Calcification is a detrimental process in vascular ageing and in diseases such as atherosclerosis and arthritis. In particular, small calcium phosphate (CaP) crystal deposits are associated with inflammation and atherosclerotic plaque de-stabilisation. We previously reported that CaP particles caused human vascular smooth muscle cell (VSMC) death and that serum reduced the toxic effects of the particles. Here, we found that the serum proteins fetuin-A and albumin (≥1 µM) reduced intracellular Ca2+ elevations and cell death in VSMCs in response to CaP particles. In addition, CaP particles functionalised with fetuin-A, but not albumin, were less toxic than naked CaP particles. Electron microscopic studies revealed that CaP particles were internalised in different ways; via macropinocytosis, membrane invagination or plasma membrane damage, which occurred within 10 minutes of exposure to particles. However, cell death did not occur until approximately 30 minutes, suggesting that plasma membrane repair and survival mechanisms were activated. In the presence of fetuin-A, CaP particle-induced damage was inhibited and CaP/plasma membrane interactions and particle uptake were delayed. Fetuin-A also reduced dissolution of CaP particles under acidic conditions, which may contribute to its cytoprotective effects after CaP particle exposure to VSMCs. These studies are particularly relevant to the calcification observed in blood vessels in patients with kidney disease, where circulating levels of fetuin-A and albumin are low, and in pathological situations where CaP crystal formation outweighs calcification-inhibitory mechanisms.

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.

The calcium ion (Ca 2+ ) is a versatile intracellular messenger. It provides dynamic regulation of vast array of cellular processes such as gene transcription, differentiation and contraction. Ca 2+ signals range from microsecond, nanoscopic events to intercellular waves lasting for many seconds. This diversity of Ca 2+ signals arises from the wide assortment of Ca 2+ transport and Ca 2+ buffering processes employed by cells. Additional diversity in Ca 2+ signalling stems from the ability of cells to utilise different sources of Ca 2+ . The cytosol is the principal Ca 2+ signalling compartment. When Ca 2+ ions enter the cytosol they interact with numerous Ca 2+ ‐binding proteins, thereby leading to activation, or inhibition, of cellular processes. Specificity is achieved by regulating the spatial and kinetic properties of Ca 2+ signal. In this way, many concurrent Ca 2+ ‐sensitive cellular processes can be discretely regulated. A number of pathologies have been related to the breakdown of cellular Ca 2+ homoeostasis or to aberrant Ca 2+ signalling. Key Concepts: Calcium is a critical intracellular signal that controls key cell fate decisions. Calcium signals derive from multiple sources that are accessed by a diverse range of transport mechanisms. The kinetics and spatial properties of calcium signals determine their impact on cellular activity. Ca 2+ signals are tissue‐specific and are appropriate to regulate the physiological functions of individual cell types.

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