Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release

Yana Dautova,Alexander N Kapustin,Kevin Pappert,Matthias Epple,Hanneke Okkenhaug,Simon J Cook,Catherine M Shanahan,Martin D Bootman,Diane Proudfoot

doi:10.1016/j.yjmcc.2017.12.007

Abstract

AimsCalcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs. Methods and resultsUsing ELISA to measure IL-1β release from VSMCs, we demonstrated that CaP particles stimulated IL-1β release from proliferating and senescent human VSMCs, but with substantially greater IL-1β release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1β release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1β release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1β and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1β in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1β release. ConclusionsCaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1β, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification.

Highlights

Vascular calcification is a regulated process occurring in aging and diseased blood vessels that correlates positively with cardiovascular deaths
To establish if calcium phosphate (CaP) particles influenced IL-1β release from human vascular smooth muscle cells (VSMC), cells were exposed to CaP particles for 16 h and IL-1β levels were measured in cell supernatants
We report that CaP particles induce the release of IL-1β from human VSMCs via activation of spleen tyrosine kinase (SYK) and caspase-1

Summary

Introduction

Vascular calcification is a regulated process occurring in aging and diseased blood vessels that correlates positively with cardiovascular deaths. The very small calcium phosphate (CaP) particles that appear as ‘speckled’ deposits in human atherosclerotic plaques are associated with plaque instability, increasing the likelihood of plaque rupture and subsequent thrombus formation [1,2,3]. Recent studies suggest that small CaP particles may be damaging due to effects on mechanical stress in the fibrous cap [4,5], or that CaP particles can be engulfed by macrophages leading to secretion of pro-inflammatory cytokines [6]. Our own studies suggest that small CaP particles are taken up into vascular smooth muscle cells (VSMCs) via endocytosis, macropinocytosis and plasma membrane damage, causing transient intracellular Ca2+ rises and inducing cell death in subsets of VSMCs [7,8].

Methods

Results

Conclusion