Altered vascular expression of EphrinB2 and EphB4 in a model of oxygen‐induced retinopathy

EphrinB2 ligands and EphB4 receptors are expressed on endothelial cells (EC) of arteries and veins, respectively, and are essential for vascular development. To understand how these molecules regulate retinal neovascularization (NV), we evaluated their expression in a model of oxygen‐induced retinopathy (OIR). EphrinB2 and EphB4 were expressed on arterial and venous trunks, respectively, and on a subset of deep capillary vessels. EphB4 expression was reduced following hyperoxia, while ephrinB2 expression remained unaltered. In addition, a subset of EphB4‐positive veins regressed in a caspase‐3‐dependent manner during hyperoxia. Arteriovenous malformations were also observed with loss of arterial‐venous boundaries. Finally, both ephrinB2 and EphB4 were expressed on a subset of neovascular tufts following hyperoxia. These data confirm the contribution of ECs from both venous and arterial origins to the development of retinal NV. Developmental Dynamics 239:1695–1707, 2010. © 2010 Wiley‐Liss, Inc.     

EphB3 marks delaminating endocrine progenitor cells in the developing pancreas

Background: Understanding the process by which pancreatic beta‐cells acquire their “fate” is critical to the development of in vitro directed differentiation protocols for cell replacement therapies for diabetics. To date, these efforts are hampered by a paucity of markers that distinguish pancreatic endocrine cells at different stages of differentiation. Results: Here, we identify EphB3 as a novel pro‐endocrine marker and use its expression to track delaminating islet lineages. First, we provide a detailed developmental expression profile for EphB3 and other EphB family members in the embryonic pancreas. We demonstrate that EphB3 transiently marks endocrine cells as they delaminate from the pancreatic epithelium, prior to their differentiation. Using a Tet‐inducible EphB3^rtTA‐lacZ reporter line, we show that short‐term pulse‐labeled EphB3⁺ cells co‐express Pdx1, Nkx6.1, Ngn3, and Synaptophysin, but not insulin, glucagon, or other endocrine hormones. Prolonged labeling tracks EphB3⁺ cells from their exit from the epithelium to their differentiation. Conclusions: These studies demonstrate that pro‐endocrine cell differentiation during late gestation, from delamination to maturation, takes approximately 2 days. Together, these data introduce EphB3 as a new biomarker to identify beta‐cells at a critical step during their step‐wise differentiation and define the timeframe of endocrine differentiation. Developmental Dynamics 241:1008–1019, 2012. © 2012 Wiley Periodicals, Inc.     

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin–Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.     

Characterisation of human induced pluripotent stem cell-derived endothelial cells under shear stress using an easy-to-use microfluidic cell culture system

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.     

Down-regulation of endothelial ephrinB2 expression by laminar shear stress.

The EphB receptors and their ephrinB ligands are involved in vascular assembly and differentiation. In this study, the authors analyzed the regulation of ephrinB2 and EphB4 in response to laminar shear stress in human endothelial cells. In order to simulate different flow conditions in vitro, human endothelial cells were exposed to laminar shear stress (1 to 50 dyn/cm2 for up to 24 h) in a cone-and-plate viscometer. EphrinB2 mRNA expression is down-regulated by arterial, but not by venous, laminar shear stress in a dose-dependent manner in primary cultures of human umbilical vein endothelial cells (HUVECs) (maximum at 30 dyn/cm2, 24 h: 46% +/- 4%of internal control without shear stress, n = 16, p < .05). The down-regulation of ephrinB2 by arterial shear stress is blocked by the protein kinase C inhibitor RO-31-8220. A similar shear stress-dependent down-regulation of ephrin-B2 can be found in human coronary artery endothelial cells (HCAECs). Chronic application of laminar shear stress does not affect EphB4 expression in venous and arterial endothelial cells. The down-regulation of ephrinB2 in response to laminar shear stress may contribute to the differentiation of endothelial cells into a nonactivated phenotype.     

Expression of the hyaluronan receptor LYVE-1 is not restricted to the lymphatic vasculature; LYVE-1 is also expressed on embryonic blood vessels

Expression of the hyaluronan receptor LYVE-1 is one of few available criteria used to discriminate lymphatic vessels from blood vessels. Until now, endothelial LYVE-1 expression was reported to be restricted to lymphatic vessels and to lymph node, liver, and spleen sinuses. Here, we provide the first evidence that LYVE-1 is expressed on blood vessels of the yolk sac during mouse embryogenesis. LYVE-1 is ubiquitously expressed in the yolk sac capillary plexus at E9.5, then becomes progressively down-regulated on arterial endothelium during vascular remodelling. LYVE-1 is also expressed on intra-embryonic arterial and venous endothelium at early embryonic stages and on endothelial cells of the lung and endocardium throughout embryogenesis. These findings have important implications for the use of LYVE-1 as a specific marker of the lymphatic vasculature during embryogenesis and neo-lymphangiogenesis. Our data are also the first demonstration, to our knowledge, that the mouse yolk sac is devoid of lymphatic vessels.     

