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A Differential Role for CD248 (Endosialin) in PDGF-Mediated Skeletal Muscle Angiogenesis

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A Differential Role for CD248 (Endosialin) in PDGF-Mediated Skeletal Muscle Angiogenesis A Differential Role for CD248 (Endosialin) in PDGF-Mediated Skeletal Muscle Angiogenesis Amy J. Naylor1", Helen M. McGettrick1,2", William D. Maynard1, Philippa May1, Francesca Barone1, Adam P. Croft1, Stuart Egginton3, Christopher D. Buckley1,2* 1 Rheumatology Research Group, Centre for Translational Inflammation Research, University of Birmingham, Birmingham, West Midlands, United Kingdom, 2 Systems Science for Health, University of Birmingham, Birmingham, West Midlands, United Kingdom, 3 Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire, United Kingdom Abstract CD248 (Endosialin) is a type 1 membrane protein involved in developmental and pathological angiogenesis through its expression on pericytes and regulation of PDGFRb signalling. Here we explore the function of CD248 in skeletal muscle angiogenesis. Two distinct forms of capillary growth (splitting and sprouting) can be induced separately by increasing microcirculatory shear stress (chronic vasodilator treatment) or by inducing functional overload (extirpation of a synergistic muscle). We show that CD248 is present on pericytes in muscle and that CD248-/- mice have a specific defect in capillary sprouting. In contrast, splitting angiogenesis is independent of CD248 expression. Endothelial cells respond to pro- sprouting angiogenic stimulus by up-regulating gene expression for HIF1a, angiopoietin 2 and its receptor TEK, PDGF-B and its receptor PDGFRb; this response did not occur following a pro-splitting angiogenic stimulus. In wildtype mice, defective sprouting angiogenesis could be mimicked by blocking PDGFRb signalling using the tyrosine kinase inhibitor Imatinib mesylate. We conclude that CD248 is required for PDGFRb-dependant capillary sprouting but not splitting angiogenesis, and identify a new role for CD248 expressed on pericytes in the early stages of physiological angiogenesis during muscle remodelling. Citation: Naylor AJ, McGettrick HM, Maynard WD, May P, Barone F, et al. (2014) A Differential Role for CD248 (Endosialin) in PDGF-Mediated Skeletal Muscle Angiogenesis. PLoS ONE 9(9): e107146. doi:10.1371/journal.pone.0107146 Editor: Atsushi Asakura, University of Minnesota Medical School, United States of America Received March 17, 2014; Accepted August 12, 2014; Published September 22, 2014 Copyright: ß 2014 Naylor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: AJN was supported by Arthritis Research UK Programme Grant (19791), HMM was supported by Arthritis Research UK Career Development Fellowship (19899) and the Systems Science for Health Initiative, University of Birmingham (5212). WDM and PM were supported by the Arthur Thomson Trust. SE was supported by a grant from the British Heart Foundation PG/08/018/24599. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] " These authors are joint first authors on this work. Introduction platelet-derived growth factor receptor beta (PDGFRb), neuron glial antigen 2 (NG2), alpha smooth muscle actin (aSMA) or Angiogenesis is the physiological process through which new CD248 (endosialin) in close proximity to CD31 positive endothe- blood vessels are formed from cells of the existing vasculature. This lial cells (reviewed in [1,8]). can be achieved by one of two processes, splitting or sprouting, During physiological angiogenesis, platelet-derived growth that require different levels of pericyte involvement (reviewed in factor-B (PDGF-B) secreted by endothelial cells dimerises and [1–2]). Splitting angiogenesis depends on the reorganisation of activates pericyte PDGFRb, which ultimately induces their existing cell populations, where opposing vascular endothelial cells proliferation and migration towards the newly developing vessel extend lamellipodia into the lumen until they contact with the [5]. Importantly, lack of PDGFRb or PDGF-BB, and therefore opposite side of the lumen, effectively splitting the vessel in two. In lack of pericyte function, results in the formation of disorganised contrast, sprouting angiogenesis is characterised by the migration vessels leading to perinatally lethal haemorrhaging and oedema and proliferation of endothelial cells towards an angiogenic [9–11]. stimulus in the tissue. CD248 (endosialin; tumour endothelial marker 1) is a C-type Pericytes play an essential role in stabilising the developing lectin-like domain family member highly expressed