Signaling by transforming growth factor (TGF)-␤ family members is mediated by Smad proteins that regulate gene transcription through functional cooperativity and association with other DNA-binding proteins. The hypoxia-inducible factor (HIF)-1 is a transcriptional complex that plays a key role in oxygen-regulated gene expression. We demonstrate that hypoxia and TGF-␤ cooperate in the induction of the promoter activity of vascular endothelial growth factor (VEGF), which is a major stimulus in the promotion of angiogenesis. This cooperation has been mapped on the human VEGF promoter within a region at ؊1006 to ؊954 that contains functional DNA-binding sequences for HIF-1 and Smads. Optimal HIF-1␣-dependent induction of the VEGF promoter was obtained in the presence of Smad3, suggesting an interaction between these proteins. Consistent with this, co-immunoprecipitation experiments revealed that HIF-1␣ physically associates with Smad3. These results demonstrate that both TGF-␤ and hypoxia signaling pathways can synergize in the regulation of VEGF gene expression at the transcriptional level.

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Endoglin is a transforming growth factor-␤ (TGF-␤) co-receptor expressed mainly on endothelial cells and involved in cardiovascular development, angiogenesis, and vascular remodeling. This is illustrated by the fact that mutations in the endoglin gene give rise to hereditary hemorrhagic telangiectasia type 1, a dominant vascular disease with clinical manifestations that originate by a mechanism of haploinsufficiency. Thus, studies on the regulated expression of endoglin are crucial to devising therapeutic strategies for hereditary hemorrhagic telangiectasia type 1. Endoglin is highly expressed in the neovasculature associated with hypoxia such as ischemic tissues and tumors, but the molecular mechanism of this up-regulation is unknown. Here, we have investigated the possible regulation of endoglin expression by hypoxia. Surface protein, transcript, and promoter activity levels of endoglin were found to be up-regulated by hypoxia, indicating that the regulation takes place at the transcriptional level. A hypoxia-responsive element downstream of the main transcription start site of the endoglin gene was functionally characterized. Whereas hypoxia alone moderately stimulated endoglin transcription, addition of TGF-␤ under hypoxic conditions resulted in transcriptional cooperation between both signaling pathways, leading to marked stimulation of endoglin expression. Because basal endoglin transcription is sustained by Sp1, and TGF-␤ and hypoxia signaling pathways are mediated by Smad proteins and hypoxia-inducible factor-1 (HIF-1), respectively, the involvement of these transcription factors was analyzed. Functional and co-immunoprecipitation experiments demonstrated the existence of a multiprotein complex (Sp1⅐Smad3⅐HIF-1) on the endoglin promoter, mediating the cooperation between the hypoxia and TGF-␤ pathways. Within this multiprotein complex, Smad3 appears to function not only as a coactivator factor, but also as an adaptor between HIF-1 and Sp1. We propose that basal endoglin transcription (highly dependent on Sp1) may switch from a constitutive to an inducible state through Sp1 interaction with HIF-1 and Smad transcription factors, induced by hypoxia and TGF-␤, respectively.

Hereditary hemorrhagic telangiectasia (HHT), the most common inherited vascular disorder, is caused by mutations in genes involved in the transforming growth factor beta (TGF-β) signaling pathway (ENG, ACVRL1, and SMAD4). Yet, approximately 15% of individuals with clinical features of HHT do not have mutations in these genes, suggesting that there are undiscovered mutations in other genes for HHT and possibly vascular disorders with overlapping phenotypes. The genetic etiology for 191 unrelated individuals clinically suspected to have HHT was investigated with the use of exome and Sanger sequencing; these individuals had no mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9 (also known as GDF2) were identified in three unrelated probands. These three individuals had epistaxis and dermal lesions that were described as telangiectases but whose location and appearance resembled lesions described in some individuals with RASA1-related disorders (capillary malformation-arteriovenous malformation syndrome). Analyses of the variant proteins suggested that mutations negatively affect protein processing and/or function, and a bmp9-deficient zebrafish model demonstrated that BMP9 is involved in angiogenesis. These data confirm a genetic cause of a vascular-anomaly syndrome that has phenotypic overlap with HHT.

Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-␤ (TGF-␤) signaling receptors to bind TGF-␤ family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-␤, TGF-␤ signaling receptors, and TGF-␤-stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglin promoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated the endoglin promoter and those of collagen ␣1(I), urokinase-type plasminogen activator, TGF-␤1, and TGF-␤ receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the Cterminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-␤ signaling complex in vascular repair. IntroductionCoordinated gene expression is a crucial requirement in the response to tissue injury. Extracellular matrix proteins, 1-3 growth factors such as transforming growth factor-␤ (TGF-␤), 1,4,5 and proteases such as urokinase-type plasminogen activator (uPA) 6,7 are jointly regulated. In particular, the TGF-␤ family plays a central role in the injury response based on the following: (1) TGF-␤1 expression is up-regulated after injury; 8,9 (2) infusion of TGF-␤ polypeptide or transfection of cDNA into injured arteries increases extracellular matrix production; 1,10 and (3) antibodies to TGF-␤ reduce intimal hyperplasia. 11 Members of the TGF-␤ superfamily exert their biologic functions through membrane receptors known as type 1 (T␤RI) and type 2 (T␤RII) serine/threonine kinases. After ligand binding, T␤RII recruits and phosphorylates T␤RI, which initiates the signaling pathway by phosphorylating the Smad family of proteins. 12,13 Endoglin is a homodimeric membrane glycoprotein that functions, in association with T␤RI and T␤RII, as an auxiliary receptor for TGF-␤1, TGF-␤3, activin, bone morphogenetic protein 2 (BMP-2), and BMP-7. [14][15][16] It is highly expressed by endothelial cells 17,18 and, at lower levels, by activated monocytes/ macrophages 19 and by mesenchymal cells, including...

