Publications by authors named "Selina McHarg"

12 Publications

  • Page 1 of 1

Increased circulating levels of Factor H-Related Protein 4 are strongly associated with age-related macular degeneration.

Nat Commun 2020 02 7;11(1):778. Epub 2020 Feb 7.

Division of Evolution and Genomic Sciences, Faculty of Biology Medicine and Health, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value = 7.1 × 10), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value = 2.2 × 10), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-14499-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005798PMC
February 2020

C-reactive protein and pentraxin-3 binding of factor H-like protein 1 differs from complement factor H: implications for retinal inflammation.

Sci Rep 2018 01 26;8(1):1643. Epub 2018 Jan 26.

Division of Evolution and Genomic Science, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

Retinal inflammation plays a key role in the progression of age-related macular degeneration (AMD), a condition that leads to loss of central vision. The deposition of the acute phase pentraxin C-reactive protein (CRP) in the macula activates the complement system, thereby contributing to dysregulated inflammation. The complement protein factor H (FH) can bind CRP and down-regulate an inflammatory response. However, it is not known whether a truncated form of FH, called factor H-like protein 1 (FHL-1), which plays a significant regulatory role in the eye, also interacts with CRP. Here, we compare the binding properties of FHL-1 and FH to both CRP and the related protein pentraxin-3 (PTX3). We find that, unlike FH, FHL-1 can bind pro-inflammatory monomeric CRP (mCRP) as well as the circulating pentameric form. Furthermore, the four-amino acid C-terminal tail of FHL-1 (not present in FH) plays a role in mediating its binding to mCRP. PTX3 was found to be present in the macula of donor eyes and the AMD-associated Y402H polymorphism altered the binding of FHL-1 to PTX3. Our findings reveal that the binding characteristics of FHL-1 differ from those of FH, likely underpinning independent immune regulatory functions in the context of the human retina.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-18395-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786067PMC
January 2018

Bruch's Membrane Compartmentalizes Complement Regulation in the Eye with Implications for Therapeutic Design in Age-Related Macular Degeneration.

Front Immunol 2017 19;8:1778. Epub 2017 Dec 19.

Division of Evolution and Genomic Medicine, Faculty of Biology Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, United Kingdom.

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world and affects nearly 200 million people globally. Local inflammation driven by complement system dysregulation is currently a therapeutic target. Bruch's membrane (BrM) is a sheet of extracellular matrix that separates the retina from the underlying choroid, a highly vascularized layer that supplies oxygen and nutrition to the outer retina. Here, we show that most complement proteins are unable to diffuse through BrM, although FHL-1, factor D and C5a can. AMD-associated lipid deposition in BrM decreases FHL-1 diffusion. We show that this impermeability of BrM creates two separate semi-independent compartments with respect to complement activation and regulation. Complement proteins synthesized locally on either side of BrM, or on the choroidal side if derived from the circulation, predominantly remain on their side of origin. As previous studies suggest that complement activation in AMD is confined to the choroidal side of BrM, we propose a model whereby complement activation in the choriocapillaris layer of the choroid generates C5a, which crosses BrM to interact with its specific receptor on RPE cells to initiate an inflammatory response in the retina. Understanding mechanisms underpinning AMD is essential for developing therapeutics that target the right molecule in the right anatomical compartment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.01778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742201PMC
December 2017

Elevation of brain glucose and polyol-pathway intermediates with accompanying brain-copper deficiency in patients with Alzheimer's disease: metabolic basis for dementia.

Sci Rep 2016 06 9;6:27524. Epub 2016 Jun 9.

School of Biological Sciences, and Maurice Wilkins Centre for Molecular Biodiscovery, Faculty of Science, University of Auckland, New Zealand.

Impairment of brain-glucose uptake and brain-copper regulation occurs in Alzheimer's disease (AD). Here we sought to further elucidate the processes that cause neurodegeneration in AD by measuring levels of metabolites and metals in brain regions that undergo different degrees of damage. We employed mass spectrometry (MS) to measure metabolites and metals in seven post-mortem brain regions of nine AD patients and nine controls, and plasma-glucose and plasma-copper levels in an ante-mortem case-control study. Glucose, sorbitol and fructose were markedly elevated in all AD brain regions, whereas copper was correspondingly deficient throughout (all P < 0.0001). In the ante-mortem case-control study, by contrast, plasma-glucose and plasma-copper levels did not differ between patients and controls. There were pervasive defects in regulation of glucose and copper in AD brain but no evidence for corresponding systemic abnormalities in plasma. Elevation of brain glucose and deficient brain copper potentially contribute to the pathogenesis of neurodegeneration in AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep27524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899713PMC
June 2016

Enrichment of Bruch's Membrane from Human Donor Eyes.

J Vis Exp 2015 Nov 15(105). Epub 2015 Nov 15.

