A novel bioaction of PAF: Induction of microbicidal activity in guinea pig bone marrow cells

When guinea pig bone marrow cells were incubated in the presence of 10⁻⁸ to 10⁻⁶ M platelet activating factor (PAF) for 24 to 72 hr, microbicidal activity againstCandida parapsilosis of cells was augmented. This augmentation was inhibited by PAF-specific antagonists, CV6209 or FR900452. PAF-specific binding sites with a high affinity were found on guinea pig bone marrow cells. Carrageenan or 2-chloroadenosine, reagents known to be preferentially cytotoxic to macrophages, abolished the microbicidal activity of PAF-treated bone marrow cells. Macrophages prepared from the peritoneal cavity, however, acquired no appreciable microbicidal action by treatment with PAF. These observations suggest that PAF may affect a class of guinea pig bone marrow cells through binding to receptors specific to PAF, resulting in activation and/or induction of differentiation of monocyte-macrophage lineage cells.     

Biological response of guinea pig peritoneal macrophages to platelet-activating factor

We investigated the effects of platelet-activating factor (PAF) on guinea pig peritoneal macrophages. Specific and high-affinity binding sites for PAF were detected on guinea pig peritoneal macrophages. Scatchard analysis of PAF binding revealed high affinity binding sites (7.9×10⁴/cell) with a dissociation constant of 2.3×10⁻¹⁰ M. When treated with 10⁻⁹−10⁻⁵ M PAF, guinea pig peritoneal macrophages released hydrogen peroxide into the medium in a time-dependent manner. The release reaction upon stimulation with 10⁻⁵ M PAF reached a plateau within 30 min and the extent of release was twice as high as that when stimulated byN-formyl-L-methionyl-leucyl-L-phenylalanine (fMLP; 2 μM)-treated cells. Neither lysoPAF nor the PAF enantiomer was effective. PAF-induced H₂O₂ release was inhibited specifically by PAF antagonists, suggesting that PAF activated macrophages through binding to specific sites. Lysosomal enzyme (N-acetyl-β-D-glucosaminidase) was released from guinea pig peritoneal macrophages upon treatment with 10⁻⁵ M PAF for 60 min. Guinea pig peritoneal macrophages were treated with PAF for 8 hr and the conditioned medium was examined for cytokines. The medium exhibited cytocidal activity against mouse fibroblast L929 cells [tumor necrosis factor (TNF) activity], and this activity was comparable to that detected after treatment of cells with the bacterial lipopolysaccharide (LPS). Furthermore, the same conditioned medium also showed colony-stimulating factor (CSF) activity. Generation of these cytokines was stereospecific. Our findings suggest that PAF is a unique macrophage activator that potentiates both respiratory burst/lysosomal enzyme release (early-phase response) and monokine production/glucose consumption (late-phase response). Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Effect of the hetrazepinoic platelet-activating factor antagonist bepafant (WEB 2170) in models of active and passive anaphylaxis in mice and guinea pigs

The selective hetrazepinoic platelet-activating factor (PAF) antagonist WEB 2170 (Bepafant) was used to study the pathophysiological role of PAF in several models of anaphylaxis in mice and guinea pigs. In actively sensitized mice, the PAF antagonist WEB 2170 (1.0–10 mg/kgp.o.) protected mice from anaphylactic death in a dosedependent manner when the anaphylactic response was potentiated by the beta-receptor antagonist propranolol. When active anaphylaxis in guinea pigs was induced intravenously by 100 mg/kg ovalbumin (OA) in the presence of small doses of the antihistamine mepyramine, additional treatment with oral or intravenous WEB 2170 protected the guinea pigs from anaphylactic death. Also, the remaining anaphylactic bronchoconstriction and blood pressure changes (including anaphylactic hypotension) were attenuated. When guinea pigs were passively sensitized with a heterologous antibodyvia the tracheal route and then challenged by ovalbumin (100 mg/kgi.v.) 24 hr after sensitization in the presence of 0.003 mg/kgi.v. mepyramine, additional treatment with tracheal WEB 2170 at 0.1–1 mg/kg protected the guinea pigs dosedependently not only from anaphylactic death but also from a further decrease of respiratory flow and changes of blood pressure. Increased levels of PAF-like activity (20–50 ng PAF/whole lung) were detected in lungs removed from antigen-challenged animals. The results suggest a causative role for PAF in active and passive anaphylaxis. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Determination of platelet-activating factor by a chemiluminescence method and its application to stimulated guinea pig neutrophils

