Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis

Using the zebrafish, we previously identified a central function for perlecan during angiogenic blood vessel development. Here, we explored the nature of perlecan function during developmental angiogenesis. A close examination of individual endothelial cell behavior revealed that perlecan is required for proper endothelial cell migration and proliferation. Because these events are largely mediated by VEGF-VEGFR2 signaling, we investigated the relationship between perlecan and the VEGF pathway. We discovered that perlecan knockdown caused an abnormal increase and redistribution of total VEGF-A protein suggesting that perlecan is required for the appropriate localization of VEGF-A. Importantly, we linked perlecan function to the VEGF pathway by efficiently rescuing the perlecan morphant phenotype by microinjecting VEGF-A₁₆₅ protein or mRNA. Combining the strategic localization of perlecan throughout the vascular basement membrane along with its growth factor-binding ability, we hypothesized a major role for perlecan during the establishment of the VEGF gradient which provides the instructive cues to endothelial cells during angiogenesis. In support of this hypothesis we demonstrated that human perlecan bound in a heparan sulfate-dependent fashion to VEGF-A₁₆₅. Moreover, perlecan enhanced VEGF mediated VEGFR2 activation of human endothelial cells. Collectively, our results indicate that perlecan coordinates developmental angiogenesis through modulation of VEGF-VEGFR2 signaling events. The identification of angiogenic factors, such as perlecan, and their role in vertebrate development will not only enhance overall understanding of the molecular basis of angiogenesis, but may also provide new insight into angiogenesis-based therapeutic approaches.     

Endorepellin,a novel inhibitor of angiogenesis derived from the C terminus of perlecan

Perlecan, a ubiquitous basement membrane heparan sulfate proteoglycan, plays key roles in blood vessel growth and structural integrity. We discovered that the C terminus of perlecan potently inhibited four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. The C terminus of perlecan was active at nanomolar concentrations and blocked endothelial cell adhesion to fibronectin and type I collagen, without directly binding to either protein; henceforth we have named it "endorepellin." We also found that endothelial cells possess a significant number of high affinity (K(d) of 11 nm) binding sites for endorepellin and that endorepellin binds endostatin and counteracts its anti-angiogenic effects. Thus, endorepellin represents a novel anti-angiogenic product, which may retard tumor neovascularization and hence tumor growth in vivo.     

Different Regulation of Physiological and Tumor Angiogenesis in Zebrafish by Protein Kinase D1 (PKD1)

Protein kinase D isoenzymes (PKDs, Prkds) are serine threonine kinases that belong to the CAMK superfamily. PKD1 is expressed in endothelial cells and is a major mediator of biological responses downstream of the VEGFRs that are relevant for angiogenesis such as endothelial cell migration, proliferation and tubulogenesis in vitro. PKDs also play a critical role in tumor development and progression, including tumor angiogenesis. However, given the plethora of signaling modules that drive angiogenesis, the precise role of PKD1 in both physiological and tumor angiogenesis in vivo has not been worked out so far. This study aimed at dissecting the contribution of PKD1 to physiological blood vessel formation, PKD1 was found to be widely expressed during zebrafish development. As far as physiological angiogenesis was concerned, morpholino-based silencing of PKD1 expression moderately reduced the formation of the intersomitic vessels and the dorsal longitudinal anastomotic vessel in tg(fli1:EGFP) zebrafish. In addition, silencing of PKD1 resulted in reduced formation of the parachordal lymphangioblasts that serves as a precursor for the developing thoracic duct. Interestingly, tumor angiogenesis was completely abolished in PKD1 morphants using the zebrafish/tumor xenograft angiogenesis assay. Our data in zebrafish demonstrate that PKD1 contributes to the regulation of physiological angiogenesis and lymphangiogenesis during zebrafish development and is essential for tumor angiogenesis.     

Integrin alpha2beta1 is the required receptor for endorepellin angiostatic activity

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the alpha2beta1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the alpha2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of alpha2beta1. Microvascular endothelial cells from alpha2beta1(-/-) mice, but not those isolated from either wild-type or alpha1beta1(-/-) mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in alpha2beta1(-/-) mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin alpha2beta1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the alpha2beta1 integrin receptor.     

Betaglycan knock‐down causes embryonic angiogenesis defects in zebrafish

Angiogenesis is an essential requirement for embryonic development and adult homeostasis. Its deregulation is a key feature of numerous pathologies and many studies have shown that members of the transforming growth factor beta (TGF‐β) family of proteins play important roles in angiogenesis during development and disease. Betaglycan (BG), also known as TGF‐β receptor type III, is a TGF‐β coreceptor essential for mice embryonic development but its role in angiogenesis has not been described. We have cloned the cDNA encoding zebrafish BG, a TGF‐β‐binding membrane proteoglycan that showed a dynamic expression pattern in zebrafish embryos, including the notochord and cells adjacent to developing vessels. Injection of antisense morpholinos decreased BG protein levels and morphant embryos exhibited impaired angiogenesis that was rescued by coinjection with rat BG mRNA. In vivo time‐lapse microscopy revealed that BG deficiency differentially affected arterial and venous angiogenesis: morphants showed impaired pathfinding of intersegmental vessels migrating from dorsal aorta, while endothelial cells originating from the caudal vein displayed sprouting and migration defects. Our results reveal a new role for BG during embryonic angiogenesis in zebrafish, which has not been described in mammals and pose interesting questions about the molecular machinery regulating angiogenesis in different vertebrates. genesis 53:583–603, 2015. © 2015 Wiley Periodicals, Inc.     

