Human Endoglin Research Articles

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbβ3 integrin

BackgroundThe 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 αIIbβ3, thereby compromising platelet binding to fibrinogen and thrombus stability. MethodsIn 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. ResultsUnder 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 αIIbβ3 and sEng and molecular modeling showed a good fitting between αIIbβ3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbβ3/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. ConclusionsOur results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbβ3, suggesting its involvement in primary hemostasis control.

Enhancing the Anti-Hepatocarcinoma Efficacy of Cytokine Induced Killer Cells by TLS11a-Lipo-hENG scFv/hIP-10 Nanocapsules

Developing a novel therapeutic approach to prevent and treat hepatocellular carcinoma (HCC) is essential. As an adoptive cell therapy candidate, cytokine-induced killer (CIK) cell immunotherapy is ideal. However, for efficacy in vivo delivery of CIK cells conjugate with antibody antineoplastic agents into the cancer cells, various biological obstacles including rapid degradation in vivo and the absence of active targeting capability need to be overcome. Previously, we developed a fusion antibody-chemokine protein which contained human Endoglin antibodies and human Interferon-gamma-induced protein 10 (hENG scFv/hIP-10). To this end, we present that a novel synthesized TLS11a-Lipo-hENG scFv/hIP-10 nanocapsules, which combine the advantages of prolonged half-life and good stability liposomes and HCC-specific targeting aptamer “TLS11a,” would be able to realize efficient and effective in vivo therapy. Also, we find that in combination with CIK cells possess a greater anti-hepatocarcinoma efficacy. Combined with TLS11a-Lipo-hENG scFv/hIP-10 nanocapsules and CIK cells, they could suppress tumor growth and prolong the survival of hepatoma-bearing mice through recruiting endogenous CIK cells in tumor tissues, inhibiting cell proliferation in tumors while promoting apoptosis and producing IFN-γ level. It appears that TLS11a-Lipo-hENG scFv/hIP-10 nanocapsules can enhance the antitumor activity of CIK cells against human hepatocellular carcinoma.

Regulation and Release of Vasoactive Endoglin by Brain Endothelium in Response to Hypoxia/Reoxygenation in Stroke.

In large vessel occlusion stroke, recanalization to restore cerebral perfusion is essential but not necessarily sufficient for a favorable outcome. Paradoxically, in some patients, reperfusion carries the risk of increased tissue damage and cerebral hemorrhage. Experimental and clinical data suggest that endothelial cells, representing the interface for detrimental platelet and leukocyte responses, likely play a crucial role in the phenomenon referred to as ischemia/reperfusion (I/R)-injury, but the mechanisms are unknown. We aimed to determine the role of endoglin in cerebral I/R-injury; endoglin is a membrane-bound protein abundantly expressed by endothelial cells that has previously been shown to be involved in the maintenance of vascular homeostasis. We investigated the expression of membranous endoglin (using Western blotting and RT-PCR) and the generation of soluble endoglin (using an enzyme-linked immunosorbent assay of cell culture supernatants) after hypoxia and subsequent reoxygenation in human non-immortalized brain endothelial cells. To validate these in vitro data, we additionally examined endoglin expression in an intraluminal monofilament model of permanent and transient middle cerebral artery occlusion in mice. Subsequently, the effects of recombinant human soluble endoglin were assessed by label-free impedance-based measurement of endothelial monolayer integrity (using the xCELLigence DP system) and immunocytochemistry. Endoglin expression is highly inducible by hypoxia in human brain endothelial monolayers in vitro, and subsequent reoxygenation induced its shedding. These findings were corroborated in mice during MCAO; an upregulation of endoglin was displayed in the infarcted hemispheres under occlusion, whereas endoglin expression was significantly diminished after transient MCAO, which is indicative of shedding. Of note is the finding that soluble endoglin induced an inflammatory phenotype in endothelial monolayers. The treatment of HBMEC with endoglin resulted in a decrease in transendothelial resistance and the downregulation of VE-cadherin. Our data establish a novel mechanism in which hypoxia triggers the initial endothelial upregulation of endoglin and subsequent reoxygenation triggers its release as a vasoactive mediator that, when rinsed into adjacent vascular beds after recanalization, can contribute to cerebral reperfusion injury.

Trophoblast Differentiation Affects Crucial Nutritive Functions of Placental Membrane Transporters.