Key Points• Endothelial endoglin has a regulatory role in leukocyte trafficking through vascular endothelia.• Leukocytes and endothelial cells interact via integrin receptors and endoglin, being this cell adhesion process stimulated by inflammatory stimuli.Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng ؉/؊ ) and their wild-type siblings Eng ؉/؉ treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng ؉/؊ than in Eng ؉/؉ mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglincoated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin ␣5␤1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin ␣5␤1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking. (Blood. 2013;121(2):403-415) IntroductionThe vascular endothelium controls the transit of white blood cells into and out of the bloodstream. The migration of leukocytes involves the adhesive interaction of cell surface receptors with ligands expressed on endothelial cells in a process regulated by inflammatory stimuli. Stromal-derived factor 1 (SDF1␣), renamed CXCL12, is a potent chemoattractant for a variety of cells including lymphocytes, monocytes, dendritic cells, and hematopoietic stem cells. 1 CXCL12 and its receptor CXCR4 play relevant roles in immune and inflammatory responses, including leukocyte migration and recruitment, as well as integrin-dependent adhesion and transendothelial migration. 1 CXCL12 is a critical activator of endothelial progenitors by inducing a proangiogenic phenotype and increasing migration and rolling mediated by ␣4 and ␣M integrin subunits. 2 The process of leukocyte migration through the endothelial cell monolayer involves an interaction between leukocytes' integrins and endothelial-cell receptors, both acting as adhesion molecules. Integrins most relevant to leukocytes belong to the ␤ 1 -integrin and the ␤ 2 -integrin subfamilies. Classic chemoattractants and chemokines are the most powerful physiologic activat...

Endoglin is a component of the transforming growth factor-␤ receptor complex abundantly expressed at the surface of endothelial cells and plays an important role in cardiovascular development and vascular remodeling. By using the cytoplasmic domain of endoglin as a bait for screening protein interactors, we have identified ZRP-1 (zyxin-related protein 1), a 476-amino acid member that belongs to a family of LIM containing proteins that includes zyxin and lipoma-preferred partner. The endoglin interacting region was mapped within the three double zinc finger LIM domains of the ZRP-1 C terminus. Analysis of the subcellular distribution of ZRP-1 demonstrated that in the absence of endoglin, ZRP-1 mainly localizes to focal adhesion sites, whereas in the presence of endoglin ZRP-1 is found along actin stress fibers. Because the LIM family of proteins has been shown to associate with the actin cytoskeleton, we investigated the possibility of a regulatory role for endoglin with regard to this structure. Expression of endoglin resulted in a dramatic reorganization of the actin cytoskeleton. In the absence of endoglin, F-actin was localized to dense aggregates of bundles, whereas in the presence of endoglin, expressed in endothelial cells, F-actin was in stress fibers and colocalized with ZRP-1. Furthermore, small interfering RNA-mediated suppression of endoglin or ZRP-1, or clustering of endoglin in endothelial cells, led to mislocalization of Factin fibers. These results suggest a regulatory role for endoglin, via its interaction with ZRP-1, in the actin cytoskeletal organization.

Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in endoglin (ENG; HHT1) or ACVRL1/ALK1 (HHT2) genes and is an autosomal dominant vascular dysplasia. Clinically, HHT is characterized by epistaxis, telangiectases and arteriovenous malformations in some internal organs such as the lung, brain or liver. Endoglin and ALK1 proteins are specific endothelial receptors of the transforming growth factor (TGF)-β superfamily that are essential for vascular integrity. Genetic studies in mice and humans have revealed the pivotal role of TGF-β signaling during angiogenesis. Through binding to the TGF-β type II receptor, TGF-β can activate two distinct type I receptors (ALK1 and ALK5) in endothelial cells, each one leading to opposite effects on endothelial cell proliferation and migration.The recent isolation and characterization of circulating endothelial cells from HHT patients has revealed a decreased endoglin expression, impaired ALK1-and ALK5-dependent TGF-β signaling, disorganized cytoskeleton and the failure to form cord-like structures which may lead to the fragility of small vessels with bleeding characteristic of HHT vascular dysplasia or to disrupted and abnormal angiogenesis after injuries and may explain the clinical symptoms associated with this disease.