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

Background: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular‐specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. Results: We present a mouse knockout allele for Paladin with a β‐galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular‐restricted expression in human brain, astrocytomas, and glioblastomas. Conclusions: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells. Developmental Dynamics 241:770–786, 2012. © 2012 Wiley Periodicals, Inc.     

Avian embryonic coronary arterio‐venous patterning involves the contribution of different endothelial and endocardial cell populations

Background: Coronary vasculature irrigates the myocardium and is crucial to late embryonic and adult heart function. Despite the developmental significance and clinical relevance of these blood vessels, the embryonic origin and the cellular and molecular mechanisms that regulate coronary arterio‐venous patterning are not known in detail. In this study, we have used the avian embryo to dissect the ontogenetic origin and morphogenesis of coronary vasculature. Results: We show that sinus venosus endocardial sprouts and proepicardial angioblasts pioneer coronary vascular formation, invading the developing heart simultaneously. We also report that avian ventricular endocardium has the potential to contribute to coronary vessels, and describe the incorporation of cardiac distal outflow tract endothelial cells to the peritruncal endothelial plexus to participate in coronary vascular formation. Finally, our findings indicate that large sinus venosus‐independent sections of the forming coronary vasculature develop without connection to the systemic circulation and that coronary arterio‐venous shunts form a few hours before peritruncal arterial endothelium connects to the aortic root. Conclusions: Embryonic coronary vasculature is a developmental mosaic, formed by the integration of vascular cells from, at least, four different embryological origins, which assemble in a coordinated manner to complete coronary vascular development. Developmental Dynamics 247:686–698, 2018. © 2017 Wiley Periodicals, Inc.     

EphB3 protein is associated with histological grade and FIGO stage in ovarian serous carcinomas

Eph (Erythropoietin‐producing human hepatocellular carcinoma cell) is the largest subfamily of receptor tyrosine kinases. Eph receptors and their ephrin ligands are involved in embryonic development and physiological processes. Aberrant expression of Eph/ephrin may contribute to a variety of diseases including cancer. EphB3 is a member of Eph receptors and has been found to play roles in carcinogenesis of some types of human cancer. But, its expression and clinical significance in ovarian serous carcinoma have not been well investigated and are unknown. In this study, a set of ovarian tissues including normal fallopian tube, serous borderline tumor, and serous carcinoma were subjected to immunohistochemistry using a specific polyclonal antibody for EphB3. The relationship between EphB3 expression and clinicopathological parameters was statistically analyzed. EphB3 was strongly expressed in all fallopian tube specimens (19/19, 100%). EphB3 was negatively or weekly expressed in 1 of 17 (5.8%) in borderline tumors and 26 of 50 (52.0%) in serous carcinomas, moderately expressed in 7 of 17 (41.2%) in borderline tumors and 14 of 50 (28%) in serous carcinomas, and strongly expressed in 9 17 (52.9%) in borderline tumors and 10 of 50 (20%) in serous carcinomas. EphB3 expression is significantly reduced in serous carcinomas compared with normal fallopian tubes and borderline tumors (p < 0.001). EphB3 expression is negatively associated with histological grade (p < 0.001, r_s = ?0.613) and FIGO stage (p = 0.001, r_s = ?0.464) of serous carcinomas. Our results show EphB3 protein lost in ovarian serous carcinoma and is associated with tumor grade and FIGO stage, which indicate EphB3 protein may play a role in carcinogenesis of ovarian serous carcinoma and may be used as a molecular marker for prognosis.     

Activin receptor-like kinase 1 is essential for placental vascular development in mice

Activin receptor-like kinase 1 (ALK1) is involved in the pathogenesis of hereditary hemorrhagic telangiectasia type II (HHT2) and pulmonary arterial hypertension. We have previously shown that Alk1 is predominantly expressed in the arterial endothelium and plays a pivotal role in the formation of embryonic blood vessels. At present, however, little is known about the precise expression pattern and function of ALK1 during extra-embryonic vascular development. Using previously generated lacZ reporter lines, we sought to examine the expression pattern and role of Alk1 during placental development in mice. Alk1 expression was restricted to endothelial cells of fetal vessels from the emergence of chorioallantoic fusion to the late gestational period, and no detectable Alk1 expression was observed in syncytiotrophoblasts or spongiotrophoblasts. Predominant arterial expression was observed in the umbilical and fetal placental vessels as well as in embryonic vessels. Morphological analysis of Alk1-null embryos indicates that Alk1 is essential for the development of distinct umbilical arteries and veins. The invasion of chorioallantoic mesoderm into the forming labyrinth layer was largely unaffected in the Alk1-null placenta, but chorioallantoic vessels appeared to be severely dilated and fused. Results from this study provide valuable information regarding the role of ALK1 in the development of placental vasculature as well as insights into the pathogenesis of HHT.     

