Endoglin Expression Research Articles

Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and is known to have low immunogenicity and an immunosuppressive microenvironment. It is also characterized by high accumulation of dense stroma, composed of mostly cancer-associated fibroblasts (CAFs). Multiple subsets of CAFs are described, with one of them expressing the transforming growth factor (TGF)-β co-receptor endoglin. In previous work, we and others have shown that endoglin-expressing CAFs stimulate tumor progression and metastasis. Therefore, in this study, we set out to investigate the role of endoglin-expressing CAFs in pancreatic cancer progression.MethodsFirst, we investigated the expression of endoglin on CAFs in both human tissues as well as a mouse model for PDAC. Since CAF-specific endoglin expression was high, we targeted endoglin by using the endoglin neutralizing antibody TRC105 in the murine KPC model for PDAC.ResultsAlthough some signs of immune activation were observed, TRC105 did not affect tumor growth. Since 90% of the CD8+ T-cells expressed the immune checkpoint PD-1, we investigated the combination with a PD1 checkpoint inhibitor, which did not enhance therapeutic responses. Finally, genetic deletion of endoglin from collagen 1a1 expressing cells also did not affect the growth of the mouse KPC tumors.ConclusionOur results show that although endoglin is highly expressed on PDAC-CAFs and signaling is efficiently inhibited by TRC105, this does not result in decreased tumor growth in the KPC model for pancreatic cancer.

Study of expression of endoglin (CD105) in oral squamous cell carcinoma.

Context:Oral cancer is the 8th most common cancer in the world. An important feature of carcinogenesis is angiogenesis. Endoglin is a powerful marker of neovascularization in solid malignancies. This study was done to ascertain its role as an indicator of metastasis and prognosis.Aim:This study aimed to evaluate and compare the expression of endoglin (CD105) in metastatic primary tumor, lymph node of the metastasized tumor, nonmetastatic primary tumor and in normal buccal mucosa immunohistochemically.Settings and Design:The total sample size comprised 45 formalin-fixed paraffin-embedded tissue blocks, n = 10 metastasized primary tumor, n = 10 lymph nodes of metastasized primary tumor, n = 20 nonmetastasized oral squamous cell carcinoma and n = 5 normal buccal mucosa were studied.Subjects and Methods:Immunohistochemistry for endoglin was performed and microvessel density (MVD) was determined by hot spot method. Microvessel density was compared between the groups.Statistical Analysis:Statistical analysis was used using one-way ANOVA. P < 0.05 was statistically significant.Results:Endoglin expression in metastatic cases (0.68 + 0.10) was higher than nonmetastatic cases (0.45 + 0.20) and the difference was statistically significant (P = 0.002).Conclusion:This study shows that presence of endoglin determines the metastatic potential of the tumor and its prognosis, thus, could be considered as a potential target of therapy.

Thresholds of Endoglin Expression in Endothelial Cells Explains Vascular Etiology in Hereditary Hemorrhagic Telangiectasia Type 1.

Hereditary Hemorrhagic Telangiectasia type 1 (HHT1) is an autosomal dominant inherited disease characterized by arteriovenous malformations and hemorrhage. HHT1 is caused by mutations in ENDOGLIN, which encodes an ancillary receptor for Transforming Growth Factor-β/Bone Morphogenetic Protein-9 expressed in all vascular endothelial cells. Haploinsufficiency is widely accepted as the underlying mechanism for HHT1. However, it remains intriguing that only some, but not all, vascular beds are affected, as these causal gene mutations are present in vasculature throughout the body. Here, we have examined the endoglin expression levels in the blood vessels of multiple organs in mice and in humans. We found a positive correlation between low basal levels of endoglin and the general prevalence of clinical manifestations in selected organs. Endoglin was found to be particularly low in the skin, the earliest site of vascular lesions in HHT1, and even undetectable in the arteries and capillaries of heterozygous endoglin mice. Endoglin levels did not appear to be associated with organ-specific vascular functions. Instead, our data revealed a critical endoglin threshold compatible with the haploinsufficiency model, below which endothelial cells independent of their tissue of origin exhibited abnormal responses to Vascular Endothelial Growth Factor. Our results support the development of drugs promoting endoglin expression as potentially protective.

