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Dickkopf‑related protein 1 induces angiogenesis by upregulating vascular endothelial growth factor in the synovial fibroblasts of patients with temporomandibular joint disorders.

Jiang SJ, Li W, Li YJ, Fang W, Long X - Mol Med Rep (2015)

Bottom Line: The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC).In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration.Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‑MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, P.R. China.

ABSTRACT
Angiogenesis has an important role in the progression of temporomandibular joint disorders (TMD). The aim of the present study was to explore the association between dickkopf‑related protein 1 (DKK‑1) and angiogenesis in TMD. The expression levels of DKK‑1 and vascular endothelial growth factor (VEGF) were quantified by an ELISA assay of the synovial fluid from patients with TMD. The correlation between DKK‑1 and VEGF was analyzed by Pearson correlation test. Synovial fibroblasts were isolated from patients with TMD and were subsequently treated with recombinant human DKK‑1, anti‑DKK‑1 antibody, hypoxia inducible factor‑1α (HIF‑1α), or small interfering RNA (siRNA). The expression levels of DKK‑1, HIF‑1α, and VEGF were subsequently quantified. The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC). Increased expression levels of DKK‑1 were concordant with increased expression levels of VEGF in the synovial fluid from patients with TMD. In the synovial fibroblasts, DKK‑1 increased the expression levels of VEGF, and promoted HIF‑1α nuclear localization. In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration. The results of the present study indicate that DKK‑1 is associated with angiogenesis in the synovial fluid of patients with TMD. Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

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Dickkopf-related protein 1 (DKK-1) increased the expression levels of inflammation-induced vascular endothelial growth factor (VEGF) in the synovial fibroblasts. (A) Treatment with interleukin (IL)-1β for 24 h dose-dependently increased the protein expression levels of both VEGF and DKK-1, as determined by western blot analysis. (B) Synovial fibroblasts treated with IL-1β (0, 5, or 10 µg/ml) for 24 h increased the expression levels of DKK-1, as demonstrated by immunofluorescence. The images of each group (0, 5, and 10 µg/ml IL-1β) were captured using the same parameters. Scale bars, 25 µm. (C) Treatment with IL-1β at the indicated concentrations (0. 5, and, 10 µg/ml IL-1β) increased the mRNA expression levels of DKK-1 and VEGF. The data are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001, vs. the vehicle group, as determined by one-way analysis of variance. (D) Synovial fibroblasts treated with 10 µg/ml recombinant human (rh)DKK-1 for 24 h exhibited increased expression levels of VEGF, and synovial fibroblasts treated with 10 µg/ml anti-DKK-1 monoclonal antibody exhibited decreased expression levels of VEGF, even when combined with IL-1β treatment.
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f2-mmr-12-04-4959: Dickkopf-related protein 1 (DKK-1) increased the expression levels of inflammation-induced vascular endothelial growth factor (VEGF) in the synovial fibroblasts. (A) Treatment with interleukin (IL)-1β for 24 h dose-dependently increased the protein expression levels of both VEGF and DKK-1, as determined by western blot analysis. (B) Synovial fibroblasts treated with IL-1β (0, 5, or 10 µg/ml) for 24 h increased the expression levels of DKK-1, as demonstrated by immunofluorescence. The images of each group (0, 5, and 10 µg/ml IL-1β) were captured using the same parameters. Scale bars, 25 µm. (C) Treatment with IL-1β at the indicated concentrations (0. 5, and, 10 µg/ml IL-1β) increased the mRNA expression levels of DKK-1 and VEGF. The data are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001, vs. the vehicle group, as determined by one-way analysis of variance. (D) Synovial fibroblasts treated with 10 µg/ml recombinant human (rh)DKK-1 for 24 h exhibited increased expression levels of VEGF, and synovial fibroblasts treated with 10 µg/ml anti-DKK-1 monoclonal antibody exhibited decreased expression levels of VEGF, even when combined with IL-1β treatment.

Mentions: To assess the expression levels of DKK-1 in inflammatory primary synovial fibroblasts, the fibroblasts were initially stimulated with inflammatory IL-1β. Previous studies have suggested that the expression levels of IL-1β are increased in the synovial fluid of TMD patients, and that IL-1β is regarded as a key inflammatory response regulator (3,19,24). Following treatment with IL-1β, both the mRNA and protein expression levels of DKK-1 increased, as compared with the control (Fig. 2A–C). The fluorescence intensity of DKK-1 in the synovial fibroblasts following treatment with IL-1β are shown in Fig. 2B. Images of each group (0. 5, and 10 µg/ml IL-1β) were captured using the same parameters. In the untreated group, fluorescence intensity was lower, as compared with that of the IL-1β-treated group. The expression levels of DKK-1 were dose-dependently elevated in both the cytoplasm and nucleus of the synovial fibroblasts following treatment with IL-1β. These results were concordant with the results obtained from the ELISA. The present study also detected the expression levels of VEGF following treatment with IL-1β. IL-1β dose-dependently increased both the protein and mRNA expression levels of VEGF (Fig. 2A and C). To determine whether DKK-1 increased the expression levels of VEGF, the synovial fibroblasts were co-treated with IL-1β and anti-DKK-1 antibody. Treatment with anti-DKK-1 antibody inhibited the stimulatory effects of IL-1β on VEGF protein expression (Fig. 2D). Furthermore, the expression levels of VEGF were induced by rhDKK-1. These results suggest that DKK-1 is upregulated during inflammation, and that DKK-1 induces VEGF expression in synovial fibroblast cells.


Dickkopf‑related protein 1 induces angiogenesis by upregulating vascular endothelial growth factor in the synovial fibroblasts of patients with temporomandibular joint disorders.