Cytotrophoblasts are progenitor cells that proliferate and fuse to form the multinucleated syncytiotrophoblast layer, implicated in placental endocrine and transport functions. While membrane transporters play a critical role in the distribution of nutrients, hormones, and xenobiotics at the maternal-fetal interface, their selectivity to the syncytiotrophoblast layer is poorly characterized. We aimed to evaluate the regulation of placental transporters in response to trophoblast differentiation in vitro. Experiments were carried out in isolated primary human trophoblast cells before and after syncytialization. Gene expression of six molecular markers and thirty membrane transporters was investigated by qPCR analysis. Subsequently, functional expression was evaluated for proteins involved in the transplacental transfer of essential nutrients i.e., cholesterol (ABCA1, ABCG1), glucose (SLC2A1), leucine (SLC3A2, SLC7A5), and iron (transferrin receptor, TfR1). We identified that human chorionic gonadotropin, placental lactogen, endoglin, and cadherin-11 serve as optimal gene markers for the syncytialization process. We showed that trophoblast differentiation was associated with differential gene expression (mostly up-regulation) of several nutrient and drug transporters. Further, we revealed enhanced protein expression and activity of ABCG1, SLC3A2, SLC7A5, and TfR1 in syncytialized cells, with ABCA1 and GLUT1 displaying no change. Taken together, these results indicate that the syncytiotrophoblast has a dominant role in transporting essential nutrients cholesterol, leucine, and iron. Nonetheless, we present evidence that the cytotrophoblast cells may also be linked to transport functions that could be critical for the cell fusion processes. Our findings collectively yield new insights into the cellular functions associated with or altered by the trophoblast fusion. Importantly, defective syncytialization could lead to nutrient transfer imbalance, ultimately compromising fetal development and programming.

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 β (TGF-β)/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.

A Single-Chain Variable Fragment Antibody/Chemokine Fusion Protein Targeting Human Endoglin to Enhance the Anti-Tumor Activity of Cytokine-Induced Killer Cells.

Cytokine-induced killer cell immunotherapy is an ideal candidate for adoptive cell transfer therapy. However, therapeutic approaches to enhance the anti-tumor activity of cytokine-induced killer cells remain to be explored. Here, we described the successful development of a novel antibody-chemokine fusion protein containing the anti-human Endoglin antibody in the single-chain variable fragment format and human interferon-gamma-induced protein 10 (hENG scFv/hIP-10). Its anti-Endoglin immunoreactivity and chemotactic activity against the cytokine-induced killer cells were characterized in vitro. To evaluate the anti-tumor effect in vivo, cytokine-induced killer cells were intravenously injected into human hepatocellular carcinoma-bearing nude mice, together with intratumoral administration of the fusion protein hENG scFv/hIP-10 as an enhancer. The tumor volume and survival time of the mice were monitored, whilst the tumor-infiltrating cytokine-induced killer cells, serum levels of interferon-gamma, tumor cell proliferation, apoptosis, and angiogenesis were measured. The results demonstrated that hENG scFv/hIP-10 and cytokine-induced killer cells synergistically inhibited tumor growth and prolonged survival of tumor-bearing mice. Moreover, the number of tumor-infiltrating cytokine-induced killer cells, serum levels of interferon-gamma, and tumor cell apoptosis were increased, accompanied with decreased tumor proliferation and angiogenesis. Thus, our study suggests that hENG scFv/hIP-10 could enhance the anti-tumor activity of cytokine-induced killer cells against human hepatocellular carcinoma.

Human endoglin-CD3 bispecific T cell engager antibody induces anti-tumor effect in vivo.

Rationale: Endoglin, also known as CD105, is a homo-dimeric membrane glycoprotein required for angiogenesis and serves as a marker for cancer vasculature. In this study, we constructed a bispecific T-cell engager (BiTE) antibody that targets human endoglin and CD3 (hEND-CD3/BiTE). We examined BiTE binding to endoglin-expressing cells and its effects on the cytolytic activity of T cells and cancer development.Methods: The in vitro effects of hEND-CD3/BiTE, including binding to target cells, T-cell activation, proliferation, and cytotoxicity, were examined in endoglin-expressing 293T cells, human umbilical vascular endothelial cells, tumor-derived endothelial cells, and CD3+ T cells. An in vivo xenograft tumor model was established using A549 human lung cancer cells. The therapeutic efficacy of hEND-CD3/BiTE was assessed by monitoring tumor growth, angiogenesis, and mouse survival.Results: hEND-CD3/BiTE specifically bound to endoglin-expressing cells and CD3+ T cells in vitro and stimulated T-cell activation, proliferation, and Th1 cytokine secretion, and promoted T-cell-mediated cytolysis of endoglin-expressing cells. The hEND-CD3/BiTE in vivo caused minimal toxicity to major organs, reduced tumor neoangiogenesis, inhibited tumor growth, and significantly improved mouse survival.Conclusions: Our study demonstrated the therapeutic potential of hEND-CD3/BiTE and provided a novel approach to clinical cancer treatment.