Carotuximab effects on endoglin expression and signaling in 7-ketocholesterol induced endothelial dysfunction in HAECs

Background and Aims: Endoglin (Eng) is a transmembrane glycoprotein that serves as an accessory receptor in TGF-β pathway. We previously showed that stimulation of endothelial cells with 7-ketocholesterol (simulating oxidized LDL) resulted in increased levels of endoglin and cell adhesion molecules (ICAM-1, P/E-selectin), as well as increased adhesion and transmigration of monocytes through endothelial monolayer. Carotuximab (TRC105) is a monoclonal antibody that binds endoglin with high affinity and blocks/inhibits endoglin effects. We hypothesized that carotuximab mediated inhibition of endoglin will affect 7-ketocholesterol induced endothelial dysfunction.

Hhex inhibits cell migration via regulating RHOA/CDC42-CFL1 axis in human lung cancer cells

BackgroundHhex(human hematopoietically expressed homeobox), also known as PRH, is originally considered as a transcription factor to regulate gene expression due to its homebox domain. Increasing studies show that Hhex plays a significant role in development, including anterior–posterior axis formation, vascular development and HSCs self-renewal etc. Hhex is linked to many diseases such as cancers, leukemia, and type-2 diabetes. Although Hhex is reported to inhibit cell migration and invasion of breast and prostate epithelial cells by upregulating Endoglin expression, the effect and molecular mechanism for lung cancer cell motility regulation remains elusive.MethodsHuman non-small cell lung cancer cells and HEK293FT cells were used to investigate the molecular mechanism of Hhex regulating lung cancer cell migration by using Western blot, immunoprecipitation, wound-healing scratch assay, laser confocal.ResultsOur data indicated that Hhex could inhibit cell migration and cell protrusion formation in lung cancer cells. In addition, Hhex inhibited CFL1 phosphorylation to keep its F-actin-severing activity. RHOGDIA was involved in Hhex-induced CFL1 phosphorylation regulation. Hhex enhanced RHOGDIA interaction with RHOA/CDC42, thus maintaining RHOA/CDC42 at an inactive form.ConclusionCollectively, these data indicate that Hhex inhibited the activation of RHOA/CDC42 by enhancing interaction of RHOGDIA with RHOA/CDC42, and then RHOA/ CDC42-p-CFL1 signaling pathway was blocked. Consequently, the formation of Filopodium and Lamellipodium on the cell surface was suppressed, and thus the ability of lung cancer cells to migrate was decreased accordingly. Our findings show Hhex plays an important role in regulating migration of lung cancer cells and may provide a potential target for lung cancer therapy.FSYn6j34J4ZMCghGVb9zTbVideo abstract

Heterogenous expression of endoglin marks advanced renal cancer with distinct tumor microenvironment fitness.

Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS‐RC‐2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin‐positive (ENG+) population exhibited growth dependency on endoglin in anchorage‐independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor β and bone morphogenetic protein‐9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer‐initiating cells, the OS5K‐3 ENG+ population did not enrich other reported cancer‐initiating cell markers or differentiate into the ENG– population. Mouse tumor inoculation models revealed that the tumor‐forming capabilities of OS5K‐3 ENG+ and ENG– cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell‐centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.

Identification and Molecular Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Niche Factor

