Fetuin-A and Albumin Alter Cytotoxic Effects of Calcium Phosphate Nanoparticles on Human Vascular Smooth Muscle Cells

Abstract

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.

Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells

“…At the same time, Na 3 Cit and Et 2 Cit effectively reduced cell damage. Dautova et al 14 found that nano-calcium phosphate crystals cause cell membrane damage, which can be effectively reduced after the addition of calcification inhibitors, such as Fetuin-A and albumin.…”

“…This condition leads to an imbalance in cell self-repair and steady-state mechanism, thereby leading to cell necrosis. 14,39 The addition of Na 3 Cit and Et 2 Cit could inhibit the increase in the intracellular Ca 2+ concentration induced by nano-HAp and lysosomal damage.…”

“…14,44 This occurrence slows the release of Ca 2+ to the cytosol and reduces intracellular calcium increase, lysosomal damage, and necrosis of MOVASs. In addition, whether nano-HAp bound to Et 2 Cit and Na 3 Cit enters endosomes or lysosomes in MOVASs has yet to be determined.…”

“…These effects may lead to atherosclerotic plaque instability and plaque rupture, which eventually result in myocardial infarction or stroke. 14 Several calcification inhibitors, such as Fetuin-A and albumin, can inhibit the VC by reducing the death of calcium phosphate-induced VSMCs. 11,14 However, the damage mechanism of calcium deposition on VSMCs and the inhibition mechanism of calcification inhibitors remain unclear.…”

“…14,30 Fluo-4/AM can bind with intracellular free Ca 2+ to produce strong green fluorescence instead of binding with extracellular Ca…”