Fig 1.
The expression of VEGF and RANKL in the synovial fluid, serum, and synovial tissues of RA patients.
(A) The synovial fluid samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. (B) The serum samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. Each dot expresses the results from an individual patient. (C) The synovial tissues of patients with RA and osteoarthritis (OA) were simultaneously labeled with anti-VEGF (green), anti-RANKL (red), and CD55 (white) antibodies and then photographed under appropriate filters. The merged image shows co-localization of the three markers (yellow). Sections were counterstained with DAPI staining. The figures are representative of three independent experiments (original magnification 400×).
Fig 2.
VEGF-induced RANKL expression in RA synovial fibroblasts.
(A) After RA synovial fibroblasts were cultured with 0–50 ng/ml of VEGF for 72 h, the RANKL mRNA expression determined by RT-PCR. Data were normalized to beta-actin and reported in relative expression units. The figure is representative of three experiments. (B) RA synovial fibroblasts were cultured with VEGF for 72 h, and RANKL concentration in the cultured media was measured by sandwich ELISA. (C) RA synovial fibroblasts were cultured with VEGF for 72 h and then stained with anti-RANKL antibodies (red) (original magnification 400×). The figures are representative of three independent experiments. (D) Triplicate wells of RA synovial fibroblasts were transfected with 1 μg of pGL3-RANKL reporter plasmids and 1 μg of pRLTk control plasmid. Both firefly and renilla luminescence were measured after 24 h incubation with 20ng/ml of VEGF. (E) After RA synovial fibroblasts were cultured with VEGF for 72 h, the concentrations of IL-1β, TNF-α, and IL-6 in the cultured media was determined by sandwich ELISA. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.
Fig 3.
The signaling pathways involved in the VEGF-induced RANKL expression in RA synovial fibroblasts.
(A) RA synovial fibroblasts were pretreated with anti-VEGFR1 (20 ng/ml), anti-VEGFR2 (20 ng/ml), SB203580, a p38 MAPK inhibitor (10 nM), Src inhibitor (10 nM), or PKC inhibitor (5 nM) for 1 h, and then cultured with 20 ng/ml VEGF for 72 h. The expression of RANKL mRNA was determined by real time-PCR. Data were normalized to beta-actin and reported in relative expression units. (B) RA synovial fibroblasts were stimulated with 20 ng/ml VEGF, the phosphorylated forms of Src, PKC, and ERK were detected by western blotting. The figures are representative of three independent experiments. (C) Stimulation of RA synovial fibroblasts with VEGF activated the phosphorylation of p-Src, Src, p-PKC, PKC, p-ERK and ERK as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. Data were normalized to beta-actin and reported in relative expression units. The figure represents one of three independent experiments. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.
Fig 4.
VEGF-induced osteoclast differentiation from CD14+ monocytes isolated from peripheral blood.
(A) CD14+ monocytes isolated from peripheral blood of RA patients were cultured with 25 ng/ml M-CSF and 0–50 ng/ml VEGF or 30 ng/ml RANKL. After maximal 21 days of culturing, TRAP-positive multinucleated cells were counted. The figures represent one of three independent experiments. (B) The gene expression of osteoclast markers such as TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.
Fig 5.
The signaling pathways for VEGF-induced osteoclast differentiation from peripheral blood monocytes.
(A) CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of 20ng/ml of anti-VEGFR1, 20ng/ml of anti-VEGFR2, or 10nM of p38 MAPK inhibitor. After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. (B) CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of Src inhibitor (10 nM), or PKC inhibitor (5 nM). After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. (C) The gene expression of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts was measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.
Fig 6.
Induction of osteoclastogenesis by VEGF-pretreated RA synovial fibroblasts.
(A) RA synovial fibroblasts were cultured with 20 ng/ml of VEGF for 72 h and RANKL production was quantified using ELISA in the cultured media. (B) RA synovial fibroblasts were preincubated with 20 ng/ml of VEGF for 72 h and Src inhibitor (10 nM) or PKC inhibitor (5 nM) and then cocultured with CD14+ monocytes from the peripheral blood in the presence of M-CSF. After 21 days of culturing, TRAP-positive multinucleated cells were counted. The figure represents one of three independent experiments. (B) The gene expressions of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.