Dennis Pfaff

Emerging roles of the Angiopoietin‐Tie and the ephrin‐Eph systems as regulators of cell trafficking

Vascular receptor tyrosine kinases (RTK) have been identified as critical regulatory signaling molecules of developmental and adult vascular morphogenic processes [vascular endothelial growth factor (VEGF) receptors=sprouting; EphB receptors=assembly; Tie2 receptor=maturation and quiescence]. It is intriguing that the same molecules that control the growth of blood and lymphatic vessels play critical roles in the adult to regulate maintenance functions related to vascular homeostasis. VEGF is among the most potent inducers of vascular permeability. The second vascular RTK system, the interaction of paracrine‐acting Angiopoietin‐1 with its cognate receptor Tie2, acts as an endothelial maintenance and survival‐mediating molecular system, which stabilizes the vessel wall and controls endothelial cell quiescence. The third vascular RTK system, the interaction of Eph receptors with their Eph family receptor‐interacting protein (ephrin) ligands, transduces positional guidance cues on outgrowing vascular sprouts, which are critical for proper arteriovenous assembly and establishment of blood flow. As such, Eph‐ephrin interactions act as an important regulator of cell–cell interactions, exerting propulsive and repulsive functions on neighboring cells and mediating adhesive functions. This review summarizes recent findings related to the roles of the Angiopoietin‐Tie and the Eph‐ephrin systems as regulators of cell trafficking in the vascular system. The recognition of vascular homeostatic functions of vascular RTKs marks an important change of paradigm in the field of angiogenesis research as it relates angiogenesis‐inducing molecules to vascular maintenance functions in the adult. This may also broaden the scope of vascular RTK‐targeted therapies.

Role of ephrinB2 expression in endothelial cells during arteriogenesis: impact on smooth muscle cell migration and monocyte recruitment

Expression of the arterial marker molecule ephrinB2 in endothelial cells is a prerequisite for adequate remodeling processes of the developing or angiogenic vasculature. Although its role in these processes has been extensively studied, the impact of ephrinB2 on the remodeling of adult arteries is largely unknown. To this end, we analyzed its expression during a biomechanically induced arteriolar remodeling process known as arteriogenesis and noted a significant increase in ephrinB2 expression under these conditions. By examining those biomechanical forces presumed to drive arteriogenesis, we identified cyclic stretch as a critical inducer of ephrinB2 expression in endothelial cells. Subsequent functional analyses in vitro revealed that endothelial cells expressing ephrinB2 limit the migration of smooth muscle cells, thereby enhancing segregation of both cell types. Moreover, MCP-1 induced transmigration of monocytes through a monolayer of endothelial cells overexpressing a truncated variant of ephrinB2 was clearly impeded. Taken together, these data suggest that expression of ephrinB2 in adult endothelial cells is up-regulated during arterial remodeling and controlled by cyclic stretch, a well-known inducer of such processes. This stretch-induced ephrinB2 expression may be pivotal for arteriogenesis as it limits smooth muscle cell migration within defined borders and controls monocyte extravasation.

Involvement of endothelial ephrin-B2 in adhesion and transmigration of EphB-receptor-expressing monocytes

The vascular endothelium is a crucial interface that controls the recruitment of circulating leukocytes. Based on the luminal expression of the ephrin-B2 ligand by endothelial cells (ECs) and the expression of EphB receptors (EphBRs) by circulating monocytes, we hypothesized that EphBR-ephrinB interactions are involved in monocyte adhesion. Adhesion experiments with monocytic cells were performed on ECs that overexpressed either full-length ephrin-B2 or cytoplasmically truncated ephrin-B2 (ΔC-ephrin-B2). Atomic force microscopy confirmed similar adhesive strengths of EphBR-expressing J774 cells to ECs that either overexpressed full-length ephrin-B2 or truncated ΔC-ephrin-B2 (1-minute interaction). Yet, adhesion experiments under static or flow conditions for 30 minutes demonstrated the preferential adhesion of monocytic cells to ECs that overexpressed full-length ephrin-B2 but not to ΔC-ephrin-B2 or to ECs that had been mock transduced. Adhesion was blocked by ephrin-B2-specific and EphBR-specific antibodies. Correspondingly, adhesion of EphB4-receptor-overexpressing monocytes to ephrin-B2-positive ECs was further augmented. Trafficking experiments of cell-surface molecules revealed that, prior to internalization, the resulting EphB4-receptor–ephrin-B2 complex translocated from the luminal surface to inter-endothelial junctions. Lastly, full-length ephrin-B2 in ECs was also involved in monocyte transmigration. Collectively, our study identifies a role of EphBR-ephrinB interactions as a new step in the cascade of events leading to monocyte adhesion and transmigration through the vascular endothelium.

