Porphyromonas gingivalis lipopolysaccharide regulates ephrin/Eph signalling in human periodontal ligament fibroblasts.

OBJECTIVE: EphrinA2-EphA2 and ephrinB2-EphB4 critically engage in bidirectional signalling to modulate alveolar bone remodelling. The present study aimed to investigate the effects of lipopolysaccharides (LPS) derived from Porphyromonas gingivalis on ephrin/Eph signalling in periodontal ligament fibroblasts (PDLFs). MATERIAL AND METHODS: The primary cultured PDLFs were incubated in the absence (as a control) or presence of P. gingivalisLPS at 0.001-10 µg/mL for 24 hours. The PDLFs were then stimulated with P. gingivalisLPS at the optimal concentration (0.1 µg/mL) for different periods (6-48 hours). The expression of ephrinA2, ephrinB2, EphA2 and EphB4 was assessed by quantitative reverse-transcription real-time polymerase chain reaction and western blotting. The osteoblastic markers alkaline phosphatase, osteocalcin and Runt-related transcription factor 2 (Runx2), and the osteoclastogenesis-related factors receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin were also evaluated. RESULTS: The ephrinA2 and EphA2 expression was upregulated and EphB4 expression was downregulated by stimulation of P. gingivalisLPS. EphrinA2 mRNA expression in the PDLFs was significantly upregulated from 12 to 48 hours (P<.05), whereas EphA2 exhibited no change for the first 24 hours, after which there was a significant increase at 48 hours (P<.05). EphB4 exhibited lower mRNA expression at 12 and 24 hours than did the control (P<.05), but the change was insignificant at 48 hours. In contrast, the expression of ephrinB2 remained unchanged. The expressions of ephrinA2, EphA2, ephrinB2 and EphB4 at the protein level showed a similar pattern to that at the mRNA level. The expression of Runx2 and osteocalcin significantly decreased, whereas that of RANKL/osteoprotegerin increased. CONCLUSION: The present study suggest that P. gingivalisLPS would contribute to a dysregulation of bone remodelling, whereby ephrinA2/EphA2 expression is stimulated and EphB4 expression is inhibited.

[Moderate Angiogenic Effect of Xuefu Zhuyu Capsule by Regulating EphB4/EphrinB2].

Objective To observe moderate angiogenic effect of Xuefu Zhuyu Capsule (XFZYC) on human microvascular endothelial cell line 1 ( HMEC-1) , and its regulation effect on expression of EphB4/EphrinB2. Methods The moderate angiogenic effect of XFZYC was clarified by detecting XFZYC containing serum on cell viability, cell cycle, migration, adhesion and in vitro angiogenesis. Its effects on expressions of EphB4/EphrinB2 were detected by Real-time quantitative PCR and Western blot. Results XFZYC containing serum (XFZYC-CS) had no effect on the cell viability or cell ratio in phase S endothelial cells. Cell migration was significantly improved by 1.25% XFZYC-CS after 24, 48, and 72 h of action 2. 50% XFZYC-CS inhibited cell migration at the primary 24 h, but it significantly promoted cell migration at 48 and 72 h afterwards. It showed just an opposite tendency to 5. 00% XFZYC-CS. Cellular adhesion number was significantly reduced by 1. 25% XFZYC-CS at 72 h. Cellular adhesion number was significant- ly increased by 2. 50% XFZYC-CS at 24 and 48 h, but inhibited at 72 h 5. 00% XFZYC-CS showed inhibition at 24 h, but turned to promotion, and disappeared afterwards. In vessel formation aspect, only 2.50% XFZYC-CS showed vessel formation promotion 5. 00% XFZYC-CS showed inhibition on vessel formation at 48 and 72 h. Results of Real-time quantitative PCR and Western blot in 2. 50% XFZYC-CS showed EphB4 expression was up-regulated at 12 h; EphB4 expression was down-regulated while EphrinB2 expression was up-regulated at 24 h. Conclusions Only 2. 50% XFZYC-CS at 48 h had promotion of migration, adhe- sion, and in vitro angiogenesis of HMEC-1 , which was the optimal condition for vessel growth. These re- sults suggested XFZYC promoted angiogenesis in certain conditional limitations. But it regulated the ex- pression of EphB4/EphrinB2, which might be one of important factors.

Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling.

Recent studies have demonstrated that erythropoietin (EPO) has extensive nonhematopoietic biological functions. However, little is known about how EPO regulates bone formation, although several studies suggested that EPO can affect bone homeostasis. In this study, we investigated the effects of EPO on the communication between osteoclasts and osteoblasts through the ephrinB2/EphB4 signaling pathway. We found that EPO slightly promotes osteoblastic differentiation with the increased expression of EphB4 in ST2 cells. However, EPO increased the expression of Nfatc1 and ephrinB2 but decreased the expression of Mmp9 in RAW264.7 cells, resulting in an increase of ephrinB2-expressing osteoclasts and a decrease in resorption activity. The stimulation of ephrinB2/EphB4 signaling via ephrinB2-Fc significantly promoted EPO-mediated osteoblastic differentiation in ST2 cells. EphB4 knockdown through EphB4 shRNA inhibited EPO-mediated osteoblastic phenotypes. Furthermore, in vivo assays clearly demonstrated that EPO efficiently induces new bone formation in the alveolar bone regeneration model. Taken together, these results suggest that ephrinB2/EphB4 signaling may play an important role in EPO-mediated bone formation.

VEGFR2-dependent angiogenic capacity of pericyte-like dental pulp stem cells.

Dental pulp stem cells (DPSCs) have previously demonstrated potential pericyte-like topography and function. However, the mechanisms regulating their pericyte function are still unknown. In this study, murine DPSC angiogenic and pericyte function were investigated. Tie2-GFP mouse DPSCs were negative for GFP, indicating the absence of endothelial cells in DPSC cultures. Endothelial cells co-cultured with DPSCs formed more mature in vitro tube-like structures as compared with those co-cultured with bone marrow stromal cells (BMSCs). Many DPSCs were located adjacent to vascular tubes, assuming a pericyte location. Subcutaneous DPSC transplants in mice with matrigel (MG) (DPSC-MG) induced more vessel formation than BMSC-MG. Soluble Flt (sFlt), an angiogenic inhibitor that binds VEGF-A, significantly decreased the amount of blood vessels in DPSC-MG, but not in BMSC-MG. sFlt inhibited VEGFR2 and downstream ERK signaling in DPSCs. Similar to sFlt inhibition, VEGFR2 knockdown in DPSCs resulted in down-regulation of Vegfa, Vegf receptors, and EphrinB2 and decreased angiogenic induction of DPSCs in vivo. Therefore, the capacity of DPSCs to induce angiogenesis is VEGFR2-dependent. DPSCs enhance angiogenesis by secreting VEGF ligands and associating with vessels resembling pericyte-like cells. This study provides first insights into the mechanism(s) of DPSC angiogenic induction and their function as pericytes, crucial aspects for DPSC use in tissue regeneration.