ABSTRACT
OBJECTIVE: Understanding the mechanisms regulating normal and pathological angiogenesis is of great scientific and clinical interest. In this report, we show that mutations in 2 different aminoacyl-transfer RNA synthetases, threonyl tRNA synthetase (tars(y58)) or isoleucyl tRNA synthetase (iars(y68)), lead to similar increased branching angiogenesis in developing zebrafish. APPROACH AND RESULTS: The unfolded protein response pathway is activated by aminoacyl-transfer RNA synthetase deficiencies, and we show that unfolded protein response genes atf4, atf6, and xbp1, as well as the key proangiogenic ligand vascular endothelial growth factor (vegfaa), are all upregulated in tars(y58) and iars(y68) mutants. Finally, we show that the protein kinase RNA-like endoplasmic reticulum kinase-activating transcription factor 4 arm of the unfolded protein response pathway is necessary for both the elevated vegfaa levels and increased angiogenesis observed in tars(y58) mutants. CONCLUSIONS: Our results suggest that endoplasmic reticulum stress acts as a proangiogenic signal via unfolded protein response pathway-dependent upregulation of vegfaa.
Subject(s)
Isoleucine-tRNA Ligase/deficiency , Neovascularization, Physiologic , Threonine-tRNA Ligase/deficiency , Unfolded Protein Response , Zebrafish Proteins/deficiency , Activating Transcription Factor 4/genetics , Activating Transcription Factor 4/metabolism , Activating Transcription Factor 6/genetics , Activating Transcription Factor 6/metabolism , Animals , Animals, Genetically Modified , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Gene Expression Regulation, Developmental , Genotype , Isoleucine-tRNA Ligase/genetics , Mutation , Phenotype , Regulatory Factor X Transcription Factors , Signal Transduction , Threonine-tRNA Ligase/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , X-Box Binding Protein 1 , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
The cerebral vasculature provides the massive blood supply that the brain needs to grow and survive. By acquiring distinctive cellular and molecular characteristics it becomes the blood-brain barrier (BBB), a selectively permeable and protective interface between the brain and the peripheral circulation that maintains the extracellular milieu permissive for neuronal activity. Accordingly, there is great interest in uncovering the mechanisms that modulate the formation and differentiation of the brain vasculature. By performing a forward genetic screen in zebrafish we isolated no food for thought (nft (y72)), a recessive late-lethal mutant that lacks most of the intracerebral central arteries (CtAs), but not other brain blood vessels. We found that the cerebral vascularization deficit of nft (y72) mutants is caused by an inactivating lesion in reversion-inducing cysteine-rich protein with Kazal motifs [reck; also known as suppressor of tumorigenicity 15 protein (ST15)], which encodes a membrane-anchored tumor suppressor glycoprotein. Our findings highlight Reck as a novel and pivotal modulator of the canonical Wnt signaling pathway that acts in endothelial cells to enable intracerebral vascularization and proper expression of molecular markers associated with BBB formation. Additional studies with cultured endothelial cells suggest that, in other contexts, Reck impacts vascular biology via the vascular endothelial growth factor (VEGF) cascade. Together, our findings have broad implications for both vascular and cancer biology.
Subject(s)
Blood-Brain Barrier/cytology , Brain/embryology , Cerebrovascular Circulation/genetics , GPI-Linked Proteins/genetics , Neovascularization, Physiologic/genetics , Wnt Signaling Pathway/genetics , Zebrafish Proteins/genetics , Animals , Animals, Genetically Modified , Brain/blood supply , Cell Line , Cerebrovascular Circulation/physiology , Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells , Humans , Mutation/genetics , Vascular Endothelial Growth Factor A/metabolism , Zebrafish/embryology , Zebrafish Proteins/metabolismABSTRACT
Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis.
Subject(s)
Diacylglycerol Cholinephosphotransferase/metabolism , Phosphatidylinositols/metabolism , Vascular Endothelial Growth Factor A/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Base Sequence , Blood Vessels/embryology , Blood Vessels/metabolism , DNA, Complementary/genetics , Diacylglycerol Cholinephosphotransferase/genetics , Humans , Mutation , Neovascularization, Physiologic/genetics , RNA, Small Interfering/genetics , Signal Transduction , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
We identified four mutants in two distinct loci exhibiting similar trunk vascular patterning defects in an F3 genetic screen for zebrafish vascular mutants. Initial vasculogenesis is not affected in these mutants, with proper specification and differentiation of endothelial cells. However, all four display severe defects in the growth and patterning of angiogenic vessels in the trunk, with ectopic branching and disoriented migration of intersegmental vessels. The four mutants are allelic to previously characterized mutants at the fused-somites (fss) and beamter (bea) loci, and they exhibit comparable defects in trunk somite boundary formation. The fss locus has been shown to correspond to tbx24; we show here that bea mutants are defective in the zebrafish dlC gene. Somitic expression of known vascular guidance factors efnb2a, sema3a1, and sema3a2 is aberrantly patterned in fss and bea mutants, suggesting that the vascular phenotype is due to loss of proper guidance cues provided by these factors.
