RESUMO
Angiogenic sprouting can contribute adaptively, or mal-adaptively, to a myriad of conditions including ischemic heart disease and cancer. While the cellular and molecular systems that regulate tip versus stalk endothelial cell (EC) specification during angiogenesis are known, those systems that regulate their distinct actions remain poorly understood. Pre-clinical and clinical findings support sustained adrenergic signaling in promoting angiogenesis, but links between adrenergic signaling and angiogenesis are lacking; importantly, adrenergic agents alter the activation status of the cAMP signaling system. Here, we show that the cAMP effector, PKA, acts in a cell autonomous fashion to constitutively reduce the in vitro and ex vivo angiogenic sprouting capacity of ECs. At a cellular level, we observed that silencing or inhibiting PKA in human ECs increased their invasive capacity, their generation of podosome rosettes and, consequently, their ability to degrade a collagen matrix. While inhibition of either Src-family kinases or of cdc42 reduced these events in control ECs, only cdc42 inhibition, or silencing, significantly impacted them in PKA(Cα)-silenced ECs. Consistent with these findings, cell-based measurements of cdc42 activity revealed that PKA activation inhibits EC cdc42 activity, at least in part, by promoting its interaction with the inhibitory regulator, guanine nucleotide dissociation inhibitor-α (RhoGDIα).
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Células Endoteliais/metabolismo , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Podossomos , Proteína cdc42 de Ligação ao GTP/fisiologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Células Endoteliais/citologia , Células Endoteliais/patologia , Humanos , Neovascularização Fisiológica/fisiologia , Podossomos/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/fisiologiaRESUMO
The Hippo pathway is a central regulator of tissue development and homeostasis, and has been reported to have a role during vascular development. Here we develop a bioluminescence-based biosensor that monitors the activity of the Hippo core component LATS kinase. Using this biosensor and a library of small molecule kinase inhibitors, we perform a screen for kinases modulating LATS activity and identify VEGFR as an upstream regulator of the Hippo pathway. We find that VEGFR activation by VEGF triggers PI3K/MAPK signaling, which subsequently inhibits LATS and activates the Hippo effectors YAP and TAZ. We further show that the Hippo pathway is a critical mediator of VEGF-induced angiogenesis and tumor vasculogenic mimicry. Thus, our work offers a biosensor tool for the study of the Hippo pathway and suggests a role for Hippo signaling in regulating blood vessel formation in physiological and pathological settings.