Publications by authors named "Tailoi Chan-Ling"

51 Publications

Novel morphometric analysis of higher order structure of human radial peri-papillary capillaries: relevance to retinal perfusion efficiency and age.

Sci Rep 2019 09 17;9(1):13464. Epub 2019 Sep 17.

University of Sydney, Department of Anatomy, Bosch Institute, Sydney, 2006, Australia.

We apply novel analyses to images of superficial capillaries that are located near and around the optic disc of the human retina: the radial peri-papillary capillaries (RPCs). Due to their unique perfusion of the nerve fibre layer the RPCs are particularly significant for optic-neuropathies. The inputs to the analysis were z-stacks from 3D confocal fluorescence microscopy from 62 human retinas aged 9 to 84 years. Our aim was to find morphometric correlates of age. The retinas had no ophthalmic history. The analysis was undertaken in two stages: (1) converting the z-stacks to 3D tubular networks of vessels, and (2) characterizing the tubular networks using features derived from the Minkowski functionals (MFs). The MFs measure: the capillary volume, surface area, mean breadth, and Euler number. The mean breadth is related to tortuosity, wall shear stress and resistance to flow, and the Euler number is related to the density of loops (collaterals). Features derived from the surface area, mean breadth and Euler number were most related to age (all p ≤ 0.006). The results indicate the importance of pressure-equalizing loops and tortuosity as quantitative measures related to perfusion efficiency. The novel morphometric analysis could quantify disease-related accelerated aging and vessel malformation.
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http://dx.doi.org/10.1038/s41598-019-49443-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748979PMC
September 2019

P2X7 receptor signaling during adult hippocampal neurogenesis.

Neural Regen Res 2019 Oct;14(10):1684-1694

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Queensland; Bosch Institute, The University of Sydney, Sydney, New South Wales; School of Environment and Science, Griffith University, Brisbane, Queensland, Australia.

Neurogenesis is a persistent and essential feature of the adult mammalian hippocampus. Granular neurons generated from resident pools of stem or progenitor cells provide a mechanism for the formation and consolidation of new memories. Regulation of hippocampal neurogenesis is complex and multifaceted, and numerous signaling pathways converge to modulate cell proliferation, apoptosis, and clearance of cellular debris, as well as synaptic integration of newborn immature neurons. The expression of functional P2X7 receptors in the central nervous system has attracted much interest and the regulatory role of this purinergic receptor during adult neurogenesis has only recently begun to be explored. P2X7 receptors are exceptionally versatile: in their canonical role they act as adenosine triphosphate-gated calcium channels and facilitate calcium-signaling cascades exerting control over the cell via calcium-encoded sensory proteins and transcription factor activation. P2X7 also mediates transmembrane pore formation to regulate cytokine release and facilitate extracellular communication, and when persistently stimulated by high extracellular adenosine triphosphate levels large P2X7 pores form, which induce apoptotic cell death through cytosolic ion dysregulation. Lastly, as a scavenger receptor P2X7 directly facilitates phagocytosis of the cellular debris that arises during neurogenesis, as well as during some disease states. Understanding how P2X7 receptors regulate the physiology of stem and progenitor cells in the adult hippocampus is an important step towards developing useful therapeutic models for regenerative medicine. This review considers the relevant aspects of adult hippocampal neurogenesis and explores how P2X7 receptor activity may influence the molecular physiology of the hippocampus, and neural stem and progenitor cells.
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http://dx.doi.org/10.4103/1673-5374.257510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585562PMC
October 2019

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells.

J Vis Exp 2019 04 3(146). Epub 2019 Apr 3.

Florey Institute of Neuroscience and Mental Health, University of Melbourne;

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.
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http://dx.doi.org/10.3791/59313DOI Listing
April 2019

Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models.

Diabetologia 2019 02 15;62(2):322-334. Epub 2018 Nov 15.

Centre for the Endothelium Vascular Biology Program Centenary Institute, The University of Sydney, Locked bag #6, Newtown, NSW, 2042, Australia.

Aims/hypothesis: A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy.

Methods: CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-β) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line.

Results: CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B).

Conclusions/interpretation: CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.
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http://dx.doi.org/10.1007/s00125-018-4770-4DOI Listing
February 2019

P2X7 Receptors Regulate Phagocytosis and Proliferation in Adult Hippocampal and SVZ Neural Progenitor Cells: Implications for Inflammation in Neurogenesis.

Stem Cells 2018 11 17;36(11):1764-1777. Epub 2018 Sep 17.

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.

