Table 1.
PCR primer sequences.
Figure 1.
TEM analysis of serum-derived and synthetic CPP.
A , cryo-TEM image of CPP isolated from uraemic human serum showing a heterogeneous population of elongate, spindle-shaped particles, with an electron-dense core, ranging in size from approximately 80 to 250 nm (bar = 100 nm). B, immunogold labeling of fetuin-A showing irregular, but dense decoration of gold (10 nm) at particle surface, consistent with a fetuin-A containing proteinacious shell (bar = 200 nm). C, electron diffraction pattern from a typical CPP comprising a series of concentric rings, consistent with a polycrystalline material. D, electron dispersive x-ray spectroscopy (EDX) of CPP showing strong peaks for O, P and Ca giving a Ca/P− of 1.67±0.03, consistent with calcium apatite-like material. E, cryo-TEM image of synthetic CPP isolated from precipitation mix after 24 h incubation at 37°C, showing similar morphology to serum-derived CPP (bar = 100 nm for E). F, electron diffraction analysis of a synthetic CPP showing a similar pattern of concentric rings to serum-derived particle.
Figure 2.
CPP induce TNF-α and IL-1β expression and secretion from macrophages at high levels.
Murine RAW 264.7 cells were treated with synthetic CPP-containing culture medium for 24 h at the concentrations indicated. CPP exposure has a dose-dependent effect on A, TNF-α and B, IL-1β gene expression by qPCR (P for trend both <0.01, ANOVA). mRNA levels were normalized to B2 M and expressed relative to control medium (1: dashed line). Panels C and D, show the dose-dependent effect of synthetic CPP exposure on TNF-α and IL-1β secretion, respectively (P for trend both <0.01, ANOVA). TNF-α and IL-1β released into culture supernatants were analyzed by ELISA. Determinations were made in quadruplicate in 4 independent experiments and are expressed as mean ± SD. Pairwise comparisons were made using the unpaired t-test; ns, not significant (P>0.05); *P<0.05; **P<0.01.
Figure 3.
Serum-derived CPP induce less TNF-α secretion from macrophages than synthetic CPP or naked hydroxyapatite crystals.
Murine RAW 264.7 cells were treated with synthetic CPP, serum-derived CPP or hydroxyapatite (HAP)-containing culture medium for 24 h at the concentrations indicated. A, Comparison of the effects of synthetic (grey) and serum-derived (black) CPP on TNF-α secretion by macrophages (P for trend, both <0.01) B, Comparison of the effects of synthetic CPP (grey) and HAP nanocrystals (black) of equivalent size (100–200 nm) and calcium content on TNF-α secretion by macrophages. TNF-α released into culture supernatants was analyzed by ELISA (P for trend, both <0.01). Determinations were made in quadruplicate, in 3 independent experiments and are expressed as mean ± SD. Pairwise comparisons were made using the unpaired t-test; ns, not significant (P>0.05); *P<0.05; **P<0.01.
Figure 4.
CPP and hydroxyapatite crystals induce iNOS expression and intracellular 8-iso-PGF2α production in macrophage.
Murine RAW 264.7 cells were treated with CPP- or hydroxyapatite (HAP)-containing culture medium for 24 h at the concentrations indicated. A, shows the dose-dependent effect of CPP or HAP exposure on iNOS gene expression by qPCR (P for trend = 0.01, ANOVA). mRNA levels were normalized to B2 M and expressed relative to control medium (1: dashed line). B, shows the dose-dependent effect of CPP or HAP exposure on intracellular 8-iso-PGF2α production (P for trend both<0.01, ANOVA). Whole cell lysates were analyzed by ELISA and expressed as a ratio to total protein (BCA assay). Determinations were made in triplicate, in 3 independent experiments and are expressed as mean ± SD. Pairwise comparisons were made using the unpaired t-test; ns, not significant (P>0.05); *P<0.05; **P<0.01.
Figure 5.
The effect of CPP and hydroxyapatite crystals on macrophage viability and apoptosis.
Murine RAW 264.7 cells cultured in hydroxyapatite (HAP)- or CPP-containing medium showed a dose-related reduction in A, viability using MTT assay after 24 h compared to cells treated with control medium (100%, dashed line) and B, reduction in cell numbers (proliferation) after 48 h incubation (n = 6 for each treatment). Apoptosis was detected in macrophages treated with >40 µg/mL HAP or >80 µg/mL CPP- containing culture medium using C, a microplate-based TUNEL assay (n = 8 for each treatment) and D, ssDNA levels measured by ELISA (n = 8 for each treatment). E, the dose-dependent effect of CPP or HAP exposure on caspase-3 gene expression by qPCR (P for trend both <0.01, ANOVA). mRNA levels were normalized to B2 M and expressed relative to control medium (1: dashed line) (n = 4 for each treatment). F, pre-treatment of cells with the pan-caspase inhibitor z-VAD-fmk (20 µmol/L) for 1 h prior to addition of CPP- or HAP-containing culture medium (both 80 µg/mL) for a further 24 h, nullified the effect of either nanocrystal preparation on macrophage viability as assessed by MTT assay (n = 6 for each treatment). G, whole cell lysate caspase-3 protease activity was measured by monitoring the cleavage of the synthetic fluorescent substrate, Z-DEVD-AMC and was increased in cells treated with µg/mL HAP or >80 µg/mL CPP- containing culture medium for 24 h and 72 h (n = 4 for each treatment). Determinations were made in 3 independent experiments and are expressed as mean ± SD. Pairwise comparisons were made using the unpaired t-test; ns, not significant (P>0.05); *P<0.05; **P<0.01.
