Endoglin (OMIM 131195), also known as cluster of differentiation CD105, was originally identified in endothelial cells by immunofluorescence using a monoclonal antibody (mAb 44G4) raised against a human pre-B leukemia cell line (Gougos and Letarte 1988). Later it was shown that endoglin is also expressed outside the endothelium in pro-fibrogenic and immune cells (Meurer et al. 2014; Ojeda-Fernandez et al. 2016). It is an integral membrane-bound disulfide-linked 180 kDa homodimeric receptor that acts as an auxiliary receptor for ligands of the transforming growth factor-β (TGF-β) superfamily. Endoglin interacts with the TGF-β signaling receptors and influences Smad-dependent and Smad-independent effects. The endoglin gene produces two splice variants (i.e., S- and L-endoglin) which cause a different response outcome due to the sequence variation of their cytoplasmic tails. Regulated ectodomain shedding by matrix metalloprotease-14 (MMP-14) converts endoglin into a soluble form (i.e., sol-Eng). In humans, several hundred mutations of the endoglin gene are known which lead to a haploinsufficiency and give rise to an autosomal dominant bleeding disorder termed hereditary hemorrhagic telangiectasia type 1 (HHT1). HHT1 is characterized by localized angiodysplasia, arteriovenous malformations (AVMs), and various vascular lesions, mainly on the face, lips, hands, and gastrointestinal mucosa. In experimental endoglin knockout mice models it was shown that homozygous endoglin loss is embryonically lethal due to cardiovascular abnormalities. Therefore, it was initially proposed that endoglin function is mainly associated with the cardiovascular system and vascular remodeling. However, it was also demonstrated that endoglin is a crucial mediator during liver fibrogenesis that enhances TGF-β-driven Smad1/5 phosphorylation and α-smooth muscle actin production during hepatic insult (Meurer et al. 2011).
Chromosomal Localization, Structure, and ENG Gene Variants
The human ENG gene contains 15 exons numbered 1 to 14, where exon 9 is split into 9a and 9b (McAllister et al. 1994). Beside the full length endoglin (FL-Eng), a splice variant has been identified, i.e., short-endoglin (S-Eng), that is characterized by the retention of intron 14 in the mature mRNA causing a shortened C-terminal tail with a different amino acid sequence (Bellon et al. 1993). The expression of S-Eng is increased in senescent endothelial cells in which the splicing factor-2 (ASF/SF2) interferes with the process of splicing (Blanco and Bernabeu, 2011). In contrast to human and mouse, splicing of endoglin in rat results in a longer S-Eng protein product with a peptide insertion in the regular C-terminus. Moreover, the usage of alternative polyadenylation sites generates two mRNAs with different sizes of yet unknown function (Meurer et al. 2011).
Since it has been realized that endoglin mutations are causative for HHT1 (McAllister et al. 1994), a wealth of different ENG mutations and variants causing altered expression or formation of aberrant protein products have been identified, and the number is still growing. Actually, the ENG database that is hosted by the Department of Pathology at the University of Utah (http://arup.utah.edu/database/ENG/ENG_welcome.php) contains 500 entries of genetic variants (retrieved on December 1, 2016) that have different genotype-phenotype correlations with regard to clinical significance (benign, suspected benign, uncertain, suspected pathogenic, pathogenic). Nevertheless, mutations are not spread randomly in the genomic sequence, and mutations are preferentially found in the orphan domain and in the N-terminal part of the zona pellucida (ZP) domain in which three highly conserved cysteines (Cys363, Cys382, and Cys412) are exceptionally prone to mutations (Llorca et al. 2007).
Endoglin: A Type I Transmembrane Receptor
Among TGF-β-family receptors, endoglin and betaglycan constitute the TGF-β type III receptor family. Both receptors share a high degree of similarity, especially in their intracellular domain that is also the most conserved region between endoglin from different species (cf. Fig. 2), implying that this region has important functions (Jovine et al. 2005). Especially because both receptors lack enzymatic activities in their short C-terminal domains, they participate in TGF-β signaling (see below) via protein-protein interactions which are either direct or modulated by endoglin phosphorylation (see below). Compared to betaglycan, the signaling specificity of endoglin is majorly determined by its ECD (Letamendia et al. 1998).
