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Endoglin, an Ancillary TGF?? Receptor, Is Required for Extraembryonic Angiogenesis and Plays a Key Role in Heart Development
Abstract
Endoglin (CD105) is expressed on the surface of endothelial and haematopoietic cells in mammals and binds TGFbeta isoforms 1 and 3 in combination with the signaling complex of TGFbeta receptors types I and II. Endoglin expression increases during angiogenesis, wound healing, and inflammation, all of which are associated with TGFbeta signaling and alterations in vascular structure. The importance of endoglin for normal vascular architecture is further indicated by the association of mutations in the endoglin gene with the inherited disorder Hereditary Haemorrhagic Telangiectasia Type 1 (HHT1), a disease characterised by bleeding from vascular malformations. In order to study the role of endoglin in vivo in more detail and to work toward developing an animal model of HHT1, we have derived mice that carry a targeted nonsense mutation in the endoglin gene. Studies on these mice have revealed that endoglin is essential for early development. Embryos homozygous for the endoglin mutation fail to progress beyond 10.5 days postcoitum and fail to form mature blood vessels in the yolk sac. This phenotype is remarkably similar to that of the TGFbeta1 and the TGFbeta receptor II knockout mice, indicating that endoglin is needed in vivo for TGFbeta1 signaling during extraembryonic vascular development. In addition, we have observed cardiac defects in homozygous endoglin-deficient embryos, suggesting endoglin also plays a role in cardiogenesis. We anticipate that heterozygous mice will ultimately serve as a useful disease model for HHT1, as some individuals have dilated and fragile blood vessels similar to vascular malformations seen in HHT patients.
... Complete loss of the Eng gene in the mouse leads to a failure of cardiovascular development and embryonic lethality pointing to its essential role in the formation of heart and blood vessels (Arthur et al., 2000). Eng knockout embryos also show reduced numbers of atrioventricular cardiac cushion mesenchyme cells at E9.5 and a failure of vessel muscularisation (Arthur et al., 2000;Li et al., 1999). ...
... Complete loss of the Eng gene in the mouse leads to a failure of cardiovascular development and embryonic lethality pointing to its essential role in the formation of heart and blood vessels (Arthur et al., 2000). Eng knockout embryos also show reduced numbers of atrioventricular cardiac cushion mesenchyme cells at E9.5 and a failure of vessel muscularisation (Arthur et al., 2000;Li et al., 1999). Using an Eng-LacZ reporter mouse, we showed that the Eng promoter drives gene expression in the primitive heart forming region at E7.5 (Jonker and Arthur, 2002). ...
... However, the dynamics of ENG expression in the coronary vasculature has not yet been placed in the context of recent major advances in understanding of coronary vessel development (Sharma et al., 2017). ENG protein is also expressed in cardiac cushion mesenchymal cells during human, mouse and chick heart development (Arthur et al., 2000;Jonker and Arthur, 2002;Mercado-Pimentel et al., 2007;Qu et al., 1998) although the dynamic change in ENG protein levels as endocardium de-differentiates to form cushion mesenchyme has not previously been quantified. A similar reprogramming process occurs when a subset of epicardial cells undergo mesenchymal transition, and although ENG expression has been shown in epicardial derived mesenchymal cells in vitro (Bax et al., 2011), this has not yet been investigated in vivo. ...
Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.
Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.
ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.
In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.
... We found a high EdU ratio in the CD105+ population, which also showed a high expression level of HAND1 (Figures S7A-S7D). CD105 is a TGFb receptor (Arthur et al., 2000). Therefore, we investigated whether TGFb signaling is associated with cell cycle activity by adding SB431542 on days 16-18 to the isolated CMs. ...
... CD105 is highly expressed in endothelial and mesenchymal cells and also a TGFb signaling receptor. Its deficiency in mouse is embryonic lethal as a result of cardiovascular abnormalities (Arthur et al., 2000), but little is known about CD105 function in human CMs. In the present study, TGFb signaling inhibition did not change the proliferation capacity of CMs, suggesting that CD105 has little functional contribution to the proliferation as a TGFb signaling receptor even though it can act as marker of proliferative CMs. ...
Hand1 and Hand2 are transcriptional factors, and knockout mice of these genes show left and right ventricular hypoplasia, respectively. However, their function and expression in human cardiogenesis are not well studied. To delineate their expressions and assess their functions in human cardiomyocytes (CMs) in vitro, we established two triple-reporter human induced pluripotent stem cell lines that express HAND1mCherry, HAND2EGFP and either MYH6-driven iRFP670 or tagBFP constitutively and investigated their expression dynamics during cardiac differentiation. On day 5 of the differentiation, HAND1 expression marked cardiac progenitor cells. We profiled the CM subpopulations on day 20 with RNA sequencing and found that mCherry+ CMs showed higher proliferative ability than mCherry− CMs and identified a gene network of LEF1, HAND1, and HAND2 to regulate proliferation in CMs. Finally, we identified CD105 as a surface marker of highly proliferative CMs.
... Endoglin (ENG) is highly expressed in endothelial cells (ECs), where it regulates development and maintenance of blood vessels [1][2][3]. Endoglin is a high affinity co-receptor for BMP9 and BMP10 ligands of the TGF-β superfamily [4,5]. It behaves as a BMP9/10 ligand reservoir on the EC surface [6] and promotes ligand-induced ALK1 phosphorylation of SMAD1/5/8 [7]. ...
... Genetic mouse models have been developed to study HHT [12]. Endoglin-null mice die by gestational day 11.5 due to defective blood vessel and heart development [1,13]. Loss of endothelial ENG in neonatal mice leads to retinal AVMs [2] that result from an abnormal increase in endothelial proliferation and reduced EC migration against blood flow during angiogenesis [2,3]. ...
Rationale: Hereditary haemorrhagic telangiectasia (HHT) is an inherited bleeding disorder characterised by arteriovenous malformations (AVMs). Such AVMs affect lungs, liver and brain, whilst telangiectases in mucocutaneous tissues are prone to haemorrhage. HHT type I is caused by loss-of-function endoglin (ENG) mutations. Evidence suggests AVMs result from abnormal responses to VEGF signalling.
Objective: We therefore characterised the vascular abnormalities in eng mutant zebrafish and investigated whether these are prevented by inhibiting different pathways downstream of VEGF signalling.
Methods and Results: We used light sheet fluorescence microscopy to visualise the vasculature in engmu130 mutant zebrafish. In addition to previously described significantly enlarged dorsal aorta and posterior cardinal vein at 3d post fertilisation, engmu130 embryos had an enlarged basilar artery (BA), and increased formation of endothelial kugeln on cerebral vessels. Adult engmu130 fish developed skin AVMs, retinal vascular abnormalities, and an enlarged heart. Tivozanib (AV951), a VEGF receptor tyrosine kinase inhibitor, prevented development of the abnormally enlarged major vessels and normalised the number of kugeln in engmu130 embryos. Inhibiting discrete signalling pathways downstream of VEGFR2 in engmu130 embryos gave further insights. Inhibiting TOR or MEK prevented the abnormal trunk and cerebral vasculature phenotype, whilst targeting NOS and MAPK had no effect. Combining subtherapeutic TOR and MEK inhibition prevented the vascular phenotype, suggesting synergy between TOR and MEK/ERK signalling pathways.
Conclusions: These results indicate the HHT- like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling, and implicate combination low dose ERK and TOR pathway inhibitors as a therapeutic strategy in HHT.
... These animals have a defective vascular remodeling, which makes vessels fragile and easily broken, resulting in internal bleeding. They also present alterations in the cardiac development, malformations in cardiac valves and in the heart partition [96][97][98]. In the case of zebrafish, homozygous mutants of endoglin are viable and survive to adulthood but have vascular malformations due to the incorrect union of the dorsal artery and cardinal vein [99]. ...
... Mural cell recruitment is also affected by reduced endoglin levels. In fact, a poor association of vascular smooth muscle cells to the endothelium is one of the causes of death in Eng +/− mice during the embryonic development [96,98,127]. Although the retinas of Eng +/− mice do not show differences in the number of mural cells with the control mice [106], maybe they cannot adhere correctly to the endothelium. ...
Simple Summary
The prognosis and response to immunotherapy depends largely on the composition of the tumor microenvironment (TME). So-called cold tumors are rich in cells and molecules that inhibit the antitumor response and, therefore, are associated with a worse prognosis. In contrast, hot tumors are rich in antitumor cells and respond well to immunotherapy. The creation of one type of TME or another is highly dependent on angiogenesis, inflammation, and cancer-associated fibroblast (CAF) accumulation. Endoglin (CD105) is a protein involved in these three processes, making it a possible target for the conversion of cold tumors into hot tumors. In this review we summarize the role of endoglin in these processes and present the anti-endoglin therapies already under study that could be applied for vascular normalization.
Abstract
Tumors are complex masses formed by malignant but also by normal cells. The interaction between these cells via cytokines, chemokines, growth factors, and enzymes that remodel the extracellular matrix (ECM) constitutes the tumor microenvironment (TME). This TME can be determinant in the prognosis and the response to some treatments such as immunotherapy. Depending on their TME, two types of tumors can be defined: hot tumors, characterized by an immunosupportive TME and a good response to immunotherapy; and cold tumors, which respond poorly to this therapy and are characterized by an immunosuppressive TME. A therapeutic strategy that has been shown to be useful for the conversion of cold tumors into hot tumors is vascular normalization. In this review we propose that endoglin (CD105) may be a useful target of this strategy since it is involved in the three main processes involved in the generation of the TME: angiogenesis, inflammation, and cancer-associated fibroblast (CAF) accumulation. Moreover, the analysis of endoglin expression in tumors, which is already used in the clinic to study the microvascular density and that is associated with worse prognosis, could be used to predict a patient’s response to immunotherapy.
... At E9.5 a reduction of erythroblasts (CD71 + /Ter119 + ) in the yolk sacs of Eng −/− embryos accompanied by a lower β-globin expression confirmed impaired primitive erythropoiesis in the absence of Eng [86]. Co-expression of Endoglin with fetal liver kinase-1 (Flk-1) is detected in the primitive streak (PS) in mesodermal cells of blood islands at E7.5 [87]. FACS analysis revealed that only Endoglin expressing cells are endowed with hematopoietic potential at E7.5. ...
... They found that Endoglin is already expressed in ES cells and represents together with Flk-1 a marker for BL-CFC (hemangioblast). Endoglin deficiency reduces the number and size of BL-CFC as well as their hematopoietic potential (erythropoiesis), which is compatible with the anemia seen in Eng −/− embryos [87,89]. In contrast, the endothelial developmental potential is even promoted in the absence of Endoglin in BL-CFC, whereas the branching ability in a sprouting assay is reduced similar to that seen in Eng −/− embryos [89,90]. ...
Transforming growth factor-β1 (TGF-β1) is a pleiotropic factor sensed by most cells. It regulates a broad spectrum of cellular responses including hematopoiesis. In order to process TGF-β1-responses in time and space in an appropriate manner, there is a tight regulation of its signaling at diverse steps. The downstream signaling is mediated by type I and type II receptors and modulated by the ‘accessory’ receptor Endoglin also termed cluster of differentiation 105 (CD105). Endoglin was initially identified on pre-B leukemia cells but has received most attention due to its high expression on activated endothelial cells. In turn, Endoglin has been figured out as the causative factor for diseases associated with vascular dysfunction like hereditary hemorrhagic telangiectasia-1 (HHT-1), pre-eclampsia, and intrauterine growth restriction (IUPR). Because HHT patients often show signs of inflammation at vascular lesions, and loss of Endoglin in the myeloid lineage leads to spontaneous inflammation, it is speculated that Endoglin impacts inflammatory processes. In line, Endoglin is expressed on progenitor/precursor cells during hematopoiesis as well as on mature, differentiated cells of the innate and adaptive immune system. However, so far only pro-monocytes and macrophages have been in the focus of research, although Endoglin has been identified in many other immune system cell subsets. These findings imply a functional role of Endoglin in the maturation and function of immune cells. Aside the functional relevance of Endoglin in endothelial cells, CD105 is differentially expressed during hematopoiesis, arguing for a role of this receptor in the development of individual cell lineages. In addition, Endoglin expression is present on mature immune cells of the innate (i.e., macrophages and mast cells) and the adaptive (i.e., T-cells) immune system, further suggesting Endoglin as a factor that shapes immune responses. In this review, we summarize current knowledge on Endoglin expression and function in hematopoietic precursors and mature hematopoietic cells of different lineages.
... Endoglin is predominantly expressed by activated endothelial cells [1] and plays a crucial role in (developmental) angiogenesis. In mice, a complete loss of endoglin is embryonically lethal around embryonic day 10.5, primarily due to impaired development of the vascular plexus into a mature vascular network, causing hampered low and osmotic imbalance, disturbing normal cardiac development [2,3]. Part of the cardiac abnormality is caused by pericardial effusion due to disturbed osmotic balance [2]. ...
... In mice, a complete loss of endoglin is embryonically lethal around embryonic day 10.5, primarily due to impaired development of the vascular plexus into a mature vascular network, causing hampered low and osmotic imbalance, disturbing normal cardiac development [2,3]. Part of the cardiac abnormality is caused by pericardial effusion due to disturbed osmotic balance [2]. This indicates the pivotal role that endoglin plays in developmental angiogenesis. ...
