Novel vascular roles of human endoglin in pathophysiology.

Endoglin, alias CD105, is a human membrane glycoprotein highly expressed in vascular endothelial cells. It is involved in angiogenesis and angiogenesis-related diseases, including the rare vascular pathology known as hereditary hemorrhagic telangiectasia type 1. Although endoglin acts as an accessory receptor for members of the transforming growth factor-ß family, in recent years, emerging evidence has shown a novel functional role for this protein beyond the transforming growth factor-ß system. In fact, endoglin has been found to be an integrin counterreceptor involved in endothelial cell adhesion processes during pathological inflammatory conditions and primary hemostasis. Furthermore, a circulating form of endoglin, also named as soluble endoglin, whose levels are abnormally increased in different pathological conditions, such as preeclampsia, seems to act as an antagonist of membrane-bound endoglin and as a competitor of the fibrinogen-integrin interaction in platelet-dependent thrombus formation. These studies suggest that membrane-bound endoglin and circulating endoglin are important components involved in vascular homeostasis and hemostasis.

Correlation Between Endoglin and Malignant Phenotype in Human Melanoma Cells: Analysis of hsa-mir-214 and hsa-mir-370 in Cells and Their Extracellular Vesicles.

Endoglin (CD105) is an auxiliary receptor of transforming growth factor (TGF)-ß family members that is expressed in human melanomas. It is heterogeneously expressed by primary and metastatic melanoma cells, and endoglin targeting as a therapeutic strategy for melanoma tumors is currently been explored. However, its involvement in tumor development and malignancy is not fully understood. Here, we find that endoglin expression correlates with malignancy of primary melanomas and cultured melanoma cell lines. Next, we have analyzed the effect of ectopic endoglin expression on two miRNAs (hsa-mir-214 and hsa-mir-370), both involved in melanoma tumor progression and endoglin regulation. We show that compared with control cells, overexpression of endoglin in the WM-164 melanoma cell line induces; (i) a significant increase of hsa-mir-214 levels in small extracellular vesicles (EVs) as well as an increased trend in cells; and (ii) significantly lower levels of hsa-mir-370 in the EVs fractions, whereas no significant differences were found in cells. As hsa-mir-214 and hsa-mir-370 are not just involved in melanoma tumor progression, but they can also target endoglin-expressing endothelial cells in the tumor vasculature, these results suggest a complex and differential regulatory mechanism involving the intracellular and extracellular signaling of hsa-mir-214 and hsa-mir-370 in melanoma development and progression.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbß3 integrin.

BACKGROUND: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbß3, thereby compromising platelet binding to fibrinogen and thrombus stability. METHODS: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. RESULTS: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbß3 and sEng and molecular modeling showed a good fitting between αIIbß3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbß3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. CONCLUSIONS: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbß3, suggesting its involvement in primary hemostasis control.

Monoclonal anti-endoglin antibody TRC105 (carotuximab) prevents hypercholesterolemia and hyperglycemia-induced endothelial dysfunction in human aortic endothelial cells.

Endoglin (Eng) is a co-receptor of the transforming growth factor ß superfamily playing an important role in endothelial dysfunction. TRC105 (carotuximab) is a monoclonal antibody that blocks Eng and its downstream Smad signaling pathway. Here we have investigated for the first time the effects of TRC105 treatment on the development of endothelial dysfunction induced by 7-ketocholesterol (7K) or high glucose (HG), focusing on Eng expression, signaling, and function. In the hypercholesterolemia study, human aortic endothelial cells (HAoECs) were treated with TRC105 (300 µg/ml) for 1 h, followed by the addition of 7K (10 µg/ml) for another 12 h. In the hyperglycemia study, HAoECs were exposed to HG (45 mM) for 60 h, followed by the addition of TRC105 for another 12 h, and cells treated with 5mM glucose and 40 mM mannitol served as control. Protein levels, adhesion, and transmigration of monocytes were assessed by flow cytometry, mRNA expression was measured by qRT-PCR. 7K and HG treatment increased protein levels of NF-κB and Eng and adhesion and transmigration of monocytes through HAoECs monolayer. TRC105 pretreatment reduced the 7K- or HG-induced Eng protein levels and pSmad1/5 and pSmad2/3 signaling. Despite increased protein levels of P-selectin and VCAM-1, TRC105 mediated blockage of Eng prevented 7K- and HG-induced adhesion and transmigration of monocytes through endothelial monolayers. These results suggest that TRC105-mediated Eng blockage can counteract the hypercholesterolemia- and hyperglycemia-induced endothelial dysfunction in HAoECs, suggesting that Eng might be a potential therapeutic target in disorders associated with elevated cholesterol and glucose levels.

