Satoshi Akiba

Inhibitory effect of the leaf extract of Ginkgo biloba L. on oxidative stress‐induced platelet aggregation

The effect of the leaf extract of Ginkgo biloba L. on platelet aggregation induced by oxidative stress was studied. The extract caused a dose‐dependent inhibition of platelet aggregation stimulated with tert‐butyl hydroperoxide (t‐BHP) and Fe2+. Similar inhibitory activity was observed when platelets were exposed to H2O2 and Fe2+. Synergistic aggregation induced by a combination of t‐BHP and Fe2+ or H2O2 and Fe2+ in association with suboptimal concentration of collagen or U46619, was prevented by the extract. However, the extract failed to inhibit aggregation in response to collagen, thrombin or U46619. Ginkgolides A, B and C inhibited platelet‐activating factor‐induced aggregation, but not oxidant‐induced aggregation. These data suggest that the suppressive effect of the extract is specific on platelet aggregation stimulated by oxidative stress, and that this effect is involved in the mechanism related to its protective effect upon cerebral or myocardial injuries.

Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF‐1α‐dependent expression of thrombospondin‐1

The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF‐1α‐dependent expression of thrombospondin‐1 (TSP‐1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP‐1, and mRNA levels of integrin β3 were increased with the increase in HIF‐1α protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti‐TSP‐1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin β3. Furthermore, the migration was completely suppressed in HIF‐1α‐knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF‐1α‐overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP‐1 of which induction is fully dependent on the stabilization of HIF‐1α, in autocrine regulation. Thus we suggest that HIF‐1α plays an important role in the pathogenesis of atherosclerosis. J. Cell. Biochem. 104: 1918–1926, 2008.

Alleviation of High-Fat Diet-Induced Fatty Liver Damage in Group IVA Phospholipase A2-Knockout Mice

Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A2 (IVA-PLA2), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA2-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA2-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA2-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA2-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA2-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E2, which has a fat storage effect, was lower in IVA-PLA2-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA2 alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA2 metabolites, such as prostaglandin E2. IVA-PLA2 could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.

Inhibition of Ca2+-independent phospholipase A2 by bromoenol lactone attenuates prostaglandin generation induced by interleukin-1β and dibutyryl cAMP in rat mesangial cells

Cytokine‐induced prostaglandin generation in rat mesangial cells has been suggested to be dependent on the expression of secretory phospholipase A2 (sPLA2). In the present study, we investigated the possible involvement of Ca2+‐independent phospholipase A2 (iPLA2) in the generation. The results showed that bromoenol lactone, a relatively selective iPLA2 inhibitor, significantly attenuated prostaglandin E2 generation induced by interleukin‐1β and dibutyryl cAMP in parallel with the inhibition of iPLA2 activity. However, the inhibitor did not affect sPLA2 release upon stimulation, activities of sPLA2 or cytosolic phospholipase A2, or Ca2+ ionophore‐induced arachidonic acid liberation. These results suggest that prostaglandin E2 generation upon stimulation may be partially mediated by iPLA2 in addition to sPLA2.

Protein kinase Cα-dependent increase in Ca2+-independent phospholipase A2 in membranes and arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells

We previously reported that zymosan-stimulated, protein kinase C (PKC)-dependent arachidonic acid liberation occurs with association of Ca2+-independent phospholipase A2 (iPLA2) with the membranes of macrophage-like P388D1 cells. In the present study, the possible involvement of PKC isoforms (alpha, beta, delta, and epsilon) on the increase in iPLA2 was examined. Stimulation of P388D1 cells with zymosan induced increases in iPLA2 activity and protein in the membranes and liberation of arachidonic acid. In the stimulated cells, PKCalpha, PKCdelta, and PKCepsilon, but not PKCbeta, were increased in the membranes. The zymosan-induced increase in iPLA2 activity was suppressed by pretreatment with 4beta-phorbol 12-myristate 13-acetate for 10 hr, by which PKCalpha and PKCdelta, but not PKCbeta and PKCepsilon, were depleted, and by Gö6976, a PKCalpha inhibitor, but not rottlerin, a PKCdelta inhibitor. The zymosan-induced release of arachidonic acid was also reduced by the PKC depletion and Gö6976. However, stimulation with 4beta-phorbol 12-myristate 13-acetate alone did not increase iPLA2 activity in the membranes. Furthermore, the depletion of intracellular Ca2+ also impaired the zymosan-induced increase in iPLA2 activity in the membranes. However, no increase in iPLA2 activity was observed upon stimulation with Ca2+-mobilizing agents (ionomycin or thapsigargin). Cytochalasin D, an inhibitor of actin polymerization, suppressed the zymosan-induced increases in iPLA2 activity and protein in the membranes and the release of arachidonic acid. These results suggest that zymosan stimulates an increase in iPLA2 in the membranes of P388D1 cells probably through activation of PKCalpha in concert with cytochalasin D-sensitive events.

