Determination of critical shear stress for maturation of human pluripotent stem cell-derived endothelial cells towards an arterial subtype.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.

The in vivo effect of prophylactic subchondral bone protection of osteoarthritic synovial membrane in bone-specific Ephb4-overexpressing mice.

Osteoarthritis (OA) is characterized by progressive joint destruction, including synovial membrane alteration. EphB4 and its ligand ephrin-B2 were found in vitro to positively affect OA subchondral bone and cartilage. In vivo in an experimental mouse model overexpressing bone-specific Ephb4 (TgEphB4), a protective effect was found on both the subchondral bone and cartilage during OA. We investigated in the TgEphB4 mouse model the in vivo effect on synovial membrane during OA. Knee OA was surgically induced by destabilization of the medial meniscus (DMM). Synovial membrane was evaluated using histology, histomorphometry, IHC, and real-time PCR. Compared to DMM-wild-type (WT) mice, DMM-TgEphB4 mice had a significant decrease in synovial membrane thickness, vascular endothelial growth factor, and the profibrotic markers fibrin, type 1 procollagen, type 3 collagen, connective tissue growth factor, smooth muscle actin-α, cartilage oligomeric matrix protein, and procollagen-lysine, and 2-oxoglutarate 5-dioxygenase 2. Moreover, factors known to modulate transforming growth factor-ß signaling, transforming growth factor receptor 1/ALK1, phosphorylated Smad-1, and heat shock protein 90ß were significantly decreased in DMM-TgEphB4 compared with DMM-WT mice. Ephb4 overexpression also exhibited a protective effect on synovial membrane thickness of aged (24-month-old) mice. Overexpression of bone-specific Ephb4 clearly demonstrated prevention of the development and/or progression of fibrosis in OA synovial membrane, reinforcing the hypothesis that protecting the subchondral bone prophylactically and during OA reduces the pathologic changes in other articular tissues.

EphB4 Expressing Stromal Cells Exhibit an Enhanced Capacity for Hematopoietic Stem Cell Maintenance.

The tyrosine kinase receptor, EphB4, mediates cross-talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin-B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over-expressing EphB4 under the control of collagen type-1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long-term culture-initiating cells (LTC-IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC-IC in vitro, where blocking EphB4/ephrin-B2 interactions decreased LTC-IC output. Similarly, short hairpin RNA-mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin-B2 expressing CD34(+) HSC in LTC-IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin-1, IL-6, FLT-3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal-mediated support of hematopoiesis, and constitute a novel component of the HSC niche.

EphB4 promotes the proliferation, invasion, and angiogenesis of human colorectal cancer.

OBJECTIVE: Eph/Ephrin signalling plays an important role in tumorigenesis, neovascularization, and vasculogenesis. However, studies concerning the role of EphB4 in colorectal cancer (CRC) show inconsistent results, and the function of EphB4 in the formation of CRC-related blood vessels is not fully understood. The aim of this study is to investigate the EphB4 expression in CRC and the role of EphB4 in tumour angiogenesis. MATERIALS AND METHODS: EphB4 and EphrinB2 expressions were detected in 200 CRC samples and 50 paired colorectal mucosae by immunohistochemistry. Xenograft animal models were established by stable knockdown and stable overexpression of EphB4, and control cell lines were used to investigate the role of EphB4 in CRC. Microvessels were stained with anti-CD34, and microvessel density (MVD) was assessed. RESULTS: EphB4 protein was more highly expressed in CRC tissues compared with adjacent normal mucosae (P<0.05), while EphrinB2 levels were unchanged. Modulation of EphB4 levels in colon cancer cell line SW480 resulted in significant effects on tumour growth and invasion in vivo, with stable overexpression of EphB4 associated with faster growth and invasion. Furthermore, microvessel density values in xenograft tumours were significantly correlated with EphB4 (P<0.05). CONCLUSION: EphB4 acts as a tumour promoter associated with proliferation, invasion, and angiogenesis, and may be used as a potential CRC therapeutic target.

EphB4 regulates the growth and migration of pancreatic cancer cells.

