Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification

Malgorzata Furmanik, Martijn Chatrou, Rick van Gorp, Asim Akbulut, Brecht Willems, Harald Schmidt, Guillaume van Eys, Marie-Luce Bochaton-Piallat, Diane Proudfoot, Erik Biessen, Ulf Hedin, Ljubica Perisic, Barend Mees, Catherine Shanahan, Chris Reutelingsperger, Leon Schurgers*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

46 Citations (Web of Science)
136 Downloads (Pure)

Abstract

Rationale: Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive, precluding mechanism-based therapies. Objective: Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent reactive oxygen species (ROS)-forming Nox5 (NADPH oxidase 5). Methods and Results: In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN-1 (calponin 1), alpha-SMA (alpha-smooth muscle actin), and SM22-alpha (smooth muscle protein 22 alpha) and an increase in synthetic marker S100A4 (S100 calcium binding protein A4) compared with contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca2+. Phenotypic switching was accompanied by increased levels of ROS and Ca2+-dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knockdown of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca2+-dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca(2+)loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca2+. Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca(2+)and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs. Conclusions: We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca(2+)uptake via EVs and show that Ca(2+)induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodeling and calcification in the context of mineral imbalance.

Original languageEnglish
Pages (from-to)911-927
Number of pages17
JournalCirculation Research
Volume127
Issue number7
DOIs
Publication statusPublished - 11 Sep 2020

Keywords

  • calcium
  • extracellular vesicles
  • NADPH oxidase 5
  • phenotype
  • vascular calcification
  • OXIDATIVE STRESS
  • MATRIX VESICLES
  • ARTERIAL CALCIFICATION
  • CALCIUM
  • MECHANISM
  • DIFFERENTIATION
  • OXIDASE
  • PHAGOCYTOSIS
  • EXPRESSION
  • APOPTOSIS

Cite this