Mariko Kawai

Ectopic Bone Formation by Human Bone Morphogenetic Protein-2 Gene Transfer to Skeletal Muscle Using Transcutaneous Electroporation

Therapy using recombinant human bone morphogenetic protein-2 (rhBMP-2) is expected to promote bone healing and regeneration. Previous studies using protein or virus vectors for direct clinical application had problems, including a lack of efficiency, safety, and simplicity of the delivery system, and required an expensive protein, carrier matrix, or antigenic viral vector. In vivo gene transfer by electroporation is a simple and inexpensive method that only requires a plasmid and an electroporation device. Here, we created a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of rats after transferring different doses of this plasmid (25 microg, 100 microg, and 400 microg) by transcutaneous electroporation (8 electrical pulses of 100 V and 50 msec, in 1 to 5 sessions). First, we verified the gene transfer by transcutaneous electroporation using pCAGGS-lacZ. Next, the BMP-2 gene transfer and the production and localization of BMP-2 were identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blots, and immunohistochemistry. Ectopic bone formation was verified by radiography, histologic and immunohistochemical analyses, and quantitative examination. Ectopic bone formation, consisting of active osteoblasts and osteoclasts, was observed in all rats treated with electroporation. Thus, transcutaneous electroporation with pCAGGS-BMP-2 induced ectopic bone formation in the skeletal muscle of rats. This supports the possibility of applying human BMP-2 gene transfer using transcutaneous electroporation clinically.

Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression.

BackgroundTranscutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS.MethodsFirst, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec.ResultsIn the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers.ConclusionThe simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.

Simple and effective osteoinductive gene therapy by local injection of a bone morphogenetic protein-2-expressing recombinant adenoviral vector and FK506 mixture in rats

We have previously utilized a human bone morphogenetic protein-2 (BMP-2)-expressing recombinant adenoviral vector (AxCAOBMP-2) for osteoinductive gene therapy in rats. However, immunosuppression is essential for osteoinduction by AxCAOBMP-2 and this is one of the major impediments to its clinical use. Injection of AxCAOBMP-2 together with the immunosuppressant FK506 made it possible to markedly reduce the dose of the immunosuppressive agent and still induce ectopic bone reliably. We injected AxCAOBMP-2 and FK506 into the right calf muscle of rats, while the same number of plaque forming units of AxCAOBMP-2 and the same dose of FK506 placebo (vehicle) were injected into the left calf muscle. At 1, 3, 5, 7, 9 days after injection, BMP-2 mRNA expression was significantly higher in the right calf muscle than in the left calf muscle. At 21 days after injection, significantly more ectopic bone was observed in the right calf muscle than in the left calf muscle. These results indicate that coinjection of FK506 significantly promotes osteoinduction. In addition, local injection of FK506 may also make it possible to prevent a decrease of gene expression with other adenoviral vector.

Simple strategy for bone regeneration with a BMP-2/7 gene expression cassette vector

Bone morphogenetic protein (BMP) is one of the most promising candidates for bone regeneration therapy. Heterodimers of BMP family proteins, such as BMP-2/4 or BMP-2/7, are well known to have stronger osteoinduction activity than BMP homodimers. Here, we constructed a double gene cassette vector encoding BMP-2 and BMP-7, pCAGGS-BMP-2/7, and examined its potential for osteoinduction in vitro and in vivo. Expression of the pCAGGS-BMP-2/7 vector induced osteogenic differentiation in various cell lines with the same efficiency as BMP-2 and BMP-7 co-expressed from separate vectors. Moreover, the pCAGGS-BMP-2/7 vector strongly induced bone formation in rat skeletal muscle when introduced by in vivo electroporation, compared with BMP-2 or BMP-7 alone. Thus, our BMP-2/7 double gene cassette vector, or some variation of it, may be applicable for the future clinical induction of bone formation, because it does not require multiple vectors or complicated preparation.

Involvement of reactive oxygen intermediate in the enhanced expression of virulence-associated genes of Listeria monocytogenes inside activated macrophages.

Listeriolysin O encoded by 1,587 bp hly is the essential virulence factor of Listeria monocytogenes that replicates in the cytosolic space after escaping from phagosome of macrophages. By using murine macrophage‐like J774.1 cells with or without activation by IFN‐γ plus LPS, the expression of both hly and its positive regulator prfA was monitored by means of RT‐PCR. In activated J774.1 cells, the level of hly expression was enhanced although the multiplication of bacteria was significantly suppressed. The elevated expression of hly inside activated macrophage was abolished by addition of SOD and catalase, suggesting that reactive oxygen intermediates contribute to the upregulation of prfA and hly transcriptions. Moreover, we found that exposure of L. monocytogenes to H2O2 dramatically enhanced the expression of both prfA and hly mRNAs. Spontaneous ONOO– generator, SIN‐1, also promoted the transcription to a certain level. These results suggested that oxygen radicals generated in activated macrophages provide a positive signal for up‐regulation of virulence genes in L. monocytogenes.

Differential expression patterns of the tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor

Tight junctions (TJs) function primarily as a barrier against paracellular transport between epithelial cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members that show tissue- or cell-specific expression. Ameloblasts, which originate from the oral epithelium, form enamel, and enamel proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemically examined the distribution patterns of TJs in ameloblasts by observing the expression patterns of OLD and CLDs (CLD-1 to CLD-10). Secretory ameloblasts contained OLD and CLD-1, -8, and -9 at the distal end of the cell. In mature ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were present mainly at both the distal and proximal ends of the cell, regardless of whether the ameloblasts were ruffle-ended or smooth-ended. Mature ameloblasts in which only the proximal ends were stained for OLD and CLDs were also found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change drastically between the secretory and mature ameloblast stages, suggesting that these patterns reflect the different functions of these cells, specifically in the transport of proteins and ions for enamel formation.

