VEGF-induced osteoclast differentiation from CD14+ monocytes isolated from peripheral blood.

<p><b>(A)</b> 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>(B)</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.</p

VEGF-induced RANKL expression in RA synovial fibroblasts.

<p><b>(A)</b> 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>(B)</b> RA synovial fibroblasts were cultured with VEGF for 72 h, and RANKL concentration in the cultured media was measured by sandwich ELISA. <b>(C)</b> 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. <b>(D)</b> 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. <b>(E)</b> 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.</p

The expression of VEGF and RANKL in the synovial fluid, serum, and synovial tissues of RA patients.

<p><b>(A)</b> The synovial fluid samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. <b>(B)</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. <b>(C)</b> 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×).</p

Induction of osteoclastogenesis by VEGF-pretreated RA synovial fibroblasts.

<p><b>(A)</b> 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>(B)</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>(B)</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.</p

The signaling pathways involved in the VEGF-induced RANKL expression in RA synovial fibroblasts.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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.</p

The signaling pathways for VEGF-induced osteoclast differentiation from peripheral blood monocytes.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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.</p

Transcriptional profiles of <i>Crassostrea gigas</i> heat shock protein (<i>Hsp</i>) superfamily in response to different concentrations of TBT, diuron, and irgarol.

<p>A) Heat map analysis for effects of TBT, diuron, and irgarol on transcriptional expressions of <i>Hsp</i> superfamily. B) Expression profiles are represented by a heat map with hierarchical clustering analysis.</p

Effects of three antifouling biocides, TBT, diuron, and irgarol on acetylcholinesterase (AChE) in the gill of <i>Crassostrea gigas</i>.

<p>A) Effects of TBT, diuron, and irgarol on enzymatic activity of AChE. B) Effects of TBT, diuron, and irgarol on mRNA expression of <i>AChE</i> gene. Data are presented as the mean ± standard deviation (S.D.). Significant difference compared with control value is indicated by an asterisk (*) on the data bar (<i>p</i> < 0.05).</p

Effects of three antifouling biocides, TBT, diuron, and irgarol on antioxidant defense system in the gill of <i>Crassostrea gigas</i>.

<p>A) Results of TBT-exposed oysters. B) Results of diuron-exposed oysters. C) Results of irgarol-exposed oysters. The remaining activities were recorded as percentages relative to the control. Data are presented as the mean ± standard deviation (S.D.). Significant difference compared with control value is indicated by an asterisk (*) on the data bar (<i>p</i> < 0.05).</p

Effects of Antifouling Biocides on Molecular and Biochemical Defense System in the Gill of the Pacific Oyster <i>Crassostrea gigas</i>

<div><p>Antifouling biocides such as organotin compounds and their alternatives are potent toxicants in marine ecosystems. In this study, we employed several molecular and biochemical response systems of the Pacific oyster <i>Crassostrea gigas</i> to understand a potential mode of action of antifouling biocides (i.e. tributyltin (TBT), diuron and irgarol) after exposure to different concentrations (0.01, 0.1, and 1 μg L^-1) for 96 h. As a result, all the three antifouling biocides strongly induced the antioxidant defense system. TBT reduced both enzymatic activity and mRNA expression of Na⁺/K⁺-ATPase and acetylcholinesterase (AChE). Lower levels of both Na⁺/K⁺-ATPase activity and <i>AChE</i> mRNA expression were observed in the diuron-exposed oysters compared to the control, while the irgarol treatment reduced only the transcriptional expression of <i>AChE</i> gene. We also analyzed transcript profile of heat shock protein (<i>Hsp</i>) superfamily in same experimental conditions. All antifouling biocides tested in this study significantly modulated mRNA expression of <i>Hsp</i> superfamily with strong induction of <i>Hsp70</i> family. Taken together, overall results indicate that representative organotin TBT and alternatives have potential hazardous effects on the gill of <i>C</i>. <i>gigas</i> within relatively short time period. Our results also suggest that analyzing a series of molecular and biochemical parameters can be a way of understanding and uncovering the mode of action of emerging antifouling biocides. In particular, it was revealed that Pacific oysters have different sensitivities depend on the antifouling biocides.</p></div