Timothy J. A. Chico

Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish

The zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186 , in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.

Modeling Cardiovascular Disease in the Zebrafish

The zebrafish possesses a host of advantages that have established it as an excellent model of vertebrate development. These include ease of genetic manipulation and transgenesis, optical clarity, and small size and cost. Biomedical researchers are increasingly exploiting these advantages to model human disease mechanisms. Here we review the use of the zebrafish for cardiovascular research. We summarize previous studies with the use of this organism to model such processes as thrombosis, collateral vessel development, inflammation, cardiomyopathy, and cardiac regeneration, evaluate its promise for novel drug discovery, and consider where the zebrafish fits into the framework of existing cardiovascular models.

Ischemia is not required for arteriogenesis in zebrafish embryos.

Objective— The role of ischemia in collateral vessel development (arteriogenesis) is a contentious issue that cannot be addressed using mammalian models. To investigate this, we developed models of arteriogenesis using the zebrafish embryo, which gains sufficient oxygenation via diffusion to prevent ischemia in response to arterial occlusion. Methods and Results— We studied gridlock mutant embryos that suffer a permanently occluded aorta and show that these restore aortic blood flow by collateral vessels. We phenocopied gridlock mutants by laser-induced proximal aortic occlusion in transgenic Fli1:eGFP/GATA1:dsRED embryos. Serial imaging showed these restore aortic blood flow via collateral vessels by recruitment of preexisting endothelium in a manner similar to gridlocks. Collateral aortic blood flow in gridlock mutants was dependent on both nitric oxide and myeloid cells. Confocal microscopy of transgenic gridlock/Fli1:eGFP mutants demonstrated no aberrant angiogenic response to the aortic occlusion. qPCR of HIF1&agr; expression confirmed the absence of hypoxia in this model system. Conclusions— We conclude that NO and myeloid cell-dependent collateral vessel development is an evolutionarily ancient response to arterial occlusion and is able to proceed in the absence of ischemia.

The Role of the Transcription Factor KLF2 in Vascular Development and Disease

The zinc-finger transcription factor KLF2 transduces the physical forces exerted by blood flow into molecular signals responsible for a wide range of biological responses. Following its initial recognition as a flow-responsive endothelial transcription factor, KLF2 is now known to be expressed in a range of cell types and to participate in a number of processes during development and disease such as endothelial homeostasis, vasoregulation, vascular growth/remodeling, and inflammation. In this review, we summarize the current understanding about KLF2 with a focus on its effects on vascular biology.

Blood flow suppresses vascular Notch signalling via dll4 and is required for angiogenesis in response to hypoxic signalling

Aims The contribution of blood flow to angiogenesis is incompletely understood. We examined the effect of blood flow on Notch signalling in the vasculature of zebrafish embryos, and whether blood flow regulates angiogenesis in zebrafish with constitutively up-regulated hypoxic signalling. Methods and results Developing zebrafish (Danio rerio) embryos survive via diffusion in the absence of circulation induced by knockdown of cardiac troponin T2 or chemical cardiac cessation. The absence of blood flow increased vascular Notch signalling in 48 h post-fertilization old embryos via up-regulation of the Notch ligand dll4. Despite this, patterning of the intersegmental vessels is not affected by absent blood flow. We therefore examined homozygous vhl mutant zebrafish that have constitutively up-regulated hypoxic signalling. These display excessive and aberrant angiogenesis from 72 h post-fertilization, with significantly increased endothelial number, vessel diameter, and length. The absence of blood flow abolished these effects, though normal vessel patterning was preserved. Conclusion We show that blood flow suppresses vascular Notch signalling via down-regulation of dll4. We have also shown that blood flow is required for angiogenesis in response to hypoxic signalling but is not required for normal vessel patterning. These data indicate important differences in hypoxia-driven vs. developmental angiogenesis.

Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response

ABSTRACT Interleukin-1 (IL-1), the ‘gatekeeper’ of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo.

Microarray profiling reveals CXCR4a is downregulated by blood flow in vivo and mediates collateral formation in zebrafish embryos

The response to hemodynamic force is implicated in a number of pathologies including collateral vessel development. However, the transcriptional effect of hemodynamic force is extremely challenging to examine in vivo in mammals without also detecting confounding processes such as hypoxia and ischemia. We therefore serially examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since they obtain sufficient oxygenation by diffusion. Morpholino antisense knock-down of cardiac troponin T2 (tnnt2) prevented cardiac contraction without affecting vascular development. Gene expression in whole embryo RNA from tnnt2 or control morphants at 36, 48, and 60 h postfertilization (hpf) was assessed using Affymetrix GeneChip Zebrafish Genome Arrays (>14,900 transcripts). We identified 308 differentially expressed genes between tnnt2 and control morphants. One such (CXCR4a) was significantly more highly expressed in tnnt2 morphants at 48 and 60 hpf than controls. In situ hybridization localized CXCR4a upregulation to endothelium of both tnnt2 morphants and gridlock mutants (which have an occluded aorta preventing distal blood flow). This upregulation appears to be of functional significance as either CXCR4a knock-down or pharmacologic inhibition impaired the ability of gridlock mutants to recover blood flow via collateral vessels. We conclude absence of hemodynamic force induces endothelial CXCR4a upregulation that promotes recovery of blood flow.

Effect of Selective or Combined Inhibition of Integrins αIIbβ3 and αvβ3 on Thrombosis and Neointima After Oversized Porcine Coronary Angioplasty

Background—Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin αIIbβ3 and integrin αvβ3 in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective αIIbβ3 antagonist (lamifiban), a selective αvβ3 antagonist (VO514), and a combined αIIbβ3/αvβ3 antagonist (G3580). Methods and Results—In vitro, both αvβ3 inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective αIIbβ3 inhibition had no effect. Intravenous infusions of either αIIbβ3 inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective αvβ3 inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were adm...

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 (klf2a sh317) 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 klf2a sh317 mutants. Homozygous klf2a sh317 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 klf2a sh317 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 klf2a sh317 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 klf2a sh317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2a sh317 mutants. Conclusions The klf2a sh317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

Dietary Phosphate Modulates Atherogenesis and Insulin Resistance in Apolipoprotein E Knockout Mice—Brief Report

Objective— Epidemiological studies link higher serum phosphate and the phosphatonin fibroblast growth factor 23 with cardiovascular events and atheroma, and they link lower serum phosphate with insulin resistance and the metabolic syndrome. We investigated whether manipulating dietary phosphate influences atherogenesis or insulin sensitivity in mice. Methods and Results— Apolipoprotein E knockout mice were fed an atherogenic diet with low (0.2%), standard (0.6%), or high (1.6%) phosphate content. Serum phosphate and fibroblast growth factor 23 significantly increased with increasing dietary phosphate intake, but lipid profile and blood pressure were unaffected. After 20 weeks, mice on the higher phosphate diet had significantly more atheroma at the aortic sinus (42±1.9% versus 30±1.5% for high versus low phosphate, P<0.01). Compared with standard and high-phosphate diet groups, mice on a low-phosphate diet had more adipose tissue and a 4-fold increase in insulin resistance measured by homeostatic model assessment (43.7±9.3 versus 8.9±0.7 for low versus high phosphate, P<0.005). Conclusion— A high-phosphate diet accelerates atherogenesis in apolipoprotein E−/− mice, whereas low phosphate intake induces insulin resistance. These data indicate for the first time that controlling dietary phosphate intake may influence development of both atherosclerosis and the metabolic syndrome.