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

PLOS ONE, Feb 2019

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-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.

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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 - 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)


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Bradley M. Wertheim, Yi-Dong Lin, Ying-Yi Zhang, Andriy O. Samokhin, George A. Alba, Elena Arons, Paul B. Yu, Bradley A. Maron. Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials, PLOS ONE, 2019, Volume 14, Issue 2, DOI: 10.1371/journal.pone.0211909