A simple, rapid and sensitive chemiluminescence method has been developed to measure platelet-activating factor (PAF). Hydrogen peroxide generated from PAF, upon phospholipase D cleavage, by choline oxidase is determined as chemiluminescence by a luminol-microperoxidase system. The detection limit of PAF by this method is 5 pmol/tube. The method is reproducible with a 5.5% coefficient of variation at 10 pmol of PAF (n=5). Lipids were extracted from guinea pig neutrophils after stimulation with cytochalasin B andN-formyl-methionyl-leucylphenylalanine, and PAF was isolated by high-performance liquid chromatography and determined by chemiluminescence measurements. The amount of PAF detected was 96.1±39.7 (mean ± SD, n=7) pmol/10⁸ cells. This highly sensitive method could be useful for the determination of PAF generated under pathophysiological conditions. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Regulation of the biosynthesis of platelet-activating factor in alveolar macrophages

Activities of enzymes which metabolize lysoplatelet-activating factor (lysoPAF) and platelet-activating factor (PAF) were studied in rabbit alveolar macrophage lysates. Substantial acetyltransferase activity was noted in the presence of 100 μM acetyl-coenzyme A (CoA), and this activity was increased in A23187-stimulated cell lysate. On the other hand, in the absence of exogenous acetyl-CoA, lysoPAF was mainly acylated through a transacylation pathway rather than by acetyltransferase in both control and A23187-stimulated cell lysates. We confirmed that the intracellular concentration of acetyl-CoA is relatively low. The observations suggest that the transacylation system may play an equally important role in the regulation of the availability of lysoPAF in intact cells. Intracellular lysoPAF was also maintained at relatively low levels. Interestingly, large amounts of PAF were produced even in unstimulated cells upon addition of an excess of exogenous lysoPAF, suggesting that generation of an adequate amount of lysoPAF within cells may be sufficient to trigger PAF synthesis in this type of cells. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor and granulocyte-mediated oxidative stress. Strategy forin vivo oxyradical visualization

Platelet-activating factor (PAF) and leukotriene B₄ are potent lipid mediators of endothelium-granulocyte interaction which results in granulocyte-dependent increased microvascular permeability. The specific mechanism by which PAF induces granulocyte-mediated endothelial injury has not been fully investigated. Digital imaging photonic intensified microscopy has revealed that PAF effectively induces granulocyte-mediated oxidative stress on microvascular beds. The method has made it possible to visualize luminol-dependent photonic burst released from PAF-treated microvascular beds in the rat mesentery. The photonic activities clearly corresponded to the localization of sticking granulocytes in post-capillary venules. By contrast, no significant chemilumigenic response could be detected in the leukotriene B₄-induced activation of endothelium-granulocyte interactionin vivo. The present findings suggest that oxygen radicals are not prerequisites for granulocyte adhesion in the microcirculation and provide evidence for a dissociation ofin vivo granulocyte function between leukotactic and oxidative activation in leukotriene B₄-induced microvascular changes. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

骨髓间充质干细胞向视网膜神经节样细胞的分化

目的探讨乳鼠视网膜细胞条件分化液诱导骨髓间充质干细胞(BMSCs)的神经分化情况,以期为视网膜退行性疾病提供治疗方案。方法体外分离培养Wistar大鼠乳鼠BMSCs,观察BMSCs的增殖情况并进行鉴定;制备乳鼠视网膜细胞条件分化液,以其诱导BMSCs,观察BMSCs的神经分化情况,并行免疫组化鉴定。结果体外培养获得了较纯的BMSCs;在乳鼠视网膜细胞条件分化液的环境中,诱导后72h,BMSCs胞体收缩成锥形或球形,细胞突起变细、变长,呈神经细胞的典型形态;免疫组化结果显示,部分细胞呈神经元特异性烯醇化酶(NSE)、巢蛋白(nestin)和Thy1.1阳性反应。结论乳鼠视网膜细胞条件分化液可诱导BMSCs分化成视网膜神经节样细胞。     

Compounds biologically similar to platelet activating factor are present in stored blood components

Agents which prime the neutrophil NADPH oxidase develop during routine storae of whole blood and packed red blood cells. This plasma priming activity can be inhibited by bepafant (WEB 2170), a specific platelet activating factor (PAF) receptor antagonist. Quantitation of the priming agent(s), by a commercially available radioimmunoassay for PAF, reproducibly demonstrated high levels of PAF activity. However, analysis of these plasma samples from stored blood components by gas chromatography/mass spectroscopy did not reveal any 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. We conclude that the polyclonal antibody to PAF used in these studies may have recognized different epitopes of a family of heterogeneous, biologically active lipids that manifest their effects through the PFA receptor.     