Developmental platelet endothelial cell adhesion molecule expression suggests multiple roles for a vascular adhesion molecule

Platelet endothelial cell adhesion molecule (PECAM) is used extensively as a murine vascular marker. PECAM interactions have been implicated in both vasculogenesis and angiogenesis. To better understand the role of PECAM in mammalian development, PECAM expression was investigated during differentiation of murine embryonic stem (ES) cells and in early mouse embryos. Undifferentiated ES cells express PECAM, and as in vitro differentiation proceeds previously unidentified PECAM-positive cells that are distinct from vascular endothelial cells appear. PECAM expression is gradually restricted to endothelial cells and some hematopoietic cells of differentiated blood islands. In embryos, the preimplantation blastocyst contains PECAM-positive cells. PECAM expression is next documented in the postimplantation embryonic yolk sac, where clumps of mesodermal cells express PECAM before the development of mature blood islands. The patterns of PECAM expression suggest that undifferentiated cells, a prevascular cell type, and vascular endothelial cells express this marker during murine development. PECAM expression in blastocysts and by ES cells suggests that PECAM may function outside the vascular/hematopoietic lineage.     

Loss of expression of EphB1 protein in serous carcinoma of ovary associated with metastasis and poor survival

Aberrant expression of receptors tyrosine kinase of Eph gene in human cancers is extensively documented. We previously found that EphB1 subtype is down-regulated in gastric cancer and colorectal cancer. Fore the more, decreased expression of EphB1 is related to invasion and metastasis in cancers. There is no published data regarding the role of EphB1 in ovarian cancer, which is the focus of the present study. The expression of EphB1 protein was determined in tissues from 74 patients with serous ovarian carcinoma and 12 normal ovarian epithelial tissues. The expression level of EphB1 protein in serous ovarian carcinoma was analyzed with respect to clinicopathological parameters and survival. EphB1 protein was positively stained in 12 normal ovarian epithelial samples, and negatively stained in 32 out of 74 (43.2%) serous ovarian cancers. Loss of expression of EphB1 protein was associated with higher tumor grade (P = 0.006), metastasis (P = 0.049) and high proliferative index Ki67 expression (P = 0.022), but not with FIGO stage (P = 0.0937), age at diagnosis (P = 0.624), and diameter of carcinoma (P = 0.108). In addition, loss of EphB1 protein in serous ovarian carcinoma was associated with a significantly worse overall survival (P = 0.015). Our data indicate that loss of EphB1 protein is associated with metastasis and poorer survival in patients with serous ovarian cancer. EphB1 may be used as a prognostic marker and a therapeutic target in serous ovarian carcinoma.     

Decreased expression of receptor tyrosine kinase of EphB1 protein in renal cell carcinomas

Receptors tyrosine kinase of Eph superfamily plays an important role in human cancers. We previously found that EphB1 subtype is down-regulated in gastric cancer, colorectal cancer and ovary serous carcinoma. Fore the more, the decreased expression of EphB1 is related to invasion and metastasis in cancers. Although EphB1 has been revealed as an important receptor in cancers, our understanding of its roles in renal cell carcinoma (RCC) is limited. In the present study, using specific anit-EphB1 polyclonal antibody and immunohistochemistry, we evaluated EphB1 protein expression levels in RCC specimens surgically resected from 82 patients (including 62 conventional clear-cell RCC, 10 papillary, and 10 chromophobic RCC cases). We found EphB1 protein is positively expressed in the epithelium of renal tubules. Decreased expression of EphB1 was found in all RCC carcinomas compared with expression in the normal epithelium of renal tubules. EphB1 protein moderately expressed in chromophobic RCC, weakly expressed in clear-cell RCC and negatively expressed in papillary RCC. Our results indicate that EphB1 may be involved in carcinogenesis of RCC, the molecular mechanisms of down-regulation of EphB1 including genetic and epigenetic alterations and the dedicated roles of EphB1 in occurrence and progress of RCC need to be explicated in next step.     