Pancreatic cancer is a serious threat to human life. Moreover, its treatment is complicated and its prognosis is very poor. Therefore, a new method for the diagnosis and treatment of pancreatic cancer is very essential. In this study, a eukaryotic expression plasmid targeting EphB4 was constructed and transfected into PANC-1 pancreatic cancer cells to investigate the inhibition of cell growth and the progression of iRNA against EphB4. This study provides the basis for the gene therapy of pancreatic cancer. The recombinant eukaryotic EphB4 expression plasmid, pSIREN-RetroQ-ZsGreen-EphB4 and a negative control plasmid, pSIREN-RetroQ-ZsGreen-N, were constructed. At 48 h after transfection, the relative messenger RNA (mRNA) and protein levels of EphB4 were measured by RT-PCR and western blot. The proliferation of the transfected cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while cell migration ability was analyzed using the scratch migration assay. At 48 h after transient transfection, EphB4 mRNA expression was significantly decreased in transfected PANC-1 cells as compared to the control group (P < 0.01). In vitro, inhibition of EphB4 expression weakened the proliferation and cell migration ability of PANC-1 cells compared to the control group. The small interfering RNA (siRNA) eukaryotic expression plasmid vector targeting EphB4 was successfully constructed and effectively transfected into PANC-1 cells. The recombinant plasmid can inhibit the expression of EphB4 mRNA and protein in PANC-1 cells, as well as cell growth and migration.

Down-regulation of EphB4 phosphorylation is necessary for esophageal squamous cell carcinoma tumorigenecity.

Eph/ephrin signaling system plays a very important role in the tumorigenesis and the formation of blood vessel. However, the function of EphB4 and its ligand ephrin B2 in the carcinogenesis of esophageal squamous cell carcinoma (ESCC) is not fully understood. Here, it was found that the expression of EphB4 was up-regulated in ESCC tissues compared with the paired normal tissues, while ephrin B2 was down-regulated in ESCC samples. Phosphorylation of EphB4 induced by its ligand ephrin B2-Fc inhibited the growth, migration and colony formation of ESCC cells. Moreover, over-expression of EphB4 or EphB4 kinase dead mutant (EphB4 KD) in ESCC cells promoted cell growth and migration, suggesting EphB4 promoted cell growth and migration independent of its kinase activity. Furthermore, we found that EphB4 interacted with the adaptor protein RACK1 and RACK1 decreased the phosphorylation level of EphB4. Taken together, our study revealed the important function and regulation of EphB4 in the progression of ESCC and suggested EphB4 as a novel target for the treatment of ESCC.

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.

EphB4 is overexpressed in gliomas and promotes the growth of glioma cells.

Glioma is one of the most common solid tumors, and the molecular mechanism for this disease is poorly understood. EphB4 tyrosine kinase receptor has been involved in various physiologic and pathologic processes, and the role of EphB4 in tumorigenesis has recently attracted much interest. However, its function in glioma remains largely unknown. In this study, we explored the function of EphB4 in glioma. We found that the expression of EphB4 was significantly upregulated in clinical glioma samples. Overexpression of EphB4 in glioma cell lines accelerated cell growth and tumorigenesis. In contrast, downregulation of EphB4 inhibited cell growth. Furthermore, we showed that EphB4 promoted cell growth by promoting EGFR signaling. Taken together, our findings suggest that EphB4 plays an important role in the progression of glioma by stimulating cell growth and EphB4 might be a potential therapeutic target for glioma.

Arterial shear stress augments the differentiation of endothelial progenitor cells adhered to VEGF-bound surfaces.

Our ongoing studies show that vascular endothelial cell growth factor (VEGF)-bound surfaces selectively capture endothelial progenitor cells (EPCs) in vitro and in vivo, and that surface-bound VEGF stimulates intracellular signal transduction pathways over prolonged culture periods, resulting in inductive differentiation of EPCs. In this article, we investigated whether simulated arterial shear stress augments the differentiation of EPCs adhered to a VEGF-bound surface. Human peripheral blood-derived mononuclear cells adhered to a VEGF-bound surface were exposed to 1 day of shear stress (15 dynes/cm(2), corresponding to shear load in arteries). Shear stress suppressed the expression of mRNAs encoding CD34 and CD133, which are markers for EPCs, and augmented the expression of mRNAs encoding CD31 and von Willebrand factor (vWF) as well as vWF protein, which are markers for endothelial cells (ECs). Shear stress enhanced expression of ephrinB2 mRNA, a marker for arterial ECs, but did not significantly change expression of EphB4 mRNA, a marker for venous ECs. Focused protein array analysis showed that mechanotransduction by shear stress activated the p38 and MAPK pathways in EPCs. Thus, arterial shear stress, in concert with surface-bound VEGF, augments the differentiation of EPCs. These results strongly support previous observation of rapid differentiation of EPCs captured on VEGF-bound stents in a porcine model.