Three-Dimensional Structure of Endoglin
Nevertheless, respective domains have important functional implications for the interaction with the signaling receptors. Recent experiments using the fluorescence recovery after photobleaching technique have underpinned the view that endoglin forms stable dimers that function as a scaffold for binding TβRII, ALK5, and ALK1 and that ALK5 bind to endoglin with differential dependence on TβRII playing a crucial role in recruiting ALK5 to the receptor complex (Pomeraniec et al. 2015).
Endoglin Phosphorylation and TGF-β Signaling
Phosphorylation of endoglin occurs primarily on serine residues and to a lesser extent on threonine residues, whereas tyrosine involvement is questionable (Lastres et al. 1994). This posttranslational modification is performed principally by TGF-β receptors (Koleva et al. 2006). FL-Eng is phosphorylated at Ser646/Ser649 by ALK5 (Ray et al. 2010), Thr640/Thr654 by ALK1 (Koleva et al. 2006), and Ser634/Ser635 by TβRII, respectively (Koleva et al. 2006). Functionally, FL-Eng itself inhibits autophosphorylation of TβRII but enhances phosphorylation of ALK5 by TβRII leading to a stronger Smad2 transcriptional activity (Guerrero-Esteo et al. 2002). Moreover, ALK1 phosphorylation and binding of endoglin was observed only in the presence of TGF-β1, and this phosphorylation leads to loss of FL-Eng from focal adhesions (Koleva et al. 2006). This functional association of endoglin and ALK1 potentiates TGF-β signaling via the ALK1 branch and significantly modulates proliferative and adhesive properties of endothelial cells (Blanco et al. 2005). On the other side, the transient overexpression of endoglin inhibits TGF-β/ALK5 signaling and enhances BMP-7/Smad1/Smad5 pathway (Scherner et al. 2007). In addition, phosphorylation of threonine Thr650 in the C-terminus of endoglin regulates the interaction with the scaffolding protein β-arrestin-2 (Lee and Blobe 2007). Moreover, the PDZ domain found at the C-terminal end of endoglin is not only a binding motive for GIPC (Lee et al. 2008) but also regulates the phosphorylation of serine residues in the C-terminus by the type I TGF-β receptors (Koleva et al. 2006). Recently it was shown that tyrosine phosphorylation of the membrane proximal intracellular 612Tyr-Ile-Tyr614-motive regulates endoglin trafficking. Direct phosphorylation of this motive by src leads to internalization of endoglin – and the interacting receptors ALK1/TβRII – and lysosomal degradation (Pan et al. 2014).
Endoglin in Health and Disease
Gene mutations that affect human endoglin function are inherited as autosomal dominant disorders and may cause AVMs in different organs, including brain, lung, and liver. These are highly characteristic in hereditary hemorrhagic telangiectasia (HHT, Osler-Weber-Rendu syndrome), and the formation of AVMs occurs in both small and large blood vessels. This leads to epistaxis, gastrointestinal bleeding, and microcytic anemia due to iron deficiency, along with characteristic mucocutaneous telangiectasia. AVMs are found in pulmonary, hepatic, and cerebral vascular tissue.
Nevertheless, in cases of suspected patients without a clear HHT diagnosis and without mutations in the known HHT genes, additional biomarkers for the diagnosis of the disease are needed. Promising candidates are next-generation sequencing (NGS) and potential biomarkers detected in plasma and serum. These include proteins involved in vascular biology as well as regulatory microRNAs and long noncoding RNAs (reviewed by Botella et al. 2015). A new approach relies on IR-spectroscopy of peripheral blood plasma to analyze the “metabolic change pattern” which turned out to be significantly different between HHT patients and individuals from the control group (Lux et al. 2013).
Furthermore, symptomatic treatment with angiogenesis inhibitors or antihormonal agents that only affects the symptoms and not the cause are widely applied (Meurer et al. 2014). In some patients, the use of thalidomide, lenalidomide, and humanized anti-VEGF monoclonal antibody (bevacizumab) that possess antiangiogenic activities are suitable to reduce the incidence of nasal and gastrointestinal bleedings. Likewise, the β-receptor blocker propanolol used in prophylaxis of esophageal variceal bleeding in patients with liver cirrhosis is beneficial when locally administered in the nose mucosa to control epitaxis. Other studies showed that the estrogen receptor antagonist tamoxifen and the selective estrogen receptor modulator raloxifene can both reduce episodes of epistaxis and transfusion requirements in patients suffering from nasal vascular malformations. However, these therapies are limited by severe side effects and the need to administer respective drugs for long periods.