Figure 6.
Internalization of CPP by RAW 264.7 cells.
A, RAW 264.7 macrophage were seeded on coverslips and incubated with 100 µg/mL AlexaFluor488-labeled fetuin-A-containing synthetic CPP (green) in DMEM culture medium supplemented with 10% FBS for 30 minutes at 37°C, with (right-hand side panel) or without (left-hand side panel), quenching of cell-surface fluorescence using anti-AlexaFluor488 IgG (80 µg/mL), or PBS as control, for 1 h at 4°C. Cells were then washed, fixed, mounted in ProLong Gold antifade reagent and visualized by confocal microscopy. B, TEM of murine RAW 264.7 macrophage treated with 100 µg/mL CPP-containing culture medium supplemented with 10% FBS for 1 h at 37°C. The dashed box outlines a vacuole containing CPP (bar = 1 µm). C, magnified image of box shown in A (bar = 200 nm). Black arrowheads indicate position of vacuolated CPP near the cell surface.
Figure 7.
The effect of fetuin-A phosphorylation on CPP uptake, induction of TNF-α expression and apoptosis in RAW 264.7 macrophages.
A, adherent RAW 264.7 cells were incubated for 15 to 120 minutes with AlexaFluor 488-labeled fetuin-A-containing CPP (100 µg/mL) at 37°C in the presence or absence of monomeric free fetuin-A or albumin at the concentrations indicated. Cell-associated fluorescence was measured using a microplate reader (n = 12 for each time-point). B, Cells incubated with CPP synthesized using either AlexaFluor 488-labeled natively phosphorylated (np) or dephosphorylated (dp) fetuin-A-containing CPP (both at 100 µg/mL), showed no significant difference in cell-associated fluorescence (n = 8 for each treatment, F-test, P = 0.149). C, CPP internalization was calculated as the ratio of quenched signal (intracellular CPP) to unquenched signal (combined cell-surface and intracellular CPP) after correction for the unquenchable fluorescence at each time point. Cell-surface fluorescence was quenched using anti-AlexaFluor488 IgG (80 µg/mL) or PBS as control for 1 h at 4°C. Internalization was unaffected by the presence of free fetuin-A and albumin (data not shown). D, Increased apoptosis of RAW 264.7 cells incubated in npFet-A-containing CPP medium compared to dpFet-A-CPP after 24 h treatment. Apoptosis was detected by measuring ssDNA levels by ELISA (n = 8 for each treatment). E, shows the dose-dependent effect of npFet-A or dpFet-A-containing CPP exposure on TNF-α gene expression assessed by qPCR, after 24 h incubation (P for trend both <0.01, ANOVA). mRNA levels were normalized to B2 M and expressed relative to control medium (1: dashed line). npFet-A-containing CPP induced a greater increase in TNF-α expression than dpFet-A-containing CPP at 60 µg/mL and above (n = 4 for each treatment). Determinations were made in 3 independent experiments and are expressed as mean ± SD. Pairwise comparisons were made using the unpaired t-test; ns, not significant (P>0.05); *P<0.05; **P<0.01.
Figure 8.
SR-AI/II pathway is a major route of CPP uptake and mediates downstream effects on TNF-α expression in RAW 264.7 macrophages.
Adherent RAW 264.7 cells were pre-incubated with A–B, polyinosinic acid, at the concentrations indicated, or C–D, SR-AI/II blocking antibody (or IgG2b isotype control, both at 10 µg/mL) prior to addition of Alexa Fluor 488-labeled fetuin-A-containing CPP (100 µg/mL) for a further 30 min. Cell-associated fluorescence and internalization was determined as before (n = 12 for each time-point). Both pre-treatments significantly reduced cell-associated fluorescence and CPP uptake compared to controls, consistent with SR-AI/II pathway being a major route of CPP clearance. E, pre-incubation of cells with SR-AI/II blocking antibody (10 µg/mL) also resulted in a significant reduction in CPP-induced TNF-α expression after 24 h treatment with CPP-containing culture medium relative to pre-treatment with IgG2b isotype antisera (10 µg/mL). mRNA levels were normalized to B2 M and expressed relative to control medium (1: dashed line). Determinations were made in 3 independent experiments and are expressed as mean ± SD. Pairwise comparisons were made using the unpaired t-test; ns, not significant (P>0.05); *P<0.05; **P<0.01.
Figure 9.
CPP induce SR-AI/II expression in RAW 264.7 macrophages.
Murine RAW 264.7 cells were treated with CPP-containing culture medium for 24, 48 and 72 h, and at the concentrations indicated. A, shows the dose-dependent effect of CPP exposure on SR-AI/II gene expression in cells treated for 24 h (P for trend = 0.01, ANOVA). mRNA levels were normalized to B2 M and expressed relative to control medium (1: dashed line). B, representative Western blot of whole-cell lysate SR-AI protein levels 12 to 72 h after incubation in culture medium containing 60 µg/mL CPP. C, SR-AI protein levels normalized to β-actin and expressed relative to control levels, with or without pre-treatment of cells with 20 µmol/L z-VAD-fmk (pan-caspase inhibitor). All determinations were made in triplicate and in 3 independent experiments and are expressed as mean ± SD. Pairwise comparisons were made using the unpaired t-test; ns, not significant (P>0.05); *P<0.05; **P<0.01.