Identifying the signaling mechanisms that regulate adult neurogenesis is essential to understanding how the brain may respond to neuro-inflammatory events. P2X7 receptors can regulate pro-inflammatory responses, and in addition to their role as cation channels they can trigger cell death and mediate phagocytosis. How P2X7 receptors may regulate adult neurogenesis is currently unclear. Here, neural progenitor cells (NPCs) derived from adult murine hippocampal subgranular (SGZ) and cerebral subventricular (SVZ) zones were utilized to characterize the roles of P2X7 in adult neurogenesis, and assess the effects of high extracellular ATP, characteristic of inflammation, on NPCs. Immunocytochemistry found NPCs in vivo and in vitro expressed P2X7, and the activity of P2X7 in culture was demonstrated using calcium influx and pore formation assays. Live cell and confocal microscopy, in conjunction with flow cytometry, revealed P2X7 NPCs were able to phagocytose fluorescent beads, and this was inhibited by ATP, indicative of P2X7 involvement. Furthermore, P2X7 receptors were activated with ATP or BzATP, and 5-ethynyl-2'-deoxyuridine (EdU) used to observe a dose-dependent decrease in NPC proliferation. A role for P2X7 in decreased NPC proliferation was confirmed using chemical inhibition and NPCs from P2X7 mice. Together, these data present three distinct roles for P2X7 during adult neurogenesis, depending on extracellular ATP concentrations: (a) P2X7 receptors can form transmembrane pores leading to cell death, (b) P2X7 receptors can regulate rates of proliferation, likely via calcium signaling, and (c) P2X7 can function as scavenger receptors in the absence of ATP, allowing NPCs to phagocytose apoptotic NPCs during neurogenesis. Stem Cells 2018;36:1764-1777.
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http://dx.doi.org/10.1002/stem.2894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635745PMC
November 2018

Increased Indoleamine 2,3-Dioxygenase and Quinolinic Acid Expression in Microglia and Müller Cells of Diabetic Human and Rodent Retina.

Invest Ophthalmol Vis Sci 2017 10;58(12):5043-5055

Department of Ophthalmology, the Eugene and Marilyn Glick Eye Institute, Indiana University, Indianapolis, Indiana, United States.

Purpose: We investigated the relationship between inflammation, neuronal loss, and expression of indoleamine 2, 3-dioxygenase (IDO) and quinolinic acid (QUIN) in the retina of subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) and in the retina of rats with T1D.

Methods: Retinas from T1D (n = 7), T2D (n = 13), and 20 age-matched nondiabetic human donors and from T1D (n = 3) and control rats (n = 3) were examined using immunohistochemistry for IDO, QUIN, cluster of differentiation 39 (CD39), ionized calcium-binding adaptor molecule (Iba-1, for macrophages and microglia), Vimentin (VIM; for Müller cells), neuronal nuclei (NeuN; for neurons), and UEA1 lectin (for blood vessels).

Results: Based on morphologic criteria, CD39+/ionized calcium binding adaptor molecule 1(Iba-1+) resident microglia and CD39-/Iba-1+ bone marrow-derived macrophages were present at higher density in T1D (13% increase) and T2D (26% increase) human retinas when compared with controls. The density and brightness of IDO+ microglia were increased in both T1D and T2D human retinas. The intensity of QUIN+ expression on CD39+ microglia and VIM+ Müller cells was greatly increased in both human T1D and T2D retinas. T1D retinas showed a 63% loss of NeuN+ neurons and T2D retinas lost approximately 43% when compared with nondiabetic human retinas. Few QUIN+ microglia-like cells were seen in nondiabetic retinas, but the numbers increased 18-fold in T1D and 7-fold in T2D in the central retina. In T1D rat retinas, the density of IDO+ microglia increased 2.8-fold and brightness increased 2.1-fold when compared with controls.

Conclusions: Our findings suggest that IDO and QUIN expression in the retinas of diabetic rats and humans could contribute to the neuronal degeneration that is characteristic of diabetic retinopathy.
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http://dx.doi.org/10.1167/iovs.17-21654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633007PMC
October 2017

Pathophysiology, screening and treatment of ROP: A multi-disciplinary perspective.

Prog Retin Eye Res 2018 01 27;62:77-119. Epub 2017 Sep 27.

Department of Paediatrics, University of Otago, Christchurch, New Zealand. Electronic address:

The population of infants at risk for retinopathy of prematurity (ROP) varies by world region; in countries with well developed neonatal intensive care services, the highest risk infants are those born at less than 28 weeks gestational age (GA) and less than 1 kg at birth, while, in regions where many aspects of neonatal intensive and ophthalmological care are not routinely available, more mature infants up to 2000 g at birth and 37 weeks GA are also at risk for severe ROP. Treatment options for both groups of patients include standard retinal laser photocoagulation or, more recently, intravitreal anti-VEGF drugs. In addition to detection and treatment of ROP, this review highlights new opportunities created by telemedicine, where screening and diagnosis of ROP in remote locations can be undertaken by non-ophthalmologists using digital fundus cameras. The ophthalmological care of the ROP infant is undertaken in the wider context of neonatal care and general wellbeing of the infant. Because of this context, this review takes a multi-disciplinary perspective with contributions from retinal vascular biologists, pediatric ophthalmologists, an epidemiologist and a neonatologist. This review highlights the latest insights regarding cellular and molecular mechanisms in the formation of the retinal vasculature in the human infant, pathogenesis of ROP, detection and treatment of severe ROP, the risks and benefits of anti-VEGF therapy, the identification of new therapies over the horizon, and the optimal neonatal care regimen for best ROP outcomes, and the benefits and pitfalls of telemedicine in the remote screening and diagnosis of ROP, all of which have the potential to improve ROP outcomes.
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http://dx.doi.org/10.1016/j.preteyeres.2017.09.002DOI Listing
January 2018

Insulin-like growth factor binding protein-3 links obesity and breast cancer progression.

