Involvement of the phosphoinositide 3-kinase/Akt signaling pathway in bone morphogenetic protein 9-stimulated osteogenic differentiation and stromal cell-derived factor 1 production in human periodontal ligament fibroblasts.

Recent studies have shown that bone morphogenetic protein 9 (BMP-9) can induce osteogenic differentiation in human periodontal stem cells and human periodontal ligament fibroblasts (PDLFs). Bone morphogenetic protein 9 may be used in periodontal tissue regeneration because of its potent osteoinductive ability. Human periodontal ligament cells also have been demonstrated to produce stromal cell-derived factor 1 (SDF-1), which is important for stem-cell homing and recruitment to injured sites. In the present study, we examined the involvement of the phosphoinositide 3-kinase (PI3K)/Akt signaling axis in osteogenic differentiation and SDF-1 production in human PDLFs stimulated with BMP-9 in osteogenic medium supplemented with dexamethasone and ascorbic acid. Pretreatment of the cells with LY294002, a PI3K-specific inhibitor, suppressed not only BMP-9-enhanced alkaline phosphatase activity but also expression of a BMP-response gene (inhibitor of DNA binding 1) and osteogenic marker genes (runt-related transcription factor 2, osterix, bone sialoprotein, and osteopontin). In addition, BMP-9 up-regulated SDF-1 production, and the production of SDF-1 was suppressed by LY294002. The protein SDF-1-alpha was identified as a major isoform of SDF-1 that was regulated by BMP-9. Our data suggest involvement of the PI3K/Akt pathway in BMP-9-stimulated osteogenic differentiation and SDF-1 production in PDLFs cultured in osteogenic medium.

An exploratory study on the efficacy of rat dedifferentiated fat cells (rDFATs) with a poly lactic-co-glycolic acid/hydroxylapatite (PLGA/HA) composite for bone formation in a rat calvarial defect model.

In the last two decades, tissue-engineering approaches using scaffolds, growth factors, and cells, or their combination, have been developed for the regeneration of periodontal tissue and bone. The aim of this study was to examine the effects of rat dedifferentiated fat cells (rDFATs) with a poly lactic-co-glycolic acid/hydroxylapatite (PLGA/HA) composite on bone formation in rat calvarial defects. Twenty animals surgically received two calvarial defects (diameter, 5 mm) bilaterally in each parietal bone. The defects were treated by one of the following procedures: PLGA/HA+osteo-differentiated rDFATs implantation (PLGA/HA+rDFATs (OD)); PLGA/HA+rDFATs implantation (PLGA/HA+rDFATs); PLGA/HA implantation (PLGA/HA); no implantation as a control. The animals were euthanized at 8 weeks after the surgery for histological evaluation. The PLGA/HA composite was remarkably resorbed and the amounts of residual PLGA/HA were very slight at 8 weeks after the surgery. The PLGA/HA-implanted groups (PLGA/HA+rDFATs (OD), PLGA/HA+rDFATs and PLGA/HA) showed recovery of the original volume and contour of the defects. The newly formed bone area was significantly larger in the PLGA/HA group (42.10 ± 9.16 %) compared with the PLGA/HA+rDFATs (21.35 ± 13.49 %) and control (22.17 ± 13.08 %) groups (P < 0.05). The percentage of defect closure (DC) by new bone in the PLGA/HA+rDFATs (OD) group (83.16 ± 13.87 %) was significantly greater than that in the control group (40.61 ± 29.62 %) (P < 0.05). Furthermore, the PLGA/HA+rDFATs (OD) group showed the highest level of DC among all the groups. The present results suggest that the PLGA/HA composite is a promising scaffold and that PLGA/HA+DFATs (OD) may be effective for bone formation.

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts.

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor-ß1 (TGF-ß1), or fibroblast growth factor 2 (FGF-2), with or without antibodies to TGF-ß1 or FGF-2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen-activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal-regulated kinase (ERK) inhibitor], SP600125 [a c-Jun N-terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor] on EMD-induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose- and time-dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF-ß1 or FGF-2 significantly decreased EMD-induced VEGF production, whereas the addition of exogenous TGF-ß1 and FGF-2 stimulated VEGF production. Enamel matrix derivative-induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF-ß1 and FGF-2 in human gingival fibroblasts and that EMD-induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative-induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.

Involvement of angiotensin II type 1 receptors in interleukin-1ß-induced interleukin-6 production in human gingival fibroblasts.

The renin-angiotensin system is thought to be involved in inflammatory processes such as periodontitis. However, its precise role is still unclear. Therefore, in the present study the expression of the angiotensin II type 1 receptor (AT1R) was investigated in inflamed human gingival tissue, and the possible involvement of the AT1R in interleukin-1ß (IL-1ß)-induced interleukin-6 (IL-6) production by cultured human gingival fibroblasts (HGFs) was also studied. Immunohistochemical staining revealed that inflammatory cells and fibroblast-like cells were positive for the AT1R. However, in healthy gingival tissue, AT1R staining was very weak. The levels of AT1R mRNA and AT1R protein increased in HGFs after stimulation with IL-1ß. The levels of IL-1ß-induced IL6 mRNA and IL-6 protein were significantly reduced in AT1R gene-silenced HGFs compared with control HGFs. The data suggest that the AT1R may be involved in the regulation of gingival inflammation by modulating IL-1ß-induced IL-6 production in HGFs.

