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

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-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 results: Using 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. Conclusions: CaP 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 procalcific potential of microcalcification

Atrial arrhythmogenesis in wild-type and Scn5a+/Δ 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+/Δ (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 (APD90), 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 APD90 in both WT and KPQ groups, giving negative CIs. Quinidine similarly exerted anti-arrhythmic effects, prolonged AERP over corresponding APD90 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 APD90 over AERP gave positive CI values and increased atrial arrhythmogenicity whereas lengthening of AERP over APD90 reduced such CI values and produced the opposite effect

Effects of CaP particles on intracellular Ca²⁺ in the presence and absence of fetuin-A.

<p>Experiments were conducted either in the absence of fetuin-A (A), with 1 µM fetuin-A (B) or 0.1 µM fetuin-A (C). A-C(i) are representative traces showing intracellular Ca²⁺ changes in individual fura-2-loaded VSMCs on addition of 25 µg/mL CaP particles (arrow). A(i) Peak amplitude (PA) is indicated after addition of CaP particles. Note that fura-2 loss coincided with PI uptake, indicating time of cell death. Intracellular Ca²⁺ changes seen after cell death did not correspond to genuine Ca²⁺ signals (also explained in Fig. 3A and B). Incubation with 1 µM fetuin-A (B(i)) but not 0.1 µM fetuin-A (C(i)) silenced intracellular Ca²⁺ activity and prevented cell death. A-C(ii) Summary of intracellular Ca²⁺ activity in all VSMCs used for each condition. Each dot represents the area under the curve calculated for each 2.5 minute interval post CaP particle addition from individual cells. ‘B’ on the x-axis of these graphs represents the baseline, or 2.5 minutes prior to addition of CaP particles. These graphs display the range and magnitude of intracellular Ca²⁺ elevations after CaP particle addition from several individual experiments and also show that after addition of 1 µM fetuin-A, Ca²⁺ activity was vastly reduced and delayed (B(ii)).</p

Explanation of particle abbreviations.

<p>Explanation of particle abbreviations.</p

TEM analysis of CaP particle/VSMC interactions in the presence of fetuin-A.

<p>A. Images taken at random (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097565#pone-0097565-g006" target="_blank">Figure 6</a>) were assessed for various features including: VSMC plasma membrane interaction; plasma membrane damage; CaP particles seen within cells and CaP particles seen within discrete intracellular compartments (vesicles) (<i>n</i> ≥9 for each condition/time point). Controls were samples exposed to serum-free physiological buffer without particles. A clear trend was observed where the presence of fetuin-A reduced CaP interactions with cells up to 10 minutes of exposure. However, the presence of fetuin-A did not appear to influence CaP interactions/uptake after 60 minutes of exposure. B. As for A, but the effect of CaP particles on VSMC interactions/uptake was compared with CaP/F (<i>n</i> ≥9). As for fetuin-A added in solution (A), the presence of fetuin-A on CaP particles appeared to delay early interactions with VSMC.</p

Effect of fetuin-A or albumin on CaP-induced VSMC death.

<p>VSMCs in 96-well plates were exposed to either control serum-free medium (no additions) or CaP particles (CaP, 25 µg/mL) in serum-free medium with or without different concentrations of fetuin-A (A) or albumin (B) for 1 hour. Both fetuin-A and albumin inhibited CaP-particle-induced VSMC death in a concentration-dependent manner, as measured by PI uptake (fluorescence intensity with blanks subtracted, <i>n</i> = 4, means ± S.D, *P<0.01, **P<0.0001, ***P<0.00001.).</p

TEM images of CaP particle/VSMC interactions in the presence of fetuin-A.

<p>TEM images show ultrastructural features observed after treatment of VSMCs with CaP particles (12.5 µg/mL) ± fetuin-A (1 µM) or with CaP/F (12.5 µg/mL). Control images from cells exposed to physiological buffer without CaP particles are shown in Ai and Aii. After 5 minutes of particle exposure (middle panel), CaP/plasma membrane interactions were observed (Bi) but these interactions were not commonly observed in the presence of fetuin-A (Bii) or with CaP/F particles (Biii). After 10 minutes (lower panel, Ci–iii), CaP particle interactions with the plasma membrane were detected for each condition. Bar = 500 nm.</p

Ultrastructural analysis of CaP particle-exposure to VSMCs.

<p>VSMCs were incubated with CaP particles (12.5 µg/mL) for specific times (as indicated) before fixing and processing for TEM analysis. A. Evidence for macropinocytosis of clusters of CaP particles was often observed after 5 minutes of particle exposure and uptake of individual particles was also seen at this early timepoint (indicated by arrows in A). Clathrin-like pits were also often observed after CaP particle exposure (B). After 10 minutes of CaP particle exposure, plasma membrane damage was observed in association with membrane protrusions (C) or ingression (D). Discrete CaP particles were also seen aligning at the plasma membrane surface (arrow in D). After 30 minutes, areas of plasma membrane damage were observed and these areas contained electron-dense particles (indicated by arrow in E). B. At 60 minutes, intracellular particle accumulation in clusters or isolated particles (indicated by arrow) were observed and large areas of plasma membrane rupture (F). Bar = 500 nm.</p