Mammalian bone marrow (BM) regeneration after myeloablation requires orchestrated interplay between osteogenesis, angiogenesis and hematopoiesis. Specific subsets of endothelial cells (ECs) have been identified in mice, including sinusoidal ECs (Hooper et al., 2009) and H-endothelium (Kusumbe et al., 2014, 2016; Ramasamy et al., 2014), that drive and coordinate these processes. Their relevance to human biology, however, remains largely elusive.Here, using a combinatory approach of cell purification and transcriptomics, we identify and characterize EC subsets associated with BM niche formation during ontogeny and regeneration in humans.Massive parallel RNA sequencing of FACS-purified BM ECs (CD45-CD235-7AAD-CD31+CD9+) from AML patients in the regenerating phase after chemotherapy (n=3) revealed upregulated endoglin (CD105) expression, in comparison to ECs at diagnosis or from normal BM (n=7) (FPKM fold-change increase (FCI): 10.1 ± 4.9; GLM LRT Edge R; p=0.001). FACS analysis confirmed a strong enrichment of endoglin-expressing ECs in regenerative marrow (n=48) compared to normal BM (n=9) (10.81% ± 2.14 vs 0.48% ± 0.24 of CD31+CD9+ cells; p=0.043).Similarly, in human fetal BM at 15-20 weeks after gestation (n=15), when angiogenesis, osteogenesis and hematopoiesis are tightly coupled, a striking predominance of endoglin-expressing ECs (62.8% ± 5.9 of CD31+CD9+ cells) was found, confirmed by in situ immunohistochemistry in comparison to adult core hip bone biopsies. Comprehensive transcriptional characterization by RNAseq of these endoglin-expressing ‘human regeneration-associated EC’ (hREC) from fetal bone (n=7) in comparison to endoglin- ECs revealed a remarkable phenotypic and molecular similarity to murine H-endothelium as well as activation of pathways and factors implicated in hematopoiesis, osteogenesis and angiogenesis. These included upregulated expression of CD31 (PECAM1) (FPKM FCI: 4.7 ± 1.0; p=0.04), mucin-like sialoglycoprotein endomucin (EMCN) (FPKM FCI: 435 ± 112; p <0.001), stromal cell-derived factor 1 (CXCL12) (FPKM FCI: 108 ± 34; p <0.001) and transcriptional activation of NOTCH (Normalized Enrichment score (NES): 1.51, False Discovery Rate-adjusted q-value (FDR): <0.10) and HIF1a (NES: 1.60, FDR <0.10) signaling pathways.IL-33 was found to be highly and significantly overexpressed in both fetal and regenerative marrow ECs in comparison to steady-state normal BM ECs (FPKM FCI: 587 ± 128 and 174 ± 86, respectively, both p <0.001). Expression of IL-33 in endoglin-expressing ECs was confirmed at the protein level in both humans and mice. Recombinant human IL-33 (1) promoted the expansion of distinct subsets of hematopoietic precursor cells, specifically immunophenotypic multipotent progenitors (2.5-FCI ± 0.43; p<0.05) and multilymphoid progenitors (2.7-FCI ± 0.2; p<0.01), (2) increased metabolic activity in HUVECs (PrestoBlue Cell Viability assay; mean fluorescence intensity 1.40-FCI ± 0.02; p=0.03), and (3) promoted osteogenic differentiation of BM-derived mesenchymal cells (Alizarin red-staining and colorimetric assessment of calcium deposition (3.61-FCI ± 0.63; p<0.02)). Repressing IL-33 in HUVEC by shRNAs impaired their expansion in culture dramatically (FC decrease 6-7 fold; p=0.01). In vivo, enrichment of an endoglin-expressing subset of BM ECs was observed in mice exposed to a myeloablative dose of 5-fluorouracil (n=5) compared to steady-state mice (n=3) (FCI: 9.0 ± 1.7; p=0.01). Administration of recombinant murine IL-33 to C57BL/6 wild-type mice recapitulated the expansion of hematopoietic progenitors with myeloid and lymphoid potential (HPC1 subset) (FCI 2.6 ± 0.5; p=0.003), and resulted in a relative increase in CD31+CD105+ BM ECs (FCI 3.5 ± 0.3; p=0.002) as well as bone progenitor (Lin-Ter119-CD51+Sca-) cells (FCI 2.9 ± 0.7; p=0.06) within the niche.Collectively, the data reveal the existence of an endothelial subset associated with BM regeneration and ontogeny in humans and identify IL-33 as a potential driver of marrow recovery. The molecular characterization of hREC is, thus, anticipated to instruct the discovery of novel determinants of EC-driven regeneration of the hematopoietic system after injury. DisclosuresRaaijmakers:Novartis: Consultancy.

Angio-Phenotype of Pediatric Acute Lymphoblastic Leukemia and Pharmacodynamic Evaluation of the Antiangiogenic Activity of Corticosteroids.

