The role of nitric oxide in myocardial ischaemia/reperfusion injury

The management of acute myocardial infarction requires rapid restoration of blood flow to attenuate cell death. In recent years, the possibility of fortifying the myocardium against ischaemia/reperfusion injury has emerged with the identification of signalling pathways that promote cellular survival, modalities that include preconditioning and ischaemia/reperfusion injury salvage (IRIS). Preconditioning and IRIS are defined as the protection triggered by a stimulus either before the insult or upon reperfusion respectively. Nitric oxide has been implicated as a potential mediator of cell survival and cell death; whether the cell lives or dies may be critically dependent upon the synthesis of this second messenger. Therefore to determine the role of nitric oxide in the mediation/ protection from ischaemia/reperfusion injury, the aim of this thesis is to elucidate the role of nitric oxide in preconditioning and IRIS paradigms. In early ischaemic preconditioning, an immediate onset phase of protection after the preconditioning stimulus, nitric oxide was found to lower the preconditioning threshold significantly; in eNOS knockout animals, the ischaemic preconditioning threshold is at least twice that in eNOS wild type animals. Delayed pharmacological preconditioning, whereby transient adenosine A1 receptor activation with 2-chloro N6 cyclopentyl adenosine results in robust protection 24 hours later, was found to be dependent upon the synthesis of nitric oxide. Interestingly, whilst previous studies indicated that delayed ischaemic preconditioning was dependent upon the induction of the inducible isoform of nitric oxide synthase, this study implicates the endothelial nitric oxide synthase (eNOS) as having an important role in mediating the protection observed. IRIS is a novel form of myocardial protection that we have hypothesised is dependent upon the activation of a reperfusion injury salvage kinase (RISK) pathway to attenuate necrotic injury. To activate the RISK pathway we used bradykinin, which was found to trigger protection that was reliant upon the activity of eNOS. Exogenous nitric oxide was administered to hearts subjected to ischaemia/reperfusion injury. In this study, a clear dose-response relationship was found between the concentration of nitric oxide and myocardial protection that peaks at 2 μM. At higher concentrations, protection against ischaemic injury was lost. The protection mediated by nitric oxide was found to be closely linked to the opening of the mitochondrial ATP sensitive potassium channel (KATP), as indicated by studies in isolated mitochondria and in the whole heart subjected to ischaemia/reperfusion injury. Thus, this thesis provides evidence that (i) nitric oxide is involved in all facets of myocardial resistance to injury, (ii) eNOS is a far more important source of nitric oxide than previously thought, and (iii) nitric oxide has a concentration dependent influence upon infarct size, possibly via a direct action upon mitochondrial KATP channels

Scanning precession electron tomography for three-dimensional nanoscale orientation imaging and crystallographic analysis.

Three-dimensional (3D) reconstructions from electron tomography provide important morphological, compositional, optical and electro-magnetic information across a wide range of materials and devices. Precession electron diffraction, in combination with scanning transmission electron microscopy, can be used to elucidate the local orientation of crystalline materials. Here we show, using the example of a Ni-base superalloy, that combining these techniques and extending them to three dimensions, to produce scanning precession electron tomography, enables the 3D orientation of nanoscale sub-volumes to be determined and provides a one-to-one correspondence between 3D real space and 3D reciprocal space for almost any polycrystalline or multi-phase material.A.S.E. and P.A.M acknowledge financial support from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 291522-3DIMAGE, the Seventh Framework Programme of the European Commission: ESTEEM2, contract number 312483, EPSRC grant number EP/H017712/1 and the Royal Society. R.K. acknowledges financial support from Rolls-Royce, EPSRC and the BMWi under EP/H022309/1, EP/H500375/1 and grant number 20T0813. We are grateful to Professor Edgar Rauch for valuable discussion on the use of the Astar system, to Dr Cathie Rae and Dr Mark Hardy of Rolls-Royce for supply of the superalloy samples and valuable discussion about their microstructure, Dr Zineb Saghi for help with the tomographic reconstructions and Dr Francisco de la Peña for help with the NMF decompositions.This is the final version. It was first published by NPG at http://www.nature.com/ncomms/2015/150601/ncomms8267/full/ncomms8267.html

