Murine sca1/flk1-positive cells are not endothelial progenitor cells, but B2 lymphocytes

Eva Steffen,Andreas Zietzer,Sven Wassmann,Tienush Rassaf,Nikos Werner,Wolfgang Bernd Edziu Mayer Von Wittgenstein,Sebastian Zimmer,Marie Hennig,Georg Nickenig,Sven Thomas Niepmann,Martin Steinmetz

doi:10.1007/s00395-020-0774-6

Abstract

Circulating sca1+/flk1+ cells are hypothesized to be endothelial progenitor cells (EPCs) in mice that contribute to atheroprotection by replacing dysfunctional endothelial cells. Decreased numbers of circulating sca1+/flk1+ cells correlate with increased atherosclerotic lesions and impaired reendothelialization upon electric injury of the common carotid artery. However, legitimate doubts remain about the identity of the putative EPCs and their contribution to endothelial restoration. Hence, our study aimed to establish a phenotype for sca1+/flk1+ cells to gain a better understanding of their role in atherosclerotic disease. In wild-type mice, sca1+/flk1+ cells were mobilized into the peripheral circulation by granulocyte-colony stimulating factor (G-CSF) treatment and this movement correlated with improved endothelial regeneration upon carotid artery injury. Multicolor flow cytometry analysis revealed that sca1+/flk1+ cells predominantly co-expressed surface markers of conventional B cells (B2 cells). In RAG2-deficient mice and upon B2 cell depletion, sca1+/flk1+ cells were fully depleted. In the absence of monocytes, sca1+/flk1+ cell levels were unchanged. A PCR array focused on cell surface markers and next-generation sequencing (NGS) of purified sca1+/flk1+ cells confirmed their phenotype to be predominantly that of B cells. Finally, the depletion of B2 cells, including sca1+/flk1+ cells, in G-CSF-treated wild-type mice partly abolished the endothelial regenerating effect of G-CSF, indicating an atheroprotective role for sca1+/flk1+ B2 cells. In summary, we characterized sca1+/flk1+ cells as a subset of predominantly B2 cells, which are apparently involved in endothelial regeneration.

Highlights

Damage of the endothelium is a main feature of a variety of vascular diseases including atherosclerosis or upon percutaneous transluminal angioplasty
Mouse-specific subset of circulating endothelial progenitor cells (EPCs) is defined as sca1+/flk1+ cells
We inflicted an electric injury of the common carotid artery on some of the mice to assess endothelial regeneration

Summary

Introduction

Damage of the endothelium is a main feature of a variety of vascular diseases including atherosclerosis or upon percutaneous transluminal angioplasty. Such damage leads to endothelial dysfunction and the deterioration of the endothelial cells, which can further result in plaque progression or restenosis following interventional treatment [5, 12]. It has been hypothesized that the endothelium is restored in loco by proliferation of neighboring endothelial cells (ECs) This paradigm has been challenged by Asahara and a plethora of later studies, which proposed that circulating endothelial progenitor cells (EPCs) significantly contribute to reendothelialization [1]. Those putative EPCs have been associated with endothelial restoration and have been shown

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