Increased endothelial cell permeability in endoglin-deficient cells.

Endoglin (ENG) is a TGF-ß superfamily coreceptor essential for vascular endothelium integrity. ENG mutations lead to a vascular dysplasia associated with frequent hemorrhages in multiple organs, whereas ENG null mouse embryos die at midgestation with impaired heart development and leaky vasculature. ENG interacts with several proteins involved in cell adhesion, and we postulated that it regulates vascular permeability. The current study assessed the permeability of ENG homozygous null (Eng(-/-)), heterozygous (Eng(+/-)), and normal (Eng(+/+)) mouse embryonic endothelial cell (EC) lines. Permeability, measured by passage of fluorescent dextran through EC monolayers, was increased 2.9- and 1.7-fold for Eng(-/-) and Eng(+/-) ECs, respectively, compared to control ECs and was not increased by TGF-ß1 or VEGF. Prolonged starvation increased Eng(-/-) EC permeability by 3.7-fold with no effect on control ECs; neutrophils transmigrated faster through Eng(-/-) than Eng(+/+) monolayers. Using a pull-down assay, we demonstrate that Ras homolog gene family (Rho) A is constitutively active in Eng(-/-) and Eng(+/-) ECs. We show that the endothelial barrier destabilizing factor thrombospondin-1 and its receptor-like protein tyrosine phosphatase are increased, whereas stabilizing factors VEGF receptor 2, vascular endothelial-cadherin, p21-activated kinase, and Ras-related C3 botulinum toxin substrate 2 are decreased in Eng(-/-) cells. Our findings indicate that ENG deficiency leads to EC hyperpermeability through constitutive activation of RhoA and destabilization of endothelial barrier function.

Novel protein interactions with endoglin and activin receptor-like kinase 1: potential role in vascular networks.

Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-ß) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-ß receptor type 2 and involved in TGF-ß signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-ß receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-ß dependent and independent ones, critical for vascular function and potentially defective in HHT.

Rho-kinase inhibitor prevents bleomycin-induced injury in neonatal rats independent of effects on lung inflammation.

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation dominated by neutrophils and macrophages, inhibited distal airway and vascular development, and pulmonary hypertension, similar to human infants with severe bronchopulmonary dysplasia. Rho-kinase (ROCK) is known to mediate lung injury in adult animals via stimulatory effects on inflammation. We therefore hypothesized that inhibition of ROCK may ameliorate bleomycin-induced lung injury in the neonatal rat. Pups received daily intraperitoneal bleomycin or saline from Postnatal Days 1 through 14 with or without Y-27632, a ROCK inhibitor. Treatment with Y-27632 prevented bleomycin-induced pulmonary hypertension, as evidenced by normalized pulmonary vascular resistance, decreased right-ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. Bleomycin-induced changes in distal lung architecture, including septal thinning, inhibited alveolarization, and decreased numbers of peripheral arteries and capillaries, were partially or completely normalized by Y-27632. Treatment with Y-27632 or a CXCR2 antagonist, SB265610, also abrogated tissue neutrophil influx, while having no effect on macrophages. However, treatment with SB265610 did not prevent bleomycin-induced lung injury. Lung content of angiostatic thrombospondin-1 (TSP1) was increased significantly in the lungs of bleomycin-exposed animals, and was completely attenuated by treatment with Y-27632. Thrombin-stimulated TSP1 production by primary cultured rat pulmonary artery endothelial cells was also attenuated by Y-27632. Taken together, our findings suggest a preventive effect of Y-27632 on bleomycin-mediated injury by a mechanism unrelated to inflammatory cells. Our data suggest that improvements in lung morphology may have been related to indirect stimulatory effects on angiogenesis via down-regulation of TSP1.

