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

Atrial arrhythmogenic properties in wild-type and Scn5a+/- murine hearts.

Loss- and gain-of-function cardiac Na(+) channel (SCN5A) mutations are associated with the Brugada and long QT type 3 syndromes (LQT3), respectively. Both result in ventricular arrhythmias, but have differing atrial effects. We have recently reported decreased incidences of atrial arrhythmias in a murine Scn5a+/Delta cardiac system used to model LQT3. We now explore for atrial arrhythmias in a murine Brugada syndrome model. Programmed electrical stimulation and burst pacing were applied to 10 wild-type (WT) and 10 Scn5a+/- Langendorff-perfused murine hearts. These induced arrhythmias in similar proportions of the Scn5a+/- and WT hearts, in contrast to their decreased incidences previously reported for Scn5a+/Delta. Almost half of the Scn5a+/- hearts displayed spontaneous atrial arrhythmias never observed in WT. Both these WT and Scn5a+/- hearts showed positive critical intervals, given by the difference between action potential durations at 90% recovery (APD(90)) and atrial effective refractory periods, before pharmacological intervention. Both flecainide and quinidine then prevented both induced and spontaneous atrial arrhythmias in all the arrhythmic WT and Scn5a+/- hearts, in contrast to their differing ventricular effects. However, flecainide prolonged APD(90) in WT but not Scn5a+/-, whereas quinidine prolonged APD(90) in both WT and Scn5a+/-. Nevertheless, addition of both agents markedly increased atrial effective refractory periods to values exceeding the corresponding values of APD(90). This resulted in negative critical intervals in both WT and Scn5a+/-. These findings demonstrate a clear-cut relationship between critical interval and atrial arrhythmic tendency for the first time and extend previous reports that had related critical interval to ventricular arrhythmia.

Atrial arrhythmogenesis in wild-type and Scn5a+/delta murine hearts modelling LQT3 syndrome.

Long QT(3) (LQT3) syndrome is associated with abnormal repolarisation kinetics, prolonged action potential durations (APD) and QT intervals and may lead to life-threatening ventricular arrhythmias. However, there have been few physiological studies of its effects on atrial electrophysiology. Programmed electrical stimulation and burst pacing induced atrial arrhythmic episodes in 16 out of 16 (16/16) wild-type (WT) and 7/16 genetically modified Scn5a+/Delta (KPQ) Langendorff-perfused murine hearts modelling LQT3 (P < 0.001 for both), and in 14/16 WT and 1/16 KPQ hearts (P < 0.001 for both; Fisher's exact test), respectively. The arrhythmogenic WT hearts had significantly larger positive critical intervals (CI), given by the difference between atrial effective refractory periods (AERPs) and action potential durations at 90% recovery (APD(90)), compared to KPQ hearts (8.1 and 3.2 ms, respectively, P < 0.001). Flecainide prevented atrial arrhythmias in all arrhythmogenic WT (P < 0.001) and KPQ hearts (P < 0.05). It prolonged the AERP to a larger extent than it did the APD(90) in both WT and KPQ groups, giving negative CIs. Quinidine similarly exerted anti-arrhythmic effects, prolonged AERP over corresponding APD(90) in both WT and KPQ groups. These findings, thus, demonstrate, for the first time, inhibitory effects of the KPQ mutation on atrial arrhythmogenesis and its modification by flecainide and quinidine. They attribute these findings to differences in the CI between WT and mutant hearts, in the presence or absence of these drugs. Thus, prolongation of APD(90) over AERP gave positive CI values and increased atrial arrhythmogenicity whereas lengthening of AERP over APD(90) reduced such CI values and produced the opposite effect.

Fetuin-A and albumin alter cytotoxic effects of calcium phosphate nanoparticles on human vascular smooth muscle cells.

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.