Fig 2: Immunohistochemistry experiments were used to compare symptomatic carotid plaques (SC) and asymptomatic carotid plaques (AsC).Asymptomatic lesions presented higher OPG infltration, p<0.001 (A), higher CD146+ pericyte infiltration, p<0.001 (B), and higher CD31+ endothelial cell infiltration, p = 0.23 (C). Representative images are on the left with corresponding quantification on the right.

Fig 3: Inhibitory activity of osteoprotegerin (OPG) on osteoclastogenesis is suppressed by HtrA1. a RAW 264.7 cells were seeded at 1 × 104 cells per well in 24-well plates. OPG at 100 ng mL-1 was pretreated with HtrA1 (0.1 ng mL-1, 10 ng mL-1) for 60 min at 37 °C. Pretreated OPG (100 ng mL-1) together with or without RANKL (100 ng mL-1) was then added to RAW 264.7 cell cultures (1 × 104 cells per well). After culture for 4 days, TRAP activity of the culture medium was measured. Data are expressed as means ± SD (n = 3). HtrA1 (0.1): t6 = 10.3, p = 9.0 × 10-5. HtrA1 (10): t6 = 11.4, p = 5.0 × 10-5. ***p < 0.001; by one-way ANOVA with Dunnett’s test. b, c RAW 264.7 cells (2 × 104 cells per well in 24-well plates) were transfected with Htra1 siRNA (20 pmol) or control scramble siRNA (Sc. siRNA, 20 pmol) and cultured for 4 days in the presence of RANKL (100 ng mL-1). b Htra1 mRNA expression was determined by quantitative RT-PCR. Data are expressed as means ± SD (n = 3). t4 = 26.9, p = 1.1 × 10-5, 95% confidence interval (CI) [22.9, 28.1]. ***p < 0.001; by Student’s t-test. c Amounts of HtrA1 protein in the culture medium was determined by western blotting. The blots were scanned and analyzed by densitometry. The data shown are a representative result of three independently performed experiments (Supplementary Fig. 9). d RAW 264.7 cells were seeded at 2 × 104 cells per well in 24-well plates. Cells were then transfected with either Htra1 siRNA (20 pmol) or control Sc. siRNA (20 pmol). Transfected cells were cultured in the presence of RANKL (100 ng mL-1) and OB-CM (0.5 mL per well). After culture for 4 days, the concentration of OPG in the culture medium was quantified by ELISA. Data are expressed as means ± SD (n = 3). t4 = -14.4, p = 1.3 × 10-4, 95% CI [-0.08, -0.054]. ***p < 0.001; by Student’s t-test

Fig 4: HtrA1 recognizes the three-dimensional structure and cleaves osteoprotegerin (OPG). a OPG22–196 (2 µg) was incubated with HtrA1 (0.5 µg) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT, 10 mM) and iodoacetamide (IAA). Conventional treatment after incubation (-) was compared with reducing OPG22–196 by pre-treatment with DTT and IAA before incubation (+). OPG fragment sequences were identified using sequence analysis software (Protein Pilot). Amino-acid residues contained in the detected peptides were counted. b OPG22–196 (2 µg) was incubated with HtrA1 (0.5 µg) at 37 °C for 5 min. The reaction mixture was treated with DTT and IAA, and subjected to MALDI-TOF MS. The measurement of native OPG22–196 was shown at 0 min. The measurement of reduced carbamidomethylated OPG fragments were shown after the incubation with HtrA1 for 5 min. m/z indicates the mass-to-charge ratio. Intact OPG22–196 and reduced carbamidomethylated OPG22–196 were detected before and after the incubation with HtrA1 (m/z 19778 and 20750, respectively). Before the incubation, a doubly charged ion of OPG (m/z 9909) was also detected (0 min). Two characteristic OPG fragment peaks (OPG22–90, OPG91–196, m/z 8673 and 12205) derived from OPG22–196 were detected 5 min after the reaction. c After the incubation of OPG22–196 with HtrA1, the reaction mixture was treated with DTT and IAA. The sequences of OPG fragments were identified using Protein Pilot. Detections of the C- and N-terminal residues in OPG fragments were counted. Leucine 90 showed the highest peak at 5 min (arrow).

Fig 5: Degradation of osteoprotegerin (OPG) in murine co-cultures. a–c Primary osteoblastic cells (3 × 104 cells per well) and bone marrow cells (4 × 105 cells per well) were co-cultured in 24-well plates in the presence or absence of 1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3; 1 × 10-8 M]. The culture medium was changed every 2 days. a OPG concentration in the culture medium during differentiation. Day 2: t4 = 8.2, p = 0.0012, 95% confidence interval (CI) [0.41, 0.82]; Day 4: t4 = 10.8, p = 4.1 × 10-4 95% CI [0.69, 1.2]; Day 6: t4 = 26.0, p = 1.3 × 10-5, 95% CI [1.1, 1.4]). b, c Cells were stained for tartrate-resistant acid phosphatase (TRAP). TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts (OCs). b TRAP-positive osteoclast (OC) counts. Day 2: t4 = -1.9, p = 0.13, and 95% CI [-13.2, 2.6]; Day 4: t4 = -8.9, p = 9.0 × 10-4, and 95% CI [-124.8, -65.2]; Day 6: t4 = -23.7, p = 1.9 × 10-5, 95% CI [-319.5, -252.5]. c TRAP-positive OCs (arrows) only formed in the presence of 1a,25(OH)2D3. Bar: 100 µm. d OPG (arrow) was detected in the culture medium on day 6. Coomassie brilliant blue staining is shown as loading control. e OPG concentration in RANKL-treated bone marrow-derived macrophages (BMMs). Cells were prepared in 24-well plates. Conditioned medium obtained from osteoblastic cell cultures (OB-CM) was used as the source of mouse OPG. BMMs were cultured for 3 days with OB-CM (0.5 mL per well) in the presence of M-CSF (100 ng mL-1) with or without RANKL (100 ng mL-1). t10 = 21.4, p = 1.1 × 10-9 and 95% CI [0.48, 0.59]. f OPG concentration in RANKL-treated RAW 264.7 cells. Cells were cultured for 2 days with OB-CM (0.5 mL per well) in the presence of RANKL (100 ng mL-1) in 24-well plates (1 × 104 cells per well). t4 = 8.1, p = 0.0012, and 95% CI [0.37, 0.75]. Data are expressed as means ± SD (n = 3 - 6). **p < 0.01, ***p < 0.001; by Student’s t-test. OPG concentrations were measured by ELISA