Therapeutic targeting of vascular malformation in a zebrafish model of hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) causes arteriovenous malformations (AVMs) in multiple organs to cause bleeding, neurological and other complications. HHT is caused by mutations in the BMP co-receptor endoglin. We characterised a range of vascular phenotypes in embryonic and adult endoglin mutant zebrafish and the effect of inhibiting different pathways downstream of Vegf signalling. Adult endoglin mutant zebrafish developed skin AVMs, retinal vascular abnormalities and cardiac enlargement. Embryonic endoglin mutants developed an enlarged basilar artery (similar to the previously described enlarged aorta and cardinal vein) and larger numbers of endothelial membrane cysts (kugeln) on cerebral vessels. Vegf inhibition prevented these embryonic phenotypes, leading us to investigate specific Vegf signalling pathways. Inhibiting mTOR or MEK pathways prevented abnormal trunk and cerebral vasculature phenotypes, whereas inhibiting Nos or Mapk pathways had no effect. Combined subtherapeutic mTOR and MEK inhibition prevented vascular abnormalities, confirming synergy between these pathways in HHT. These results indicate that the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of Vegf signalling. Combined low-dose MEK and mTOR pathway inhibition could represent a novel therapeutic strategy in HHT.

The effect of hyperglycemia on neurovascular coupling and cerebrovascular patterning in zebrafish.

Neurovascular coupling (through which local cerebral blood flow changes in response to neural activation are mediated) is impaired in many diseases including diabetes. Current preclinical rodent models of neurovascular coupling rely on invasive surgery and instrumentation, but transgenic zebrafish coupled with advances in imaging techniques allow non-invasive quantification of cerebrovascular anatomy, neural activation, and cerebral vessel haemodynamics. We therefore established a novel non-invasive, non-anaesthetised zebrafish larval model of neurovascular coupling, in which visual stimulus evokes neuronal activation in the optic tectum that is associated with a specific increase in red blood cell speed in tectal blood vessels. We applied this model to the examination of the effect of glucose exposure on cerebrovascular patterning and neurovascular coupling. We found that chronic exposure of zebrafish to glucose impaired tectal blood vessel patterning and neurovascular coupling. The nitric oxide donor sodium nitroprusside rescued all these adverse effects of glucose exposure on cerebrovascular patterning and function. Our results establish the first non-mammalian model of neurovascular coupling, offering the potential to perform more rapid genetic modifications and high-throughput screening than is currently possible using rodents. Furthermore, using this zebrafish model, we reveal a potential strategy to ameliorate the effects of hyperglycemia on cerebrovascular function.

Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish.

We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

Venous identity requires BMP signalling through ALK3.

Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-ß and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4, with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

INTRODUCTION AND OBJECTIVES: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development. METHODS AND RESULTS: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants. CONCLUSIONS: The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

Macrophages are required for dendritic cell uptake of respiratory syncytial virus from an infected epithelium.

We have previously shown that the respiratory syncytial virus [RSV] can productively infect monocyte derived dendritic cells [MoDC] and remain dormant within the same cells for prolonged periods. It is therefore possible that infected dendritic cells act as a reservoir within the airways of individuals between annual epidemics. In the present study we explored the possibility that sub-epithelial DCs can be infected with RSV from differentiated bronchial epithelium and that in turn RSV from DCs can infect the epithelium. A dual co-culture model was established in which a differentiated primary airway epithelium on an Air Liquid Interface (ALI) was cultured on a transwell insert and MoDCs were subsequently added to the basolateral membrane of the insert. Further experiments were undertaken using a triple co-culture model in which in which macrophages were added to the apical surface of the differentiated epithelium. A modified RSV [rr-RSV] expressing a red fluorescent protein marker of replication was used to infect either the MoDCs or the differentiated epithelium and infection of the reciprocal cell type was assessed using confocal microscopy. Our data shows that primary epithelium became infected when rr-RSV infected MoDCs were introduced onto the basal surface of the transwell insert. MoDCs located beneath the epithelium did not become infected with virus from infected epithelial cells in the dual co-culture model. However when macrophages were present on the apical surface of the primary epithelium infection of the basal MoDCs occurred. Our data suggests that RSV infected dendritic cells readily transmit infection to epithelial cells even when they are located beneath the basal layer. However macrophages appear to be necessary for the transmission of infection from epithelial cells to basal dendritic cells.

Advances in fish vaccine delivery.

