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miRNA-27a-3p and miRNA-222-3p as Novel Modulators of Phosphodiesterase 3a (PDE3A) in Cerebral Microvascular Endothelial Cells

  • S. Yasmeen
  • S. Kaur
  • A. H. Mirza
  • B. Brodin
  • F. Pociot
  • C. KruuseEmail author
Article

Abstract

Endothelial dysfunction is a key element in cerebral small vessel disease (CSVD), which may cause stroke and cognitive decline. Cyclic nucleotide signaling modulates endothelial function. The cyclic adenosine monophosphate-degrading enzyme phosphodiesterase 3 (PDE3) is an important treatment target which may be modulated by microRNAs (miRNAs) important for regulating gene expression. We aimed to identify PDE3-targeting miRNAs to highlight potential therapeutic targets for endothelial dysfunction and CSVD. PDE3-targeting miRNAs were identified by in silico analysis (TargetScan, miRWalk, miRanda, and RNA22). The identified miRNAs were ranked on the basis of TargetScan context scores and their expression (log2 read counts) in a human brain endothelial cell line (hCMEC/D3) described recently. miRNAs were subjected to co-expression meta-analysis (CoMeTa) to create miRNA clusters. The pathways targeted by the miRNAs were assigned functional annotations via the KEGG pathway and COOL. hCMEC/D3 cells were transfected with miRNA mimics miR-27a-3p and miR-222-3p, and the effect on PDE3A protein expression was analyzed by Western blotting. Only PDE3A is expressed in hCMEC/D3 cells. The in silico prediction identified 67 PDE3A-related miRNAs, of which 49 were expressed in hCMEC/D3 cells. Further analysis of the top two miRNA clusters (miR-221/miR-222 and miR-27a/miR-27b/miR-128) indicated a potential link to pathways relevant to cerebral and vascular integrity and repair. hCMEC/D3 cells transfected with miR-27a-3p and miR-222-3p mimics had reduced relative expression of PDE3A protein. PDE3A-related miRNAs miR-221/miR-222 and miR-27a/miR-27b/miR-128 are potentially linked to pathways essential for immune regulation as well as cerebral and vascular integrity/function. Furthermore, relative PDE3A protein expression was reduced by miR27a-3p and miR-222-3p.

Keywords

Small vessel disease microRNA PDE3 Stroke Endothelial cells 

Notes

Author Contributions

Study concept and design: CK, AHM, SY, SK, and FP; acquisition of data: SK, AHM and SY; analysis and interpretation of data: SY, SK, CK, FP and BB; drafting of the manuscript: SY; critical revision of the manuscript for important intellectual content: SY, SK, CK, FP, AHM, and BB; approval of final manuscript: CK, SY, SK, FP, BB and AHM; obtained funding: SY and CK; study supervision, CK, FP and BB.

Funding Information

This work was funded by the Herlev Research Council. S.Y. is supported by the Department of Neurology, Herlev University Hospital. Running costs are supported by the Aase and Ejnar Danielsens Foundation, the Fonden for Lægevidenskabens Fremme, the Novo Nordic Foundation, and Direktør Jacob Madsen og Hustru Olga Madsens Fond.

Compliance with Ethical Standards

Conflicts of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2018_1446_MOESM1_ESM.pdf (43 kb)
Supplementary Figures 1 and 2 (PDF 42.9 kb)
12035_2018_1446_MOESM2_ESM.xlsx (253 kb)
Supplementary Table 1 (XLSX 253 kb)
12035_2018_1446_MOESM3_ESM.pdf (42 kb)
Supplementary Table 2 (PDF 41 kb)

