Induced changes of leukocyte slow rolling in an in flow pharmacological model of adhesion to endothelial cells.

BACKGROUND: Rolling of leukocytes at the surface of the vascular endothelium is a prerequisite for a subsequent firm adhesion, particularly the slow rolling appearing on ELAM CD62E. Therefore, it may be considered that increasing the rolling velocities should be a precise therapeutic target in clinical situations where leukocytes accumulate, mainly venous and arterial ischaemia. METHODS: Human neutrophils were allowed to flow on endothelial HUVECs, with and without 4 hours interleukin-1alpha activation, the cells having or not been incubated with INO5042 anti-inflammatory drug. Under a mean shear-stress of 2 dyn/cm(2), rollers and stickers were identified and quantified, using a video-camera and picture analysing software. RESULTS: When the drug had been added to endothelial cells a shift of velocities was observed towards fast speeds (from 3-5 to 7-11 microm/sec). The same results was significantly found when neutrophils, alone or along with endothelium, had been submitted to the drug, the number of stickers and rollers beeing reduced as well. Finally, such a precise pharmacological method proved efficient to detect the exact mechanism of INO5042 on white cell adhesion.

Vascular remodeling in varicose veins.

The present study describes the histopathologic aspects of varicose (n=29; mean age, 52 +/- 12 years) and normal saphenous veins (n=17; mean age, 51 +/- 12 years) of patients from a similar age group. We focused on the changes that occur in the circular layer of the venous wall. We examined the venous walls by light microscopy and transmission electronmicroscopy. A semiquantitative grading system was used to assess the smooth muscle cell (SMC) hypertrophy and the change that occurs in the elastin pattern. The volume densities (Vv) of SMC and collagen were measured as well as the diameter of the SMC, and the nuclei of SMC per fixed area were counted. The varicose vein wall differed from the normal saphenous vein by the presence of hypertrophic SMC as well as disorganized elastin patterns. A correlation between the hypertrophic SMC and an abnormal elastin pattern was observed (r=0.658, p<0.001). Ultrastructurally, the SMC show prominent microherniations and vesicles that bud from the cell. These vesicles contain microfilaments and microtubuli, although no other organelles could be detected. The elastin fibers are disrupted from the hypertrophic SMC. No significant difference could be detected in both the Vv of SMC and the Vv of collagen. The diameter of the SMC in the varicose vein (d=9.45 +/- 1.22 microm) differs significantly from that in the normal saphenous vein (d=6.22 +/- 1.47 microm) (p<0.001). Also, the nuclei of SMC per fixed area differs significantly between the varicose (87 +/- 18) and nonvaricose (117 +/- 24) veins (p<0.001). We conclude that the cellular hypertrophy of the SMC and the microherniations could be the basis for disruption of the elastin fibers connected to the SMC in varicose veins. Disrupted connections between SMC and elastin fibers could in turn induce the weakness of the venous wall observed in varicose vein disease.

Ecto-phosphorylation on aortic endothelial cells. Exquisite sensitivity to staurosporine.

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.

Enhancement of the endothelial production of prostacyclin by inhibitors of protein synthesis.

1. Pretreatment of bovine aortic endothelial cells with cycloheximide enhanced their capacity to release prostacyclin in response to adenosine 5'-triphosphate (ATP) and bradykinin. 2. The action of cycloheximide was time-dependent; it became detectable after a 1 h exposure to the cells and was maximal after 3 h. 3. Puromycin mimicked the effect of cycloheximide. For these two agents, the enhancement of prostacyclin release was obtained at concentrations producing a partial inhibition of protein synthesis. 4. Cycloheximide increased the mobilization of free arachidonic acid induced by ATP in bovine aortic endothelial cells. 5. In conclusion, the synthesis of new proteins is not involved in the stimulatory action of ATP and bradykinin on prostacyclin production by bovine aortic endothelial cells. Despite the short half-life of prostaglandin H synthase in endothelial cells, cycloheximide and puromycin enhanced the release of prostacyclin induced by agonists. Our data suggest that this release might be under the control of rapidly turning-over phospholipase inhibitory proteins.

Increased levels of c-fos and c-myc mRNA in ATP-stimulated endothelial cells.

In bovine aortic endothelial cells, ATP induced a transient and sequential accumulation of c-fos and c-myc mRNA, which was detected after 1 hour and 3 hours, respectively. The effect of ATP on c-fos mRNA was stronger than that of TNF and bFGF. Both ATP and bFGF increased c-myc mRNA after a 3 hour treatment, whereas TNF did not. If none of the 3 agonists tested induced a selective expression of c-fos or c-myc, each of them was associated with a different quantitative combination of the 2 signals, which might be related to the distinct responses that they trigger in endothelial cells.

Amiloride analogs induce the phosphorylation of elongation factor-2 in vascular endothelial cells.

5-(N-Ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of Na+/H+ antiport, reduced [35S]methionine incorporation in proteins and induced the phosphorylation of a Mr 95,000 protein in bovine aortic endothelial cells. This protein was previously shown to become phosphorylated in response to ATP, bradykinin, and A23187 (1) and was identified as elongation factor-2 (2). The action of EIPA was independent of changes in cytosolic pH, because it was neither mimicked by sodium acetate nor inhibited by ammonium chloride, and it was reproduced by 2',4'-dimethylbenzamil, an analog of amiloride that is inactive on the Na+/H+ antiport. Furthermore, EIPA enhanced the Ca2(+)-dependent phosphorylation of a similar Mr 95,000 protein in a cell-free system, rabbit reticulocyte lysate, where an inhibitory effect of amiloride on protein synthesis has already been described (3). Because phosphorylation decreases the activity of elongation factor-2, our observation might explain why amiloride analogs inhibit protein synthesis.

Nicotine increases basal prostacyclin production and DNA synthesis of human endothelial cells in primary cultures.

In order to better understand the direct effects of nicotine on the metabolism of human vascular endothelial cells we studied the effect of the drug on prostacyclin (PGI2) production and cellular proliferation. This study was performed in an in vitro model of primary cultures of human umbilical vein endothelial cells. PGI2 level was measured with a radioimmunoassay of 6-keto-PGF1 alpha secreted into culture medium. We observed that incubating the cells with nicotine resulted in a dose-dependent increase of the basal level of PGI2; however, at high doses, nicotine, tended to decrease the capacity of production obtained by thrombin stimulation. Endothelial cell growth was stimulated by nicotine with a maximal effect at 0.05 microgram/ml nicotine. We conclude that nicotine appeared, in some experimental conditions, to stimulate some parameters of endothelial cell metabolism and particularly prostacyclin production. Our results stress the importance of the experimental conditions, and may provide an explanation for the disparities of results in the literature. The results are discussed in relation to smoking induced vascular alteration.

Nicotine alters fibronectin and factor VIII/vWF in human vascular endothelial cells.

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.