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

doi:10.3389/fcell.2021.695684

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

Front Cell Dev Biol. 2021 Aug 12:9:695684. doi: 10.3389/fcell.2021.695684. eCollection 2021.

Authors

Renée J H A Tillie¹, Jenny De Bruijn¹, Javier Perales-Patón^{2

3}, Lieve Temmerman¹, Yanal Ghosheh⁴, Kim Van Kuijk¹, Marion J Gijbels^{1

5

6}, Peter Carmeliet^{7

8

9}, Klaus Ley^{4

10}, Julio Saez-Rodriguez^{2

3}, Judith C Sluimer^{1

11}

Affiliations

¹ Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Center, Maastricht, Netherlands.
² Faculty of Medicine, Institute for Computational Biomedicine, Heidelberg University Hospital, Heidelberg University, Heidelberg, Germany.
³ Institute of Experimental Medicine and Systems Biology, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany.
⁴ La Jolla Institute for Immunology, San Diego, CA, United States.
⁵ Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, Netherlands.
⁶ Department of Medical Biochemistry, Experimental Vascular Biology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.
⁷ Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, Center for Cancer Biology, Vlaams Instituut voor Biotechnologie (VIB), Leuven Cancer Institute, KU Leuven, Leuven, Belgium.
⁸ State Key Laboratory of Ophthalmology, Zhongshan Opthalmic Center, Sun Yat-sen University, Guangzhou, China.
⁹ Department of Biomedicine, Aarhus University, Aarhus, Denmark.
¹⁰ Department of Bioengineering, University of California, San Diego, San Diego, CA, United States.
¹¹ British Heart Foundation (BHF) Centre for Cardiovascular Sciences (CVS), University of Edinburgh, Edinburgh, United Kingdom.

Abstract

Background: The 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^+/-Pfkfb3 ^fl/fl (Pfkfb3 ^fl/fl ) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr^-/-LysMCre^+/-Pfkfb3 ^wt/wt control mice (Pfkfb3 ^wt/wt ).

Methods and results: Analysis 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. Pfkfb3 ^wt/wt and Pfkfb3 ^fl/fl mice were fed a 0.25% cholesterol diet for 12 weeks. Pfkfb3 ^fl/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 Pfkfb3 ^fl/fl compared to Pfkfb3 ^wt/wt BMDMs. Unexpectedly, plaque and necrotic core size, as well as macrophage (MAC3), neutrophil (Ly6G) and collagen (Sirius Red) content were unchanged in advanced Pfkfb3 ^fl/fl lesions. Similarly, early lesion plaque and necrotic core size and total plaque burden were unaffected.

Conclusion: Partial 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.

Keywords: PFKFB3; atherosclerosis; dendritic cell; glycolysis; glycolysis inhibition; macrophage; myeloid cells; neutrophil.