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1.  TIE2-expressing monocytes/macrophages regulate revascularization of the ischemic limb 
EMBO Molecular Medicine  2013;5(6):858-869.
A third of patients with critical limb ischemia (CLI) will eventually require limb amputation. Therapeutic neovascularization using unselected mononuclear cells to salvage ischemic limbs has produced modest results. The TIE2-expressing monocytes/macrophages (TEMs) are a myeloid cell subset known to be highly angiogenic in tumours. This study aimed to examine the kinetics of TEMs in patients with CLI and whether these cells promote neovascularization of the ischemic limb. Here we show that there are 10-fold more circulating TEMs in CLI patients, and removal of ischemia reduces their numbers to normal levels. TEM numbers in ischemic muscle are two-fold greater than normoxic muscle from the same patient. TEMs from patients with CLI display greater proangiogenic activity than TIE2-negative monocytes in vitro. Using a mouse model of hindlimb ischemia, lentiviral-based Tie2 knockdown in TEMs impaired recovery from ischemia, whereas delivery of mouse macrophages overexpressing TIE2, or human TEMs isolated from CLI patients, rescued limb ischemia. These data suggest that enhancing TEM recruitment to the ischemic muscle may have the potential to improve limb neovascularization in CLI patients.
PMCID: PMC3779448  PMID: 23653322
angiogenesis; limb ischemia; macrophages; monocytes; TIE2
2.  Optimisation of Embryonic and Larval ECG Measurement in Zebrafish for Quantifying the Effect of QT Prolonging Drugs 
PLoS ONE  2013;8(4):e60552.
Effective chemical compound toxicity screening is of paramount importance for safe cardiac drug development. Using mammals in preliminary screening for detection of cardiac dysfunction by electrocardiography (ECG) is costly and requires a large number of animals. Alternatively, zebrafish embryos can be used as the ECG waveform is similar to mammals, a minimal amount of chemical is necessary for drug testing, while embryos are abundant, inexpensive and represent replacement in animal research with reduced bioethical concerns. We demonstrate here the utility of pre-feeding stage zebrafish larvae in detection of cardiac dysfunction by electrocardiography. We have optimised an ECG recording system by addressing key parameters such as the form of immobilization, recording temperature, electrode positioning and developmental age. Furthermore, analysis of 3 days post fertilization (dpf) zebrafish embryos treated with known QT prolonging drugs such as terfenadine, verapamil and haloperidol led to reproducible detection of QT prolongation as previously shown for adult zebrafish. In addition, calculation of Z-factor scores revealed that the assay was sensitive and specific enough to detect large drug-induced changes in QTc intervals. Thus, the ECG recording system is a useful drug-screening tool to detect alteration to cardiac cycle components and secondary effects such as heart block and arrhythmias in zebrafish larvae before free feeding stage, and thus provides a suitable replacement for mammalian experimentation.
PMCID: PMC3620317  PMID: 23579446
3.  G6f-Like Is an ITAM-Containing Collagen Receptor in Thrombocytes 
PLoS ONE  2012;7(12):e52622.
Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution.
PMCID: PMC3528668  PMID: 23285115
4.  A Systems Biology Approach Identifies Molecular Networks Defining Skeletal Muscle Abnormalities in Chronic Obstructive Pulmonary Disease 
PLoS Computational Biology  2011;7(9):e1002129.
Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory process of the lung inducing persistent airflow limitation. Extensive systemic effects, such as skeletal muscle dysfunction, often characterize these patients and severely limit life expectancy. Despite considerable research efforts, the molecular basis of muscle degeneration in COPD is still a matter of intense debate. In this study, we have applied a network biology approach to model the relationship between muscle molecular and physiological response to training and systemic inflammatory mediators. Our model shows that failure to co-ordinately activate expression of several tissue remodelling and bioenergetics pathways is a specific landmark of COPD diseased muscles. Our findings also suggest that this phenomenon may be linked to an abnormal expression of a number of histone modifiers, which we discovered correlate with oxygen utilization. These observations raised the interesting possibility that cell hypoxia may be a key factor driving skeletal muscle degeneration in COPD patients.
Author Summary
Chronic Obstructive Pulmonary Disease (COPD) is a major life threatening disease of the lungs, characterized by airflow limitation and chronic inflammation. Progressive reduction of the body muscle mass is a condition linked to COPD that significantly decreases quality of life and survival. Physical exercise has been proposed as a therapeutic option but its utility is still a matter of debate. The mechanisms underlying muscle wasting are also still largely unknown. The results presented in this paper show that diseased muscles are largely unable to coordinate the expression of muscle remodelling and bioenergetics pathways and that the cause of this phenomena may be tissue hypoxia. These findings contrast with current hypotheses based on the role of chronic inflammation and show that a mechanism based on an oxygen driven, epigenetic control of these two important functions may be an important disease mechanism.
