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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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Confocal micrographs identifying apoptosis and necrosis in cultured vascular smooth muscle cells treated with CNPs.Notes: Prior to confocal imaging, CNP-treated (A–F,H) and vehicle-treated control (G) cells were incubated with calcein-AM, EthD-1, and annexin V-AlexaFluor® 647 conjugate (Life Technologies, Carlsbad, CA, USA) to label live cells (green), dead (red) cells, and apoptotic bodies (cyan), respectively. After several days in culture, monolayer cells (m) contracted into ridges (between white lines) with associated apoptotic bodies (A). Ridges later developed into nodules (arrows) comprising a core of dead cells, with live cells and apoptotic bodies peripherally (B). Maximum-intensity projection of 14 optical sections through the mid-portion of a nodule shows live (C), dead (D), and apoptotic (E) cells; (F) shows a composite image. Representative maximum-intensity projections of 76 optical sections through whole individual nodules of vehicle-treated cells (G) typically displayed less labeling for fragmented nuclei and apoptotic bodies than did a comparable projection of CNP-treated cells (H). Dotted outlines (G,H) encompass one nodule. Monolayer cell viability at day 28, determined by live/dead cell counting, was significantly reduced by CNPs and by 2 mM P; cells preexposed to 70% methanol served as dead cell control (I). *Significant difference (P<0.05) from control; n=9–10 each.Abbreviations: CNP, calcifying nanoparticle; EthD-1, ethidium homodimer-1; P, phosphate; AM, acetoxymethyl ester.
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f5-ijn-9-2689: Confocal micrographs identifying apoptosis and necrosis in cultured vascular smooth muscle cells treated with CNPs.Notes: Prior to confocal imaging, CNP-treated (A–F,H) and vehicle-treated control (G) cells were incubated with calcein-AM, EthD-1, and annexin V-AlexaFluor® 647 conjugate (Life Technologies, Carlsbad, CA, USA) to label live cells (green), dead (red) cells, and apoptotic bodies (cyan), respectively. After several days in culture, monolayer cells (m) contracted into ridges (between white lines) with associated apoptotic bodies (A). Ridges later developed into nodules (arrows) comprising a core of dead cells, with live cells and apoptotic bodies peripherally (B). Maximum-intensity projection of 14 optical sections through the mid-portion of a nodule shows live (C), dead (D), and apoptotic (E) cells; (F) shows a composite image. Representative maximum-intensity projections of 76 optical sections through whole individual nodules of vehicle-treated cells (G) typically displayed less labeling for fragmented nuclei and apoptotic bodies than did a comparable projection of CNP-treated cells (H). Dotted outlines (G,H) encompass one nodule. Monolayer cell viability at day 28, determined by live/dead cell counting, was significantly reduced by CNPs and by 2 mM P; cells preexposed to 70% methanol served as dead cell control (I). *Significant difference (P<0.05) from control; n=9–10 each.Abbreviations: CNP, calcifying nanoparticle; EthD-1, ethidium homodimer-1; P, phosphate; AM, acetoxymethyl ester.

Mentions: Annexin V labeling, indicating exposed phosphatidylserine, was negative in monolayer cells of all cultures examined. However, annexin V-labeled structures up to about 300 nm in size, most likely representing apoptotic bodies, were typically distributed sparsely among the cells. Numerous apoptotic bodies, as well as an increased proportion of dead cells, colocalized within ridges formed by retracted monolayer cells (Figures 5A and B). Multicellular nodules that subsequently formed from the ridge cells contained a necrotic core of EthD-1-labeled nuclear fragments surrounded by a layer of live cells with associated apoptotic bodies (Figures 5C–F). It was not possible to quantify cell viability within multicellular nodules using this methodology. However, nodules from CNP-treated cells typically displayed more intense nucleic-acid staining of fragmented cells and greater numbers of apoptotic bodies than was observed in control cells (Figures 5G and H). After 21 days, monolayer cell viability, determined by calcein AM/EthD-1 labeling, was 97.9%±0.5% in control cultures, and was slightly but significantly reduced to 96.2%±1.0% and 95.6%±0.7% in CNP-treated and 2 mM P-treated cultures, respectively; n=9–10 each (Figure 5I).


