The present study describes

The expression of cytochrome P450 (CYP) enzymes and cyclo-oxygenases (COX) was investigated in human saphenous veins by reverse transcription-polymerase chain reaction analysis. Non-varicose veins were obtained from patients undergoing aortocoronary bypass grafting, whereas varicose veins were obtained from patients undergoing stripping removal of varicose saphenous veins. In non-varicose veins, CYP1B1, CYP2C, CYP2E1 and CYP4A11 were detected, whereas CYP2J2, CYP3A5, COX-1 and COX-2 were detected almost exclusively in varicose veins. CYP4F2 was not detectable. Except for CYP4A11, the levels of individual CYP mRNA were higher in varicose veins than in control veins. Smooth muscle cell volume, determined by a colour image-analysis system, was increased approximately 1.5-fold in varicose veins. Because CYPs and COXs produce various vasoactive compounds, increased expression of these enzymes could be involved in the impairment of vascular tone and may contribute to varicose pathology. Then, CYP or COX modulators may be potentially active in the treatment of chronic venous insufficiency.

Primary cultured human endothelial cells derived from umbilical cord vein were exposed during the growth of the culture to medium containing nicotine at various concentrations (0.5-200 micrograms/ml). Patterns of cellular fibronectin and factor VIII/vWF were compared to control by immunofluorescence technique. The levels of glycoproteins released in the culture medium were quantified by ELISA method. Treated cells showed an important decrease in fibronectin content with fragmentation of the fibronectin pericellular filaments, whereas the levels of secreted fibronectin were reduced in a dose-dependent manner. This reduction of fibronectin availability was correlated with an elongation of cell shape as revealed with phase contrast microscopy. By immunofluorescence, factor VIII/vWF cytoplasmic granules appeared drastically reduced whereas the secretion of the protein was significantly increased. As shown by electron microscopy, there was a concomitant reduction in the number and size of Weibel-Palade bodies. These studies indicate that nicotine modifies fibronectin and factor VIII/vWF distributions but in different ways.

One- and two-dimensional gel electrophoresis of proteins from bovine aortic endothelial cells (BAEC) incubated with [gamma-32P]ATP revealed the preferential labelling of a cell-associated 21 kDa substrate. The labelling of this band was detectable within 30 s, increased up to 30 min and was stable for at least 3 h following the wash-out of the ATP. This protein was also labelled after incubation of the cells with [gamma-35S]ATP. Incorporation of radioactivity into the 21 kDa band did not occur if the endothelial cells were treated with low concentrations of trypsin (0.01%) before or after the labelling period. The pattern of BAEC protein phosphorylation by [gamma-32P]ATP was completely different from that of the fetal calf serum used for the cell culture. The presence of serum during the incubation of BAEC with [gamma-32P]ATP did not modify qualitatively the labelling pattern and, in particular, did not enhance the phosphorylation of the 21 kDa substrate; this suggests that neither the kinase nor the 21 kDa substrate are adsorbed serum proteins. Staurosporine, a protein kinase inhibitor with low specificity, decreased the labelling of the 21 kDa protein with an IC50 of 2 nM. In contrast, at 100 nM, staurosporine did not decrease the accumulation of inositol phosphates induced by ATP via the activation of P2y receptors. These data indicate the presence of aortic endothelial cells of an ecto-kinase which uses extracellular ATP to produce the selective and long-lived phosphorylation of a 21 kDa endothelial substrate. Ecto-phosphorylation of this protein might play a role in the modulation of endothelial cell functions by ATP, in addition to the P2y receptors [Boeynaems & Pearson (1990) Trends Pharmacol. Sci. 11, 34-37]. The exquisite sensitivity of ecto-phosphorylation to inhibition by staurosporine and its specific inhibition by some isoquinolinesulphonamide compounds provide potential pharmacological tools to investigate this hypothesis.

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