Characterization of the coronary vascular responses to platelet-activating factor in the isolated perfused heart

Platelet-activating factor (PAF) is a potent phospholipid mediator with diversein vivo andin vitro coronary vascular effects. In the present study, the coronary vascular responses to bolus injections of PAF were compared in rat hearts perfused under constant flow and under constant pressure. Low levels of PAF (1 pmol) produced vasodilatation only, while higher PAF concentrations (100 pmol) produced initial vasodilatation which was followed by a vasoconstriction under both experimental conditions. To determine species differences in PAF action, the effect of PAF was tested on perfused guinea pig hearts. Unlike in perfused rat hearts, only a dosedependent vasoconstrictor response was observed in perfused guinea pig hearts following a bolus injection of 1 fmol to 10 pmol of PAF. The results from repeated injections of PAf indicated that depletion of vasoactive mediators induced by PAF or receptor desensitization may explain a failure of a second injection of PAF to initiate a vasoconstrictor response. After PAF injection, the coronary vascular response to leukotriene was not altered, indicating that the reduced vasoconstrictor effect of a second injection of PAF cannot be due to a reduced ability of the smooth muscle to constrict. The study demon-strates that similar coronary vascular responses to PAF are observed in perfused rat hearts under either constant flow rate or constant pressure and that some of the variable coronary vascular responses reported may be due to the difference between animal species.     

The role of platelet-activating factor (PAF) in experimental glomerular injury

Platelet-activating factor (PAF) is a potent autacoid that participates in inflammation and other pathophysiological processes. In this review we deal with recent evidence suggesting that PAF is a mediator that is released early during glomerular injury. PAF can be synthesized in the glomerulus by infiltrating intrinsic glomerular cells. Normal glomeruli produce PAF upon stimulation, and glomerular PAF synthesis is increased in a variety of experimental glomerulopathies. The local infusion of PAF into the renal artery of isolated blood-free kidneys induces proteinuria. PAF attracts and activates inflammatory cells. Glomerular mesangial, endothelial and epithelial cells are also targets for PAF. Therapy with specific PAF receptor antagonists has prevented or reduced proteinuria and improved glomerular inflammation in several experimental models of proliferative glomerulonephritis and minimal change nephrosis. However, the beneficial effect of administration of PAF antagonists once proteinuria is fully developed has been minimal. PAF may also play a role in the recruitment of inflammatory interstitial cells. Based on a paper presented at the Third International Conference on Platet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Role of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes

The effect of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes (PMN) was studied. Cypridina luciferin analog (CLA) dependent chemiluminescence was used to detect superoxide anion radicals. PAF induced superoxide generation in human PMN in a dose-dependent manner. Preincubation with a small amount of PAF (5 x 10⁻⁹ M) enhanced PMN superoxide release induced by various stimuli, such as phorbol myristate acetate (PMA), opsonized zymosan (OZ), calcium ionophore (A23187) andN-formyl-methionyl-leucyl-phenylalanine (FMLP). The PAF antagonist, CV-6209, inhibited superoxide production induced by PAF, but not that induced by other stimuli. These findings would indicate that PAF may play an important role at inflammatory reaction sites and that CV-6209 may inhibit excessive inflammatory reaction.     

