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

Aims: Calcium 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-1 beta (IL-1 beta). 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 results: Using ELISA to measure IL-1 beta release from VSMCs, we demonstrated that CaP particles stimulated IL-1 beta release from proliferating and senescent human VSMCs, but with substantially greater IL-1 beta 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-1 beta 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-1 beta 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-1 beta and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1 beta 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-1 beta release. Conclusions: CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1 beta, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro calcific potential of microcalcification.