Corticosteroids are essential in the treatment of pediatric acute lymphoblastic leukaemia (ALL). Corticosteroid anti-angiogenic activity is postulated but unproven.Objectives: To assess the kinetics of cellular and plasma angiogenic surrogate markers during 7-day prednisone pre-phase/induction therapy in the AIEOP ALL 2000 protocol.Methods: 17 diagnosed common ALL and 12 normal controls were enrolled. Total and viable circulating endothelial cells (CECs) and progenitors (CEPs), vascular endothelial growth factor receptors (VEGFR2, R3), endoglin (CD105), VECadherin (CD144) expression were analyzed by four color flow-cytometry on peripheral blood. Plasma circulating VEGF, VEGFRs and endoglin were analyzed by ELISA. Gene expression of VE-Cadherin, Nanog, RGS5, VEGFR2 and R3 were quantified by Real Time RT-PCR.Results: Total CECs (31 ±8cells/uL), viable CECs (12 ± 5 cells/uL) and CEPs (0.91 ±0.91 cells/uL) were found to be significantly higher in pediatric controls than in adult. At baseline patients demonstrated a statistically higher number of CECs (CD45− CD31+ CD146+ cells) and CEPs (CD133+ cells) p=0.01 and 0.03, compared to controls. CD105, VEGFR-3 and VEGFR-2 expression on leukemic blasts was 91%, 27% and 0% of patients, respectively.After 7 days of prednisone, a statistically significant reduction of total and viable CECs (p= 0.01) and CEPs ( p<0.0001) as well as endothelial cells expressing VEGFR2, R3, and CD105 (p=0.01, p=0.03, p=0.02) was observed. Plasma VEGFR2, VEGFR3, and endoglin showed a trend to decrease at day 8 and 33 but did not reach significance. A statistically significant reduction of blast was also observed.The pro-apototic effect of prednisone was greater on blast compared to lymphocytes.VEGFR-3 gene expression was significantly increased in patients at diagnosis compared to pediatric controls, and significantly reduced on day 8.Conclusions:Increase in CECs, CEPS confirm a pro-angiogenic activity at diagnosis in common ALL. The anti-leukemic effects of prednisone may be linked to its pro-apoptotic and anti-angiogenic activities. Total and viable CECs, CEPs, expression of endoglin and VEGFR3 on blast appear to be suitable angiogenic surrogate biomarkers.

The functional role of decorin in corneal neovascularization in vivo

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn−/−) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn−/− and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn−/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn−/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn−/− mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn−/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn−/− mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn−/− mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn−/− mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.

Systematic Analysis of the Transcriptome Profiles and Co-Expression Networks of Tumour Endothelial Cells Identifies Several Tumour-Associated Modules and Potential Therapeutic Targets in Hepatocellular Carcinoma.

Simple SummaryEndothelial cells, the innermost layer of blood vessels, play an essential role in the progression of cancer, particularly liver cancer. To develop cancer therapies targeting those cells, the investigation of gene co-expression networks is of great importance. In this study, we investigated the gene expression profile of tumour endothelial cells. We compared it to endothelial cells from non-tumour liver-tissue. Using gene-network based methods, we could identify genes that may play a unique role in liver cancer progression or be a target for cancer therapy. Additionally, we provided a framework for similar analyses in other cancers.Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer-related death, with tumour associated liver endothelial cells being thought to be major drivers in HCC progression. This study aims to compare the gene expression profiles of tumour endothelial cells from the liver with endothelial cells from non-tumour liver tissue, to identify perturbed biologic functions, co-expression modules, and potentially drugable hub genes that could give rise to novel therapeutic targets and strategies. Gene Set Variation Analysis (GSVA) showed that cell growth-related pathways were upregulated, whereas apoptosis induction, immune and inflammatory-related pathways were downregulated in tumour endothelial cells. Weighted Gene Co-expression Network Analysis (WGCNA) identified several modules strongly associated to tumour endothelial cells or angiogenic activated endothelial cells with high endoglin (ENG) expression. In tumour cells, upregulated modules were associated with cell growth, cell proliferation, and DNA-replication, whereas downregulated modules were involved in immune functions, particularly complement activation. In ENG+ cells, upregulated modules were associated with cell adhesion and endothelial functions. One downregulated module was associated with immune system-related functions. Querying the STRING database revealed known functional-interaction networks underlying the modules. Several possible hub genes were identified, of which some (for example FEN1, BIRC5, NEK2, CDKN3, and TTK) are potentially druggable as determined by querying the Drug Gene Interaction database. In summary, our study provides a detailed picture of the transcriptomic differences between tumour and non-tumour endothelium in the liver on a co-expression network level, indicates several potential therapeutic targets and presents an analysis workflow that can be easily adapted to other projects.

The effect of aflibercept and arsenic trioxide on the proliferation, migration and apoptosis of oral squamous cell carcinoma in vitro.