Jiang SJ, Li W, Li YJ, Fang W, Long X - Mol Med Rep (2015)

Dickkopf-related protein 1 (DKK-1) increased the expression levels of inflammation-induced vascular endothelial growth factor (VEGF) in the synovial fibroblasts. (A) Treatment with interleukin (IL)-1β for 24 h dose-dependently increased the protein expression levels of both VEGF and DKK-1, as determined by western blot analysis. (B) Synovial fibroblasts treated with IL-1β (0, 5, or 10 µg/ml) for 24 h increased the expression levels of DKK-1, as demonstrated by immunofluorescence. The images of each group (0, 5, and 10 µg/ml IL-1β) were captured using the same parameters. Scale bars, 25 µm. (C) Treatment with IL-1β at the indicated concentrations (0. 5, and, 10 µg/ml IL-1β) increased the mRNA expression levels of DKK-1 and VEGF. The data are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001, vs. the vehicle group, as determined by one-way analysis of variance. (D) Synovial fibroblasts treated with 10 µg/ml recombinant human (rh)DKK-1 for 24 h exhibited increased expression levels of VEGF, and synovial fibroblasts treated with 10 µg/ml anti-DKK-1 monoclonal antibody exhibited decreased expression levels of VEGF, even when combined with IL-1β treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581813&req=5

f2-mmr-12-04-4959: Dickkopf-related protein 1 (DKK-1) increased the expression levels of inflammation-induced vascular endothelial growth factor (VEGF) in the synovial fibroblasts. (A) Treatment with interleukin (IL)-1β for 24 h dose-dependently increased the protein expression levels of both VEGF and DKK-1, as determined by western blot analysis. (B) Synovial fibroblasts treated with IL-1β (0, 5, or 10 µg/ml) for 24 h increased the expression levels of DKK-1, as demonstrated by immunofluorescence. The images of each group (0, 5, and 10 µg/ml IL-1β) were captured using the same parameters. Scale bars, 25 µm. (C) Treatment with IL-1β at the indicated concentrations (0. 5, and, 10 µg/ml IL-1β) increased the mRNA expression levels of DKK-1 and VEGF. The data are presented as the mean ± standard error of the mean. **P<0.01, and ***P<0.001, vs. the vehicle group, as determined by one-way analysis of variance. (D) Synovial fibroblasts treated with 10 µg/ml recombinant human (rh)DKK-1 for 24 h exhibited increased expression levels of VEGF, and synovial fibroblasts treated with 10 µg/ml anti-DKK-1 monoclonal antibody exhibited decreased expression levels of VEGF, even when combined with IL-1β treatment.
Mentions: To assess the expression levels of DKK-1 in inflammatory primary synovial fibroblasts, the fibroblasts were initially stimulated with inflammatory IL-1β. Previous studies have suggested that the expression levels of IL-1β are increased in the synovial fluid of TMD patients, and that IL-1β is regarded as a key inflammatory response regulator (3,19,24). Following treatment with IL-1β, both the mRNA and protein expression levels of DKK-1 increased, as compared with the control (Fig. 2A–C). The fluorescence intensity of DKK-1 in the synovial fibroblasts following treatment with IL-1β are shown in Fig. 2B. Images of each group (0. 5, and 10 µg/ml IL-1β) were captured using the same parameters. In the untreated group, fluorescence intensity was lower, as compared with that of the IL-1β-treated group. The expression levels of DKK-1 were dose-dependently elevated in both the cytoplasm and nucleus of the synovial fibroblasts following treatment with IL-1β. These results were concordant with the results obtained from the ELISA. The present study also detected the expression levels of VEGF following treatment with IL-1β. IL-1β dose-dependently increased both the protein and mRNA expression levels of VEGF (Fig. 2A and C). To determine whether DKK-1 increased the expression levels of VEGF, the synovial fibroblasts were co-treated with IL-1β and anti-DKK-1 antibody. Treatment with anti-DKK-1 antibody inhibited the stimulatory effects of IL-1β on VEGF protein expression (Fig. 2D). Furthermore, the expression levels of VEGF were induced by rhDKK-1. These results suggest that DKK-1 is upregulated during inflammation, and that DKK-1 induces VEGF expression in synovial fibroblast cells.

Bottom Line: The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC).In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration.Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‑MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, P.R. China.

ABSTRACT
Angiogenesis has an important role in the progression of temporomandibular joint disorders (TMD). The aim of the present study was to explore the association between dickkopf‑related protein 1 (DKK‑1) and angiogenesis in TMD. The expression levels of DKK‑1 and vascular endothelial growth factor (VEGF) were quantified by an ELISA assay of the synovial fluid from patients with TMD. The correlation between DKK‑1 and VEGF was analyzed by Pearson correlation test. Synovial fibroblasts were isolated from patients with TMD and were subsequently treated with recombinant human DKK‑1, anti‑DKK‑1 antibody, hypoxia inducible factor‑1α (HIF‑1α), or small interfering RNA (siRNA). The expression levels of DKK‑1, HIF‑1α, and VEGF were subsequently quantified. The present study also investigated the effects of DKK‑1 on the migration of human umbilical vein endothelial cells (HUVEC). Increased expression levels of DKK‑1 were concordant with increased expression levels of VEGF in the synovial fluid from patients with TMD. In the synovial fibroblasts, DKK‑1 increased the expression levels of VEGF, and promoted HIF‑1α nuclear localization. In addition, DKK‑1 induced HUVEC migration, and HIF‑1α siRNA inhibited DKK‑1‑induced cell migration. The results of the present study indicate that DKK‑1 is associated with angiogenesis in the synovial fluid of patients with TMD. Furthermore, HIF‑1α may be associated with DKK‑1‑induced HUVEC activation.

Show MeSH
Related in: MedlinePlus