Endoglin, a type-III accessory receptor for the Transforming Growth Factor (TGF)-β superfamily pathway, is known for its crucial role during angiogenesis. Extensive work has shown the important roles that endoglin plays in balancing the TGF-β signaling pathway, thereby regulating endothelial cell proliferation and migration. However, recent work indicates a far more widespread role for endoglin beyond the endothelial cells. In this review, we will provide a summary of recent publications on endoglin expression on epithelial (cancer) cells, cancer-associated fibroblasts, and mesenchymal stem cells. Additionally, we will discuss the role of endoglin in innate and adaptive immunity. Finally, we will discuss the results of clinical trials using the endoglin targeting antibody (TRC105), focusing on the effects observed beyond the endothelium. In conclusion, although endoglin was initially identified as an endothelial marker, additional roles for endoglin on other cell types have been shown, although the number of studies is still limited, with sometimes conflicting data. Future studies will further establish the roles of endoglin beyond the endothelium.
... are involved in heart development (Arthur et al., 2000;Boulter et al., 2001;James et al., 1999;Poltavski et al., 2019). EIF3A plays a role in intestinal development (Liu et al., 2007). ...
Many organisms can adjust their development according to environmental conditions, including the presence of conspecifics. Although this developmental plasticity is common in amphibians, its underlying molecular mechanisms remain largely unknown. Exposure during development to either ‘cannibal cues’ from older conspecifics, or ‘alarm cues’ from injured conspecifics, causes reduced growth and survival in cane toad (Rhinella marina) tadpoles. Epigenetic modifications, such as changes in DNA methylation patterns, are a plausible mechanism underlying these developmental plastic responses. Here we tested this hypothesis, and asked whether cannibal cues and alarm cues trigger the same DNA methylation changes in developing cane toads. We found that exposure to both cannibal cues and alarm cues was associated with local changes in DNA methylation patterns. These DNA methylation changes affected genes putatively involved in developmental processes, but in different genomic regions for different conspecific-derived cues. Genetic background explains most of the epigenetic variation among individuals. Overall, the molecular mechanisms triggered by exposure to cannibal cues seem to differ from those triggered by alarm cues. Studies linking epigenetic modifications to transcriptional activity are needed to clarify the proximate mechanisms that regulate developmental plasticity in cane toads.
... Especially focusing on cardiac development, the TGFβ signalling pathway has already been reported to be involved by several studies. Interestingly, the cardiac progenitor marker endoglin (CD105) is also known to be an ancillary TGFβ receptor and has been shown to be required for extraembryonic angiogenesis and heart development [46]. In several species, bone morphogenic proteins (BMPs), members of the TGFβ superfamily, have also been found to mediate cardiac development. ...
The ageing phenotype is strongly driven by the exhaustion of adult stem cells (ASCs) and the accumulation of senescent cells. Cardiovascular diseases (CVDs) and heart failure (HF) are strongly linked to the ageing phenotype and are the leading cause of death. As the human heart is considered as an organ with low regenerative capacity, treatments targeting the rejuvenation of human cardiac stem cells (hCSCs) are of great interest. In this study, the beneficial effects of human blood serum on proliferation and senescence of hCSCs have been investigated at the molecular level. We show the induction of a proliferation-related gene expression response by human blood serum at the mRNA level. The concurrent differential expression of the TGFβ target and inhibitor genes indicates the participation of TGFβ signalling in this context. Surprisingly, the application of TGFβ1 as well as the inhibition of TGFβ type I and type II receptor (TGFβRI/II) signalling strongly increased the proliferation of hCSCs. Likewise, both human blood serum and TGFβ1 reduced the senescence in hCSCs. The protective effect of serum on senescence in hCSCs was enhanced by simultaneous TGFβRI/II inhibition. These results strongly indicate a dual role of TGFβ signalling in terms of the serum-mediated effects on hCSCs. Further analysis via RNA sequencing (RNA-Seq) revealed the participation of Ras-inactivating genes wherefore a prevention of hyperproliferation upon serum-treatment in hCSCs via TGFβ signalling and Ras-induced senescence is suggested. These insights may improve treatments of heart failure in the future.
... Endoglin, or CD105, is a type I transmembrane protein which serves as a BMP9/10 co-receptor and TGF-β signaling auxiliary receptor that is expressed primarily on vascular endothelial cells [69,70]. It is important for maintaining the proper structure of vasculature and is indispensable for heart development [71]. Furthermore, together with CD90 and CD44, CD105 is a marker of mesenchymal stem cells [55]. ...
Heart failure remains a major cause of death worldwide. There is a need to establish new management options as current treatment is frequently suboptimal. Clinical approaches based on autologous stem cell transplant is potentially a good alternative. The heart was long considered an organ unable to regenerate and renew. However, several reports imply that it may possess modest intrinsic regenerative potential. To allow for detailed characterization of cell cultures, whole transcriptome profiling was performed after 0, 7, 15, and 30 days of in vitro cell cultures (IVC) from the right atrial appendage and right atrial wall utilizing microarray technology. In total, 4239 differentially expressed genes (DEGs) with ratio > abs |2| and adjusted p-value ≤ 0.05 for the right atrial wall and 4662 DEGs for the right atrial appendage were identified. It was shown that a subset of DEGs, which have demonstrated some regulation of expression levels with the duration of the cell culture, were enriched in the following GO BP (Gene Ontology Biological Process) terms: “stem cell population maintenance” and “stem cell proliferation”. The results were validated by RT-qPCR. The establishment and detailed characterization of in vitro culture of myocardial cells may be important for future applications of these cells in heart regeneration processes.
... These studies revealed enriched expression of genes important for cell migration and cytoskeletal remodeling, including Adam10, a metalloproteinase that promotes EC permeability, 74 and Itga2 and Itga4, components of the integrin receptors for collagen and fibronectin at P1. By P7, genes related to vascular development were upregulated, including Eng, an ancillary TGFb receptor essential for extraembryonic angiogenesis, 75 Tspan12, a tetraspanin that regulates retinal vascular development by augmenting Norrin signaling, 76 iScience Article vascular development. 77 By P21, these and other genes important for blood vessel morphogenesis were downregulated, whereas genes regulating antigen processing and presentation were increased, including upregulation of Cd36 as seen in CAP1, a gene that functions as a scavenger receptor involved in the presentation of MHC class II antigens, 78 and H2-D1 and H2-K1, genes encoding MHC Class I molecules. ...
At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2) contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury that impairs angiogenesis induced both common and unique endothelial gene signatures, dysregulated capillary EC crosstalk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.
... Male-upregulated placental receptors include ENG, ERBB2 and INSR, and upregulated maternal decidual ligands include COL1A1, HSPA5, and TGFB1. Males showed endoglin (ENG) upregulation in bulk CV as well [35], a TGF-beta-related integral membrane component that plays an important role in developmental tissue invasion and vascular remodeling [69]. In line with other observations in bulk CV, the male trophoblast transcriptome appeared to be enriched for protein translation, mitochondrial and ribosomal functions [34]. ...
The fetal placenta is a source of hormones and immune factors that play a vital role in maintaining pregnancy and facilitating fetal growth. Cells in this extraembryonic compartment match the chromosomal sex of the embryo itself. Sex differences have been observed in common gestational pathologies, highlighting the importance of maternal immune tolerance to the fetal compartment. Over the past decade, several studies examining placentas from term pregnancies have revealed widespread sex differences in hormone signaling, immune signaling, and metabolic functions. Given the rapid and dynamic development of the human placenta, sex differences that exist at term (37–42 weeks gestation) are unlikely to align precisely with those present at earlier stages when the fetal–maternal interface is being formed and the foundations of a healthy or diseased pregnancy are established. While fetal sex as a variable is often left unreported in studies performing transcriptomic profiling of the first-trimester human placenta, four recent studies have specifically examined fetal sex in early human placental development. In this review, we discuss the findings from these publications and consider the evidence for the genetic, hormonal, and immune mechanisms that are theorized to account for sex differences in early human placenta. We also highlight the cellular and molecular processes that are most likely to be impacted by fetal sex and the evolutionary pressures that may have given rise to these differences. With growing recognition of the fetal origins of health and disease, it is important to shed light on sex differences in early prenatal development, as these observations may unlock insight into the foundations of sex-biased pathologies that emerge later in life.
... Overall, we found that HHT1 c.1678C>T -hiPSC-ECs showed multiple similarities to Eng +/mutant mice (Arthur et al., 2000;Carvalho et al., 2004;Lebrin et al., 2010), although there were some differences. These included the formation of narrower vessels with fewer ECs by HHT1 c.1678C>T -hiPSC-ECs than healthy controls. ...
Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by weak blood vessels. HHT1 is caused by mutations in the ENDOGLIN (ENG) gene. Here, we generated induced pluripotent stem cells (hiPSCs) from a patient with rare mosaic HHT1 with tissues containing both mutant (ENGc.1678C>T) and normal cells, enabling derivation of isogenic diseased and healthy hiPSCs, respectively. We showed reduced ENG expression in HHT1 endothelial cells (HHT1-hiPSC-ECs), reflecting haploinsufficiency. HHT1c.1678C>T-hiPSC-ECs and the healthy isogenic control behaved similarly in two-dimensional (2D) culture, forming functionally indistinguishable vascular networks. However, when grown in 3D organ-on-chip devices under microfluidic flow, lumenized vessels formed in which defective vascular organization was evident: interaction between inner ECs and surrounding pericytes was decreased, and there was evidence for vascular leakage. Organs on chip thus revealed features of HHT in hiPSC-derived blood vessels that were not evident in conventional 2D assays.
... Research has indicated that endoglin knockout mice could develop embryonic lethality in an animal study. Therefore, endoglin is essential for angiogenesis in the development phase [30]. Previous preclinical research and clinical trials indicate that endoglin is an important biomarker of angiogenesis. ...
Background:
Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway.
Methods:
Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5.
Results:
Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation.
Conclusions:
The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.
... In addition to the role of VEGF as a master regulator of angiogenesis, several studies have supported the importance of TGF-β1 in angiogenic sprouting (Hirota et al., 2015;Siqueira et al., 2018), as well as in the induction of vascular endothelial cell specializations and stabilization of the BBB (Ferrari et al., 2009;Nguyen et al., 2011;Pepper, 1997). Deficits in components of the TGF-β signaling pathway result in death during the embryonic period, due to impaired development of the vasculature, characterized by the presence of hyper-dilated vessels and intracerebral haemorrhages (Arthur et al., 2000;Larsson et al., 2001;Li et al., 1999;Nguyen et al., 2011). In addition, Anderson et al. (2011) showed that TGF-β1 can up-regulate Gpr124 expression in CNS blood vessels, suggesting that this cytokine is important in the early stages of vasculature formation. ...
Ethanol consumption during pregnancy or lactation permanently impairs the development of the central nervous system (CNS), resulting in the spectrum of fetal alcohol disorders (FASD). FASD is a general term that covers a set of deficits in the embryo caused by gestational alcohol exposure, with fetal alcohol syndrome (FAS) considered the most serious. The clinical features of FAS include facial abnormalities, short stature, low body weight, and evidence of structural and/or functional damage to the central nervous system (CNS). The prevalence of FAS carriers worldwide is about 15 for every 10,000 live births (about 119,000 children with APS born per year). Epidemiological data in the US show that the incidence of FAS exceeds other congenital syndromes such as Down syndrome and spina bifida. The deleterious effects of ethanol appear in different brain regions, varying according to the dose and period of neural development when the embryo was exposed, and include: 1) microcephaly; 2) abnormalities in cortical development, with a significant decrease in gyrification; 3) agenesis or hypoplasia of the corpus callosum; and 4) cognitive and behavioral deficits (such as impaired memory and learning, speech difficulties, and hyperactivity). Current evidence indicates that CNS blood vessels are particularly affected by teratogenic ethanol. The CNS vasculature is composed of specialized endothelial cells that establish intimate interactions with astrocytes, pericytes, and microglia, constituting the neurovascular unit of the blood-brain barrier (BBB). Together with the fact that BBB exert protective function, it can prevent the passage of substances and drugs to treat diseases that affect the CNS. Pathological changes in the BBB, such as drug abuse during pregnancy, congenital infections, or ageing processes can drastically alter the molecular structure and vascular stability, disrupting the BBB and aggravating certain neurodegenerative and neurological diseases. In this review, we address the effects of alcohol exposure on the formation of the BBB, specifically describing the cellular and molecular events induced by ethanol in the physiology of endothelial cells and glial cells, as well as their interaction during CNS development.
... Studies have shown that mice lacking endoglin die from defective vascular development [8,29,30]. Endoglin has been found to be expressed in cardiac fibroblasts and to modulate the profibrogenic actions of angiotensin II (Ang II) [4,6]. Several studies have reported that the circulating maternal autoantibody agonist of the angiotensin II type-I receptor agonistic autoantibody (AT1-AA) has an important role in preeclampsia pathophysiology and that tumour necrosis factor (TNF)-α is an essential mediator for this autoantibody. ...
... Several investigations were conducted to elucidate the specific mechanisms on how ENG contributes to maintain proper arteriovenous development. ENG knocked-out mice, which were generated in separate laboratories, showed the formation of AVM and died at around midgestation with some grossly defective vasculature (Arthur et al., 2000;Bourdeau et al., 1999;Li et al., 1999). Mice lacking functional endoglin showed a reduction of smooth muscle coverage of the dorsal aorta in early embryonic development (Bourdeau et al., 1999;Gougos and Letarte, 1990;Li et al., 1999). ...
