5-week-old male C57BL/6.KOR/StmSlc-ApoE^shl and C57BL/6 mice were obtained from Japan SLC (Shizuoka, Japan). After 1 week of adaptation, mice were divided and fed a western-type diet (20% Fat; 0.15% cholesterol (w/w)) containing 0.6% calcium, and 0.6%, 1.2% or 1.8% phosphorus (w/w) respectively for 7 weeks for flow cytometry or 20 weeks for atherosclerotic formation analysis. All procedures were approved by the guidelines for animal experimentation of the University of Tokushima.

A blood sample was obtained via abdominal inferior vena cava with heparin, immediately centrifuged at 10,000 rpm for 3 min at room temperature and the plasma was stored at −80°C until measurement. Urine was collected over a 24-h period and was also stored at −80°C until measurement. Total-Cholesterol (T-Cho), triglycerides (TG), inorganic P, Ca and creatinine levels in the plasma and/or urine were measured by using commercially available kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). ELISA was performed for measuring plasma FGF23 (KAINOS LABORATORIES, Inc., Tokyo, Japan), intact parathyroid hormone (iPTH) (Immunotopics International, San Clemente, CA) and MCP-1 (Thermo Fisher Scientific, Inc., Rockford, IL). Tissue plasminogen activator inhibitor-1 (tPAI-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured with a mouse adipokine plasma multiplex assay system (Millipore Japan, Tokyo, Japan). 1,25-dihydroxyvitamin D (1,25-(OH)₂D) was measured by the radioreceptor assay (SRL, Inc., Tokyo, Japan).

The aorta was perfused with phosphate buffered saline (PBS) from the left ventricle of the heart and used for en face aorta analysis. The aorta from iliac bifurcation to aortic arch was removed and external fatty deposits outside the aorta were completely removed. The aorta was cut open by longitudinally and fixed with 4% (w/v) formalin for 30 min. After Rinsing in distilled water and 60% (v/v) isopropanol, the specimen was stained with oil red O for 5 min, and then rinsed in 60% (v/v) isopropanol and distilled water. Picture was taken by TS-CA-200M camera (SUGITOH Co., Ltd., Tokyo, Japan). Image analysis was performed with Image Pro Plus (version 6.0; Media Cybernetics, Bethesda, MD). The amount of lipid deposits in each sample was measured relative to total area.

Anti-mouse Gr-1 (Ly-6G) antibody conjugated with eFluor®780, anti-CD11b (M1/70) antibody conjugated with PE(phycoerythrin)-APC-Cy7 and anti-F4/80 (BM8) antibody conjugated with eFluor®450 were obtained from eBioscience, Inc. (San Diego, CA). Peripheral blood leucocytes (500 µl whole blood per a mouse) were collected via inferior vena cava and erythrocytes were lysed. Samples were incubated with FcγIII/II receptor (2.4G2) antibody to block nonspecific antibody binding for 15 min at 4°C, stained with antibody mixture for 15 min at 4°C then stained for 7-amino-actinomycin D (7-AAD) (Sigma Aldrich Japan, Tokyo, Japan) to detect dead cells. Cells were analyzed on FACS Canto II (Japan Becton Dickinson, Tokyo, Japan). Up to 100,000 events were acquired.

To collect the peritoneal macrophages, non-treatment C57BL/6 mice were injected i.p. with 4.05% (w/v) thioglycolate medium, and after 3 days, mice were euthanized and i.p. macrophages were collected with 0.9% (w/v) saline. After washing with saline, macrophages were centrifuged for 5 min at 1,000 rpm, resuspended with Dulbecco’s modified Eagle’s medium (DMEM) including 10% (v/v) of fetal calf serum (FCS) and seeded in 96-well plates (5 × 10⁴ cells per well) for AlamarBlue assay and on coverslip in 24-well plates (5 × 10⁵ cells per well) for TUNEL staining. After incubation in humidified 37°C, 5% (v/v) CO₂ incubator for several hours, non-attached cells were removed then macrophages were used for each experiment.

Macrophages were starved fresh medium without FCS overnight and medium was changed 2 h before stimulation. To evaluate effect of phosphate concentrations in medium on cellular viability, macrophages were incubated with DMEM including 10% (v/v) of AlamarBlue reagent (Invitrogen Japan, Tokyo, Japan) for 4 h. We added appropriate amounts of sodium phosphate buffer (0.1 M Na₂HPO₄/NaH₂PO₄, pH 7.4). Delta absorbance (570–600 nm) was measured.

Apoptosis in situ detection kit (Wako) was used for TUNEL assay. Macrophages on coverslip were fixed with 4% (w/v) formalin and permeablized with permeablization solution (0.1% (w/v) sodium citric acid and 0.1% (v/v) Triton X-100). Applying TdT reaction solution for 10 min at 37°C, washing with PBS, peroxidase (POD)-conjugated antibody for 10 min at 37°C, washing with PBS then positive staining cells were visualized with diaminobenzidine (DAB) solution.

