Hidefumi Komura

Metformin, an Antidiabetic Agent, Suppresses the Production of Tumor Necrosis Factor and Tissue Factor by Inhibiting Early Growth Response Factor-1 Expression in Human Monocytes in Vitro

Metformin, an antidiabetic agent, has been shown to reduce atherothrombotic disease in diabetic patients independent of antihyperglycemic effect. Recent studies have demonstrated that metformin attenuates the proinflammatory responses in human vascular wall cells and macrophages. However, the detailed molecular mechanisms underlying these therapeutic effects remain unclear. In the present study, we investigated the effects of metformin on tumor necrosis factor (TNF) production and tissue factor (TF) expression in isolated human monocytes stimulated with lipopolysaccharide (LPS) or oxidized low-density lipoprotein (oxLDL). Metformin significantly inhibited both TNF production and TF expression in isolated human monocytes stimulated with LPS or oxLDL. Metformin also significantly inhibited TNF and TF mRNA in human monocytes stimulated with LPS. Although metformin did not inhibit the activation of either nuclear factor-κB or activator protein-1, it inhibited the expression of early growth response factor-1 (Egr-1) and phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2 in monocytes stimulated with LPS or oxLDL. These results suggest that metformin may attenuate the inflammatory responses, at least in part, by suppressing the production of both TNF and TF through the inhibition of the ERK1/2-Egr-1 pathway in human monocytes.

Protective effect of milk fat globule-epidermal growth factor-factor Viii after renal ischemia-reperfusion injury in mice*

Objectives:Renal ischemia-reperfusion injury causes acute renal failure, and the hallmarks of renal ischemia-reperfusion injury are inflammation, apoptosis, necrosis, and capillary dysfunction. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, is produced by immune cells and reported to participate in multiple physiologic processes associated with tissue remodeling. We have recently shown that MFG-E8 treatment attenuates organ injury, inflammatory responses, and survival after sepsis through the enhancement of phagocytosis of apoptotic cells. The purpose of this study was to determine whether administration of MFG-E8 attenuates renal ischemia-reperfusion injury. Design:Prospective, controlled, and randomized animal study. Setting:A research institute laboratory. Subjects:Male C57BL/6J mice (20–25 g). Interventions:Renal ischemia-reperfusion injury with bilateral renal pedicle clamping for 45 mins, followed by reperfusion. A recombinant murine MFG-E8 (0.4 &mgr;g/20 g) was given intraperitoneally at the beginning of reperfusion. Measurements and Main Results:MFG-E8 levels, organ injury variables, inflammatory responses, histology, apoptosis, and capillary functions were assessed at 1.5 and 20 hrs after reperfusion. A 60-hr survival study was conducted in MFG-E8−/− and recombinant murine MFG-E8-treated wild-type mice. After renal ischemia-reperfusion injury, MFG-E8 mRNA and protein expressions were significantly decreased in the kidneys and spleen. Treatment with recombinant murine MFG-E8 recovered renal dysfunction, significantly suppressed inflammatory responses, apoptosis, necrosis, and improved capillary functions in the kidneys. In the survival study, MFG-E8−/− mice showed a significant deterioration and, in contrast, recombinant murine MFG-E8-treated wild-type mice showed a significant improvement of survival compared with vehicle-treated wild-type mice. Conclusions:MFG-E8 can be developed as novel treatment for renal ischemia-reperfusion injury. This protective effect appears to be mediated through the enhancement of apoptotic cell clearance and improvement of capillary functions in the kidneys.