during vessel in sprouting angiogenesis [3]. Indeed, a loss or lack of embryogenesis [12], and upregulated on pericytes in vascularised pericytes results in weaknesses in the capillaries, and has been brain tumours and sarcomas [13–14]. Despite confusion in early associated with micro-aneurysms and loss of sight [4–5]. Pericytes literature on the subject (eg. [15]), CD248 expression is not seen are a heterogeneous population of perivascular cells located in on endothelial cells, but rather is a marker of neighbouring close proximity to endothelial cells beneath a common basement pericytes and stromal cells where it has the potential to influence membrane [2,6–7]. To date, no specific pan-pericyte marker has the process of angiogenesis [16–17]. For example, tumour growth been identified. Pericytes are defined as cells expressing either PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e107146 CD248 Is Required for Sprouting Angiogenesis and large vessel formation in vivo are markedly reduced by genetic or (ii) liquid nitrogen for gene expression analysis. All samples were deletion of CD248 [18] or removal of its cytoplasmic tail [19]. stored at -80uC until use. Similarly, antibody blockade of CD248 interfered with pericyte migration and tube formation in vitro [12]. CD248 has also been Immunofluorescence shown to regulate the kinetics of vessel pruning during develop- Transverse 8 mm cryosections of the EDL muscle were cut at mental vasculogenesis, ensuring only correctly organised and 220uC, air dried for 1 hr, fixed in acetone at 4uC for 20 minutes viable vessels survive [20]. and stored at 220uC until use. Sections were rehydrated and Recent evidence suggests that CD248 may exert its effects blocked at room temperature for 30 minutes in phosphate buffered through regulation of the PDGF pathway as PDGF-BB-induced saline (PBS) with 1% bovine serum albumin (PBSA; Sigma- phosphorylation of extracellular signal-regulated kinase (ERK), Aldrich, UK) for capillary: fibre ratio studies, or for 15–20 minutes but not phosphorylation of PDGFRb itself, was markedly first in 0.05% Avidin, then 0.005% biotin and finally 10% horse diminished in CD248-deficient pericytes [21]. Thus, CD248 serum diluted in PBS (all from Sigma) for pericyte imaging. may function to enhance or modify PDGF-BB signalling acting Sections were incubated with the following primary antibodies for downstream of PDGFRb but upstream of ERK1/2 by an as yet 1 hr at room temperature or overnight at 4uC: anti-CD31 (1:100: unknown mechanism. AbD Serotec, BioRad, USA; clone 2H8); anti-CD248 (1:400; CD248 is required for pathological angiogenesis and develop- clone p13, gift from Claire Isacke, The Institute of Cancer mental vasculogenesis [18–20]; however little is known about its Research, London, UK); anti-NG2 (1:75; Upstate, Merck function in physiological angiogenesis and whether this differs Millipore, USA; clone 132.38); anti-aSMA (1:100; Neomarkers, according to the form of capillary growth elicited (splitting vs. ThermoScientific, USA; clone 1A4); anti-PDGFRb biotin (1:100; sprouting). Here we have examined the role of CD248 within eBioscience, USA; clone APB5), anti-collagen IV (1:200; Abcam, skeletal muscle. Skeletal muscle displays a highly organised Life Technologies, UK; polyclonal; ab19808), anti-phospho ERK microvascular network with a consistent relationship between (1:100; Cell Signaling Technologies, UK; Phospho-p44/42 the number of capillaries and the number of muscle fibres; any MAPK (Erk1/2) (Thr202/Tyr204) Antibody #9101). Subse- increase in the capillary to fibre ratio (C:F) strongly indicates an quently, slides were incubated with secondary and then tertiary angiogenic response [22–23]. We explored this relationship and antibodies and Hoechst 33342 (10 mg/ml; Invitrogen, UK) for 30– demonstrated that CD248-deficient mice display a specific defect 60 min each before mounting with either Prolong Gold Antifade in sprouting, but not splitting, angiogenesis in skeletal muscle and (Molecular Probes, Life Technologies, UK) or Dabco (Sigma- demonstrate for the first time that CD248-positive pericytes are Aldrich, UK). required during the early stages of physiological angiogenesis. In some cases (Figure 1A, B and C), in order to visualise all pericytes at once, tissues were incubated with a cocktail of primary b a Methods antibodies to PDGFR , NG2 and SMA which were detected with specific secondary antibodies that all shared the same In vivo studies of angiogenesis fluorophore so that all pericytes, regardless of protein expression Male C57Bl/6 WT (Harlan,
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