TY - JOUR TI - Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification AU - Furmanik Malgorzata AU - Chatrou Martijn AU - van Gorp Rick H AU - Akbulut Asim AU - Willems Brecht AU - Schmidt Harald HHW AU - van Eys Guillaume AU - Bochaton-Piallat Marie-Luce AU - Proudfoot Diane AU - Biessen Erik AL AU - Hedin Ulf AU - Matic Ljubica AU - Mees Barend AU - Shanahan Catherine M AU - Reutelingsperger Chris AU - Schurgers Leon J T2 - Circulation Research AB - 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 ROS-forming NADPH oxidase 5 (Nox5). Methods and Results: In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN1, αSMA and SM22α and an increase in synthetic marker S100A4 compared to 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 reactive oxygen species (ROS) and Ca2+-dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knock-down 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 Ca2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca2+. Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca2+ 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 Ca2+ uptake via EVs and show that Ca2+ 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 remodelling and calcification in the context of mineral imbalance. DO - 10.1161/CIRCRESAHA.119.316159 DP - ahajournals.org (Atypon) VL - 0 IS - 0 J2 - Circulation Research UR - https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.119.316159 Y2 - 2020/06/22/18:16:27 ER -