ABSTRACT
The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.
Subject(s)
Endothelial Cells/metabolism , Neoplasms/physiopathology , Neovascularization, Pathologic/metabolism , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/metabolism , Animals , Blotting, Western , Cell Proliferation , Computational Biology , DNA Primers/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Female , Fibroblast Growth Factor 2/metabolism , Flow Cytometry , Fluorescent Antibody Technique , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Neoplasms/metabolism , RNA, Small Interfering/genetics , Receptors, G-Protein-Coupled/genetics , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
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.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Neovascularization, Pathologic/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Receptor, EphB4/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Vascular Endothelial Growth Factor A/metabolism , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacokinetics , Animals , Biological Assay , Humans , Lung/drug effects , Lung/metabolism , Mice , Neovascularization, Pathologic/enzymology , Organ Size/drug effects , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrimidines/chemistry , Pyrimidines/pharmacokinetics , Receptor, EphB4/metabolism , Signal Transduction/drug effects , Solubility/drug effectsABSTRACT
Both thrombin and tryptase have been shown to induce smooth muscle cell proliferation in vitro. We have used cultured primary guinea-pig tracheal smooth muscle in order to define pharmacologically the receptors involved in this effect. Tryptase, a protease-activated receptor (PAR)-2 agonist, induced DNA synthesis up to the second passage of the cells, thereafter the response waned. In contrast, thrombin, a PAR-1 agonist, and the PAR-1 activating peptide (SFLLRN) induced DNA synthesis starting from the third passage only. Thrombin and tryptase responses were dose-dependently inhibited by leupeptin. The selective PAR-2 activating peptide (SLIGRL-NH(2)) was unable to induce DNA synthesis in cells from passages 1 to 6. In agreement with the functional data, mRNA expression for PAR-1 was increased in cells in later passages. In contradiction with the functional data, however, equal mRNA expression for PAR-2 was found in all passages. These results suggest that thrombin induces guinea-pig tracheal smooth muscle DNA synthesis through activation of PAR-1. However, the differential effect of tryptase and SLIGRL-NH(2) suggests that tryptase might exert some of its effect via a non-PAR-2 receptor.