References

  1. 1.
    Nezu T, Hosomi N, Aoki S, Kubo S, Araki M, Mukai T, Takahashi T, Maruyama H et al (2015) Endothelial dysfunction is associated with the severity of cerebral small vessel disease. Hypertens Res 38(4):291–297PubMedCrossRefGoogle Scholar
  2. 2.
    Patterson CE, Lum H, Schaphorst KL, Verin AD, Garcia JN (2000) Regulation of endothelial barrier function by the cAMP-dependent protein kinase. Endothelium 7(4):287–308PubMedCrossRefGoogle Scholar
  3. 3.
    Surapisitchat J, Beavo JA (2011) Regulation of endothelial barrier function by cyclic nucleotides: the role of phosphodiesterases. Handb Exp Pharmacol 204:193–210CrossRefGoogle Scholar
  4. 4.
    Birk S, Edvinsson L, Olesen J, Kruuse C (2004) Analysis of the effects of phosphodiesterase type 3 and 4 inhibitors in cerebral arteries. Eur J Pharmacol 489(1–2):93–100PubMedCrossRefGoogle Scholar
  5. 5.
    de Donato G, Setacci F, Galzerano G, Mele M, Ruzzi U, Setacci C (2016) The use of cilostazol in patients with peripheral arterial disease: results of a national physician survey. J Cardiovasc Surg 57(3):457–465Google Scholar
  6. 6.
    Dinicolantonio JJ, Lavie CJ, Fares H, Menezes AR, O'Keefe JH, Bangalore S, Messerli FH (2013) Meta-analysis of cilostazol versus aspirin for the secondary prevention of stroke. Am J Cardiol 112(8):1230–1234PubMedCrossRefGoogle Scholar
  7. 7.
    Matsumoto M (2005) Cilostazol in secondary prevention of stroke: impact of the cilostazol stroke prevention study. Atheroscler Suppl 6(4):33–40PubMedCrossRefGoogle Scholar
  8. 8.
    Fukuhara S, Sakurai A, Sano H, Yamagishi A, Somekawa S, Takakura N, Saito Y, Kangawa K et al (2005) Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway. Mol Cell Biol 25(1):136–146PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Horai S, Nakagawa S, Tanaka K, Morofuji Y, Couraud PO, Deli MA, Ozawa M, Niwa M (2013) Cilostazol strengthens barrier integrity in brain endothelial cells. Cell Mol Neurobiol 33(2):291–307PubMedCrossRefGoogle Scholar
  10. 10.
    Sugiura Y, Morikawa T, Takenouchi T, Suematsu M, Kajimura M (2014) Cilostazol strengthens the endothelial barrier of postcapillary venules from the rat mesentery in situ. Phlebology 29(9):594–599PubMedCrossRefGoogle Scholar
  11. 11.
    Malik R, Dichgans M (2018) Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes. Nat Genet 50(4):524–537PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Arboix A (2011) Lacunar infarct and cognitive decline. Expert Rev Neurother 11(9):1251–1254PubMedCrossRefGoogle Scholar
  13. 13.
    Teng Z, Dong Y, Zhang D, An J, Lv P (2017) Cerebral small vessel disease and post-stroke cognitive impairment. Int J Neurosci 127(9):824–830PubMedCrossRefGoogle Scholar
  14. 14.
    Lee SJ, Lee JS, Choi MH, Lee SE, Shin DH, Hong JM (2017) Cilostazol improves endothelial function in acute cerebral ischemia patients: a double-blind placebo controlled trial with flow-mediated dilation technique. BMC Neurol 17(1):169PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Wang J, Chen J, Sen S (2016) MicroRNA as biomarkers and diagnostics. J Cell Physiol 231(1):25–30PubMedCrossRefGoogle Scholar
  16. 16.
    Rupaimoole R, Slack FJ (2017) MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov 16(3):203–222PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Kuehbacher A, Urbich C, Zeiher AM, Dimmeler S (2007) Role of dicer and Drosha for endothelial MicroRNA expression and angiogenesis. Circ Res 101(1):59–68PubMedCrossRefGoogle Scholar
  18. 18.
    Suarez Y, Fernandez-Hernando C, Pober JS, Sessa WC (2007) Dicer dependent microRNAs regulate gene expression and functions in human endothelial cells. Circ Res 100(8):1164–1173PubMedCrossRefGoogle Scholar
  19. 19.
    Keravis T, Lugnier C (2012) Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in various diseases and perspectives for future therapeutic developments. Br J Pharmacol 165(5):1288–1305PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Lugnier C (2006) Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents. Pharmacol Ther 109(3):366–398PubMedCrossRefGoogle Scholar
  21. 21.