Endoglin and activin receptor-like kinase 1 heterozygous mice have a distinct pulmonary and hepatic angiogenic profile and response to anti-VEGF treatment.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia associated with dysregulated angiogenesis and arteriovascular malformations. The disease is caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase 1 (ALK1; HHT2) genes, coding for transforming growth factor ß (TGF-ß) superfamily receptors. Vascular endothelial growth factor (VEGF) has been implicated in HHT and beneficial effects of anti-VEGF treatment were recently reported in HHT patients. To investigate the systemic angiogenic phenotype of Endoglin and Alk1 mutant mice and their response to anti-VEGF therapy, we assessed microvessel density (MVD) in multiple organs after treatment with an antibody to mouse VEGF or vehicle. Lungs were the only organ showing an angiogenic defect, with reduced peripheral MVD and secondary right ventricular hypertrophy (RVH), yet distinctly associated with a fourfold increase in thrombospondin-1 (TSP-1) in Eng (+/-) versus a rise in angiopoietin-2 (Ang-2) in Alk1 (+/-) mice. Anti-VEGF treatment did reduce lung VEGF levels but interestingly, led to an increase in peripheral pulmonary MVD and attenuation of RVH; it also normalized TSP-1 and Ang-2 expression. Hepatic MVD, unaffected in mutant mice, was reduced by anti-VEGF therapy in heterozygous and wild type mice, indicating a liver-specific effect of treatment. Contrast-enhanced micro-ultrasound demonstrated a reduction in hepatic microvascular perfusion after anti-VEGF treatment only in Eng (+/-) mice. Our findings indicate that the mechanisms responsible for the angiogenic imbalance and the response to anti-VEGF therapy differ between Eng and Alk1 heterozygous mice and raise the need for systemic monitoring of anti-angiogenic therapy effects in HHT patients.

Anti-VEGF therapy reduces intestinal inflammation in Endoglin heterozygous mice subjected to experimental colitis.

Chronic intestinal inflammation is associated with pathological angiogenesis that further amplifies the inflammatory response. Vascular endothelial growth factor (VEGF), is a major angiogenic cytokine that has been implicated in chronic colitis and inflammatory bowel diseases. Endoglin (CD105), a transforming growth factor-ß superfamily co-receptor expressed on endothelial and some myeloid cells, is a modulator of angiogenesis involved in wound healing and potentially in resolution of inflammation. We showed previously that Endoglin heterozygous (Eng (+/-)) mice subjected to dextran sodium sulfate developed severe colitis, abnormal colonic vessels and high tissue VEGF. We therefore tested in the current study if treatment with a monoclonal antibody to VEGF could ameliorate chronic colitis in Eng (+/-) mice. Tissue inflammation and microvessel density (MVD) were quantified on histological slides. Colonic wall thickness, microvascular hemodynamics and targeted MAdCAM-1(+) inflamed vessels were assessed in vivo by ultrasound. Mediators of angiogenesis and inflammation were measured by Milliplex and ELISA assays. Colitic Eng (+/-) mice showed an increase in intestinal inflammation, MVD, colonic wall thickness, microvascular hemodynamics and the number of MAdCAM-1(+) microvessels relative to colitic Eng (+/+) mice; these parameters were all attenuated by anti-VEGF treatment. Of all factors up-regulated in the inflamed gut, granulocyte colony-stimulating factor (G-CSF) and amphiregulin were further increased in colitic Eng (+/-) versus Eng (+/+) mice. Anti-VEGF therapy decreased tissue VEGF and inflammation-induced endoglin, IL-1ß and G-CSF in colitic Eng (+/-) mice. Our results suggest that endoglin modulates intestinal angiogenic and inflammatory responses in colitis. Furthermore, contrast-enhanced ultrasound provides an excellent non-invasive imaging modality to monitor gut angiogenesis, inflammation and responses to anti-angiogenic treatment.

A critical role for endoglin in the emergence of blood during embryonic development.

Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here, we show that endoglin (Eng), a receptor for the TGFß superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng(+)Flk1(+) mesodermal precursor population at embryonic day 7.5 (E7.5), a cell fraction also endowed with endothelial potential. In Eng-knockout embryos, hematopoietic colony activity and numbers of CD71(+)Ter119(+) erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key bone morphogenic protein (BMP) target genes, including the hematopoietic regulators Scl, Gata1, Gata2, and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng(+)Flk-1(+) progenitors coexpressed TGFß and BMP receptors and target genes. Furthermore, Eng(+)Flk-1(+) cells presented high levels of phospho-SMAD1/5, indicating active TGFß and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of Eng-expressing cells was dependent on the TGFß superfamily ligands BMP4, BMP2, or TGF-ß1. These data demonstrate that the E(+)F(+) fraction at E7.5 represents mesodermal cells competent to respond to TGFß1, BMP4, or BMP2, shaping their hematopoietic development, and that Eng acts as a critical regulator in this process by modulating TGF/BMP signaling.

Impaired resolution of inflammation in the Endoglin heterozygous mouse model of chronic colitis.