Glucose is necessary for stabilization of hypoxia-inducible factor-1α under hypoxia: Contribution of the pentose phosphate pathway to this stabilization

In this study, we observed that low glucose or fructose reduces the increase in hypoxia‐inducible factor‐1α (HIF‐1α) protein under hypoxic conditions. 6‐Aminonicotinamide (6‐AN), an inhibitor of the pentose phosphate pathway (PPP), also inhibited the increase of HIF‐1α protein under hypoxic conditions, while the reduced protein levels of HIF‐1α by low glucose were apparently recovered by the addition of MG‐132 or NADPH. Moreover, siRNA for glucose‐6‐phosphate dehydrogenase, which produces NADPH, reduced the increase in HIF‐1α protein. On the other hand, cobalt‐induced expression of HIF‐1α protein was not affected by low glucose or 6‐AN under normoxic conditions. In conclusion, glucose metabolism through the PPP, but not in glycolysis, plays an important role in the stabilization of HIF‐1α protein under hypoxic conditions.

Transforming growth factor-α stimulates prostaglandin generation through cytosolic phospholipase A2 under the control of p11 in rat gastric epithelial cells

The regulatory effects of transforming growth factor (TGF)‐α on phospholipase A2 (PLA2) isozymes contributing to prostaglandin generation in rat gastric epithelial RGM1 cells were examined. Stimulation with TGF‐α for 24 h time‐dependently induced prostaglandin E2 generation with an increase in cyclo‐oxygenase‐2 protein. The TGF‐α‐induced prostaglandin E2 generation was suppressed by NS‐398, a cyclo‐oxygenase‐2 inhibitor. TGF‐α stimulated the activity and the protein synthesis of cytosolic PLA2 (cPLA2). A time‐dependent increase in cPLA2 protein occurred in parallel with PGE2 generation, which was inhibited by methyl arachidonyl fluorophosphonate (MAFP), a cPLA2 inhibitor. However, no change in activity of secretory PLA2 or Ca+2‐independent PLA2 was observed in the TGF‐α‐stimulated cells. Stimulation with the Ca2+ ionophore A23187 for 10 min induced MAFP‐sensitive arachidonic acid liberation. Interestingly, preincubation with TGF‐α for 24 h diminished A23187‐stimulated arachidonic acid liberation despite the increase in cPLA2 protein. Under the conditions, TGF‐α was found to increase p11, an endogenous cPLA2 suppressor, also known as annexin II light chain. The TGF‐α‐induced increase in p11 was suppressed by tyrphostin AG1478, an inhibitor of tyrosine kinase of epidermal growth factor receptor, which was also found to restore the inhibition by TGF‐α of A23187‐stimulated arachidonic acid liberation. However, TGF‐α did not alter protein levels of annexin II heavy chain. These results suggest that TGF‐α stimulates prostaglandin generation through an increase in cPLA2, the hydrolytic action of which may be under the control of p11.

