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

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.

Oral Administration of Bovine Milk‐Derived Extracellular Vesicles Alters the Gut Microbiota and Enhances Intestinal Immunity in Mice

“…To investigate the loss of mEVs during the extraction process, a bead‐based platform was demonstrated that allowed a semi‐quantitative analysis of EVs by flow‐cytometry. [ 27,28 ] The EVs membrane marker‐CD9 could be detected ( Figure 2 C), and the FACS signals of EVs‐CD9 isolated by CHT method were significantly higher than those obtained via UNT and HAT methods (Figure 2D). It was suggested that there was the low loss of EVs in CHT method.…”

Signaling pathways involved in vascular smooth muscle cell calcification during hyperphosphatemia

“…Phosphate complexes with calcium and calcium phosphate nanoparticles are able to activate pro-calcific intracellular signaling pathways [ 25 , 26 ]. Increased calcium phosphate product levels [ 27 ] and calcium phosphate–protein complexes, known as calciprotein particles (CPPs) [ 28 – 30 ], are associated with the development of vascular calcification in CKD.…”

“…Exposure to calcium phosphate crystals induces IL-1β release via the activation of spleen tyrosine kinase (SYK), apparently independent from inflammasome activation [ 25 ]. Nonetheless, inflammasome activation is required for vascular calcification during hyperphosphatemia [ 137 ], effects presumably involving TNFα [ 139 ].…”

Foam cell formation and cholesterol trafficking and metabolism disturbances in atherosclerosis

“…48 Besides, VSMcs can acquire pro-inflammatory cell phenotype and release inflammatory cytokines, including il-1β, il-6 and McP-1 and express inflammatory molecules, such as icAM-1 and VcAM-1. [50][51][52][53][54] Moreover, they were shown to release related to plaque vulnerability MMP-9 upon loading with lipids. 49 in atherosclerotic plaque, icAM-1 is also upregulated on the surface of VSMcs and promotes adhesion to monocyte/macrophages and t cells.…”