Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles

Malgorzata Furmanik, Rick van Gorp, Meredith Whitehead, Sadia Ahmad, Jayanta Bordoloi, Alexander Kapustin, Leon J Schurgers*, Catherine M Shanahan*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


OBJECTIVE: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release.

CONCLUSIONS: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

Original languageEnglish
Pages (from-to)898-914
Number of pages17
JournalArteriosclerosis Thrombosis and Vascular Biology
Issue number2
Publication statusPublished - Feb 2021


  • Activating Transcription Factor 4/genetics
  • Adolescent
  • Adult
  • Aged
  • Animals
  • Cells, Cultured
  • Disease Models, Animal
  • Endoplasmic Reticulum Stress/drug effects
  • Extracellular Vesicles/drug effects
  • Female
  • Gene Expression Regulation
  • Heat-Shock Proteins/genetics
  • Humans
  • Male
  • Middle Aged
  • Muscle, Smooth, Vascular/drug effects
  • Myocytes, Smooth Muscle/metabolism
  • Rats, Sprague-Dawley
  • Signal Transduction
  • Vascular Calcification/chemically induced
  • Warfarin/toxicity
  • Young Adult
  • eIF-2 Kinase/genetics
  • vascular calcification
  • arteries
  • warfarin
  • aging
  • endoplasmic reticulum


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