High expression of EphB6 protein in tongue squamous cell carcinoma is associated with a poor outcome

EphB6 is a member in the receptor tyrosine kinase Eph family in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In this study, we examined the expression of EphB6 protein in 54 of tissue specimens of tongue squamous cell carcinoma by using a specific polyclonal anti-EphB6 antibody. The relationship between expression of EphB6 and clinical pathologic parameters was analyzed. The expression level of EphB6 in carcinoma cells from 34 out of 54 (63%) specimens was no alterative compared with normal squamous cells in same patient. The level of EphB6 protein staining was increased in carcinoma cells in 20 out of 54 (37%) specimens compared with normal squamous cells in same patient. The high-expression of EphB6 was significantly associated with age (P=0.021), tumor TNM stage (P=0.026) and lymph node metastasis (P=0.046). Patients with high expressed EphB6 protein had a high mortality (P=0.057). No significant relationship between expression of EphB6 and sex, tumor grade, HPV infection, relapse and smoke was found. We showed that patients with high expression of EphB6 had a significantly poor overall survival (OS) compared to patients with negative or weak expression (P=0.042). Our results indicated that EphB6 protein may be used as a new marker for prognosis for tongue squamous cell carcinoma.     

Low Expression of EphB2, EphB3, and EphB4 in Bladder Cancer: Novel Potential Indicators of Muscular Invasion

PurposeEph receptors are differentially expressed in numerous malignant tumors. This study intended to analyze the roles of EphB receptors (EphB2, B3, and B4) in urinary bladder cancer.Materials and MethodsTissue microarray-based immunohistochemical analysis was used to investigate the expression patterns of EphB2, EphB3, and EphB4 in 154 bladder cancer specimens. Immunohistochemical staining was conducted examining the extent of stained cells and staining intensity. EphB was considered to be highly expressed when the intensity of staining was more than moderate in >25% of cells in the tissue section. Small interfering RNA (siRNA) was used to knock down EphB expression in bladder cancer cell lines (T24, 5637) to determine the effects of EphB on tumor cell invasion, proliferation, and migration.ResultsEphB receptors (B2, B3, and B4) were detected in 40.9% (EphB2, 63/154), 71.4% (EphB3, 110/154), and 53.2% (EphB4, 82/154) of bladder cancer specimens. Low expression of EphB2, B3, and B4 receptors were significantly associated with higher tumor grade (EphB2, p<0.001; EphB3, p=0.032; EphB4, p<0.001) and muscular invasion (EphB2, p=0.002; EphB3, p=0.009; EphB4, p<0.001). No obvious correlation was observed with other clinicopathological variables, such as age, sex, recurrence, lymph node involvement, metastasis, and overall survival. Inactivation of EphB receptors by siRNA transfection increased cell viability, tumor cell invasion, proliferation, and migration in comparison with untransfected cancer cells.ConclusionLow expression of EphB receptors (B2, B3, and B4) can be a predictive marker for muscular invasion of bladder cancer.     

Systematic assessment of protein phenotypes characterizing high‐grade tumour budding in mismatch repair‐proficient colorectal cancer

Karamitopoulou E, Lugli A, Panayiotides I, Karakitsos P, Peros G, Rallis G, Patsouris E S, Terracciano L & Zlobec I (2010) Histopathology 57 , 233–243
Systematic assessment of protein phenotypes characterizing high‐grade tumour budding in mismatch repair‐proficient colorectal cancer Aims: A tumour bud is defined as a single tumour cell or tumour cell cluster of up to five cells at the invasive tumour front. Significant differences in survival have been detected in colorectal cancer patients with low‐ compared to high‐grade budding. The aim of this study was to identify potential multi‐marker phenotypes characterizing low‐ and high‐grade budding in mismatch repair (MMR)‐proficient colorectal cancer. Methods and results: Established and promising prognostic proteins such as epidermal growth factor receptor (EGFR), pERK, RHAMM, RKIP, β‐catenin, E‐cadherin, pAKT, p16, p21, Ki67, Bcl‐2, vascular endothelial growth factor (VEGF), apoptotic protease activating factor‐1 (APAF‐1), MUC1, EphB2, matrix metalloproteinase 7, pSMAD2, CDX2, laminin5γ2 and MST1 were analysed on 208 MMR‐proficient colorectal cancers with complete clinicopathological data. The most accurate markers for predicting high‐grade budding (more than six tumour buds) were EphB2 (P < 0.001), Bcl‐2 (P < 0.001), RKIP (P < 0.001), E‐cadherin (P = 0.004), laminin5γ2 (P = 0.004) and APAF‐1 (P = 0.005). On multivariable analysis, only loss of Bcl‐2 (P < 0.001) and EphB2 (P < 0.001) were independent predictors of high‐grade budding. Bcl‐2?/EphB2? tumours were more frequently poorly differentiated (P < 0.001), of advanced pT stage (P = 0.002), lymph node positive (P = 0.023), presented vascular (P = 0.053) and lymphatic invasion (P = 0.005) and had a negative impact on patient survival (P = 0.012). Conclusions: The multi‐marker phenotype EphB2?/Bcl‐2? is an independent predictor of high‐grade budding and implies increased aggressive behaviour in MMR‐proficient colorectal cancer.