Genetic compensation is a remarkable biological concept to explain the genetic robustness in an organism to maintain its fitness and viability if there is a disruption occurred in the genetic variation by mutation. However, the underlying mechanism in genetic compensation remain unsolvable. The initial concept of genetic compensation has been studied in model organisms when there was a discrepancy between knockout-mediated and knockdown-mediated phenotypes. In the zebrafish model, several studies have reported that zebrafish mutants did not exhibit severe phenotype as shown in zebrafish morphants for the same genes. This phenomenon in zebrafish mutants but not morphants is due to the response of genetic compensation. In 2019, two amazing works partially uncovered genetic compensation could be triggered by the upregulation of compensating genes through regulating NMD and/or PTC-bearing mRNA in collaboration with epigenetic machinery in mutant zebrafish. In this review, we would like to update the recent advances and future perspectives of genetic compensation studies, which including the hypothesis of time-dependent involvement and addressing the discrepancy between knockout-mediated and knockdown-mediated to study gene function in the zebrafish model. At last, the study of genetic compensation could be a potential therapeutic strategy to treat human genetic disorder related diseases.
... 50 Endoglin is critical for embryogenesis as endoglin-knockout mouse models fail to progress beyond 10.5 days of embryonic development, displaying vascular and cardiac abnormalities and anemia of the yolk sac. 51 On the other hand, doxycycline-controlled upregulation of endoglin in ES cells enhances primitive hematopoietic differentiation and suppresses cardiac differentiation. 46 Enforced expression of endoglin in ES cells hampers GSK3b activity that is dependent on Wnt/b-catenin signaling. ...
Myeloid dysplastic syndrome (MDS) reflects a preleukemic BM disorder with limited treatment options and poor disease survival1. As only a minority of MDS patients is eligible to curative hematopoietic stem cell (HSC) transplantation, there is an urgent need to develop alternative treatment options. Chronic activation of Wnt/β-catenin has been implicated to underlie MDS formation and recently assigned to drive MDS transformation to acute myeloid leukemia (AML). Wnt/β-catenin signaling therefore may harbor a pharmaceutical target to treat MDS and/or prevent leukemia formation. However, targeting the Wnt/β-catenin pathway will also affect healthy hematopoiesis in MDS patients.The control of Wnt/β-catenin on healthy hematopoiesis is poorly understood. Whereas Wnt/β-catenin is dispensable for steady-state erythropoiesis, its activity is essential for stress erythropoiesis in response to BM injury and anemia. Manipulation of Wnt/β-catenin signaling in MDS may therefore deregulate stress erythropoiesis and even increase anemia severity. Here we provide a comprehensive overview of the most recent and established insights in the field to acquire more insight into the control of Wnt/β-catenin signaling on healthy and inefficient erythropoiesis as seen in MDS.
... In initial studies, total loss of HHT genes Eng (Eng -− ) or Alk1 (Alk1 −/− ) resulted in embryonic lethality in mice [40,[73][74][75]. Both Eng −/− and Alk1 −/− yolk sacs contained enlarged, fragile vessels. ...
Arteriovenous malformations are a vascular anomaly typically present at birth, characterized by an abnormal connection between an artery and a vein (bypassing the capillaries). These high flow lesions can vary in size and location. Therapeutic approaches are limited, and AVMs can cause significant morbidity and mortality. Here, we describe our current understanding of the pathogenesis of arteriovenous malformations based on preclinical and clinical findings. We discuss past and present accomplishments and challenges in the field and identify research gaps that need to be filled for the successful development of therapeutic strategies in the future.
... Endoglin (ENG or CD105) is a homodimeric glycoprotein, expressed in endothelial cells, bone marrow cells, and macrophages, and involved in embryogenesis, angiogenesis, and vascular establishment as well as homeostasis [59,60]. In tumors its presence is associated with neo-angiogenesis, which represents a key feature of malignant cancer. ...
Sarcomas are one of the most difficult type of cancer to manage and treat because of their extremely heterogeneous molecular and morphological features. Despite the progress made over the years in the establishment of standard protocols for high and low grading/staging sarcoma patients, mostly with chemotherapy and/or radiotherapy, 50% of treated patients experience relapse episodes. Because of this, in the last 20 years, new therapeutic approaches for sarcoma treatment have been evaluated in preclinical and clinical studies. Among them, antibody-based therapies have been the most studied. Immunoconjugates consist of a carrier portion, frequently represented by an antibody, linked to a toxic moiety, i.e., a drug, toxin, or radionuclide. While the efficacy of immunoconjugates is well demonstrated in the therapy of hematological tumors and more recently also of epithelial ones, their potential as therapeutic agents against sarcomas is still not completely explored. In this paper, we summarize the results obtained with immunoconjugates targeting sarcoma surface antigens, considering both preclinical and clinical studies. To date, the encouraging results obtained in preclinical studies allowed nine immunoconjugates to enter clinical trials, demonstrating the validity of immunotherapy as a promising pharmacological tool also for sarcoma therapy.
... Endoglin (CD105) is also known as a TGFβ type III receptor that modulates TGFβ signaling by forming a complex with type I and type II receptors (Cheifetz et al., 1992;Lastres et al., 1996;Zhang et al., 1996). Endoglin is primarily known to be associated with angiogenesis and vascular development and has been identified as a genetic factor for hereditary hemorrhagic telangiectasis (Arthur et al., 2000;Bourdeau et al., 1999;Li et al., 1999). Subsequently, endoglin expression on interstitial fibroblasts (St-Jacques et al., 1994), interstitial and peritoneal macrophages (Lastres et al., 1992;Ojeda-Fernández et al., 2016), mesenchymal stem cells (Jones et al., 2002) and osteogenic populations in common skeletal progenitor cells (Chan et al., 2013) has been reported. ...
Bone morphogenic protein‐9 (BMP9), a member of the transforming growth factor β (TGFβ) superfamily, plays important roles in the development and maintenance of various cell lineages via complexes of type I and type II TGFβ receptors. Endoglin is a coreceptor for several TGFβ family members, including BMP9, which is highly expressed in a particular stage of differentiation in erythroid cells as well as in endothelial cells. Whereas the importance of the interaction between BMP9 and endoglin for endothelial development has been reported, the contribution of BMP9 to endoglin‐expressing erythroid cells remains to be clarified. To address this point, we prepared an anti‐BMP9 antibody that blocks the BMP9‐endoglin interaction. Of note, challenge with the antibody promotes erythropoiesis in wild‐type mice but not in a mouse model of renal anemia in which erythropoietin (EPO) production in the kidneys is genetically ablated. While endoglin‐positive erythroid progenitors are mainly maintained as progenitors when bone marrow‐derived lineage‐negative and cKit‐positive cells are cultured in the presence of EPO and stem cell factor, the erythroid‐biased accumulation of progenitors is impeded by the presence of BMP9. Our findings uncover an unrecognized role for BMP9 in attenuating erythroid differentiation via its interaction with endoglin on erythroid progenitors
... Several investigations were conducted to elucidate the specific mechanisms on how ENG contributes to maintain proper arteriovenous development. ENG knocked-out mice, which were generated in separate laboratories, showed the formation of AVM and died at around midgestation with some grossly defective vasculature (Arthur et al., 2000;Bourdeau et al., 1999;Li et al., 1999). Mice lacking functional endoglin showed a reduction of smooth muscle coverage of the dorsal aorta in early embryonic development (Bourdeau et al., 1999;Gougos and Letarte, 1990;Li et al., 1999). ...
Arteriovenous malformations are congenital vascular lesions characterized by a direct and tangled connection between arteries and veins, which disrupts oxygen circulation and normal blood flow. Arteriovenous malformations often occur in the patient with hereditary hemorrhagic telangiectasia. The attempts to elucidate the causative factors and pathogenic mechanisms of arteriovenous malformations are now still in progress. Some studies reported that shear stress in blood flow is one of the factors involved in arteriovenous malformations manifestation. Through several mechanotransducers harboring the endothelial cells membrane, the signal from shear stress is transduced towards the responsible signaling pathways in endothelial cells to maintain cell homeostasis. Any disruption in this well-established communication will give rise to abnormal endothelial cells differentiation and specification, which will later promote arteriovenous malformations. In this review, we discuss the update of several mechanotransducers that have essential roles in shear stress-induced signaling pathways, such as activin receptor-like kinase 1, Endoglin, Notch, vascular endothelial growth factor receptor 2, Caveolin-1, Connexin37, and Connexin40. Any disruption of these signaling potentially causes arteriovenous malformations. We also present some recent insights into the fundamental analysis, which attempts to determine potential and alternative solutions to battle arteriovenous malformations, especially in a less invasive and risky way, such as gene treatments.
... Mice carrying mutations on both alleles of Eng or Alk1 genes were embryonic lethal and showed obvious defects in angiogenesis and cardiac development. 73,74 However, mice with heterozygous mutations in either of these genes can survive to adult stage and recapitulate relatively mild phenotypes seen in HHT patients, 74,75 suggesting that additional factors, such as mutation on the other allele (second hit) and environmental mediators are required for bAVM development. Moreover, morpholino-induced knockdown of Eng or Alk1 in zebrafish models recapitulate the morphologic, functional, and molecular defects seen in human AVMs, allowing visualization of precise spatiotemporal patterns during vascular development. ...
Brain arteriovenous malformations (bAVM) are an important cause of intracranial hemorrhage (ICH), especially in younger patients. The pathogenesis of bAVM are largely unknown. Current understanding of bAVM etiology is based on studying genetic syndromes, animal models, and surgically resected specimens from patients. The identification of activating somatic mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene and other mitogen-activated protein kinase ( MAPK) pathway genes has opened up new avenues for bAVM study, leading to a paradigm shift to search for somatic, de novo mutations in sporadic bAVMs instead of focusing on inherited genetic mutations. Through the development of new models and understanding of pathways involved in maintaining normal vascular structure and functions, promising therapeutic targets have been identified and safety and efficacy studies are underway in animal models and in patients. The goal of this paper is to provide a thorough review or current diagnostic and treatment tools, known genes and key pathways involved in bAVM pathogenesis to summarize current treatment options and potential therapeutic targets uncovered by recent discoveries.
... Heterozygous mutations of Eng or Alk1 genes give rise to vascular lesions forming at low frequencies and later in life, making them inconvenient models for further study [86,87]. Constitutive inactivation of Eng, Alk1 and Smad4 genes in mice leads to embryonic lethality due to multiple cardiovascular defects, again rendering the study of molecular mechanisms difficult [58,88,89]. However, postnatal tamoxifen-inducible, EC-specific homozygous deletion of any of these genes induces HHT-like vascular malformations, including excessive angiogenesis, enlarged veins and AV shunts in the neonatal retinal vascularization model [84,85,90,91]. ...
There are two vascular networks in mammals that coordinately function as the main supply and drainage systems of the body. The blood vasculature carries oxygen, nutrients, circulating cells, and soluble factors to and from every tissue. The lymphatic vasculature maintains interstitial fluid homeostasis, transports hematopoietic cells for immune surveillance, and absorbs fat from the gastrointestinal tract. These vascular systems consist of highly organized networks of specialized vessels including arteries, veins, capillaries, and lymphatic vessels that exhibit different structures and cellular composition enabling distinct functions. All vessels are composed of an inner layer of endothelial cells that are in direct contact with the circulating fluid; therefore, they are the first responders to circulating factors. However, endothelial cells are not homogenous; rather, they are a heterogenous population of specialized cells perfectly designed for the physiological demands of the vessel they constitute. This review provides an overview of the current knowledge of the specification of arterial, venous, capillary, and lymphatic endothelial cell identities during vascular development. We also discuss how the dysregulation of these processes can lead to vascular malformations, and therapeutic approaches that have been developed for their treatment.
... In mammals, it is important for vasculogenesis, as TβRIII knockout mice are embryonic lethal at day 14.5 due to defective vasculogenesis (Compton et al., 2007). Endoglin, the most abundant TGF-β superfamily coreceptor in ECs, regulates differentiation and angiogenesis (Li et al., 1999;Arthur et al., 2000), and mutations in endoglin cause HHT1 (McAllister et al., 1994;Lebrin et al., 2005). At the cell surface, endoglin can bind TGF-β1/3 and BMP9/10 (and other BMPs to a lesser extent) and can interact with signaling TGF-β superfamily receptors (Bernabeu et al., 2009;Alt et al., 2012). ...
Complex formation and endocytosis of transforming growth factor-β (TGF-β) receptors play important roles in signaling. However, their inter-dependence remained unexplored. Here, we demonstrate that ALK1, a TGF-β type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-β receptors (TβRIII).We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-β receptor (TβRII), endoglin or TβRIII. These complexes regulate the endocytosis of the TGF-β receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TβRIII and endoglin, while ALK5 and TβRII mildly enhance endoglin, but not TβRIII, internalization. Conversely, the slowly-endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5 or TβRII, while TβRIII has a differential effect, slowing down the internalization of ALK5 and TβRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-β receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications on signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.
... Different Eng or Acvrl1 genetic mouse models of HHT have been described by several groups during the last three decades [45]. Mice lacking functional endoglin [46][47][48] or ALK1 [49,50] were generated by germline gene-targeting. In all cases, global embryonic loss of endoglin or ALK1 expression leads to cardiovascular defects, enlarged fragile vessels, and embryonic lethality by mid-gestation. ...
