Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials
February
Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials
Bradley M. WertheimID 0 1
Yi-Dong Lin 1
Ying-Yi Zhang 1
Andriy O. Samokhin 1
George A. Alba 1
Elena Arons 1
Paul B. Yu 1
Bradley A. Maron 1
0 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital , Boston, MA , United States of America, 2 Department of Medicine, Division of Cardiovascular Medicine, Brigham and Women's Hospital , Boston, MA , United States of America, 3 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital , Boston, MA , United States of America
1 Editor: James West, Vanderbilt University Medical Center , UNITED STATES
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Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
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-?-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-activated cell sorting. Heterogeneity in the validity and reproducibility of results using
commercial antibodies against endothelial surface markers corresponded to a substantial
burden on laboratory time, labor, and scientific budget. We demonstrate a novel protocol for
the culture-free isolation and transcriptomic analysis of rat PMVECs with translational
relevance to PAH. In doing so, we highlight wide variability in the quality of commonly used
biological reagents, which emphasizes the importance of investigator-initiated validation of
commercial biomaterials.
Foundation, Cardiovascular Medicine Educational
Research Foundation [CMREF]). 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.
Abbreviations: a.u., arbitrary units; Ab, antibody;
AF 488, Alexa Fluor 488; AF 647, Alexa Fluor 647;
DAB, 3, 3?-diaminobenzidine; GS-IB4, isolectin 1-B4
from Griffonia simplicifolia; HLF, human lung
fibroblast; HPAEC, human pulmonary artery
endothelial cell; HPASMC, human pulmonary artery
smooth muscle cell; ICC, immunocytochemistry;
IF, immunofluorescence; PAH, pulmonary arterial
hypertension; PE, phycoerythrin; PMVEC, rat
pulmonary microvascular endothelial cell; MCT,
monocrotaline; RECA-1, rat endothelial cell
antigen-1; RIN, RNA integrity number; RLF, rat
lung fibroblast; RPAEC, rat pulmonary artery
endothelial cell; RPASMC, rat pulmonary artery
smooth muscle cell; S.E., standard error; vWF, von
Willebrand factor; ?-SMA, ?-smooth muscle actin.
Introduction
Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by
dysregulated transcriptional mechanisms that promote endothelial dysfunction [
1
]. Studying
pulmonary artery endothelial cells (PAECs) from PAH patients is optimal, but access is limited,
in part, by low disease prevalence and technical obstacles [
2,3
]. Therefore, studying PAECs
from PAH animal models offers an important and well-established alternative approach to
analyzing disease-specific pathobiological mechanisms [4]. Protocols for isolating primary PAECs
from PAH models have been reported previously, but these strategies require passaging cells in
vitro to ensure a sufficient population for further analysis [
5?19
]. However, sequential passaging
may alter the phenotype and molecular program of cells [
20
]. Effective cell isolation without
serial passaging is possible,[
16
] but has not been reported for rodent PAECs.
Limited reproducibility of published scientific results has led to an emerging initiative
among funding sponsors, including the National Institutes of Health, that emphasizes data
quality [
21,22
]. The widespread availability of commercial bio (...truncated)