Inhibition of α4-integrin stimulates epicardial–mesenchymal transformation and alters migration and cell fate of epicardially derived mesenchyme

AbstractEpithelial–mesenchymal transformation of the embryonic epicardium produces the subepicardial mesenchyme that is essential for normal coronary vascular development. Gene targeting experiments in mice have demonstrated an essential role for α4-integrin in normal epicardial development, but the precise cellular consequences of α4-integrin loss remain uncertain. To better understand the function of α4-integrin in epicardial development, we constructed a replication-incompetent adenovirus (AdlacZα4AS) that expresses antisense chicken α4-integrin as the 3′ untranslated region of a lacZ reporter gene. This construct effectively labeled cells while greatly reducing levels of α4-integrin mRNA and protein. In quail chick chimeras, transplanted epicardial cells infected with AdlacZα4AS adhered to the heart and were incorporated into the epicardium, but 4 days after grafting, were largely absent from the epicardial epithelium, recapitulating the defect in α4-null mice. This did not result from epicardial cell apoptosis or anomalous migration of epicardial cells to extracardiac sites. Rather, AdlacZα4AS-infected epicardial cells were particularly invasive, being three to four times more likely to migrate to the interstitium of the myocardium than AdlacZ-infected epicardial cells. Accelerated epicardial–mesenchymal transformation and migration of α4-negative epicardium was observed in an organ culture system that does not require prior culture of epicardial cells. Remarkably, AdlacZα4AS infection also prevented targeting of epicardially derived mesenchyme to the media of developing coronary vasculature in the myocardial interstitium. This study provides evidence that epicardial α4-integrin normally restrains epicardial–mesenchymal transformation, invasion, and migration and is essential for correct targeting of epicardially derived mesenchyme to the developing coronary vasculature

Expression and Function of Ccbe1 in the Chick Early Cardiogenic Regions Are Required for Correct Heart Development

During the course of a differential screen to identify transcripts specific for chick heart/hemangioblast precursor cells, we have identified Ccbe1 (Collagen and calcium-binding EGF-like domain 1). While the importance of Ccbe1 for the development of the lymphatic system is now well demonstrated, its role in cardiac formation remained unknown. Here we show by whole-mount in situ hybridization analysis that cCcbe1 mRNA is initially detected in early cardiac progenitors of the two bilateral cardiogenic fields (HH4), and at later stages on the second heart field (HH9-18). Furthermore, cCcbe1 is expressed in multipotent and highly proliferative cardiac progenitors. We characterized the role of cCcbe1 during early cardiogenesis by performing functional studies. Upon morpholino-induced cCcbe1 knockdown, the chick embryos displayed heart malformations, which include aberrant fusion of the heart fields, leading to incomplete terminal differentiation of the cardiomyocytes. cCcbe1 overexpression also resulted in severe heart defects, including cardia bifida. Altogether, our data demonstrate that although cardiac progenitors cells are specified in cCcbe1 morphants, the migration and proliferation of cardiac precursors cells are impaired, suggesting that cCcbe1 is a key gene during early heart development.FCT [SFRH/BD/65628/2009, SFRH/BPD/86497/2012, SFRH/BPD/41081/2007]; F.C.T.B.I. fellowship [PTDC/SAU-BID/114902/ 2009]; FCT; Institute for Biotechnology Bioengineering (Centro Biomedicina Molecular e Celular (IBB/CBME), Laboratorio Associado (LA) in the frame of Project [PestOE/EQB/LA0023/2013]info:eu-repo/semantics/publishedVersio

The Peritoneum Is Both a Source and Target of TGF-β in Women with Endometriosis

Transforming growth factor-β (TGF-β) is believed to play a major role in the aetiology of peritoneal endometriosis. We aimed to determine if the peritoneum is a source of TGF-β and if peritoneal TGF-β expression, reception or target genes are altered in women with endometriosis. Peritoneal fluid, peritoneal bushings and peritoneal biopsies were collected from women with and without endometriosis. TGF-β1, 2 and 3 protein concentrations were measured in the peritoneal fluid. TGF-β1 was measured in mesothelial cell conditioned media. Control peritoneum and peritoneum prone to endometriosis (within Pouch of Douglas) from women without disease (n = 16) and peritoneum distal and adjacent to endometriosis lesions in women with endometriosis (n = 15) and were analysed for TGF-β expression, reception and signalling by immunohistochemistry, qRT-PCR and a TGF-β signalling PCR array. TGF-β1 was increased in the peritoneal fluid of women with endometriosis compared to those without disease (P<0.05) and peritoneal mesothelial cells secrete TGF-β1 in-vitro. In women with endometriosis, peritoneum from sites adjacent to endometriosis lesions expressed higher levels of TGFB1 mRNA when compared to distal sites (P<0.05). The TGF-β-stimulated Smad 2/3 signalling pathway was active in the peritoneum and there were significant increases (P<0.05) in expression of genes associated with tumorigenesis (MAPK8, CDC6), epithelial-mesenchymal transition (NOTCH1), angiogenesis (ID1, ID3) and neurogenesis (CREB1) in the peritoneum of women with endometriosis. In conclusion, the peritoneum, and in particular, the peritoneal mesothelium, is a source of TGF-β1 and this is enhanced around endometriosis lesions. The expression of TGF-β-regulated genes is altered in the peritoneum of women with endometriosis and this may promote an environment favorable to lesion formation

On the Origin of Cells

While non-blood cell lineage has been studied for decades by developmental biologists, only recently has it been considered in disease. This is partly due to a lack of suitable reagents in experimental models, but it is also the result of a failure to understand the ability of cells to move or differentiate in pathological environments. This Editorial gives a quick overview of the Special Issue “Cell Fate Decisions in Development and Disease” and underscores the importance of understanding the mechanisms of cell fate determination and lineage