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder that presents with telangiectases in skin and mucosae, and arteriovenous malformations (AVMs) in internal organs such as lungs, liver, and brain. Mutations in ENG (endoglin), ACVRL1 (ALK1), and MADH4 (Smad4) genes account for over 95% of HHT. Localized telangiectases and AVMs are present in different organs, with frequencies which differ among affected individuals. By itself, HHT gene heterozygosity does not account for the focal nature and varying presentation of the vascular lesions leading to the hypothesis of a “second-hit” that triggers the lesions. Accumulating research has identified a variety of triggers that may synergize with HHT gene heterozygosity to generate the vascular lesions. Among the postulated second-hits are: mechanical trauma, light, inflammation, vascular injury, angiogenic stimuli, shear stress, modifier genes, and somatic mutations in the wildtype HHT gene allele. The aim of this review is to summarize these triggers, as well as the functional mechanisms involved.
... We previously reported that intraplaque hypoxia accelerates abnormal microvessel formation, which in turn promotes IPH [11]. Other studies have also suggested that reduced CD105 gene expression in human vascular ECs inhibits angiogenesis, and that CD105-null mice exhibit impaired vasculature formation [2,14]. The results of the present study align with these previous findings that a high ratio of endothelial expression of endoglin (CD105) and Ki67 characterizes dilated microvessels, and that dilation is a marker of active angiogenesis. ...
Background:
The purpose of the present study was to clarify the characteristics of endothelial cell (EC) proliferation in intraplaque microvessels in vulnerable plaques and impact on clinical results.
Methods:
The present study included 76 patients who underwent carotid endarterectomy. Patients were classified into three groups based on their symptoms: asymptomatic, symptomatic without recurrent ischemic event, and symptomatic with recurrent ischemic event. MR plaque imaging was performed and surgical specimens underwent immunohistochemical analysis. The number of CD31+ microvessels, and Ki67+ and CD105+ ECs in the carotid plaques was quantified, as measurements of maximum CD31+ microvessel diameter.
Results:
MR plaque imaging yielded 41 subjects (54.0%) diagnosed with plaque with intraplaque hemorrhage (IPH), 14 subjects (18.4%) diagnosed with fibrous plaques, and 21 (27.6%) subjects diagnosed with lipid-rich plaques. The average largest diameter of microvessel in fibrous plaques, lipid-rich plaques, and plaque with IPH was 12.7 ± 4.1 μm, 31.3 ± 9.3 μm, and 56.4 ± 10.0 μm, respectively (p < 0.01). Dilated microvessels (>40 μm) were observed in 9.6% of plaques with IPH but only in 2.8% of lipid-rich plaques and 0% of fibrous plaques (p < 0.01). Ki67+/CD31+ ECs were identified in 2.8 ± 1.2% of fibrous plaques, 9.6 ± 6.9% of lipid-rich plaques, and in 19.5 ± 5.9% of plaques with IPH (p < 0.01). The average largest diameter of microvessels in the asymptomatic group was 17.1 ± 8.7 μm, 32.3 ± 10.8 μm in the symptomatic without recurrence group, and 55.2 ± 13.2 μm in the symptomatic with recurrence group (p < 0.01).
Conclusion:
Dilated microvessels with proliferative ECs may play a key role in IPH pathogenesis. Furthermore, dilated microvessels are likely related to clinical onset and the recurrence of ischemic events. The purpose of the present study was to clarify the characteristics of EC proliferation in intraplaque microvessels in vulnerable plaques and their impact on clinical results, focusing on dilated intraplaque microvessels.
... Elle joue un rôle complexe de modulateur. Un déficit complet est fatal pour l'embryon par défaut d'angiogénèse et du développement cardiovasculaire (Li et al., 1999;Arthur et al., 2000), alors qu'à l'inverse, un excès d'endogline se retrouve dans les situations de néovascularisation (tumeur, inflammation, …) (Torsney et al., 2002). Pendant la grossesse, les STs ainsi que les monocytes expriment fortement l'endogline (St-Jacques et al., 1994). ...
Résumé : Le sFlt1 semble être un bon marqueur de la prééclampsie (PE). Nous avons évalué dans le cadre d’un essai randomisé contrôlé (MOMA), l’impact sur l’issue de grossesse d’un suivi rapproché de femmes identifiées comme « à risque » par un taux élevé de sFlt1 plasmatique entre 24 et 29 SA. Dans cet essai 939 nullipares ont été incluses en 2 bras (sflt1 connu ou non connu). Nos résultats ne montrent pas d’amélioration de l’issue de grossesse et suggèrent que l’inefficience de l’intervention (suivi rapproché) en est la cause la plus probable. Aussi la mesure du sFlt1 pour prédire la survenue d'une PE ne semble pas utile tant qu’un traitement efficace n’est pas à disposition. Des anomalies de la stéroïdogénèse placentaire ont été retrouvées associées à la PE. 90 échantillons issus de la cohorte MOMA répartis en trois groupes (25 PE, 25 retard de croissance intra utérin (RCIU) sans PE et 40 contrôles) ont été utilisés pour évaluer le profil stéroïdes par la technique fiable de GC/MS (entre 24 et 29 SA) - soit avant les signes cliniques -. Nous avons mis en évidence dans le groupe PE un défaut d'aromatisation des androgènes par le calcul du ratio estradiol/Δ4-Dione (sang) et un défaut d’expression de l’aromatase (placenta). Tout comme les taux significativement bas de sulfate de prégnénolone retrouvés, ce défaut d’aromatisation semble spécifique de la PE car ils n’ont pas été observés dans le groupe RCIU. Ces modifications s'inscrivent dans une dérégulation du profil stéroïdien global. Nous avons en effet observé dans les groupes PE et RCIU, un excés de 20α-dihydro-progestérone (20α-DHP) et un ratio 20α-DHP/Progestérone significativement élevé. Les stéroïdes sexuels partagent des voies de signalisation communes avec les facteurs angiogéniques, faisant inscrire aisément l’hypothèse « anomalies de la stéroïdogénèse » dans les concepts actuels de la physiopathologie de la PE. Les liens entre perturbations de la stéroïdogénèse et la prééclampsie sont discutés.
... Endoglin can bind to and modulate activity of many type I and type II Tgf-β receptors [55]. Endoglin null embryos display abnormally enlarged ventricles, suggesting an important role for Endoglin in cardiac development, and potentially in inhibition of CM proliferation [56]. However, the effect of Endoglin on Tgf-β stimulation varies substantially by cell type. ...
Purpose of review:
The loss of contractile function after heart injury remains one of the major healthcare issues of our time. One strategy to deal with this problem would be to increase the number of cardiomyocytes to enhance cardiac function. In the last couple of years, reactivation of cardiomyocyte proliferation has repeatedly demonstrated to aid in functional recovery after cardiac injury.
Recent findings:
The Tgf-β superfamily plays key roles during development of the heart and populating the embryonic heart with cardiomyocytes. In this review, we discuss the role of Tgf-β signaling in regulating cardiomyocyte proliferation during development and in the setting of cardiac regeneration. Although various pathways to induce cardiomyocyte proliferation have been established, the extent to which cardiomyocyte proliferation requires or involves activation of the Tgf-β superfamily is not entirely clear. More research is needed to better understand cross-talk between pathways that regulate cardiomyocyte proliferation.
... Dans un premier temps, les premiers modèles furent des souris invalidées pour soit Acvrl1 -/ou Eng -/-. La perte totale d'Endogline conduisait à des défauts cardiovasculaires et la mort embryonnaire en cours de gestation (Bourdeau, 1999;Li, 1999;Arthur, 2000), montrant l'importance de l'Endogline dans l'angiogénèse et la mise en place du système cardiaque. On constate des phénotypes semblables chez des souris invalidées en Acvrl1 (Oh, 2000). ...
BMP9 est un ligand de la famille TGF-β et considéré comme un facteur de quiescence vasculaire. Différents acteurs de la voie de signalisation de BMP9 comme les récepteurs ALK1, et BMPR2 sont mutés dans deux pathologies vasculaires rares, la maladie de Rendu-Osler ou Télangiectasie Hémorragique Héréditaire et l’Hypertension Artérielle Pulmonaire, qui sont liées à une dysfonction endothéliale. Les traitements actuels visent à améliorer les symptômes des patients, sans apporter de réelle guérison de la maladie. Aussi, il est primordial aujourd'hui de trouver de nouveaux traitements étiologiques rétablissant cette voie de signalisation. Pour ce faire, j'ai utilisé le criblage à haut débit afin de repositionner de potentiels candidats-médicaments venant des chimiothèques Prestwick et TargetMol, comprenant au total 2133 molécules approuvées par FDA et EMA. Ces criblages ont été effectués sur des modèles de cellules endothéliales rapportant la voie BMP9 grâce à un promoteur sensible aux BMPs (BRE, BMP Response Element). J’ai développé et minaturisé un premier modèle de cellules endothéliales HMEC-1 secrétant une Metridia Luciférase sous le contrôle de BRE, puis un deuxième modèle utilisant une double transfection permettant l’expression de la Firefly Luciférase sous le contrôle de BRE et de la Renilla Luciférase comme contrôle de transfection. Le premier modèle n’a révelé que des faux positifs. Par contre, le deuxième modèle m’a permis d’identifier plusieurs molécules qui ont la particularité d’activer la voie de l’AMPc mais dont l’action n’a pas pu être confirmée au niveau moléculaire. Un dernier crible utilisant des cellules non endothéliales (myoblastes C2C12BRA) a permis d’identifier deux autres molécules qui sont en cours de caractérisation.
... Given that many types of structural and physiological anomalies can lead to deficiencies in cardiac function in utero, our demonstration that cardiac function promotes OFT expansion by regulating both endocardial proliferation and endothelial addition provides new models to consider for the origins of multiple types of OFT defects. Moreover, our identification of a previously unappreciated role for Acvrl1 during OFT morphogenesis raises interest in the potential conservation of this role in mammals, and it is intriguing to note that mice lacking the Acvrl1 co-receptor endoglin exhibit misshapen OFT morphology (Arthur et al., 2000). Altogether, our work provides an important step forward in connecting cardiac function to specific mechanosensitive pathways and the cell behaviors that they regulate, and significantly advances our understanding of the many ways in which form follows function during organogenesis. ...
Physical forces are important participants in the cellular dynamics that shape developing organs. During heart formation, for example, contractility and blood flow generate biomechanical cues that influence patterns of cell behavior. Here, we address the interplay between function and form during the assembly of the cardiac outflow tract (OFT), a crucial connection between the heart and vasculature that develops while circulation is underway. In zebrafish, we find that the OFT expands via accrual of both endocardial and myocardial cells. However, when cardiac function is disrupted, OFT endocardial growth ceases, accompanied by reduced proliferation and reduced addition of cells from adjacent vessels. The flow-responsive TGFβ receptor Acvrl1 is required for addition of endocardial cells, but not for their proliferation, indicating distinct modes of function-dependent regulation for each of these essential cell behaviors. Together, our results indicate that cardiac function modulates OFT morphogenesis by triggering endocardial cell accumulation that induces OFT lumen expansion and shapes OFT dimensions; moreover, these morphogenetic mechanisms provide new perspectives regarding the potential causes of cardiac birth defects.
... Genetic mouse models of bAVM have been mostly developed by manipulating Eng or Alk1 genes. Eng +/− or Alk1 +/− heterozygous knockout (KO) mice are viable and show HHT phenotypes during adulthood, while Eng −/− or Alk1 −/− homozygous KO mice are embryonic lethal [38,[54][55][56][57][58][59]. bAVMs, including AV shunts and niduses of dilated vessels, occurred in only 30% of Eng +/− mice aged 25 to 40 weeks with incomplete penetrance [56]. ...
Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.
For monogenic diseases caused by pathogenic loss-of-function DNA variants, attention focuses on dysregulated gene-specific pathways, usually considering molecular subtypes together within causal genes. To better understand phenotypic variability in hereditary hemorrhagic telangiectasia (HHT), we sub-categorized pathogenic DNA variants in ENG/endoglin, ACVRL1/ALK1, and SMAD4 if they generated premature termination codons (PTCs) subject to nonsense mediated decay. In three pre-phenotyped patient cohorts, a PTC-based classification system explained some previously puzzling hemorrhage variability. In blood outgrowth endothelial cells (BOECs) derived from ACVRL1+/PTC, ENG+/PTC, and SMAD4+/PTC patients, PTC-containing RNA transcripts persisted at low levels (8-23% expected, varying between replicate cultures); genes differentially expressed to Bonferroni p<0.05 in HHT+/PTC BOECs clustered significantly only to generic protein terms ('isopeptide-bond'/'ubiquitin-like conjugation') and pulse chase experiments detected subtle protein maturation differences, but no evidence for PTC-truncated protein. BOECs displaying highest PTC persistence were discriminated in unsupervised hierarchical clustering of 'invariant' housekeeper genes, with patterns compatible with higher cellular stress in BOECs with >11% PTC persistence. To test directionality, we used a HeLa reporter system to detect induction of activating transcription factor (ATF)4 which controls expression of stress-adaptive genes, and showed that ENG Q436X but not ENG R93X directly induced ATF4. AlphaFold accurately modelled relevant ENG domains, with AlphaMissense suggesting that readthrough substitutions would be benign for ENG R93X and other "less rare" ENG nonsense variants, but more damaging for Q436X. We conclude that PTCs should be distinguished from other loss-of-function variants, PTC transcript levels increase in stressed cells, and readthrough proteins and mechanisms provide promising research avenues.
