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We have previously observed a stimulatory effect of fluid shear stress on the regeneration of cultured endothelial cell layers after mechanical denudation. In this study we examined how fluid shear stress affects endothelial cell DNA synthesis during regeneration. Following mechanical denudation of narrow linear areas, monolayers of bovine aortic endothelial cells cultured on plastic dishes were subjected to shear stress of 1.3–4.1 dynes/cm2for 24–48 hours in a specially designed apparatus. After the application of shear stress, cells were stained with propidium iodide, and its fluorescence intensity, reflecting cellular DNA content, was measured using photometric fluorescence microscopy. The DNA content of cells exposed to shear stress increased significantly more than that of paired, static control cells (p < 0.005 to p < 0.001). The DNA histogram showed that cells exposed to shear stress contained a relatively high proportion of cells located in the S, G2, and M phases of the cell cycle as compared with the static control. These data suggest that fluid shear stress enhances endothelial cell DNA synthesis during the repair of mechanical denudation.

Unsaturated fatty acids, including n-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (C22:6, DHA) and eicosapentaenoic acid (C20:5, EPA), and a series of n-6 PUFAs were investigated for their anti-tumour and antimetastatic effects in a subcutaneous (s.c.) implanted highly metastatic colon carcinoma 26 (Co 26Lu) model. EPA and DHA exerted significant inhibitory effects on tumour growth at the implantation site and significantly decreased the numbers of lung metastatic nodules. Oleic acid also significantly inhibited lung metastatic nodules. Treatment with arachidonic acid showed a tendency for reduction in colonization. However, treatment with high doses of fatty acids, especially linoleic acid, increased the numbers of lung metastatic nodules. DHA and EPA only inhibited lung colonizations when administered together with the tumour cells, suggesting that their incorporation is necessary for an influence to be exerted. Chromatography confirmed that contents of fatty acids in both tumour tissues and plasma were indeed affected by the treatments. Tumour cells pretreated with fatty acids in vivo, in particular DHA, also showed a low potential for lung colony formation when transferred to new hosts. Thus, DHA treatment exerted marked antimetastatic activity associated with pronounced change in the fatty acid component of tumour cells. The results indicate that uptake of DHA into tumour cells results in altered tumour cell membrane characteristics and a decreased ability to metastasize.

We studied the responses of cultured endothelial cells to mechanical shearing force directly applied to those cells in vitroto determine changes in the concentration of intracellular calcium ion (Ca++), one of the factors that transfers information within the cell. Cultured bovine fetal aortic endothelial cells containing the Ca++fluorescence indicator, Fura-2, were rubbed with a latex balloon in a specially designed system, and changes in the fluorescence of Fura-2 caused by this shear stimulation were determined by photometric fluorescence microscopy. Immediately after shear stimulation, the concentration of Ca++in the cells was increased and reached a peak (511 ± 165 nM, n = 12) within 15 seconds after stimulation. After the peak, the concentration was gradually restored to the resting level (55 ± 17 nM, n = 12). The magnitude of the Ca++response was dependent on the intensity of the shear force applied. Analysis of fluorescence images of Fura-2 revealed that the cells showed this Ca++reaction without being injured or desquamated, although there were slight differences in the degree and duration of reaction among cells. This reaction appeared even when the cells were placed in the air with no contact with the fluid. This result suggests that neither the fluid flow associated with the balloon movement nor chemical substances in the fluid are involved in the reaction, but that pure physical force alone is responsible for the Ca++reaction. Further, it suggests that endothelial cells have the ability to perceive such physical stimulation as shear force and to transfer this information to the interior of the cell viachanges in the intracellular Ca++concentration.

To determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phospholipase D (PLD) activity in osteoblast-like UMR-106 cells, we used cells prelabeled with [3H] myristic acid or [3H] arachidonic acid, which were preferentially incorporated to phosphatidylcholine. The treatment of [3H] myristate-labeled cells with TPA in the presence of 1% ethanol caused a dose-dependent formation of [3H] phosphatidylethanol (PEt), a product specific to PLD, suggesting an activation of this enzyme. Pretreatment of the cells with protein kinase C (PKC) inhibitors (GF109203X, staurosporine or H-7) abolished the TPA-dependent formation of PEt. The PEt formation in response to TPA treatment was not observed after the pretreatment of the cells with TPA to downregulate PKC. These results suggest the involvement of PKC in the TPA-induced activation of PLD. With [3H] arachidonate-labeled cells, TPA treatment in the absence of ethanol resulted in the liberation of [3H] arachidonic acid, which was gradually converted to prostaglandin E2(PGE2), but the accumulations of [3H] phosphatidic acid (PA) and [3H] diacylglycerol (DAG) were very small and temporary. In contrast, PA was linearly accumulated following TPA treatment, when the cells were pretreated with an inhibitor of phosphatidate phosphohydrolase (PAP), propranolol, with no accumulation of either DAG or arachidonic acid. The TPA treatment of the cells pretreated with a DAG lipase inhibitor, RHC-80267, caused the generation of DAG after a lag period of approximately 5 min, with a very small and temporary accumulation of PA. The TPA treatment of cells pretreated with a cyclooxygenase (COX) inhibitor, indomethacin, blocked the PGE2production. The TPA-induced PGE2production was not affected by the pretreatment of cells with a phospholipase A2inhibitor, p-bromophenacylbromide, or with a phospholipase C inhibitor, D-609. TPA also stimulated PGE2production in osteoblastic cells that were enzymatically isolated from adult rat calvaria, and the experiments with lipid metabolizing enzyme inhibitors gave the same profile of inhibition of TPA-induced PGE2production as was observed in UMR-106 cells. These results suggest that PA formed as a consequence of the activation of PLD by TPA is rapidly converted to arachidonic acid via a PAP/DAG lipase pathway, followed by a gradual conversion of arachidonic acid to PGE2by COX in both UMR-106 cells and isolated adult osteoblastic cells, and that neither phospholipase A2nor phospholipase C is involved in the TPA-induced PGE2production. To the best of our knowledge, this is the first report that shows that the activation of PKC in osteoblastic cells leads to the production of PGE2via a PLD/PAP/DAG lipase/COX pathway.