From the Biochemistry (M.F., M.C., R.v.G., A.A., B.W., G.v.E., C.R., L.S.) and Pathology (E.B.), Cardiovascular Research Institute Maastricht, Pharmacology and Personalised Medicine, Faculty of Health, Medicine and Life Sciences (H.S.), Maastricht University, The Netherlands; Pathology and Immunology, Faculty of Medicine, University of Geneva, Switzerland (M.-L.B.-P.); Signalling Programme, Babraham Institute, Cambridge, United Kingdom (D.P.); Molecular Medicine and Surgery, Vascular Surgery Division, Karolinska Institute, Stockholm, Sweden (U.H., L.P.M.); Vascular Surgery, Maastricht University Medical Centre, The Netherlands (B.M.); and British Heart Foundation Centre of Excellence, School of Cardiovascular Medicine and Sciences, King's College London, United Kingdom (C.S.).
Circ Res. 2020 Sep 11;127(7):911-927. doi: 10.1161/CIRCRESAHA.119.316159. Epub 2020 Jun 22.
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.
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).
In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN-1 (calponin 1), α-SMA (α-smooth muscle actin), and SM22-α (smooth muscle protein 22α) 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 Ca. Phenotypic switching was accompanied by increased levels of ROS and Ca-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 Ca-dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca. Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs.
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 uptake via EVs and show that Ca 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. Graphic Abstract: A graphic abstract is available for this article.
血管钙化是指血管壁中钙磷酸盐晶体的形成，由血管平滑肌细胞（VSMCs）介导。然而，潜在的分子机制仍不清楚，这使得基于机制的治疗方法难以实现。
表型转换表示收缩蛋白的丧失和迁移及增殖的增加，此时 VSMCs 被称为合成型。我们研究了 VSMC 表型转换如何影响血管钙化以及独特的钙依赖性活性氧（ROS）形成 Nox5（NADPH 氧化酶 5）的可能作用。
与收缩型 VSMCs 相比，体外培养的合成型 VSMCs 中收缩标志物 CNN-1（钙调蛋白 1）、α-SMA（α-平滑肌肌动蛋白）和 SM22-α（平滑肌蛋白 22α）的表达降低，而合成标志物 S100A4（S100 钙结合蛋白 A4）的表达增加。这与高细胞外 Ca 刺激下合成细胞的钙化增加有关。表型转换伴随着合成型 VSMCs 中 ROS 和 Ca 依赖性 Nox5 水平的增加。Nox5 本身调节 VSMC 表型，因为 Nox5 的 siRNA 敲低增加了收缩标志物的表达并减少了钙化，而过表达 Nox5 则降低了收缩标志物的表达。合成型 VSMCs 中的 ROS 产生依赖于细胞质 Ca，这与 Nox5 介导的情况一致。用 Ca 负载的细胞外囊泡（EVs）处理 VSMCs 会导致细胞质 Ca 增加。用 dynasore 抑制 EV 内吞作用可阻断细胞质 Ca 的增加和 VSMC 的钙化。增加的 ROS 产生导致 EV 释放增加和 VSMCs 的吞噬作用减少。
我们在这里表明，收缩型 VSMCs 不易发生钙化，并确定 Nox5 是 VSMC 表型转换的关键调节剂。此外，我们描述了一种通过 EV 摄取 Ca 的新机制，并表明 Ca 通过 Nox5 在 VSMCs 中诱导 ROS 产生。ROS 产生是 EV 释放所必需的，而 EV 促进钙化。鉴定控制 Nox5 和 VSMC 衍生 EV 的分子途径为调节矿物质失衡背景下的血管重塑和钙化提供了潜在靶点。