Antithrombin inhibits lipopolysaccharide-induced tumor necrosis factor-α production by monocytes in vitro through inhibition of Egr-1 expression

Background: Antithrombin (AT) improves the outcome of septic patients with intravascular coagulation. However, the mechanisms underlying the therapeutic benefits of AT are not fully understood. Tumor necrosis factor‐α (TNF‐α) plays a critical role in the development of organ failure and intravascular coagulation in sepsis.Aim: This study aimed to elucidate a molecular mechanism by which AT inhibits TNF‐α productionMethods: Human peripheral monocyte was stimulated by lipopolysaccharide (LPS) and TNF‐α concentration in media was measured. Levels of phosphorylation of extracellular signal‐regulated protein kinases (ERK) 1/2 and early growth response factor‐1 (Egr‐1) were estimated by western blotting or by electrophoretic mobility shift assay.Results: Antithrombin (3 U mL–1) inhibited TNF‐α production by monocytes stimulated with LPS. Conversely, chemically modified AT that lacks affinity for heparin did not. AT inhibited the phosphorylation of ERK 1/2 and decreased the expression of Egr‐1 in LPS‐stimulated monocytes. However, it did not affect the activation of either nuclear factor‐κB or activator protein‐1. Pretreatment with KT5720, a protein kinase A inhibitor, reversed the inhibitory effect of AT on the LPS‐induced phosphorylation of ERK1/2. Although 2 U mL–1 AT slightly inhibited TNF‐α production by LPS‐stimulated monocytes, it significantly inhibited TNF‐α production in the presence of a low concentration of beraprost, a stable derivative of prostacyclin. Conclusions: These observations suggest that AT might inhibit LPS‐induced production of TNF‐α by inhibiting the increase in Egr‐1 expression in monocytes via interaction with heparin‐like substances expressed on the cell surface.

Modulation of Procarboxypeptidase R (ProCPR) Activation by Complementary Peptides to Thrombomodulin

We designed complementary peptides (C‐peptides) using a novel computer program (MIMETIC), which generates a series of peptides designed to interact with a target peptide sequence. Carboxypeptidase R (CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is stable. CPR is generated from its precursor form (proCPR) by trypsinlike enzymes, and its activation is mediated by thrombin generated in the coagulation cascade. The efficiency of activation is enhanced approximately 1,200‐fold when thrombin (T) is bound to thrombomodulin (TM). We attempted to generate C‐peptides which recognize the T‐binding site within TM assuming that some of these might interfere with the generation of T and TM complexes (T‐TM). Among three peptides designed, two inhibited the enhancement in activation of proCPR by T in the presence of TM. One of the peptides at 16 μM reduced the activation of proCPR to the level obtained by T alone.

Effect of anticoagulants in colorimetric assay for basic carboxypeptidases.

Carboxypeptidases (CP) in plasma and sera serve as regulators of anaphylatoxins such as C3a and C5a. The activity of CP can be measured by determining hippuric acid after cleavage of the small synthetic substrate hippuryl‐l‐arginine. Although a colorimetric assay is convenient for determining hippuric acid generated by CP, we noticed that some anticoagulants, such as citrate, interfere with the color development of the reagents used. EDTA and heparin provide an appropriate value. EGTA used as anticoagulant also provides an appropriate value. Therefore, concentration of citrate in samples should be controlled to be constant for background subtraction.

Heat Stability of Carboxypeptidase R of Experimental Animals

Carboxypeptidase N (CPN) and carboxypeptidase R (CPR) are present in fresh serum, and cleave C‐terminal arginine or lysine residues from bioactive peptides such as anaphylatoxins and kinins resulting in regulation of peptide activity. Although CPN is present in the active form in plasma, CPR is generated from proCPR by trypsin‐like enzymes such as thrombin. CPR regulates not only inflammatory peptides but also restricts fibrinolysis. To elucidate the complex role of CPN and CPR in vivo, studies in animal models will be essential. CPR of guinea pig, rat and rabbit decayed at 37 C rapidly as in the case of human CPR. However, at 25 C, CPR of those species decayed to some extent, although human serum CPR did not decay within 60 min. In the presence of thrombin inhibitor, CPR in the sera of animals tested decayed more rapidly than CPR in serum without thrombin inhibitor suggesting that additional generation of CPR may have been prevented during decay evaluation. However, human serum CPR decayed more rapidly in the absence of thrombin inhibitor indicating that thrombin may accelerate the decay in human serum.