Oncotarget 2016 Aug;7(34):55491-55505

Hormones and Cancer Laboratories, Kolling Institute, University of Sydney, Royal North Shore Hospital, Sydney, New South Wales 2065, Australia.

Obesity is associated epidemiologically with poor breast cancer prognosis, but the mechanisms remain unclear. Since IGF binding protein-3 (IGFBP-3) influences both breast cancer growth and adipocyte maturation, it may impact on how obesity promotes breast oncogenesis. This study investigated the role of endogenous IGFBP-3 on the development of obesity and subsequently on breast tumor growth. Wild-type (WT) C57BL/6 or IGFBP-3-null (BP3KO) mice were fed a high-fat diet (HFD) or control chow-diet for 15 weeks before orthotopic injection with syngeneic EO771 murine breast cancer cells. When the largest tumor reached 1000 mm3, tissues and tumors were excised for analysis. Compared to WT, BP3KO mice showed significantly reduced weight gain and mammary fat pad mass (contralateral to tumor) in response to HFD, despite similar food intake. EO771 tumor weight and volume were increased by HFD and decreased by BP3KO. Despite differences in tumor size, tumors in BP3KO mice showed no differences from WT in the number of mitotically active (Ki67+) and apoptotic (cleaved caspase-3+) cells, but had greater infiltration of CD3+ T-cells. These data suggest that endogenous (circulating and/or stromal) IGFBP-3 is stimulatory to adipose tissue expansion and enhances mammary tumor growth in immune-competent mice, potentially by suppressing T-cell infiltration into tumors.
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http://dx.doi.org/10.18632/oncotarget.10675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342431PMC
August 2016

The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome.

Sci Rep 2016 07 11;6:29514. Epub 2016 Jul 11.

EMBL Australia, The Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.

Copy number variations to chromosome 21 (HSA21) cause intellectual disability and Down Syndrome, but our understanding of the HSA21 genetic factors which contribute to fetal brain development remains incomplete. Here, we focussed on the neurodevelopmental functions for EURL (also known as C21ORF91, Refseq Gene ID:54149), a protein-coding gene at the centromeric boundary of the Down Syndrome Critical Region (DSCR) of HSA21. We report that EURL is expressed during human and mouse cerebral cortex development, and we report that alterations to EURL mRNA levels within the human brain underlie Down Syndrome. Our gene perturbation studies in mice demonstrate that disruptions to Eurl impair progenitor proliferation and neuronal differentiation. Also, we find that disruptions to Eurl impair the long-term positioning and dendritic spine densities of cortical projection neurons. We provide evidence that EURL interacts with the coiled-coil domain-containing protein CCDC85B so as to modulate β-catenin levels in cells. Further, we utilised a fluorescent reporter (8xTOPFLASHd2EGFP) to demonstrate that disruptions to Eurl alter β-catenin signalling in vitro as well as in vivo. Together, these studies highlight EURL as an important new player in neuronal development that is likely to impact on the neuropathogenesis of HSA21-related disorders including Down Syndrome.
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http://dx.doi.org/10.1038/srep29514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941730PMC
July 2016

Author Response: Sufficient Evidence for Lymphatics in the Developing and Adult Human Choroid?

Invest Ophthalmol Vis Sci 2015 Oct;56(11):6711-3

School of Optometry, University of New South Wales, Sydney, New South Wales, Australia; and 5Save Sight Institute, University of Sydney, Sydney, New South Wales, Australia.

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http://dx.doi.org/10.1167/iovs.15-18011DOI Listing
October 2015

Differential expression of sirtuins in the aging rat brain.

Front Cell Neurosci 2015 8;9:167. Epub 2015 May 8.

Neuropharmacology Group, MND and Neurodegenerative Diseases Research Centre, Macquarie University North Ryde NSW, Australia.

