Calcifying nanoparticles promote mineralization in vascular smooth muscle cells: implications for atherosclerosis
Larry W Hunter,1 Jon E Charlesworth,2 Sam Yu,3,4 John C Lieske,5,6 Virginia M Miller1,7
1Department of Surgery, 2Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA; 3Lincoln University, Christchurch, New Zealand; 4Izon Science Ltd., Christchurch, New Zealand; 5Department of Internal Medicine, Division of Nephrology and Hypertension; 6Department of Laboratory Medicine and Pathology; 7Department of Physiology
and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA
Background: Nano-sized complexes of calcium phosphate mineral and proteins (calcifying nanoparticles [CNPs]) serve as mineral chaperones. Thus, CNPs may be both a result and cause of soft tissue calcification processes. This study determined if CNPs could augment calcification of arterial vascular smooth muscle cells in vitro.
Methods: CNPs 210 nm in diameter were propagated in vitro from human serum. Porcine aortic smooth muscle cells were cultured for up to 28 days in medium in the absence (control) or presence of 2 mM phosphate ([P] positive calcification control) or after a single 3-day exposure to CNPs. Transmission electron-microscopy was used to characterize CNPs and to examine their cellular uptake. Calcium deposits were visualized by light microscopy and von Kossa staining and were quantified by colorimetry. Cell viability was quantified by confocal microscopy of live-/dead-stained cells and apoptosis was examined concurrently by fluorescent labeling of exposed phosphatidylserine.
Results: CNPs, as well as smaller calcium crystals, were observed by transmission electron-microscopy on day 3 in CNP-treated but not P-treated cells. By day 28, calcium deposits were visible in similar amounts within multicellular nodules of both CNP- and P-treated cells. Apoptosis increased with cell density under all treatments. CNP treatment augmented the density of apoptotic bodies and cellular debris in association with mineralized multicellular nodules.
Conclusion: Exogenous CNPs are taken up by aortic smooth muscle cells in vitro and potentiate accumulation of smooth-muscle-derived apoptotic bodies at sites of mineralization. Thus, CNPs may accelerate vascular calcification.
Keywords: biomineralization, hydroxyapatite, vascular calcification, protein-mineral complex
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