Biologic nanoparticles induce calcification of vascular smooth muscle cells (VSMCs) in vitro

Abstract

Biologic nanoparticles (NPs) isolated from sites of ectopic mineralization in humans propagate and form protein/mineral complexes when placed under cell culture conditions. When NPs were injected into animals, vascular calcification developed at sites of endothelial denudation. Our aim was to begin to investigate how NPs induce mineralization of VSMCs. Porcine VSMCs were cultured in 4-well plates (Medium-199/10% FBS) until 80–100% confluent (day 0). Medium was then supplemented with either 10 mM β-glycerophosphate (BGP; to induce mineralization), with NPs (50 or 150 nephelometric turbidity units; NTU) or with vehicle. After 15 days, cell nodules formed in all cultures. Nodules from control wells were negative for calcification (von Kossa), whereas 26.4%, 16.9% and 45.2% of those from BGP-treated, 50 NTU and 150 NTU NP-treated wells, respectively, were positive. Immunofluorescent labeling/confocal microscopy detected the mineralization-protective antigen γ-carboxylated matrix Gla protein (cMGP) in monolayer cells of all treatments at days 0; and 15; the non-functional uncarboxylated MPG isomer was present at lower levels in monolayer cells. Neither isoform was detected in nodules of any group. Thus, NPs induce mineralization of VSMCs in vitro by a mechanism that may not involve regulation of MGP. (Supported by grants from the Fetzer Foundation, NIH HL88988 and Mayo Clinic).