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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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Hypoxia inducible factor-1α (HIF-1α) small interfering (si)RNA inhibits dickkopf-related protein 1 (DKK-1)-induced increase of human umbilical vein endothelial cells (HUVEC) migration. To collect the conditioned medium (CM), the synovial fibroblasts were treated with the indicated agents, prior to being cultured in fresh serum-deprived medium for 24 h, followed by collection of the CM. (A) Recombinant human (rh)DKK-1 accelerated the migration of endothelial cells as visualized by the wound-healing, transwell (8 µm pore size; scale bars, 50 µm), and tube formation assays. Pre-treatment with HIF-1α siRNA significantly inhibited the upregulatory effects of rhDKK-1. Quantification of HUVEC migration and tube formation in each group. The data are presented as the mean ± standard error of the mean. ###P<0.001, vs. the control group (CTR) or scramble group, and ***P<0.001, vs. the rhDKK-1 treatment group; one-way analysis of variance. (B) Wound-healing assay, (C) transwell assay, and (D) tube formation assay.
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f4-mmr-12-04-4959: Hypoxia inducible factor-1α (HIF-1α) small interfering (si)RNA inhibits dickkopf-related protein 1 (DKK-1)-induced increase of human umbilical vein endothelial cells (HUVEC) migration. To collect the conditioned medium (CM), the synovial fibroblasts were treated with the indicated agents, prior to being cultured in fresh serum-deprived medium for 24 h, followed by collection of the CM. (A) Recombinant human (rh)DKK-1 accelerated the migration of endothelial cells as visualized by the wound-healing, transwell (8 µm pore size; scale bars, 50 µm), and tube formation assays. Pre-treatment with HIF-1α siRNA significantly inhibited the upregulatory effects of rhDKK-1. Quantification of HUVEC migration and tube formation in each group. The data are presented as the mean ± standard error of the mean. ###P<0.001, vs. the control group (CTR) or scramble group, and ***P<0.001, vs. the rhDKK-1 treatment group; one-way analysis of variance. (B) Wound-healing assay, (C) transwell assay, and (D) tube formation assay.

Mentions: The synovial fibroblasts were treated with rhDKK-1 and/or HIF-1α siRNA, following which the CM was collected and, the cells were cultured in fresh serum-deprived medium for 24 h. To further assess the biological role of DKK-1 in angiogenesis, the effects of DKK-1 on HUVECs were investigated using a wound healing assay. As shown in Fig. 4A, treatment of the HUVECs with CM from synovial fibroblasts pretreated with rhDKK-1 increased migration 6-fold, as compared with the control. Furthermore, similar results were observed in the transwell and tube formation assays (Fig. 4A–D). In order to reveal the precise molecular mechanism underlying DKK-1-induced angiogenesis induction, the present study investigated whether HIF-1α, a key regulator of angiogenesis, mediated DKK-1-induced endothelial cell function. As expected, the CM from the synovial fibroblasts pretreated with HIF-1α siRNA significantly suppressed HUVEC migration, as compared with rhDKK-1 treatment alone, or scramble alone. These results were also demonstrated by the wound-healing assay, transwell assay, and tube formation (Fig. 4A–D).


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)

Hypoxia inducible factor-1α (HIF-1α) small interfering (si)RNA inhibits dickkopf-related protein 1 (DKK-1)-induced increase of human umbilical vein endothelial cells (HUVEC) migration. To collect the conditioned medium (CM), the synovial fibroblasts were treated with the indicated agents, prior to being cultured in fresh serum-deprived medium for 24 h, followed by collection of the CM. (A) Recombinant human (rh)DKK-1 accelerated the migration of endothelial cells as visualized by the wound-healing, transwell (8 µm pore size; scale bars, 50 µm), and tube formation assays. Pre-treatment with HIF-1α siRNA significantly inhibited the upregulatory effects of rhDKK-1. Quantification of HUVEC migration and tube formation in each group. The data are presented as the mean ± standard error of the mean. ###P<0.001, vs. the control group (CTR) or scramble group, and ***P<0.001, vs. the rhDKK-1 treatment group; one-way analysis of variance. (B) Wound-healing assay, (C) transwell assay, and (D) tube formation assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-mmr-12-04-4959: Hypoxia inducible factor-1α (HIF-1α) small interfering (si)RNA inhibits dickkopf-related protein 1 (DKK-1)-induced increase of human umbilical vein endothelial cells (HUVEC) migration. To collect the conditioned medium (CM), the synovial fibroblasts were treated with the indicated agents, prior to being cultured in fresh serum-deprived medium for 24 h, followed by collection of the CM. (A) Recombinant human (rh)DKK-1 accelerated the migration of endothelial cells as visualized by the wound-healing, transwell (8 µm pore size; scale bars, 50 µm), and tube formation assays. Pre-treatment with HIF-1α siRNA significantly inhibited the upregulatory effects of rhDKK-1. Quantification of HUVEC migration and tube formation in each group. The data are presented as the mean ± standard error of the mean. ###P<0.001, vs. the control group (CTR) or scramble group, and ***P<0.001, vs. the rhDKK-1 treatment group; one-way analysis of variance. (B) Wound-healing assay, (C) transwell assay, and (D) tube formation assay.
Mentions: The synovial fibroblasts were treated with rhDKK-1 and/or HIF-1α siRNA, following which the CM was collected and, the cells were cultured in fresh serum-deprived medium for 24 h. To further assess the biological role of DKK-1 in angiogenesis, the effects of DKK-1 on HUVECs were investigated using a wound healing assay. As shown in Fig. 4A, treatment of the HUVECs with CM from synovial fibroblasts pretreated with rhDKK-1 increased migration 6-fold, as compared with the control. Furthermore, similar results were observed in the transwell and tube formation assays (Fig. 4A–D). In order to reveal the precise molecular mechanism underlying DKK-1-induced angiogenesis induction, the present study investigated whether HIF-1α, a key regulator of angiogenesis, mediated DKK-1-induced endothelial cell function. As expected, the CM from the synovial fibroblasts pretreated with HIF-1α siRNA significantly suppressed HUVEC migration, as compared with rhDKK-1 treatment alone, or scramble alone. These results were also demonstrated by the wound-healing assay, transwell assay, and tube formation (Fig. 4A–D).

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