Highly accumulated platelet vascular endothelial growth factor in coagulant thrombotic region.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm. OBJECTIVES: To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis. METHODS: The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed. RESULTS AND CONCLUSIONS: Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-alpha, and TGF-beta.

The agonist of the protease-activated receptor-1 (PAR) but not PAR3 mimics thrombin-induced vascular endothelial growth factor release in human smooth muscle cells.

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, induces vascular endothelial growth factor-(VEGF) release. However, the molecular mechanism of thrombin-induced VEGF release is largely unknown. Anagonist of protease-activated receptor-i (PARI), SFLL-RNPNDKYEPF, mimicked thrombin-induced VEGF release in human vascular smooth muscle (HVSM) cells, as determined by enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and Northern blotting. In contrast, the agonist of PAR3, TFR- GAP, did not affect VEGF release or expression. SFLL-RNPNDKYEPF, but not TFRGAP, up-regulated [Ca2-]i.Moreover, the calcium ionophone A23187 was found to trigger VEGF release in HVSM cells. Thrombin-inducedVEGF release was blocked by anti-thrombin, heparin, a synthetic thrombin receptor inhibitor E5510, the calcium chelator BAPTA, the protein kinase C inhibitor calphostin C, and the MEK1/2 inhibitor U0126. Thus, our data show that thrombin caused VEGF release via PARI activation in a manner dependent on [Ca2+]i and p44/42 downstream from the receptor activation.

Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts.

Anandamide triggers various cellular activities by binding to cannabinoid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-beta cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.

Bioactivity of the vascular endothelial growth factor trapped in fibrin clots: production of IL-6 and IL-8 in monocytes by fibrin clots.

The blood coagulation cascade is activated following vascular-wall injury. The serine protease thrombin is the final protease in this cascade that causes the formation of fibrin from fibrinogen. Thrombin also causes the activation of platelets, which are trapped in a fibrin net followed by hemostasis. Platelets gathered into fibrin clots release several growth factors such as platelet-derived growth factor and transforming growth factor beta. In the present study, we demonstrated that the vascular endothelial growth factor (VEGF) could be bound to fibrin clots in the plasma, and that incubation of the endothelial cells with these VEGF-bound fibrin clots induced proliferation of endothelial cells. Thus, it suggests that clot-bound VEGF may play a role in wound healing through the proliferation of endothelial cells and vascular smooth-muscle cells. On the other hand, a noticeable migration of monocytes was observed when they were cultured on dishes in the presence of VEGF-bound fibrin clots. Moreover, peripheral blood monocytes incubated in the presence of VEGF-bound fibrin clots strikingly increased the production of IL-6 and IL-8, demonstrating that VEGF trapped in fibrin clots not only induces proliferation of human umbilical vein endothelial cells and migration of monocytes but also enhances secretion of IL-6 and IL-8. Thus, our data suggest that fibrin clots that contain several growth factors act as a bioactive reservoir and may play an important role in hemostasis as well as wound healing.

Inhibitory effect of epigallocatechin-gallate on brain tumor cell lines in vitro.

We investigated the effect of epigallocatechin-gallate (EGCG), the main constituent of green tea polyphenols, on human glioblastoma cell lines U-373 MG and U-87 MG, rat glioma cell line C6, and rat nonfunctioning pituitary adenoma cell line MtT/E. Cell viability was determined by assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the extent of apoptosis was studied by flow cytometric analysis. Apoptosis was also characterized by morphology using fluorescent microscopy. The role of insulin-like growth factor-I (IGF-I) was studied by assay with MTT, immunohistochemistry, and immunoradiometric assay. After 72-h exposure, a statistically significant loss of viability (P = < 0.0001) was observed at concentrations of 12.5, 25, 50, and 100 microg/ml in U-373 MG cells and U-87 MG cells. EGCG at concentrations of 50 microg/ml and higher significantly reduced the viability of C6 cells. EGCG inhibited viability of MtT/E cells only at a concentration of 100 microg/ml. Quantitative study by flow cytometry demonstrated that lower doses of EGCG (12.5, 25, 50 microg/ml) induced apoptosis in U-373 MG, U-87 MG, and C6 cells; however, only the highest dose (100 microg/ml) induced apoptosis in MtT/E cells. Compared with other cell lines, MtT/E cells showed stronger IGF-I immunoreactivity. Neutralization of IGF-I with an antihuman IGF-I antibody reduced viability of the cell lines. It can be concluded that EGCG has an inhibitory effect on malignant brain tumors, and IGF-I may be involved in the effects of EGCG.

Anandamide induces apoptosis of PC-12 cells: involvement of superoxide and caspase-3.

Anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand has been suggested to have physiological role in mammalian nervous system. However, little is known about the role of anandamide on neuronal cells. Here, we demonstrate that anandamide causes death of PC-12 cells, showing marked DNA condensation and fragmentation, appearance of cells at sub-G(0)/G(1) and redistribution of phosphatidyl serine, the hallmark features of apoptosis. Anandamide raised intracellular superoxide level and CPP32-like protease activity in PC-12 cells markedly. Furthermore, antioxidant N-acetyl cysteine prevented anandamide-induced superoxide anion formation and cell death, implying that intracellular superoxide is a novel mediator of anandamide-induced apoptosis of PC-12 cells.

Polymyxin B binds to anandamide and inhibits its cytotoxic effect.

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Inhibition of caspase-3 activation by SB 203580, p38 mitogen-activated protein kinase inhibitor in nitric oxide-induced apoptosis of PC-12 cells.

Mitogen-activated protein kinase (MAPK) p38 plays pivotal role in cell proliferation, differentiation, and apoptosis when cysteine protease caspase induces apoptosis in different cell systems. SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole) is widely used as a specific inhibitor of p38 MAPK, and prevents apoptosis induced by various agents. The effect of SB 203580 on nitric oxide(NO)- or peroxynitrite-induced cell death is not known. Western blotting results indicate that p38 MAPK was activated significantly in NO- or peroxynitrite-induced cell death in a time-dependent manner, and subsequently this cell death was markedly inhibited by SB 203580, as determined by fluorescence-activated cell sorting (FACS)-can analyzer. Furthermore, NO/peroxynitrite-induced caspase-3 activation was notably inhibited by SB 203580, however, phosphorylation of either p38 MAPK or p44/42 was not influenced by SB 203580. Thus, it is likely that SB 203580 prevents NO/peroxynitrite-induced cell death by inhibiting caspase-3 activation in PC-12 cells.

Inhibition of thrombin-induced neuronal cell death by recombinant thrombomodulin and E5510, a synthetic thrombin receptor signaling inhibitor.

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a pivotal role in thrombosis and haemostasis. Thrombin acts as a mitogen and apoptosis inducer in a dose-dependent fashion. We have previously shown that thrombin caused proliferation of vascular smooth muscle cells (VSMCs). Here, we show that a low concentration of thrombin caused proliferation of mouse neuroblastoma (Neuro-2a) and human neuroblastoma (NB-1) cells, while higher concentrations affected cell viability in a time-dependent manner. Similar effects were observed when thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) was applied. The dying cells showed nuclear condensation and fragmentation, suggesting that cell death occurred by apoptosis. The extent to which thrombin induced cell death was significantly attenuated by recombinant thrombomodulin (rTM), or by a minimum functional domain of TM, termed E456. Furthermore, a synthetic compound that inhibits signaling from the thrombin receptor, 4-cyano-5,5-bis (4-methoxyphenyl)-4-pentanoic acid (E5510), and the antioxidant N-acetyl L-cysteine (NAC), efficiently prevented thrombin-induced Neuro-2a cell death. Thus, thrombin inhibitors and antioxidant appear to neutralize thrombin toxicity.

Recombinant thrombomodulin inhibits thrombin-induced vascular endothelial growth factor production in neuronal cells.

Thrombin is a serine protease which is generated from its precursor prothrombin by the activation of the blood coagulation cascade. Thrombin converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a central role in thrombosis and hemostasis by regulating platelet aggregation and blood coagulation. Here, we show that thrombn enhanced vascular endothelial growth factor (VEGF) production in a dose- and time-dependent manner in the supernatant of cultured PC-12 cells, as determined by enzyme-linked immunosorbent assay (ELISA). Thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) exerted an effect similar to thrombin on VEGF production. Thrombin-induced VEGF production was significantly attenuated by recombinant human thrombomodulin (rTM) and its minimal functional domain E456. Furthermore, the antioxidant N-acetyl-L-cysteine (NAC) markedly inhibited thrombin-induced VEGF production. Thus, rTM and NAC apparently inhibited the effect of thrombin on VEGF production in neuronal cells.

In vitro propagation of Rauvolfia serpentina through tissue culture.

Multiple shoots were induced from nodal segments and shoot apices of Rauvolfia serpentina and MS medium containing 1.0 mg/l BA and 0.1 mg/l NAA was found to give the best shoot proliferation rate. Callus formed at cut bases of the explants which produced shoots when subcultured on media containing low concentration of BA (0.5 or 0.1 mg/l) and NAA (0.1 mg/l). The in vitro proliferated shoots were rooted and later transferred to the soil.