The Influence of Monoclonal Antibodies to Human Endoglin on the Functional Characteristics of EA.hy926 Endothelial Cells

The endothelium of blood vessels plays an important role in the regulation of normal and pathological processes occurring in the tissues. Monoclonal antibodies (MAbs) to endoglin (CD105), a marker of endothelial cells, are considered as potential agents to affect the functional characteristics of endothelial cells. Here, we report the ability of MAbs against several epitopes of endoglin to modify the functional characte-ristics of human EA.hy926 endothelial cells in vitro. Different MAbs were found to slow down cell proliferation or migration, to increase the adhesion of monocyte-like cells, or to reduce the rate of soluble endoglin accumulation in a culture medium. Two MAbs of the eight studied reagents were able to affect several functional characteristics of endothelial cells.

Comparison of p38 MAPK, soluble endoglin and endothelin-1 level in severe preeclampsia and HELLP syndrome patients

Objective: To compare the levels of p38 mitogen-activated protein kinase (MAPK), soluble endoglin and endothelin-1 in the serum of patients with severe preeclampsia, hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome and normal pregnancies.Methods: This study was an observational analytic cross-sectional study performed at Wahidin Sudirohusodo Hospital, Makassar, Indonesia, in the period of 5th February 2016 to 20th January 2017. P38 MAPK, soluble endoglin and endothelin-1 levels of patients with normal pregnancies, severe preeclampsia and HELLP syndrome were measured by enzyme-linked immunoabsorbentassay technique, using kits of human soluble endoglin, endothelin-1 and p38 MAPK, Quantikine immunoassay: R&D System Inc.Results: Level of serum p38 MAPK in HELLP syndrome group was higher than in severe preeclampsia and normal pregnancy groups. Soluble endoglin and endothelin-1 levels in pregnancies with severe preeclampsia and HELLP syndrome were higher than normal pregnancy but there was no significant difference between these two groups (P>0.05). Levels of p38 MAPK, soluble endoglin and endothelin-1 also had a positive linear correlation with systolic and diastolic blood pressures (P<0.05).Conclusions: P38 MAPK in serum may be a marker for evidence of the severe hypoxia and its application may be considered for the diagnosis of HELLP syndrome.

Abstract 2900: Evaluation of endoglin/CD105 as a tumor vasculature target with antibody drug conjugates