Crystallographic Approach to Regenerated and Pathological Hard Tissues

A dominant inorganic substance in hard tissue is known to be a biological apatite (BAp)^nano-crystal which basically crystallizes in an anisotropic hcp lattice, and the BAp c-axis is parallel to extended collagen fibrils. We applied the microbeam X-ray diffractometer system with an incident beam spot 100µm or 50µm in diameter to the original, regenerated and pathological hard tissues in order to analyze the preferential alignment of the BAp c-axis as a parameter of bone quality closely relating to the mechanical function. We conclude that the BAp orientational distribution in the hard tissues is a new measure to evaluate stress distribution in vivo, nano-scale microstructure and the related mechanical function, healing process of the regenerated bone and progress of the bone diseases.

Tensile stress stimulates the expression of osteogenic cytokines/growth factors and matricellular proteins in the mouse cranial suture at the site of osteoblast differentiation

Mechanical stress promotes osteoblast proliferation and differentiation from mesenchymal stem cells (MSCs). Although numerous growth factors and cytokines are known to regulate this process, information regarding the differentiation of mechanically stimulated osteoblasts from MSCs in in vivo microenvironment is limited. To determine the significant factors involved in this process, we performed a global analysis of differentially expressed genes, in response to tensile stress, in the mouse cranial suture wherein osteoblasts differentiate from MSCs. We found that the gene expression levels of several components involved in bone morphogenetic protein, Wnt, and epithelial growth factor signalings were elevated with tensile stress. Moreover gene expression of some extracellular matrices (ECMs), such as cysteine rich protein 61 (Cyr61)/CCN1 and galectin-9, were upregulated. These ECMs have the ability to modulate the activities of cytokines and are known as matricellular proteins. Cyr61/CCN1 expression was prominently increased in the fibroblastic cells and preosteoblasts in the suture. Thus, for the first time we demonstrated the mechanical stimulation of Cyr61/CCN1 expression in osteogenic cells in an ex vivo system. These results suggest the importance of matricellular proteins along with the cytokine-mediated signaling for the mechanical regulation of MSC proliferation and differentiation into osteoblastic cell lineage in vivo.

Histogenetic Study of Verruciform Xanthoma of the Gingiva

Verruciform xanthoma is characterized macroscopically by papillomatous or verrucous hyperplasia of the mucosal epithelium and histopathologically by papillary epithelial hyperplasia and foam cell accumulation in the lamina propria between epithelial processes. This relatively rare lesion does not appear to be a true tumor, but rather an inflammatory event. Here, verruciform xanthoma of the gingiva was subjected to histopathological and immunohistochemical analyses. Notably, the cortical layer of the lesion exhibited a verrucous and granular outer surface, similar to a papilloma. Within the lesion, keratinized stratified squamous epithelium and epithelial process extensions were observed, and the connective tissue between clubbed epithelial processes was filled with foam cells. These foam cells exhibited strong cytoplasmic and membrane expression of CD68, α1-antitrypsin, and macrophage scavenger receptor-1 (MSR-1), as well as human leukocyte antigen (HLA)-DR and oxidized low-density lipoprotein cholesterol (ox-LDL). The epithelial cells also expressed HLA-DR in the cytoplasm and cell membrane. By contrast, the expression of S-100 and CD1a in Langerhans cells was clearly reduced in the epithelium of the verruciform xanthoma, while the inflammatory infiltrating cell population comprised of mainly CD3or CD8-positive cells, with few CD20or CD4-positive cells. The increased lipid content of the cell membrane and concomitant epithelial hyperplasia causes cellular injury and leakage into the connective tissue consistent with dysregulated cellular immunity in the stratified squamous epithelium. Accordingly, it may be concluded that macrophages phagocytose these lipids and differentiate to foam cells.

Alveolar bone regeneration during three weeks following transfer of BMP-2/7 gene via in vivo electroporation

Alveolar bone is not spontaneously regenerated following trauma or periodontitis. We previously proposed an animal model for new alveolar bone regeneration therapy based on the non-viral BMP-2/7 gene expression vector and in vivo electroporation, which induced the formation of new alveolar bone over the course of a week. Here, we analysed alveolar bone during a period of three weeks following gene transfer to periodontal tissue. Non-viral plasmid vector pCAGGS-BMP-2/7 or pCAGGS control was injected into palatal periodontal tissue of the first molar of the rat maxilla and immediately electroporated with 32 pulses of 50 V for 50 msec. Over the following three weeks, rats were double bone-stained by calcein and tetracycline every three days and mineral apposition rates (MAR) were measured. Double bonestaining revealed that MAR of alveolar bone was at similar level three days before BMP-2/7 gene transfer as three days after gene transfer. However, from 3 to 6 days, 6 to 9 days, 9 to 12 days, 12 to 15 days, 15 to 18 days, and 18 to 20 days after, MARs were significantly higher than prior to gene transfer. Our proposed gene therapy for alveolar bone regeneration combining nonviral BMP-2/7 gene expression vector and in vivo electroporation could increase alveolar bone regeneration potential in the targeted area for up to three weeks.