Endoglin in Liver Fibrosis and Hepatocellular Carcinoma
During liver fibrosis and cirrhosis, the excessive accumulation of extracellular matrix (ECM) proteins promotes hepatic scarring and eventually leads to organ failure. In all these processes, TGF-β is the most effective fibrogenic cytokine that induces fibrosis through multiple mechanisms, including direct activation of hepatic stellate cells (HSC), stimulation of ECM production, as well as prompting the synthesis of tissue inhibitors of matrix metalloproteinases (TIMPs) that prevent ECM degradation.
Endoglin inhibits the ALK5-Smad2/3 signaling branch and promotes ALK1-Smad1/5 signaling thereby promoting pro-fibrogenic activities of TGF-β (Meurer et al. 2011). In addition, endoglin expression is increased in activated HSC in vitro and in murine models of liver injury in vivo (Meurer et al. 2005). Since HSC are the major source for ECM production in liver fibrosis, the increase of endoglin is a critical factor that significantly contributes to the development of hepatic fibrosis. This could be underscored in myofibroblast-like HSC cell lines in which transient overexpression of endoglin leads to an increased expression of the activation marker α-SMA and the matricellular protein connective tissue growth factor (CTGF) (Meurer et al. 2011; Meurer et al. 2013). In addition, membrane-bound endoglin is expressed in a subset of newly formed microvessels in hepatocellular carcinoma (HCC) and has therefore been suggested as a complementary biomarker that may be used as a parameter to distinguish benign from malignant liver nodules (Segatelli et al. 2014), and to estimate angiogenesis in HCC (Yao et al. 2007). In addition, it was shown that the detection of soluble endoglin in the serum might be used as a complementary biomarker to assess the development of HCC in cirrhotic patients (Yagmur et al. 2007).
Experimental Models in Endoglin Research
Primary cultures of endothelial cells that are generated from heterozygous mice carrying only one functional Eng allele are another experimental tool that is suitable to investigate biological functions of endoglin in vascular pathology. Similarly, other studies have shown that the application of siRNA targeting endoglin expression in human and murine endothelial cells is suitable to reduce the levels of endoglin mRNA and protein and to develop a good antiangiogenic therapeutic potential in vivo.
There is also the possibility to transiently or stably overexpress the different splice variants (i.e., S- and L-Eng) in vitro and in vivo (Lastres et al. 1994). In a pioneering study this approach was used to demonstrate that S-Eng has antiangiogenic properties in cancer development (Pérez-Gómez et al. 2005).
Endoglin, also known as cluster of differentiation CD105, was originally identified in 1988 as a novel marker of endothelial cells. Later it was shown that endoglin is also expressed in pro-fibrogenic cells, including mesangial cells, cardiac and scleroderma fibroblasts, and hepatic stellate cells. It is an integral membrane-bound disulfide-linked 180 kDa homodimeric receptor that acts as an auxiliary TGF-β receptor. In humans, a large variety of mutations and variants of the endoglin gene are identified that give rise to an autosomal dominant bleeding disorder that is characterized by localized angiodysplasia and AVMs. This disease, termed hereditary hemorrhagic telangiectasia type I, induces various vascular lesions, mainly on the face, lips, hands, and gastrointestinal mucosa. Mice lacking functional Eng gene show embryonic lethality at day 10–11.5 post coitum due to major defects in angiogenesis and heart development. For experimental endoglin research, mice that are heterozygous for mutations or conditional knockout mice that allow cell-specific inactivation of the Eng gene at different times during development have become attractive tools in endoglin research. Two variants of endoglin (i.e., S- and L-endoglin) are formed by alternative splicing that distinguishes from each other in the length of their cytoplasmic tails. Moreover, a soluble form of endoglin (sol-Eng) is formed by shedding mediated by MMP-14 that cleaves within the extracellular juxtamembrane region, which is competent to modulate signaling and bears potential as a complementary biomarker in some pathological settings. Endoglin interacts with the TGF-β signaling receptors and influences Smad-dependent and Smad-independent effects. Recent work has demonstrated that endoglin is a crucial mediator during liver fibrogenesis that critically controls the activity of the different Smad branches.