Centre for Ophthalmology & Vision Sciences, Institute of Human Development, University of Manchester; Centre for Advanced Discovery and Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre;

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch's membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch's membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch's membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch's membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch's membrane biochemistry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/53382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692728PMC
November 2015

Age-related macular degeneration and the role of the complement system.

Mol Immunol 2015 Sep 21;67(1):43-50. Epub 2015 Mar 21.

Centre for Ophthalmology & Vision Sciences, Institute of Human Development, University of Manchester, Manchester, UK; Centre for Advanced Discovery & Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK; Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK. Electronic address:

Age-related macular degeneration (AMD) is a leading cause of visual impairment. It is characterised by damage to a tissue complex composed of the retinal pigment epithelium, Bruch's membrane and choriocapillaris. In early AMD extracellular debris including drusen accumulates in Bruch's membrane and then in late AMD geographic atrophy and/or neovascularisation develop. Variants in genes encoding components of the alternative pathway of the complement cascade have a major influence on AMD risk, especially at the RCA locus on chromosome 1, which contains CFH and the CFHR genes. Immunohistochemical studies have demonstrated complement components in unaffected and AMD macular tissue. Whilst other factors, including oxidative stress, play important roles in AMD pathogenesis, evidence for the central role played by complement dysregulation is discussed in this review.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molimm.2015.02.032DOI Listing
September 2015

Deficient copper concentrations in dried-defatted hepatic tissue from ob/ob mice: A potential model for study of defective copper regulation in metabolic liver disease.

Biochem Biophys Res Commun 2015 May 20;460(3):549-54. Epub 2015 Mar 20.

Centre for Advanced Discovery and Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust (CMFT), Manchester M13 9WL, United Kingdom; Centre for Endocrinology & Diabetes, Institute of Human Development, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom; School of Biological Sciences, Faculty of Science, and Department of Medicine, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand; Department of Pharmacology, Medical Sciences Division, University of Oxford, Oxford OX1 3QT, United Kingdom. Electronic address:

Ob/ob mice provide an animal model for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) in patients with obesity and type-2 diabetes. Low liver copper has been linked to hepatic lipid build-up (steatosis) in animals with systemic copper deficiency caused by low-copper diets. However, hepatic copper status in patients with NAFLD or NASH is uncertain, and a validated animal model useful for the study of hepatic copper regulation in common forms of metabolic liver disease is lacking. Here, we report parallel measurements of essential metal levels in whole-liver tissue and defatted-dried liver tissue from ob/ob and non-obese control mice. Measurements in whole-liver tissue from ob/ob mice at an age when they have developed NAFLD/NASH, provide compelling evidence for factitious lowering of copper and all other essential metals by steatosis, and so cannot be used to study hepatic metal regulation in this model. By marked contrast, metal measurements in defatted-dried liver samples reveal that most essential metals were actually normal and indicate specific lowering of copper in ob/ob mice, consistent with hepatic copper deficiency. Thus ob/ob mice can provide a model useful for the study of copper regulation in NAFLD and NASH, provided levels are measured in defatted-dried liver tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2015.03.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427106PMC
May 2015

Down-regulation of desmosomes in cultured cells: the roles of PKC, microtubules and lysosomal/proteasomal degradation.

PLoS One 2014 7;9(10):e108570. Epub 2014 Oct 7.

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108570PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188543PMC
June 2015

Diabetic cardiomyopathy is associated with defective myocellular copper regulation and both defects are rectified by divalent copper chelation.

Cardiovasc Diabetol 2014 Jun 14;13:100. Epub 2014 Jun 14.

The School of Biological Sciences, Faculty of Science, University of Auckland, Auckland, New Zealand.

Background: Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). The present study sought to determine how myocardial copper status and key copper-proteins might become impaired by diabetes, and how they respond to treatment with the Cu (II)-selective chelator triethylenetetramine (TETA) in DCM.

Methods: Experiments were performed in Wistar rats with streptozotocin (STZ)-induced diabetes with or without TETA treatment. Cardiac function was analyzed in isolated-perfused working hearts, and myocardial total copper content measured by particle-induced x-ray emission spectroscopy (PIXE) coupled with Rutherford backscattering spectrometry (RBS). Quantitative expression (mRNA and protein) and/or activity of key proteins that mediate LV-tissue-copper binding and transport, were analyzed by combined RT-qPCR, western blotting, immunofluorescence microscopy, and enzyme activity assays. Statistical analysis was performed using Student's t-tests or ANOVA and p-values of < 0.05 have been considered significant.

