Kinetics of thrombomodulin release and endothelial cell injury by neutrophil-derived proteases and oxygen radicals

Abstract

Thrombomodulin is a transmembranous glycoprotein of endothelial cells. In vitro it is a marker of endothelial cell injury. In vivo the levels of serum thrombomodulin are regarded as a parameter of activity in vasculitides. The latter are pathophysiologically determined by neutrophil-derived inflammation and endothelial cell injury caused by secretion of proteases and hydrogen peroxide. It was the objective of this study to determine whether thrombomodulin is only a late marker of advanced endothelial cell injury or whether it indicates also earlier stages of cell alterations. Over 24 hr endothelial cell cultures were incubated with hydrogen peroxide or the neutrophil proteases proteinase-3, elastase and cathepsin G. The time-dependent increase of thrombomodulin in the supernatant was determined by enzyme-linked immunosorbent assay and immunoblot. In addition the viability (eosin, tetrazolium dye assay), detachment (crystal-violet assay), and apoptosis (4',6-diamine-2'-phenylindole-dihydrochloride assay) of the respective endothelial cells were determined for adherent and non-adherent cells. A rapid thrombomodulin increase was found under all experimental conditions. The additional immunoblotting analysis showed the pattern of proteolytic cleavage caused by the protease reactivity. In case of hydrogen peroxide the thrombomodulin increase was closely correlated with the loss of cell viability and lysis. The incubation of endothelial cells with the different proteases resulted in a time-dependent detachment of primarily viable cells. In addition to cell necrosis apoptotic cell death was found in the subgroup of detached endothelial cells after prolonged incubation over 24 hr with proteinase-3 (23%), elastase (31%), and cathepsin G (19%). In contrast, still adhering cells did not show any signs of necrosis or apoptosis. In summary these studies confirm in vitro that soluble thrombomodulin is not only a parameter of advanced endothelial cell destruction itself but also in addition an early marker of initial endothelial cell membrane changes induced by neutrophil derived proteases and oxygen radicals.

Figure 1

The increase of thrombomodulin in the supernatant of endothelial cell cultures is shown after 0·5, 1, 4, and 24 hr of incubation. 10 µg/ml proteinase-3, 5 µg/ml elastase, and 5 µg cathepsin G as well as 0·5% hydrogen peroxide in serum free medium was used. The thrombomodulin values were determined by ELISA. Standard cultures were used as controls. The mean of three experiments is shown. For more details see Table 1.

Figure 2

The immunoblot analysis of the respective culture supernatants confirms the rapid loss of thrombomodulin from the endothelial cells. The different blots show the typical pattern of proteolytic thrombomodulin cleavage for the experiments with the three neutrophil proteases proteinase-3, elastase, and cathepsin G. Background staining is found in the standard control culture only. A representative blot is shown.

Figure 3

The DNA agarose gel electrophoresis shows no signs of DNA degradation for the subsets of adherent endothelial cells under all experimental condition. However, DNA degradation occurs time-dependent in the subgroups of these nonadherent endothelial cells incubated with one the three proteases which were tested; proteinase-3, elastase, and cathepsin G. In addition, indications of a typical DNA ladder pattern were additionally found in these three protease related DNA degradation smears when these were directly viewed under the ultra violet lamp.

Figure 4

After 24 hr of incubation the fluorescence microscopy of detached endothelial cells in the supernatant shows the typical apoptotic pattern of dense chromatin granules after staining with DAPI (preparation on slides using cytospin technique; for more details see the method section). This pattern is due to DNA condensation. The apoptotic pattern is found in the subgroups of endothelial cells treated with proteases (proteinase-3, elastase, cathepsin G) but not with hydrogen radicals. No detectable apoptotic pattern occurred under any culture conditions among the endothelial cell, which still remained adherent (not shown; more results in Table 2) (magnification ×40).