scholarly journals The Latent Transforming Growth Factor-β–binding Protein-1 Promotes In Vitro Differentiation of Embryonic Stem Cells into Endothelium

2000 ◽  
Vol 11 (12) ◽  
pp. 4295-4308 ◽  
Author(s):  
Anna Gualandris ◽  
Justin P. Annes ◽  
Marco Arese ◽  
Irene Noguera ◽  
Vladimir Jurukovski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transforming Growth Factor Β ◽  
Cell Types ◽  
Embryoid Bodies

The latent transforming growth factor-β–binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-β–masking protein, LTBP-1 is involved both in the sequestration of latent TGF-β in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-β or the latency associated peptide, which neutralize TGF-β. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-β1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-β–dependent mechanism.

scholarly journals CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor β transcriptional responses in endothelial cells

1998 ◽  
Vol 95 (16) ◽  
pp. 9506-9511 ◽  
Author(s):  
James N. Topper ◽  
Maria R. DiChiara ◽  
Jonathan D. Brown ◽  
Amy J. Williams ◽  
Dean Falb ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Transforming Growth Factor Β ◽  
Cell Types ◽  
Creb Binding Protein ◽  
Transcriptional Responses ◽  
Smad Proteins ◽  
Smad Protein

The transforming growth factor-β (TGF-β) superfamily of growth factors and cytokines has been implicated in a variety of physiological and developmental processes within the cardiovascular system. Smad proteins are a recently described family of intracellular signaling proteins that transduce signals in response to TGF-β superfamily ligands. We demonstrate by both a mammalian two-hybrid and a biochemical approach that human Smad2 and Smad4, two essential Smad proteins involved in mediating TGF-β transcriptional responses in endothelial and other cell types, can functionally interact with the transcriptional coactivator CREB binding protein (CBP). This interaction is specific in that it requires ligand (TGF-β) activation and is mediated by the transcriptional activation domains of the Smad proteins. A closely related, but distinct endothelial-expressed Smad protein, Smad7, which does not activate transcription in endothelial cells, does not interact with CBP. Furthermore, Smad2,4–CBP interactions involve the COOH terminus of CBP, a region that interacts with other regulated transcription factors such as certain signal transduction and transcription proteins and nuclear receptors. Smad–CBP interactions are required for Smad-dependent TGF-β-induced transcriptional responses in endothelial cells, as evidenced by inhibition with overexpressed 12S E1A protein and reversal of this inhibition with exogenous CBP. This report demonstrates a functional interaction between Smad proteins and an essential component of the mammalian transcriptional apparatus (CBP) and extends our insight into how Smad proteins may regulate transcriptional responses in many cell types. Thus, functional Smad–coactivator interactions may be an important locus of signal integration in endothelial cells.

scholarly journals Topically Applied Etamsylate: A New Orphan Drug for HHT-Derived Epistaxis (Antiangiogenesis through FGF Pathway Inhibition)

TH Open ◽  
2019 ◽  
Vol 03 (03) ◽  
pp. e230-e243 ◽  
Author(s):  
Virginia Albiñana ◽  
Guillermo Giménez-Gallego ◽  
Angela García-Mato ◽  
Patricia Palacios ◽  
Lucia Recio-Poveda ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Orphan Drug ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Life Threatening ◽  
Vascular Dysplasia ◽  
Pathway Inhibition

AbstractHereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by recurrent and spontaneous epistaxis (nose bleeds), telangiectases on skin and mucosa, internal organ arteriovenous malformations, and dominant autosomal inheritance. Mutations in Endoglin and ACVRL1/ALK1, genes mainly expressed in endothelium, are responsible in 90% of the cases for the pathology. These genes are involved in the transforming growth factor-β(TGF-β) signaling pathway. Epistaxis remains as one of the most common symptoms impairing the quality of life of patients, becoming life-threatening in some cases. Different strategies have been used to decrease nose bleeds, among them is antiangiogenesis. The two main angiogenic pathways in endothelial cells depend on vascular endothelial growth factor and fibroblast growth factor (FGF). The present work has used etamsylate, the diethylamine salt of the 2,5-dihydroxybenzene sulfonate anion, also known as dobesilate, as a FGF signaling inhibitor. In endothelial cells, in vitro experiments show that etamsylate acts as an antiangiogenic factor, inhibiting wound healing and matrigel tubulogenesis. Moreover, etamsylate decreases phosphorylation of Akt and ERK1/2. A pilot clinical trial (EudraCT: 2016–003982–24) was performed with 12 HHT patients using a topical spray of etamsylate twice a day for 4 weeks. The epistaxis severity score (HHT-ESS) and other pertinent parameters were registered in the clinical trial. The significant reduction in the ESS scale, together with the lack of significant side effects, allowed the designation of topical etamsylate as a new orphan drug for epistaxis in HHT (EMA/OD/135/18).

Expression and function of CD105 during the onset of hematopoiesis from Flk1+ precursors

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3635-3642 ◽  
Author(s):  
Sarah K. Cho ◽  
Annie Bourdeau ◽  
Michelle Letarte ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transforming Growth Factor Β ◽  
Hematopoietic Development ◽  
Mesoderm Specification ◽  
Molecular Events ◽  
Regulated Expression ◽  
And Function

