The TGFß Family in Human Placental Development at the Fetal-Maternal Interface.

Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFß) family as it has been shown that the TGFß family has an important role in human placental development and disease.

EGFL7 Mediates BMP9-Induced Sprouting Angiogenesis of Endothelial Cells Derived from Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) are instrumental in characterizing the molecular mechanisms of human vascular development and disease. Bone morphogenetic proteins (BMPs) play a pivotal role in cardiovascular development in mice, but their importance for vascular cells derived from hESCs has not yet been fully explored. Here, we demonstrate that BMP9 promotes, via its receptor ALK1 and SMAD1/5 activation, sprouting angiogenesis of hESC-derived endothelial cells. We show that the secreted angiogenic factor epidermal growth factor-like domain 7 (EGFL7) is a downstream target of BMP9-SMAD1/5-mediated signaling, and that EGFL7 promotes expansion of endothelium via interference with NOTCH signaling, activation of ERK, and remodeling of the extracellular matrix. CRISPR/Cas9-mediated deletion of EGFL7 highlights the critical role of EGFL7 in BMP9-induced endothelial sprouting and the promotion of angiogenesis. Our study illustrates the complex role of the BMP family in orchestrating hESC vascular development and endothelial sprouting.

BMP-SMAD signaling: From pluripotent stem cells to cardiovascular commitment.

Human pluripotent stem cells (hPSCs) can form all somatic cells of the body. They thus offer opportunities for understanding (i) the basic steps of early human development, (ii) the pathophysiology in human degenerative diseases and (iii) approaches to regenerative medicine and drug development. Methods for improving their differentiation to defined mesodermal derivatives in particular will benefit their use in all of these areas but most particularly applications that require cardiac and vascular tissue. However, the molecular mechanisms that regulate mesodermal development in humans are still poorly understood. Gene ablation studies in mice have shown that the signaling pathways activated by the transforming growth factor beta (TGFß) superfamily, including the bone morphogenetic proteins (BMP), play crucial roles in mesoderm differentiation and patterning the early embryo. Understanding their interplay and interaction with other signaling pathways, how they activate and inhibit transcription factors and epigenetic regulators during self-renewal, maintenance and exit from pluripotency and differentiation could provide vital information for a range of applications. This includes disease modeling when the hPSCs are derived from patients or drug screens for diseases of mesodermal organs. Here, we review the role of the BMP-SMAD signaling pathway in pluripotent stem cells and during mesoderm differentiation with focus on the cells that make up the cardiovascular system.

[Breakthrough in research on pluripotent stem cells and their application in medicine]. / Straumhvörf í rannsóknum á fjölhæfum stofnfrumum og notagildi theirra í læknavísindum.

Embryonic stem cells are, as the name indicates, isolated from embryos. They are pluripotent cells which can be maintained undifferentiated or induced to differentiate into any cell type of the body. In 1998 the first isolation of human embryonic stem cells was successful and they became an interesting source for stem cell regenerative medicine. Only 8 years later pluripotent stem cells were generated by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This was a revolution in the way people thought of cell commitment during development. Since then, a lot of research has been done in understanding the molecular biology of pluripotent stem cells. iPSCs can be generated from somatic cells of a patient and therefore have the same genome. Hence, iPSCs have great potential application in medicine, as they can be utilized in disease modelling, drug screening and cell replacement therapy.

BMP4 promotes EMT and mesodermal commitment in human embryonic stem cells via SLUG and MSX2.

Bone morphogenetic proteins (BMPs) initiate differentiation in human embryonic stem cells (hESCs) but the exact mechanisms have not been fully elucidated. We demonstrate here that SLUG and MSX2, transcription factors involved in epithelial-mesenchymal transitions, essential features of gastrulation in development and tumor progression, are important mediators of BMP4-induced differentiation in hESCs. Phosphorylated Smad1/5/8 colocalized with the SLUG protein at the edges of hESC colonies where differentiation takes place. The upregulation of the BMP target SLUG was direct as shown by the binding of phosphorylated Smad1/5/8 to its promoter, which interrupted the formation of adhesion proteins, resulting in migration. Knockdown of SLUG by short hairpin RNA blocked these changes, confirming an important role for SLUG in BMP-mediated mesodermal differentiation. Furthermore, BMP4-induced MSX2 expression leads to mesoderm formation and then preferential differentiation toward the cardiovascular lineage.

VEGF and inhibitors of TGFbeta type-I receptor kinase synergistically promote blood-vessel formation by inducing alpha5-integrin expression.

Vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGFbeta) are potent regulators of angiogenesis. How VEGF and TGFbeta signaling pathways crosstalk is not well understood. Therefore, we analyzed the effects of the TGFbeta type-I-receptor inhibitors (SB-431542 and LY-2157299) and VEGF on endothelial cell (EC) function and angiogenesis. We show that SB-431542 dramatically enhances VEGF-induced formation of EC sheets from fetal mouse metatarsals. Sub-optimal doses of VEGF and SB-431542 synergistically induced EC migration and sprouting of EC spheroids, whereas overexpression of a constitutively active form of TGFbeta type-I receptor had opposite effects. Using quantitative PCR, we demonstrated that VEGF and SB-431542 synergistically upregulated the mRNA expression of genes involved in angiogenesis, including the integrins alpha5 and beta3. Specific downregulation of alpha5-integrin expression or functional blocking of alpha5 integrin with a specific neutralizing antibody inhibited the cooperative effect of VEGF and SB-431542 on EC sprouting. In vivo, LY-2157299 induced angiogenesis and enhanced VEGF- and basic-fibroblast-growth-factor-induced angiogenesis in a Matrigel-plug assay, whereas adding an alpha5-integrin-neutralizing antibody to the Matrigel selectively inhibited this enhanced response. Thus, induction of alpha5-integrin expression is a key determinant by which inhibitors of TGFbeta type-I receptor kinase and VEGF synergistically promote angiogenesis.

Smad7 and protein phosphatase 1alpha are critical determinants in the duration of TGF-beta/ALK1 signaling in endothelial cells.

BACKGROUND: In endothelial cells (EC), transforming growth factor-beta (TGF-beta) can bind to and transduce signals through ALK1 and ALK5. The TGF-beta/ALK5 and TGF-beta/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-beta signaling is an important specificity determinant for signaling responses. TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently. RESULTS: The temporal activation of TGF-beta-induced Smad1/5 phosphorylation in ECs was found to be affected by de novo protein synthesis, and ALK1 and Smad5 expression levels determined signal strength of TGF-beta/ALK1 signaling pathway. Smad7 and protein phosphatase 1alpha (PP1alpha) mRNA expression levels were found to be specifically upregulated by TGF-beta/ALK1. Ectopic expression of Smad7 or PP1alpha potently inhibited TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs. Conversely, siRNA-mediated knockdown of Smad7 or PP1alpha enhanced TGF-beta/ALK1-induced signaling responses. PP1alpha interacted with ALK1 and this association was further potentiated by Smad7. Dephosphorylation of the ALK1, immunoprecipitated from cell lysates, was attenuated by a specific PP1 inhibitor. CONCLUSION: Our results suggest that upon its induction by the TGF-beta/ALK1 pathway, Smad7 may recruit PP1alpha to ALK1, and thereby control TGF-beta/ALK1-induced Smad1/5 phosphorylation.

Functions of the TGFbeta superfamily in human embryonic stem cells.

The establishment of human embryonic stem (ES) cells has opened possibilities for cell replacement therapy to treat diseases such as diabetes, Parkinson's disease and cardiac myopathies. Self-renewal is one of the essential defining characteristics of stem cells. If stem cells are to have widespread therapeutic applications, it is essential to identify the extrinsic and intrinsic factors maintaining self-renewal, particularly in culture. Insight into the regulation of known self-renewal transcription factors and cross-talk between their upstream signalling pathways is important for a better understanding of how stem cell self-renewal and differentiation are related to downstream target genes. This may lead to the establishment of protocols for obtaining a large supply of ES cells. Here, we review the role that TGFbeta superfamily members are thought to play in self-renewal and differentiation of human and mouse ES cells. We focus on the prototype TGFbeta, TGFbeta1, activin A, nodal and bone morphogenetic proteins and their expression, activity and function in embryonic stem cells.

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction.

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells.

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP-Jkappa/CBF-1 and GC-rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1-induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.

Controlling cell fate by bone morphogenetic protein receptors.

Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the fate of different cell types, including mesenchymal and endothelial cells. BMPs inhibit myogenic differentiation, but promote the differentiation of mesenchymal cells into osteoblasts. Furthermore, endothelial migration and tube formation are stimulated by BMPs. Like other members of the transforming growth factor-beta (TGF-beta) superfamily, BMPs elicit their cellular effects via specific types I and II serine/threonine receptors. The activated BMP type I receptor phosphorylates specific receptor-regulated (R)-Smad proteins, which assemble into heteromeric complexes with common partner (Co)-Smad4. Heteromeric Smad complexes efficiently translocate into the nucleus, where they regulate the transcription of target genes. Inhibitors of differentiation (Id) are genes that are specifically induced by BMPs in tissues of different origin. Promoter analysis of Id1 indicates three distinct sequence elements that are sufficient and essential for efficient BMP-induced activation. Furthermore, recent studies reveal an important effector function for Id1 in various BMP-induced biological responses.

