title: The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating.
abstract: Nephropathy involves pathophysiological changes to the glomerulus. The primary glomerular endothelial cells (GEnCs) have emerged as an important tool for studying glomerulosclerotic mechanisms and in the screening process for drug-candidates. The success of the studies is dependent on the quality of the cell model. Therefore, we set out to establish an easy, reproducible model of the quiescent endothelial monolayer with the use of commercially available extracellular matrices (ECMs).
  
  
  
  Primary hGEnCs were seeded on various ECMs. Cell adhesion was monitored by an impedance sensing system. The localization of junctional proteins was assessed by immunofluorescence and the barrier function by passage of fluorescent dextrans and magnitude of VEGF response.
  
  
  
  All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70kDa dextrans which was 82% tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions (AJ).
  
  
  
  We recommend culturing hGEnCs on the mature glomerular basement membrane laminin - rhLN521 - which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined AJs and physiological perm-selectivity.
authors.affiliation: Global Research, Novo Nordisk A/S, Måløv, Denmark; Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark. Electronic address: kpaj@novonordisk.com.
authors.forename: Kamilla
authors.initials: K
authors.lastname: Pajęcka
authors.affiliation: Global Research, Novo Nordisk A/S, Måløv, Denmark.
authors.forename: Malik Nygaard
authors.initials: MN
authors.lastname: Nielsen
authors.affiliation: Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Troels Krarup
authors.initials: TK
authors.lastname: Hansen
authors.affiliation: Global Research, Novo Nordisk A/S, Måløv, Denmark.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Williams
mesh_terms: D002199:Capillary Permeability; D002448:Cell Adhesion; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible; D024141:Collagen Type IV / Q000737:chemistry; D003470:Culture Media; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology; D005109:Extracellular Matrix / Q000737:chemistry*; D005353:Fibronectins / Q000737:chemistry; D006801:Humans; D007678:Kidney Glomerulus / Q000098:blood supply; D007797:Laminin / Q000737:chemistry; D055806:Microvessels / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D020099:Coated Materials, Biocompatible; D024141:Collagen Type IV; D003470:Culture Media; D005353:Fibronectins; D007797:Laminin; D042461:Vascular Endothelial Growth Factor A
keywords: Endothelial; Glomerular; Laminin-521; Permeability; Quiescent; VE-cadherin