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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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Light micrographs of cultured vascular smooth muscle cells to identify calcium deposits by von Kossa staining.Notes: Calcium deposits identified by von Kossa staining (black). Cells were stained with Eosin-Y (red). On experiment day 3 (top row), vehicle-treated (A), CNP-treated (B), and P-treated (C) cells were morphologically similar and nearly confluent. Some had begun retracting into ridges (white arrows), which by day 28 (bottom) had developed into dense multicellular nodules (blue arrows). Ca deposits were identified within nodular matrix of CNP- and P-treated cultures, but were absent in controls; (E, F and D, respectively). Insets show higher magnification of nodules with Eosin-Y omitted.Abbreviations: CNP, calcifying nanoparticle; P, phosphate.
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f2-ijn-9-2689: Light micrographs of cultured vascular smooth muscle cells to identify calcium deposits by von Kossa staining.Notes: Calcium deposits identified by von Kossa staining (black). Cells were stained with Eosin-Y (red). On experiment day 3 (top row), vehicle-treated (A), CNP-treated (B), and P-treated (C) cells were morphologically similar and nearly confluent. Some had begun retracting into ridges (white arrows), which by day 28 (bottom) had developed into dense multicellular nodules (blue arrows). Ca deposits were identified within nodular matrix of CNP- and P-treated cultures, but were absent in controls; (E, F and D, respectively). Insets show higher magnification of nodules with Eosin-Y omitted.Abbreviations: CNP, calcifying nanoparticle; P, phosphate.

Mentions: After 3 days in culture, cells formed a monolayer of nearly-confluent cells (Figures 2A–C). Subsequently, groups of cells formed ridges, which ultimately developed into multicellular nodules (Figures 2D–F). Under all culture conditions, Ca deposition was not detected at day 3 by von Kossa staining. However, by day 28, von Kossa-positive crystals were present only in nodules of CNP-treated and 2 mM P-treated cultures. Crystals in CNP-treated and P-treated cells averaged about 1.7 μm and 8.4 μm in diameter, respectively (Figures 2E and F). Cells cultured in control media lacked crystals, even though there were no significant differences in the number of nodules per coverslip between control, CNP-, and P-treated cells (61.5±1.3, 48.8±6.2, and 57.5±4.6, respectively; n=4 each; Figure 3A). Among CNP- and P-treated cells, 81.0%±2.0% and 37.6%±4.2% of nodules were mineralized, respectively (Figure 3B). Ca was not detected in the HCl demineralizing solution from any of the control cultures at any time-point (Figure 3C), or in that of CNP-treated or P-treated cells after 3 days. However, it was present at 20.03±3.41 and 27.45±4.77 μg/mg protein in CNP-treated and P-treated cells, respectively, at experiment completion (Figure 3C).


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)

Light micrographs of cultured vascular smooth muscle cells to identify calcium deposits by von Kossa staining.Notes: Calcium deposits identified by von Kossa staining (black). Cells were stained with Eosin-Y (red). On experiment day 3 (top row), vehicle-treated (A), CNP-treated (B), and P-treated (C) cells were morphologically similar and nearly confluent. Some had begun retracting into ridges (white arrows), which by day 28 (bottom) had developed into dense multicellular nodules (blue arrows). Ca deposits were identified within nodular matrix of CNP- and P-treated cultures, but were absent in controls; (E, F and D, respectively). Insets show higher magnification of nodules with Eosin-Y omitted.Abbreviations: CNP, calcifying nanoparticle; P, phosphate.
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijn-9-2689: Light micrographs of cultured vascular smooth muscle cells to identify calcium deposits by von Kossa staining.Notes: Calcium deposits identified by von Kossa staining (black). Cells were stained with Eosin-Y (red). On experiment day 3 (top row), vehicle-treated (A), CNP-treated (B), and P-treated (C) cells were morphologically similar and nearly confluent. Some had begun retracting into ridges (white arrows), which by day 28 (bottom) had developed into dense multicellular nodules (blue arrows). Ca deposits were identified within nodular matrix of CNP- and P-treated cultures, but were absent in controls; (E, F and D, respectively). Insets show higher magnification of nodules with Eosin-Y omitted.Abbreviations: CNP, calcifying nanoparticle; P, phosphate.
Mentions: After 3 days in culture, cells formed a monolayer of nearly-confluent cells (Figures 2A–C). Subsequently, groups of cells formed ridges, which ultimately developed into multicellular nodules (Figures 2D–F). Under all culture conditions, Ca deposition was not detected at day 3 by von Kossa staining. However, by day 28, von Kossa-positive crystals were present only in nodules of CNP-treated and 2 mM P-treated cultures. Crystals in CNP-treated and P-treated cells averaged about 1.7 μm and 8.4 μm in diameter, respectively (Figures 2E and F). Cells cultured in control media lacked crystals, even though there were no significant differences in the number of nodules per coverslip between control, CNP-, and P-treated cells (61.5±1.3, 48.8±6.2, and 57.5±4.6, respectively; n=4 each; Figure 3A). Among CNP- and P-treated cells, 81.0%±2.0% and 37.6%±4.2% of nodules were mineralized, respectively (Figure 3B). Ca was not detected in the HCl demineralizing solution from any of the control cultures at any time-point (Figure 3C), or in that of CNP-treated or P-treated cells after 3 days. However, it was present at 20.03±3.41 and 27.45±4.77 μg/mg protein in CNP-treated and P-treated cells, respectively, at experiment completion (Figure 3C).

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