Endothelial EphrinB2 Is Controlled by Microenvironmental Determinants and Associates Context-Dependently With CD31

Objective—The EphB ligand ephrinB2 has been identified as a critical determinant of arterial endothelial differentiation and as a positive regulator of invading endothelial cells during angiogenesis. This study was aimed at identifying determinants of endothelial cell ephrinB2 expression. Methods and Results—Arteriovenous asymmetrical endothelial cell ephrinB2 expression in vivo is lost on transfer into culture with aortic endothelial cells becoming partially ephrinB2-negative and saphenous vein endothelial cells becoming partially ephrinB2-positive. Contact with smooth muscle cells and angiogenic stimulation by vascular endothelial growth factor lead to an increased endothelial cell ephrinB2 expression. Quiescent, smooth muscle-contacting endothelial cells express ephrinB2 uniformly on their luminal surface. In contrast, monolayer endothelial cells translocate ephrinB2 to interendothelial cell junctions, which is strongly enhanced by EphB4-Fc-mediated receptor body activation. Junctional ephrinB2 colocalizes and coimmunoprecipitates with CD31. Conclusions—This study identifies distinct regulatory mechanisms of endothelial ephrinB2 expression and cellular distribution in quiescent and activated endothelial cells. The data demonstrate that endothelial cell ephrinB2 expression is controlled by microenvironmental determinants rather than being an intrinsic endothelial cell differentiation marker.

Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K

Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O‐glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel‐associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin‐deficient mice have unraveled a critical role of endosialin in site‐specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac‐2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C‐terminal fragment of Mac‐2 BP/90K, which was shown to contain binding sites for galectin‐3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac‐2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up‐regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac‐2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss‐of‐function adhesion experiments of Mac‐2 BP/90K‐expressing tumor cells on endosialin‐expressing fibroblasts revealed a repulsive outcome of the Mac‐2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac‐2 BP/90K on tumor cells.—Becker, R., Lenter, M. C., Vollkommer, T., Boos, A. M., Pfaff, D., Augustin, H. G., Christian, S. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis‐related protein Mac‐2 BP/90K. FASEB J. 22, 3059–3067 (2008)

EphB4 Promotes Site-Specific Metastatic Tumor Cell Dissemination by Interacting with Endothelial Cell–Expressed EphrinB2

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4–expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. Mol Cancer Res; 8(10); 1297–309. ©2010 AACR.

Ephrin-B2 expression critically influences Nipah virus infection independent of its cytoplasmic tail

BackgroundCell entry and cell-to-cell spread of the highly pathogenic Nipah virus (NiV) requires binding of the NiV G protein to cellular ephrin receptors and subsequent NiV F-mediated fusion. Since expression levels of the main NiV entry receptor ephrin-B2 (EB2) are highly regulated in vivo to fulfill the physiological functions in axon guidance and angiogenesis, the goal of this study was to determine if changes in the EB2 expression influence NiV infection.ResultsSurprisingly, transfection of increasing EB2 plasmid concentrations reduced cell-to-cell fusion both in cells expressing the NiV glycoproteins and in cells infected with NiV. This effect was attributed to the downregulation of the NiV glycoproteins from the cell surface. In addition to the influence on cell-to-cell fusion, increased EB2 expression significantly reduced the total amount of NiV-infected cells, thus interfered with virus entry. To determine if the negative effect of elevated EB2 expression on virus entry is a result of an increased EB2 signaling, receptor function of a tail-truncated and therefore signaling-defective ΔcEB2 was tested. Interestingly, ΔcEB2 fully functioned as NiV entry and fusion receptor, and overexpression also interfered with virus replication.ConclusionOur findings clearly show that EB2 signaling does not account for the striking negative impact of elevated receptor expression on NiV infection, but rather that the ratio between the NiV envelope glycoproteins and surface receptors critically influence cell-to-cell fusion and virus entry.

FGF2 induces RANKL gene expression as well as IL1β regulated MHC class II in human bone marrow-derived mesenchymal progenitor stromal cells

OBJECTIVEnHuman bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1β and IFNγ.nnnMETHODSnRANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1β and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR.nnnRESULTSnIn hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1β and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1β induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1β through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1β in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts.nnnCONCLUSIONSnRANKL expression and IL1β regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1β and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration.