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)

Confocal micrographs identifying apoptosis and necrosis in cultured vascular smooth muscle cells treated with CNPs.Notes: Prior to confocal imaging, CNP-treated (A–F,H) and vehicle-treated control (G) cells were incubated with calcein-AM, EthD-1, and annexin V-AlexaFluor® 647 conjugate (Life Technologies, Carlsbad, CA, USA) to label live cells (green), dead (red) cells, and apoptotic bodies (cyan), respectively. After several days in culture, monolayer cells (m) contracted into ridges (between white lines) with associated apoptotic bodies (A). Ridges later developed into nodules (arrows) comprising a core of dead cells, with live cells and apoptotic bodies peripherally (B). Maximum-intensity projection of 14 optical sections through the mid-portion of a nodule shows live (C), dead (D), and apoptotic (E) cells; (F) shows a composite image. Representative maximum-intensity projections of 76 optical sections through whole individual nodules of vehicle-treated cells (G) typically displayed less labeling for fragmented nuclei and apoptotic bodies than did a comparable projection of CNP-treated cells (H). Dotted outlines (G,H) encompass one nodule. Monolayer cell viability at day 28, determined by live/dead cell counting, was significantly reduced by CNPs and by 2 mM P; cells preexposed to 70% methanol served as dead cell control (I). *Significant difference (P<0.05) from control; n=9–10 each.Abbreviations: CNP, calcifying nanoparticle; EthD-1, ethidium homodimer-1; P, phosphate; AM, acetoxymethyl ester.
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Related In: Results  -  Collection

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f5-ijn-9-2689: Confocal micrographs identifying apoptosis and necrosis in cultured vascular smooth muscle cells treated with CNPs.Notes: Prior to confocal imaging, CNP-treated (A–F,H) and vehicle-treated control (G) cells were incubated with calcein-AM, EthD-1, and annexin V-AlexaFluor® 647 conjugate (Life Technologies, Carlsbad, CA, USA) to label live cells (green), dead (red) cells, and apoptotic bodies (cyan), respectively. After several days in culture, monolayer cells (m) contracted into ridges (between white lines) with associated apoptotic bodies (A). Ridges later developed into nodules (arrows) comprising a core of dead cells, with live cells and apoptotic bodies peripherally (B). Maximum-intensity projection of 14 optical sections through the mid-portion of a nodule shows live (C), dead (D), and apoptotic (E) cells; (F) shows a composite image. Representative maximum-intensity projections of 76 optical sections through whole individual nodules of vehicle-treated cells (G) typically displayed less labeling for fragmented nuclei and apoptotic bodies than did a comparable projection of CNP-treated cells (H). Dotted outlines (G,H) encompass one nodule. Monolayer cell viability at day 28, determined by live/dead cell counting, was significantly reduced by CNPs and by 2 mM P; cells preexposed to 70% methanol served as dead cell control (I). *Significant difference (P<0.05) from control; n=9–10 each.Abbreviations: CNP, calcifying nanoparticle; EthD-1, ethidium homodimer-1; P, phosphate; AM, acetoxymethyl ester.
Mentions: Annexin V labeling, indicating exposed phosphatidylserine, was negative in monolayer cells of all cultures examined. However, annexin V-labeled structures up to about 300 nm in size, most likely representing apoptotic bodies, were typically distributed sparsely among the cells. Numerous apoptotic bodies, as well as an increased proportion of dead cells, colocalized within ridges formed by retracted monolayer cells (Figures 5A and B). Multicellular nodules that subsequently formed from the ridge cells contained a necrotic core of EthD-1-labeled nuclear fragments surrounded by a layer of live cells with associated apoptotic bodies (Figures 5C–F). It was not possible to quantify cell viability within multicellular nodules using this methodology. However, nodules from CNP-treated cells typically displayed more intense nucleic-acid staining of fragmented cells and greater numbers of apoptotic bodies than was observed in control cells (Figures 5G and H). After 21 days, monolayer cell viability, determined by calcein AM/EthD-1 labeling, was 97.9%±0.5% in control cultures, and was slightly but significantly reduced to 96.2%±1.0% and 95.6%±0.7% in CNP-treated and 2 mM P-treated cultures, respectively; n=9–10 each (Figure 5I).

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