    Houslay M (2015) Hypertension linked to PDE3A activation. Nat Genet 47(6):562–563PubMedCrossRefGoogle Scholar
  22. 22.
    Maass PG, Aydin A, Luft FC, Schachterle C, Weise A, Stricker S, Lindschau C, Vaegler M et al (2015) PDE3A mutations cause autosomal dominant hypertension with brachydactyly. Nat Genet 47(6):647–653PubMedCrossRefGoogle Scholar
  23. 23.
    Harndahl L, Wierup N, Enerback S, Mulder H, Manganiello VC, Sundler F, Degerman E, Ahren B et al (2004) Beta-cell-targeted overexpression of phosphodiesterase 3B in mice causes impaired insulin secretion, glucose intolerance, and deranged islet morphology. J Biol Chem 279(15):15214–15222PubMedCrossRefGoogle Scholar
  24. 24.
    Kwon SU, Cho YJ, Koo JS, Bae HJ, Lee YS, Hong KS, Lee JH, Kim JS (2005) Cilostazol prevents the progression of the symptomatic intracranial arterial stenosis: the multicenter double-blind placebo-controlled trial of cilostazol in symptomatic intracranial arterial stenosis. Stroke 36:782–786PubMedCrossRefGoogle Scholar
  25. 25.
    Gotoh F, Tohgi H, Hirai S, Terashi A, Fukuuchi Y, Otomo E, Shinohara Y, Itoh E et al (2000) Cilostazol stroke prevention study: a placebo-controlled double-blind trial for secondary prevention of cerebral infarction. J Stroke Cerebrovasc Dis 9(4):147–157PubMedCrossRefGoogle Scholar
  26. 26.
    Netherton SJ, Maurice DH (2005) Vascular endothelial cell cyclic nucleotide phosphodiesterases and regulated cell migration: implications in angiogenesis. Mol Pharmacol 67(1):263–272PubMedCrossRefGoogle Scholar
  27. 27.
    Hori M (2007) The phosphodiesterase 4D gene for early onset ischemic stroke among normotensive patients. Stroke 5(2):436–438Google Scholar
  28. 28.
    Lum H, Malik AB (1996) Mechanisms of increased endothelial permeability. Can J Physiol Pharmacol 74(7):787–800PubMedGoogle Scholar
  29. 29.
    Cosin-Tomas M, Antonell A, Llado A, Alcolea D, Fortea J, Ezquerra M, Lleo A, Marti MJ et al (2017) Plasma miR-34a-5p and miR-545-3p as early biomarkers of Alzheimer’s disease: potential and limitations. Mol Neurobiol 54(7):5550–5562PubMedCrossRefGoogle Scholar
  30. 30.
    Baulina N, Kulakova O, Kiselev I, Osmak G, Popova E, Boyko A, Favorova O (2018) Immune-related miRNA expression patterns in peripheral blood mononuclear cells differ in multiple sclerosis relapse and remission. J Neuroimmunol 317:67–76PubMedCrossRefGoogle Scholar
  31. 31.
    Topol A, Zhu S, Hartley BJ, English J, Hauberg ME, Tran N, Rittenhouse CA, Simone A et al (2017) Dysregulation of miRNA-9 in a subset of schizophrenia patient-derived neural progenitor cells. Cell Rep 20(10):2525PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Dweep H, Sticht C, Pandey P, Gretz N (2011) miRWalk – database: prediction of possible miRNA binding sites by “walking” the genes of three genomes. J Biomed Inform 44(5):839–847PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Gennarino VA, D'Angelo G, Dharmalingam G, Fernandez S, Russolillo G, Sanges R, Mutarelli M, Belcastro V et al (2012) Identification of microRNA-regulated gene networks by expression analysis of target genes. Genome Res 22(6):1163–1172PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Li J, Zhao Y, Lu Y, Ritchie W, Grau G, Vadas MA, Gamble JR (2016) The poly-cistronic miR-23-27-24 complexes target endothelial cell junctions: differential functional and molecular effects of miR-23a and miR-23b. Mol Ther–Nucleic Acids 5(8):e354PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Cerutti C, Edwards LJ, de Vries HE, Sharrack B, Male DK, Romero IA (2017) MiR-126 and miR-126* regulate shear-resistant firm leukocyte adhesion to human brain endothelium. Sci Rep 7:45284PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H, Nicotra A et al (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19(13):1872–1874PubMedCrossRefGoogle Scholar
  37. 37.
    Agarwal V, Bell GW, Nam J-W, Bartel DP (2015) Predicting effective microRNA target sites in mammalian mRNAs. eLife 4:e05005PubMedCentralCrossRefGoogle Scholar
  38. 38.
