Michelle Letarte

Soluble endoglin contributes to the pathogenesis of preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Maternal endothelial dysfunction mediated by excess placenta-derived soluble VEGF receptor 1 (sVEGFR1 or sFlt1) is emerging as a prominent component in disease pathogenesis. We report a novel placenta-derived soluble TGF-β coreceptor, endoglin (sEng), which is elevated in the sera of preeclamptic individuals, correlates with disease severity and falls after delivery. sEng inhibits formation of capillary tubes in vitro and induces vascular permeability and hypertension in vivo. Its effects in pregnant rats are amplified by coadministration of sFlt1, leading to severe preeclampsia including the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and restriction of fetal growth. sEng impairs binding of TGF-β1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-β signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.

Endoglin Is an Accessory Protein That Interacts with the Signaling Receptor Complex of Multiple Members of the Transforming Growth Factor-β Superfamily

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.

A murine model of hereditary hemorrhagic telangiectasia

Endoglin (CD105), an accessory protein of the TGF-beta receptor superfamily, is highly expressed on endothelial cells. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the Endoglin gene, leading to haploinsufficiency. To generate a disease model and ascertain the role of endoglin in development, we generated mice lacking 1 or both copies of the gene. Endoglin null embryos die at gestational day 10.0-10.5 due to defects in vessel and heart development. Vessel formation appears normal until hemorrhage occurs in yolk sacs and embryos. The primitive vascular plexus of the yolk sac fails to mature into defined vessels, and vascular channels dilate and rupture. Internal bleeding is seen in the peritoneal cavity, implying fragile vessels. Heart development is arrested at day 9.0, and the atrioventricular canal endocardium fails to undergo mesenchymal transformation and cushion-tissue formation. These data suggest that endoglin is critical for both angiogenesis and heart valve formation. Some heterozygotes, either with an inbred 129/Ola or mixed C57BL/6-129/Ola background, show signs of HHT, such as telangiectases or recurrent nosebleeds. In this murine model of HHT, it appears that epigenetic factors and modifier genes, some of which are present in 129/Ola, contribute to disease heterogeneity.

Hereditary haemorrhagic telangiectasia: current views on genetics and mechanisms of disease

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterised by epistaxis, telangiectases, and multiorgan vascular dysplasia. The two major types of disease, HHT1 and HHT2, are caused by mutations in the ENG (endoglin) and ACVRL1 genes, respectively. The corresponding endoglin and ALK-1 proteins are specific endothelial receptors of the transforming growth factor β superfamily essential for maintaining vascular integrity. Many mutations have been identified in ENG and ACVRL1 genes and support the haploinsufficiency model for HHT. Two more genes have recently been implicated in HHT: MADH4 mutated in a combined syndrome of juvenile polyposis and HHT (JPHT), and an unidentified HHT3 gene linked to chromosome 5. Current knowledge on the genetics of HHT is summarised, including the pathways that link the genes responsible for HHT and the potential mechanisms underlying the pathogenesis of the disease.

A Role for Endoglin in Coupling eNOS Activity and Regulating Vascular Tone Revealed in Hereditary Hemorrhagic Telangiectasia

Decreased endothelial NO synthase (eNOS)-derived NO bioavailability and impaired vasomotor control are crucial factors in cardiovascular disease pathogenesis. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a vascular disorder associated with ENDOGLIN (ENG) haploinsufficiency and characterized by venous dilatations, focal loss of capillaries, and arteriovenous malformations (AVMs). We report that resistance arteries from Eng+/− mice display an eNOS-dependent enhancement in endothelium-dependent dilatation and impairment in the myogenic response, despite reduced eNOS levels. We have found that eNOS is significantly reduced in endoglin-deficient endothelial cells because of decreased eNOS protein half-life. We demonstrate that endoglin can reside in caveolae and associate with eNOS, suggesting a stabilizing function of endoglin for eNOS. After Ca2+-induced activation, endoglin-deficient endothelial cells have reduced eNOS/Hsp90 association, produce less NO, and generate more eNOS-derived superoxide (O2−), indicating that endoglin also facilitates eNOS/Hsp90 interactions and is an important regulator in the coupling of eNOS activity. Treatment with an O2− scavenger reverses the vasomotor abnormalities in Eng+/− arteries, suggesting that uncoupled eNOS and resulting impaired myogenic response represent early events in HHT1 pathogenesis and that the use of antioxidants may provide a novel therapeutic modality.

Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative.

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.

Endoglin Null Endothelial Cells Proliferate Faster and Are More Responsive to Transforming Growth Factor β1 with Higher Affinity Receptors and an Activated Alk1 Pathway

Endoglin is an accessory receptor for transforming growth factor β (TGFβ) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFβ type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFβ pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFβ, Eng-/- cells displayed a potent anti-proliferative response. TGFβ-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFβ treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFβ concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFβ type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFβ receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFβ signaling in endothelial cells by regulating surface TGFβ receptors and suppressing Smad1 activation. Thus an altered balance in TGFβ receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.

Ectopic expression of human and feline CD9 in a human B cell line confers beta 1 integrin-dependent motility on fibronectin and laminin substrates and enhanced tyrosine phosphorylation

Few molecules have been shown to confer cell motility. Although the motility-arresting properties of anti-CD9 monoclonal antibody (mAb) suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a motorgenic signal, gene transfection studies have failed to confirm this hypothesis. We report here that ectopic expression of human CD9 (CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human B cell line Raji dramatically enhances migration across fibronectin- and laminin-coated polycarbonate filters. Migration of Raji/CD9h and Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19 and by the anti-β1 integrin mAb AP-138. Migration of Raji/CD9h on laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji cells adhered to fibronectin as effectively as mock transfectants, expression of CD9 enhanced motility, but not adhesion. CD9-enhanced migration was inhibited by the protein tyrosine kinase inhibitor herbimycin A suggesting that tyrosine phosphorylation played a role in the generation of a motorgenic signal. Raji/CD9h transfectants adherent to fibronectin expressed 6-fold higher levels of phosphotyrosine than Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine more effectively than Raji including a protein of 110 kDa which was phosphorylated on the motility-inducing substrates laminin and fibronectin, but not on bovine serum albumin. Our results support a role for CD9 in the amplification of a motorgenic signal in B cells involving β1 integrins and the activation of protein tyrosine kinases.

Endoglin Expression Is Reduced in Normal Vessels but Still Detectable in Arteriovenous Malformations of Patients with Hereditary Hemorrhagic Telangiectasia Type 1

Endoglin is predominantly expressed on endothelium and is mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1). We report the analysis of endoglin in tissues of a newborn (family 2), who died of a cerebral arteriovenous malformation (CAVM), and in a lung specimen surgically resected from a 78-year-old patient (family 5), with a pulmonary AVM (PAVM). The clinically affected father of the newborn revealed a novel mutation that was absent in his parents and was identified as a duplication of exons 3 to 8, by quantitative multiplex polymerase chain reaction. The corresponding mutant protein (116-kd monomer) and the missense mutant protein (80-kd monomer) present in family 5 were detected only as transient intracellular species and were unreactive by Western blot analysis and immunostaining. Normal endoglin (90-kd monomer) was reduced by 50% on peripheral blood-activated monocytes of the HHT1 patients. When analyzed by immunostaining and densitometry, presumed normal blood vessels of the newborn lung and brain and vessels adjacent to the adult PAVM showed a 50% reduction in the endoglin/PECAM-1 ratio. A similar ratio was observed in the CAVM and PAVM, suggesting that all blood vessels of HHT1 patients express reduced endoglin in situ and that AVMs are not attributed to a focal loss of endoglin.

VE‐cadherin is a critical endothelial regulator of TGF‐β signalling

VE‐cadherin is an endothelial‐specific transmembrane protein concentrated at cell‐to‐cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE‐cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE‐cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE‐cadherin expression and junctional clustering are required for optimal transforming growth factor‐β (TGF‐β) signalling in endothelial cells (ECs). TGF‐β antiproliferative and antimigratory responses are increased in the presence of VE‐cadherin. ECs lacking VE‐cadherin are less responsive to TGF‐β/ALK1‐ and TGF‐β/ALK5‐induced Smad phosphorylation and target gene transcription. VE‐cadherin coimmunoprecipitates with all the components of the TGF‐β receptor complex, TβRII, ALK1, ALK5 and endoglin. Clustered VE‐cadherin recruits TβRII and may promote TGF‐β signalling by enhancing TβRII/TβRI assembly into an active receptor complex. Taken together, our data indicate that VE‐cadherin is a positive and EC‐specific regulator of TGF‐β signalling. This suggests that reduction or inactivation of VE‐cadherin may contribute to progression of diseases where TGF‐β signalling is impaired.