Vascular endothelial growth factor (VEGF) and its receptor family including VEGFR-1 (Fit-1) were recently shown to be involved in pathological angiogenesis including tumor angiogenesis, tumor metastases, inflam-matory disease such as rheumatoid arthritis, psoriasis, and atherosclerosis. Rheumatoid arthritis (RA) is a chronic systemic disease characterized by an inflammatory erosive synovicitis and a pannus of inflammatory vascular tissue, leading to irreversible cartilage and bone destruction. A few cytokines such as TNF-α, IL-1 and IL-6 are known to be involved in RA. VEGF-A was reported to be highly expressed in synovial fluid in human RA, suggesting a role in RA progression. We have recently shown that VEGFR-1 is expressed not only in vascular endothelial cells but also in inflammatory cells, especially in monocyte/macrophage. However, the molecular basis of their actions on RA is not fully understood. Here we report that in a murine model of RA, deletion of the tyrosine kinase (TK) domain of VEGFR-1 (Vegfr-1 tk-/-) decreased the incidence and clinical symptoms of RA. Pathological symptoms, such as synovial hyperplasia, inflammatory infiltrates, pannus formation and cartilage/bone destruction became milder in Vegfr-1 tk-/- mice compared with Wild-type (Wt) mice in the Human T-cell Leukemia Virus-1 pX (HTLV-1 pX) induced chronic models. VEGFR-1 TK-deficient bone marrow cells showed a suppression of multi-lineage colony formation. Furthermore, macrophages induced to differentiate in vitro showed a decrease in phagocytosis and the secretion of Interleukin-6 (IL-6) and VEGF-A. Treatment of this RA model with a small molecule inhibitor for VEGFR TK, KRN951, also attenuated the arthritis. These results indicate that the VEGFR-1 TK signaling modulates the proliferation of bone marrow hematopoietic cells and immunity of monocyte/macrophages, and promotes chronic inflammation, which may be a new target in the treatment of RA.

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Regenerative medical therapy has been expected as the third therapy following the reconstructive surgery and organ transplantation to compensate for the technological and methodological disadvantages. There are three therapeutic objectives. The first is to create a new therapeutic strategy. The second is to enlarge the clinical applications of the conventional therapy. The third is to suppress the progressive deterioration of diseases. For every objective of regenerative therapy, basic idea is to induce and accelerate the regeneration and repairing of defective and damaged tissues based on the natural healing potential of patients them-selves. For the successful tissue regeneration and repairing, it is indispensable to provide cells with a local environment which enables them to efficiently proliferate and differentiate, resulting in cell-induced tissue regeneration. Biomaterials play an important role in the creation of this regeneration environment in terms of the cells scaffold of artificial extracellular matrix and the delivery technology of bio-signaling molecules to enhance the cells potential for tissue regeneration. In addition, biomaterials give cells culture conditions suitable for their in vitro proliferation and differentiation to obtain a large number of cells with a high quality for cell transplantation therapy. Cells can be genetically engineered to activate the biological functions by using the non-viral carrier of biomaterials. Several examples of in vivo tissue regeneration and basic researches for stem cells with the technology of cell scaffold and drug delivery system (DDS) of growth factors and genes are introduced to emphasize significance of biomaterials in the full realization of regenerative medical therapy.

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Embryonic stem (ES)-like cells can be induced from either mouse embryonic or adult fibroblasts by introducing four factors (Oct3/4, Sox2, c-Myc, and Klf4) and by selection for Fbx15 expression. These Fbx15 PS (induced pluripotent stem) cells are similar to ES cells in their morphology, proliferation and teratoma formation. Fbx15 PS cells, however, showed a different gene expression pattern from ES cells and they failed to produce adult chimeras. More ES-like PS cells might be generated by using a more ES specific marker than Fbx15. Based on this hypothesis, we focused on Nanog in this study, because of its essential role in pluripotency. Embryonic fibroblasts (MEF) were isolated from Nanog reporter mice, and Nanog PS cells were established by retrovirus-mediated transfection of the aforementioned four factors. Nanog PS cells showed morphology, proliferation, and teratoma formation similar to those of ES cells. These Nanog PS cells highly expressed ES cell markers, such as Nanog, Fbx15, ERas, and ESG1. Transgene expression of the four factors was strongly silenced in Nanog PS cells. When transplanted into blastocysts, adult chimeric mice were obtained from Nanog PS cells. Moreover, Nanog PS cells were transmitted through the germline to the next generation from the chimeric mice. Around 20% of the offspring, however, formed tumors, in which the reactivation of retroviral c-Myc was observed. These data demonstrated that germ-line competent PS cells can be obtained from MEF by the four factors and Nanog-selection. However, the retroviral transduction of c-Myc should be replaced by other methods prior to future medical application.