PMCID: PMC3164707  PMID: 21909251
5.  Angiogenic potential of endothelial progenitor cells and embryonic stem cells 
Vascular Cell  2011;3:11.
Endothelial progenitor cells (EPCs) are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC) tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).
The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.
We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.
PMCID: PMC3108917  PMID: 21569302
6.  Loss of Akt activity increases circulating soluble endoglin release in preeclampsia: identification of inter-dependency between Akt-1 and heme oxygenase-1 
European Heart Journal  2011;33(9):1150-1158.
Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-β (TGF-β) signalling, which is known to be elevated in preeclampsia.
Methods and results
Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng.
The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.
PMCID: PMC3341632  PMID: 21411816
Endothelium; Soluble endoglin; HO-1; PI3K/Akt; HO-1; Preeclampsia
7.  Evolution of muscle phenotype for extreme high altitude flight in the bar-headed goose 
Bar-headed geese migrate over the Himalayas at up to 9000 m elevation, but it is unclear how they sustain the high metabolic rates needed for flight in the severe hypoxia at these altitudes. To better understand the basis for this physiological feat, we compared the flight muscle phenotype of bar-headed geese with that of low altitude birds (barnacle geese, pink-footed geese, greylag geese and mallard ducks). Bar-headed goose muscle had a higher proportion of oxidative fibres. This increased muscle aerobic capacity, because the mitochondrial volume densities of each fibre type were similar between species. However, bar-headed geese had more capillaries per muscle fibre than expected from this increase in aerobic capacity, as well as higher capillary densities and more homogeneous capillary spacing. Their mitochondria were also redistributed towards the subsarcolemma (cell membrane) and adjacent to capillaries. These alterations should improve O2 diffusion capacity from the blood and reduce intracellular O2 diffusion distances, respectively. The unique differences in bar-headed geese were much greater than the minor variation between low altitude species and existed without prior exercise or hypoxia exposure, and the correlation of these traits to flight altitude was independent of phylogeny. In contrast, isolated mitochondria had similar respiratory capacities, O2 kinetics and phosphorylation efficiencies across species. Bar-headed geese have therefore evolved for exercise in hypoxia by enhancing the O2 supply to flight muscle.
PMCID: PMC2817306  PMID: 19640884
oxygen transport cascade; high altitude adaptation; physiological evolution; exercise performance; phylogenetically independent contrasts
8.  The role of vascular myoglobin in nitrite-mediated blood vessel relaxation 
Cardiovascular Research  2010;89(3):560-565.
This work investigates the role of myoglobin in mediating the vascular relaxation induced by nitrite. Nitrite, previously considered an inert by-product of nitric oxide metabolism, is now believed to play an important role in several areas of pharmacology and physiology. Myoglobin can act as a nitrite reductase in the heart, where it is plentiful, but it is present at a far lower level in vascular smooth muscle—indeed, its existence in the vessel wall is controversial. Haem proteins have been postulated to be important in nitrite-induced vasodilation, but the specific role of myoglobin is unknown. The current study was designed to confirm the presence of myoglobin in murine aortic tissue and to test the hypothesis that vascular wall myoglobin is important for nitrite-induced vasodilation.
Methods and results
Aortic rings from wild-type and myoglobin knockout mice were challenged with nitrite, before and after exposure to the haem-protein inhibitor carbon monoxide (CO). CO inhibited vasodilation in wild-type rings but not in myoglobin-deficient rings. Restitution of myoglobin using a genetically modified adenovirus both increased vasodilation to nitrite and reinstated the wild-type pattern of response to CO.
Myoglobin is present in the murine vasculature and contributes significantly to nitrite-induced vasodilation.
PMCID: PMC3028970  PMID: 20889759
Vasodilation; Nitrite; Nitric oxide; Myoglobin
9.  A new procedure for determining the genetic basis of a physiological process in a non-model species, illustrated by cold induced angiogenesis in the carp 
BMC Genomics  2009;10:490.
Physiological processes occur in many species for which there is yet no sequenced genome and for which we would like to identify the genetic basis. For example, some species increase their vascular network to minimise the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures. Since many angiogenic and endothelial genes have been discovered in man, functional homolog relationships between carp, zebrafish and human were used to predict the genetic basis of cold-induced angiogenesis in Cyprinus Carpio (carp). In this work, carp sequences were collected and built into contigs. Human-carp functional homolog relationships were derived via zebrafish using a new Conditional Stepped Reciprocal Best Hit (CSRBH) protocol. Data sources including publications, Gene Ontology and cDNA libraries were then used to predict the identity of known or potential angiogenic genes. Finally, re-analyses of cold carp microarray data identified carp genes up-regulated in response to low temperatures in heart and muscle.
