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Calcifying nanoparticles promote mineralization in vascular smooth muscle cells: implications for atherosclerosis.

Hunter LW, Charlesworth JE, Yu S, Lieske JC, Miller VM - Int J Nanomedicine (2014)

Bottom Line: Thus, CNPs may be both a result and cause of soft tissue calcification processes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Mayo Clinic, Rochester, MN, USA.

ABSTRACT

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.

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Characterization of CNPs.Notes: Scanning electron microscopy of particles (A). EDX analysis identified Ca and P (B). The size of CNPs ranged from 20 nm to about 1 μm; most CNPs averaged 210 nm (C). The largest proportion of particles relative to the total population was near the 220 nm size-range (TOF) (D). CNP demineralization revealed smaller structures (E), shown by SDS-PAGE/silver staining to be comprised of numerous proteins (F). Incorporation of 45Ca into CNPs under culture conditions in the absence of cells was nearly linear (mean ± SEM, n=6–8) (G).Abbreviations:45Ca, radiolabeled calcium; CNPs, calcifying nanoparticles; EDX, energy-dispersive elemental analysis; P, phosphate; TOF, time of flight; (SDS-PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis.
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f1-ijn-9-2689: Characterization of CNPs.Notes: Scanning electron microscopy of particles (A). EDX analysis identified Ca and P (B). The size of CNPs ranged from 20 nm to about 1 μm; most CNPs averaged 210 nm (C). The largest proportion of particles relative to the total population was near the 220 nm size-range (TOF) (D). CNP demineralization revealed smaller structures (E), shown by SDS-PAGE/silver staining to be comprised of numerous proteins (F). Incorporation of 45Ca into CNPs under culture conditions in the absence of cells was nearly linear (mean ± SEM, n=6–8) (G).Abbreviations:45Ca, radiolabeled calcium; CNPs, calcifying nanoparticles; EDX, energy-dispersive elemental analysis; P, phosphate; TOF, time of flight; (SDS-PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Mentions: CNP isolates were spheroid structures less than 1.2 μm in diameter with Ca and P components (Figures 1A and B). Their sizes ranged from 20 nm to about 1 μm. The average size was 210 nm (Figure 1C) and the largest proportion of particles, relative to the total population, was found in a size range less than 220 nm (Figure 1D). Demineralization of CNPs with HCl revealed smaller structures (Figure 1E) that were comprised of numerous proteins (Figure 1F), consistent with previous published work by our group.27 CNPs adhered to collagen-coated cover-slips in the absence of cells and accumulated 45Ca from the media over the study period of 15 days (Figure 1G).


Calcifying nanoparticles promote mineralization in vascular smooth muscle cells: implications for atherosclerosis.

Hunter LW, Charlesworth JE, Yu S, Lieske JC, Miller VM - Int J Nanomedicine (2014)

Characterization of CNPs.Notes: Scanning electron microscopy of particles (A). EDX analysis identified Ca and P (B). The size of CNPs ranged from 20 nm to about 1 μm; most CNPs averaged 210 nm (C). The largest proportion of particles relative to the total population was near the 220 nm size-range (TOF) (D). CNP demineralization revealed smaller structures (E), shown by SDS-PAGE/silver staining to be comprised of numerous proteins (F). Incorporation of 45Ca into CNPs under culture conditions in the absence of cells was nearly linear (mean ± SEM, n=6–8) (G).Abbreviations:45Ca, radiolabeled calcium; CNPs, calcifying nanoparticles; EDX, energy-dispersive elemental analysis; P, phosphate; TOF, time of flight; (SDS-PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043721&req=5

f1-ijn-9-2689: Characterization of CNPs.Notes: Scanning electron microscopy of particles (A). EDX analysis identified Ca and P (B). The size of CNPs ranged from 20 nm to about 1 μm; most CNPs averaged 210 nm (C). The largest proportion of particles relative to the total population was near the 220 nm size-range (TOF) (D). CNP demineralization revealed smaller structures (E), shown by SDS-PAGE/silver staining to be comprised of numerous proteins (F). Incorporation of 45Ca into CNPs under culture conditions in the absence of cells was nearly linear (mean ± SEM, n=6–8) (G).Abbreviations:45Ca, radiolabeled calcium; CNPs, calcifying nanoparticles; EDX, energy-dispersive elemental analysis; P, phosphate; TOF, time of flight; (SDS-PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Mentions: CNP isolates were spheroid structures less than 1.2 μm in diameter with Ca and P components (Figures 1A and B). Their sizes ranged from 20 nm to about 1 μm. The average size was 210 nm (Figure 1C) and the largest proportion of particles, relative to the total population, was found in a size range less than 220 nm (Figure 1D). Demineralization of CNPs with HCl revealed smaller structures (Figure 1E) that were comprised of numerous proteins (Figure 1F), consistent with previous published work by our group.27 CNPs adhered to collagen-coated cover-slips in the absence of cells and accumulated 45Ca from the media over the study period of 15 days (Figure 1G).

Bottom Line: Thus, CNPs may be both a result and cause of soft tissue calcification processes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Mayo Clinic, Rochester, MN, USA.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus