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*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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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.

Original languageEnglish
Article number959457
JournalFrontiers in Cardiovascular Medicine
Volume9
DOIs
Publication statusPublished - 20 Sept 2022

Bibliographical note

Funding Information:
This work was funded by the “Deutsche Forschungsgemein
schaft” (DFG, German Research Foundation) [GO1801/5-1 to
CG and Transregional Collaborative Research Centre (TRR
219; Project-ID 322900939) to CG, WJ-D, RK, and NM],
a research grant from the European Union’s Horizon 2020
research and innovation program under the Marie SkłodowskaCurie grant agreement No. 722609, INTRICARE (to CG, WJ-D,
and NM), and a grant from the European Research Area
Network on Cardiovascular Diseases (ERA-NET CVD JTC2017,
Microexploration project; BMBF 01KL1801) to CG and DM.
EvdV received financial support from the Interdisciplinary
Center for Clinical Research within the faculty of Medicine at
the RWTH Aachen University. TQ received financial support
from the Fondation de l’Avenir (Paris, France), the University
Hospital of Nantes (Nantes, France), and the Fédération
Française de Cardiologie (Paris, France).


Publisher Copyright:
Copyright © 2022 Heuschkel, Babler, Heyn, van der Vorst, Steenman, Gesper, Kappel, Magne, Gouëffic, Kramann, Jahnen-Dechent, Marx, Quillard and Goettsch.

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