RESUMO
Engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmo1/Dock180) is a bipartite guanine nucleotide exchange factor for the monomeric GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). Elmo1/Dock180 regulates Rac1 activity in a specific spatiotemporal manner in endothelial cells (ECs) during zebrafish development and acts downstream of the Netrin-1/Unc5-homolog B (Unc5B) signaling cascade. However, mechanistic details on the pathways by which Elmo1/Dock180 regulates endothelial function and vascular development remained elusive. In this study, we aimed to analyze the vascular function of Elmo1 and Dock180 in human ECs and during vascular development in zebrafish embryos. In vitro overexpression of Elmo1 and Dock180 in ECs reduced caspase-3/7 activity and annexin V-positive cell number upon induction of apoptosis. This protective effect of Elmo1 and Dock180 is mediated by activation of Rac1, p21-activated kinase (PAK) and AKT/protein kinase B (AKT) signaling. In zebrafish, Elmo1 and Dock180 overexpression reduced the total apoptotic cell and apoptotic EC number and promoted the formation of blood vessels during embryogenesis. In conclusion, Elmo1 and Dock180 protect ECs from apoptosis by the activation of the Rac1/PAK/AKT signaling cascade in vitro and in vivo. Thus, Elmo1 and Dock180 facilitate blood vessel formation by stabilization of the endothelium during angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Vasos Sanguíneos/metabolismo , Neovascularização Fisiológica , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/genética , Vasos Sanguíneos/crescimento & desenvolvimento , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas rac de Ligação ao GTP/biossíntese , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Respiratory distress syndrome (RDS) caused by preterm delivery is a major clinical problem with limited mechanistic insight. Late-stage embryonic lung development is driven by hypoxia and the hypoxia-inducible transcription factors Hif-1α and Hif-2α, which act as important regulators for lung development. Expression of the BTB-and kelch-domain-containing (BTB-kelch) protein KLEIP (Kelch-like ECT2-interacting protein; also named Klhl20) is controlled by two hypoxia response elements, and KLEIP regulates stabilization and transcriptional activation of Hif-2α. Based on the available data, we hypothesized an essential role for KLEIP in murine lung development and function. Therefore, we have performed a functional, histological, mechanistic and interventional study in embryonic and neonatal KLEIP(-/-) mice. Here, we show that about half of the KLEIP(-/-) neonates die due to respiratory failure that is caused by insufficient aeration, reduced septal thinning, reduced glycogenolysis, type II pneumocyte immaturity and reduced surfactant production. Expression analyses in embryonic day (E) 18.5 lungs identified KLEIP in lung capillaries, and showed strongly reduced mRNA and protein levels for Hif-2α and VEGF; such reduced levels are associated with embryonic endothelial cell apoptosis and lung bleedings. Betamethasone injection in pregnant females prevented respiratory failure in KLEIP(-/-) neonates, normalized lung maturation, vascularization, aeration and function, and increased neonatal Hif-2α expression. Thus, the experimental study shows that respiratory failure in KLEIP(-/-) neonates is determined by insufficient angiocrine Hif-2α-VEGF signaling and that betamethasone activates this newly identified signaling cascade in late-stage embryonic lung development.
