Beneficial effect of ultra-low-dose aspirin in platelet activity alterations and haemorrhage observed in experimental portal hypertension.

Ultra-low-dose aspirin has shown a prothrombotic effect in the laser-induced thrombosis model. Several studies of our laboratory have shown a positive effect in rats with two different experimental models of portal hypertension: portal vein ligation, a model with an almost normal liver, and 30 days of bile duct ligation, a model with cirrhosis and presence of ascitis. In both models of portal hypertensive rats, bleeding time was prolonged and thrombi formation, in a laser-induced model of thrombi production, decreased. The hypotheses of the presented studies were that ultra-low-dose aspirin could decrease the bleeding complications in these models and that the mechanism for these effects could act thorough the COX pathway. In different studies, ultra-low dose of aspirin normalized the induced hemorrhage time, thrombi production, and platelet-endothelial cell interaction. The possible beneficial role of these doses of aspirin and mechanism of COX 2 inhibition are discussed.

Is there a role of platelet-activating factor in human lung cancer?

Platelet-activating factor (PAF) is a lipid mediator that stimulates the in vitro growth of various human tumour cell lines and that enhances the effect of vascular endothelial growth factor that plays a key role during angiogenesis of human cancer. In this study, we assessed the levels of PAF and of the acetylhydrolase activity (AHA, the PAF degrading enzyme) in patients with lung cancer. Results indicated no significant differences between blood PAF amounts of lung cancer patients (91+/-33 pg/ml, n=31) and a control group of patients with chronic obstructive pulmonary disease (COPD) induced by habitual smoking (117+/-28 pg/ml, n=10). Similarly, their serum AHA levels were not different (67.9+/-3.0 nmol/min/ml as compared to 68.3+/-5.2 nmol/min/ml for lung cancer patients and controls, respectively). In contrast, PAF amounts were markedly (P=0.01, t-test for paired data) reduced in the lung tumour tissues (77+/-29 pg/g, n=10) as compared to the non-tumour tissues (208+/-67 pg/g, n=10). These low levels of PAF were not related to a lower amounts of the lyso-PAF precursor but to an elevated (P=0.01, t-test for paired data) AHA in the tumour tissues (37.0+/-4.9 nmol/min/g, n=10) as compared to the non-tumour tissues (24.6+/-2.6 nmol/min/ml, n=10). Reverse transcriptase polymerase chain reaction experiments showed the presence of the PAF receptor (PAF-R) transcript 1 but not transcript 2 in blood mononuclear cells of lung cancer patients and COPD patients. Flow cytometry experiments did not highlight differences in the number and the distribution of PAF-R on their circulating leukocytes. In conclusion, this clinical study highlights no evidence for a potential important role of PAF during human lung cancer.

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human blood CD34(+) progenitors.

The influence of lipoxygenase metabolites of arachidonic acid on proliferation and differentiation of CD34(+) cells was studied. Their effects on the CFU-GM and BFU-E progenitors were investigated by culture of CD34(+) cells in liquid or semisolid medium. Only 12-HETE (1 microM) stimulated the [(3)H]thymidine as well as BrdU incorporation and increased the number of cell divisions (PKH2 tracking). Addition of 12-HETE and 15-HETE but not of LXA(4), LXB(4), LTB(4), and LTC(4) to liquid cultures of CD34(+) cells for 3 and 8 days reduced in a time-dependent manner the number of CFU-GM and BFU-E. Both HETEs also increased the percentage of glycophorin A(+) cells while they reduced the percentage of CD34(-)/CD33(+) cells after 3 and 5 days of liquid cultures. These results show that HETE treatment stimulates proliferation and accelerates the differentiation of CD34(+) cells, mostly toward the erythroid lineage.

Metabolism and effect of platelet-activating factor on the growth of human myeloma cell lines.

In this study we have investigated the presence of PAF receptor (PAF-R) on 5 myeloma cell lines (U266, L363, IM9, OPM2 and XG1), their metabolism of PAF and lyso PAF, and the effect of PAF on their growth. All myeloma cell lines express a PAF acetylhydrolase activity and metabolize [3H]PAF and [3H]lyso PAF in 1-alkyl-2-acyl analogue of phosphatidylcholine. Polymerase chain reaction on reverse transcript (RT-PCR) experiments indicate that OPM2, U266, IM9, XG1 and L363 cells express the PAF-R transcript 1 but not the PAF-R transcript 2. Flow cytometry experiments reveal that PAF-R are present on these myeloma cell lines. PAF and the non-metabolizable PAF agonist 1-O-hexadecyl-2-N-methycarbamyl-glycero-3-phosphocholine have no effect on the growth of OPM2, U266, IM9, XG1 and L363 assessed by [3H]thymidine incorporation into DNA. As a positive control of PAF effect on myeloma cells, PAF (1 microM) enhances by 100% the immunoglobulin synthesis by IM9 cells cultured for 48 h. In conclusion the five myeloma cell lines used in this study metabolize PAF through the deacetylation/reacylation pathway. They express membrane PAF-R through the PAF-R mRNA transcript 1 but PAF does not affect their growth.