Controlling the angiogenic switch: a balance between two distinct TGF-b receptor signaling pathways.

Biochemical studies in endothelial cells (ECs) and genetic studies in mice and humans have yielded major insights into the role of transforming growth factor beta (TGF-beta) and its downstream Smad effectors in embryonic vascular morphogenesis and in the establishment and maintenance of vessel wall integrity. These studies showed that TGF-beta signaling is of critical importance for normal vascular development and physiology. They also indicated the involvement of two distinct TGF-beta signaling cascades within ECs, namely the activin receptor-like kinase 5 (ALK5)-Smad2/3 pathway and the ALK1-Smad1/5 pathway. Aberrant TGF-beta signaling forms the basis for several vascular disorders such as hereditary hemorrhagic telengiectasia and primary pulmonary hypertension as well as neovascularization during tumorigenesis. This review describes the role of TGF-beta in angiogenesis and some of the controversial issues concerning TGF-beta signaling through ALK1 and ALK5 in ECs.

Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling.

Transforming growth factor-beta (TGFbeta) regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways. Activin receptor-like kinase-1 (ALK1) induces Smad1/5 phosphorylation, leading to an increase in endothelial cell proliferation and migration, while ALK5 promotes Smad2/3 activation and inhibits both processes. Here, we report that ALK5 is important for TGFbeta/ALK1 signaling; endothelial cells lacking ALK5 are deficient in TGFbeta/ALK1-induced responses. More specifically, we show that ALK5 mediates a TGFbeta-dependent recruitment of ALK1 into a TGFbeta receptor complex and that the ALK5 kinase activity is required for optimal ALK1 activation. TGFbeta type II receptor is also required for ALK1 activation by TGFbeta. Interestingly, ALK1 not only induces a biological response opposite to that of ALK5 but also directly antagonizes ALK5/Smad signaling.

Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells.

BACKGROUND: Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the proliferation, differentiation, and migration of a large variety of cell types. Like other members of the transforming growth factor-beta family, BMPs elicit their cellular effects through activating specific combinations of type I and type II serine/threonine kinase receptors and their downstream effector proteins, which are termed Smads. In the present study, we investigated BMP receptor/Smad expression and signaling in endothelial cells (ECs) and examined the effects of BMP on EC behavior. METHODS AND RESULTS: Immunohistochemical analysis of tissue sections of human colon and mouse heart and aorta showed that BMP receptors are expressed in ECs in vivo. Bovine aortic ECs and mouse embryonic ECs were found to express BMP receptors and their Smads. BMP receptor activation induced the phosphorylation of specific Smad proteins and promoted EC migration and tube formation. Id1 was identified as a BMP/Smad target in ECs. Ectopic expression of Id1 mimicked BMP-induced effects. Importantly, specific interference with Id1 expression blocked BMP-induced EC migration. CONCLUSIONS: The BMP/Smad pathway can potently activate the endothelium. Id1 expression is strongly induced by BMP in ECs. Ectopic expression of Id1 induces EC migration and tube formation. Moreover, Id1 played a critical role in mediating BMP-induced EC migration.

Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors.

The generation of mice lacking specific components of the transforming growth factor-beta (TGF-beta) signal tranduction pathway shows that TGF-beta is a key player in the development and physiology of the cardiovascular system. Both pro- and anti-angiogenic properties have been ascribed to TGF-beta, for which the molecular mechanisms are unclear. Here we report that TGF-beta can activate two distinct type I receptor/Smad signalling pathways with opposite effects. TGF-beta induces phosphorylation of Smad1/5 and Smad2 in endothelial cells and these effects can be blocked upon selective inhibition of ALK1 or ALK5 expression, respectively. Whereas the TGF-beta/ALK5 pathway leads to inhibition of cell migration and proliferation, the TGF-beta/ALK1 pathway induces endothelial cell migration and proliferation. We identified genes that are induced specifically by TGF-beta-mediated ALK1 or ALK5 activation. Id1 was found to mediate the TGF-beta/ALK1-induced (and Smad-dependent) migration, while induction of plasminogen activator inhibitor-1 by activated ALK5 may contribute to the TGF-beta-induced maturation of blood vessels. Our results suggest that TGF-beta regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signalling.

Transient disruption of autocrine TGF-beta signaling leads to enhanced survival and proliferation potential in single primitive human hemopoietic progenitor cells.

Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-beta negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-beta type II receptor (TbetaRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-beta signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TbetaRIIDN and green fluorescence protein in the same CD34(+)CD38(-)Lin(-) cells were constructed. Overexpression of TbetaRIIDN specifically disrupted TGF-beta-mediated signaling. Autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34(+)CD38(-)Lin(-) cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-beta-negative regulatory loop of primitive hemopoietic progenitor cells.