    Kalari KR, Thompson KJ, Nair AA, Tang X, Bockol MA, Jhawar N, Swaminathan SK, Lowe VJ et al (2016) BBBomics-human blood brain barrier transcriptomics hub. Front Neurosci 10:71PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Ortega FJ, Mercader JM, Moreno-Navarrete JM, Rovira O, Guerra E, Esteve E, Xifra G, Martínez C et al (2014) Profiling of circulating microRNAs reveals common microRNAs linked to type 2 diabetes that change with insulin sensitization. Diabetes Care 37(5):1375–1383PubMedCrossRefGoogle Scholar
  40. 40.
    Karolina DS, Tavintharan S, Armugam A, Sepramaniam S, Pek SL, Wong MT, Lim SC, Sum CF et al (2012) Circulating miRNA profiles in patients with metabolic syndrome. J Clin Endocrinol Metab 97(12):E2271–E2276PubMedCrossRefGoogle Scholar
  41. 41.
    Wang L, Bao H, Wang KX, Zhang P, Yao QP, Chen XH, Huang K, Qi YX et al (2017) Secreted miR-27a induced by cyclic stretch modulates the proliferation of endothelial cells in hypertension via GRK6. Sci Rep 7:41058PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Erener S, Marwaha A, Tan R, Panagiotopoulos C, Kieffer TJ (2017) Profiling of circulating microRNAs in children with recent onset of type 1 diabetes. JCI Insight 2(4):e89656PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Herrera BM, Lockstone HE, Taylor JM, Ria M, Barrett A, Collins S, Kaisaki P, Argoud K et al (2010) Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes. Diabetologia 53(6):1099–1109PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Meng S, Cao JT, Zhang B, Zhou Q, Shen CX, Wang CQ (2012) Downregulation of microRNA-126 in endothelial progenitor cells from diabetes patients, impairs their functional properties, via target gene Spred-1. J Mol Cell Cardiol 53(1):64–72PubMedCrossRefGoogle Scholar
  45. 45.
    Nielsen LB, Wang C, Sorensen K, Bang-Berthelsen CH, Hansen L, Andersen ML, Hougaard P, Juul A et al (2012) Circulating levels of microRNA from children with newly diagnosed type 1 diabetes and healthy controls: evidence that miR-25 associates to residual beta-cell function and glycaemic control during disease progression. Exp Diabetes Res 2012:896362PubMedPubMedCentralGoogle Scholar
  46. 46.
    Topakian R, Barrick TR, Howe FA, Markus HS (2010) Blood-brain barrier permeability is increased in normal-appearing white matter in patients with lacunar stroke and leucoaraiosis. J Neurol Neurosurg Psychiatry 81(2):192–197PubMedCrossRefGoogle Scholar
  47. 47.
    Wardlaw JM, Farrall A, Armitage PA, Carpenter T, Chappell F, Doubal F, Chowdhury D, Cvoro V et al (2008) Changes in background blood-brain barrier integrity between lacunar and cortical ischemic stroke subtypes. Stroke 39(4):1327–1332PubMedCrossRefGoogle Scholar
  48. 48.
    Urabe T (2013) Cilostazol strengthens barrier integrity in brain endothelial cells. Neurosci Res 33(2):291–307Google Scholar
  49. 49.
    Liu S, Yu C, Yang F, Paganini-Hill A, Fisher MJ (2012) Phosphodiesterase inhibitor modulation of brain microvascular endothelial cell barrier properties. J Neurol Sci 320(1–2):45–51PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Uchiyama S (2009) Stroke prevention by cilostazol in patients with atherothrombosis: meta-analysis of placebo-controlled randomized trials. J Stroke Cerebrovasc Dis 18(6):482–90Google Scholar
  51. 51.
    Shi L, Pu J, Xu L, Malaguit J, Zhang J, Chen S (2014) The efficacy and safety of cilostazol for the secondary prevention of ischemic stroke in acute and chronic phases in Asian population- an updated meta-analysis. BMC Neurol 14:251PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Liu X, Cheng Y, Zhang S, Lin Y, Yang J, Zhang C (2009) A necessary role of miR-221 and miR-222 in vascular smooth muscle cell proliferation and neointimal hyperplasia. Circ Res 104(4):476–487PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Poliseno L, Tuccoli A, Mariani L, Evangelista M, Citti L, Woods K, Mercatanti A, Hammond S et al (2006) MicroRNAs modulate the angiogenic properties of HUVECs. Blood 108(9):3068–3071PubMedCrossRefGoogle Scholar
  54. 54.
    Zhu N, Zhang D, Chen S, Liu X, Lin L, Huang X, Guo Z, Liu J et al (2011) Endothelial enriched microRNAs regulate angiotensin II-induced endothelial inflammation and migration. Atherosclerosis 215(2):286–293PubMedCrossRefGoogle Scholar
  55. 55.
