Shousanli (LI 10). TUNEL staining was measured to assess apoptosis in AF cells, immunohistochemistry was used to examine Bcl-2 and Bax expression, colorimetric assays were used to determine caspase 9 and caspase 3 activities and RT-PCR and western blotting were used to assess the mRNA and protein expression of Crk and ERK2. Treatment with EA reduced the number of AF-positive cells in TUNEL staining, increased Bcl-2-positive cells and decreased Bax-positive cells in immunohistochemical staining, significantly inhibited the activation of caspases-9 and −3, and enhanced the mRNA and protein expression of Crk and ERK2. Our data show that EA inhibits AF cell apoptosis via the mitochondria-dependent pathway and up-regulates Crk and ERK2 expression. These results suggest that treatment with may be a good alternative therapy for preventing cervical spondylosis.     

DDX6 localizes to nuage structures and the annulus of mammalian spermatogenic cells

The localization of DEAD (Asp-Glu-Ala-Asp) box helicase 6 (DDX6) in spermatogenic cells from the mouse, rat, and guinea pig was studied by immunofluorescence (IF) and immunoelectron microscopy (IEM). Spermatogenic cells from these species yielded similar DDX6 localization pattern. IF microscopy results showed that DDX6 localizes to both the nucleus and cytoplasm. In the cytoplasm of spermatogenic cells, diffuse cytosolic and discrete granular staining was observed, with the staining pattern changing during cell differentiation. IEM revealed that DDX6 localized to the five different types of nuage structures and non-nuage structures, including small granule aggregate and late spermatid annuli. Nuclear labeling was strongest in leptotene and zygotene spermatocytes and moderately strong in the nuclear pocket of late spermatids. DDX6 also localized to the surface of outer dense fibers, which comprise of flagella. The results show that DDX6 is present in nuage and non-nuage structures as well as nuclei, suggesting that DDX6 has diverse functions in spermatogenic cells.     

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells

Background

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.

Methods

Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.

Results

The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.

Conclusions

Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.     

Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton

Abstract The epicardium is embryologically formed by outgrowth of proepicardial cells over the naked heart tube. Epicardium-derived cells (EPDCs) migrate into the myocardium, contributing to myocardial architecture, valve development, and the coronary vasculature. Defective EPDC formation causes valve malformations, myocardial thinning, and coronary defects. In the atrioventricular (AV) valves and the fibrous heart skeleton isolating atrial from ventricular myocardium, EPDCs colocalize with periostin, a matrix molecule involved in remodeling. We investigated whether proepicardial outgrowth inhibition affected periostin expression and how this related to development of the AV valves and fibrous heart skeleton.
Periostin expression by epicardium and EPDCs was confirmed in vitro in primary cultures of human and quail EPDCs. Disturbing EPDC formation in quail embryos reduced periostin expression in the endocardial cushions and AV junction. Disturbed fibrous tissue development resulted in AV myocardial connections reflected by preexcitation electrocardiographic patterns.
We conclude that EPDCs are local producers of periostin. Disturbance of EPDC formation results in decreased cardiac periostin levels and hampers the development of fibrous tissue in AV junction and the developing AV valves. The resulting cardiac anomalies might link to Wolff–Parkinson White syndrome with persistent AV myocardial connections.     

Graded mesoderm assembly governs cell fate and morphogenesis of the early mammalian heart

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Quantifying the contribution of actin networks to the elastic strength of fibroblasts

The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., K?s, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767-784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of approximately 220 Pa for normal fibroblasts and approximately 185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural response.     

Genetic pathways to mammalian heart development: Recent progress from manipulation of the mouse genome

Mammalian heart development requires multiple genetic networks, only some of which are becoming known in all their complexity. Substantial new information has become available thanks to an expanding toolkit that offers more and more mouse gene manipulation options, and that is taking the mouse closer to more powerful invertebrate genetic models. We review examples of recent data with a cardiac-lineage-based view of heart development, especially outflow tract and right ventricle. The medical significance of these studies is not only relevant to congenital heart disease, but also to the biology of cardiac cell regeneration.     

CX3CL1/fractalkine shedding by human hepatic stellate cells: contribution to chronic inflammation in the liver

Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-γ induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro . We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.     

Activation propetties of the inward-recifying potassium channel on mammalian heart cells

Summary The early phase of activation of the inward-rectifying potassium channel is studied on single cells from guinea-pig heart. The current is quasi-instantaneous when it is outward, but activates with time when it is inward. This relaxation is exponential and its time-constant decreases with hyperpolarization. TheI/V curve reflects a strong inward rectification and has a negative slope conductance on depolarization. Similar results were recorded in the absence of sodium, calcium, chloride ions and in isotonic potassium. Cesium slows down the phase of activation, and eventually appears to block the channels by suppression of the activation. Barium, conversely, does not affect the activation, but promotes an inactivation of this current, which blocks it. These results are independent on the cells' dissociation method. They suggest that this current is the inward rectifier, calledI _K1 on heart. Its activation curve suggests that the inward and outward currents are flowing through the same channels. The inward rectifier is time-and voltage-dependent on heart as on other tissues. The effects of cesium and barium are also similar. The importance of its negative slope conductance is discussed.     

