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. 2020 Dec 10;41(2):898–914. doi: 10.1161/ATVBAHA.120.315506

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© 2020 The Authors.

Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.

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Figure 5. — Warfarin-mediated endoplasmic reticulum (ER) stress induces extracellular vesicle (EV) release in a PERK (PKR [protein kinase RNA]-like ER kinase)-dependent manner. A, Real-time polymerase chain reaction analysis of ER stress markers in vascular smooth muscle cells (VSMCs) treated with 10 μM warfarin for 8 d in M119 with 5% FBS. Statistical significance was tested with 1-sample t tests. B and C, Western blot showing that warfarin induces Grp78 (glucose-regulated protein, 78 kDa) and Grp94 expression in human VSMCs in vitro. Cells were treated with 10 μM warfarin for 24 h in M199 with 0.5% FBS. Statistical significance was tested with ANOVA. D and E, Western blot showing that warfarin induces PERK phosphorylation in human VSMCs in vitro. Cells were treated with 50 μM warfarin for 1 h in M199 with 0.5% FBS. Statistical significance was tested with t test. F, ER stress inhibitor 4-phenylbutyric acid (PBA) decreased warfarin-enhanced calcification of VSMCs. Cells were treated with 2.7 mmol/L Ca²⁺, 2.5 mmol/L PO₄^3-, 0.5 mmol/L PBA and 10 μM warfarin for 8 days in M199 with 5% FBS. Statistical significance was tested with ANOVA. G, Quantification of EV release using a bead capture assay. Cells were treated with 50 μM warfarin with or without ER stress inhibitors GSK (10 µmol/L GSK2656157) or CB53 (25 µmol/L CB5305630) for 24 h in M199 with 0.5% FBS. Statistical significance was tested with ANOVA. H–J, Western blot showing that GSK, but not CB53, can inhibit a warfarin-induced Grp78 increase. Statistical significance was tested with ANOVA. All graphs show mean+SD and individual data points. ATF indicates activating transcription factor; AU, arbitrary units; CHOP, C/EBP-homologous protein; CTRL, control; IRE, inositol-requiring protein; pPERK, phosphorylated PKR (protein kinase RNA)-like ER kinase; TM, tunicamycin; and Warf, warfarin. Dots denote individual data points, *P<0.05, **P<0.01, ***P<0.001.