Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials

PloS One
Bradley M WertheimBradley A Maron

Abstract

Transcriptomic analysis of pulmonary microvascular endothelial cells from experimental models offers insight into pulmonary arterial hypertension (PAH) pathobiology. However, culturing may alter the molecular profile of endothelial cells prior to analysis, limiting the translational relevance of results. Here we present a novel and validated method for isolating RNA from pulmonary microvascular endothelial cells (PMVECs) ex vivo that does not require cell culturing. Initially, presumed rat PMVECs were isolated from rat peripheral lung tissue using tissue dissociation and enzymatic digestion, and cells were cultured until confluence to assess endothelial marker expression. Anti-CD31, anti-von Willebrand Factor, and anti-α-smooth muscle actin immunocytochemistry/immunofluorescence signal was detected in presumed rat PMVECs, but also in non-endothelial cell type controls. By contrast, flow cytometry using an anti-CD31 antibody and isolectin 1-B4 (from Griffonia simplicifolia) was highly specific for rat PMVECs. We next developed a strategy in which the addition of an immunomagnetic selection step for CD31+ cells permitted culture-free isolation of rat PMVECs ex vivo for RNA isolation and transcriptomic analysis using fluorescence-...Continue Reading

References

Jan 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·D E MaddoxI J Goldstein
Oct 1, 1994·The American Journal of Physiology·J C MageeK S Guice
Jun 4, 2008·Annals of the New York Academy of Sciences·Peter Baluk, Donald M McDonald
Jun 13, 2008·Nature Protocols·Judy R van BeijnumArjan W Griffioen
Aug 2, 2008·Molecular & Cellular Proteomics : MCP·Lisa BerglundMathias Uhlén
Mar 24, 2009·American Journal of Physiology. Lung Cellular and Molecular Physiology·Melane L FehrenbachHorace M Delisser
Mar 24, 2010·Journal of Immunological Methods·Kazuaki KajimotoHideyoshi Harashima
Jun 15, 2010·European Journal of Medicinal Chemistry·Marcin Sobczak
Dec 24, 2010·Journal of Visualized Experiments : JoVE·Magdalena SobczakMagdalena Chrzanowska-Wodnicka
Aug 27, 2011·Circulation Research·Rasa TamosiunieneMark R Nicolls
Nov 1, 2011·Annual Review of Physiology·Ching-Pin Chang, Benoit G Bruneau
Feb 4, 2012·Journal of Molecular and Cellular Cardiology·Jinghai Chen, Da-Zhi Wang
Mar 20, 2012·American Journal of Respiratory Cell and Molecular Biology·Suzy A A ComhairSerpil C Erzurum
May 21, 2013·The Journal of Heart and Lung Transplantation : the Official Publication of the International Society for Heart Transplantation·Jonathan B PollettMichael J Passineau
Feb 1, 2014·Nature·Francis S Collins, Lawrence A Tabak
Mar 31, 2015·American Journal of Physiology. Lung Cellular and Molecular Physiology·Mikko H HeleniusTero-Pekka Alastalo
Oct 9, 2015·Nature Protocols·Rajesh S AlphonseBernard Thébaud
Jan 14, 2016·American Journal of Respiratory and Critical Care Medicine·Robert SzulcekHarm J Bogaard
Jul 11, 2016·Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology·Constantin AdamsJoachim Riethmueller
Oct 28, 2016·Angiogenesis·Aigul MoldobaevaElizabeth M Wagner
Mar 24, 2017·Biological Procedures Online·Yan LuLing/L Xu
Mar 31, 2017·F1000Research·Jan L A Voskuil
Jul 7, 2017·Pulmonary Circulation·Christa Gaskill, Susan M Majka
Feb 2, 2018·American Journal of Physiology. Lung Cellular and Molecular Physiology·Karthik SureshLarissa A Shimoda
Jun 15, 2018·Science Translational Medicine·Andriy O SamokhinBradley A Maron

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Citations

Dec 12, 2019·Frontiers in Cellular Neuroscience·Xiaojuan SuDezhi Mu
Jan 19, 2020·The Journal of Heart and Lung Transplantation : the Official Publication of the International Society for Heart Transplantation·Andriy O SamokhinBradley A Maron

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Methods Mentioned

BETA
flow cytometry
ELISA
fluorescence activated cell sorting
phase contrast microscopy
confocal microscopy
enzymatic dissociation
FACS
fluorescence-activated cell sorting
flow cytometry gating

Software Mentioned

Image J
Origin Pro

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