A Simplified Procedure to Isolate Endothelial Cells From Pulmonary Arterial Blood

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Abstract RATIONALE. Pulmonary arterial hypertension (PAH) is a progressive condition marked by increased pressures in the pulmonary arteries, which can lead to right heart failure and death. Research into the mechanisms of PAH is challenging since a) animal models fail to precisely recapitulate the disease, providing limited insight into its pathobiology, and b) cellular material from PAH patients represent end-stage disease, i.e. donor lungs obtained during autopsies or transplants. Consequently, there is a pressing need for tools that can clarify the mechanisms of PAH in individual patients, which is essential for tailoring treatment strategies. METHODS. We have developed and refined methods to isolate circulating endothelial cells (CEC) from PAH patients who undergo right heart catheterization for the diagnosis or follow-up of the disease. Our simplified procedure requires less than an hour of processing time. CECs are isolated from the buffy coats of about 12 ml of blood obtained from the pulmonary artery. The CECs are then cultured under adequate conditions. We tested the endothelial phenotype with flow cytometry, tube formation and transcriptomics. RESULTS. The CECs isolated from PAH patients are CD31+ / CD144+ and produce capillary-like structures, i.e. tubes, when cultured on a basement membrane extract hydrogel. We compared the transcriptomic analysis of these CECs with commercially available endothelial cells from pulmonary arteries of “healthy” lungs (cPAEC) and endothelial cells from pulmonary arteries of PAH patients (PAH-PAECs) obtained by our team from explanted lungs. CECs showed expression of typical endothelial cell markers such as PECAM1, VWF, VEGFR2 and CLDN5. In addition, we observed the characteristic phenotype associated with PAH, including higher expression of NEDD9, DKK1, IL-8 and ENTRD1. CONCLUSIONS. Using a relatively simple methodology we isolated CECs from over 100 patients with PAH, with about 80% success rate. CECs carry the characteristic phenotypic abnormalities seen in other cell models of PAH. These CECs are a promising model to test critical mechanisms of disease ex-vivo, that could help better understand the pathogenesis and guide therapeutic decisions in PAH.

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