Endoglin and MMP14 Contribute to Ewing Sarcoma Spreading by Modulation of Cell-Matrix Interactions.

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell-matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.

Functional Alterations Involved in Increased Bleeding in Hereditary Hemorrhagic Telangiectasia Mouse Models.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal-dominant genetic disorder involving defects in two predominant genes known as endoglin (ENG; HHT-1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT-2). It is characterized by mucocutaneous telangiectases that, due to their fragility, frequently break causing recurrent epistaxis and gastrointestinal bleeding. Because of the severity of hemorrhages, the study of the hemostasis involved in these vascular ruptures is critical to find therapies for this disease. Our results demonstrate that HHT patients with high bleeding, as determined by a high Epistaxis Severity Score (ESS), do not have prolonged clotting times or alterations in clotting factors. Considering that coagulation is only one of the processes involved in hemostasis, the main objective of this study was to investigate the overall mechanisms of hemostasis in HHT-1 (Eng +/-) and HHT-2 (Alk1 +/-) mouse models, which do not show HHT vascular phenotypes in the meaning of spontaneous bleeding. In Eng +/- mice, the results of in vivo and in vitro assays suggest deficient platelet-endothelium interactions that impair a robust and stable thrombus formation. Consequently, the thrombus could be torn off and dragged by the mechanical force exerted by the bloodstream, leading to the reappearance of hemorrhages. In Alk1 +/- mice, an overactivation of the fibrinolysis system was observed. These results support the idea that endoglin and Alk1 haploinsufficiency leads to a common phenotype of impaired hemostasis, but through different mechanisms. This contribution opens new therapeutic approaches to HHT patients' epistaxis.

Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein.

Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin.

Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/- mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFα and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFα-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/-, and MLEC+/- compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

Homozygous GDF2 nonsense mutations result in a loss of circulating BMP9 and BMP10 and are associated with either PAH or an "HHT-like" syndrome in children.

BACKGROUND: Disrupted endothelial BMP9/10 signaling may contribute to the pathophysiology of both hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH), yet loss of circulating BMP9 has not been confirmed in individuals with ultra-rare homozygous GDF2 (BMP9 gene) nonsense mutations. We studied two pediatric patients homozygous for GDF2 (BMP9 gene) nonsense mutations: one with PAH (c.[76C>T];[76C>T] or p.[Gln26Ter];[Gln26Ter] and a new individual with pulmonary arteriovenous malformations (PAVMs; c.[835G>T];[835G>T] or p.[Glu279Ter];[Glu279Ter]); both with facial telangiectases. METHODS: Plasma samples were assayed for BMP9 and BMP10 by ELISA. In parallel, serum BMP activity was assayed using an endothelial BRE-luciferase reporter cell line (HMEC1-BRE). Proteins were expressed for assessment of secretion and processing. RESULTS: Plasma levels of both BMP9 and BMP10 were undetectable in the two homozygous index cases and this corresponded to low serum-derived endothelial BMP activity in the patients. Measured BMP9 and BMP10 levels were reduced in the asymptomatic heterozygous p.[Glu279Ter] parents, but serum activity was normal. Although expression studies suggested alternate translation can be initiated at Met57 in the p.[Gln26Ter] mutant, this does not result in secretion of functional BMP9. CONCLUSION: Collectively, these data show that homozygous GDF2 mutations, leading to a loss of circulating BMP9 and BMP10, can cause either pediatric PAH and/or "HHT-like" telangiectases and PAVMs. Although patients reported to date have manifestations that overlap with those of HHT, none meet the Curaçao criteria for HHT and seem distinct from HHT in terms of the location and appearance of telangiectases, and a tendency for tiny, diffuse PAVMs.

Pregnancy-Induced High Plasma Levels of Soluble Endoglin in Mice Lead to Preeclampsia Symptoms and Placental Abnormalities.