Results: Left-ventricular (LV) copper levels and function were severely depressed in rats following 16-weeks' diabetes, but both were unexpectedly normalized 8-weeks after treatment with TETA was instituted. Localized myocardial copper deficiency was accompanied by decreased expression and increased polymerization of the copper-responsive transition-metal-binding metallothionein proteins (MT1/MT2), consistent with impaired anti-oxidant defences and elevated susceptibility to pro-oxidant stress. Levels of the high-affinity copper transporter-1 (CTR1) were depressed in diabetes, consistent with impaired membrane copper uptake, and were not modified by TETA which, contrastingly, renormalized myocardial copper and increased levels and cell-membrane localization of the low-affinity copper transporter-2 (CTR2). Diabetes also lowered indexes of intracellular (IC) copper delivery via the copper chaperone for superoxide dismutase (CCS) to its target cuproenzyme, superoxide dismutase-1 (SOD1): this pathway was rectified by TETA treatment, which normalized SOD1 activity with consequent bolstering of anti-oxidant defenses. Furthermore, diabetes depressed levels of additional intracellular copper-transporting proteins, including antioxidant-protein-1 (ATOX1) and copper-transporting-ATPase-2 (ATP7B), whereas TETA elevated copper-transporting-ATPase-1 (ATP7A).

Conclusions: Myocardial copper deficiency and defective cellular copper transport/trafficking are revealed as key molecular defects underlying LV impairment in diabetes, and TETA-mediated restoration of copper regulation provides a potential new class of therapeutic molecules for DCM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1475-2840-13-100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070334PMC
June 2014

An improved and reliable method for isolation of microvascular endothelial cells from human omentum.

Microcirculation 2011 Nov;18(8):635-45

Institute of Biomedical and Clinical Science, Peninsula Medical School, University of Exeter, St Luke's Campus, Exeter, Devon, UK.

Objectives: Despite an increasing research demand for human microvascular endothelial cells, isolation of primary endothelial cells from human tissue remains difficult. The omentum, a highly vascular visceral adipose tissue, could provide an excellent source of these cells.

Methods: A reliable method to isolate HOMECs has been developed. It consists of initial enzymatic digestion (to deplete cell contaminants), followed by further digestion, selective filtration, and immunoselection using Dynabeads coated with CD31 antibody. Cultures were characterized for expression of endothelial cell markers and their ability to undergo VEGF-dependent in vitro tube structure formation.

Results: Omental-derived cultures of microvascular endothelial cells were achieved with <5% contamination of other cell types. The endothelial origin of cells was confirmed by the constitutive expression of a range of vascular endothelial markers (CD31, CD105, vWF) and internalization of DiI-AcLDL. Furthermore, cultures were negative for lymphatic endothelial markers, underwent in vitro angiogenesis, and exhibited typical endothelial morphology.

Conclusions: This isolation method produces homogeneous HOMEC cultures that can be maintained in vitro for at least six passages without loss of cellular features characterizing endothelial cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1549-8719.2011.00128.xDOI Listing
November 2011

Desmosomes: adhesive strength and signalling in health and disease.

Biochem J 2010 Aug;429(3):419-33

University of Manchester, UK.

Desmosomes are intercellular junctions whose primary function is strong intercellular adhesion, known as hyperadhesion. In the present review, we discuss how their structure appears to support this function as well as how they are assembled and down-regulated. Desmosomal components also have signalling functions that are important in tissue development and remodelling. Their adhesive and signalling functions are both compromised in genetic and autoimmune diseases that affect the heart, skin and mucous membranes. We conclude that much work is required on structure-function relationships within desmosomes in vivo and on how they participate in signalling processes to enhance our knowledge of tissue homoeostasis and human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20100567DOI Listing
August 2010

TILRR, a novel IL-1RI co-receptor, potentiates MyD88 recruitment to control Ras-dependent amplification of NF-kappaB.

J Biol Chem 2010 Mar 25;285(10):7222-32. Epub 2009 Nov 25.

Units of Cell Biology, University of Sheffield, Sheffield S102RX, United Kingdom.

Host defense against infection is induced by Toll-like and interleukin (IL)-1 receptors, and controlled by the transcription factor NF-kappaB. Our earlier studies have shown that IL-1 activation impacts cytoskeletal structure and that IL-1 receptor (IL-1RI) function is substrate-dependent. Here we identify a novel regulatory component, TILRR, which amplifies activation of IL-1RI and coordinates IL-1-induced control with mechanotransduction. We show that TILRR is a highly conserved and widely expressed enhancer of IL-1-regulated inflammatory responses and, further, that it is a membrane-bound glycosylated protein with sequence homology to members of the FRAS-1 family. We demonstrate that TILRR is recruited to the IL-1 receptor complex and magnifies signal amplification by increasing receptor expression and ligand binding. In addition, we show that the consequent potentiation of NF-kappaB is controlled through IL-1RI-associated signaling components in coordination with activation of the Ras GTPase. Using mutagenesis, we demonstrate that TILRR function is dependent on association with its signaling partner and, further, that formation of the TILRR-containing IL-1RI complex imparts enhanced association of the MyD88 adapter during ligand-induced activation of NF-kappaB. We conclude that TILRR is an IL-1RI co-receptor, which associates with the signaling receptor complex to enhance recruitment of MyD88 and control Ras-dependent amplification of NF-kappaB and inflammatory responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.073429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2844171PMC
March 2010