ABSTRACT
Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.
Subject(s)
Endothelial Cells/metabolism , Glycolysis , Neovascularization, Physiologic , Phosphofructokinase-2/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Endothelial Cells/cytology , Female , Gene Deletion , Gene Silencing , Humans , Male , Mice , Mice, Inbred C57BL , Phosphofructokinase-2/genetics , Pseudopodia/metabolism , ZebrafishABSTRACT
BACKGROUND: Normal growth and development of organisms requires maintenance of a dynamic balance between systems that promote cell survival and those that induce apoptosis. The molecular mechanisms that regulate these processes remain poorly understood, and thus further in vivo study is required. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that uniquely also promotes mitosis and cell proliferation. Postnatally, survivin is hardly detected in most tissues, but is upregulated in all cancers, and as such, is a potential therapeutic target. Prenatally, survivin is also highly expressed in several tissues. Fully delineating the properties of survivin in vivo in mice has been confounded by early lethal phenotypes following survivin gene inactivation. RESULTS: To gain further insights into the properties of survivin, we used the zebrafish model. There are 2 zebrafish survivin genes (Birc5a and Birc5b) with overlapping expression patterns during early development, prominently in neural and vascular structures. Morpholino-induced depletion of Birc5a causes profound neuro-developmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. Similar abnormalities, all less severe except for hematopoiesis, were evident with suppression of Birc5b. The phenotypes induced by morpholino knockdown of one survivin gene, were rescued by overexpression of the other, indicating that the Birc5 paralogs may compensate for each. The potent vascular endothelial growth factor (VEGF) also entirely rescues the phenotypes induced by depletion of either Birc5a and Birc5b, highlighting its multi-functional properties, as well as the power of the model in characterizing the activities of growth factors. CONCLUSION: Overall, with the zebrafish model, we identify survivin as a key regulator of neurogenesis, vasculo-angiogenesis, hematopoiesis and cardiogenesis. These properties of survivin, which are consistent with those identified in mice, indicate that its functions are highly conserved across species, and point to the value of the zebrafish model in understanding the role of this IAP in the pathogenesis of human disease, and for exploring its potential as a therapeutic target.
Subject(s)
Embryo, Nonmammalian/metabolism , Inhibitor of Apoptosis Proteins/genetics , Microtubule-Associated Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Apoptosis/genetics , Apoptosis/physiology , Blood Vessels/embryology , Blood Vessels/metabolism , Embryo, Nonmammalian/embryology , Gene Expression Regulation, Developmental , Genetic Complementation Test , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Heart/embryology , Hematopoiesis/genetics , In Situ Hybridization , In Situ Nick-End Labeling , Microinjections , Microscopy, Fluorescence , Microtubule-Associated Proteins/physiology , Molecular Sequence Data , Myocardium/metabolism , Neurogenesis/genetics , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/genetics , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Survivin , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/physiology , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/physiologyABSTRACT
Acute renal failure (ARF) is a major worldwide cause of morbidity and mortality, lacking specific targeted, effective therapies. Renal tubular cell apoptosis has been recognized to play a critical role in the pathogenesis of ARF, yet few studies have evaluated whether intervention in apoptotic pathways can ameliorate the deterioration in renal function associated with ARF. Using transgenic mice with diminished levels of the inhibitor of apoptosis protein, survivin, we show that survivin is required to protect the kidney from apoptosis, to suppress renal expression of p53, and to ameliorate renal dysfunction in two models of ARF. Gene delivery of survivin to wild-type mice and mice with 50% levels of survivin, prior to or at the time of induction of ARF, interferes with the deterioration of renal function and preserves the integrity of the kidneys and the renal tubular cells by inhibiting activation of apoptotic pathways in the kidneys and suppressing expression of p53. These results encourage further evaluation of survivin, its active structural domains, and other inhibitors of apoptosis proteins, for preventing and/or treating acute renal failure.