Effect of platelet-activating factor on lipoprotein lipase and blood lipids

We investigated the effect of platelet-activating factor (PAF) and of the PAF specific antagonist CV-6209 on plasma lipid metabolism, and particularly on post-heparin plasma lipolytic activity in male Wistar rats. Lipoprotein lipase (LPL) activity was enhanced by intravenous injection of PAF before intravenous injection of heparin when the PAF dose was low (0.2 μg/kg). PAF activated hepatic triacylglycerol lipase (HTGL) activity dose-dependently. Plasma triacylglycerols (TG) significantly decreased with the activation of LPL and/or HTGL. Plasma total cholesterol (TC) and phospholipid (PL) levels decreased at a low dose of PAF (0.2 μg/kg), but increased when higher doses were used. The PAF antagonist CV-6209 partially reversed the PAF induced effects on HTGL, TC and PL. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Production of platelet-activating factor by human normodense and hypodense eosinophils

Normodense eosinophils and neutrophils from normal donors produced considerable amounts of plateletactivating factor (PAF) when stimulated with ionophore A23187. PAF produced by eosinophils appeared to be degraded more rapidly than PAF formed by neutrophils, suggesting a higher activity of PAF-degrading enzyme in eosinophils. Substantial proportions of PAF newly formed by both eosinophils and neutrophils were shown to be cell-associated. By comparison, hypodense eosinophils obtained from a patient with idiopathic hypereosinophilic syndrome produced an extremely large amount of PAF and released much of it into the incubation medium. The accelerated formation of PAF in hypodense eosinophils may be related to various cardiovascular complications associated with hypereosinophilic syndrome. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

S100 Calcium-Binding Protein P Secreted from Megakaryocytes Promotes Osteoclast Maturation

Megakaryocytes (MKs) differentiate from hematopoietic stem cells and produce platelets at the final stage of differentiation. MKs directly interact with bone cells during bone remodeling. However, whether MKs are involved in regulating bone metabolism through indirect regulatory effects on bone cells is unclear. Here, we observed increased osteoclast differentiation of bone marrow-derived macrophages (BMMs) cultured in MK-cultured conditioned medium (MK CM), suggesting that this medium contains factors secreted from MKs that affect osteoclastogenesis. To identify the MK-secreted factor, DNA microarray analysis of the human leukemia cell line K562 and MKs was performed, and S100 calcium-binding protein P (S100P) was selected as a candidate gene affecting osteoclast differentiation. S100P was more highly expressed in MKs than in K562 cells, and showed higher levels in MK CM than in K562-cultured conditioned medium. In BMMs cultured in the presence of recombinant human S100P protein, osteoclast differentiation was promoted and marker gene expression was increased. The resorption area was significantly larger in S100P protein-treated osteoclasts, demonstrating enhanced resorption activity. Overall, S100P secreted from MKs promotes osteoclast differentiation and resorption activity, suggesting that MKs indirectly regulate osteoclast differentiation and activity through the paracrine action of S100P.     

Antitumor activity of synthetic alkylphospholipids with or without PAF activity

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) has been reported to possess definite antitumor activity in vivo. Twenty-two alkyl lysophospholipid analogs were chemically synthesized, and their antitumor activity against mouse experimental tumors (Sarcoma 180, MM46, P388) was examined. Among them, 1-O-octadecyl-2-O-acetoacetyl-rac-glycerol-3-phosphocholine was found to show antitumor activity similar to ET-18-OMe with less acute toxicity. Intravenous injection of the ET-18-OMe withsn-3 configuration retarded the subcutaneous growth of Sarcoma 180 cells effectively, while the growth inhibition by thesn-1 isomer was much less effective. This stereospecificity was similar to that observed in their activities as platelet-activating factor (PAF) agonists. The acetoacetyl compound, another PAF agonist, showed similar stereospecific antitumor action in vivo. These findings suggest that some alkyl lysophospholipids may activate host cells to a cytostatic stage against tumor cells in vivo through binding to a PAF receptor. Our preliminary results indicated that the responsible cells under these conditions might be primarily immature macrophages present in the bone marrow. No appreciable or even adverse stereospecificity was observed in the different sets of experiments where the activity of ET-18-OMe against MM46 tumor cells in vivo or the direct cytotoxicity against human promyelocytic leukemia HL-60 cells in vitro was examined. Under, some conditions, the antitumor activity of ET-18-OMe in vivo may be revealed through direct cytotoxicity and/or modulation of the host defense system by “nonspecific” mechanisms. Some alkylphospholipids without PAF activity may also show antitumor activity through similar, “nonspecific” mechanisms.     