The CSRBH approach outperformed all other methods and attained 8,726 carp to human functional homolog relationships for 16,650 contiguous sequences. This represented 3,762 non-redundant genes and 908 of them were predicted to have a role in angiogenesis. The total number of up-regulated differentially expressed genes was 698 and 171 of them were putatively angiogenic. Of these, 5 genes representing the functional homologs NCL, RHOA, MMP9, GRN and MAPK1 are angiogenesis-related genes expressed in response to low temperature.
We show that CSRBH functional homologs relationships and re-analyses of gene expression data can be combined in a non-model species to predict genes of biological interest before a genome sequence is fully available. Programs to run these analyses locally are available from .
PMCID: PMC2771047  PMID: 19852815
10.  A Novel Role for PECAM-1 (CD31) in Regulating Haematopoietic Progenitor Cell Compartmentalization between the Peripheral Blood and Bone Marrow 
PLoS ONE  2008;3(6):e2338.
Although the expression of PECAM-1 (CD31) on vascular and haematopoietic cells within the bone marrow microenvironment has been recognized for some time, its physiological role within this niche remains unexplored. In this study we show that PECAM-1 influences steady state hematopoietic stem cell (HSC) progenitor numbers in the peripheral blood but not the bone marrow compartment. PECAM-1−/− mice have higher levels of HSC progenitors in the blood compared to their littermate controls. We show that PECAM-1 is required on both progenitors and bone marrow vascular cells in order for efficient transition between the blood and bone marrow to occur. We have identified key roles for PECAM-1 in both the regulation of HSC migration to the chemokine CXCL12, as well as maintaining levels of the matrix degrading enzyme MMP-9 in the bone marrow vascular niche. Using intravital microscopy and adoptive transfer of either wild type (WT) or PECAM-1−/− bone marrow precursors, we demonstrate that the increase in HSC progenitors in the blood is due in part to a reduced ability to migrate from blood to the bone marrow vascular niche. These findings suggest a novel role for PECAM-1 as a regulator of resting homeostatic progenitor cell numbers in the blood
PMCID: PMC2394654  PMID: 18523558
11.  Hibernation in an Antarctic Fish: On Ice for Winter 
PLoS ONE  2008;3(3):e1743.
Active metabolic suppression in anticipation of winter conditions has been demonstrated in species of mammals, birds, reptiles and amphibians, but not fish. This is because the reduction in metabolic rate in fish is directly proportional to the decrease in water temperature and they appear to be incapable of further suppressing their metabolic rate independently of temperature. However, the Antarctic fish (Notothenia coriiceps) is unusual because it undergoes winter metabolic suppression irrespective of water temperature. We assessed the seasonal ecological strategy by monitoring swimming activity, growth, feeding and heart rate (fH) in N. coriiceps as they free-ranged within sub-zero waters. The metabolic rate of wild fish was extrapolated from fH recordings, from oxygen consumption calibrations established in the laboratory prior to fish release. Throughout the summer months N. coriiceps spent a considerable proportion of its time foraging, resulting in a growth rate (Gw) of 0.18±0.2% day−1. In contrast, during winter much of the time was spent sedentary within a refuge and fish showed a net loss in Gw (−0.05±0.05% day−1). Whilst inactive during winter, N. coriiceps displayed a very low fH, reduced sensory and motor capabilities, and standard metabolic rate was one third lower than in summer. In a similar manner to other hibernating species, dormancy was interrupted with periodic arousals. These arousals, which lasted a few hours, occurred every 4–12 days. During arousal activity, fH and metabolism increased to summer levels. This endogenous suppression and activation of metabolic processes, independent of body temperature, demonstrates that N. coriiceps were effectively ‘putting themselves on ice’ during winter months until food resources improved. This study demonstrates that at least some fish species can enter a dormant state similar to hibernation that is not temperature driven and presumably provides seasonal energetic benefits.
PMCID: PMC2254195  PMID: 18320061
12.  Does respiratory sinus arrhythmia occur in fishes? 
Biology Letters  2005;1(4):484-487.
The hypothesis that respiratory modulation of heart rate variability (HRV) or respiratory sinus arrhythmia (RSA) is restricted to mammals was tested on four Antarctic and four sub-Antarctic species of fish, that shared close genotypic or ecotypic similarities but, due to their different environmental temperatures, faced vastly different selection pressures related to oxygen supply. The intrinsic heart rate (fH) for all the fish species studied was ∼25% greater than respiration rate (fV), but vagal activity successively delayed heart beats, producing a resting fH that was synchronized with fV in a progressive manner. Power spectral statistics showed that these episodes of relative bradycardia occurred in a cyclical manner every 2–4 heart beats in temperate species but at >4 heart beats in Antarctic species, indicating a more relaxed selection pressure for cardio-respiratory coupling. This evidence that vagally mediated control of fH operates around the ventilatory cycle in fish demonstrates that influences similar to those controlling RSA in mammals operate in non-mammalian vertebrates.
PMCID: PMC1626384  PMID: 17148239
notothenioids; Antarctic fish; power spectral analysis; cardio-respiratory coupling

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