Aflibercept and arsenic trioxide drugs apply a cytotoxic effect on some human cancer cell lines. However, no more study has followed the effects of both drugs, especially arsenic trioxide, on oral squamous cell carcinoma (OCC). We used three OCC lines as a model to show the effect of these drugs on the genetically complex disease and investigate its targeted therapy. In this study, three human OCC cell lines were used from different patients. We treated cell lines with both medications to detect the effect and relevant molecular basis. First, methyl thiazolyl tetrazolium (MTT) assay was performed to detect the cytotoxicity effect and cell growth. Second, flow cytometry, gene and protein expression were performed to evaluate the anti-angiogenic effect on OCC lines. Next apoptosis was analyzed by flow cytometry. Finally, clonogenesis capacity and cell migration were assessed by colony formation assay and wound healing, respectively. Aflibercept had no cytotoxic effect on the three OCC cell lines but decreased cell growth rate. Arsenic trioxide had a significant cytotoxic effect on three cell lines. Our results demonstrated that both drugs significantly decreased endoglin, VEGFA, and VEGFB expression. In addition, Migration and colony formation assays confirmed that these drugs have significant anti-proliferative and anti-migration effect on oral carcinoma cells. These results revealed that both medications might be a potential drug for the management of oral cancer patients.

Hot and Cold Tumors: Is Endoglin (CD105) a Potential Target for Vessel Normalization?

Simple SummaryThe prognosis and response to immunotherapy depends largely on the composition of the tumor microenvironment (TME). So-called cold tumors are rich in cells and molecules that inhibit the antitumor response and, therefore, are associated with a worse prognosis. In contrast, hot tumors are rich in antitumor cells and respond well to immunotherapy. The creation of one type of TME or another is highly dependent on angiogenesis, inflammation, and cancer-associated fibroblast (CAF) accumulation. Endoglin (CD105) is a protein involved in these three processes, making it a possible target for the conversion of cold tumors into hot tumors. In this review we summarize the role of endoglin in these processes and present the anti-endoglin therapies already under study that could be applied for vascular normalization.Tumors are complex masses formed by malignant but also by normal cells. The interaction between these cells via cytokines, chemokines, growth factors, and enzymes that remodel the extracellular matrix (ECM) constitutes the tumor microenvironment (TME). This TME can be determinant in the prognosis and the response to some treatments such as immunotherapy. Depending on their TME, two types of tumors can be defined: hot tumors, characterized by an immunosupportive TME and a good response to immunotherapy; and cold tumors, which respond poorly to this therapy and are characterized by an immunosuppressive TME. A therapeutic strategy that has been shown to be useful for the conversion of cold tumors into hot tumors is vascular normalization. In this review we propose that endoglin (CD105) may be a useful target of this strategy since it is involved in the three main processes involved in the generation of the TME: angiogenesis, inflammation, and cancer-associated fibroblast (CAF) accumulation. Moreover, the analysis of endoglin expression in tumors, which is already used in the clinic to study the microvascular density and that is associated with worse prognosis, could be used to predict a patient’s response to immunotherapy.

The Role of Endoglin in Hepatocellular Carcinoma.

Endoglin (CD105) is a type-1 integral transmembrane glycoprotein and coreceptor for transforming growth factor-β (TGF-β) ligands. The endoglin/TGF-β signaling pathway regulates hemostasis, cell proliferation/migration, extracellular matrix (ECM) synthesis and angiogenesis. Angiogenesis contributes to early progression, invasion, postoperative recurrence, and metastasis in hepatocellular carcinoma (HCC), one of the most widespread malignancies globally. Endoglin is overexpressed in newly formed HCC microvessels. It increases microvessel density in cirrhotic and regenerative HCC nodules. In addition, circulating endoglin is present in HCC patients, suggesting potential for use as a diagnostic or prognostic factor. HCC angiogenesis is dynamic and endoglin expression varies by stage. TRC105 (carotuximab) is an antibody against endoglin, and three of its clinical trials were related to liver diseases. A partial response was achieved when combining TRC105 with sorafenib. Although antiangiogenic therapy still carries some risks, combination therapy with endoglin inhibitors or other targeted therapies holds promise.

