Distinct role of mitochondrial function and protein kinase C in intimal and medial calcification in vitro

Marina A Heuschkel; Anne Babler; Jonas Heyn; Emiel P C van der Vorst; Marja Steenman; Maren Gesper; Ben A Kappel; David Magne; Yann Gouëffic; Rafael Kramann; Willi Jahnen-Dechent; Nikolaus Marx; Thibaut Quillard; Claudia Goettsch

doi:10.3389/fcvm.2022.959457

Distinct role of mitochondrial function and protein kinase C in intimal and medial calcification in vitro

Front Cardiovasc Med. 2022 Sep 20:9:959457. doi: 10.3389/fcvm.2022.959457. eCollection 2022.

Authors

Marina A Heuschkel¹, Anne Babler², Jonas Heyn¹, Emiel P C van der Vorst^{3

4

5

6}, Marja Steenman⁷, Maren Gesper¹, Ben A Kappel¹, David Magne⁸, Yann Gouëffic⁹, Rafael Kramann^{2

10

11}, Willi Jahnen-Dechent¹², Nikolaus Marx¹, Thibaut Quillard^{7

13}, Claudia Goettsch¹

Affiliations

¹ Department of Internal Medicine I-Cardiology, Medical Faculty, RWTH Aachen University, Aachen, Germany.
² Institute of Experimental Medicine and Systems Biology, University Hospital, RWTH Aachen, Aachen, Germany.
³ Interdisciplinary Center for Clinical Research, Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, Germany.
⁴ Department of Pathology, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, Netherlands.
⁵ Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University Munich, Munich, Germany.
⁶ DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany.
⁷ L'institut Du Thorax, Inserm UMR 1087, CNRS, INSERM, France and Nantes Université, Nantes, France.
⁸ ICBMS UMR CNRS 5246, Université Claude Bernard Lyon 1, Villeurbanne, France.
⁹ Department of Vascular Surgery, Vascular Center, Groupe Hospitalier Paris Saint-Joseph, Paris, France.
¹⁰ Department of Nephrology and Clinical Immunology, University Hospital RWTH Aachen, Aachen, Germany.
¹¹ Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, Rotterdam, Netherlands.
¹² Biointerface Laboratory, Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.
¹³ PHY-OS Laboratory, INSERM UMR 1238, Nantes University of Medicine, Nantes, France.

Abstract

Introduction: Vascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways.

Materials and methods and results: Human coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs-a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease-a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). In vitro validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP.

Conclusion: Our direct comparison results of two in vitro calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification in vitro. We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in in vitro studies using different calcification protocols.

Keywords: drug repurposing; matrix mineralization; mitochondrial function; protein kinase C; vascular calcification; vascular smooth muscle cells.