Genetic Analysis of theDrosophilaβ3-Tubulin Gene Demonstrates That the Microtubule Cytoskeleton in the Cells of the Visceral Mesoderm Is Required for Morphogenesis of the Midgut Endoderm

AbstractWe have investigated the cellular basis for lethality of mutant alleles of theDrosophila melanogasterβ3-tubulin gene,βTub60D.Lethal β3 mutations can be grouped into two classes: the most severe mutations (Class I alleles) cause death during the first larval instar, while weaker alleles (Class II) cause death in later larval stages or in early pupal development. Since β3 is not expressed during larval development, lethality of the Class I mutations must reflect essential functions of β3 in embryogenesis. β3-tubulin is zygotically expressed during midembryogenesis in the developing mesoderm, and the major site of β3 accumulation is in the developing muscles during myogenesis. We show that the embryonic pattern of β3 expression, including accumulation in the developing musculature, is conserved in otherDrosophilaspecies. However, we found that loss of β3 function does not cause discernible defects in either the ultrastructure or function of the larval muscle. Thus β3-tubulin is dispensable in its highest site of accumulation. Rather, the essential site of function of β3 in embryos is in cells of the visceral mesoderm. Lethality of Class I alleles is caused by defects in midgut morphogenesis and failure of gut function. Although the folding pattern is irregular and the gut is smaller than normal, a complete folded gut forms in mutant larvae, and the visceral muscle functions normally to move food through the gut. However, mutant larvae cannot absorb nutrients across the gut wall. Thus loss of β3 function in the mesoderm results in defects in the underlying endodermally derived layer of the gut. Our data provide an assay for cellular interactions between mesoderm and endodermal tissues and reveal a role for the microtubule cytoskeleton of the visceral mesodermal cells in differentiation of the endodermal cell layer of the larval gut

Testosterone Modifies Alterations to Detrusor Muscle after Partial Bladder Outlet Obstruction in Juvenile Mice

Lower urinary tract symptoms secondary to posterior urethral valves (PUV) arise in boys during adolescence. The reasons for this have previously been attributed to increased urine output as boys experience increased growth. Additionally, there are few choices for clinicians to effectively treat these complications. We formed the new hypothesis that increased androgen levels at this time of childhood development could play a role at the cellular level in obstructed bladders. To test this hypothesis, we investigated the role of testosterone on bladder detrusor muscle following injury from partial bladder outlet obstruction (PO) in mice. A PO model was surgically created in juvenile male mice. A group of mice were castrated by bilateral orchiectomy at time of obstruction (CPO). Testosterone cypionate was administered to a group of castrated, obstructed mice (CPOT). Bladder function was assessed by voiding stain on paper (VSOP). Bladders were analyzed at 7 and 28 days by weight and histology. Detrusor collagen to smooth muscle ratio (Col/SM) was calculated using Masson’s trichrome stain. All obstructed groups had lower max voided volumes (MVV) than sham mice at 1 day. Hormonally intact mice (PO) continued to have lower MVV at 7 and 28 days while CPO mice improved to sham levels at both time points. In accordance, PO mice had higher bladder-to-body weight ratios than CPO and sham mice demonstrating greater bladder hypertrophy. Histologically, Col/SM was lower in sham and CPO mice. When testosterone was restored in CPOT mice, MVV remained low at 7 and 28 days compared to CPO and bladder-to-body weight ratios were also greater than CPO. Histologic changes were also seen in CPOT mice with higher Col/SM than sham and CPO mice. In conclusion, our findings support a role for testosterone in the fibrotic changes that occur after obstruction in male mice. This suggests that while other changes may occur in adolescent boys that cause complication in boys with PUV, the bladder itself responds to testosterone at the cellular level. This opens the door to a new understanding of pathways that influence bladder fibrosis and could lead to novel approaches to treat boys with PUV

Soluble VCAM-1 Alters Lipid Phosphatase Activity in Epicardial Mesothelial Cells: Implications for Lipid Signaling During Epicardial Formation

Epicardial formation involves the attachment of proepicardial (PE) cells to the heart and the superficial migration of mesothelial cells over the surface of the heart. Superficial migration has long been known to involve the interaction of integrins expressed by the epicardium and their ligands expressed by the myocardium; however, little is understood about signals that maintain the mesothelium as it migrates. One signaling pathway known to regulate junctional contacts in epithelia is the PI3K/Akt signaling pathway and this pathway can be modified by integrins. Here, we tested the hypothesis that the myocardially expressed, integrin ligand VCAM-1 modulates the activity of the PI3K/Akt signaling pathway by activating the lipid phosphatase activity of PTEN. We found that epicardial cells stimulated with a soluble form of VCAM-1 (sVCAM-1) reorganized PTEN from the cytoplasm to the membrane and nucleus and activated PTEN’s lipid phosphatase activity. Chick embryonic epicardial mesothelial cells (EMCs) expressing a shRNA to PTEN increased invasion in collagen gels, but only after stimulation by TGFβ3, indicating that loss of PTEN is not sufficient to induce invasion. Expression of an activated form of PTEN was capable of blocking degradation of junctional complexes by TGFβ3. This suggested that PTEN plays a role in maintaining the mesothelial state of epicardium and not in EMT. We tested if altering PTEN activity could affect coronary vessel development and observed that embryonic chick hearts infected with a virus expressing activated human PTEN had fewer coronary vessels. Our data support a role for VCAM-1 in mediating critical steps in epicardial development through PTEN in epicardial cells