Abstract Purpose: Solid tumor growth and metastasis requires continuous formation of new blood vessels. Endoglin/CD105 is a well-acknowledged endothelial cell proliferation marker, which is strongly expressed in tumor-associated vasculature. Vasculature targeting agents induce their anti-tumor effects through selective damage to the vasculature and reduction of tumor blood perfusion. Here, we evaluated endoglin as a potential vascular target for antibody-drug conjugate (ADC) therapy. Anti-endoglin ADCs were prepared with two classes of potent cytotoxic payloads, using either the microtubule disrupting maytansine derivatives DM1 and DM4, or the highly potent indolinobenzodiazepine DNA-alkylating payload DGN549. The pharmacokinetics, safety, and anti-tumor activities of the anti-endoglin ADCs were evaluated in rats. Experimental Design: Humanized monoclonal antibodies against endoglin were generated and screened for binding to both human and rat endoglin. Payloads were conjugated to the lead antibody through lysine residues. Binding and cytotoxicity of ADCs were tested in vitro on human umbilical vein endothelial cells (HUVECs). The antibody was administered to Sprague Dawley rats, and the circulation half-life determined by ELISA of the plasma samples. In vivo efficacy of anti-endoglin-DM and anti-endoglin-DGN549 ADCs were tested in multiple xenograft tumor models in Taconic female rnu/rnu rats. Results: A humanized anti-endoglin antibody, huRH105, which binds to both human and rat endoglin with high affinity was generated. The pharmacokinetic profile of huRH105 in rat demonstrated dose-dependency with the low dose (0.5 mg/kg) cohort having a high plasma clearance rate. ADCs of huRH105 were prepared with three different payloads: DM1, DM4 and DGN549. All three ADCs bound with similar sub-nanomolar affinity to endoglin-expressing HUVEC cells, and exhibited potent specific cytotoxicity against these cells in vitro. huRH105-SMCC-DM1 was tolerated at a single dose of 10-20 mg/kg in rats, with no discernable impact on body weight. This ADC displayed only modest anti-tumor activity against human A2780 ovarian tumor xenografts at 5 mg/kg in rats. To identify optimal drug linkers for more potent anti-tumor activity, two other ADC constructs, huRH105-sSPDB-DM4 and huRH105-DGN549 were evaluated against A2780 tumor xenografts grown subcutaneously. huRH105-sSPDB-DM4 was not active at 5 mg/kg; huRH105-DGN549 displayed modest anti-tumor activity at 0.33 mg/kg, eliciting tumor growth delay without notable toxicities. Conclusion: Endoglin targeted ADCs, huRH105-DM and huRH105-DGN549 conjugates were prepared and evaluated for their in vitro cytotoxicity and in vivo anti-tumor activity/tolerability. The ADCs exhibited modest anti-tumor activity and therapeutic indices in rat models. Citation Format: Rui Wu, Cristina Gavrilescu, Yimao Liu, Valerie Chamberlain Santos, Katharine C. Lai, Luke Harris, Prerak Shah, Kerry Donahue, Ravi Chari, Richard Gregory, Thomas Chittenden, Cynthia Guidi, Thomas A. Keating. Evaluation of endoglin/CD105 as a tumor vasculature target with antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2900.

Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes

BackgroundEndoglin (CD105) is overexpressed on tumor cells and tumor vasculatures, making it a potential target for diagnostic imaging and therapy of different neoplasms. Therefore, studies on nanocarrier systems designed for endoglin-directed diagnostic and drug delivery purposes would expose the feasibility of targeting endoglin with therapeutics. MethodsLiposomes carrying high concentrations of a near-infrared fluorescent dye in the aqueous interior were prepared by the lipid film hydration and extrusion procedure, then conjugated to single chain antibody fragments either selective for murine endoglin (termed mEnd-IL) or directed towards human endoglin (termed hEnd-IL). A combination of Dynamic Light Scattering, electron microscopy, cell binding and uptake assays, confocal microscopy and in vivo fluorescence imaging of mice bearing xenografted human breast cancer and human fibrosarcoma models were implemented to elucidate the potentials of the liposomes. ResultsThe mEnd-IL and hEnd-IL were highly selective for the respective murine- and human endoglin expressing cells in vitro and in vivo. Hence, the hEnd-IL bound distinctly to the tumor cells and enabled suitable fluorescence imaging of the tumors, whereas the mEnd-IL bound the tumor vasculature, but also to the liver, kidney and lung vasculature of mice. ConclusionsThe work highlights key differences between targeting vascular (murine) and neoplastic (human) endoglin in animal studies, and suggests that the hEnd-IL can serve as a delivery system that targets human endoglin overexpressed in pathological conditions. General significanceThe endoglin-targeting liposomes presented herewith represent strategic tools for the future implementation of endoglin-directed neoplastic and anti-angiogenic therapies.

Circulating soluble endoglin modifies the inflammatory response in mice.