Endoglin is a coreceptor of the TGF-ß superfamily predominantly expressed on the vascular endothelium and selective subsets of immune cells. We previously demonstrated that Endoglin heterozygous (Eng (+/-)) mice subjected to dextran sulfate sodium (DSS) developed persistent gut inflammation and pathological angiogenesis. We now report that colitic Eng (+/-) mice have low colonic levels of active TGF-ß1, which was associated with reduced expression of thrombospondin-1, an angiostatic factor known to activate TGF-ß1. We also demonstrate dysregulated expression of BMPER and follistatin, which are extracellular regulators of the TGF-ß superfamily that modulate angiogenesis and inflammation. Heightened colonic levels of the neutrophil chemoattractant and proangiogenic factor, CXCL1, were also observed in DSS-treated Eng (+/-) mice. Interestingly, despite increased macrophage and neutrophil infiltration, a gut-specific reduction in expression of the key phagocytic respiratory burst enzymes, NADPH oxidase 2 (Nox-2) and myeloperoxidase, was seen in Eng (+/-) mice undergoing persistent inflammation. Taken together, these findings suggest that endoglin is required for TGF-ß superfamily mediated resolution of inflammation and fully functional myeloid cells.

Reduced endoglin activity limits cardiac fibrosis and improves survival in heart failure.

BACKGROUND: Heart failure is a major cause of morbidity and mortality worldwide. The ubiquitously expressed cytokine transforming growth factor-ß1 (TGFß1) promotes cardiac fibrosis, an important component of progressive heart failure. Membrane-associated endoglin is a coreceptor for TGFß1 signaling and has been studied in vascular remodeling and preeclampsia. We hypothesized that reduced endoglin expression may limit cardiac fibrosis in heart failure. METHODS AND RESULTS: We first report that endoglin expression is increased in the left ventricle of human subjects with heart failure and determined that endoglin is required for TGFß1 signaling in human cardiac fibroblasts using neutralizing antibodies and an siRNA approach. We further identified that reduced endoglin expression attenuates cardiac fibrosis, preserves left ventricular function, and improves survival in a mouse model of pressure-overload-induced heart failure. Prior studies have shown that the extracellular domain of endoglin can be cleaved and released into the circulation as soluble endoglin, which disrupts TGFß1 signaling in endothelium. We now demonstrate that soluble endoglin limits TGFß1 signaling and type I collagen synthesis in cardiac fibroblasts and further show that soluble endoglin treatment attenuates cardiac fibrosis in an in vivo model of heart failure. CONCLUSION: Our results identify endoglin as a critical component of TGFß1 signaling in the cardiac fibroblast and show that targeting endoglin attenuates cardiac fibrosis, thereby providing a potentially novel therapeutic approach for individuals with heart failure.

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-beta 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-beta1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-beta signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.

VE-cadherin is a critical endothelial regulator of TGF-beta 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-beta (TGF-beta) signalling in endothelial cells (ECs). TGF-beta antiproliferative and antimigratory responses are increased in the presence of VE-cadherin. ECs lacking VE-cadherin are less responsive to TGF-beta/ALK1- and TGF-beta/ALK5-induced Smad phosphorylation and target gene transcription. VE-cadherin coimmunoprecipitates with all the components of the TGF-beta receptor complex, TbetaRII, ALK1, ALK5 and endoglin. Clustered VE-cadherin recruits TbetaRII and may promote TGF-beta signalling by enhancing TbetaRII/TbetaRI assembly into an active receptor complex. Taken together, our data indicate that VE-cadherin is a positive and EC-specific regulator of TGF-beta signalling. This suggests that reduction or inactivation of VE-cadherin may contribute to progression of diseases where TGF-beta signalling is impaired.

Spontaneous adult-onset pulmonary arterial hypertension attributable to increased endothelial oxidative stress in a murine model of hereditary hemorrhagic telangiectasia.