Hereditary hemorrhagic telangiectsia (HHT) is an inherited vascular disorder with highly variable expressivity, affecting up to 1 in 5,000 individuals. This disease is characterized by small arteriovenous malformations (AVMs) in mucocutaneous areas (telangiectases) and larger visceral AVMs in the lungs, liver, and brain. HHT is caused by loss-of-function mutations in the BMP9-10/ENG/ALK1/SMAD4 signaling pathway. This Review presents up-to-date insights on this mutated signaling pathway and its crosstalk with proangiogenic pathways, in particular the VEGF pathway, that has allowed the repurposing of new drugs for HHT treatment. However, despite the substantial benefits of these new treatments in terms of alleviating symptom severity, this not-so-uncommon bleeding disorder still currently lacks any FDA- or European Medicines Agency–approved (EMA-approved) therapies.
Hereditary Haemorrhagic Telangiectasia (HHT) causes arteriovenous malformations (AVMs) in multiple organs to cause bleeding, neurological and other complications. HHT is caused by mutations in the BMP co-receptor endoglin. We characterised a range of vascular phenotypes in embryonic and adult endoglin mutant zebrafish and the effect of inhibiting different pathways downstream of VEGF signalling.
Adult endoglin mutant zebrafish developed skin AVMs, retinal vascular abnormalities, and cardiac enlargement. Embryonic endoglin mutants develop an enlarged basilar artery (similar to the previously described enlarged aorta and cardinal vein) and larger numbers of endothelial membrane cysts (kugeln) on cerebral vessels. VEGF inhibition prevented these embryonic phenotypes, leading us to investigate specific VEGF-signalling pathways. Inhibiting TOR or MEK pathways prevented abnormal trunk and cerebral vasculature phenotypes, while inhibiting NOS or MAPK pathways had no effect. Combined subtherapeutic TOR and MEK inhibition prevented vascular abnormalities, confirming synergy between these pathways in HHT.
These results indicate the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling. Combined low dose MEK and TOR pathway inhibition may represent a novel therapeutic strategy in HHT.
Bone morphogenetic proteins (BMPs) are secretory proteins belonging to the transforming growth factor-β (TGF-β) superfamily. These proteins play important roles in embryogenesis, bone morphogenesis, blood vessel remodeling and the development of various organs. In recent years, as research has progressed, BMPs have been found to be closely related to cardiovascular diseases, especially atherosclerosis, vascular calcification, cardiac remodeling, pulmonary arterial hypertension (PAH) and hereditary hemorrhagic telangiectasia (HHT). In this review, we summarized the potential roles and related mechanisms of the BMP family in the cardiovascular system and focused on atherosclerosis and PAH.
Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by vessel dilatation, such as telangiectasia in skin and mucosa and arteriovenous malformations (AVM) in internal organs such as the gastrointestinal tract, lungs, and brain. AVMs are fragile and tortuous vascular anomalies that directly connect arteries and veins, bypassing healthy capillaries. Mutations in transforming growth factor β (TGFβ) signaling pathway components, such as ENG (ENDOGLIN), ACVRL1 (ALK1), and SMAD4 (SMAD4) genes, account for most of HHT cases. 10–20% of HHT patients develop brain AVMs (bAVMs), which can lead to vessel wall rupture and intracranial hemorrhages. Though the main mutations are known, mechanisms leading to AVM formation are unclear, partially due to lack of animal models. Recent mouse models allowed significant advances in our understanding of AVMs. Endothelial-specific deletion of either Acvrl1, Eng or Smad4 is sufficient to induce AVMs, identifying endothelial cells (ECs) as primary targets of BMP signaling to promote vascular integrity. Loss of ALK1/ENG/SMAD4 signaling is associated with NOTCH signaling defects and abnormal arteriovenous EC differentiation. Moreover, cumulative evidence suggests that AVMs originate from venous ECs with defective flow-migration coupling and excessive proliferation. Mutant ECs show an increase of PI3K/AKT signaling and inhibitors of this signaling pathway rescue AVMs in HHT mouse models, revealing new therapeutic avenues. In this review, we will summarize recent advances and current knowledge of mechanisms controlling the pathogenesis of bAVMs, and discuss unresolved questions.
Blood vessels expand via sprouting angiogenesis, and this process involves numerous endothelial cell behaviors, such as collective migration, proliferation, cell–cell junction rearrangements, and anastomosis and lumen formation. Subsequently, blood vessels remodel to form a hierarchical network that circulates blood and delivers oxygen and nutrients to tissue. During this time, endothelial cells become quiescent and form a barrier between blood and tissues that regulates transport of liquids and solutes. Bone morphogenetic protein (BMP) signaling regulates both proangiogenic and homeostatic endothelial cell behaviors as blood vessels form and mature. Almost 30 years ago, human pedigrees linked BMP signaling to diseases associated with blood vessel hemorrhage and shunts, and recent work greatly expanded our knowledge of the players and the effects of vascular BMP signaling. Despite these gains, there remain paradoxes and questions, especially with respect to how and where the different and opposing BMP signaling outputs are regulated. This review examines endothelial cell BMP signaling in vitro and in vivo and discusses the paradox of BMP signals that both destabilize and stabilize endothelial cell behaviors.
OBJECTIVE
Hereditary hemorrhagic telangiectasia is the only condition associated with multiple inherited brain arteriovenous malformations (AVMs). Therefore, a mouse model was developed with a genetics-based approach that conditionally deleted the causative activin receptor-like kinase 1 ( Acvrl1 or Alk1 ) gene. Radiographic and histopathological findings were correlated, and AVM stability and hemorrhagic behavior over time were examined.
METHODS
Alk1 -floxed mice were crossed with deleter mice to generate offspring in which both copies of the Alk1 gene were deleted by Tagln -Cre to form brain AVMs in the mice. AVMs were characterized using MRI, MRA, and DSA. Brain AVMs were characterized histopathologically with latex dye perfusion, immunofluorescence, and Prussian blue staining.
RESULTS
Brains of 55 Tagln -Cre ⁺ ; Alk1 2f/2f mutant mice were categorized into three groups: no detectable vascular lesions (group 1; 23 of 55, 42%), arteriovenous fistulas (AVFs) with no nidus (group 2; 10 of 55, 18%), and nidal AVMs (group 3; 22 of 55, 40%). Microhemorrhage was observed on MRI or MRA in 11 AVMs (50%). AVMs had the angiographic hallmarks of early nidus opacification, a tangle of arteries and dilated draining veins, and rapid shunting of blood flow. Latex dye perfusion confirmed arteriovenous shunting in all AVMs and AVFs. Microhemorrhages were detected adjacent to AVFs and AVMs, visualized by iron deposition, Prussian blue staining, and macrophage infiltration using CD68 immunostaining. Brain AVMs were stable on serial MRI and MRA in group 3 mice (mean age at initial imaging 2.9 months; mean age at last imaging 9.5 months).
CONCLUSIONS
Approximately 40% of transgenic mice satisfied the requirements of a stable experimental AVM model by replicating nidal anatomy, arteriovenous hemodynamics, and microhemorrhagic behavior. Transgenic mice with AVFs had a recognizable phenotype of hereditary hemorrhagic telangiectasia but were less suitable for experimental modeling. AVM pathogenesis can be understood as the combination of conditional Alk1 gene deletion during embryogenesis and angiogenesis that is hyperactive in developing and newborn mice, which translates to a congenital origin in most patients but an acquired condition in patients with a confluence of genetic and angiogenic events later in life. This study offers a novel experimental brain AVM model for future studies of AVM pathophysiology, growth, rupture, and therapeutic regression.
At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2), contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury which impairs angiogenesis, induced both common and unique endothelial gene signatures, dysregulated capillary EC cross-talk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.
Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor β1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphor-AKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and -2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis.
Inhibins and activins are dimeric ligands belonging to the TGFβ superfamily with emergent roles in cancer. Inhibins contain an α-subunit ( INHA ) and a β-subunit (either INHBA or INHBB ), while activins are mainly homodimers of either β A ( INHBA ) or β B ( INHBB ) subunits. Inhibins are biomarkers in a subset of cancers and utilize the coreceptors betaglycan ( TGFBR3 ) and endoglin ( ENG ) for physiological or pathological outcomes. Given the array of prior reports on inhibin, activin and the coreceptors in cancer, this study aims to provide a comprehensive analysis, assessing their functional prognostic potential in cancer using a bioinformatics approach. We identify cancer cell lines and cancer types most dependent and impacted, which included p53 mutated breast and ovarian cancers and lung adenocarcinomas. Moreover, INHA itself was dependent on TGFBR3 and ENG/CD105 in multiple cancer types. INHA , INHBA , TGFBR3 , and ENG also predicted patients’ response to anthracycline and taxane therapy in luminal A breast cancers. We also obtained a gene signature model that could accurately classify 96.7% of the cases based on outcomes. Lastly, we cross-compared gene correlations revealing INHA dependency to TGFBR3 or ENG influencing different pathways themselves. These results suggest that inhibins are particularly important in a subset of cancers depending on the coreceptor TGFBR3 and ENG and are of substantial prognostic value, thereby warranting further investigation.
Transforming growth factor‐β (TGF‐β) superfamily signaling via their cognate receptors is frequently modified by TGF‐β superfamily co‐receptors. Signaling through SMAD‐mediated pathways may be enhanced or depressed depending on the specific co‐receptor and cell context. This dynamic effect on signaling is further modified by the release of many of the co‐receptors from the membrane to generate soluble forms that are often antagonistic to the membrane‐bound receptors. The co‐receptors discussed here include TβRIII (betaglycan), endoglin, BAMBI, CD109, SCUBE proteins, neuropilins, Cripto‐1, MuSK, and RGMs. Dysregulation of these co‐receptors can lead to altered TGF‐β superfamily signaling that contributes to the pathophysiology of many cancers through regulation of growth, metastatic potential, and the tumor microenvironment. Here we describe the role of several TGF‐β superfamily co‐receptors on TGF‐β superfamily signaling and the impact on cellular and physiological functions with a particular focus on cancer, including a discussion on recent pharmacological advances and potential clinical applications targeting these co‐receptors.
A spotlight has been shone on endoglin in recent years due to that fact of its potential to serve as both a reliable disease biomarker and a therapeutic target. Indeed, endoglin has now been assigned many roles in both physiological and pathological processes. From a molecular point of view, endoglin mainly acts as a co-receptor in the canonical TGFβ pathway, but also it may be shed and released from the membrane, giving rise to the soluble form, which also plays important roles in cell signaling. In cancer, in particular, endoglin may contribute to either an oncogenic or a non-oncogenic phenotype depending on the cell context. The fact that endoglin is expressed by neoplastic and non-neoplastic cells within the tumor microenvironment suggests new possibilities for targeted therapies. Here, we aimed to review and discuss the many roles played by endoglin in different tumor types, as well as the strong evidence provided by pre-clinical and clinical studies that supports the therapeutic targeting of endoglin as a novel clinical strategy.
Approximately 30 years ago, endoglin was identified as a transforming growth factor (TGF)-β coreceptor with a crucial role in developmental biology and tumor angiogenesis. Its selectively high expression on tumor vessels and its correlation with poor survival in cancer patients led to the exploration of endoglin as a therapeutic target for cancer. The endoglin neutralizing antibody TRC105 (Carotuximab®, Tracon Pharmaceuticals (San Diego, CA, USA) was subsequently tested in a wide variety of preclinical cancer models before being tested in phase I-III clinical studies in cancer patients as both a monotherapy and in combination with other chemotherapeutic and anti-angiogenic therapies. The combined data of these studies have revealed new insights into the role of endoglin in angiogenesis and its expression and functional role on other cells in the tumor microenvironment. In this review, we will summarize the preclinical work, clinical trials and biomarker studies of TRC105 and explore what these studies have enabled us to learn and what questions remain unanswered.
Liver cancer is a global problem and hepatocellular carcinoma (HCC) accounts for about 85% of this cancer. In the USA, etiologies and risk factors for HCC include chronic hepatitis C virus (HCV) infection, diabetes, non-alcoholic steatohepatitis (NASH), obesity, excessive alcohol drinking, exposure to tobacco smoke, and genetic factors. Chronic HCV infection appears to be associated with about 30% of HCC. Chronic HCV infection induces multistep changes in liver, involving metabolic disorders, steatosis, cirrhosis and HCC. Liver carcinogenesis requires initiation of neoplastic clones, and progression to clinically diagnose malignancy. Tumor progression associates with profound exhaustion of tumor-antigen-specific CD8⁺ T cells, and accumulation of PD-1hi CD8⁺ T cells and Tregs. In this chapter, we provide a brief description of HCV and environmental/genetic factors, immune regulation, and highlight mechanisms of HCV associated HCC. We also underscore HCV treatment and recent paradigm of HCC progression, highlighted the current treatment and potential future therapeutic opportunities.
Inhibins and activins are dimeric ligands belonging to the TGFb superfamily with emergent roles in cancer. Inhibins contain an alpha subunit (INHA) and a beta subunit (either INHBA or INHBB), while activins are mainly homodimers of either betaA (INHBA) or betaB (INHBB) subunits. Inhibins are biomarkers in a subset of cancers and utilize the coreceptors betaglycan (TGFBR3) and endoglin (ENG) for physiological or pathological outcomes. Given the array of prior reports on inhibin, activin and the coreceptors in cancer, this study aims to provide a comprehensive analysis, assessing their functional prognostic potential in cancer using a bioinformatics approach. We identify cancer cell lines and cancer types most dependent and impacted, which included p53 mutated breast and ovarian cancers and lung adenocarcinomas. Moreover, INHA itself was dependent on TGFBR3 and ENG in multiple cancer types. INHA, INHBA, TGFBR3, and ENG also predicted patients response to anthracycline and taxane therapy in luminal A breast cancers. We also obtained a gene signature model that could accurately classify 96.7% of the cases based on outcomes. Lastly, we cross compared gene correlations revealing INHA dependency to TGFBR3 or ENG influencing different pathways themselves. These results suggest that inhibins are particularly important in a subset of cancers depending on the coreceptor TGFBR3 and ENG and are of substantial prognostic value, thereby warranting further investigation.
