Angiogenesis - the formation of new blood vessels from pre-existing blood vessels - is vital for normal development in embryos and for tissue repair in adults. Elucidating the mechanisms of angiogenesis has been subject to intense research following the discovery that angiogenesis is critical for the progression of several pathologies, including cancer. The mitogen-activated protein kinase (MAPK), extracellular signalregulated kinase 5 (ERKS) is activated in response to a variety of extracellular stimuli, including pro-angiogenic stimuli in endothelial cells. Ablation of the ErkS gene in mice is embryonically lethal due to disruption of cardiovascular development and loss of vascular integrity. In this study, the potential role of ERKS in mediating various stages of vascular endothelial growth factor (VEGF)-stimulated angiogenesis in primary human dermal microvascular endothelial cells (HDMECs) was investigated. It was found that VEGF stimulated ERKS activation in HDMECs via VEGF receptor-2 (VEGFR-2) and protein kinase C-delta (PKCo). Small interfering RNA (siRNA)-mediated silencing of
ERKS expression revealed that ERKS was required for HDMEC survival during VEGFstimulated differentiation of HDMECs to form capillary structures within a 3-D collagen gel matrix, but not for VEGF-stimulated proliferation or migration. Furthermore, constitutive activation of ERKS induced tubular morphogenesis in the absence of VEGF, and facilitated sustained survival of HDMEC capillary structures in the presence of VEGF. Analysis of intracellular signalling pathways showed that ERKS mediated VEGFinduced endothelial cell survival via regulation of AKT activation and subsequent inactivation of the pro-apoptotic protein BAD, whilst concomitantly facilitating VEGFstimulated expression of the anti-apoptotic protein BCL2, resulting in decreased caspase-3 activity and suppression of apoptosis. To further investigate the role of
ERKS in angiogenesis, a HDMEC/NHDF (normal human dermal fibroblast) co-culture in vitro angiogenesis assay was developed, revealing that chronic treatment with ERKSspecific siRNA prevented VEGF and fibroblast growth factor-2 (FGF-2)-stimulated angiogenesis by specifically inducing apoptosis of HDMECs, but not NHDFs. Together, this study identifies ERKS as a critical mediator of VEGF-induced human endothelial cell survival during a specific stage of angiogenesis, and suggests that inhibition of ERKS activity may prevent aberrant angiogenesis in vivo.