RW is supported by grants from the German Research Foundation (SFB/TRR57, P13/Q3) and a grant from the Interdisciplinary Centre for Clinical Research within the Faculty of Medicine at the RWTH Aachen University (IZKF Aachen, Project E7-6). The authors are grateful to Sabine Weiskirchen for preparing line drawings, Dr. J. MacDonald (Department of Radiology, Hereditary Hemorrhagic Telangiectasia Center, University of Utah, Salt Lake City, UT, USA) and Dr. U. Geisthoff (Department of Otorhinolaryngology, Essen University Hospital, Essen, Germany) for providing photographs. In addition, the authors would like to thank the colleagues Prof. Frank Gaillard, Dr. Andrew Dixon, and Dr. Nasir Siddiqui that deposited radiology images in the Radiopaedia.org resource and allowed us to use them in our work.
- Ali BR, Ben-Rebeh I, John A, Akawi NA, Milhem RM, Al-Shehhi NA, Al-Ameri MM, Al-Shamisi SA, Al-Gazali L. Endoplasmic reticulum quality control is involved in the mechanism of endoglin-mediated hereditary haemorrhagic telangiectasia. PLoS One. 2011;6(10):e26206. doi: 10.1371/journal.pone.0026206.PubMedPubMedCentralCrossRefGoogle Scholar
- Bellón T, Corbí A, Lastres P, Calés C, Cebrián M, Vera S, Cheifetz S, Massague J, Letarte M, Bernabéu C. Identification and expression of two forms of the human transforming growth factor-β-binding protein endoglin with distinct cytoplasmic regions. Eur J Immunol. 1993;23:2340–5. doi: 10.1002/eji.1830230943.PubMedCrossRefGoogle Scholar
- Blanco FJ, Santibanez JF, Guerrero-Esteo M, Langa C, Vary CP, Bernabeu C. Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factor-β receptor complex. J Cell Physiol. 2005;204:574–84. doi: 10.1002/jcp.20311.PubMedCrossRefGoogle Scholar
- McAllister KA, Grogg KM, Johnson DW, Gallione CJ, Baldwin MA, Jackson CE, Helmbold EA, Markel DS, McKinnon WC, Murrell J. Endoglin, a TGF-β binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet. 1994;8:345–51. doi: 10.1038/ng1294-345.PubMedCrossRefGoogle Scholar
- Meurer SK, Alsamman M, Sahin H, Wasmuth HE, Kisseleva T, Brenner DA, Trautwein C, Weiskirchen R, Scholten D. Overexpression of endoglin modulates TGF-β1-signalling pathways in a novel immortalized mouse hepatic stellate cell line. PLoS One. 2013;8(2):e56116. doi: 10.1371/journal.pone.0056116.PubMedPubMedCentralCrossRefGoogle Scholar
- Ojeda-Fernández L, Recio-Poveda L, Aristorena M, Lastres P, Blanco FJ, Sanz-Rodríguez F, Gallardo-Vara E, de Las Casas-Engel M, Corbí Á, Arthur HM, Bernabeu C, Botella LM. Mice lacking endoglin in macrophages show an impaired immune response. PLoS Genet. 2016;12(3):e1005935. doi: 10.1371/journal.pgen.1005935.PubMedPubMedCentralCrossRefGoogle Scholar
- Shovlin CL, Guttmacher AE, Buscarini E, Faughnan ME, Hyland RH, Westermann CJ, Kjeldsen AD, Plauchu H. Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome). Am J Med Genet. 2000;91:66–7. doi:10.1002/(SICI)1096-8628(20000306)91:1<66::AID-AJMG12>3.0.CO;2-P.PubMedCrossRefGoogle Scholar
- Xu G, Barrios-Rodiles M, Jerkic M, Turinsky AL, Nadon R, Vera S, Voulgaraki D, Wrana JL, Toporsian M, Letarte M. Novel protein interactions with endoglin and activin receptor-like kinase 1: potential role in vascular networks. Mol Cell Proteomics. 2014;13:489–502. doi: 10.1074/mcp.M113.033464.PubMedCrossRefGoogle Scholar
- Yagmur E, Rizk M, Stanzel S, Hellerbrand C, Lammert F, Trautwein C, Wasmuth HE, Gressner AM. Elevation of endoglin (CD105) concentrations in serum of patients with liver cirrhosis and carcinoma. Eur J Gastroenterol Hepatol. 2007;19:755–61. doi: 10.1097/MEG.0b013e3282202bea.PubMedCrossRefGoogle Scholar