Objective:
Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in AVM development, we sought to identify targets differentially regulated by BMP9 and shear stress. Approach and Results: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused AVM-like vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for SMAD1/5, addition of TNFα (tumor necrosis factor-α), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFα and endothelial-specific compound heterozygosity for SMAD1/5 caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions.
Conclusions:
Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.
Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with beta-glycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with I-125-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta1 (K(D) approximately 50 pM) and TGF-beta3 with high affinity but fails to bind TGF-beta2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent haemorrhage. Linkage for some families has been established to chromosome 9q33-q34. In the present study, endoglin, a transforming growth factor beta (TGF-beta) binding protein, was analysed as a candidate gene for the disorder based on chromosomal location, expression pattern and function. We have identified mutations in three affected individuals: a C to G substitution converting a tyrosine to a termination codon, a 39 base pair deletion and a 2 basepair deletion which creates a premature termination codon. We have identified endoglin as the HHT gene mapping to 9q3 and have established HHT as the first human disease defined by a mutation in a member of the TGF-beta receptor complex.
Transforming growth factor–β (TGF-β) signaling is mediated by a complex of type I (TBRI) and type II (TBRII) receptors. The
type III receptor (TBRIII) lacks a recognizable signaling domain and has no clearly defined role in TGF-β signaling. Cardiac
endothelial cells that undergo epithelial-mesenchymal transformation express TBRIII, and here TBRIII-specific antisera were
found to inhibit mesenchyme formation and migration in atrioventricular cushion explants. Misexpression of TBRIII in nontransforming
ventricular endothelial cells conferred transformation in response to TGF-β2. These results support a model where TBRIII localizes
transformation in the heart and plays an essential, nonredundant role in TGF-β signaling.
To delineate specific developmental roles of transforming growth factor beta_1 (TGF-beta_1) we have disrupted its cognate gene in mouse embryonic stem cells by homologous recombination to generate TGF-beta_1 null mice. These mice do not produce detectable amounts of either TGF-beta_1 RNA or protein. After normal growth for the first 2 weeks they develop a rapid wasting syndrome and die by 3-4 weeks of age. Pathological examination revealed an excessive inflammatory response with massive infiltration of lymphocytes and macrophages in many organs, but primarily in heart and lungs. Many lesions resembled those found in autoimmune disorders, graft-vs.-host disease, or certain viral diseases. This phenotype suggests a prominent role for TGF-beta_1 in homeostatic regulation of immune cell proliferation and extravasation into tissues.
The growth and differentiation factor transforming growth factor-beta2 (TGFbeta2) is thought to play important roles in multiple developmental processes. Targeted disruption of the TGFbeta2 gene was undertaken to determine its essential role in vivo. TGFbeta2-null mice exhibit perinatal mortality and a wide range of developmental defects for a single gene disruption. These include cardiac, lung, craniofacial, limb, spinal column, eye, inner ear and urogenital defects. The developmental processes most commonly involved in the affected tissues include epithelial-mesenchymal interactions, cell growth, extracellular matrix production and tissue remodeling. In addition, many affected tissues have neural crest-derived components and simulate neural crest deficiencies. There is no phenotypic overlap with TGFbeta1- and TGFbeta3-null mice indicating numerous non-compensated functions between the TGFbeta isoforms.
A broad spectrum of biological activities has been proposed for transforming growth factor−3(TGF− 3). To study TGF−3function in development, TGF−3 null mutant mice were generated by gene−targeting. Within 20 hours of birth, homozygous TGF−3-/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis. Unlike other null mutants with cleft palate, TGF−3-/- mice lack other concomitant craniofacial abnormalities. This study demonstrates an essential function for TGF−3 in the normal morphogenesis of palate and lung, and directly implicates this cytokine in mechanisms of epithelial−mesenchymal interaction.
Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF-β binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant-negative model of receptor–complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice-site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT. Hum Mutat 11:286–294, 1998. © 1998 Wiley-Liss, Inc.
The reverse genetics technologies that have recently been developed for mice have provided new tools to probe gene function in vivo. Unfortunately these powerful systems often require the analysis of large numbers of DNA samples. The gene-targeting technology requires screening of embryonic stem-cell clones and later of the mice themselves, the latter also being the case for standard transgenic technology. It is not always possible or desirable to rely on PCR analyses, necessitating the isolation of large numbers of DNA samples of sufficient quality for Southern blot analysis. We have simplified the standard mammalian DNA isolation procedure with the aim of minimizing the number of manipulations required for each sample. The basic procedure applied to cultured cells does not require any centrifugation steps or organic solvent extractions.
Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.
Using immunohistochemical methods, we have investigated the role of transforming growth factor-beta (TGF-beta) in the development of the mouse embryo. For detection of TGF-beta in 11-18-d-old embryos, we have used a polyclonal antibody specific for TGF-beta type 1 and the peroxidase-antiperoxidase technique. Staining of TGF-beta is closely associated with mesenchyme per se or with tissues derived from mesenchyme, such as connective tissue, cartilage, and bone. TGF-beta is conspicuous in tissues derived from neural crest mesenchyme, such as the palate, larynx, facial mesenchyme, nasal sinuses, meninges, and teeth. Staining of all of these tissues is greatest during periods of morphogenesis. In many instances, intense staining is seen in mesenchyme when critical interactions with adjacent epithelium occur, as in the development of hair follicles, teeth, and the submandibular gland. Marked staining is also seen when remodeling of mesenchyme or mesoderm occurs, as during formation of digits from limb buds, formation of the palate, and formation of the heart valves. The presence of TGF-beta is often coupled with pronounced angiogenic activity. The histochemical results are discussed in terms of the known biochemical actions of TGF-beta, especially its ability to control both synthesis and degradation of both structural and adhesion molecules of the extracellular matrix.
Mice lacking TGF-beta 3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-beta 3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.
Transforming growth factor beta 1 (TGF beta 1) is shown here to be required for yolk sac haematopoiesis and endothelial differentiation. Mice with a targeted mutation in the TGF beta 1 gene were examined to determine the cause of prenatal lethality, which occurs in 50% of homozygous TGF beta 1 null (TGF beta 1-/-) conceptions. 50% of TGF beta 1-/- and 25% of TGF beta 1-+-) conceptions. 50% of TGF beta 1-/- and 25% of TGF beta 1+/- conceptuses were found to die at around 10.5 dpc. The primary defects were restricted to extraembryonic tissues, namely the yolk sac vasculature and haematopoietic system. The embryos per se showed developmental retardation, oedema and necrosis, which were probably secondary to the extraembryonic lesions. The defect in vasculogenesis appeared to affect endothelial differentiation, rather than the initial appearance and outgrowth of endothelial cells. Initial differentiation of yolk sac mesoderm to endothelial cells occurred, but defective differentiation resulted in inadequate capillary tube formation, and weak vessels with reduced cellular adhesiveness. Defective haematopoiesis resulted in a reduced erythroid cell number within the yolk sac. Defective yolk sac vasculogenesis and haematopoiesis were present either together, or in isolation of each other. The phenotypes are consistent with the observation of abundant TGF beta 1 gene expression in both endothelial and haematopoietic precursors. The data indicate that the primary effect of loss of TGF beta 1 function in vivo is not increased haematopoietic or endothelial cell proliferation, which might have been expected by deletion of a negative growth regulator, but defective haematopoiesis and endothelial differentiation.
Epithelial-mesenchymal transformation is a critical event in the development of many organ systems including the heart. Descriptive studies have implicated a number of factors in mediating this transformation, including transforming growth factor beta (TGFbeta). We now report that disruption of a TGFbeta signal transduction complex by antibodies directed against the Type II TGFbeta receptor blocks both the endocardial cell activation and subsequent migration that constitute transformation in the chick atrioventricular (AV) cushion. The Type II receptor was localized to both endothelial and endocardial cells of the chick embryo. Incubation of AV cushion explants from Stage 14, 16, and 18 embryos with antibody resulted in a blockade of AV endocardial cell transformation by greater than 50% as measured by mesenchyme formation. Similarly, the appearance of procollagen Type I, a marker of endocardial cell transformation, was blocked. In addition, within 2 hr after the incubation of activated Stage 18 explants with Type II antibody the rate of migration of transformed cells was decreased by 50%. These data suggest that TGFbeta acts directly on AV cushion endocardial cells to stimulate epithelial-mesenchymal transformation and that TGFbeta mediates at least two distinct components of AV cushion transformation, activation and migration.
Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S-endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF-beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L-endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.
The growth and differentiation factor transforming growth factor-beta2 (TGFbeta2) is thought to play important roles in multiple developmental processes. Targeted disruption of the TGFbeta2 gene was undertaken to determine its essential role in vivo. TGFbeta2-null mice exhibit perinatal mortality and a wide range of developmental defects for a single gene disruption. These include cardiac, lung, craniofacial, limb, spinal column, eye, inner ear and urogenital defects. The developmental processes most commonly involved in the affected tissues include epithelial-mesenchymal interactions, cell growth, extracellular matrix production and tissue remodeling. In addition, many affected tissues have neural crest-derived components and simulate neural crest deficiencies. There is no phenotypic overlap with TGFbeta1- and TGFbeta3-null mice indicating numerous non-compensated functions between the TGFbeta isoforms.
Endoglin (CD105), a component of the TGF-beta 1 receptor complex, is the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). We have identified a novel endoglin splice site mutation, leading to an in-frame deletion of exon 3, in a new-born from a family with HHT. Expression of normal and mutant endoglin proteins was analyzed in umbilical vein endothelial cells from this baby and in activated monocytes from the affected father. In both samples, only normal dimeric endoglin (160 kD) was observed at the cell surface, at 50% of control levels. Despite an intact transmembrane region, mutant protein was only detectable by metabolic labeling, as an intracellular homodimer of 130 kD. In monocytes from three clinically affected HHT1 patients, with known mutations creating premature stop codons in exons 8 and 10, surface endoglin was also reduced by half and no mutant was detected. Overexpression into COS-1 cells of endoglin cDNA truncated in exons 7 and 11, revealed their intracellular expression, inability to be secreted and to form heterodimers at the cell surface. These results indicate that mutated forms of endoglin are transiently expressed intracellularly and not likely to act as dominant negative proteins, as proposed previously. A reduction in the level of functional endoglin is thus involved in the generation of HHT1, and associated arteriovenous malformations.
The recent identification of the SMAD family of signal transducer proteins has unravelled the mechanisms by which transforming growth factor-beta (TGF-beta) signals from the cell membrane to the nucleus. Pathway-restricted SMADs are phosphorylated by specific cell-surface receptors that have serine/threonine kinase activity, then they oligomerize with the common mediator Smad4 and translocate to the nucleus where they direct transcription to effect the cell's response to TGF-beta. Inhibitory SMADs have been identified that block the activation of these pathway-restricted SMADs.
Endoglin (CD105) is a transmembrane glycoprotein that binds transforming growth factor (TGF)-β1 and -β3, and coprecipitates
with the Ser/Thr kinase signaling receptor complex by affinity labeling of endothelial and leukemic cells. The present study
shows that in addition to TGF-β1 and -β3, endoglin interacts with activin-A, bone morphogenetic protein (BMP)-7, and BMP-2
but requires coexpression of the respective ligand binding kinase receptor for this association. Endoglin cannot bind ligands
on its own and does not alter binding to the kinase receptors. It binds TGF-β1 and -β3 by associating with the TGF-β type
II receptor and interacts with activin-A and BMP-7 via activin type II receptors, ActRII and ActRIIB, regardless of which
type I receptor partner is coexpressed. However, endoglin binds BMP-2 by interacting with the ligand binding type I receptors,
ALK3 and ALK6. The formation of heteromeric signaling complexes was not altered by the presence of endoglin, although it was
coprecipitated with these complexes. Endoglin did not interact with BMP-7 through complexes containing the BMP type II receptor,
demonstrating specificity of its action. Our data suggest that endoglin is an accessory protein of multiple kinase receptor
complexes of the TGF-β superfamily.
von Willebrand factor (vWF) is frequently used as a biochemical marker for endothelial cells (ECs). Despite this, little is known about the relative level of expression and regulation of this hemostatic factor in ECs in different vascular beds in vivo. In the present study, we used quantitative reverse transcription polymerase chain reaction and in situ hybridization analysis to study vWF gene expression in murine tissues. Large differences in the level of vWF mRNA were observed when comparing highly vascularized tissues, with the lung and brain containing 5 to 50 times higher concentrations of vWF mRNA than the kidney and liver. In this regard, ECs of small vessels and some microvessels in the lung and brain expressed abundant vWF mRNA, whereas ECs of similar sized vessels in the liver and kidney expressed relatively low levels. In general, significantly higher levels of vWF mRNA and antigen were demonstrated in ECs of larger vessels compared with microvessels and in venous ECs compared with arterial ECs. Although intraperitoneal administration of endotoxin (or tumor necrosis factor-) increased plasma vWF levels, it had variable effects on the steady-state level of vWF mRNA in murine tissues (ie, it decreased vWF mRNA in many tissues, increased it in others, and had little effect on still others). These results indicate that vWF is differentially expressed and regulated in ECs present in different tissues and within the same vascular bed.