Notch ligand Delta-like 1 is essential for postnatal arteriogenesis.

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.

EphB4 overexpression in B16 melanoma cells affects arterial-venous patterning in tumor angiogenesis.

EphB4 receptor and its ligand ephrinB2 play an important role in vascular development during embryogenesis. In blood vessels, ephrinB2 is expressed in arterial endothelial cells (EC) and mesenchymal supporting cells, whereas EphB4 is only expressed in venous ECs. Previously, we reported that OP9 stromal cells, which support the development of both arterial and venous ECs, in which EphB4 was overexpressed, could inhibit ephrinB2-positive (ephrinB2+) EC development in an embryonic tissue organ culture system. Although the EphB4 receptor is expressed in a variety of tumor cells, its exact function in regulating tumor progression has not been clearly shown. Here we found that overexpression of EphB4 in B16 melanoma cells suppressed tumor growth in a s.c. transplantation tumor model. Histologic examination of these tumors revealed that EphB4 overexpression in B16 cells selectively suppressed arterial ephrinB2+ EC development. By coculturing ephrinB2-expressing SV40-transformed mouse ECs (SVEC) with EphB4-overexpressing B16 cells, we found that EphB4 induced the apoptosis of SVECs. However, ephrinB2 did not induce the apoptosis of EphB4-overexpressing B16 cells. Based on results from these experiments, we concluded that EphB4 overexpression in B16 tumor cells suppresses the survival of arterial ECs in tumors by a reverse signaling via ephrinB2.

Core Element Cloning, Cis-Element Mapping and Serum Regulation of the Human EphB4 Promoter: A Novel TATA-Less Inr/MTE/DPE-Like Regulated Gene.

The EphB4 gene encodes for a transmembrane tyrosine kinase receptor involved in embryonic blood vessel differentiation and cancer development. Although EphB4 is known to be regulated at the post-translational level, little is known about its gene regulation. The present study describes the core promoter elements' identification and cloning, the cis-regulatory elements' mapping and the serum regulation of the human EphB4 gene promoter region. Using bioinformatic analysis, Sanger sequencing and recombinant DNA technology, we analyzed the EphB4 gene upstream region spanning +40/-1509 from the actual transcription start site (TSS) and proved it to be a TATA-less gene promoter with dispersed regulatory elements characterized by a novel motif-of-ten element (MTE) at positions +18/+28, and a DPE-like motif and a DPE-like-repeated motif (DRM) spanning nt +27/+30 and +32 +35, respectively. We also mapped both proximal (multiple Sp1) and distal (HoxA9) trans-activating/dispersed cis-acting transcription factor (TF)-binding elements on the region we studied and used a transient transfection reporter assay to characterize its regulation by serum and IGF-II using EphB4 promoter deletion constructs with or without the identified new DNA-binding elements. Altogether, these findings shed new light on the human EphB4 promoter structure and regulation, suggesting mechanistic features conserved among Pol-II TATA-less genes phylogenetically shared from Drosophila to Human genomes.

Compartments with predominant ephrin-B1 and EphB2/B4 expression are present alternately along the excurrent duct system in the adult mouse testis and epididymis.

BACKGROUND: Ephrin receptors (Eph) and ligands are membrane-bound cell-cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown. OBJECTIVES: We aimed to investigate the expression of several EphB receptors and ephrin-B ligands in the testis and epididymis of adult mice. MATERIALS AND METHODS: mRNA and protein expression was detected via reverse transcription-polymerase chain reaction amplification and immunostaining, respectively. RESULTS: Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin-B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin-B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine-phosphorylated in the testis and/or epididymis. DISCUSSION: Receptor localization and phosphorylation patterns suggested that EphB/ephrin-B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin-B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation. CONCLUSION: Overall, this study represents the first analysis of EphB receptor and ephrin-B ligand expression in the normal adult testis and epididymis.

EPHB4 and survival of colorectal cancer patients.