Although there are seven mammalian sirtuins (SIRT1-7), little is known about their expression in the aging brain. To characterize the change(s) in mRNA and protein expression of SIRT1-7 and their associated proteins in the brain of "physiologically" aged Wistar rats. We tested mRNA and protein expression levels of rat SIRT1-7, and the levels of associated proteins in the brain using RT-PCR and western blotting. Our data shows that SIRT1 expression increases with age, concurrently with increased acetylated p53 levels in all brain regions investigated. SIRT2 and FOXO3a protein levels increased only in the occipital lobe. SIRT3-5 expression declined significantly in the hippocampus and frontal lobe, associated with increases in superoxide and fatty acid oxidation levels, and acetylated CPS-1 protein expression, and a reduction in MnSOD level. While SIRT6 expression declines significantly with age acetylated H3K9 protein expression is increased throughout the brain. SIRT7 and Pol I protein expression increased in the frontal lobe. This study identifies previously unknown roles for sirtuins in regulating cellular homeostasis and healthy aging.
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http://dx.doi.org/10.3389/fncel.2015.00167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424846PMC
May 2015

Loss of survival factors and activation of inflammatory cascades in brain sympathetic centers in type 1 diabetic mice.

Am J Physiol Endocrinol Metab 2015 Apr 24;308(8):E688-98. Epub 2015 Feb 24.

Department of Ophthalmology, The Eugene and Marilyn Glick Eye Institute, Indiana University, Indianapolis, Indiana

Neuroinflammation and neurodegeneration have been observed in the brain in type 1 diabetes (T1D). However, little is known about the mediators of these effects. In T1D mice with 12- and 35-wk duration of diabetes we examined two mechanisms of neurodegeneration, loss of the neuroprotective factors insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and changes in indoleamine 2,3-dioxygenase (IDO) expression in the brain, and compared the response to age-matched controls. Furthermore, levels of matrix metalloproteinase-2 (MMP-2), nucleoside triphosphate diphosphohydrolase-1 (CD39), and ionized calcium-binding adaptor molecule 1 (Iba-1) were utilized to assess inflammatory changes in astrocytes, microglia, and blood vessels. In the diabetic hypothalamus (HYPO), we observed 20% reduction in neuronal soma diameter (P<0.05) and reduced neuronal expression of IGFBP-3 (-32%, P<0.05) and IGF-I (-15%, P<0.05) compared with controls at 35 wk. In diabetic HYPO, MMP-2 expression was increased in astrocytes (46%, P<0.01), and IDO⁺ cell density rose by (62%, P<0.05). CD39 expression dropped by 30% (P<0.05) in microglia and blood vessels. With 10 wk of systemic treatment using minocycline, an anti-inflammatory agent that crosses the blood-brain barrier, MMP-2, IDO, and CD39 levels normalized (P<0.05). Our results suggest that increased IDO and early loss of CD39⁺ protective cells lead to activation of inflammation in sympathetic centers of the CNS. As a downstream effect, the loss of the neuronal survival factors IGFBP-3 and IGF-I and the neurotoxic products of the kynurenine pathway contribute to the loss of neuronal density observed in the HYPO in T1D.
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http://dx.doi.org/10.1152/ajpendo.00504.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398829PMC
April 2015

Evidence for lymphatics in the developing and adult human choroid.

Invest Ophthalmol Vis Sci 2015 Jan 13;56(2):1310-27. Epub 2015 Jan 13.

Discipline of Anatomy, School of Medical Sciences, Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia.

Purpose: Lymphatics subserve many important functions in the human body including maintenance of fluid homeostasis, immune surveillance, and tumor metastasis. Our aim was to provide structural and phenotypic evidence of lymphatic-like structures in the human choroid, including details of its development.

Methods: Using multiple-marker immunohistochemistry (IHC), choroids from human fetal eyes (8-26 weeks gestation) and adults (17-74 years) were examined with lymphatic- and vascular-specific markers: prospero homeobox-1 (PROX-1), lymphatic vascular endothelium receptor-1 (LYVE-1), podoplanin, D2-40, endomucin, VEGF-C, vascular endothelial growth factor receptor-3 (VEGFR-3 or Flt4), UEA lectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), CD34, and CD39. Transmission electron microscopy (TEM) was used to establish evidence for choroidal lymphatics, and to provide details of stratification and relative frequency of lymphatics compared to choroidal blood vessels.

Results: Immunohistochemistry and TEM indicated a central-to-peripheral topography of lymphatic formation, with numerous blind-ended lymph sacs just external to the choriocapillaris, as well as the presence of infrequent precollector and collector lymphatic channels. Characteristic ultrastructural features of lymphatics in adult human choroid included anchoring filaments, luminal flocculent protein but absence of erythrocytes, fragmented and/or absent basal lamina, absence of intracellular Weibel-Palade bodies, infrequent pericyte ensheathment, and lack of fenestrae.

Conclusions: The system of blind-ended initial lymphatic segments seen just external to the fenestrated vessels of the choriocapillaris is ideally placed for recirculating extracellular fluid and strategically placed for immune surveillance. The presence of a system of lymphatic-like channels in the human choroid provides an anatomical basis for antigen presentation in the posterior eye, with a possible route from the eye to the sentinel lymph nodes, similar to that already described for anterior eye lymphatics.
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http://dx.doi.org/10.1167/iovs.14-15705DOI Listing
January 2015

P2X7 receptors mediate innate phagocytosis by human neural precursor cells and neuroblasts.

Stem Cells 2015 Feb;33(2):526-41

Discipline of Anatomy and Histology, Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia; Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia.