© 1998 by The American Society of Hematology.
Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S-endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF-beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L-endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.
The early embryonic heart consists of two cell types. The cells form an inner epithelial tube of endocardium within an outer tube of myocardium separated by a cell-free extracellular matrix. A crucial process in heart development is the production of cushion mesenchyme in the atrioventricular (AV) canal and outflow tract (OT). Cushion mesenchyme differentiates from the endocardium in response to signaling molecules produced by the adjacent myocardium. In chicken hearts, both transforming growth factor-β3 (TGF-β3) and TGF-β2 are present and have been identified as being important in the production of cushion mesenchyme. We were interested in how the signals from these two similar molecules may be differentiated during early heart development. To this end, we examined the expression of endoglin, a TGF-β receptor molecule, in the developing chick heart. Endoglin is typically located on endothelial cell layers and binds tightly to TGF-β1 and TGF-β3 but not well to TGF-β2. We show that during the formation of the primitive heart tube, endoglin is found at relatively high levels in both presumptive myocardium and endocardium. However, as myocardium differentiates and development proceeds, endoglin expression is progressively reduced. At stage 20 in the heart, endoglin expression is most readily seen in the AV canal and the OT. This pattern of expression is similar to the reported TGF-β3 expression patterns in the heart. Dev. Dyn. 1998;213:237–247. © 1998 Wiley-Liss, Inc.
TGF-β signaling is mediated through two types of serine/threonin kinase-containing receptors, type I (TGF-βRI) and type II (TGF-βRII), which form a heteromeric complex. In this signaling complex, ligand binding TGF-βRII phosphorylates and thereby activates the TGF-βRI to signal downstream pathways. To determine the role of TGF-βRII in embryogenesis, we have generated a TGF-βRII gene (Tgfbr2) knockout mouse line. The heterozygousTgfbr2knockout mice are developmentally normal. The homozygousTgfbr2mutation causes defects in the yolk sac hematopoiesis and vasculogenesis, resulting in an embryonic lethality around 10.5 days of gestation. This phenotype is indistinguishable from the previously reported embryonic lethality by the homozygous TGF-β1 gene (Tgfb1) null mutation. In addition, we have generated chimeric mice using aTgfbr2(−/−) embryonic stem cell line. Some chimeric mice showed several types of congenital anomalies, suggesting that TGF-βRII is important for normal development in a variety of organs.
Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional growth factor that has profound regulatory effects on many developmental and physiological processes. Disruption of the TGF-beta 1 gene by homologous recombination in murine embryonic stem cells enables mice to be generated that carry the disrupted allele. Animals homozygous for the mutated TGF-beta 1 allele show no gross developmental abnormalities, but about 20 days after birth they succumb to a wasting syndrome accompanied by a multifocal, mixed inflammatory cell response and tissue necrosis, leading to organ failure and death. TGF-beta 1-deficient mice may be valuable models for human immune and inflammatory disorders, including autoimmune diseases, transplant rejection and graft versus host reactions.
We describe the primary structure of rat betaglycan, a polymorphic membrane-anchored proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). As deduced from its cDNA sequence, the 853 amino acid core protein of betaglycan has an extracellular domain with clustered sites for potential attachment of glycosaminoglycan chains. These chains are dispensable for TGF-beta binding to the core protein. The transmembrane region and the short cytoplasmic tail of betaglycan are very similar to these regions in human endoglin, an endothelial cell membrane glycoprotein involved in intercellular recognition. The ectodomain of betaglycan can be released as a soluble proteoglycan; a potential cleavage site near the transmembrane region is identical to the highly regulated cleavage site of the membrane-anchored transforming growth factor-alpha precursor. The unique features of betaglycan suggest important roles in cell interaction with TGF-beta.
Bandeiraea simplicifolia B4 isolectin (BSLB4) and polyclonal antisera against von Willebrand factor (VWF) were used to study the origin of endothelial cells and their organization into blood vessels in the postimplantation mouse embryo. Examination of BSLB4-stained whole mounted and sectioned embryos revealed intense staining of the endothelium, highlighting large vessels, capillaries, and many individual cells. Dorsal aorta formation was first obvious at E7 when many lectin-positive cells appeared in paraxial and lateral plate mesoderm. As development proceeded to E8, BSLB4-positive cells became organized into craniocaudal lines destined to become the aorta proper. At E9, BSLB4 stained all vessels of the embryo including the dorsal aorta, the intersomitic arteries, and the endocardium. VWF expression was not detected until E8 when BSLB4/VWF double-stained sections revealed the dorsal aortae as the first VWF-positive vessels, while other endothelium visible with BSLB4 remained negative for VWF immunostaining. By E12 many other vessels became VWF-positive, including the aortic arches, the intersomitic arteries, and the cardinal veins. However, many angioblasts and capillaries remained VWF-negative, reflecting the heterogeneous expression of VWF among endothelium that has been reported in adults of other species. The histochemical data reported here support the conclusions of earlier avian studies by showing distinct vascular patterns in the initial formation of vessels from isolated angioblasts (vasculogenesis), followed by the extension and organization of the initial vascular structures (angiogenesis). Moreover, our data suggest that the endothelium arises from distinct VWF-positive sources associated with the dorsal aorta, as well as VWF-negative sources associated with other vessels in the embryo.
The hematopoietic system of vertebrates is derived from the mesodermal germ layer in early embryogenesis. Various animal models have been used for the study of hematopoiesis, from early stages in the visceral yolk sac or its analog, to the later stages where hematopoiesis is observed in intraembryonic areas surrounding the aorta, genital ridge and pro/mesonephros. Using the mouse as a model, we describe what is known about mammalian embryonic hematopoiesis and put it in the context of hematopoietic cell formation in avian, amphibian and fish embryos. Evolutionary comparisons and recent experimental evidence show that there are two embryonic sites of developing hematopoietic activity in the mouse before fetal liver hematopoiesis and suggest that, during ontogeny, two successive waves of hematopoietic activity may contribute to the blood system of the adult.
The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.
Hereditary hemorrhagic telangiectasia is a rare autosomal dorminant disease that features abnormal and fragile vascular dilations of terminal vessels in skin and mucous membranes, as well as arteriovenous malformations of internal organs, particularly lungs, brain, and liver. Often patients have not been diagnosed with HHT for a long time, and undiagnosed HHT patients unnecessarily develop serious complications such as severe life-threatening hemorrhage, stroke or brain abscess. Therefore, early detection and appropriate screening is very important. Early detection of HHT allows the appropriate screening for the presence of silent disease such as AVMs in the lungs, liver, or brain, and preventive treatment in the patient and their affected family members. Dentists should be familiar with HHT because the telangiectases on skin and oral mucosa are often the most dramatic and most easily identified component of HHT. Recently, we experienced a case of HHT. We present the case with a review of the literature.
Endoglin, a glycoprotein that is expressed by human endothelial cells, binds TGF-beta 1 and -beta 3 with high affinity. It was originally identified with the 44G4 mAb that was produced against a human pre-B cell line. We now report that another anti-pre-B cell mAb, 29-G8, reacts with pro-B and pre-B leukemic cells, but not with mature B and T cells, and recognizes a different epitope of endoglin. The 29-G8 mAb bound specifically to recombinant endoglin and immunoprecipitated a phosphorylated homodimeric glycoprotein with subunits of M(r) 95,000 from the 697 pre-B cell line. This new Ab removed all of the molecules identified by the prototypic 44G4 anti-endoglin Ab, but the reverse was not true. A subpopulation of 29-G8+ endoglin molecules on this pre-B cell line was unreactive with the 44G4 mAb, thus suggesting that these anti-endoglin Abs see different epitopes that may discriminate different species of endoglin molecules. Flow cytometric analysis with the 29-G8 mAb revealed two endoglin-positive subpopulations in fetal bone marrow: early B-lineage precursor cells (CD19+ and CD34+), and proerythroblasts (CD71+ and glycophorin A+). In adult bone marrow, only the proerythroblast subpopulation was observed. Stromal cells derived from fetal bone marrow also reacted strongly with the 29-G8 and 44G4 Abs, and these cells responded with enhanced proliferation after stimulation with either TGF-beta 1 or the anti-endoglin Abs. Thus, endoglin, a specialized component of the TGF-beta receptor system, may play a physiologic role in the stromal-hemopoietic cell interactions occurring during development.
Maternal sources of transforming growth factor-beta 1 (TGF-beta 1) are shown here to contribute to the normal appearance and
perinatal survival of TGF-beta 1 null newborn mice. Labeled TGF-beta 1 crossed the placenta and was recovered intact from
various tissues after oral administration to mouse pups. TGF beta-1 protein was also detected in cells recovered from breast
milk. In immunohistochemical analyses, TGF-beta 1 null embryos and null newborn pups born to TGF-beta 1 heterozygotes stained
positive for TGF-beta 1, whereas those born to a null female were negative and had severe cardiac abnormalities. These results
suggest an important role for maternal sources of TGF-beta 1 during development and, more generally, provide evidence for
maternal rescue of targeted gene disruption in the fetus.
The rat monoclonal antibody, MJ7/18, which reacts selectively with the endothelium of blood vessels in mouse was used to screen a cDNA library derived from a transformed mouse brain endothelial cell line. The sequence of a cDNA encoding the cell surface MJ7/18 antigen revealed homology to human endoglin, a homodimeric transforming growth factor-beta (TGF-beta)-binding cell-surface glycoprotein expressed predominantly on vascular endothelial cells. Northern blot analysis shows a 3.4-kb single transcript of the mouse endoglin. The mouse endoglin is a type-I integral membrane protein of 653 amino acids (aa). The human and mouse sequences display 71% aa sequence identity with almost identical transmembrane and cytoplasmic domains. Like its human counterpart, mouse endoglin displays significant sequence homology to the type-III TGF-beta receptor in two extracellular domains, as well as striking similarity in the transmembrane and cytoplasmic regions. One of the extracellular regions of homology with TGF-beta receptor III represents a truncated version of a homology unit defining a novel gene family including uromodulin, the pancreatic granule protein gp2, and zona pellucida receptors for sperm. However, unlike its human counterpart, mouse endoglin does not contain an RGD tripeptide which has been suggested as a ligand of integrins.
Endoglin is an integral membrane glycoprotein predominantly expressed on human endothelial cells and recently shown to bind transforming growth factor-beta 1 (TGF beta 1) with high affinity. We now report the cloning and sequencing of a full-length murine endoglin complementary DNA of 2902 base pairs which hybridizes specifically with a single messenger RNA (mRNA) species. The polypeptide of 653 amino acids has an overall identity of 72% with human and porcine endoglin. The transmembrane and cytoplasmic domains of all three proteins differ by two to four amino acids and are 70% identical to the corresponding regions of the TGF beta binding protein, betaglycan. Relative levels of murine endoglin mRNA were estimated by polymerase chain reaction and found to be high in ovary and uterus, intermediate in heart and muscle, and low in placenta and spleen. In situ hybridization and immunofluorescence confirmed that murine endoglin, like its human counterpart, is present in blood vessels and capillaries in all tissues examined. In addition, the stromal cells in the connective tissue of intestine, stomach, heart, muscle, uterus, ovary, and testis were strongly and specifically reactive with complementary RNA probes and with a polyclonal antibody to endoglin; epithelial cell layers were distinctly unreactive. This distribution is similar to that of extracellular TGF beta 1, particularly in heart and uterus, and suggests that endoglin on stromal fibroblast-like cells might be regulating access of TGF beta 1 to the signaling receptor complex. NCTC-2071 fibroblasts in culture were shown to express high levels of endoglin mRNA by polymerase chain reaction. After chemical cross-linking with [125I]TGF beta 1 and immunoprecipitation with the polyclonal antihuman endoglin serum, a radiolabeled band of mol wt 180,000 corresponding to dimeric endoglin was observed under nonreducing conditions, whereas a single band of mol wt 90,000 was seen under reducing conditions. Thus murine fibroblast endoglin is capable of binding TGF beta 1. Future studies should establish the specialized role of endoglin in the TGF beta receptor complex of endothelial and stromal cells.
Transforming growth factor beta (TGF beta) signals through a heteromeric protein kinase receptor that has a limited ability to bind ligand. This limitation is overcome by the action of betaglycan (TGF beta type III receptor), a separate TGF beta-binding membrane protein of previously unknown function. Betaglycan presents TGF beta directly to the kinase subunit of the signaling receptor, forming a high affinity ternary complex. Membrane betaglycan increases TGF beta binding to the signaling receptor, enhances cell responsiveness to TGF beta, and eliminates marked biological differences between TGF beta isoforms. Thus, betaglycan is a direct regulator of TGF beta access to the signaling receptors.
Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber (ORW) syndrome, is an autosomal dominant vascular dysplasia. So far, two loci have been demonstrated for ORW. Linkage studies established an ORW locus at chromosome 9q3; endoglin was subsequently identified as the ORW1 gene. A second locus, designated ORW2, was mapped to chromosome 12. Here we report a new 4 cM interval for ORW2 that does not overlap with any previously defined. A 1.38-Mb YAC contig spans the entire interval. It includes the activin receptor like kinase 1 gene (ACVRLK1 or ALK1), a member of the serine-threonine kinase receptor family expressed in endothelium. We report three mutations in the coding sequence of the ALK1 gene in those families which show linkage of the ORW phenotype to chromosome 12. Our data suggest a critical role for ALK1 in the control of blood vessel development or repair.