Incorporation and effect of arachidonic acid on the growth of the human K562 cell line.

Lipoxygenase inhibitors reduce the growth of K562 cells (chronic myelogenous human leukaemia blasts) suggesting a role for endogenous lipoxygenase products of arachidonic acid (AA) in their proliferation. The objectives of this work are to investigate the incorporation of AA into K562 cells and to assess the effects of the exogenous addition of AA and lipoxygenase products on their growth. The mechanism of acylation of [3H]-AA indicates that K562 cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]-AA is incorporated unmodified into K562 phospholipids and is linked by an ester bond. Prelabelling-chase experiments indicate a transfer of labelled AA from phosphatidylcholine to phosphatidylethanolamine. The addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin B4, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE) has no effect on K562 cell proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while K562 cells readily incorporate AA into their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

Expression of platelet-activating factor receptor transcript-1 but not transcript-2 by human bone marrow cells.

The presence of platelet-activating factor receptor (PAF-R) transcripts 1 and 2 was investigated in human bone marrow cells by a reverse transcriptase polymerase chain reaction (RT-PCR) procedure which detected their simultaneous presence. RT-PCR experiments reveal PAF-R transcript 1 (but not 2) in freshly isolated mononuclear marrow cells, CD34+ hematopoietic stem/progenitor cells and cultured marrow stromal cells. For these experiments, the 5637 human bladder carcinoma cell line is used as a positive control for the presence of PAF-R transcripts 1 and 2. Flow cytometry experiments confirm the presence of PAF-R on marrow stromal cells and CD34+ stem/progenitor cells. In conclusion, the expression of PAF-R transcript 1, which mainly exists in circulating leukocytes, is also found in CD34+ stem/progenitor cells and cells of the marrow microenvironment, strengthening the potential role of PAF during marrow hematopoiesis.

Arachidonic acid and freshly isolated human bone marrow mononuclear cells.

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.

Incorporation and effect of arachidonic acid on the growth of human myeloma cell lines.

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.

Arachidonic acid and human bone marrow stromal cells.

Human bone marrow stromal cells regulate the growth of marrow hematopoietic progenitors by secreting cytokines. Arachidonic acid (AA) is the fatty acid precursor of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) that modulate the growth of human bone marrow progenitors. We have investigated the incorporation of AA in human bone marrow stromal cell cultures, their production of PGE2 and LTB4 and the effect of AA on their growth. Gas chromatography analysis reveals the presence of AA in the human bone marrow plasma and in bone marrow stromal cell cultures. In stromal cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids. Prelabeling-chase experiments indicate a preferential incorporation of AA into phosphatidylethanolamine and no trafficking of labeled AA between phospholipid species. Bone marrow stromal cells release PGE2 and LTB4 in response to phorbol myristic acetate (PMA) (1 microM) and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml). Exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow stromal cells capt exogenous AA and, thus, may participate to the control of marrow AA concentrations. They may also regulate human marrow hematopoiesis by secreting AA metabolites such as PGE2 and LTB4.

Effects of lipidic mediators on the growth of human myeloid and erythroid marrow progenitors.

Freshly isolated human marrow mononuclear cells produce lipidic compounds such as PAF and leukotrienes. These lipidic molecules act on human marrow myelopoiesis and erythropoiesis by modulating the growth of committed progenitors (CFU-GM and BFU-E) in vitro. Nanomolar concentrations of leukotriene B4 and C4 stimulate the growth of human marrow CFU-GM. In contrast, micromolar concentrations of lipoxygenase inhibitors (NDGA and BW755C) decrease their growth suggesting a role for endogenous lipoxygenase metabolites in this process. Micromolar concentrations of prostaglandin E2 up-regulate and down-regulate the growth of marrow BFU-E and CFU-GM, respectively. In contrast, the other cyclooxygenase metabolites have no effect. Recent studies indicate that nanomolar concentrations of PAF decrease the growth of CFU-GM and BFU-E from purified marrow CD34+ cells. Together these results indicate that lipidic mediators act on human myelopoiesis and erythropoiesis. However at this time the mechanisms and molecular signals mediating the effects of lipidic molecules on human marrow cells are unexplored.