17β‐estradiol ameliorates age‐associated loss of fibroblast function by attenuating IFN‐γ/STAT1‐dependent miR‐7 upregulation

Age-related defects in fibroblast differentiation and functionality were previously shown to be associated with impaired hyaluronan (HA) synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, as a result of upregulated microRNA-7 (miR-7) expression. In aging fibroblasts, inhibiting miR-7 prevented the dysregulation of the HA-mediated CD44/EGFR signaling pathway. Here, we investigated transcriptional upregulation of miR-7 and implicated the age-associated over-activation of JAK/STAT1 as a primary candidate. STAT1 binding sites were identified on the putative miR-7 promoter and stimulation of fibroblasts with the inflammatory cytokine, interferon-{gamma} (IFN-{gamma}), significantly increased miR-7 transcriptional activity and resulted in upregulated miR-7 and loss of EGFR. Additionally, we demonstrated arole for the anti-inflammatory steroid, 17b-estradiol (E2), in the attenuation of miR-7 expression. E2 stimulation promoted estrogen receptor (ER) interactions with the miR-7 putative promoter and suppressed miR-7 expression. E2 also attenuated STAT1expression and activity. Furthermore, treatments with E2 restored fibroblast functionality, including proliferation, migration and differentiation, key events in effective wound healing. In light of our findings, we propose that the regulation of miR-7 by pro- and anti-inflammatory mediators plays a wider role than previously thought. The modulation of fibroblast functions and ultimately wound healing by miR-7 activators or inhibitors could provide realistic targets for the restoration of chronic wound healing capabilities in the elderly

Tumour necrosis factor-stimulated gene (TSG)-6-mediated interactions with the inter-alpha-inhibitor heavy chain 5 facilitate TGF beta1-dependent fibroblast to myofibroblast differentiation.

Fibroblasts are central to wound healing and fibrosis through Transforming Growth Factor-beta (TGF-beta1)-triggered differentiation into contractile, alpha_smooth muscle actin (alpha_sma)-positive myofibroblasts. This is mediated by accumulation of a pericellular matrix of hyaluronan (HA) and the HA-dependent co-localisation of CD44 with the Epidermal Growth Factor Receptor (EGFR). Interactions of HA with hyaladherins, such as Inter-alpha-Inhibitor (IalphaI) and Tumour Necrosis Factor-stimulated gene_6 (TSG_6) are also essential for differentiation. This study investigated the mechanisms involved. TSG6 and alpha_sma had different kinetics of induction by TGFbeta1, with TSG6 peaking before alphasma. siCD44 or EGFR inhibition prevented differentiation but had no effect on TSG-6 expression. TSG-6 was essential for differentiation, and mAb A38 (preventing IalphaI heavy chain [HC] transfer), HA-oligosaccharides, Cobalt, or siBikunin prevented TSG-6 activity, preventing differentiation. A38 also prevented the EGFR/CD44 association. This suggested that TSG6/IalphaI HC interaction was necessary for the effect of TSG-6 and that HC-stabilisation of HA initiated the CD44/EGF-R association. The newly-described HC5 was shown to be the principal HC expressed and its cell surface expression was prevented by siRNA inhibition of TSG6 or Bikunin. HC5 was released by hyaluronidase treatment, confirming its association with cell surface HA. Finally, HC5 knock down by siRNA confirmed its role in myofibroblast differentiation. The current study describes a novel mechanism linking the TSG-6 transfer of the newly-described HC5 to the HA-dependent control of cell phenotype. The interaction of HC5 with cell surface HA was essential for TGFbeta1-dependent differentiation of fibroblasts to myofibroblasts, highlighting its importance as a novel potential therapeutic target

Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype

The cell surface protein CD44 is involved in diverse physiological processes, and its aberrant function is linked to various pathologies such as cancer, immune dysregulation, and fibrosis. The diversity of CD44 biological activity is partly conferred by the generation of distinct CD44 isoforms through alternative splicing. We identified an unexpected function for the ubiquitous hyaluronan-degrading enzyme, hyaluronidase 2 (HYAL2), as a regulator of CD44 splicing. Standard CD44 is associated with fibrotic disease, and its production is promoted through serine-arginine–rich (SR) protein–mediated exon exclusion. HYAL2 nuclear translocation was stimulated by bone morphogenetic protein 7, which inhibits the myofibroblast phenotype. Nuclear HYAL2 displaced SR proteins from the spliceosome, thus enabling HYAL2, spliceosome components (U1 and U2 small nuclear ribonucleoproteins), and CD44 pre-mRNA to form a complex. This prevented double-exon splicing and facilitated the inclusion of CD44 exons 11 and 12, which promoted the accumulation of the antifibrotic CD44 isoform CD44v7/8 at the cell surface. These data demonstrate previously undescribed mechanisms regulating CD44 alternative splicing events that are relevant to the regulation of cellular phenotypes in progressive fibrosis

Hyaluronidase-2 regulates RhoA signalling, myofibroblast contractility and other key pro-fibrotic myofibroblast functions

Hyaluronidase-2 (HYAL2) is a weak, acid-active hyaluronan-degrading enzyme that is broadly expressed in somatic tissues. Aberrant HYAL2 expression is implicated in diverse pathology. However, a significant proportion of HYAL2 is enzymatically inactive, thus the mechanisms through which HYAL2 dysregulation influences pathobiology is unclear. Recently, non-enzymatic HYAL2 functions have been described and our group has shown that nuclear HYAL2 can influence mRNA splicing to prevent myofibroblast differentiation. Myofibroblasts drive fibrosis, thereby promoting progressive tissue damage and leading to multimorbidity. This study identifies a novel HYAL2 cytoplasmic function in myofibroblasts that is unrelated to its enzymatic activity. In fibroblasts and myofibroblasts HYAL2 interacts with the small GTPase signaling molecule, RhoA. Transforming Growth Factor (TGF)-β1-driven fibroblast-to-myofibroblast differentiation promotes HYAL2 cytoplasmic re-localization to bind to the actin cytoskeleton. Cytoskeletal-bound HYAL2 functions as a key regulator of downstream RhoA signaling and influences pro-fibrotic myofibroblast functions including myosin light-chain kinase (MLCK) mediated myofibroblast contractility, myofibroblast migration, myofibroblast collagen/fibronectin deposition, as well as connective tissue growth factor (CTGF/CCN2) and matrix metalloproteinase-2 (MMP2) expression. These data demonstrate that in certain biological contexts the non-enzymatic effects of HYAL2 are critical in orchestrating RhoA signaling and downstream pathways that are important for full pro-fibrotic myofibroblast functionality. In conjunction with previous data demonstrating the influence of HYAL2 on RNA splicing, these findings begin to explain the broad biological effects of HYAL2

Improving estimates of growth for pearl perch (Glaucosoma scapulare) in Queensland, Australia

The pearl perch (Glaucosoma scapulare) is endemic to the east coast of Australia in depths to 150 m. The species has a long history of exploitation, and the stock is currently depleted. Previous research indicated the species is long lived and slow growing based on fishery-dependent sampling undertaken in the late 1990s and early 2000s on traditional fishing grounds at the southern end of the species’ range. Increasing fishing power has facilitated the expansion of the fishery to areas to the north and east of traditional grounds, which has resulted in the appearance of older fish (>10 yr) in fishery-dependent samples not previously observed. The current study estimated the growth parameters using 1153 length-at-age observations from fish collected in Queensland between January 2020 and December 2021. The lack of significant numbers of individuals at either end of the age frequency distribution necessitated the estimation of growth in a Bayesian framework with informative priors for length-at-age-zero and maximum length using a multi-model approach. The von Bertalanffy growth function (VBGF) was found to best fit the observed length-at-age data and the estimated VBGF parameters were L∞ = 562 mm FL, L0 = 2.02 mm FL and k = 0.295 yr−1. The high proportion of older fish in samples, combined with prior information on relevant parameters, improves growth parameter estimation by reducing bias and facilitating improved model fits to observed length-at-age data