    Dentelli P, Rosso A, Orso F, Olgasi C, Taverna D, Brizzi MF (2010) microRNA-222 controls neovascularization by regulating signal transducer and activator of transcription 5A expression. Arterioscler Thromb Vasc Biol 30(8):1562–1568PubMedCrossRefGoogle Scholar
  56. 56.
    Urbich C, Kaluza D, Fromel T, Knau A, Bennewitz K, Boon RA, Bonauer A, Doebele C et al (2012) MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A. Blood 119(6):1607–1616PubMedCrossRefGoogle Scholar
  57. 57.
    Zhou Q, Gallagher R, Ufret-Vincenty R, Li X, Olson EN, Wang S (2011) Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23∼27∼24 clusters. Proc Natl Acad Sci 108(20):8287–8292PubMedCrossRefGoogle Scholar
  58. 58.
    Sepramaniam S, Tan J-R, Tan K-S, DeSilva DA, Tavintharan S, Woon F-P, Wang C-W, Yong F-L et al (2014) Circulating microRNAs as biomarkers of acute stroke. Int J Mol Sci 15(1):1418–1432PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Sabirzhanov B, Zhao Z, Stoica BA, Loane DJ, Wu J, Borroto C, Dorsey SG, Faden AI (2014) Downregulation of miR-23a and miR-27a following experimental traumatic brain injury induces neuronal cell death through activation of proapoptotic Bcl-2 proteins. J Neurosci 34(30):10055–10071PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Sala Frigerio C, Lau P, Salta E, Tournoy J, Bossers K, Vandenberghe R, Wallin A, Bjerke M et al (2013) Reduced expression of hsa-miR-27a-3p in CSF of patients with Alzheimer disease. Neurology 81(24):2103–2106PubMedCrossRefGoogle Scholar
  61. 61.
    Regev K, Paul A, Healy B, von Glenn F, Diaz-Cruz C, Gholipour T, Mazzola MA, Raheja R et al (2016) Comprehensive evaluation of serum microRNAs as biomarkers in multiple sclerosis. Neurol Neurophysiol Neurosci 3(5):e267Google Scholar
  62. 62.
    Sorensen SS, Nygaard AB, Carlsen AL, Heegaard NHH, Bak M, Christensen T (2017) Elevation of brain-enriched miRNAs in cerebrospinal fluid of patients with acute ischemic stroke. Biomarker Res 5:24CrossRefGoogle Scholar
  63. 63.
    Sorensen SS, Nygaard AB, Nielsen MY, Jensen K, Christensen T (2014) miRNA expression profiles in cerebrospinal fluid and blood of patients with acute ischemic stroke. Transl Stroke Res 5(6):711–718PubMedCrossRefGoogle Scholar
  64. 64.
    Dharap A, Bowen K, Place R, Li LC, Vemuganti R (2009) Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome. J Cereb Blood Flow Metab 29(4):675–687PubMedPubMedCentralCrossRefGoogle Scholar
  65. 65.
    Jeyaseelan K, Lim KY, Armugam A (2008) MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke 39(3):959–966PubMedCrossRefGoogle Scholar
  66. 66.
    Malik R, Dichgans M, A.F. Consortium, H. Cohorts for, C. Aging Research in Genomic Epidemiology, C. International Genomics of Blood Pressure, I. Consortium, Starnet, G. BioBank Japan Cooperative Hospital, C. Consortium, E.-C. Consortium, E.P.-I. Consortium, C. International Stroke Genetics, M. Consortium, C.C. Neurology Working Group of the, N.S.G. Network, U.K.Y.L.D. Study, M. Consortium, M. Consortium (2018) Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes. Nat Genet 50(4):524–537PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Daige CL, Wiggins JF, Priddy L, Nelligan-Davis T, Zhao J, Brown D (2014) Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer. Mol Cancer Ther 13(10):2352–2360PubMedCrossRefGoogle Scholar
  68. 68.
    Gebert LFR, Rebhan MAE, Crivelli SEM, Denzler R, Stoffel M, Hall J (2014) Miravirsen (SPC3649) can inhibit the biogenesis of miR-122. Nucleic Acids Res 42(1):609–621PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • S. Yasmeen
    • 1
    • 2
  • S. Kaur
    • 3
    • 4
  • A. H. Mirza
    • 2
    • 3
  • B. Brodin
    • 2
    • 5
  • F. Pociot
    • 2
    • 3
    • 4
  • C. Kruuse
    • 1
    • 2
  1. 1.Stroke Unit and Neurovascular Research Unit, Department of NeurologyHerlev and Gentofte HospitalHerlevDenmark
  2. 2.Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark
  3. 3.Pediatric DepartmentHerlev University HospitalHerlevDenmark
  4. 4.Steno Diabetes Center CopenhagenGentofteDenmark
  5. 5.CNS Drug Delivery and Barrier ModellingUniversity of CopenhagenCopenhagenDenmark

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