The family of receptor tyrosine kinases EPH and their Ephrin ligands regulate cell proliferation, migration, and attachment. An important role in colorectal carcinogenesis is emerging for some of its members. In this study, we evaluate the role of EPHB4 in colorectal cancer and its value as a prognostic marker. EPHB4 levels were assessed by immunohistochemical staining of tissue microarrays of 137 colorectal tumors and aberrant hypermethylation of the EPHB4 promoter was investigated using methylation-specific PCR. We found that EPHB4 expression is frequently reduced or lost in colorectal tumors. Patients with low EPHB4 tumor levels had significantly shorter survival than patients in the high EPHB4 group (median survival, 1.8 and >9 years, respectively; P < 0.01, log-rank test), and this finding was validated using an independent set of 125 tumor samples. In addition, we show that EPHB4 promoter hypermethylation is a common mechanism of EPHB4 inactivation. Moreover, reintroduction of EPHB4 resulted in a significant reduction in the clonogenic potential of EPHB4-deficient cells, whereas abrogation of EPHB4 in cells with high levels of this receptor lead to a significant increase in clonogenicity. In summary, we identified EPHB4 as a useful prognostic marker for colorectal cancer. In addition, we provide mechanistic evidence showing that promoter methylation regulates EPHB4 transcription and functional evidence that EPHB4 can regulate the long-term clonogenic potential of colorectal tumor cells, revealing EPHB4 as a potential new tumor suppressor gene in colorectal cancer.

New potential ligand-receptor signaling loops in ovarian cancer identified in multiple gene expression studies.

Based on the hypothesis that gene products involved in the same biological process would be coupled at transcriptional level, a previous study analyzed the correlation of the gene expression patterns of ligand-receptor (L-R) pairs to discover potential autocrine/paracrine signaling loops in different cancers (Graeber and Eisenberg. Nat Genet 2001; 29:295). By refining the starting database, a list of 511 L-R pairs was compiled, combined to eight data sets from a single pathology, epithelial ovarian cancer, and examined as a proof-of-principle of the statistical and biological validity of the correlation of the L-R gene expression patterns in cancer. Analysis revealed a Bonferroni-corrected significant correlation of 105 L-R pairs in at least one data set and, by systematic analysis, identified 39 more frequently correlated L-R pairs, 7 of which were already biologically confirmed. In four data sets examined for an L-R correlation associated with patient survival time, 15 L-R pairs were significantly correlated in short surviving patients in two of the data sets. Immunohistochemical analysis of one of the newly identified correlated L-R pairs (i.e., EFNB3-EPHB4) revealed the correlated expression of ephrin-B3 and EphB4 proteins in 45 of 55 epithelial ovarian tumor samples (P < 0.0001). Together, these data not only support the validity of cross-comparison analysis of gene expression data because known and expected correlations were confirmed but also point to the promise of such analysis in identifying new L-R signaling loops in cancer.

Expression and prognostic significance of EFNB2 and EphB4 genes in patients with oesophageal squamous cell carcinoma.

OBJECTIVE: Tyrosine kinases and its receptors play important roles in growth, migration, and invasion of malignant cells. Among those, there are only few reports examining the expression pattern of Eph/ephrin signalling system in oesophageal carcinoma. The prognostic importance of ephrin-B2 ligand (EFNB2) and its receptor EphB4, and its correlation with clinicopathologic characteristics are yet to be delineated in patients with oesophageal carcinoma. MATERIALS AND METHODS: EFNB2 gene and EphB4 receptor gene were examined of mRNA specimens in 61 patients with oesophageal squamous cell carcinoma using reverse-transcriptase polymerase chain reaction. EFNB2 protein was selectively examined using an immunohistochemical analysis. RESULTS: EFNB2 mRNA expression was detected in 38 (62.3%) and EphB4 expression was found in 44 (72.1%) out of 61 cancer tissues analysed. There was a statistically significant correlation between EFNB2 expression and number of lymph node metastasis (P<0.05), and a trend toward statistical significance for correlation between EFNB2 expression and American Joint Committee on Cancer Classification Stage (P<0.1), indicating that EFNB2 expression was up-regulated by advancement of the disease process. EFNB2 protein was strongly expressed in tumour with high mRNA EFNB2 expression and was weakly expressed in tumour with low mRNA expression in some representative tumours. The 5-year overall survival rate (23%) of patients with positive EFNB2 gene expression was significantly worse than 55% of negative expression (P<0.05). The results of multivariate analysis of prognosticators for survival showed that positive EFNB2 gene expression (P<0.01) and number of lymph node metastasis (P<0.05) were identified as significant factors indicative of a poorer survival. CONCLUSIONS: EFNB2 gene expression may be a biological marker and a useful prognostic indicator in patients with oesophageal squamous cell carcinoma.

