Regulation of angiogenesis by endocytic trafficking mediated by cytoplasmic dynein 1 light intermediate chain 1.

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

In vivo dissection of Rhoa function in vascular development using zebrafish.

The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. In this study, we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant negative, or constitutively active forms of rhoaa in ECs, pharmacological inhibitors of RHOA and ROCK1/2, and Rock1 and Rock2a/b dgRNP-injected zebrafish embryos to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis in vivo, showing that either too much or too little RHOA activity leads to vascular dysfunction.

Rapid Generation of Pigment Free, Immobile Zebrafish Embryos and Larvae in Any Genetic Background Using CRISPR-Cas9 dgRNPs.

The ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, increasing amounts of pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges for live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals, but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. In this study, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.

Chemokine mediated signalling within arteries promotes vascular smooth muscle cell recruitment.

The preferential accumulation of vascular smooth muscle cells (vSMCs) on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. In zebrafish, the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vSMCs during vascular development. We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries of zebrafish and mice in vivo. Additionally, we demonstrate that expression of the blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signalling axis leads to the differential acquisition of vSMCs at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.

Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling.

Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth. However, tumor overproduction of pro-angiogenic ligands can overcome these therapies, hampering success of this approach. To circumvent this problem, we target the resynthesis of phosphoinositides consumed during intracellular transduction of pro-angiogenic signals in endothelial cells (EC), thus harnessing the tumor's own production of excess stimulatory ligands to deplete adjacent ECs of the capacity to respond to these signals. Using zebrafish and human endothelial cells in vitro, we show ECs deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased vascular endothelial growth factor (VEGF) stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol-(4,5)-bisphosphate (PIP2), resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling. Using murine tumor allograft models, we show that systemic or EC specific suppression of phosphoinositide recycling results in reduced tumor growth and tumor angiogenesis. Our results suggest inhibition of phosphoinositide recycling provides a useful anti-angiogenic approach.

MicroRNA-mediated control of developmental lymphangiogenesis.

The post-transcriptional mechanisms contributing to molecular regulation of developmental lymphangiogenesis and lymphatic network assembly are not well understood. MicroRNAs are important post-transcriptional regulators during development. Here, we use high throughput small RNA sequencing to identify miR-204, a highly conserved microRNA dramatically enriched in lymphatic vs. blood endothelial cells in human and zebrafish. Suppressing miR-204 leads to loss of lymphatic vessels while endothelial overproduction of miR-204 accelerates lymphatic vessel formation, suggesting a critical positive role for this microRNA during developmental lymphangiogenesis. We also identify the NFATC1 transcription factor as a key miR-204 target in human and zebrafish, and show that NFATC1 suppression leads to lymphatic hyperplasia. The loss of lymphatics caused by miR-204 deficiency can be largely rescued by either endothelial autonomous expression of miR-204 or by suppression of NFATC1. Together, our results highlight a miR-204/NFATC1 molecular regulatory axis required for proper lymphatic development.

An epigenetic mechanism for cavefish eye degeneration.

Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution. However, less is known about the role of epigenetics in this process. Although previous studies have identified eye development genes associated with the loss-of-eyes phenotype in the Pachón blind cave morph of the Mexican tetra Astyanax mexicanus, no inactivating mutations have been found in any of these genes. Here, we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave A. mexicanus. By performing parallel analyses in A. mexicanus cave and surface morphs, and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development. The most significantly hypermethylated and downregulated genes in the cave morph are also linked to human eye disorders, suggesting that the function of these genes is conserved across vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.

Aminoacyl-Transfer RNA Synthetase Deficiency Promotes Angiogenesis via the Unfolded Protein Response Pathway.

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.

Viral pre-challenge increases central nervous system inflammation after intracranial interleukin-1ß injection.