During early human neurogenesis there is overproduction of neuroblasts and neurons accompanied by widespread programmed cell death (PCD). While it is understood that CD68(+) microglia and astrocytes mediate phagocytosis during target-dependent PCD, little is known of the cell identity or the scavenger molecules used to remove apoptotic corpses during the earliest stages of human neurogenesis. Using a combination of multiple-marker immunohistochemical staining, functional blocking antibodies and antagonists, we showed that human neural precursor cells (hNPCs) and neuroblasts express functional P2X7 receptors. Furthermore, using live-cell imaging, flow cytometry, phagocytic assays, and siRNA knockdown, we showed that in a serum-free environment, doublecortin(+) (DCX) neuroblasts and hNPCs can clear apoptotic cells by innate phagocytosis mediated via P2X7. We found that both P2X7(high) DCX(low) hNPCs and P2X7(high) DCX(high) neuroblasts, derived from primary cultures of human fetal telencephalon, phagocytosed targets including latex beads, apoptotic ReNcells, and apoptotic hNPC/neuroblasts. Pretreatment of neuroblasts and hNPCs with 1 mM adenosine triphosphate (ATP), 100 µM OxATP (P2X7 antagonist), or siRNA knockdown of P2X7 inhibited phagocytosis of these targets. Our results show that P2X7 functions as a scavenger receptor under serum-free conditions resembling those in early neurogenesis. This is the first demonstration that hNPCs and neuroblasts may participate in clearance of apoptotic corpses during pre target-dependent neurogenesis and mediate phagocytosis using P2X7 as a scavenger receptor.
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http://dx.doi.org/10.1002/stem.1864DOI Listing
February 2015

Ultrasmall superparamagnetic iron oxide nanoparticle prelabelling of human neural precursor cells.

Biomaterials 2014 Jul 13;35(21):5549-64. Epub 2014 Apr 13.

Department of Anatomy and Histology, Bosch Institute, Sydney Medical School, University of Sydney, NSW 2006, Australia. Electronic address:

Stem cells prelabelled with iron oxide nanoparticles can be visualised using magnetic resonance imaging (MRI). This technique allows for noninvasive long-term monitoring of migration, integration and stem cell fate following transplantation into living animals. In order to determine biocompatibility, the present study investigated the biological impact of introducing ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) into primary human fetal neural precursor cells (hNPCs) in vitro. USPIOs with a mean diameter of 10-15 nm maghemite iron oxide core were sterically stabilised by 95% methoxy-poly(ethylene glycol) (MPEG) and either 5% cationic (NH2) end-functionalised, or 5% Rhodamine B end-functionalised, polyacrylamide. The stabilising polymer diblocks were synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation. Upon loading, cellular viability, total iron capacity, differentiation, average distance of migration and changes in intracellular calcium ion concentration were measured to determine optimal loading conditions. Taken together we demonstrate that prelabelling of hNPCs with USPIOs has no significant detrimental effect on cell biology and that USPIOs, when utilised at an optimised dosage, are an effective means of noninvasively tracking prelabelled hNPCs.
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http://dx.doi.org/10.1016/j.biomaterials.2014.03.061DOI Listing
July 2014

Mapping NAD(+) metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence.

Biogerontology 2014 Apr 17;15(2):177-98. Epub 2013 Dec 17.

Centre for Healthy Brain Ageing, School of Psychiatry, University of New South Wales, Sydney, NSW, Australia.

Over the last decade, the importance of NAD(+) has expanded beyond its role as an essential cofactor for energy metabolism. NAD(+) has emerged as a major signalling molecule that serves as the sole substrate for several enzymatic reactions including the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), NAD-dependent protein deacetylases or CD38, and transcriptional factors by a new class of histone deacetylases known as sirtuins. NAD(+) levels are regulated by the metabolic status and cellular stress caused by oxidative stress and DNA damage. Since a detailed study of NAD(+) metabolism in the healthy ageing mammalian brain is nascent, we examined the effect of ageing on intracellular NAD(+) metabolism in different brain regions in female Wistar rats in young (3 months), middle aged (12 months) and older adults (24 months). Our results are the first to show a significant decline in intracellular NAD(+) levels and NAD:NADH ratio with ageing in the CNS, occurring in parallel to an increase in lipid peroxidation and protein oxidation (o- and m-tyrosine) and a decline in total antioxidant capacity. Hyperphosphorylation of H2AX levels was also observed together with increased PARP-1 and PARP-2 expression, and CD38 activity, concomitantly with reduced NAD(+) and ATP levels and SIRT1 function in the cortex, brainstem, hippocampus and cerebellum. Reduced activity of mitochondrial complex I-IV and impaired maximum mitochondrial respiration rate were also observed in the ageing rat brain. Among the multiple physiological pathways associated with NAD(+) catabolism, our discovery of CD38 as the major regulator of cellular NAD(+) levels in rat neurons indicates that CD38 is a promising therapeutic target for the treatment of age-related neurodegenerative diseases.
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http://dx.doi.org/10.1007/s10522-013-9489-5DOI Listing
April 2014

CNS inflammation and bone marrow neuropathy in type 1 diabetes.