Abstract During ontogeny, the hematopoietic system is established from mesoderm-derived precursors; however, molecular events regulating the onset of hematopoiesis are not well characterized. Several members of the transforming growth factor β (TGF-β) superfamily have been implicated as playing a role during mesoderm specification and hematopoiesis. CD105 (endoglin) is an accessory receptor for members of the TGF-β superfamily. Here it is reported that during the differentiation of murine embryonic stem (ES) cells in vitro, hematopoietic commitment within Flk1+ mesodermal precursor populations is characterized by CD105 expression. In particular, CD105 is expressed during the progression from the Flk1+CD45− to Flk1−CD45+ stage. The developmentally regulated expression of CD105 suggests that it may play a role during early hematopoiesis from Flk1+ precursors. To determine whether CD105 plays a functional role during early hematopoietic development, the potential of CD105-deficient ES cells to differentiate into various hematopoietic lineages in vitro was assessed. In the absence of CD105, myelopoiesis and definitive erythropoiesis were severely impaired. In contrast, lymphopoiesis appeared to be only mildly affected. Thus, these findings suggest that the regulated expression of CD105 functions to support lineage-specific hematopoietic development from Flk1+ precursors.

scholarly journals Role of the Latent Transforming Growth Factor β-Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In Vivo

2000 ◽  
Vol 15 (1) ◽  
pp. 68-81 ◽  
Author(s):  
Sarah L. Dallas ◽  
Douglas R. Keene ◽  
Scott P. Bruder ◽  
Juha Saharinen ◽  
Lynn Y. Sakai ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Bone Cells ◽  
Binding Protein ◽  
Transforming Growth Factor Β

scholarly journals Transforming growth factor (TGF)beta, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells

2004 ◽  
Vol 379 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Anouchka SKOUDY ◽  
Meritxell ROVIRA ◽  
Pierre SAVATIER ◽  
Franz MARTIN ◽  
Trinidad LEÓN-QUINTO ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Signalling Pathways ◽  
Fibroblast Growth ◽  
Development And Differentiation

Extracellular signalling cues play a major role in the activation of differentiation programmes. Mouse embryonic stem (ES) cells are pluripotent and can differentiate into a wide variety of specialized cells. Recently, protocols designed to induce endocrine pancreatic differentiation in vitro have been designed but little information is currently available concerning the potential of ES cells to differentiate into acinar pancreatic cells. By using conditioned media of cultured foetal pancreatic rudiments, we demonstrate that ES cells can respond in vitro to signalling pathways involved in exocrine development and differentiation. In particular, modulation of the hedgehog, transforming growth factor β, retinoid, and fibroblast growth factor pathways in ES cell-derived embryoid bodies (EB) resulted in increased levels of transcripts encoding pancreatic transcription factors and cytodifferentiation markers, as demonstrated by RT-PCR. In EB undergoing spontaneous differentiation, expression of the majority of the acinar genes (i.e. amylase, carboxypeptidase A and elastase) was induced after the expression of endocrine genes, as occurs in vivo during development. These data indicate that ES cells can undergo exocrine pancreatic differentiation with a kinetic pattern of expression reminiscent of pancreas development in vivo and that ES cells can be coaxed to express an acinar phenotype by activation of signalling pathways known to play a role in pancreatic development and differentiation.

Transforming growth factor-β1 signaling contributes to development of smooth muscle cells from embryonic stem cells

2004 ◽  
Vol 287 (6) ◽  
pp. C1560-C1568 ◽  
Author(s):  
Sanjay Sinha ◽  
Mark H. Hoofnagle ◽  
Paul A. Kingston ◽  
Mary E. McCanna ◽  
Gary K. Owens
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Model System ◽  
Embryoid Bodies ◽  
Regulatory Pathways ◽  
Tgf Β1

Knockout of transforming growth factor (TGF)-β1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-β1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle α-actin (SMαA), SM22α, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF-β with an adenovirus expressing a soluble truncated TGF-β type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-β1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF-β-Smad signaling for an early vs. late SMC marker gene in that SMαA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-β1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.

Gene-trap expression screening to identify endothelial-specific genes

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Masanori Hirashima ◽  
Alan Bernstein ◽  
William L. Stanford ◽  
Janet Rossant
Keyword(s):  
Endothelial Cells ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transforming Growth Factor Β ◽  
Gene Trap ◽  
Lacz Reporter ◽  
Vascular Differentiation ◽  
Expression Screening

AbstractThe endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene-trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. In a pilot screen, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor-β type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene-trap expression screening can thus be used to identify early endothelial-specific genes and analyze their function in mice.

scholarly journals Latent Transforming Growth Factor-β Binding Protein Domains Involved in Activation and Transglutaminase-dependent Cross-Linking of Latent Transforming Growth Factor-β

1997 ◽  
Vol 136 (5) ◽  
pp. 1151-1163 ◽  
Author(s):  
Irene Nunes ◽  
Pierre-Emmanuel Gleizes ◽  
Christine N. Metz ◽  
Daniel B Rifkin
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Noncovalent Interactions ◽  
Binding Protein ◽  
Transforming Growth Factor Β ◽  
Cell Types ◽  
Terminal Region ◽  
Cross Linking ◽  
Surface Receptors ◽  
Amino Terminal

Transforming growth factor-β (TGF-β) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-β, the TGF-β propeptide, and the latent TGF-β binding protein (LTBP). To interact with its cell surface receptors, TGF-β must be released from the latent complex by disrupting noncovalent interactions between mature TGF-β and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-β. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S–transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in crosslinking and formation of TGF-β by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-β generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (ΔN293) or 441 (ΔN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that ΔN293 LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas ΔN441 LTBP-1S was not. This suggests that residues 294–441 are critical to the transglutaminase reactivity of LTBP-1S.

Mouse embryonic stem cells with aberrant transforming growth factor β signalling exhibit impaired differentiation in vitro and in vivo

1998 ◽  
Vol 63 (3) ◽  
pp. 101-113 ◽  
Author(s):  
Marie-José Goumans ◽  
Dorien Ward-van Oostwaard ◽  
Florence Wianny ◽  
Pierre Savatier ◽  
An Zwijsen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Embryonic Stem ◽  
Transforming Growth Factor Β ◽  
Mouse Embryonic Stem Cells
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