Enhancement of phospholipase A2 activation by phosphatidic acid endogenously formed through phospholipase D action in rat peritoneal mast cell

Contribution of phosphatidic acid (PA) generated by activated phospholipase (PL) D to PLA2 activation was studied in rat peritoneal mast cells. Exogenous didecanoyl PA induced arachidonate liberation in the permeabilized cells which was inhibited by p‐bromophenacyl bromide. Upon exposure of the cells to ethanol in a high enough concentration to prevent PA formation, A23187‐induced arachidonate liberation was suppressed by 50% and the rest was completely inhibited by p‐bromophenacyl bromide. In contrast, propranolol, which enhanced PA accumulation, significantly increased the arachidonate liberation. These results suggest that A23187‐induced PLA2 activation may be potentiated, at least in part, by PA generated through PLD action.

Production of interleukin (IL)-33 in the lungs during multiple antigen challenge-induced airway inflammation in mice, and its modulation by a glucocorticoid.

Although interleukin (IL)-33 is a candidate aggravator of asthma, the cellular sources of IL-33 in the lungs during the progression of antigen-induced airway inflammation remain unclear. Furthermore, it has not been determined whether the antigen-induced production of IL-33 can be pharmacologically modulated in vivo. In this study, we examined the production of IL-33 in the lungs of sensitized mice during multiple intratracheal challenges with the antigen, ovalbumin. The 1st challenge clearly induced the IL-33 production in the lungs, and it was enhanced by the 2nd-4th challenges. IL-33 mRNA transcription was also induced after these challenges. An immunohistochemical analysis revealed that the cellular sources of IL-33 after the 1st challenge were mainly bronchial epithelial cells, while those after the 3rd challenge were not only the epithelial cells, but also inflammatory cells that infiltrated the lungs. Flow cytometric analyses indicated that approximately 20% and 10% of the IL-33-producing cells in the lungs were M2 macrophages and conventional dendritic cells, respectively. A systemic treatment with dexamethasone before the 1st challenge potently suppressed the IL-33 production. When dexamethasone was administered before the respective challenges, production of the IL-33 protein and the infiltration of IL-33-producing M2 macrophages and dendritic cells into the lungs in the 3rd challenge were also suppressed. In conclusion, the cellular sources of IL-33 in the lungs were dynamically altered during multiple challenges: not only bronchial epithelial cells, but also the M2 macrophages and dendritic cells that infiltrated the lungs produced IL-33. The production of IL-33 was susceptible to the glucocorticoid treatment.

Regulatory role of antigen-induced interleukin-10, produced by CD4(+) T cells, in airway neutrophilia in a murine model for asthma.

It has been suggested that interleukin (IL)-10 exerts immunosuppressive effects on allergic inflammation, including asthma, mainly through inhibition of Th2 cell-mediated eosinophilic airway inflammation. In a model of experimental asthma utilizing multiple intratracheal antigen challenges in sensitized mice, IL-10 production as well as eosinophilia and neutrophilia in the lung were induced by the multiple challenges. In this study, we set out to reveal the cellular source of endogenously produced IL-10, and the roles of IL-10 in airway leukocyte inflammation using an anti-IL-10 receptor monoclonal antibody. Balb/c mice were sensitized i.p. with ovalbumin+Al(OH)(3), and then challenged by intratracheal administration of ovalbumin 4 times. Flow cytometric analyses revealed that the cellular source of IL-10 was CD4(+) T cells lacking the transcription factor, forkhead box P3. Treatment with anti-IL-10 receptor monoclonal antibody prior to the 4th challenge significantly augmented airway neutrophilia as well as the production of IL-1β, and CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2, but not airway eosinophilia, Th2 cytokine (IL-4 and IL-5) production, or a late-phase increase in specific airway resistance. Approximately 40% of IL-10 receptor(+) cells expressed the macrophage marker F4/80, whereas only 3-4% of the IL-10 receptor(+) cells were granulocyte differentiation antigen (Gr)-1(high) cells (neutrophils). In conclusion, multiple airway antigen challenges induced the proliferation of IL-10-expressing CD4(+) T cells in regulating airway neutrophilia. Systemic blockade of IL-10 function coincided with increases in IL-1β and CXC chemokines. Thus, IL-1β and CXC chemokines may be targets for development of novel pharmacotherapy for neutrophilic asthma.