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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 4. — Endoplasmic reticulum (ER) stress–induced vascular smooth muscle cell (VSMC) calcification is mediated by extracellular vesicle (EV) release and Grp78 (glucose-regulated protein, 78 kDa). A, Quantification of EV release using a bead capture assay. VSMCs were treated 0.01 to 0.1 μg/mL tunicamycin (TM) or 0.01 to 0.1 μg/mL thapsigargin (TG) for 24 h in M199 with 0.5% FBS. Statistical significance was tested with ANOVA. B, SMPD3 (sphingomyelin phosphodiesterase 3) mRNA expression measured by real-time polymerase chain reaction. VSMCs were treated 0.1 μg/mL TM or 0.1 μg/mL TG for 24 h. Statistical significance was tested with 1-sample t tests. C, SMPD3 inhibitor spiroepoxide blocks ER stress-enhanced VSMC calcification. VSMCs were treated with 0.5 µmol/L spiroepoxide, 0.05 µg TM and 2.7 mmol/L Ca²⁺ and 2.5 mmol/L PO₄^3- for 5 d in 5% FBS. Statistical significance was tested with ANOVA. D, EVs from VSMCs treated with TM have a higher propensity to calcify. Pellet (100 000g) EVs were isolated by ultracentrifugation and equal amounts (protein) were calcified on a collagen matrix with 2.7 mmol/L Ca²⁺ and 2.5 mmol/L PO₄^3-. Statistical significance was tested with 1-sample t test. E and F, Western blot and quantification, showing that 4-phenylbutyric acid (PBA), which decreases ER stress-induced calcification, decreases Grp78 (glucose-regulated protein, 78 kDa) expression in VSMCs. VSMCs were treated with 0.1 μg/mL TM and 1 mmol/L PBA for 24 h. Statistical significance was tested with ANOVA. G and H, Western blot showing Grp78 is increased in EVs released from cells treated with ER stress inducers. VSMCs were treated with 0.2 μg/mL TM or TG in phenol red-free DMEM with 0.1% bovine serum albumin (FBS free); EVs were isolated from culture media by ultracentrifugation. Statistical significance was tested with 1-sample t tests. I, EVs were isolated by ultracentrifugation (100 000g), immunogold staining, and electron microscopy was carried out on sections of fixed EV pellets. Grp78/Grp94 (6 nm gold) are localized in EVs of various sizes expressing CD63 and TSG101 (tumor susceptibility gene 101 protein; both 10 nm gold). Scale bars are 100 nm. J, Dot blot showing Grp78/Grp94 is detected predominantly on the surface of EVs (samples without detergent). TSG101 and CD63 were used as positive and negative control, respectively. EVs were isolated by ultracentrifugation from primary VSMCs culture supernatants. Representative blots from n=3 experiments. K, siRNA (short interfering RNA) knock-down of Grp78 decreases calcification of VSMCs treated with 2.7 mmol/L Ca²⁺ and 2.5 mmol/L PO₄^3- for 18 h. Cells were treated with Grp78 siRNA or control siRNA 24 h before applying calcification media. Calcification was quantified with o-cresolphthalein assay. Statistical significance was tested with t test. All graphs show mean+SD and individual data points. AU indicates arbitrary units; and CTRL, control. Dots denote individual data points, *P<0.05, **P<0.01, ***P<0.001.