INTRODUCTION: Systemic inflammation has been shown to significantly worsen the outcome of neurological disease. However, after acute injuries to the brain both pre- and post-conditioning with bacterial endotoxin has been shown to reduce leukocyte recruitment to the CNS. Here, we sought to determine whether viral pre-challenge would have an effect on the outcome of acute CNS inflammation that was distinct from endotoxin. METHODS: Animals received a single intracranial microinjection of IL-1ß in the presence or absence of a viral pre-challenge 24 hours prior to surgery. Liver and brain tissue were analysed for chemokine expression by qRT-PCR and leukocyte and monocyte infiltration 12 hours, 3 days and 7 days after the IL-1ß injection. RESULTS: Here, a single injection of adenovirus prior to IL-1ß injection resulted in adhesion molecule expression, chemokine expression and the recruitment of neutrophils to the injured CNS in significantly higher numbers than in IL-1ß injected animals. The distribution and persistence of leukocytes within the CNS was also greater after pre-challenge, with neutrophils being found in both the ipsilateral and contralateral hemispheres. Thus, despite the absence of virus within the CNS, the presence of virus within the periphery was sufficient to exacerbate CNS disease. CONCLUSIONS: These data suggest that the effect of a peripheral inflammatory challenge on the outcome of CNS injury or disease is not generic and will be highly dependent on the nature of the pathogen.

Integrin-linked kinase modulates longevity and thermotolerance in C. elegans through neuronal control of HSF-1.

Integrin-signaling complexes play important roles in cytoskeletal organization and cell adhesion in many species. Components of the integrin-signaling complex have been linked to aging in both Caenorhabditis elegans and Drosophila melanogaster, but the mechanism underlying this function is unknown. Here, we investigated the role of integrin-linked kinase (ILK), a key component of the integrin-signaling complex, in lifespan determination. We report that genetic reduction of ILK in both C. elegans and Drosophila increased resistance to heat stress, and led to lifespan extension in C. elegans without majorly affecting cytoskeletal integrity. In C. elegans, longevity and thermotolerance induced by ILK depletion was mediated by heat-shock factor-1 (HSF-1), a major transcriptional regulator of the heat-shock response (HSR). Reduction in ILK levels increased hsf-1 transcription and activation, and led to enhanced expression of a subset of genes with roles in the HSR. Moreover, induction of HSR-related genes, longevity and thermotolerance caused by ILK reduction required the thermosensory neurons AFD and interneurons AIY, which are known to play a critical role in the canonical HSR. Notably, ILK was expressed in neighboring neurons, but not in AFD or AIY, implying that ILK reduction initiates cell nonautonomous signaling through thermosensory neurons to elicit a noncanonical HSR. Our results thus identify HSF-1 as a novel effector of the organismal response to reduced ILK levels and show that ILK inhibition regulates HSF-1 in a cell nonautonomous fashion to enhance stress resistance and lifespan in C. elegans.

The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans.

Autophagy is a cellular recycling process that has an important anti-aging role, but the underlying molecular mechanism is not well understood. The mammalian transcription factor EB (TFEB) was recently shown to regulate multiple genes in the autophagy process. Here we show that the predicted TFEB orthologue HLH-30 regulates autophagy in Caenorhabditis elegans and, in addition, has a key role in lifespan determination. We demonstrate that hlh-30 is essential for the extended lifespan of Caenorhabditis elegans in six mechanistically distinct longevity models, and overexpression of HLH-30 extends lifespan. Nuclear localization of HLH-30 is increased in all six Caenorhabditis elegans models and, notably, nuclear TFEB levels are augmented in the livers of mice subjected to dietary restriction, a known longevity-extending regimen. Collectively, our results demonstrate a conserved role for HLH-30 and TFEB in autophagy, and possibly longevity, and identify HLH-30 as a uniquely important transcription factor for lifespan modulation in Caenorhabditis elegans.

Liver Kupffer cells control the magnitude of the inflammatory response in the injured brain and spinal cord.