Am J Pathol 2013 Nov;183(5):1608-20

Department of Anatomy, Bosch Institute, University of Sydney, Sydney, Australia.

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.
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http://dx.doi.org/10.1016/j.ajpath.2013.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814523PMC
November 2013

Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

PLoS One 2013 14;8(3):e57038. Epub 2013 Mar 14.

Department of Anatomy, School of Medical Sciences and Bosch Institute, University of Sydney, New South Wales, Australia.

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057038PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597639PMC
September 2013

Protection of blood retinal barrier and systemic vasculature by insulin-like growth factor binding protein-3.

PLoS One 2012 6;7(7):e39398. Epub 2012 Jul 6.

Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

Previously, we showed that insulin growth factor (IGF)-1 binding protein-3 (IGFBP-3), independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR). The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB) integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1) and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP). IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO) release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab). Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB) stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs). NO release was neither associated with an increase in intracellular calcium nor decreased by Ca(2+)/calmodulin-dependent protein kinase II (CamKII) blockade; however, dephosphorylation of eNOS-Thr(495) was observed. Phosphatidylinositol 3-kinase (PI3K) activity and Akt-Ser(473) phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate vasorelaxation via activation of SRB1. This response is IGF-1- and calcium-independent, but requires PI3K/Akt activation, suggesting that IGFBP-3 has novel protective effects on retinal and systemic vasculature and may be a therapeutic candidate for ocular complications such as diabetic retinopathy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039398PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391198PMC
March 2013

The crossroads of neuroinflammation in infectious diseases: endothelial cells and astrocytes.

Trends Parasitol 2012 Aug 22;28(8):311-9. Epub 2012 Jun 22.

Vascular Immunology Unit, Sydney Medical School and Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia.

Homeostasis implies constant operational defence mechanisms, against both external and internal threats. Infectious agents are prominent among such threats. During infection, the host elicits the release of a vast array of molecules and numerous cell-cell interactions are triggered. These pleiomorphic mediators and cellular effects are of prime importance in the defence of the host, both in the systemic circulation and at sites of tissue injury, for example, the blood-brain barrier (BBB). Here, we focus on the interactions between the endothelium, astrocytes, and the molecules they release. Our review addresses these interactions during infectious neurological diseases of various origins, especially cerebral malaria (CM). Two novel elements of the interplay between endothelium and astrocytes, microparticles and the kynurenine pathway, will also be discussed.
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http://dx.doi.org/10.1016/j.pt.2012.05.008DOI Listing
August 2012

Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease.

Prog Retin Eye Res 2012 Sep 3;31(5):377-406. Epub 2012 May 3.

Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA.

We review the cellular and physiological mechanisms responsible for the regulation of blood flow in the retina and choroid in health and disease. Due to the intrinsic light sensitivity of the retina and the direct visual accessibility of fundus blood vessels, the eye offers unique opportunities for the non-invasive investigation of mechanisms of blood flow regulation. The ability of the retinal vasculature to regulate its blood flow is contrasted with the far more restricted ability of the choroidal circulation to regulate its blood flow by virtue of the absence of glial cells, the markedly reduced pericyte ensheathment of the choroidal vasculature, and the lack of intermediate filaments in choroidal pericytes. We review the cellular and molecular components of the neurovascular unit in the retina and choroid, techniques for monitoring retinal and choroidal blood flow, responses of the retinal and choroidal circulation to light stimulation, the role of capillaries, astrocytes and pericytes in regulating blood flow, putative signaling mechanisms mediating neurovascular coupling in the retina, and changes that occur in the retinal and choroidal circulation during diabetic retinopathy, age-related macular degeneration, glaucoma, and Alzheimer's disease. We close by discussing issues that remain to be explored.
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http://dx.doi.org/10.1016/j.preteyeres.2012.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418965PMC
September 2012

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats.

FEBS J 2011 Nov;278(22):4425-34

University of New South Wales, Faculty of Medicine, Sydney, NSW, Australia.

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.
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http://dx.doi.org/10.1111/j.1742-4658.2011.08366.xDOI Listing
November 2011

Free insulin-like growth factor binding protein-3 (IGFBP-3) reduces retinal vascular permeability in association with a reduction of acid sphingomyelinase (ASMase).

Invest Ophthalmol Vis Sci 2011 Oct 21;52(11):8278-86. Epub 2011 Oct 21.

Departments of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32610-0267, USA.

Purpose: To examine the effect of free insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), independent of the effect of insulin-like growth factors, in modulating retinal vascular permeability.