Endoglin Expression and Surface Renewal in Mesenchymal Stem Cells and Endothelial Cells

Endoglin (CD105) is one of the major marker antigens of mesenchymal stem cells (MSC) and endothelial cells. While the functions of endoglin in endothelial cells have been widely declared, little is known about its role in MSC biology. Here, we present a comparative study of CD105 expression, interna-lization and shedding by human endothelial cell line EA.hy926 and human adipose tissue-derived MSC from various sources. While the fraction of CD105-positive cells in all MSC cultures and EA.hy926 endothelial cells was consistently high, over 97% of the population, the density of CD105 molecules on the surface of MSC isolated from visceral and subcutaneous adipose tissue was substantially different. The total expression level of endoglin mRNA in MSC and endothelial cells was similar, whereas the contribution of the transcripts that yield a short CD105 isoform was higher in endothelial cells. Using a set of monoclonal antibodies (mAbs) directed against different endoglin epitopes, we revealed significant differences in the dynamics of CD105 metabolism on the membranes of endothelial cells and MSC. On EA.hy926 endothelial cells, CD105 molecules bound with antibodies were internalized and remained in the perinuclear space. In MSC cultures, on the contrary, the CD105-mAbs complexes were not subjected to endocytosis and remained on the cell membrane for a long time. We demonstrated that MSC, similarly to the endothelial cells, do shed the extracellular endoglin fragment into the environment as a soluble CD105 isoform. The shedding process in MSC was, however, substantially less intensive compared with the endothelial cells. Thus, we revealed, for the first time, that in MSC, unlike the endothelial cells, endoglin persists on cell surface for a long time and is not interna-lized after binding with antibodies. Endoglin shedding from MSC surface and the concomitant generation of the soluble endoglin form were also demonstrated for the first time.

Endoglin Level Is Critical for Cartilage Tissue Formation In Vitro by Passaged Human Chondrocytes.

Transforming growth factor beta (TGFβ) signaling is required for in vitro chondrogenesis. In animal models of osteoarthritis (OA), TGFβ receptor alterations are detected in chondrocytes in severe OA cartilage. It is not known whether such changes are dependent on the grade of human OA and if they affect chondrogenesis. Thus, the purpose of this study was to determine if human OA chondrocytes obtained from low-grade or high-grade disease could form cartilage tissue and to assess the role of the co-receptors, endoglin (ENG) and TGFβ receptor 3 (TGFBRIII), in the regulation of this tissue generation in vitro. We hypothesized that the grade of OA disease would not affect the ability of cells to form cartilage tissue and that the TGFβ co-receptor, ENG, would be critical to regulating tissue formation. Chondrocytes isolated from low-grade OA or high-grade OA human articular cartilage (AC) were analyzed directly (P0) or passaged in monolayer to P2. Expression of the primary TGFβ receptor ALK5, and the co-receptors ENG and TGFβRIII, was assessed by image flow cytometry. To assess the ability to form cartilaginous tissue, cells were placed in three-dimensional culture at high density and cultured in chondrogenic media containing TGFβ3. ENG knockdown was used to determine its role in regulating tissue formation. Overall, grade-specific differences in expression of ALK5, ENG, and TGFβRIII in primary or passaged chondrocytes were not detected; however, ENG expression increased significantly after passaging. Despite the presence of ALK5, P0 cells did not form cartilaginous tissue. In contrast, P2 cells derived from low-grade and high-grade OA AC formed hyaline-like cartilaginous tissues of similar quality. Knockdown of ENG in P2 cells inhibited cartilaginous tissue formation compared to controls indicating that the level of ENG protein expression is critical for in vitro chondrogenesis by passaged articular chondrocytes. This study demonstrates that it is not the grade of OA, but the levels of ENG in the presence of ALK5 that influences the ability of human passaged articular chondrocytes to form cartilaginous tissue in vitro in 3D culture. This has implications for cartilage repair therapies. Impact statement These findings are important clinically, given the limited availability of osteoarthritis (OA) cartilage tissue. Being able to use cells from all grades of OA will increase our ability to obtain sufficient cells for cartilage repair. In addition, it is possible that endoglin (ENG) levels, in the presence of ALK5 expression, may be suitable to use as biomarkers to identify cells able to produce cartilage.

Endoglin Expression and Microvessel Density as Prognostic Factors in Pediatric Rhabdomyosarcoma.

