Vascular development consists of two processes termed vasculogenesis and angiogenesis. The system of TIE 2-Angiopoietin (Ang) is involved in the later, angiogenesis. It has been demonstrated that the function of TIE 2 is associated with adhesion and dissociation between endothelial cells and mural cells, and survival, apoptosis, and chemotaxis of endothelial cells Ang 2, which is produced from endothelial cells under tissue hypoxia, has been suggested to be a key regulator for the initiation of endothelial cell sprouting from pre-existing vessels. Although Ang 2 binds to TIE 2, Ang 2 does not promote activation of TIE 2. Ang 2 produced from endothelial cells under hypoxia inhibits the binding of Ang 1 to TIE 2. Ang 1 promotes activation of TIE 2 and adhesion between endothelial cells and mural cells. Therefore, endothelial cells detouched from mural cells by Ang 2 are permitted to move to avascular area in which oxygen or nutrient is desired. We recently found that hematopoietic stem cells promote chemotaxis and nextwork formation of TIE 2 positive endothelial cells by producing angiopoietin-1. This novel function may be utilized for regeneration therapy of neovascularization in the patient by transplanting the hematopoietic stem cell into the location.

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Polymorphonuclear leukocytes (PMNL) play important roles both in host defenses and systemic inflammatory responses after severe insults. The two-hit theory specifies that initial injury primes the inflammatory system, in particular PMNL and endothelium, and that exposure to a second stimulus induces an exaggerated inflammatory response and further tissue injury. These concepts have been introduced in animal studies and have not been proved in patients following severe insults. We evaluated the systemic inflammatory response to second hits in severe trauma patients based on the functions in PMNL, and reviewed the second hit phenomenon following severe injuries. Infections as second hits further enhanced the priming of PMNL in severely injured patients, but second hit priming in PMNL did not cause systemic vascular endothelial damage. Brain deaths after severe head injuries enhanced the priming of PMNL, and induced endothelial activation and organ damage. Secondary operations following trauma suppressed the oxidative activity in primed PMNL or inhibited the phagocytosis in non-primed PMNL, but the dynamic changes in PMNL functions did not cause further tissue injury or immunosuppression. These results suggest that the impacts of second hits on PMNL functions depend on the pre-operative priming conditions and the type of secondary insult. The concepts of two-hit theory may not be true in most cases and second hit phenomenon may be limited in trauma patients.

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Fas and Fas ligand (FasL) have been assigned a pivotal role for the development and maintenance of peripheral tolerance. Mice having defects in Fas and FasL develop lupus like symptoms. In this study, we have studied FasL expression of peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE) . Expression of FasL was not or very weakly detected in the freshly isolated PBMC in normal individuals. In contrast, freshly SLE PBMC exhibits enhanced expression of FasL withoutin vitrostimulation. Not only purified T cells but also purified B cells expressed FasL on their cell surface spontaneously in patients with SLE. Anti-DNA antibody secreting B cells were purified by using biotin labeled DNA and streptavidin-bead, and the freshly isolated anti-DNA autoantibody secreting B cells express FasL spontaneously. Thus autoreactive B lymphocytes that aberrantly express FasL would kill Fas+ immunoregulatory T lymphocytes. The aberrantly expressed FasL on B cells would, at least partly, facilitate escape of the autoreactive B cells from immune tolerance system, and may contribute sustained secretion of autoantibodies in patients with SLE.

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We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneous beating cells were repeatedly screened for. The cells showed a f ibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells : they connected with adjoining cells after 1 week, formed myotube-like structures and began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. They expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres and atrial granules. These cells had several types of action potentials : sinus node like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain and α-actin, indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. These cells expressed Nkx 2.5/Csx, GATA 4, TEF-1 and MEF-2 C mRNA before 5-azacytidine treatment, and expressed MEF-2 A and MEF-2 D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

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We previously demonstrated that treatment of human gingival fibroblasts (GF) with anti-CD 44 mAb (OS/37) enhanced the adhesion between K 562 (erythroleukemia line) and GF and that anti-CD 43 mAb, 3 S-B 2, specifically inhibited this heterotypic cell adhesion. In this study, the regulatory mechanism of GF-K 562 interaction by 3 S-B 2 was investigated. In order to assess the possibility that CD 43 may function as an adhesion molecule between GF and K 562, soluble chimeric CD 43, CD 43-Ig, was added to the co-culture of K 562 and OS/37-treated GF. However, CD 43-Ig did not inhibit the heterotypic cell adhesion. This suggests that CD 43 on K 562 may not be directly involved in the adhesion to OS/37-treated GF. We then examined the possible regulatory mechanism via CD 43-cytoplasmic domain in this heterotypic cell adhesion. By transfecting full length or cytoplasmic region- deleted human CD 43 cDNA into mouse thymoma (EL-4), we established EL-4-CD 43 F or EL-4-CD 43 TM, respectively. Although these two transfectants expressed equal level of exogenous CD 43 molecules, 3 S-B 2 inhibited the binding between OS/37-treated GF and EL-4-CD 43 F, but not EL-4-CD 43 TM. These results demonstrate that acting as a signal transducing molecule, CD 43 regulates the GF-K 562 binding probably via a cytoplasmic domain of CD 43.

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The change in pain threshold level by injection of sodium hyaluronate (HA average molecular weight 600, 000 to 1, 200, 000) into the knee joint cavity was evaluated in rats using supported hind limb paw pressure measurement. Paw pressure ratio (ppr) of treated to non-treated hind limb was monitored by pressure transducers with computer analysis. HA of 0.25 to 1.0 mg/rat in constant volume (0.1 ml) was injected into the joint cavity resulting in a significant decrease in ppr with dose dependent manner. The nociceptive effect was transient and restored 10 minutes after HA was administrated. Monosodium urate crystals (MSU) injected directly into the hind limb joint cavity produced severe pain with the threshold being restored to normal after 7-day MSU-treatment. Long lasting and drastic nociceptive effect was induced with HA injected into the 7-day MSU-treated joint cavity. These results suggest that HA injected into the inflamed joint cavity produces a lowering effect of the pain threshold.

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It is a well-known fact that IgE is a key substance which induces the IgE-mediated allergic reaction in the extravascular tissue. However, it remains to be elucidated how IgE in the circulating blood transfers to the site of allergic reaction in the extravascular tissue. In this paper, the rat IgE passage through cultured rat aortic endothelial cells was examined using a dual chamber system. The effect of tranexamic acid was investigated for the supernatant of cultured mast cell-enhanced permeability of rat IgE across rat aortic endothelial cell. The permeability constant (PC) was used to evaluate the degree of the IgE passage and effect of tranexamic acid for IgE passage. PC of IgE was (0.29 ±0.13) × 10^-5cm/sec in serum free medium and it was (1.13± 0.06) × 10^-5cm/sec in the supernatant of cultured mast cell. In addition, tranexamic acid (t-AMCHA) of 5 × 10^-3M, 1 × 10^-2M, 5 × 10^-2M at the concentration in upper chamber suppressed significantly the enhanced IgE passage by the supernatant of cultured mast cell in lower chamber. It was suggested from the above-mentioned results that tranexamic acid (Transamin^®) might be useful to control IgE-mediated allergic reaction due to the inhibition of IgE transfer to extravascular site from the circulating blood.

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