Preeclampsia is a pregnancy-specific disease of high prevalence characterized by the onset of hypertension, among other maternal or fetal signs. Its etiopathogenesis remains elusive, but it is widely accepted that abnormal placentation results in the release of soluble factors that cause the clinical manifestations of the disease. An increased level of soluble endoglin (sEng) in plasma has been proposed to be an early diagnostic and prognostic biomarker of this disease. A pathogenic function of sEng involving hypertension has also been reported in several animal models with high levels of plasma sEng not directly dependent on pregnancy. The aim of this work was to study the functional effect of high plasma levels of sEng in the pathophysiology of preeclampsia in a model of pregnant mice, in which the levels of sEng in the maternal blood during pregnancy replicate the conditions of human preeclampsia. Our results show that wild type pregnant mice carrying human sEng-expressing transgenic fetuses (fWT(hsEng+)) present high plasma levels of sEng with a timing profile similar to that of human preeclampsia. High plasma levels of human sEng (hsEng) are associated with hypertension, proteinuria, fetal growth restriction, and the release of soluble factors to maternal plasma. In addition, fWT(hsEng+) mice also present placental alterations comparable to those caused by the poor remodeling of the spiral arteries characteristic of preeclampsia. In vitro and ex vivo experiments, performed in a human trophoblast cell line and human placental explants, show that sEng interferes with trophoblast invasion and the associated pseudovasculogenesis, a process by which cytotrophoblasts switch from an epithelial to an endothelial phenotype, both events being related to remodeling of the spiral arteries. Our findings provide a novel and useful animal model for future research in preeclampsia and reveal a much more relevant role of sEng in preeclampsia than initially proposed.

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.

Differential Expression of Circulating Plasma miRNA-370 and miRNA-10a from Patients with Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant, vascular disorder that presents with telangiectases and arteriovenous malformations. HHT is a genetically heterogeneous disorder, involving mutations in endoglin (ENG; HHT1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT2) genes that account for over 85% of all HHT patients. The current diagnosis of HHT patients remains at the clinical level, but many suspected patients do not have a clear HHT diagnosis or do not show pathogenic mutations in HHT genes. This situation has prompted the search for biomarkers to help in the early diagnosis of the disease. We have analyzed the plasma levels in HHT patients of selected micro-RNAs (miRNAs), small single-stranded RNAs that regulate gene expression at the transcriptional level by interacting with specific RNA targets. A total of 16 HHT1 and 17 HHT2 plasma samples from clinically confirmed patients and 16 controls were analyzed in this study. Total RNA was purified from plasma, and three selected miRNAs (miRNA-10a, miRNA-214, and miRNA-370), related to the pathobiology of cardiovascular diseases and potentially targeting ENG or ALK1, were measured by quantitative polymerase chain reaction. Compared with controls, levels of miRNA-370, whose putative target is ENG, were significantly downregulated in HHT1, but not in HHT2, whereas the levels of miRNA-10a, whose putative target is ALK1, were significantly upregulated in HHT2, but not in HHT1. In addition, the levels of miRNA-214, potentially targeting ENG and ALK1, did not change in either HHT1 or HHT2 patients versus control samples. While further studies are warranted, these results suggest that dysregulated plasma levels of miRNA-370 or miRNA-10a could help to identify undiagnosed HHT1 or HHT2 patients, respectively.

Review of Pharmacological Strategies with Repurposed Drugs for Hereditary Hemorrhagic Telangiectasia Related Bleeding.

The diagnosis of hereditary hemorrhagic telangiectasia (HHT) is based on the Curaçao criteria: epistaxis, telangiectases, arteriovenous malformations in internal organs, and family history. Genetically speaking, more than 90% of HHT patients show mutations in ENG or ACVRL1/ALK1 genes, both belonging to the TGF-ß/BMP9 signaling pathway. Despite clear knowledge of the symptoms and genes of the disease, we still lack a definite cure for HHT, having just palliative measures and pharmacological trials. Among the former, two strategies are: intervention at "ground zero" to minimize by iron and blood transfusions in order to counteract anemia. Among the later, along the last 15 years, three different strategies have been tested: (1) To favor coagulation with antifibrinolytic agents (tranexamic acid); (2) to increase transcription of ENG and ALK1 with specific estrogen-receptor modulators (bazedoxifene or raloxifene), antioxidants (N-acetylcysteine, resveratrol), or immunosuppressants (tacrolimus); and (3) to impair the abnormal angiogenic process with antibodies (bevacizumab) or blocking drugs like etamsylate, and propranolol. This manuscript reviews the main strategies and sums up the clinical trials developed with drugs alleviating HHT.

Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4.

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction. Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels (sEng+) showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male sEng+ mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4. Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in sEng+ mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners.

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-ß receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-ß receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-ß family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.

MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells.

Upon inflammation, monocyte-derived macrophages (MΦ) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and efficient initial response (GM-MΦ) and a good resolution (M-MΦ) of the inflammatory process. The functional activity of polarized MΦ is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MΦ that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MΦ secretome involved in the shedding of soluble endoglin. We find that the GM-MΦ secretome contains metalloprotease 12 (MMP-12), a GM-MΦ specific marker that may account for the anti-angiogenic activity of the GM-MΦ secretome. Cell surface endoglin is present in both GM-MΦ and M-MΦ, but soluble endoglin is only detected in GM-MΦ culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MΦ and endothelial cells. These data demonstrate a direct correlation between GM-MΦ polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.

Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction in vivo and in vitro.

Our objective was to investigate the effect of cholesterol [hypercholesterolemia and 7-ketocholesterol (7K)] on endoglin (Eng) expression and regulation with respect to endothelial or vascular dysfunction in vivo and in vitro. In vivo experiments were performed in 2-mo-old atherosclerosis-prone apolipoprotein E-deficient/LDL receptor-deficient (ApoE-/-/LDLR-/-) female mice and their wild-type C57BL/6J littermates. In in vitro experiments, human aortic endothelial cells (HAECs) were treated with 7K. ApoE-/-/LDLR-/- mice developed hypercholesterolemia accompanied by increased circulating levels of P-selectin and Eng and a disruption of NO metabolism. Functional analysis of the aorta demonstrated impaired vascular reactivity, and Western blot analysis revealed down-regulation of membrane Eng/Smad2/3/eNOS signaling in ApoE-/-/LDLR-/- mice. 7K increased Eng expression via Krüppel-like factor 6 (KLF6), liver X nuclear receptor, and NF-κB in HAECs. 7K-induced Eng expression was prevented by the treatment with 2-hydroxypropyl-ß-cyclodextrin; 8-{[5-chloro-2-(4-methylpiperazin-1-yl) pyridine-4-carbonyl] amino}-1-(4-fluorophenyl)-4, 5-dihydrobenzo[g]indazole-3-carboxamide; or by KLF6 silencing. 7K induced increased adhesion and transmigration of monocytic human leukemia promonocytic cell line cells and was prevented by Eng silencing. We concluded that hypercholesterolemia altered Eng expression and signaling, followed by endothelial or vascular dysfunction before formation of atherosclerotic lesions in ApoE-/-/LDLR-/- mice. By contrast, 7K increased Eng expression and induced inflammation in HAECs, which was followed by an increased adhesion and transmigration of monocytes via endothelium, which was prevented by Eng inhibition. Thus, we propose a relevant role for Eng in endothelial or vascular dysfunction or inflammation when exposed to cholesterol.-Vicen, M., Vitverova, B., Havelek, R., Blazickova, K., Machacek, M., Rathouska, J., Najmanová, I., Dolezelova, E., Prasnicka, A., Sternak, M., Bernabeu, C., Nachtigal, P. Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction in vivo and in vitro.

Endoglin as an Adhesion Molecule in Mature and Progenitor Endothelial Cells: A Function Beyond TGF-ß.

Endoglin (ENG) is a transmembrane glycoprotein expressed on endothelial cells that functions as a co-receptor for several ligands of the transforming growth factor beta (TGF-ß) family. ENG is also a recognized marker of angiogenesis and mutations in the endoglin gene are responsible for Hereditary Hemorrhagic Telangiectasia (HHT) type 1, a vascular disease characterized by defective angiogenesis, arteriovenous malformations, telangiectasia, and epistaxis. In addition to its involvement in the TGF-ß family signaling pathways, several lines of evidence suggest that the extracellular domain of ENG has a role in integrin-mediated cell adhesion via its RGD motif. Indeed, we have described a role for endothelial ENG in leukocyte trafficking and extravasation via its binding to leukocyte integrins. We have also found that ENG is involved in vasculogenic properties of endothelial progenitor cells known as endothelial colony forming cells (ECFCs). Moreover, the binding of endothelial ENG to platelet integrins regulate the resistance to shear during platelet-endothelium interactions under inflammatory conditions. Because of the need for more effective treatments in HHT and the involvement of ENG in angiogenesis, current studies are aimed at identifying novel biological functions of ENG which could serve as a therapeutic target. This review focuses on the interaction between ENG and integrins with the aim to better understand the role of this protein in blood vessel formation driven by progenitor and mature endothelial cells.