Lrrc10 is required for early heart development and function in zebrafish

Leucine-rich Repeat Containing protein 10 (LRRC10) has recently been identified as a cardiac-specific factor in mice. However, the function of this factor remains to be elucidated. In this study, we investigated the developmental roles of Lrrc10 using zebrafish as an animal model. Knockdown of Lrrc10 in zebrafish embryos (morphants) using morpholinos caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and embryonic lethality between day 6 and 7 post fertilization. The Lrrc10 morphants exhibited cardiac functional defects as evidenced by a decrease in ejection fraction and cardiac output. Further investigations into the underlying mechanisms of the cardiac defects revealed that the number of cardiomyocyte was reduced in the morphants. Expression of two cardiac genes was deregulated in the morphants including an increase in atrial natriuretic factor, a hallmark for cardiac hypertrophy and failure, and a decrease in cardiac myosin light chain 2, an essential protein for cardiac contractility in zebrafish. Moreover, a reduced fluorescence intensity from NADH in the morphant heart was observed in live zebrafish embryos as compared to control. Taken together, the present study demonstrates that Lrrc10 is necessary for normal cardiac development and cardiac function in zebrafish embryos, which will enhance our understanding of congenital heart defects and heart disease.     

Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos

Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6 days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.     

Diverse cell signaling events modulated by perlecan

Perlecan is a ubiquitous pericellular proteoglycan ideally placed to mediate cell signaling events controlling migration, proliferation, and differentiation. Its control of growth factor signaling usually involves interactions with the heparan sulfate chains covalently coupled to the protein core's N-terminus. However, this modular protein core also binds with relatively high affinity to a number of growth factors and surface receptors, thereby stabilizing cell-matrix links. This review will focus on perlecan-growth factor interactions and describe recent advances in our understanding of this highly conserved proteoglycan during development, cancer growth, and angiogenesis. The pro-angiogenic capacities of perlecan that involve proliferative and migratory signals in response to bound growth factors will be explored, as well as the anti-angiogenic signals resulting from interactions between the C-terminal domain known as endorepellin and integrins that control adhesion of cells to the extracellular matrix. These two somewhat diametrically opposed roles will be discussed in light of new data emerging from various fields which converge on perlecan as a key regulator of cell growth and angiogenesis.     

Opposing effects of circadian clock genes bmal1 and period2 in regulation of VEGF-dependent angiogenesis in developing zebrafish

Molecular mechanisms underlying circadian-regulated physiological processes remain largely unknown. Here, we show that disruption of the circadian clock by both constant exposure to light and genetic manipulation of key genes in zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. Using a promoter-reporter system consisting of various deleted vegf-promoter mutants, we show that Bmal1 directly binds to and activates the vegf promoter via E-boxes. Additionally, we provide evidence that knockdown of Bmal1 leads to impaired Notch-inhibition-induced vascular sprouting. These results shed mechanistic insight on the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis.     

Loss of zebrafish lgi1b leads to hydrocephalus and sensitization to pentylenetetrazol induced seizure-like behavior

Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs.     

The N-terminal acetyltransferase Naa10 is essential for zebrafish development

N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish.     

A unique role for 6-O sulfation modification in zebrafish vascular development

Heparan sulfate proteoglycans are important modulators of growth factor signaling in a variety of patterning processes. Secreted growth factors that play critical roles in angiogenesis bind to heparan sulfate, and this association is affected by 6-O-sulfation of the heparan sulfate chains. Addition of 6-O-sulfate is catalyzed by a family of sulfotransferases (HS6STs), and genetic manipulation of their function permits an assessment of their contribution to vascular assembly. We report on the biochemical activity and expression patterns of two zebrafish HS6ST genes. In situ hybridization reveals dynamic and distinct expression patterns of these two genes during development. Structural analysis of heparan sulfate from wild-type and morpholino antisense 'knockdown' embryos suggests that HS6ST-1 and HS6ST-2 have similar biochemical activity. HS6ST-2, but not HS6ST-1, morphants exhibit abnormalities in the branching morphogenesis of the caudal vein during embryonic development of the zebrafish. Our finding that HS6ST-2 is required for the branching morphogenesis of the caudal vein is the first in vivo evidence for an essential role of a gene encoding a heparan sulfate modifying enzyme in vertebrate angiogenesis. Our analysis of two zebrafish HS6ST genes suggests that a wide range of biological processes may be regulated by an array of sulfation-modifying enzymes in the vertebrate genome.     

Augmenter of liver regeneration (alr) promotes liver outgrowth during zebrafish hepatogenesis

Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.