Disease prevention is essential to the continued development of aquaculture around the world. Vaccination is the most effective method of combating disease and currently there are a number of vaccines commercially available for use in fish. The majority of aquatic vaccines are delivered by injection, which is by far the most effective method when compared to oral or immersion deliveries. However it is labor intensive, costly and not feasible for large numbers of fish under 20 g. Attempts to develop novel oral and immersion delivery methods have resulted in varying degrees of success but may have great potential for the future.

Improved grading and survival prediction of human astrocytic brain tumors by artificial neural network analysis of gene expression microarray data.

Histopathologic grading of astrocytic tumors based on current WHO criteria offers a valuable but simplified representation of oncologic reality and is often insufficient to predict clinical outcome. In this study, we report a new astrocytic tumor microarray gene expression data set (n = 65). We have used a simple artificial neural network algorithm to address grading of human astrocytic tumors, derive specific transcriptional signatures from histopathologic subtypes of astrocytic tumors, and asses whether these molecular signatures define survival prognostic subclasses. Fifty-nine classifier genes were identified and found to fall within three distinct functional classes, that is, angiogenesis, cell differentiation, and lower-grade astrocytic tumor discrimination. These gene classes were found to characterize three molecular tumor subtypes denoted ANGIO, INTER, and LOWER. Grading of samples using these subtypes agreed with prior histopathologic grading for both our data set (96.15%) and an independent data set. Six tumors were particularly challenging to diagnose histopathologically. We present an artificial neural network grading for these samples and offer an evidence-based interpretation of grading results using clinical metadata to substantiate findings. The prognostic value of the three identified tumor subtypes was found to outperform histopathologic grading as well as tumor subtypes reported in other studies, indicating a high survival prognostic potential for the 59 gene classifiers. Finally, 11 gene classifiers that differentiate between primary and secondary glioblastomas were also identified.

Genomic analysis of pilocytic astrocytomas at 0.97 Mb resolution shows an increasing tendency toward chromosomal copy number change with age.

Brain tumors are the most common solid tumors of childhood, accounting for over 20% of cancers in children under 15 years of age. Pilocytic astrocytomas (PAs), World Health Organization grade I, are one of the most frequently occurring childhood brain tumors, yet little is known about genetic changes characterizing this entity. We have used microarray comparative genomic hybridization at 0.97 Mb resolution to study a series of PAs (n = 44). No copy number abnormality was seen in 64% of cases at this resolution. However, whole chromosomal gain (median 5 chromosomes affected) occurred in 32% of tumors. The most frequently affected chromosomes were 5 and 7 (11 of 44 cases each) followed by 6, 11, 15, and 20 (greater than 10% of cases each). Findings were confirmed by fluorescence in situ hybridization and microsatellite analysis in a subset of tumors. Chromosomal gain was significantly more frequent in PAs from patients over 15 years old (p = 0.03, Fisher exact test). The number of chromosomes involved was also significantly greater in the older group (p = 0.02, Mann-Whitney U test). One case (2%) showed a region of gain on chromosome 3 and one (2%) a deletion on 6q as their sole abnormalities. This is the first genomewide study to show this nonrandom pattern of genetic alteration in pilocytic astrocytomas.

High-resolution array-based comparative genomic hybridization of medulloblastomas and supratentorial primitive neuroectodermal tumors.

Medulloblastomas and supratentorial primitive neuroectodermal tumors are aggressive childhood tumors. We report our findings using array comparative genomic hybridization (CGH) on a whole-genome BAC/PAC/cosmid array with a median clone separation of 0.97 Mb to study 34 medulloblastomas and 7 supratentorial primitive neuroectodermal tumors. Array CGH allowed identification and mapping of numerous novel, small regions of copy number change to genomic sequence in addition to the large regions already known from previous studies. Novel amplifications were identified, some encompassing oncogenes MYCL1, PDGFRA, KIT, and MYB not previously reported to show amplification in these tumors. In addition, one supratentorial primitive neuroectodermal tumor had lost both copies of the tumor-suppressor genes CDKN2A and CDKN2B. Ten medulloblastomas had findings suggestive of isochromosome 17q. In contrast to previous reports using conventional CGH, array CGH identified 3 distinct breakpoints in these cases: Ch 17: 17940393-19251679 (17p11.2, n = 6), Ch 17: 20111990-23308272 (17p11.2-17q11.2, n = 4), and Ch 17: 38425359-39091575 (17q21.31, n = 1). Significant differences were found in the patterns of copy number change between medulloblastomas and supratentorial primitive neuroectodermal tumors, providing further evidence that these tumors are genetically distinct despite their morphologic and behavioral similarities.