All data were expressed as the means ± SD. Statistical analysis was analyzed with student’s t test for non-parametric comparison between two groups, or analysis of variance (ANOVA) with post hoc test by Fisher’s protected least significant difference (PLSD) test for multiple comparisons. p<0.05 was considered significant.

ApoE-deficient mice were fed with western diet including 0.6% Ca, and 0.6%, 1.2% or 1.8% P for 20 weeks. In normal condition, serum Ca and P concentrations are strictly regulated by parathyroid hormone (PTH), Fibroblast growth factor 23 (FGF23) and 1,25-(OH)₂D, thus excess P can be immediately excreted into the urine. To confirm effects of prolonged much amount of P intake on kidney function and mineral metabolism in ApoE-deficient mice, we assayed plasma and urinary concentrations of markers related on Ca and P metabolisms. All groups did not show any significant difference in plasma P, and plasma and urinary Ca levels while urinary P level corrected by creatinine in 1.8% P group was significantly higher than that in the other groups (Table 1). Plasma intact PTH level in 1.2% P group was significantly higher than that in 0.6% P group, and plasma FGF23 in 1.8% P group was significantly higher than that in the other groups but plasma 1,25-(OH)₂D levels in all groups were not significantly different. Considering all various markers together, Ca and P metabolisms were normally regulated in ApoE-deficient mice in response to dietary P modifications.

As abnormal blood lipids are important risk factors for atherosclerosis, we examined effects of different amount of P intake on lipid metabolism. There was no significant difference among groups on plasma TG and T-Cho levels. Therefore, dietary P intake did not affect blood lipid concentrations in ApoE-deficient mice.

We evaluated atherosclerotic plaque formation by staining enface aorta with Oil red O. Despite our hypothesis which higher P intake can progress atherosclerotic formation, surprisingly, 1.8% P intake significantly suppressed the development of atherosclerotic plaque compared with lower P intake (Fig. 1a). Especially, lesions in thoracic and abdominal aorta decreased in 1.8% P group.

To elucidate the mechanism of suppression of atherosclerosis formation in 1.8% P group, we analyzed plasma tissue plasminogen activator inhibitor-1 (tPAI-1), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations. Plasma tPAI-1 levels were significantly decreased in 1.2% P and 1.8% P groups compared with 0.6% P group (Fig. 1b). Plasma MCP-1 levels were significantly decreased in 1.8% P group compared with the other groups. Plasma TNF-α and IL-6 did not show any significant difference among all groups.

As shown above, high P diet intake unexpectedly suppressed atherosclerosis formation. Next we investigated effect of dietary P intake on peripheral blood monocytes, because monocyte recruitment is a primary event for atherosclerogenesis. We fed ApoE-deficient and C57BL/6, as a control, mice with high fat diet including 0.6% or 1.8% phosphorus for 7 weeks and the blood leukocytes were analyzed by flow cytometry. Intraperitoneal CD11b positive and F4/80 positive cells, which indicate monocytes, were significantly increased in ApoE-0.6% P group than C57BL/6-0.6% P (WT-0.6% P) group (Fig. 2). On the other hand, the cell numbers in ApoE-1.8% P group were reduced as same as that in WT-0.6% P group.

As reason for alteration of peripheral blood monocyte cellularity, we took into considerations of effects of dietary P intake on haematocyte differentiation and/or monocyte apoptosis. In this study, we investigated whether dietary P intake can induce monocyte apoptosis; because there are several reports which higher dose P stimulation can induce apoptosis in some cell lines, such as endothelial cells, osteoblasts and chondrocytes.^(17–19) First, we used AlamarBlue reagent to assess whether extracellular P concentrations can affect macrophage viability. High P loading dose-dependently decreased the metabolic activity of thioglycolate-elicited macrophages (Fig. 3a). Since ApoE-deficient mice show increased oxidative stress,⁽²⁰⁾ thioglycolate-elicited macrophages were stimulated by high P loading under oxidative stress condition, and apoptosis was analyzed by TUNEL staining to confirm whether high concentrations of P can stimulate macrophage apoptosis. Macrophage apoptosis was enhanced by high P loading under oxidative stress (Fig. 3b).

Activation of monocytes plays an important role in the development of atherosclerosis. Peripheral leukocytes from 7-week fed ApoE-deficient and C57BL/6 mice were analyzed by flow cytometry. We found that CD11b positive, F4/80 positive and Gr-1 positive cells (including inflammatory monocytes) were 2-fold as large as Gr-1 negative cells (resident monocytes) in ApoE-0.6% P group but not the other groups (Fig. 4).