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Title: The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating

Abstract

Background and aims: 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). Methods: 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. Results: 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 70 kDa 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). Conclusion: We recommend culturing hGEnCs on the mature glomerular basement membrane laminin - rhLN521 – which, as the only commercially available ECM,more » promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined AJs and physiological perm-selectivity. - Highlights: • rhLN521, rhLN511 and hFN assure physiologically relevant permeability. • rhLN521 and rhLN511 ensure best cell morphology and adherens junction formation. • Collagen IV and I based coating results in disorganized hGEnC monolayer. • Physiologically relevant ECM may lead to down-regulation of self-produced matrices.« less

Authors:
 [1];  [2];  [1];  [3];  [1]
  1. Global Research, Novo Nordisk A/S, Måløv (Denmark)
  2. (Denmark)
  3. Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus (Denmark)
Publication Date:
OSTI Identifier:
22649849
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 353; Journal Issue: 1; Other Information: Copyright (c) 2017 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ADHESION; ANIMAL TISSUES; BUFFERS; CELL PROLIFERATION; COLLAGEN; COMPARATIVE EVALUATIONS; DEXTRAN; GROWTH FACTORS; IN VITRO; INDEXES; MEMBRANES; MORPHOLOGY; PERMEABILITY; PHOSPHATES; PLANT TISSUES; POLYMERASE CHAIN REACTION; SCREENING; SUBSTRATES; VENTILATION BARRIERS

Citation Formats

Pajęcka, Kamilla, E-mail: kpaj@novonordisk.com, Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Nielsen, Malik Nygaard, Hansen, Troels Krarup, and Williams, Julie M. The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating. United States: N. p., 2017. Web. doi:10.1016/J.YEXCR.2017.02.039.
Pajęcka, Kamilla, E-mail: kpaj@novonordisk.com, Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Nielsen, Malik Nygaard, Hansen, Troels Krarup, & Williams, Julie M. The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating. United States. doi:10.1016/J.YEXCR.2017.02.039.
Pajęcka, Kamilla, E-mail: kpaj@novonordisk.com, Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Nielsen, Malik Nygaard, Hansen, Troels Krarup, and Williams, Julie M. Sat . "The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating". United States. doi:10.1016/J.YEXCR.2017.02.039.
@article{osti_22649849,
title = {The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating},
author = {Pajęcka, Kamilla, E-mail: kpaj@novonordisk.com and Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus and Nielsen, Malik Nygaard and Hansen, Troels Krarup and Williams, Julie M.},
abstractNote = {Background and aims: 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). Methods: 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. Results: 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 70 kDa 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). Conclusion: 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. - Highlights: • rhLN521, rhLN511 and hFN assure physiologically relevant permeability. • rhLN521 and rhLN511 ensure best cell morphology and adherens junction formation. • Collagen IV and I based coating results in disorganized hGEnC monolayer. • Physiologically relevant ECM may lead to down-regulation of self-produced matrices.},
doi = {10.1016/J.YEXCR.2017.02.039},
journal = {Experimental Cell Research},
number = 1,
volume = 353,
place = {United States},
year = {Sat Apr 01 00:00:00 EDT 2017},
month = {Sat Apr 01 00:00:00 EDT 2017}
}