Gene transfer to corneal epithelium and keratocytes mediated by ultrasound with microbubbles.

PURPOSE: The cornea is an ideal organ for evaluating gene transfer because it can be treated noninvasively and monitored easily. The present study was performed to investigate the practical efficacy and safety of ultrasound (US) plus microbubble (MB)-mediated gene transfer to cornea. METHODS: Cultured rabbit corneal epithelial (RC-1) cells were incubated in 24-well dishes with plasmid DNA having a green fluorescent protein (GFP) gene under a cytomegalovirus promoter. The cells were exposed to US under different intensities (1 MHz; power, 0.5 approximately 2 W/cm2; duration, 15-120 seconds; duty cycle, 20%-100%). The effect of simultaneous stimulation with MBs was also examined. Gene transfer was quantified by counting the number of GFP-positive cells under microscopy. Furthermore, in vivo gene transfer was examined by GFP plasmid injection into rabbit cornea and US exposure with MBs. RESULTS: In the in vitro study, DNA exposure alone could not transfer gene into cultured RC-1 cells; US enhanced gene transfer slightly. Coexposure with MBs significantly increased gene transfer efficiency. In the in vivo study, DNA injection alone could transfer the gene to a limited degree, but plasmid injection plus US with MBs strongly increased gene transfer efficiency without apparent tissue damage, and gene transfer was achieved two dimensionally. CONCLUSIONS: US with MBs greatly increases gene transfer to in vivo and in vitro corneal cells. This noninvasive gene transfer method may be a useful tool for clinical gene therapy.

Inhibition of melanoma by ultrasound-microbubble-aided drug delivery suggests membrane permeabilization.

Ultrasound exposure-induced cavitation has been shown to accentuate cell membrane permeability, thus promoting effective drug delivery into cells, a technique that can be enhanced in the presence of microbubbles (MB). Here we applied this method as a treatment for malignant melanoma of the eyelid. The incidence of malignant melanoma in ophthalmology is relatively high, but its treatment is cosmetically difficult. A greater in vitro growth suppression of B-16 melanoma cells was achieved using ultrasound and MB in combination with the anticancer drug bleomycin than when a more concentrated dose of bleomycin alone was applied to the cell culture. Moreover, this effect was enhanced in an in vivo tumor model created by injecting B-16 melanoma cells into the lower eyelids of SCID mice. The antitumor effect of bleomycin was observed at a lower dose (0.5 mg/ ml) when the treatment was used in conjunction with ultrasound. The effect was further enhanced when MB were included, with tumor shrinkage occurring at bleomycin levels of 0.06 mg/ml. These results show that ultrasound and MB promote efficient bleomycin uptake by cells, and that the technique is a potentially useful drug delivery method.

Recombinant human growth/differentiation factor-5 (rhGDF-5) induced bone formation in murine calvariae.

OBJECTIVES: Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta superfamily, shows a close structural relationship to bone morphogenetic proteins and plays crucial roles in skeletal morphogenesis. Recombinant human (rh) GDF-5 was reported as a suitable factor for enhancing healing in bone defect and inducing ectopic bone formation. The purpose of the present study was to investigate the mechanism of bone formation induced by rhGDF-5 in murine calvariae by radiological, histological and immunohistochemical methods. Cell proliferation was also examined in vitro. MATERIAL AND METHODS: Cells including primary osteoblasts, periosteum cells and connective tissue fibroblasts were isolated enzymatically from neonatal murine calvariae or head skin. In the presence or absence of rhGDF-5, cell proliferation was estimated by tetrazolium reduction assay. To examine the mechanism of osteoinduction, rhGDF-5/atelocollagen (AC) composite or 0.01 N HCl/AC composite were injected into murine calvariae subcutaneously. Tissue was examined radiologically, histologically and immunohistochemically. RESULTS: In the presence of rhGDF-5, proliferation of primary osteoblasts, periosteum cells, and connective tissue fibroblasts was increased significantly in culture. Immunohistochemical observations showed cells at the site injected with rhGDF-5/AC displayed immunoreactivity for proliferating cell nuclear antigen (PCNA). Newly formed bone- and cartilage-like tissue contained chondrocyte osteocyte and osteoclastic cells, and were immunoreactive for both type I and II collagen. CONCLUSION: Exposure to GDF-5 promotes proliferation and differentiation of calvarial cells, which give rise to ectopic bone formation.

Synthesis of anilino-monoindolylmaleimides as potent and selective PKCbeta inhibitors.

We report herein synthesis of PKCbeta-selective inhibitors possessing the novel pharmacophore of anilino-monoindolylmaleimide. Several compounds of this series exhibited IC50's as low as 50 nM against human PKCbeta2. One of the most potent compounds, 6l, inhibited PKCbeta1 and PKCbeta2 with IC50 of 21 and 5 nM, respectively, and exhibited selectivity of more than 60-fold in favor of PKCbeta2 relative to other PKC isozymes (PKCalpha, PKCgamma, and PKCepsilon).