Traffic control: p120-catenin acts as a gatekeeper to control the fate of classical cadherins in mammalian cells

Proteins of the p120 family have been implicated in the regulation of cadherin-based cell adhesion, but their relative importance in this process and their mechanism of action have remained less clear. Three papers in this issue suggest that p120 plays a key role in maintaining normal levels of cadherin in mammalian cells, and that it may do so by regulating cadherin trafficking (Chen et al., 2003; Davis et al., 2003; Xiao et al., 2003).     

Chronic exposure to EGF affects trafficking and function of ENaC channel in cystic fibrosis cells

Using the whole-cell patch-clamp technique, we identified an amiloride (AMI)-sensitive Na(+) current in cystic fibrosis cells, JME/CF15, growing in standard medium. The reversal potential of this current depended on Na(+) concentrations and the cation selectivity was much higher for Na(+) than for K(+), indicating that the current is through ENaC channels. In contrast, cells from EGF-containing medium lacked AMI-sensitive Na(+) currents. In permeabilized cells growing in EGF-containing medium, alphaENaC was mainly detected in a perinuclear region, while in cells from standard medium it was distributed over the cell body. Western-blot analysis showed that in standard medium cells expressed fast-migrating EndoH-insensitive and slow-migrating EndoH-sensitive alphaENaC fractions, while in cells growing in the presence of EGF, alphaENaC was only detected as the fast-migrating EndoH-insensitive fraction. Long-term incubation of cells with EGF resulted in an increased basal Ca(2+) level, [Ca(2+)](i). A similar increase of [Ca(2+)](i) was also observed in the presence of 2muM thapsigargin, resulting in inhibition of ENaC function. Thus, in JME/CF15 cells inhibition of the ENaC function by chronic incubation with EGF is a Ca(2+)-mediated process that affects trafficking and surface expression of ENaC channels.     

大鼠肺纤维化形成中肺内肥大细胞结缔组织生长因子的表达

目的：观察博莱霉素（BLM）诱导肺纤维化形成中肺肥大细胞（MCs）是否表达结缔组织生长因子（CTGF）。方法：32只雄性SD大鼠,随机分为博莱霉素（BLM）组和对照（Control）组（n=16）。BLM组为气管内一次性滴注BLM（5mg／ks）;Control组为气管内滴注与BLM等容量的生理盐水（NS）。各组分别在气管滴注后第14天和第28天处死大鼠,取肺组织样本。用氯胺-T法检测肺组织羟脯氨酸含量以判断肺纤维化程度;用甲苯胺蓝染色显示肺组织切片中的MCs;免疫组化染色显示肺CTGF的表达和分布。结果：①与对照大鼠比,气管内滴注BLM后第28天大鼠的肺羟脯氨酸含量明显增高（P〈0．01）。②与对照大鼠比,气管内滴注BLM后第14天和第28天大鼠肺内MCs数明显增多（均P〈0．01）,肺内CTGF表达上调（均P〈0．01）。③对照大鼠肺内未见CTGF免疫阳性的MCs;而气管内滴注BLM后第14天和第28天大鼠肺内病灶区中有CTGF免疫阳性的MCs。结论：肺纤维化形成中肺MCs表达CTGF,这可能是MCs促进肺纤维化的作用机制之一。     

A bacterial cell to cell signal in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.     

Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia

Classic tissue recombination and in vitro lineage tracing studies suggest that condensed metanephric mesenchyme (MM) gives rise to nephronic epithelium of the adult kidney. However, these studies do not distinguish between cap mesenchyme and pre-tubular aggregates comprising the condensed MM, nor do they establish whether these cells have self-renewing capacity. To address these questions, we generated Cited1-CreER^T2 BAC transgenic mice, which express tamoxifen-regulated Cre recombinase exclusively in the cap mesenchyme. Fate mapping was performed by crossing these mice with the Rosa26R^LacZ reporter line and evaluating the location and cellular characteristics of LacZ positive cells at different time points following tamoxifen injection. These studies confirmed expected results from previous in vitro analysis of MM cell fate, and provide in vivo evidence that the cap mesenchyme does not contribute to collecting duct epithelium in the adult. Furthermore, by exploiting the temporally regulated Cre recombinase, these studies show that nephronic epithelium arising at different stages of nephrogenesis has distinct spatial distribution in the adult kidney, and demonstrate for the first time that the cap mesenchyme includes a population of self-renewing epithelial progenitor cells.     

Proteome analysis reveals adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell, membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pattern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to PAO revealed differential expression of virulence factors under non-inducing conditions. The membrane proteome of strain C showed modulation of the expression of porins involved in nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed retention of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether, our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat during the course of chronic colonization.     

Successful targeting of the mouse cystic fibrosis transmembrane conductance regulator gene in embryonal stem cells

We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.