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Non-steroidal anti-inflammatory drugs (NSAIDs) are a useful family of therapeutics. The anti-inflammatory actions of NSAIDs are mediated through their inhibitory effects on cyclooxygenase (COX) activity and resulting decrease in prostaglandins (PGs). On the other hand, NSAID use is associated with gastrointestinal complications. Although PGs have a strong protective effect on gastrointestinal mucosa, the inhibition of COX by NSAIDs is not the sole explanation for the gastrointestinal side effects of NSAIDs. In this study, we examined the COX-independent mechanism involved in NSAID-induced gastric lesions. Using DNA microarray analysis, we found that CHOP, a transcription factor with apoptosis-inducing ability is induced by NSAIDs and treatment of cells with NSAIDs caused apoptosis in wild-type cells but not in CHOP-knock out cells. We also found that NSAIDs have membrane permeabilization activity. Furthermore, intracellular Ca²⁺ chelator, BAPTA-AM inhibited the NSAID-induced apoptosis and induction of CHOP. In vivo, we proposed that both COX inhibition at gastric mucosa and direct gastric mucosal cell damage (such as induction of apoptosis) by NSAIDs are required for the production of gastric lesions. Results showed that NSAID-induced apoptosis is mediated by permeabilization of cytoplasmic membranes, increase in the intracellular Ca²⁺ levels and induction of CHOP. Furthermore, results suggest that NSAIDs without membrane permeabilizing activity have reduced aastrointestinal side effects.

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Adrenomedullin (AM) is a vasodilating peptide secreted mainly from vascular wall, and its expression is markedly enhanced after stroke. We developed novel AM-transgenic (AM-Tg) mice and performed middle cerebral artery occlusion for 20 min (20m-MCAO) to examine the effects of AM on degenerative and regenerative processes in ischemic brain. The infarct area and gliosis after 20m-MCAO was reduced in AM-Tg mice in association with suppression of leukocyte infiltration, oxidative stress, and apoptosis in the ischemic core. In addition, vascular regeneration and subsequent neurogenesis were enhanced in AM-Tg mice, preceded by increase in mobilization of CD34+ cells, which can differentiate into endothelial cells. The vasculo-neuro-regenerative actions observed in AM-Tg mice in combination with anti-inflammatory and neuro-protective effects resulted in improved recovery of motor function. In vitro, AM exerted direct anti-apoptotic and neurogenic actions on neuronal cells. In summary, this study provides a basis for new strategy to rescue ischemic brain by AM through its multiple hormonal actions which concurrently lead to anti-inflammation, neuro-protection and vasculo-neuro-regeneration.

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Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with synovitis and bone destruction. The levels of monocyte/macrophage-derived cytokines, including TNFα, interleukin-1 (IL-1), and IL-6, and the T cell-derived cytokine, IL-17, all of which are involved in the pathogenesis of RA, are elevated in the synovial fluid of RA patients.
Geranylgeranylacetone (GGA), an acyclic polyisoprenoid known as teprenone, has been widely used as an antiulcer drug. We have reported that GGA inhibits human osteoclastgenesis, and that GGA increases the bone mineral density in ovariectomized rats and tail-suspended rats. These effects are due to inhibiting the prenylation of geranylgeranylpyrophosphate (GGPP) by GGA in the mevalonate pathway. Recently, we also demonstrated that GGA induces cell death in fibroblast-like synoviocytes from patients with RA. These findings suggest that GGA may be available as a new agent for RA and osteoporosis.

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