Angiogenesis, the formation of new blood vessels from the pre-existent microvasculature, is an essential component of wound repair and tumor growth. Nonsteroidal anti-inflammatories (NSAIDs) are known to suppress the incidence and progression of malignancies including colorectal cancers, and also to delay the wound healing. However, the precise mechanisms are not fully elucidated. Recent results obtained from prostaglandin (PG) receptor knockout mice indicate that host stromal PGE type receptor signaling is crucial in tumor-associated angiogenesis. Implanted tumor growth and tumor-associated angiogenesis were markedly suppressed in EP3 receptor knockout mice (EP3^-/-), in comparison with their wild-type counterparts (WT). Tumor-associated angiogenesis in WT depends on vascular endothelial growth factor (VEGF). Major VEGF-expressing cells in stroma were CD3/Mac-1 double-negative fibroblasts, and that stromal VEGF expression was markedly reduced in EP3^-/-. An EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT. The wound healing process was significantly delayed in EP3^-/-. The bone marrow transplantation of EP3^-/- bone marrow cells revealed that the recruitment of EP3-expressing bone marrow cells to the wound granulation tissues was critical to the healing of wounds. These demonstrate the significance of EP receptor signaling to the angiogenesis in vivo. Such signaling will be a good target for controlling angiogenesis in pathological conditions.

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In macrophages, Na⁺/H⁺ exchangers (NHEs) are activated by various stimuli and regulate the functions of macrophages. The NHE inhibitors amiloride, 5-(N,N-dimethyl)-amiloride (DMA) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) inhibited the lipopolysaccharide (LPS)-induced production of prostaglandin (PG) E₂ in the mouse macrophage-like cell line RAW 264. They inhibited both the LPS-induced release of arachidonic acid from membrane phospholipids at 4 h and the LPS-induced increase in the level of cyclooxygenase (COX)-2 protein at 6 h, but did not directly inhibit the COX activity. The vacuolar-type (H⁺)-ATPase (V-ATPase) inhibitor, bafilomycin A1, which activates NHE via reducing intracellular pH, also increased the level of COX-2 protein. The bafilomycin A1-induced expression of COX-2 was inhibited by the NHE inhibitors and by the Na⁺/Ca²⁺ exchanger (NCX) inhibitor SN-6, indicating that the activation of NHE leads to COX-2 expression via, in part, functional coupling with NCX. In an air pouch-type LPS-induced inflammation model in mice, amiloride and EIPA, as well as the COX inhibitor indomethacin, significantly reduced the level of PGE₂ in the pouch fluid collected at 8 h and the vascular permeability during 4 to 8 h. The accumulation of leukocytes in the pouch fluid collected at 8 h was significantly inhibited by amiloride and EIPA but not by indomethacin. Thus, NHE inhibitors showed more effective anti-inflammatory activity than indomethacin. Therefore, NHEs might be a novel target for developing anti-inflammatory drugs that inhibit the expression of COX-2 in the activated inflammatory cells.

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Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine that triggers dendritic cell (DC)-mediated T helper 2-type inflammatory responses and is considered to be a master switch for allergic inflammation such as that in asthma and atopic dermatitis. Furthermore, proinflammatory cytokines and Toll-like receptor ligands can induce TSLP production in human bronchial epithelial cells and human keratinocytes. We first found high expression of endogenous TSLP in the epithelium of allergic rhinitis with recruitment and infiltration of DCs. In culture, the TSLP production in human nasal epithelial cells was markedly and significantly increased by treatment with the proinflammatory cytokines interleukin 1β/tumor necrosis factor-α and a Toll-like receptor 2 ligand, P₃CSK₄. Since it is also thought that TSLP expression not only activates DCs but also affects the epithelial barrier in allergic rhinitis, we investigated the effects of TSLP on tight junctions of human nasal epithelial cells and DCs in vitro. Treatment with TSLP enhanced the barrier function of human nasal epithelial cells in vitro together with an increase of the tight junction proteins claudin-1,-4,-7, and occludin. Furthermore, TSLP could exclusively induce claudin-7 expression in mouse DC line XS52, which expressed tight junction molecules claudin-1,-3,-4,-6,-7,-8, occludin and tricellulin.
These findings suggest that nasal epithelial-derived TSLP plays an important role in allergic rhinitis as well as asthma and atopic dermatitis and may control tight junctions of epithelial cells and DCs to preserve the epithelial barrier and promote direct sampling of antigens by DCs.

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Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst stage embryos that can maintain an undifferentiated state indefinitely and differentiate into derivatives of all three germ layers: the ectoderm, endoderm and mesoderm. In addition, induced pluripotent stem (iPS) cells are generated by reprogramming somatic cells through the retroviral gene transfer of four factors (Oct3/4, Sox2, Klf4, and c-Myc). Here we summarize in vitro neural differentiation of pluripotent stem cells and their differentiated progeny, with a special emphasis on extracellular patterning signals for regional specification in the developing central nervous system. We have reported two methods that potently induce neural differentiation from ES cells: stromal cell-derived inducing activity (SDIA) method and serum-free floating culture of embryoid body-like aggregates (SFEB) method. SDIA- or SFEB-treated ES cells generate naive precursors that are competent to differentiate into neuroectodermal derivatives along the rostral-caudal and dorsal-ventral axes in response to patterning signals. By modifying the SFEB method with patterning factors, we have induced retinal cells from ES cells. These findings indicate the potential of the pluripotent stem cell culture system to be used for basic and medical researches.

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Dendritic cells (DCs) play important roles as modulators of immune responses and are well known for their ability to activate T cells. Recently, we demonstrated that implantation of DCs into the injured spinal cord results in activation of endogenous neural stem/progenitor cells (NSPCs), promoting repair of the injured central nervous system (CNS). DCs are strong inducers of the proliferation and survival of NSPCs as well as producers of the neurotrophic factor, NT-3. To analyze the therapeutic efficacy of DC therapy for CNS injury in a nonhuman primate, we established a method to isolate DCs from the common marmoset (CM), becuase the CM offers many advantages for preclinical studies over other monkeys. Bone marrow (BM)-derived CD11c⁺ cells from the CM showed the characteristic features of DCs, including the typical DC morphology and the ability of the cells to undergo endocytosis, secrete IL-12, and stimulate xenogenic T cells. The BM of the CM proved to be an excellent cell source for isolating DCs intended for preclinical studies of cell therapy, for which large quantities of the cells are required. We are considering a preclinical study of cell therapy using BM-derived CD11c⁺ DCs for the treatment of SCI in the CM, to evaluate the therapeutic effects and safety of this procedure for clinical application.

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Cancer stem cells (CSCs) comprise a minor population of self-renewing, immortal tumor cells capable of differentiating into the various cell types found within the bulk tumor. However, whether CSCs are derived from normal stem or progenitor cells and the underlying mechanism whereby they are generated remain to be elucidated. To answer these queries and to investigate the process of carcinogenesis, we have developed a novel strategy to introduce genetic changes into a subset of mammary stem/progenitor cells in vitro and in vivo. We used retroviral vectors to introduce the enhanced green fluorescence protein (EGFP) reporter gene into the bulk of mouse mammary epithelial cells, and evaluated the tissue regenerative potential of these cells in vitro and in vivo. We showed that EGFP-transduced primary mouse mammary epithelial cells could regenerate duct-alveolar unit-like structures in 3-D Matrigel culture. However, we could not confirm clonogenic outgrowths of ectopic EGFP-expressing transplanted mammary epithelial cells in the recipient mouse. We present the preliminary results of these studies and discuss the significance of this model.

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