Brefeldin A inhibits thrombin receptor regeneration in human endothelial cells.

Endothelial cells, once stimulated with thrombin, are resistant to subsequent stimulation. After a recovery period of about 60 min the cells are sensitized again for activation by thrombin. The resensitization is independent of the receptor de novo synthesis. Therefore an intracellular pool of thrombin receptors that is possibly co-localized with the Golgi apparatus has been assumed. Brefeldin A (BFA) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organization of the Golgi apparatus. Accordingly we have examined the effects of BFA on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. The von Willebrand factor (VWF) release from Weibel-Palade vesicles and the intracellular calcium mobilization were used as physiological parameters of thrombin receptor activation. The addition of BFA (2 microg/ml) to endothelial cells or the reduction of the incubation temperature from 37 degrees C to 16 degrees C blocked the receptor response regeneration almost completely.

Trypsin induced von Willebrand factor release from human endothelial cells in mediated by PAR-2 activation.

Nystedt and co-workers cloned in 1994 a second protease activatable receptor (PAR-2) that could be activated by trypsin but not by thrombin (1). In this study, we investigated whether trypsin induced stimulation of endothelial cells is linked to PAR-2 activation. We have found by mRNA analysis that endothelial cells of venous and arterial origin express both protease activatable receptors. The functional thrombin receptor and the protease activated receptor-2 (PAR-2) mediate apparently the same effects in human vascular endothelial cells. Both, the activation of the thrombin receptor with thrombin or SFLLRN and the activation of the PAR-2 with trypsin or SLIGRL induced intracellular calcium mobilisation and a subsequent release of von Willebrand factor (vWf) from Weibel-Palade bodies. As a consequence, it can be concluded that endothelial cells have two different receptors mediating the same cellular responses after activation.

Effect of polyvinyl chloride plastic on the growth and physiology of human umbilical vein endothelial cells.

In this work, human umbilical vein endothelial cells were cultured on common polystyrene cell culture plates, referred to as control plates, as well as on soft polyvinyl chloride plastics (PVC). Growth of human umbilical endothelial cells (HUVEC) on PVC coated with gelatin, collagen A and heparin plasma was significantly less than that on the control plates coated with the same substance or fibronectin. Cells cultured on PVC produced up to four times as much tissue plasminogen activator than control cells. With reference to plasminogen activator inhibitor 1 (PAI-1), more PAI-1 was released from cells grown on PVC than from those on the control plates coated with gelatin and collagen A. After endotoxin stimulation, the PAI-1 release of HUVEC cultured on PVC was significantly higher than that of control cells with the exception of cells grown on the fibronectin-coated PVC that showed no difference. It is concluded that the type of plastic and coat used to culture HUVEC play a definite role in their growth and function.

Regulation of the thrombin receptor response in human endothelial cells.

At present only little information is available about the regulation of the thrombin receptor activity in human endothelial cells. The study presented was performed to clarify the problem of thrombin receptor regulation in human endothelial cells. Endothelial cells were isolated from the vein or artery of human umbilical cords. These cells were stimulated with thrombin or with the thrombin receptor activation peptides (TRAP) SFLLRN or SFLLRNPNDKYEPF and subsequently the von Willebrand factor (vWf) release was measured as a physiologically significant response regarding the thrombin receptor activation. Shortly after the first activation by thrombin or SFLLRN, the cells were desensitised to a second stimulation. After a recovery phase of 10 min, merely 35% of the receptor response was obtained by subsequent stimulation. Expanding the recovery time to 90 min resulted in a vWf release of nearly 100% of the amount measured after the first stimulation. The resensitisation rate was similar for thrombin and SFLLRN stimulated cells. Adding RNA synthesis inhibitors or protein synthesis inhibitors had no effect on the recovery of the receptor response. Therefore, a de novo synthesis of receptor protein must be excluded as a means of resensitising endothelial cells to thrombin. It was shown that the dephosphorylation of tyrosine kinase inactivated receptors is also not responsible for receptor regeneration. These results prove that endothelial cells are capable of developing full thrombin receptor activity after a relatively short recovery phase of only 60-90 min as compared to the recovery phase of 16-24 h in megacaryoblastic cell lines. Desensitisation is similar in thrombin and TRAP stimulated cells. We assumed a transport mechanism for intracellular stored vesicles for receptor resensitisation; a co-localisation with vWf in the Weibel-Palade bodies is improbable.

The tethered ligand receptor is the responsible receptor for the thrombin induced release of von Willebrand factor from endothelial cells (HUVEC).

The present study was undertaken to define clearly the receptor, which is responsible for the thrombin induced vWf release from HUVEC. Vu et al. reported that cleavage of the platelet thrombin receptor by thrombin resulted in a new N-terminus (SFLLRN...) which acts as tethered ligand (4). The free peptide activates platelets and induces rises in both cytosolic free Ca2+ and PGI2 production in HUVEC (10). HUVEC were incubated with thrombin, SFLLRN or other relevant substances. After incubation, the intracellular vWf content was compared with the vWf concentration in the supernatant. The intracellular vWf concentration was measured by microscope fluorometry and the concentrations in the supernatant with an ELISA. The thrombin stimulated cells showed 53% vWf antigen compared with control cells (100%). This result was well correlated with the 2.4 fold higher vWf concentrations measured in the supernatant of thrombin stimulated cells than in control cells. Also the addition of SFLLRN (1-60 microM) or trypsin (1-50 nM) increased vWf release from HUVEC in a dose dependent manner. These results indicate that thrombin induced vWf release from HUVEC is mediated through the activation of the tethered ligand receptor.

Quantitation of phagocytic cells in phenytoin-induced connective tissue proliferation in the rat.

This study demonstrates that, in rats, systemically administered phenytoin produces a statistically significant increase in accumulation of phagocytic and esterase positive cells in areas of phenytoin-induced connective tissue proliferation. Many of these cells are perceived to be macrophages. Since macrophages have been shown to produce chemoattractant and mitogens for fibroblasts, they may very well play a major role in the gingival hyperplasia seen in patients taking phenytoin. The results of the study show that the effects of phenytoin on fibroblasts may be more complex than previously thought.

In vivo studies on the carcinogenic potential of an orthodontic bonding resin.

Data from another laboratory have indicated that the individual components of an orthodontic bonding resin might contain a carcinogen. Since that report, the formulation of the product was changed. The purpose of this study was to determine whether the new product is safe. Three groups of rats were used for the experiment. One group served as a control, while the other groups ingested the sealant resin or sealant catalyst. The materials were suspended in an alcohol-aqueous mixture and the solutions were given to the animals as their only source of fluid. The exposure was for 1 year. After this period of time, all the rats were given tap water and observed until day 600. The animals were autopsied at time of death or at the end of the experiment. During the treatment, there were significant differences (p less than 0.01) in water intake among the three groups. The average intake per day for the animals in the control group, the resin group, and the catalyst group was 50.2 cc, 37.8 cc, and 42.2 cc, respectively. Several animals died during the experiment, but there was no significant differences in the life expectancy of the animals in the three groups. The autopsies uncovered one malignant neoplasm, an undifferentiated sarcoma, in a rat from the control group and four benign tumors in rats from the three groups. All of these results indicate that the new formulation of the orthodontic bonding resin is not carcinogenic when ingested at a dose level of 50 ppm.

Murine tumor induction by cytomegalovirus.

Cytomegalic inclusion disease can be induced in mice with relative ease. In the study reported here, grandular neoplasms were produced in four mice by means of immunosupression together with large doses of cytomegalovirus. The results of this study suggest that cytomegalovirus may well be an etiologic agent in the formation of salivary gland tumors.