Methods: We assessed the ability of a form of IGFBP-3 that does not bind IGF-1 (IGFBP-3NB), to regulate the blood retinal barrier (BRB) using two distinct experimental mouse models, laser-induced retinal vessel injury and vascular endothelial growth factor (VEGF)-induced retinal vascular permeability. Additionally, in vitro studies were conducted. In the animal models, BRB permeability was quantified by intravenous injection of fluorescein labeled serum albumin followed by digital confocal image analysis of retinal flat-mounts. Claudin-5 and vascular endothelial-cadherin (VE-cadherin) localization at interendothelial junctions was studied by immunofluorescence. In vitro changes in transendothelial electrical resistance (TEER) and flux of fluorescent dextran in bovine retinal endothelial monolayers (BREC) were measured after IGFBP-3NB treatment. Acid (ASMase) and neutral (NSMase) sphingomyelinase mRNA levels and activity were measured in mouse retinas.

Results: Four days postinjury, laser-injured mouse retinas injected with IGFBP-3NB plasmid demonstrated reduced vascular permeability compared with retinas of laser-injured mouse retinas injected with control plasmid. IGFBP-3NB administration resulted in a significant decrease in laser injury-associated increases in ASMase and NSMase mRNA and activity when compared with laser alone treated mice. In vivo, intravitreal injection of IGFBP-3NB reduced vascular leakage associated with intravitreal VEGF injection. IGFBP-3NB partially restored VEGF-induced in vivo permeability and dissociation of claudin-5 and VE-cadherin at junctional complexes. When IGFBP-3NB was applied basally to bovine retinal endothelial cells (BREC) in vitro, TEER increased and macromolecular flux decreased.

Conclusions: Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.
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http://dx.doi.org/10.1167/iovs.11-8167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208060PMC
October 2011

Quantitation of brain edema and localisation of aquaporin 4 expression in relation to susceptibility to experimental cerebral malaria.

Int J Clin Exp Pathol 2011 Aug 23;4(6):566-74. Epub 2011 Jul 23.

Vascular Immunology Unit, Dept. of Pathology, Faculty of Medicine and Bosch Institute, University of Sydney, Sydney, Australia.

The pathogenic mechanisms underlying the occurrence of cerebral malaria (CM) are still incompletely understood but, clearly, cerebral complications may result from concomitant microvessel obstruction and inflammation. The extent to which brain edema contributes to pathology has not been investigated. Using the model of P. berghei ANKA infection, we compared brain microvessel morphology of CM-susceptible and CM-resistant mice. By quantitative planimetry, we provide evidence that CM is characterized by enlarged perivascular spaces (PVS). We show a dramatic aquaporin 4 (AQP4) upregulation, selectively at the level of astrocytic foot processes, in both CM and non-CM disease, but significantly more pronounced in mice with malarial-induced neurological syndrome. This suggests that a threshold of AQP4 expression is needed to lead to neurovascular pathology, a view that is supported by significantly higher levels in mice with clinically overt CM. Numbers of intravascular leukocytes significantly correlated with both PVS enlargement and AQP4 overexpression. Thus, brain edema could be a contributing factor in CM pathogenesis and AQP4, specifically in its astrocytic location, a key molecule in this mechanism. Since experimental CM is associated with substantial brain edema, it models paediatric CM better than the adult syndrome and it is tempting to evaluate AQP4 in the former context. If AQP4 changes are confirmed in human CM, it may represent a novel target for therapeutic intervention.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160608PMC
August 2011

Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats.

PLoS One 2011 Apr 26;6(4):e19194. Epub 2011 Apr 26.

Department of Pharmacology, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia.

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082551PMC
April 2011

Novel protective properties of IGFBP-3 result in enhanced pericyte ensheathment, reduced microglial activation, increased microglial apoptosis, and neuronal protection after ischemic retinal injury.

Am J Pathol 2011 Apr;178(4):1517-28

Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610-0267, USA.

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GFP(+) hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity.
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http://dx.doi.org/10.1016/j.ajpath.2010.12.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078432PMC
April 2011

Evidence of hematopoietic differentiation, vasculogenesis and angiogenesis in the formation of human choroidal blood vessels.

Exp Eye Res 2011 May 24;92(5):361-76. Epub 2011 Feb 24.

Department of Anatomy, Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia.