EphB4 expression and biological significance in prostate cancer.

Prostate cancer is the most common cancer in men. Advanced prostate cancer spreading beyond the gland is incurable. Identifying factors that regulate the spread of tumor into the regional nodes and distant sites would guide the development of novel diagnostic, prognostic, and therapeutic targets. The aim of our study was to examine the expression and biological role of EphB4 in prostate cancer. EphB4 mRNA is expressed in 64 of 72 (89%) prostate tumor tissues assessed. EphB4 protein expression is found in the majority (41 of 62, 66%) of tumors, and 3 of 20 (15%) normal prostate tissues. Little or no expression was observed in benign prostate epithelial cell line, but EphB4 was expressed in all prostate cancer cell lines to varying degrees. EphB4 protein levels are high in the PC3 prostate cancer cell line and several folds higher in a metastatic clone of PC3 (PC3M) where overexpression was accompanied by EphB4 gene amplification. EphB4 expression is induced by loss of PTEN, p53, and induced by epidermal growth factor/epidermal growth factor receptor and insulin-like growth factor-I/insulin-like growth factor-IR. Knockdown of the EphB4 protein using EphB4 short interfering RNA or antisense oligodeoxynucleotide significantly inhibits cell growth/viability, migration, and invasion, and induces apoptosis in prostate cancer cell lines. Antisense oligodeoxynucleotide targeting EphB4 in vivo showed antitumor activity in murine human tumor xenograft model. These data show a role for EphB4 in prostate cancer and provide a rationale to study EphB4 for diagnostic, prognostic, and therapeutic applications.

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Homeobox A9 transcriptionally regulates the EphB4 receptor to modulate endothelial cell migration and tube formation.

Homeobox genes (Hox) encode for transcription factors, which regulate cell proliferation and migration and play an important role in the development of the cardiovascular system during embryogenesis. In this study, we investigated the role of HoxA9 for endothelial cell migration and angiogenesis in vitro and identified a novel target gene, the EphB4 receptor. Inhibition of HoxA9 expression decreased endothelial cell tube formation and inhibited endothelial cell migration, suggesting that HoxA9 regulates angiogenesis. Because Eph receptor tyrosine kinases importantly contribute to angiogenesis, we examined whether HoxA9 may transcriptionally regulate the expression of EphB4. Downregulation of HoxA9 reduced the expression of EphB4. Chromatin-immunoprecipitation revealed that HoxA9 interacted with the EphB4 promoter, whereas a deletion construct of HoxA9 without DNA-binding motif (Delta(aa) 206-272) did not bind. Consistently, HoxA9 wild-type overexpression activated the EphB4 promoter as determined by reporter gene expression. HoxA9 binds to the EphB4 promoter and stimulates its expression resulting in an increase of endothelial cell migration and tube forming activity. Thus, modulation of EphB4 expression may contribute to the proangiogenic effect of HoxA9 in endothelial cells.

A rapid and sensitive method for EphB4 identification as a diagnostic and therapeutic biomarker in invasive breast cancer.

BACKGROUND: In the roadmap to design diagnostic and therapeutic markers for breast cancer, EphB4 is of special interest due to its multiple roles in tumor initiation, progression and invasion. The aim of present study was to characterize a rapid and sensitive ELISA-based method to measure EphB4 level and its phosphorylation status following stimulation with its ligand, ephrinB2, in an invasive breast cancer cell line. MATERIALS AND METHODS: MDA-MB-231 breast cancer cells were lysed and EphB4 level was measured using ELISA. EphB4 level was measured in sub- and post-confluent states in culture dishes. Receptor phosphorylation was also detected by ELISA assay, using various concentrations of pre-clustered ephrinB2 for 20 minutes. RESULTS: Expression of EphB4 receptor was detected by ELISA in all samples. EphB4 level was significantly higher in post.confluent than sub.confluent cells. Phosphorylated receptor was also detectable with this method when cells were exogenously stimulated. CONCLUSIONS: Quantitative data from ELISA manifested a difference between levels of EphB4 in two states of different invasive properties. Moreover, ELISA method may be considered rapid and sensitive enough to detect even low levels of total and phosphorylated EphB4 Cost-effectiveness of this method for the detection of differential expression of EphB4 proteins in clinics is also noticeable.