Inflammation is associated with every health condition, and is an important component of many pathologies such as cardiovascular diseases. Circulating levels of soluble endoglin have been shown to be higher in the serum of patients with cardiovascular diseases with a significant inflammatory component. The aim of this study was to evaluate the implication of circulating soluble endoglin in the inflammatory response. For this purpose, a transgenic mouse expressing human soluble endoglin (sEng+) was employed, and three different inflammatory approaches were used to mimic inflammatory conditions in different tissues. This study shows that control sEng+ mice have a normal inflammatory state. The lung and kidney injury induced by the inflammatory agents was reduced in sEng+ mice, especially the intra-alveolar and kidney infiltrates, suggesting a possible reduction in inflammation induced by soluble endoglin. To deepen into this possible effect, the leukocyte number in the bronchoalveolar lavage and air pouch lavage was evaluated and a significant reduction of neutrophil infiltration in LPS-treated lungs and ischemic kidneys from sEng+ with respect to WT mice was observed. Additionally, the mechanisms through which soluble endoglin prevents inflammation were studied. We found that in sEng+ animals the increment of proinflammatory cytokines, TNFα, IL1β and IL6, induced by the inflammatory stimulus was reduced. Soluble endoglin also prevents the augmented adhesion molecules, ICAM, VCAM and E-selectin induced by the inflammatory stimulus. In addition, vascular permeability increased by inflammatory agents was also reduced by soluble endoglin. These results suggest that soluble endoglin modulates inflammatory-related diseases and open new perspectives leading to the development of novel and targeted approaches for the prevention and treatment of cardiovascular diseases.

Human endoglin as a potential new partner involved in platelet\u2013endothelium interactions

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng+/−) mice compared to Eng+/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.

A fast and effective determination of the biodistribution and subcellular localization of fluorescent immunoliposomes in freshly excised animal organs

BackgroundPreclinical research implementing fluorescence-based approaches is inevitable for drug discovery and technology. For example, a variety of contrast agents developed for biomedical imaging are usually evaluated in cell systems and animal models based on their conjugation to fluorescent dyes. Biodistribution studies of excised organs are often performed by macroscopic imaging, whereas the subcellular localization though vital, is often neglected or further validated by histological procedures. Available systems used to define the subcellular biodistribution of contrast agents such as intravital microscopes or ex vivo histological analysis are expensive and not affordable by the majority of researchers, or encompass tedious and time consuming steps that may modify the contrast agents and falsify the results. Thus, affordable and more reliable approaches to study the biodistribution of contrast agents are required. We developed fluorescent immunoliposomes specific for human fibroblast activation protein and murine endoglin, and used macroscopic fluorescence imaging and confocal microscopy to determine their biodistribution and subcellular localization in freshly excised mice organs at different time points post intravenous injection.ResultsNear infrared fluorescence macroscopic imaging revealed key differences in the biodistribution of the respective immunoliposomes at different time points post injection, which correlated to the first-pass effect as well as the binding of the probes to molecular targets within the mice organs. Thus, a higher accumulation and longer retention of the murine endoglin immunoliposomes was seen in the lungs, liver and kidneys than the FAP specific immunoliposomes. Confocal microscopy showed that tissue autofluorescence enables detection of organ morphology and cellular components within freshly excised, non-processed organs, and that fluorescent probes with absorption and emission maxima beyond the tissue autofluorescence range can be easily distinguished. Hence, the endoglin targeting immunoliposomes retained in some organs could be detected in the vascular endothelia cells of the organs.ConclusionsThe underlying work represents a quick, effective and more reliable setup to validate the macroscopic and subcellular biodistribution of contrast agents in freshly excised animal organs. The approach will be highly beneficial to many researchers involved in nanodrug design or in fluorescence-based studies on disease pathogenesis.

Rapid endoglin determination in serum samples using an amperometric magneto-actuated disposable immunosensing platform

A sensitive and rapid method for the determination of the clinically relevant biomarker human endoglin (CD105) in serum samples is presented, involving a magneto-actuated immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs). Micro-sized magnetic particles were modified with a specific antibody to selectively capture the target protein which was further sandwiched with a secondary HRP-labeled antibody. The immunocomplexes attached to the magnetic carriers were amperometrically detected at SPCEs using the hydroquinone (HQ)/H2O2/HRP system. The magneto-actuated immunosensing platform was able to detect 5 pmoles of endoglin (in 25μL of sample, 0.2μM) in 30min providing statistically similar results to those obtained using a commercial ELISA kit for the determination of endogenous content of endoglin in human serum samples.

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

0130 : Endoglin is a new partner involved in platelet – endothelium interactions: role in microvessel stability?