Improving estimates of growth for pearl perch (Glaucosoma scapulare) in Queensland, Australia

Abstract The pearl perch (Glaucosoma scapulare) is endemic to the east coast of Australia in depths to 150 m. The species has a long history of exploitation, and the stock is currently depleted. Previous research indicated the species is long lived and slow growing based on fishery-dependent sampling undertaken in the late 1990s and early 2000s on traditional fishing grounds at the southern end of the species’ range. Increasing fishing power has facilitated the expansion of the fishery to areas to the north and east of traditional grounds, which has resulted in the appearance of older fish (>10 yr) in fishery-dependent samples not previously observed. The current study estimated the growth parameters using 1153 length-at-age observations from fish collected in Queensland between January 2020 and December 2021. The lack of significant numbers of individuals at either end of the age frequency distribution necessitated the estimation of growth in a Bayesian framework with informative priors for length-at-age-zero and maximum length using a multi-model approach. The von Bertalanffy growth function (VBGF) was found to best fit the observed length-at-age data and the estimated VBGF parameters were L∞ = 562 mm FL, L0 = 2.02 mm FL and k = 0.295 yr−1. The high proportion of older fish in samples, combined with prior information on relevant parameters, improves growth parameter estimation by reducing bias and facilitating improved model fits to observed length-at-age data

Geometric reconstruction methods for electron tomography

Electron tomography is becoming an increasingly important tool in materials science for studying the three-dimensional morphologies and chemical compositions of nanostructures. The image quality obtained by many current algorithms is seriously affected by the problems of missing wedge artefacts and nonlinear projection intensities due to diffraction effects. The former refers to the fact that data cannot be acquired over the full $180^\circ$ tilt range; the latter implies that for some orientations, crystalline structures can show strong contrast changes. To overcome these problems we introduce and discuss several algorithms from the mathematical fields of geometric and discrete tomography. The algorithms incorporate geometric prior knowledge (mainly convexity and homogeneity), which also in principle considerably reduces the number of tilt angles required. Results are discussed for the reconstruction of an InAs nanowire

MicroRNA-7 inhibition rescues age-associated loss of epidermal growth factor receptor and hyaluronan-dependent differentiation in fibroblasts

Age-related defects in fibroblast differentiation were previously shown to be associated with impaired hyaluronan synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, with both required for normal fibroblast functionality. In fibroblasts, transforming growth factor-beta 1 (TGF-β1)-dependent phenotypic activation uses two distinct but co-operating pathways that involve TGF-β receptor (TGF-βR)/Smad2 activation and HA-mediated CD44-EGFR co-localization and signalling through extracellular signal-regulated kinase 1/2 (ERK1/2). The HA-mediated CD44-EGFR pathway was found to be compromised with in vitro aging, through loss of EGFR expression and a reduced movement of CD44 throughout the cellular membrane. Here, we also investigate the involvement of microRNAs (miRNAs) in age-related loss of differentiation, through investigation of miRNA-7 (miR-7) regulation of the HA-mediated EGFR-signalling pathway. The transcription of miR-7 was found to be upregulated in aged cells. In young cells, age-related loss of differentiation could be mimicked through transfection of pre-miR-7, and in aged cells, could be reversed through transfection of locked nucleic acids (LNA) targeting miR-7. Additionally, miR-7 was found to be involved in the regulation of CD44 membrane motility, which was downregulated in instances of miR-7 upregulation, and partially restorable through either miR-7 inhibition or HAS2 overexpression. The altered dynamics of CD44 in the cell membrane demonstrated a further action of miR-7 in regulating the HA-dependent CD44/EGFR pathway. We explain this novel mechanism of age-associated functional consequence due to miR-7 upregulation and demonstrate that it is reversible; highlighting miR-7 as a potential target for restoring the healing capabilities in chronic wounds in the elderly