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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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CNP-incorporation into cultured vascular smooth muscle cells.Notes: CNPs used to treat cells were sectioned for TEM, which showed concentric layers alternating in electron-density; two typical particles are shown (A and B). CNP-treated cells examined on day 3 showed intracellular structures similar in size and appearance to applied CNPs (C; area outlined by box is shown at higher magnification in D). High-density structures less than 50 nm (shown within the white circles) were present in CNP-treated cells after 3 days (E). Particles similar in morphology to the applied CNPs, but typically more electron-dense, were observed within the matrix (F; area outlined by box is shown at higher magnification in G) and within intracellular vacuoles (H) of nodules at 28 days. EDX of incorporated particles revealed Ca and P (I). Crystals (arrows) up to several μm were found within P-treated cells after 28 days (J).Abbreviations: CNP, calcifying nanoparticle; EDX, energy-dispersive elemental analysis; P, phosphate; TEM, transmission-electron microscopy.
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f4-ijn-9-2689: CNP-incorporation into cultured vascular smooth muscle cells.Notes: CNPs used to treat cells were sectioned for TEM, which showed concentric layers alternating in electron-density; two typical particles are shown (A and B). CNP-treated cells examined on day 3 showed intracellular structures similar in size and appearance to applied CNPs (C; area outlined by box is shown at higher magnification in D). High-density structures less than 50 nm (shown within the white circles) were present in CNP-treated cells after 3 days (E). Particles similar in morphology to the applied CNPs, but typically more electron-dense, were observed within the matrix (F; area outlined by box is shown at higher magnification in G) and within intracellular vacuoles (H) of nodules at 28 days. EDX of incorporated particles revealed Ca and P (I). Crystals (arrows) up to several μm were found within P-treated cells after 28 days (J).Abbreviations: CNP, calcifying nanoparticle; EDX, energy-dispersive elemental analysis; P, phosphate; TEM, transmission-electron microscopy.

Mentions: The serum-derived CNPs applied to smooth muscle cells appeared, in cross-section, to consist of concentric layers that alternated in electron density (Figures 4A and B). Structures similar in size and appearance were present within 19.6%±3.7% (n=8) of CNP-treated cells (Figures 4C and D), but not in control or P-treated cells after 3 days of treatment. At this time point, highly electron-dense crystalline structures less than 50 nm in diameter were also observed in a small proportion of CNP-treated cells (Figure 4E). After 28 days in culture, CNPs were only observed within multicellular nodules of CNP-treated cells, either incorporated into the extracellular matrix (Figures 4F and G) or, less commonly, localized within intracellular vacuoles of nodular peripheral cells (Figure 4H). Although these particles were similar in morphology to those observed after 3 days, they were typically more electron-dense. Energy-dispersive elemental analysis of intra- and extracellular particles revealed Ca and phosphorus components (Figure 4I). Cells treated with 2 mM P displayed large crystals up to several μm in size within the nodules. However, it was not possible to confirm their original localization since it appeared that the particles may have been displaced by the sectioning procedure (Figure 4J).


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)

CNP-incorporation into cultured vascular smooth muscle cells.Notes: CNPs used to treat cells were sectioned for TEM, which showed concentric layers alternating in electron-density; two typical particles are shown (A and B). CNP-treated cells examined on day 3 showed intracellular structures similar in size and appearance to applied CNPs (C; area outlined by box is shown at higher magnification in D). High-density structures less than 50 nm (shown within the white circles) were present in CNP-treated cells after 3 days (E). Particles similar in morphology to the applied CNPs, but typically more electron-dense, were observed within the matrix (F; area outlined by box is shown at higher magnification in G) and within intracellular vacuoles (H) of nodules at 28 days. EDX of incorporated particles revealed Ca and P (I). Crystals (arrows) up to several μm were found within P-treated cells after 28 days (J).Abbreviations: CNP, calcifying nanoparticle; EDX, energy-dispersive elemental analysis; P, phosphate; TEM, transmission-electron microscopy.
© Copyright Policy
Related In: Results  -  Collection

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

f4-ijn-9-2689: CNP-incorporation into cultured vascular smooth muscle cells.Notes: CNPs used to treat cells were sectioned for TEM, which showed concentric layers alternating in electron-density; two typical particles are shown (A and B). CNP-treated cells examined on day 3 showed intracellular structures similar in size and appearance to applied CNPs (C; area outlined by box is shown at higher magnification in D). High-density structures less than 50 nm (shown within the white circles) were present in CNP-treated cells after 3 days (E). Particles similar in morphology to the applied CNPs, but typically more electron-dense, were observed within the matrix (F; area outlined by box is shown at higher magnification in G) and within intracellular vacuoles (H) of nodules at 28 days. EDX of incorporated particles revealed Ca and P (I). Crystals (arrows) up to several μm were found within P-treated cells after 28 days (J).Abbreviations: CNP, calcifying nanoparticle; EDX, energy-dispersive elemental analysis; P, phosphate; TEM, transmission-electron microscopy.
Mentions: The serum-derived CNPs applied to smooth muscle cells appeared, in cross-section, to consist of concentric layers that alternated in electron density (Figures 4A and B). Structures similar in size and appearance were present within 19.6%±3.7% (n=8) of CNP-treated cells (Figures 4C and D), but not in control or P-treated cells after 3 days of treatment. At this time point, highly electron-dense crystalline structures less than 50 nm in diameter were also observed in a small proportion of CNP-treated cells (Figure 4E). After 28 days in culture, CNPs were only observed within multicellular nodules of CNP-treated cells, either incorporated into the extracellular matrix (Figures 4F and G) or, less commonly, localized within intracellular vacuoles of nodular peripheral cells (Figure 4H). Although these particles were similar in morphology to those observed after 3 days, they were typically more electron-dense. Energy-dispersive elemental analysis of intra- and extracellular particles revealed Ca and phosphorus components (Figure 4I). Cells treated with 2 mM P displayed large crystals up to several μm in size within the nodules. However, it was not possible to confirm their original localization since it appeared that the particles may have been displaced by the sectioning procedure (Figure 4J).

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