Hereditary Hemorragic Telangectasia (HHT) is characterized by a bleeding tendency that is postulated to be a consequence of microvessel fragility. HHT type 1 patients present heterozygous mutations in the endoglin gene resulting in a loss of expression of membrane endoglin in their endothelial cells (EC), while platelets in these patients display normal functions in vitro . We have reported previously that endothelial endoglin is involved, via its RGD motif, in inflammation though integrin-mediated leukocyte adhesion and transmigration. We recently found that endoglin-haplodeficient mice ( Eng+/–) have significantly higher bleeding time than Eng+/+ animals, therefore, we haypothesized that endoglin may also act as an adhesion molecule via integrin recognition in the interaction between EC and platelets during hemostasis. Coating plastic wells with the extracellular domain of endoglin enhanced platelets adhesion to the wells under static condition and this interaction persist under flow condition. Similarly, platelets could adhere on confluent EC in an endoglin-mediated process, while the silencing of endoglin expression in EC, and the addition of soluble endoglin or of anti-endoglin antibodies, inhibited stable platelet adhesion to EC. Remarkably, platelets from Glanzmann's thrombasthenia patients lacking the€αIIbβ3 integrin were defective for adhesion on EC, wheras CHO cells, ectopically expressing the human αIIbβ3 integrin acquired the capacity to adhere on rat myoblast transfectants expressing human endoglin (L6E9-E), thus confirming the interaction between endoglin and the αIIbβ3 integrin. These results establish a new critical role for endoglin in integrin-mediated adhesion of platelets to endothelium and provide a better understanding on the cellular mechanisms involved in bleeding events occurring in HHT. The author hereby declares no conflict of interest

Production and characterization of monoclonal antibodies against human endoglin

Endoglin (CD105), a component of TGF-β, BMP-9, BMP-10 binding receptor complexes, is the marker of endothelial and mesenchymal stem cells. Its expression is significantly increased in the blood vessel endothelium of ischemic tissues and growing tumors. Measurement of the endoglin soluble form in serum and urine is used in diagnostics of cancer and pregnancy disorders. The aim of this work was to produce a panel of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. A recombinant protein derived from murine myeloma cells representing the whole extracellular part of endoglin was used as the immunogen. F1(SJL/J × BALB/c) mice were the donors of immune splenocytes. Hybridoma screening was performed using E. coli-produced recombinant molecules, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight different epitopes were produced. Eight antibodies bind the membrane-associated endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by Western blot. Sandwich ELISA system was designed to measure soluble endoglin in the cell culture medium.

The impact of Ets-1 oncoprotein and human endoglin (CD105) on the recurrence of non-melanoma skin cancers.

The aim of this study was to investigate if the expression of CD105 and Ets-1 was predictive of aggressive biologic behavior of non-melanoma skin cancers (NMSC) and to evaluate indicators of local recurrence. A total of 144 patients with NMSC were included in the current study. Surgical specimens were independently examined for diagnosis confirmation and immunohistochemical expression of Ets-1 and CD105 by two dermatopathologists. The most common tumor type was basal cell carcinoma (n = 76), followed by squamous cell carcinomas (SCC) (n = 65). The most common anatomic location was the head and neck area (n = 115). The follow-up was ˃ 2 years in all examined cases. A statistically significant correlation was found between tumor local recurrence and age (P = 0.03), Ets-1 expression (P ˂ 0.0001) and CD105 expression (P ˂ 0.0001). Our data confirm that both Ets-1 and CD105 show promise as prognostic markers for local recurrence of NMSC. However, this statement is made with caution, and additional studies, with larger populations, are necessary to examine the correlation between these two markers and local recurrence. A better understanding of the pathogenesis of local recurrence in primary NMSC may result in potential therapeutic interventions.

Experimental procedures to identify and validate specific mRNA targets of miRNAs.

Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes. Since the biological roles of individual miRNAs will be dictated by the mRNAs that they regulate, the identification and validation of miRNA/mRNA target interactions is critical for our understanding of the regulatory networks governing biological processes. We promulgate the combined use of prediction algorithms, the examination of curated databases of experimentally supported miRNA/mRNA interactions, manual sequence inspection of cataloged miRNA binding sites in specific target mRNAs, and review of the published literature as a reliable practice for identifying and prioritizing biologically important miRNA/mRNA target pairs. Once a preferred miRNA/mRNA target pair has been selected, we propose that the authenticity of a functional miRNA/mRNA target pair be validated by fulfilling four well-defined experimental criteria. This review summarizes our current knowledge of miRNA biology, miRNA/mRNA target prediction algorithms, validated miRNA/mRNA target data bases, and outlines several experimental methods by which miRNA/mRNA targets can be authenticated. In addition, a case study of human endoglin is presented as an example of the utilization of these methodologies.