OBJECTIVE: Loss-of-function mutations in genes coding for transforming growth factor-beta/bone morphogenetic protein receptors and changes in nitric oxide(*) (NO(*)) bioavailability are associated with hereditary hemorrhagic telangiectasia and some forms of pulmonary arterial hypertension. How these abnormalities lead to seemingly disparate pulmonary pathologies remains unknown. Endoglin (Eng), a transforming growth factor-beta coreceptor, is mutated in hereditary hemorrhagic telangiectasia and involved in regulating endothelial NO(*) synthase (eNOS)-derived NO(*) production and oxidative stress. Because some patients with pulmonary arterial hypertension harbor ENG mutations leading to haplo insufficiency, we investigated the pulmonary vasculature of Eng(+/-) mice and the potential contribution of abnormal eNOS activation to pulmonary arterial hypertension. METHODS AND RESULTS: Hemodynamic, histological, and biochemical assessments and x-ray micro-CT imaging of adult Eng(+/-) mice indicated signs of pulmonary arterial hypertension including increased right ventricular systolic pressure, degeneration of the distal pulmonary vasculature, and muscularization of small arteries. These findings were absent in 3-week-old Eng(+/-) mice and were attributable to constitutively uncoupled eNOS activity in the pulmonary circulation, as evidenced by reduced eNOS/heat shock protein 90 association and increased eNOS-derived superoxide ((*)O(2)(-)) production in a BH(4)-independent manner. These changes render eNOS unresponsive to regulation by transforming growth factor-beta/bone morphogenetic protein and underlie the signs of pulmonary arterial hypertension that were prevented by Tempol. CONCLUSIONS: Adult Eng(+/-) mice acquire signs of pulmonary arterial hypertension that are attributable to uncoupled eNOS activity and increased (*)O(2)(-) production, which can be prevented by antioxidant treatment. Eng links transforming growth factor/bone morphogenetic protein receptors to the eNOS activation complex, and its reduction in the pulmonary vasculature leads to increased oxidative stress and pulmonary arterial hypertension.

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo.

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-beta superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.

Soluble endoglin modulates aberrant cerebral vascular remodeling.

OBJECTIVE: Brain arteriovenous malformations (AVMs) are an important cause of neurological morbidity in young adults. The pathophysiology of these lesions is poorly understood. A soluble form of endoglin (sEng) has been shown to cause endothelial dysfunction and induce preeclampsia. We tested if sEng would be elevated in brain AVM tissues relative to epilepsy brain tissues, and also investigated whether sEng overexpression via gene transfer in the mouse brain would induce vascular dysplasia and associated changes in downstream signaling pathways. METHODS: Expression levels of sEng in surgical specimens were determined by Western blot assay and enzyme-linked immunosorbent assay. Vascular dysplasia, levels of matrix metalloproteinase (MMP), and oxidative stress were determined by immunohistochemistry and gelatin zymography. RESULTS: Brain AVMs (n = 33) had higher mean sEng levels (245 +/- 175 vs 100 +/- 60, % of control, p = 0.04) compared with controls (n = 8), as determined by Western blot. In contrast, membrane-bound Eng was not significantly different (108 +/- 79 vs 100 +/- 63, % of control, p = 0.95). sEng gene transduction in the mouse brain induced abnormal vascular structures. It also increased MMP activity by 490 +/- 30% (MMP-9) and 220 +/- 30% (MMP-2), and oxidants by 260 +/- 20% (4-hydroxy-2-nonenal) at 2 weeks after injection, suggesting that MMPs and oxidative radicals may mediate sEng-induced pathological vascular remodeling. INTERPRETATION: The results suggest that elevated sEng may play a role in the generation of sporadic brain AVMs. Our findings may provide new targets for therapeutic intervention for patients with brain AVMs. Ann Neurol 2009;66:19-27.

Potential Second-Hits in Hereditary Hemorrhagic Telangiectasia.

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.

Immunology Education Without Borders.

One of the mandates of the International Union of Immunological Societies (IUIS) is to promote immunological education to young scientists across the globe, including a large focus on those from low and low-to-middle income countries (LIC and LMIC). It strives to achieve this goal through the Education Committee (EDU), which is one of ten committees of the IUIS. To this end, EDU organizes three to four one-week courses per year in close cooperation with regional immunological societies and local organizers. Initially, the focus has been on Africa, addressing the most relevant topics and health issues facing specific countries or regions in the continent. The idea was then extended to Latin America and now also includes courses in Asia. The faculty of all courses is a blend of international and local/regional experts also known for their teaching expertise. The courses are highly interactive, and include "meet-the-speakers" sessions, poster walks, and sessions on grant or PhD project writing, and on practical aspects of becoming a successful scientist. Importantly, all the IUIS-EDU courses use a combination of pre- and during-course on-line learning followed by consolidation of knowledge in a collegial setting. This "flipped" classroom approach ensures that participants have acquired the basic knowledge needed to optimize their participation in the course. Immunopaedia is the IUIS-endorsed immunology learning site used for this purpose. All faculty members are requested to contribute material related to their specific topic while students must learn the on-line material before coming in person to the course. All course participants have free access to all Immunopaedia material indefinitely. The implementation of regional immunology courses targeted to local health issues in areas of the world where PhD students, post-doctoral, and early career scientists often do not have access to open on-line resources and contact with renowned experts in the field has proven to be highly successful. The long-term impact of this structured educational program is already visible through the large number of young scientists who are now connected via Immunopaedia and who are forming networks in regions where there had been very little contact before and building new Immunological Societies.

