Partial Inhibition of the 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase-3 (PFKFB3) Enzyme in Myeloid Cells Does Not Affect Atherosclerosis.

Renée J H A Tillie,Klaus Ley,Javier Perales-Patón,Julio Saez-Rodriguez,Kim Van Kuijk,Marion J Gijbels,Yanal Ghosheh,Lieve Temmerman,Peter Carmeliet,Judith C Sluimer,Jenny De Bruijn

doi:10.3389/fcell.2021.695684

Abstract

BackgroundThe protein 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a key stimulator of glycolytic flux. Systemic, partial PFKFB3 inhibition previously decreased total plaque burden and increased plaque stability. However, it is unclear which cell type conferred these positive effects. Myeloid cells play an important role in atherogenesis, and mainly rely on glycolysis for energy supply. Thus, we studied whether myeloid inhibition of PFKFB3-mediated glycolysis in Ldlr–/–LysMCre+/–Pfkfb3fl/fl (Pfkfb3fl/fl) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr–/–LysMCre+/–Pfkfb3wt/wt control mice (Pfkfb3wt/wt).Methods and ResultsAnalysis of atherosclerotic human and murine single-cell populations confirmed PFKFB3/Pfkfb3 expression in myeloid cells, but also in lymphocytes, endothelial cells, fibroblasts and smooth muscle cells. Pfkfb3wt/wt and Pfkfb3fl/fl mice were fed a 0.25% cholesterol diet for 12 weeks. Pfkfb3fl/fl bone marrow-derived macrophages (BMDMs) showed 50% knockdown of Pfkfb3 mRNA. As expected based on partial glycolysis inhibition, extracellular acidification rate as a measure of glycolysis was partially reduced in Pfkfb3fl/fl compared to Pfkfb3wt/wt BMDMs. Unexpectedly, plaque and necrotic core size, as well as macrophage (MAC3), neutrophil (Ly6G) and collagen (Sirius Red) content were unchanged in advanced Pfkfb3fl/fl lesions. Similarly, early lesion plaque and necrotic core size and total plaque burden were unaffected.ConclusionPartial myeloid knockdown of PFKFB3 did not affect atherosclerosis development in advanced or early lesions. Previously reported positive effects of systemic, partial PFKFB3 inhibition on lesion stabilization, do not seem conferred by monocytes, macrophages or neutrophils. Instead, other Pfkfb3-expressing cells in atherosclerosis might be responsible, such as DCs, smooth muscle cells or fibroblasts.

Highlights

Myeloid cells [i.e., monocytes, macrophages, neutrophils and dendritic cells (DCs)] play an active role in atherogenesis
We studied the hypothesis that myeloid inhibition of PFKFB3-mediated glycolysis in LDL receptor knockout (Ldlr−/−)LysMCre+/−Pfkfb3f l/fl (Pfkfb3f l/fl) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr−/−LysMCre+/−Pfkfb3wt/wt control mice (Pfkfb3wt/wt)
Single-cell RNA-sequencing datasets from atherosclerotic plaques were collected from Gene Expression Omnibus (GEO) database or requested to corresponding authors: Wirka et al, 2019 (4 human specimens, GSE131780), Zernecke et al, 2020, and van Kuijk et al, 2021 (11 pooled Ldlr−/− LysMCre+/− mice, GSE150089)

Summary

Introduction

Myeloid cells [i.e., monocytes, macrophages, neutrophils and dendritic cells (DCs)] play an active role in atherogenesis. LDL is oxidized (oxLDL) by reactive oxygen species (ROS) and enzymes (Tabas et al, 2007) This results in a pro-inflammatory response that triggers myeloid cell recruitment (Moore and Tabas, 2011; Silvestre-Roig et al, 2020). Serine proteases and NETs by macrophages and neutrophils can cause fibrous cap thinning (Moore and Tabas, 2011; SilvestreRoig et al, 2020). This increases the risk of plaque rupture, which can have detrimental consequences. We studied whether myeloid inhibition of PFKFB3-mediated glycolysis in Ldlr−/−LysMCre+/−Pfkfb3f l/fl (Pfkfb3f l/fl) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr−/−LysMCre+/−Pfkfb3wt/wt control mice (Pfkfb3wt/wt)

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Discussion

Conclusion