The CNS inflammatory response is regulated by hepatic chemokine synthesis, which promotes leukocytosis and facilitates leukocyte recruitment to the site of injury. To understand the role of the individual cell populations in the liver during the hepatic response to acute brain injury, we selectively depleted Kupffer cells (KC), using clodronate-filled liposomes, and assessed the inflammatory response following a microinjection of IL-1beta into the rat brain or after a compression injury in the spinal cord. We show by immunohistochemistry that KC depletion reduces neutrophil infiltration into the IL-1beta-injected brain by 70% and by 50% into the contusion-injured spinal cord. qRT-PCR analysis of hepatic chemokine mRNA expression showed that chemokine expression in the liver after brain injury is not restricted to a single cell population. In non-depleted rats, CXCL-10, IL-1beta, CCL-2, and MIP-1alpha mRNAs were increased up to sixfold more than in KC depleted rats. However, CXCL-1 and MIP-1beta were not significantly affected by KC depletion. The reduction in chemokine mRNA expression by the liver was not associated with decreased neutrophil mobilisation as might have been expected. These findings suggest that in response to CNS injury, KC mediated mechanisms are responsible for increasing neutrophil entry to the site of CNS injury, but that neutrophil mobilisation is dependent on other non-KC mediated events. However, the suppression of KC activity may prevent secondary damage after acute brain injury.

Immunomodulatory effects of etanercept in a model of brain injury act through attenuation of the acute-phase response.

TNF-alpha has proved to be a successful target in the treatment of many peripheral inflammatory diseases, but the same interventions worsen immune-mediated CNS disease. However, anti-TNF-alpha strategies may offer promise as therapy for non-immune CNS injury. In this study, we have microinjected IL-1beta or lipopolysaccharide (LPS) into the rat brain as a simple model of brain injury and have systemically administered the TNF-alpha antagonist etanercept to discover whether hepatic TNF-alpha, produced as part of the acute-phase response to CNS injury, modulates the inflammatory response in the brain. We report a significant reduction in neutrophil numbers recruited to the IL-1beta- or LPS-challenged brain as a result of TNF-alpha inhibition. We also show an attenuation in the levels of hepatic mRNA including TNF-alpha mRNA and of TNF-alpha-induced genes, such as the chemokines CCL-2, CXCL-5, and CXCL-10, although other chemokines elevated by the injury were not significantly changed. The reduction in hepatic chemokine synthesis results in reduced numbers of circulating neutrophils, and also a reduction in the numbers recruited to the liver as a consequence of brain injury. These findings suggest that TNF-alpha inhibitors may reduce CNS inflammatory responses by targeting the hepatic acute-phase response, and thus therapies for brain injury need not cross the blood-brain barrier to be effective.

Post-conditioning with lipopolysaccharide reduces the inflammatory infiltrate to the injured brain and spinal cord: a potential neuroprotective treatment.

Systemic infection often accompanies or precedes acute brain injury, but it remains unclear how the systemic response contributes to outcome. To examine this problem we have microinjected recombinant interleukin-1beta (IL-1beta), a cytokine associated with acute brain injury, into the rat brain parenchyma and either preceded or followed this challenge with the intravenous injection of lipopolysaccharide (LPS), which mimics systemic inflammatory response syndrome. The microinjection of IL-1beta alone into the brain parenchyma gives rise to leukocyte mobilization in the blood, and to the delayed recruitment of neutrophils and monocytes to the brain with no evidence of blood-brain barrier breakdown or overt neuronal cell death. Systemic LPS pre-conditioning resulted in a dose-dependent reduction both in the number of circulating leukocytes and in the number of leukocytes recruited to the brain parenchyma after 12 h. Surprisingly, LPS given two hours after injury was equally effective in reducing the recruitment of leukocytes to the brain, which is more relevant to the management of clinical disease. In a more clinically relevant model of spinal cord injury, intravenous LPS post-conditioning also reduced the numbers of leukocytes mobilized in the blood and recruited to the spinal cord and thus limited the breakdown of the blood-spinal cord barrier. The effects appear to be specific to LPS, as they were not observed after intravenous IL-1beta pre-conditioning. Our studies suggest that individual pro-inflammatory conditioning strategies may protect the injured central nervous system from the damaging consequences of leukocyte recruitment and may provide scope for novel therapeutic intervention.