Fatty acid metabolism and cell proliferation: IV. Effect of prostanoid biosynthesis from endogenous fatty acid release with cyclosporin-A

Cyclosporin-A (Cyc-A) stimulates prostanoid (PGI₂) synthesis in confluent smooth muscle cells from guinea pig aorta through the release of endogenous fatty acid. Cyc-A, like other stimulatory agents for prostanoids, promotes smooth muscle cell proliferation and prostanoid synthesis in these proliferating cells. Indomethacin, a cyclooxygenase inhibitor, and exogenous arachidonic acid block the Cyc-A effect on cell proliferation.     

Fatty acid metabolism and cell proliferation. III. Effect of prostaglandin biosynthesis either from exogenous fatty acid or endogenous fatty acid release with hydralazine

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Cells were seeded at from 40 to 80 cells per cm2 and cloned for 8 days. Media were analyzed for PGI2 (6-keto-PGF1 alpha) using radioimmunoassay. Prostanoids were synthesized when cells were grown in media alone. Arachidonic acid stimulated prostanoid synthesis and promoted cell proliferation. Indomethacin blocked prostanoid synthesis and abolished the stimulatory effect of arachidonic acid on cell proliferation. Hydralazine stimulated fatty acid release and prostanoid synthesis in confluent cells. Hydralazine also stimulated prostanoid synthesis and promoted proliferation in growing cells. Indomethacin blocked prostanoid synthesis and abolished the stimulatory effect of hydralazine on cell proliferation.     

Pharmacological properties of YM461, a new orally active platelet-activating factor antagonist

The antagonistic effect of YM461 [1-(3-phenylpropyl)-4-[2-(3-pyridyl)thiazolidin-4-ylcarbonyl]piperazine fumarate] against platelet-activating factor (PAF) was examined in severalin vitro andin vivo systems. We found that YM461 inhibited [³H]PAF binding to rabbit platelet membranes with a pK_i value of 8.90. YM461 inhibited PAF induced rabbit and human platelet aggregation with pA₂ values of 7.52 and 7.29, respectively; the slopes of the Schild plots were 1.07 and 1.01, respectively. However, YM461 at 10⁻⁴M did not affect rabbit and human platelet aggregation induced by ADP, collagen, arachidonic acid or epinephrine. YM461 inhibited PAF induced death in mice with an ED₅₀ (50% effective dose) value of 0.35 mg/kgp.o. YM461 at doses above 0.3 mg/kgi.v. inhibited PAF induced hypotension in rats. YM461 showed a dose-dependent inhibition of PAF induced hemoconcentration in rats with ED₅₀ values of 0.15 and 0.21 mg/kgp.o., respectively, at 0.5 and 1 hr after oral administration. The anti-PAF effect of YM461 persisted more than 6 hr after 3 mg/kgp.o. in rats. YM461 inhibited the bronchoconstriction induced by PAF with an ED₅₀ value of 1.2 mg/kgp.o. in anesthetized guinea pigs. Furthermore, the compound at doses above 3 mg/kgp.o. significantly inhibited antigen-induced anaphylactic asthma in conscious guinea pigs pretreated with mepyramine and propranolol. These results indicate that YM461 is a selective, potent and orally active PAF antagonist. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Osteogenic Efficacy of Human Trophoblasts-Derived Conditioned Medium on Mesenchymal Stem Cells

Trophoblasts play an important role in the regulation of the development and function of the placenta. Our recent study demonstrated the skin regeneration capacity of trophoblast-derived extracellular vesicles (EV). Here, we aimed to determine the potential of trophoblast-derived conditioned medium (TB-CM) in enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We found that TB-CM promoted the osteogenic differentiation of MSCs in a dose-dependent manner. Furthermore, it inhibited adipogenesis of MSCs. We also found that the primary trophoblast-derived conditioned medium (PTB-CM) significantly enhanced the proliferation and osteogenic differentiation of human MSCs. Our study demonstrated the regulatory mechanisms underlying the TB-CM-induced osteogenesis in MSCs. An upregulation of genes associated with cytokines/chemokines was observed. The treatment of MSCs with TB-CM stimulated osteogenesis by activating several biological processes, such as mitogen-activated protein kinase (MAPK) and bone morphogenetic protein 2 (BMP2) signaling. This study demonstrated the proliferative and osteogenic efficacies of the trophoblast-derived secretomes, suggesting their potential for use in clinical interventions for bone regeneration and treatment.     

An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult—they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.