Deeper insights into heart failure with preserved ejection fraction through RNA-sequencing of circulating endothelial cells - advanced protocol establishment enables complete transcriptome analysis

Heart failure with preserved ejection fraction (HFpEF) continues to pose a significant challenge with a bleak prognosis and a rising incidence, particularly among the aging demographic in western countries. An in-depth comprehension of the cellular-level pathogenesis, with a specific emphasis on the pivotal role of cellular senescence in endothelial dysfunction, is imperative for the evolution of innovative therapeutic interventions. Many previous approaches to investigate the genesis of HFpEF are based only on HFpEF-mouse-models. The isolation of circulating endothelial cells from peripheral blood samples represents a feasible, less invasive way to study endothelial dysfunction, a central component of HFpEF-pathogenesis, in human material.

This study focuses on isolating circulating endothelial cells (CEC) to address endothelial dysfunction and pinpoint novel therapeutic targets through a comprehensive whole transcriptome analysis. Methodologically, CEC isolation from whole blood samples was achieved using a newly established fluorescence-activated cell sorting (FACS) panel. HFpEF patients were recruited based on their HFA-PEFF score, with non-diseased controls comprising both young and old subjects matched for sex and age. Subsequently, the isolated CECs were then subjected to transcriptome analysis.

Our new protocol is based on red blood cell lysis followed by magnetic cell sorting to deplete CD45+ cells. This is followed by staining with fluorescent-conjugated monoclonal antibodies. This FACS panel includes known endothelial-specific markers including DAPI, CD11b and CD45 as negative, CD146, CD34 and CD31 as positive markers. The prepared cells were isolated using FACS and a specially developed gating. We isolated the RNA in preparation for sequencing. Our protocol was validated by immunofluorescence staining and PCR analysis. 

In summary, we have successfully established a new, robust and highly targeted workflow for consistently isolating circulating endothelial cells from blood and performing RNA sequencing on the collected samples. This is a key advance towards a deeper understanding of the pathophysiology of heart failure with preserved ejection fraction. The application of our methodology enables the characterization of patients with HFpEF at the transcriptome level. The whole transcriptome analyses provides potential starting points for further in vitro and in vivo studies. As we extend our investigations to larger patient cohorts in the future, there is potential for the identification of novel therapeutic targets.