There is still no satisfactory explanation for the low catalytic activity of tissue factor (TF)/factor VII(a) complexes towards coagulation factor X, as found on the apical surface side of cell layers. It has been hypothesized that TF exists in a latent form. Layers of cultured human smooth muscle cells, constitutively expressing TF, were immunogold-labeled for TF in situ and processed for electron microscopy. We showed that, besides internalization and accumulation in lysosomal-like structures, TF remained associated with noncoated, flask- shaped microinvaginations of the plasma membrane. These invaginations were identified as caveolae. In regions in which intercellular contacts were interrupted, more TF-positive caveolae were observed. Enzymatically detached smooth muscle cells exhibited a similar enlargement of caveolar structures. Concomitantly, an increase of catalytic activity of apically formed TF/VIIa complexes towards factor X was found on the suspended cells. We speculate that caveolae- associated TF may function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost.

This study reveals a role for ghrelin, but not leptin, signaling within medial hypothalamus in FAA on both a population level and in single cells, identifying a subset of neurons onto which cue information and ghrelin signaling converge, possibly to drive FAA.

Endothelial cells in culture were exposed during four hours to the apoptosis inducing agents endotoxin (lipopolysaccharide, LPS) and Fas-ligand mimicking antibody in various concentrations. With addition of a deletion primer as internal standard a competitive RT-PCR was performed to measure semi-quantitatively the expression of mRNA of Vascular endothelial growth factor (VEGF). It appeared that endothelial cells survive increasing amounts of LPS and show a concentration- and time-dependent increase in the expression of VEGF-mRNA. The same effect was found with Fas-ligation, although at high concentrations Fas-ligation induced no further increase, but even a decrease of VEGF expression, possibly related to cell damage. Apoptotic cells were rarely observed after LPS-stimulation, but simultaneous incubation with a blocking antibody to VEGF resulted in a significant increase in apoptosis. We hypothesize that endothelial cells are resistant to apoptosis induction by autocrine expression of VEGF under stress conditions.

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Because there is no consensus regarding the precise distribution of induced endothelial tissue factor (TF), we studied TF activity in and on tumor necrosis factor alpha-stimulated cultured human umbilical vein endothelial cells (ECs) and their underlying matrix. TF was mainly expressed on the cell surface. Only small traces were found on the apical surface suggesting that TF is predominantly located on the basolateral side of the cell membrane. The presence of TF on the cell surface was confirmed by flow cytometry. Subendothelial TF activity appeared to be dependent upon the procedure used to remove the stimulated EC monolayer. Whereas ammonium hydroxide or hypotonic lysis resulted in relatively high levels of matrix-associated TF, virtually no TF was found on the matrix after mild enzymatic detachment of stimulated ECs. Cell removal with EDTA resulted in intermediate levels of matrix-associated TF. Neither the enzymatic treatment nor EDTA degraded or removed this TF activity. Similar patterns were observed for matrix-associated TF antigen and EC surface markers. Electron microscopic analysis showed cell fragments on the matrix after monolayer lysis. The findings strongly suggest that induced endothelial TF associated with the subendothelial matrix actually represents TF on EC remnants.