The small molecule specific EphB4 kinase inhibitor NVP-BHG712 inhibits VEGF driven angiogenesis

EphB4 and its cognitive ligand ephrinB2 play an important role in embryonic vessel development and vascular remodeling. In addition, several reports suggest that this receptor ligand pair is also involved in pathologic vessel formation in adults including tumor angiogenesis. Eph/ephrin signaling is a complex phenomena characterized by receptor forward signaling through the tyrosine kinase of the receptor and ephrin reverse signaling through various protein–protein interaction domains and phosphorylation motifs of the ephrin ligands. Therefore, interfering with EphR/ephrin signaling by the means of targeted gene ablation, soluble receptors, dominant negative mutants or antisense molecules often does not allow to discriminate between inhibition of Eph/ephrin forward and reverse signaling. We developed a specific small molecular weight kinase inhibitor of the EphB4 kinase, NVP-BHG712, which inhibits EphB4 kinase activity in the low nanomolar range in cellular assays showed high selectivity for targeting the EphB4 kinase when profiled against other kinases in biochemical as well as in cell based assays. Furthermore, NVP-BHG712 shows excellent pharmacokinetic properties and potently inhibits EphB4 autophosphorylation in tissues after oral administration. In vivo, NVP-BHG712 inhibits VEGF driven vessel formation, while it has only little effects on VEGF receptor (VEGFR) activity in vitro or in cellular assays. The data shown here suggest a close cross talk between the VEGFR and EphR signaling during vessel formation. In addition to its established function in vascular remodeling and endothelial arterio-venous differentiation, EphB4 forward signaling appears to be an important mediator of VEGF induced angiogenesis since inhibition of EphB4 forward signaling is sufficient to inhibit VEGF induced angiogenesis

Inhibition of Tumor Growth and Angiogenesis by Soluble EphB4

EphB receptors and their ephrinB ligands play a key role in the formation of a regular vascular system. Recent studies have also shown the involvement of Eph/ephrin interactions in malignant tumor progression and angiogenesis. We have generated soluble monomeric EphB4 (sEphB4)-expressing A375 melanoma cells to study the effect of dominant negatively acting sEphB4 on tumor growth and angiogenesis. Soluble EphB4-expressing A375 tumors grown subcutaneously in nude mice show dramatically reduced tumor growth compared to control tumors. The proliferative capacity of sEphB4-expressing cells in monolayer culture is not altered. Yet, sEphB4-expressing A375 cells cannot establish proper cell-cell contacts in three-dimensional spheroids. However, sEphB4 transfectants have reduced proliferation and apoptosis rates when grown in three-dimensional culture in vitro or in subcutaneous tumors in vivo. Analysis of the vascular phenotype of the tumors revealed a reduction of intratumoral microvessel density in sEphB4-expressing tumors. Corresponding to these mouse experiments, a matched pair analysis of EphB4 and ephrinB2 expression in human colon carcinomas revealed significantly upregulated levels of EphB4 expression compared to adjacent normal tissue. Taken together, the data identify dual effects of sEphB4 on the tumor and the vascular compartment that collectively inhibit tumor growth

Podocyte EphB4 signaling helps recovery from glomerular injury

Eph receptor tyrosine kinases and their ligands (ephrins) have a pivotal role in the homeostasis of many adult organs and are widely expressed in the kidney. Glomerular diseases beginning with mesangiolysis can recover, with podocytes having a critical role in this healing process. We studied here the role of Eph signaling in glomerular disease recovery following mesangiolytic Thy1.1 nephritis in rats. EphB4 and ephrinBs were expressed in healthy glomerular podocytes and were upregulated during Thy1.1 nephritis, with EphB4 strongly phosphorylated around day 9. Treatment with NPV-BHG712, an inhibitor of EphB4 phosphorylation, did not cause glomerular changes in control animals. Nephritic animals treated with vehicle did not have morphological evidence of podocyte injury or loss; however, application of this inhibitor to nephritic rats induced glomerular microaneurysms, podocyte damage, and loss. Prolonged NPV-BHG712 treatment resulted in increased albuminuria and dysregulated mesangial recovery. Additionally, NPV-BHG712 inhibited capillary repair by intussusceptive angiogenesis (an alternative to sprouting angiogenesis), indicating a previously unrecognized role of podocytes in regulating intussusceptive vessel splitting. Thus, our results identify EphB4 signaling as a pathway allowing podocytes to survive transient capillary collapse during glomerular disease

VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of the chorioallantoic membrane

AbstractWe have studied the effect of VEGF121homodimer and VEGF121/165heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2–4 μg and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU–anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension,D,of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF121is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF121application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF121) and 1.41 (VEGF121/165). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF165and VEGF121/165, VEGF121diffuses over a slightly greater distance. Usingin situhybridization, VEGF receptor-2 (flk-1/Quek1) and the homologousflt-4(Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF121application on the CAM of 11-day-old quail embryos. During normal development,flk-1expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF121application induces expression offlk-1in capillaries that normally do not express the receptor. In the normal development of the CAM,flt-4becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF121does not alterflt-4expression