Endoglin regulates cyclooxygenase-2 expression and activity.

The endoglin heterozygous (Eng(+/-)) mouse, which serves as a model of hereditary hemorrhagic telangiectasia (HHT), was shown to express reduced levels of endothelial NO synthase (eNOS) with impaired activity. Because of intricate changes in vasomotor function in the Eng(+/-) mice and the potential interactions between the NO- and prostaglandin-producing pathways, we assessed the expression and function of cyclooxygenase (COX) isoforms. A specific upregulation of COX-2 in the vascular endothelium and increased urinary excretion of prostaglandin E(2) were observed in the Eng(+/-) mice. Specific COX-2 inhibition with parecoxib transiently increased arterial pressure in Eng(+/-) but not in Eng(+/+) mice. Transfection of endoglin in L6E9 myoblasts, shown previously to stimulate eNOS expression, led to downregulation of COX-2 with no change in COX-1. In addition, COX-2 promoter activity and protein levels were inversely correlated with endoglin levels, in doxycyclin-inducible endothelial cells. Chronic NO synthesis inhibition with N(omega)-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng(+/+) mice. N(omega)-nitro-l-arginine methyl ester also increased COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells. The level of COX-2 expression following transforming growth factor-beta1 treatment was less in endoglin than in mock transfected L6E9 myoblasts and was higher in human endothelial cells silenced for endoglin expression. Our results indicate that endoglin is involved in the regulation of COX-2 activity. Furthermore, reduced endoglin levels and associated impaired NO production may be responsible, at least in part, for augmented COX-2 expression and activity in the Eng(+/-) mice.

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.

Reduced angiogenic responses in adult Endoglin heterozygous mice.

OBJECTIVE: To determine if angiogenesis is altered in adult Endoglin heterozygous (Eng(+/-)) mice, the animal model for the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). METHODS: Primary cultures of endothelial cells were generated from Eng(+/-) and Eng(+/+) mice and analyzed for proliferation, migration, and ability to form capillary-like tubes. Endothelial cells derived from umbilical veins of newborns (HUVEC) with an HHT1 genotype were also tested for capillary formation. Two in vivo models of angiogenesis were tested in the Eng(+/-) and Eng(+/+) mice: Matrigel implant-dependent angiogenesis and reperfusion following hindlimb ischemia. RESULTS: The Eng(+/-) endothelial cells displayed significantly reduced proliferation and migration, increased collagen production, and decreased NO synthase expression and vascular endothelial growth factor (VEGF) secretion. They also showed impaired capillary tube formation in vitro, as did the HHT1 HUVEC. These endothelial cell-specific abnormalities were associated with impaired Matrigel-dependent capillary tube formation in vivo and delayed reperfusion following hindlimb ischemia. CONCLUSIONS: Although vascular development is normal in Eng(+/-) mice, angiogenic abnormalities were observed in the adult mice and their isolated endothelial cells. These results suggest that a normal level of endoglin is required for full angiogenic activity.

Structural Basis of the Human Endoglin-BMP9 Interaction: Insights into BMP Signaling and HHT1.

Endoglin (ENG)/CD105 is an essential endothelial cell co-receptor of the transforming growth factor ß (TGF-ß) superfamily, mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1) and involved in tumor angiogenesis and preeclampsia. Here, we present crystal structures of the ectodomain of human ENG and its complex with the ligand bone morphogenetic protein 9 (BMP9). BMP9 interacts with a hydrophobic surface of the N-terminal orphan domain of ENG, which adopts a new duplicated fold generated by circular permutation. The interface involves residues mutated in HHT1 and overlaps with the epitope of tumor-suppressing anti-ENG monoclonal TRC105. The structure of the C-terminal zona pellucida module suggests how two copies of ENG embrace homodimeric BMP9, whose binding is compatible with ligand recognition by type I but not type II receptors. These findings shed light on the molecular basis of the BMP signaling cascade, with implications for future therapeutic interventions in this fundamental pathway.

A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia.

Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women.