Extracellular cadherin repeat domains EC1 and EC5 of T-cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells

T‐cadherin (T‐cad) promotes survival, proliferation, and migration of endothelial cells and induces angiogenesis. We aimed to identify domains of T‐cad functionally relevant to its effects on endothelial cell behavior. To specifically target the functional properties of the 5 cadherin repeat domains (EC1–EC5) of T‐cad, endothelial cells were transduced with lentivectors containing specific T‐cad‐domain‐deletion mutant constructs (ΔΙ, ΔΠ, ΔΠΙ, ΔIV, ΔV). Empty (E) lentivector‐transduced cells served as control. Similarly to overexpression of native T‐cad, cells expressing ΔΙΙ, ΔΙΙΙ, or ΔΙV displayed elevated levels of p‐Akt and p‐GSK3β and increased proliferation rates (for ΔΙΙ, ΔΙΙΙ) vs. E. ΔΙ‐ and ΔV‐transduced cells exhibited reduced levels of p‐Akt and p‐GSK3β and retarded growth rates vs. E. Stimulatory effects of native T‐cad overexpression on Akt and GSK3β phosphorylation were dose dependently inhibited by coexpression of ΔΙ or ΔV. Subsequent functional analyses compared only ΔΙ‐, ΔΙΙ‐, and ΔV‐mutant constructs with E as a negative control. Unlike ΔΙΙ cells, ΔΙ and ΔV cells failed to exhibit homophilic ligation and deadhesion responses on a substratum of T‐cad protein. In the wound assay, migration was increased for ΔΙΙ cells but impaired for ΔΙ and ΔV cells. m endothelial cell‐spheroid assay, angiogenic sprouting was augmented for ΔΙΙ cells but inhibited for ΔΙ and ΔV cells. We conclude that EC1 and EC5 domains of T‐cad are essential for its proan‐giogenic effects. ΔΙ and ΔV constructs may serve as dominant‐negative mutants and as potential tools targeting excessive angiogenesis.—Joshi, M. B., Kyriakakis, E., Pfaff, D., Rupp, K., Philippova, M., Erne, P., Resink, T. J. Extracellular cadherin repeat domains EC1 and EC5 of T‐cadherin are essential for its ability to stimulate angiogenic behavior of endothelial cells. FASEBJ. 23, 4011–4021 (2009). www.fasebj.org

EGFR and IGF-1R in regulation of prostate cancer cell phenotype and polarity: opposing functions and modulation by T-cadherin

T‐cadherin is an atypical glycosylphosphatidylinsoitol‐anchored member of the cadherin superfamily of adhesion molecules. We found that T‐cadherin over‐expression in malignant (DU145) and benign (BPH‐1) prostatic epithelial cell lines or silencing in the BPH‐1 cell line, respectively, promoted or inhibited migration and spheroid invasion in collagen I gel and Matrigel. T‐cadherin‐dependent effects were associated with changes in cell phenotype: overexpression caused cell dissemination and loss of polarity evaluated by relative positioning of the Golgi/nuclei in cell groups, whereas silencing caused formation of compact polarized epithelial‐like clusters. Epidermal growth factor receptor (EGFR) and IGF factor‐1 receptor (IGF‐1R) were identified as mediators of T‐cadherin effects. These receptors per se had opposing influences on cell phenotype. EGFR activation with EGF or IGF‐1R inhibition with NVP‐AEW541 promoted dissemination, invasion, and polarity loss. Conversely, inhibition of EGFR with gefitinib or activation of IGF‐1R with IGF‐1 rescued epithelial morphology and decreased invasion. T‐cadherin silencing enhanced both EGFR and IGF‐1R phosphorylation, yet converted cells to the morphology typical for activated IGF‐1R. T‐cadherin effects were sensitive to modulation of EGFR or IGF‐1R activity, suggesting direct involvement of both receptors. We conclude that T‐cadherin regulates prostate cancer cell behavior by tuning the balance in EGFR/IGF‐1R activity and enhancing the impact of IGF‐1R.—Maslova, K., Kyriakakis, E., Pfaff, D., Frachet, A., Frismantiene, A., Bubendorf, L., Ruiz, C., Vlajnic, T., Erne, P., Resink, T. J., Philippova, M. EGFR and IGF‐1R in regulation of prostate cancer cell phenotype and polarity: opposing functions and modulation by T‐cadherin. FASEB J. 29, 494‐507 (2015). www.fasebj.org