(1) Background: The study proposed to analyze microvessel density (MVD) in rhabdomyosarcoma (RMS) based on the expression of angiogenesis markers and define its prognostic role in this group of patients. (2) Methods: The study included forty-nine pediatric patients diagnosed with RMS. Tumor tissue expression of CD31, CD34, and CD105 was analyzed. MVD was calculated and correlated with clinical RMS prognostic parameters. (3) Results: CD31, CD34, and CD105 are expressed in all RMS cases. MVD/CD105 was significantly higher in the RMS group than in the control group. The mean and median values of MVD/CD105 in RMS were lower than MVD/CD31 and MVD/CD34. MVD/CD105 was significantly higher in patients with alveolar RMS and those with metastatic disease. Patients with higher levels of MVD/CD105 had a higher risk of death (HR = 1.009). (4) Conclusion: CD105 is a relevant angiogenesis marker in pediatric RMS, and MVD/CD105 is an independent risk factor of short overall survival in children with RMS.

Endoglin and TGF-\u03b2 signaling in glioblastoma

Microvascular proliferation is a key feature of glioblastoma and neovascularization has been implicated in tumor progression. Glioblastomas use pro-angiogenic factors such as vascular endothelial growth factor (VEGF) for new blood vessel formation. Yet, anti-VEGF therapy does not prolong overall survival so that alternative angiogenic pathways may need to be explored as drug targets. Both glioma cells and glioma-associated endothelial cells produce TGF-β superfamily ligands which bind TGF-β receptors (TGF-βR). The TGF-βR type III endoglin (CD105), is a marker of proliferating endothelium that has already been studied as a potential therapeutic target. We studied endoglin expression in glioblastoma tissue and in glioma-associated endothelial cells in a cohort of 52 newly diagnosed and 10 recurrent glioblastoma patients by immunohistochemistry and by ex vivo single-cell gene expression profiling of 6 tumors. Endoglin protein levels were similar in tumor stroma and endothelium and correlated within tumors. Similarly, endoglin mRNA determined by ex vivo single-cell gene expression profiling was expressed in both compartments. There was positive correlation between endoglin and proteins of TGF-β superfamily signaling. No prognostic role of endoglin expression in either compartment was identified. Endoglin gene silencing in T98G glioma cells and in human cerebral microvascular endothelial cells (hCMEC) did not affect constitutive or exogenous TGF-β superfamily ligand-dependent signaling, except for a minor facilitation of pSmad1/5 signaling in hCMEC. These observations challenge the notion that endoglin might become a promising therapeutic target in glioblastoma.

Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.

L-Arginine Protective Effect on Systemic Blood Pressure and Placental Expression of Endoglin, Transforming Growth Factor-β1 in the Preeclampsia Mice Model

L-Arginine Protective Effect on Systemic Blood Pressure and Placental Expression of Endoglin, Transforming Growth Factor-β1 in the Preeclampsia Mice Model

The Inhibition of Prolyl Oligopeptidase as New Target to Counteract Chronic Venous Insufficiency: Findings in a Mouse Model.

(1) Background: Chronic venous insufficiency (CVI) is a common disorder related to functional and morphological abnormalities of the venous system. Inflammatory processes and angiogenesis alterations greatly concur to the onset of varicose vein. KYP-2047 is a selective inhibitor of prolyl oligopeptidase (POP), a serine protease involved in the release of pro-angiogenic molecules. The aim of the present study is to evaluate the capacity of KYP-2047 to influence the angiogenic and inflammatory mechanisms involved in the pathophysiology of CVI. (2) Methods: An in vivo model of CVI-induced by saphene vein ligation (SVL) and a tissue block culture study were performed. Mice were subjected to SVL followed by KYP-2047 treatment (intraperitoneal, 10 mg/kg) for 7 days. Histological analysis, Masson’s trichrome, Van Gieson staining, and mast cells evaluation were performed. Release of cytokines, nitric oxide synthase production, TGF-beta, VEGF, α-smooth muscle actin, PREP, Endoglin, and IL-8 quantification were investigated. (3) Results: KYP-2047 treatment ameliorated the histological abnormalities of the venous wall, reduced the collagen increase and modulated elastin content, lowered cytokines levels and prevented mast degranulation. Moreover, a decreased expression of TGF-beta, eNOS, VEGF, α-smooth muscle actin, IL-8, and PREP was observed in in vivo study; also a reduction in VEGF and Endoglin expression was confirmed in tissue block culture study. (4) Conclusions: For the first time, this research, highlighting the importance of POP as new target for vascular disorders, revealed the therapeutic potential of KYP-2047 as a helpful treatment for the management of CVI.