Type II and type I receptor serine-threonine kinases (RSTK) are important components of the transmembrane signaling machinery that allow cells to respond to the transforming growth factor-beta (TGF-beta) superfamily of cytokines. We have cloned from rat lung and report here a 3,935-base pair (bp) cDNA encoding a type I RSTK previously identified as R-3 (rat) or ALK-1 (human). Northern blot analysis reveals that the R-3 mRNA is more abundant in lung than in other adult rat tissues. With the use of in situ hybridization, the R-3 transcripts are found exclusively in the pulmonary vessels of all sizes, as well as in aorta, vena cava, and certain blood vessels of kidney, spleen, heart and intestine. In most blood vessels, a higher level of gene expression is found in endothelium than in adjacent smooth muscle. The R-3 transcripts are also found in splenic macrophages, as well as within cells of marginal zone of the splenic lymphoid tissue. In fetal rat lung, the expression of R-3 transcripts differs from the expression patterns of two other type 1 RSTK. The R-3 is expressed in vessels; the activin type IB receptor (R-2) is preferentially expressed in putative developing airways, whereas the TGF-beta type I receptor (R-4) transcripts appear to be ubiquitous. Our data suggest that in vivo R-3 may propagate signaling of TGF-beta in selected cell types. The differential expression of multiple type I receptors within different cell lineages may therefore define cell specific responses to TGF-beta.
Medical students learning the anatomy of the human cardiovascular system recognize that the blood vessels are named mainly on the basis of luminal diameter, branching, position, and organ supplied. Students and physicians rely upon the general constancy of vascular determinants from one individual to another and take for granted that anatomy books will not go out of date. It is only when they learn that these vessels with their proper diameters and branches are formed in the embryo, mostly before the heart starts beating, that students begin to appreciate the true complexity of the genetic program that governs the development of the vascular system.
The transforming growth factor beta 1 (TGF beta 1) signalling pathway is important in embryogenesis and has been implicated in hereditary haemorrhagic telangiectasia (HHT), atherosclerosis, tumorigenesis and immunomodulation. Therefore, identification of factors which modulate TGF beta 1 bioactivity in vivo is important. On a mixed genetic background, approximately 50% Tgfb1-/- conceptuses die midgestation from defective yolk sac vasculogenesis. The other half are developmentally normal but die three weeks postpartum. Intriguingly, the vascular defects of Tgfb1-/- mice share histological similarities to lesions seen in HHT patients. It has been suggested that dichotomy in Tgfb1-/- lethal phenotypes is due to maternal TGF beta 1 rescue of some, but not all, Tgfb1-/- embryos12. Here we show that the Tgfb1-/- phenotype depends on the genetic background of the conceptus. In NIH/Ola, C57BL/6J/Ola and F1 conceptuses, Tgfb1-/- lethality can be categorized into three developmental classes. A major codominant modifier gene of embryo lethality was mapped to proximal mouse chromosome 5, using a genome scan for non-mendelian distribution of alleles in Tgfb1-/- neonatal animals which survive prenatal lethality. This gene accounts for around three quarters of the genetic effect between mouse strains and can, in part, explain the distribution of the three lethal phenotypes. This approach, using neonatal DNA samples, is generally applicable to identification of loci that influence the effect of early embryonic lethal mutations, thus furthering knowledge of genetic interactions that occur during early mammalian development in vivo.
Genetic studies have recently revealed a role for transforming growth factor-beta-1 (TGF-beta 1) and its receptors (TGF-beta Rs I and II as well as endoglin) in embryonic vascular assembly and in the establishment and maintenance of vessel wall integrity. The purpose of this review is threefold: first, to reassess previous studies on TGF-beta and endothelium in the light of these recent findings; second, to describe some of the well-established as well as controversial issues concerning TGF-beta and its regulatory role in angiogenesis; and third, to explore the notion of "context' with respect to TGF-beta and endothelial cell function. Although the focus of this review will be on the endothelium, other vascular wall cells are also likely to be important in the pathogenesis of the vascular lesions revealed by genetic studies.
To identify mutations that cause hereditary hemorrhagic telangiectasia (HHT, or Rendu-Osler-Weber syndrome), clinical evaluations and genetic studies were performed on 32 families. Linkage studies in four of eight families indicated an endoglin (ENG) gene mutation. ENG sequences of affected members of the four linked families and probands from the 24 small families were screened for mutations, by Southern blot analyses and by cycle sequencing of PCR-amplified DNA. Seven novel mutations were identified in eight families. Two mutations (a termination codon in exon 4 and a large genomic deletion extending 3' of intron 8) did not produce a stable ENG transcript in lymphocytes. Five other mutations (two donor splice-site mutations and three deletions) produce altered mRNAs that are predicted to encode markedly truncated ENG proteins. Mutations in other families are predicted to lie in ENG-regulatory regions or in one of the additional genes that may cause HHT. These data suggest that the molecular mechanism by which ENG mutations cause HHT is haploinsufficiency. Furthermore, because the clinical manifestation of disease in these eight families was similar, we hypothesize that phenotypic variation of HHT is not related to a particular ENG mutation.
ALK-1 is a type I serine/threonine kinase receptor for members of the TGF-beta superfamily of growth factors; its endogenous ligand is not known. In this study, we have analyzed the temporal and spatial expression pattern of ALK-1 mRNA in mouse embryos from the one-cell zygote until 12.5 dpc using RT-PCR and in situ hybridization. ALK-1 mRNA was first detected in the embryo at 6.5 dpc. From 7.5-8.5 dpc expression was highest at sites of vasculogenesis in both the embryonic and extraembryonic part of the conceptus, in trophoblast giant cells, and in the endothelial lining of the blood vessels in the decidua. From 9.5-12.5 dpc, ALK-1 was found to be expressed in several different tissues and organs, but was highest in blood vessels, mesenchyme of the lung, submucosal layer of the stomach and intestines, and at specific sites of epithelial-mesenchymal interactions. Its expression pattern suggests that ALK-1 is a type I receptor for TGF-beta1 in the developing mouse.
Endoglin is a component of the receptor complex for transforming growth factor (TGF)-beta1 and TGF-beta3. We analysed its expression by immunohistochemistry in human embryos at 4-8 weeks of gestation and in hearts ranging from 4-13 weeks old. We compared endoglin distribution with that of TGF-beta receptors type I (TbetaR-I), type II (TbetaR-II) and betaglycan. Endoglin was found on endothelial cells in all tissues examined, consistent with its expression in adult blood vessels. TbetaR-I, TbetaR-II and betaglycan were observed on most cell types and had an overall similar pattern of distribution. Endoglin was detected on the endocardium as early as 4 weeks, but was absent from myocardium. It was present at high levels on the endocardial cushion tissue mesenchyme from 5-8 weeks' gestation, during heart septation and valve formation, and subsequently decreased as the valves matured. Endoglin expression in heart extracts was confirmed by Western blot analysis. TbetaR-I, TbetaR-II and betaglycan were mostly found on cardiac myocytes, but were detectable at low levels on endocardium. They were expressed transiently on cushion mesenchyme, albeit at much lower levels than endoglin. All four components of the TGF-beta receptor complex were detected by RT-PCR in embryonic heart. Thus transient up-regulation of the components of the TGF-beta receptor complex, and particulartly of endoglin, is associated with heart septation and valve formation during early human development.
Endoglin, the gene linked to the autosomal dominant vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1), encodes a 95-kDa membrane-bound proteoglycan which binds TGF beta 1 and regulates signaling via the type I and II TGF beta receptors on the surface of vascular endothelial cells. Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analysis we have shown that endoglin mRNA is expressed in both cultured human VSMCs and VSMCs freshly isolated from human aortas. Northern blot analysis was also used to demonstrate that endoglin expression decreased in serum-stimulated cultured human VSMCs but could be maintained by exogenous TGF beta 1. Endoglin protein expression in human VSMCs was shown by immunocytochemistry. These data, the first describing the existence of endoglin in VSMCs, suggest that through regulating TGF beta 1 signaling endoglin may mediate the effects of TGF beta 1 on VSMC behavior in vitro and in vivo.
TGF beta family members are implicated in cardiac organogenesis, growth control, and positional information, including the direction of cardiac looping. However, genetic analysis of TGF beta signaling in mice has been confounded, in some cases, by noncardiac and generalized defects. Hence, deciphering TGF beta function in myocardium would benefit from cardiac-restricted mutations. We developed a constitutively activated type I receptor, ALK5L193A,P194A,T204D, and directed it to embryonic myocardium in transgenic mice. Expression of the activated ALK5 gene arrests looping morphogenesis and causes a linear, dilated, hypoplastic heart tube, despite normal expression of Nkx2.5 and dHAND, cardiogenic transcription factors whose absence provokes a similar phenotype. Ventricular hypoplasia was associated with precocious induction of the cyclin-dependent kinase inhibitor, p21. Thus, an ALK5-sensitive pathway mediates looping, perhaps through control of cardiac myocyte proliferation.
The transforming growth factor beta (TGF-beta) family of growth factors control the development and homeostasis of most tissues in metazoan organisms. Work over the past few years has led to the elucidation of a TGF-beta signal transduction network. This network involves receptor serine/threonine kinases at the cell surface and their substrates, the SMAD proteins, which move into the nucleus, where they activate target gene transcription in association with DNA-binding partners. Distinct repertoires of receptors, SMAD proteins, and DNA-binding partners seemingly underlie, in a cell-specific manner, the multifunctional nature of TGF-beta and related factors. Mutations in these pathways are the cause of various forms of human cancer and developmental disorders.
von Willebrand factor (vWF) is frequently used as a biochemical marker for endothelial cells (ECs). Despite this, little is known about the relative level of expression and regulation of this hemostatic factor in ECs in different vascular beds in vivo. In the present study, we used quantitative reverse transcription polymerase chain reaction and in situ hybridization analysis to study vWF gene expression in murine tissues. Large differences in the level of vWF mRNA were observed when comparing highly vascularized tissues, with the lung and brain containing 5 to 50 times higher concentrations of vWF mRNA than the kidney and liver. In this regard, ECs of small vessels and some microvessels in the lung and brain expressed abundant vWF mRNA, whereas ECs of similar sized vessels in the liver and kidney expressed relatively low levels. In general, significantly higher levels of vWF mRNA and antigen were demonstrated in ECs of larger vessels compared with microvessels and in venous ECs compared with arterial ECs. Although intraperitoneal administration of endotoxin (or tumor necrosis factor-) increased plasma vWF levels, it had variable effects on the steady-state level of vWF mRNA in murine tissues (ie, it decreased vWF mRNA in many tissues, increased it in others, and had little effect on still others). These results indicate that vWF is differentially expressed and regulated in ECs present in different tissues and within the same vascular bed.
© 1998 by The American Society of Hematology.
The early embryonic heart consists of two cell types. The cells form an inner epithelial tube of endocardium within an outer tube of myocardium separated by a cell-free extracellular matrix. A crucial process in heart development is the production of cushion mesenchyme in the atrioventricular (AV) canal and outflow tract (OT). Cushion mesenchyme differentiates from the endocardium in response to signaling molecules produced by the adjacent myocardium. In chicken hearts, both transforming growth factor-beta3 (TGF-beta3) and TGF-beta2 are present and have been identified as being important in the production of cushion mesenchyme. We were interested in how the signals from these two similar molecules may be differentiated during early heart development. To this end, we examined the expression of endoglin, a TGF-beta receptor molecule, in the developing chick heart. Endoglin is typically located on endothelial cell layers and binds tightly to TGF-beta1 and TGF-beta3 but not well to TGF-beta2. We show that during the formation of the primitive heart tube, endoglin is found at relatively high levels in both presumptive myocardium and endocardium. However, as myocardium differentiates and development proceeds, endoglin expression is progressively reduced. At stage 20 in the heart, endoglin expression is most readily seen in the AV canal and the OT. This pattern of expression is similar to the reported TGF-beta3 expression patterns in the heart.
Endoglin (CD105) is the target gene for the hereditary hemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. It shares with betaglycan a limited amino acid sequence homology and being components of the membrane transforming growth factor-beta (TGF-beta) receptor complex. Using rat myoblasts as a model system, we found that overexpression of endoglin led to a decreased TGF-beta response to cellular growth inhibition and plasminogen activator inhibitor-1 synthesis, whereas overexpression of betaglycan resulted in an enhanced response to inhibition of cellular proliferation and plasminogen activator inhibitor-1 induced expression in the presence of TGF-beta. The regulation by endoglin of TGF-beta responses seems to reside on the extracellular domain, as evidenced by the functional analysis of two chimeric proteins containing different combinations of endoglin and betaglycan domains. Binding followed by cross-linking with 125I-TGF-beta1 demonstrated that betaglycan expressing cells displayed a clear increase (about 3. 5-fold), whereas endoglin expressing cells only displayed an slight increment (about 1.6-fold) in ligand binding with respect to mock transfectants. SDS-polyacrylamide gel electrophoresis analysis of radiolabeled receptors demonstrated that expression of endoglin or betaglycan is associated with an increased TGF-beta binding to the signaling receptor complex; however, while endoglin increased binding to types I and II receptors, betaglycan increased the binding to the type II receptor. Conversely, we found that TGF-beta binding to endoglin required the presence of receptor type II as evidenced by transient transfections experiments in COS cells. These findings suggest a role for endoglin in TGF-beta responses distinct from that of betaglycan.