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Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here we investigated how CTGF and matrix metalloproteinases (MMPs) are involved in the early stage of hypoxia-induced angiogenesis using human breast cancer cell line, MDA231, and vascular endothelial cells. Hypoxic stimulation (5% O(2)) of MDA231 cells increased their steady-state level of ctgf mRNA by approximately 2-fold within 1.5 h, and the levels remained at a plateau up to 6 h, and then decreased by 12 h as compared with the cells cultured under the normoxic condition. Membrane-type 1 MMP (MT1-MMP) mRNA levels was also increased within a few hours of the exposure to hypoxia. Indeed, ELISA revealed that the CTGF protein/cell in medium conditioned by MDA231 cells exposed to hypoxia was maximally greater at 24 h than in the medium from normoxic cultures and that the secretion rate (supernatant CTGF/cell layer CTGF) increased in a time-dependent manner from 24 to 72 h of hypoxic exposure. Hypoxic induction of CTGF was also confirmed by immunohistochemical analyses. Furthermore, zymogram analysis revealed that the production of active MMP-9 was also induced in MDA231 cells incubated under hypoxic conditions. Finally, we found that recombinant CTGF also increased the expression of a number of metalloproteinases that play a role in the vascular invasive processes and decreased the expression of tissue inhibitors of metalloproteinases by vascular endothelial cells. These findings suggest that hypoxia stimulates MDA231 cells to release CTGF as an angiogenic modulator, which initiates the invasive angiogenesis cascade by modulating the balance of extracellular matrix synthesis and degradation via MMPs secreted by endothelial cells in response to CTGF. This cascade may play critical roles in the hypoxia-induced neovascularization that accompanies tumor invasion in vivo.

(HN) S U M M A R Y Cementogenesis starts with the differentiation of cementoblasts. Mature cementoblasts secrete cementum matrix. Cementum components are similar to bone; moreover, cementoblasts possess many characteristics similar to those of osteoblasts. Runx2 and osterix, the transcriptional factors for osteoblast differentiation, participate in tooth formation. However, the characteristics of Runx2 and osterix during the differentiation process of cementoblasts remain unclear. In this study, we examined the immunolocalization patterns of Runx2, osterix, and osteopontin during rat molar tooth formation. Periodontal ligament cells and osteoblasts located on the alveolar bone surface showed immunoreactivity for Runx2. Colocalization of Runx2 and osterix was detected in cementoblasts, which penetrated the ruptured Hertwig's epithelial root sheath and attached to root dentin. Moreover, osteopontin was observed in Runx2-positive cementoblasts facing the root surface. However, the cells adjacent to cementoblasts showed only Runx2 reactivity. Neither Runx2 nor osterix was seen in cementocytes. These results suggest that both Runx2 and osterix are important for differentiation into cementoblasts. Additionally, osterix may be indispensable for transcription of osteopontin expression. (J Histochem Cytochem 57:397-403, 2009)

OBJECTIVES AND DESIGN: A preceding paper has noted a detection of defensin‐1 (HNP‐1), a peptide with antimicrobial and cytotoxic properties, in the saliva of patients with oral squamous cell carcinoma. The present study deals with the presence of HNP‐1 in the saliva of patients with various oral diseases. METHODS: Whole saliva samples were obtained from the patientS. HNP‐1 in the saliva was isolated and purified by HPLC and the amino acid sequence of the peptide was determined. The molecular weight of HNP‐1 was measured by mass spectrometry. The concentration of HNP‐1 in saliva was determined by comparing the height of eluted HNP‐1 with that of a synthetic HNP‐1 standard. RESULTS: The concentrations of HNP‐1 in the saliva of patients with oral lichen planus (n= 5), leukoplakia (n= 4), and glossitis associated with iron deficiency (n= 4) were 8.3± 4.3μg ml‐1, 13.2± 7.9/w.g ml ‐1, and 11.4± 4.9 μg ml‐1, (mean± S. d.), respectively. These concentrations were significantly higher than those in healthy subjects (0.8 μg ml‐1) (P < 0.01). In contrast, salivary HNP‐1 concentrations in patients with glossodynia (n= 4) and oral discomfort (n= 4) were similar to those in healthy subjects. CONCLUSIONS: Since HNP‐1 is a non‐specific defensive peptide present in neutrophils, it may play an important role in the protection against diseases such as oral lichen planus, leukoplakia, and glossitis associated with iron deficiency.

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