We investigated the gene expression for non-collagenous proteins in periodontal ligament (PDL) by in situ hybridization histochemistry with a non-radioisotopic probe with cRNAs for osteocalcin (Osc), osteonectin (Osn), and osteopontin (Opn) in rat maxillary dento-alveolar Unit containing molars and intact PJlL. A highly intense positive sig-

No abstract

Edema occurs in some types of chronic inflammation such as nasal polyps, uterine cervical polyps and gastric hyperplastic polyps. However, the factors or cellular components involved in the development of edema in chronic inflammation remain to be clarified. Recently, the gene encoding vascular permeability factor (VPF) or vascular endothelial growth factor (VEGF) and the genes encoding its receptors (kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase-1 [fit-1]) have been cloned. VPF/VEGF induces vascular hyperpermeability and vascular endothelial proliferation through KDR or fit-1 receptors. As there is a possibility that VPF/VEGF may play a role in the development of edema in chronic inflammation, we examined the messenger (m) RNA expression of VPF/VEGF and its receptors in nasal polyp tissues, which is an example of chronic inflammation with remarkable edema. Using northern blotting, all nasal polyp tissues examined expressed mRNA of VPF/VEGF and KDR. In situ hybridization revealed that VPF/VEGF mRNA-expressing cells were scattered in the edematous stroma of nasal polyps. In the adjacent sections, these cells showed the morphological features of plasma cells and expressed mRNA of immunoglobulin light chains. Human B cell leukemia and plasmacytoma cell lines expressed VPF/VEGF mRNA but human mast-cell leukemia and T cell leukemia cell lines did not. The alternatively spliced pattern of VPF/VEGF transcripts observed in nasal polyp tissues was consistent with that in plasmacytoma cell lines. Taken together, the VPF/VEGF mRNA-expressing cells in nasal polyps appeared to be plasma cells, suggesting that plasma cells may play an important role in the development of edema in chronic inflammation through the production of VPF/VEGF.

The sensitive and high resolution method of in situ hybridization technique has been developed using digoxigenin-11 UTP labeled cRNA as a probe. The system was applied to the decalcified mineralized tissues such as bone and dentin. Using the system, the localization of the mRNA of bone extracellular matrix proteins, osteopontin (Osp), osteonectin (Osn), Osteocalcin (Osc) and matrix Gla protein (MGP) was examined in decalcified bone. Decalcified femurs and mandibulae of embryo, neonatal, 2 to 40-week old mice were used for examination.Osn and Osc mRNAs were localized at osteoblast in bone and at odontoblast in dentin. Although the distribution pattern of Osn positive cells and Osc positive cells was partially overlapped, Osc mRNA was detected at matured osteoblast and odontoblast but Osn mRNA was detected not only at matured but differentiating osteoblast such as flat osteoblast in the periosteal layer. Osp mRNA was detected at osteoblast in bone, but no apparent expression of Osp mRNA was found at odontoblast in dentin. MGP mRNA was detected in hypertrophic chondrocytes.These results indicated the usefulness of this system for identifying the cell types in bone and dentin.

To determine whether a system of ectopic bone formation induced by osteosarcoma-derived bone-inducing substance (bone morphogenetic protein-4) can be used as a model of developing bone at the molecular level, we studied the expression of bone-related protein mRNAs in the process of ectopic bone formation using non-radioisotopic in situ hybridization. Osteonectin mRNA was detected in fibroblast-like cells, which are similar to periosteal cells from the early to middle stages of bone development. The proportion of osteonectin mRNA-expressing cells was greater than that of osteopontin mRNA-expressing cells in hypertrophic chondrocytes and osteoblast-like cells. In contrast, osteopontin mRNA was localized in a limited population of hypertrophic chondrocytes, a single layer of osteoblast-like cells adjacent to the bone trabeculae in the middle stage of bone formation, and in a limited subset of osteocytes in the late stage. A strong osteocalcin mRNA signal was detected in osteoblast-like cells from the middle to late stages and in a limited subset of osteocytes in the late stage of bone development. Since the sequential gene expression pattern of bone-related proteins in the present system is comparable to that in embryonic osteogenesis, this system may be useful as a model for studying gene expression in osteogenesis.