The proline-rich tyrosine kinase (PYK2) is a focal adhesion-related tyrosine kinase that can be activated by VEGF, increased intracellular Ca²⁺ and oxidative stress. Given that the endothelial nitric oxide synthase (eNOS) plays an important role in the endothelial function and that the activation of PYK2 leads to phosphorylation of eNOS on Y657 and enzyme inhibition, we studied the effects of the PYK2 inhibitor PF4594755 on the VEGF-eNOS axis in angiogenesis.

Vascular endothelial cell growth factor (VEGF) enhanced the proliferation of cultured murine lung endothelial cells (MLEC) by a mechanism partially sensitive to the NOS inhibitor L-NAME. In MLEC from eNOS^-/- mice infected with wildtype eNOS or its non-phosphorylatable Y657F mutant, L-NAME could likewise inhibit VEGF-induced proliferation, whereas cells infected with the phosphomimetic Y657D mutant proliferated significantly slower and were not affected by L-NAME. In collagen-embedded aortic rings, the VEGF-induced sprouting of endothelial cell tubes was likewise reduced by L-NAME. Under the conditions studied, VEGF stimulated the tyrosine phosphorylation of PYK2 and eNOS, both of which were sensitive to PF4594755. PYK2 inhibition significantly increased the VEGF-induced cell proliferation as well as the total sprout length. In conclusion, the tyrosine phosphorylation of eNOS (on Y657) following the activation of PYK2 under both basal and activated conditions can be inhibited by pharmacological inhibition of PYK2. This results in further augmentation of the eNOS-dependent VEGF-induced cell proliferation and aortic sprouting.