05 March 2019

PVRI-funded manuscript published in PLOS one

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From left to right: Bradley Wertheim, Bradley Maron, Stephanie Barwick and Katharine Clapham

The PVRI is pleased to announce that our fellowship awardee, Bradley Wertheim, has published a manuscript in PLOS One under the leadership of Bradley Maron at Brigham and Women's Hospital, Harvard, USA.

A brief note from Bradley to describe his work:

As a 2017 PVRI Basic Science Fellowship awardee, I study the pathophysiological and pathobiological differences between early and advanced stage pulmonary arterial hypertension (PAH), under the mentorship of Dr Bradley Maron. The overarching objective of this project is to identify treatment targets for PAH patients early in the course of the disease. My project has converging aims:

  1. Describe the impact of mild elevation in pulmonary artery pressure on right ventricle-pulmonary vascular function and morphology, and
  2. Identify and validate novel transcriptomic mediators of endothelial dysfunction that are unique to early-stage PAH. 

 

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-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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