Human fetal eyes 8-40 weeks gestation (WG) were examined using markers to hematopoietic stem cells (HSC), vascular precursor cells (VPC), monocytes/macrophages and endothelial cells (EC). Electron microscopy and bromo-deoxyuridene labeling were undertaken to confirm the existence of solid vascular cords and to demonstrate vasculogenesis and angiogenesis in developing choroidal tissue. Our results demonstrated that the earliest incipient choroid consisted of vimentin(+) mesenchymal precursor cells which downregulated vimentin expression with maturation. Our observations lead us to conclude that these vimentin(-)/CD34(+)/CD44(+)/CD133(+) HSCs then differentiated into three distinct lineages: single isolated CD34(-)/CD39(+) VPCs that formed solid vascular cords which lumenized and became lined with CD34(+) vascular ECs; CD34(--+)/CD14(+)/CD68(+) monocytes that differentiated into tissue macrophages; and CD133(+)/CD34(--+)/α-smooth muscle actin(+) mural precursor cells that matured into smooth muscle cells and pericytes. Blood vessel formation occurred throughout the whole choroid simultaneously, indicative of in situ differentiation. Vasculogenesis, as evidenced by lumenization of solid vascular cords, was responsible for the formation of the entire choroidal area with angiogenesis, in all three layers of the choroid, only adding to vascular density. These results suggest that formation of the human choroid involves three processes: HSC differentiation, vasculogenesis and angiogenesis. Since vasculogenesis takes place independently of VEGF(165), further insights regarding the molecular mechanisms of vasculogenesis are required to better inform future treatments of choroidal neovascularization.
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http://dx.doi.org/10.1016/j.exer.2011.02.009DOI Listing
May 2011

Role of CD44+ stem cells in mural cell formation in the human choroid: evidence of vascular instability due to limited pericyte ensheathment.

Invest Ophthalmol Vis Sci 2011 Jan 21;52(1):399-410. Epub 2011 Jan 21.

Department of Anatomy, Bosch Institute, University of Sydney, Sydney, Australia.

Purpose: To examine mural cell differentiation and pericyte ensheathment during human choroidal vascular formation and into adulthood.

Methods: Triple- and double-labeled immunohistochemistry (alpha-smooth muscle actin [αSMA], desmin, NG2, calponin, caldesmon, CD44, CD34, and CD39) were applied to human fetal (8-32 weeks' gestation) and adult choroidal and retinal wholemounts and histologic cross-sections. Transmission electron microscopy (TEM) was also undertaken.

Results: Early in development CD44+ stem cells also stained with αSMA and CD39, suggesting a common precursor. At 12 weeks' gestation, αSMA+ mural precursor cells, confirmed by TEM, were found scattered and isolated over the primordial vascular tree. During development, αSMA+ cells formed a continuous sheath around large arterioles; in veins there were gaps in αSMA expression. The choriocapillaris had an extensive vascular bed but limited coverage by αSMA+ and NG2+ mural cells. Calponin was expressed only on large vessels, and no caldesmon was detected. Pericyte ensheathment of adult capillaries was 11% for choroid versus 94% for retina. Remarkably, choroidal pericytes had no visible intermediate filaments (IFs) on TEM, though IFs were present in retinal pericytes. Neither retinal nor choroidal pericytes stained with desmin.

Conclusions: CD44+ stem cells are involved in the formation of mural cells in the human choroidal vasculature. A marked reduction in pericyte ensheathment of human choroidal vessels suggests a permanently open "plasticity window" and a predisposition to vascular instability and poor autoregulatory ability.
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http://dx.doi.org/10.1167/iovs.10-5403DOI Listing
January 2011

Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.

Circ Res 2009 Oct 17;105(9):897-905. Epub 2009 Sep 17.

Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, FL 32610, USA.

Rationale: Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury.

Objective: To examine the mechanism of IGFBP-3-mediated repair following vascular injury.

Methods And Results: We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3-expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3-mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway.

Conclusion: These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.
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http://dx.doi.org/10.1161/CIRCRESAHA.109.199059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3635679PMC
October 2009

Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons.

Proc Natl Acad Sci U S A 2009 Sep 25;106(36):15489-94. Epub 2009 Aug 25.

Florey Neuroscience Institutes and Centre for Neuroscience, The University of Melbourne, Parkville, Victoria 3010, Australia.

The regulation of metal ion transport within neurons is critical for normal brain function. Of particular importance is the regulation of redox metals such as iron (Fe), where excess levels can contribute to oxidative stress and protein aggregation, leading to neuronal death. The divalent metal transporter 1 (DMT1) plays a central role in the regulation of Fe as well as other metals; hence, failure of DMT1 regulation is linked to human brain pathology. However, it remains unclear how DMT1 is regulated in the brain. Here, we show that DMT1 is regulated by Ndfip1 (Nedd4 family-interacting protein 1), an adaptor protein that recruits E3 ligases to ubiquitinate target proteins. Using human neurons we show the Ndfip1 is upregulated and binds to DMT1 in response to Fe and cobalt (Co) exposure. This interaction results in the ubiquitination and degradation of DMT1, resulting in reduced metal entry. Induction of Ndfip1 expression protects neurons from metal toxicity, and removal of Ndfip1 by shRNAi results in hypersensitivity to metals. We identify Nedd4-2 as an E3 ligase recruited by Ndfip1 for the ubiquitination of DMT1 within human neurons. Comparison of brains from Ndfip1(-/-) with Ndfip1(+/+) mice exposed to Fe reveals that Ndfip1(-/-) brains accumulate Fe within neurons. Together, this evidence suggests a critical role for Ndfip1 in regulating metal transport in human neurons.
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http://dx.doi.org/10.1073/pnas.0904880106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741278PMC
September 2009