Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation.

We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial cells. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release, respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA-mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cells, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53.

Microparticle-associated tissue factor is recycled by endothelial cells resulting in enhanced surface tissue factor activity.

In this study the uptake of tissue factor (TF)-positive microparticles by endothelial cells and the recycling of the TF component were examined. Human dermal blood endothelial cells (HDBEC) were incubated with microparticles derived from cancer cell lines for up to 6 hours. Measurement of HDBEC cell surface TF antigen revealed two distinct peaks at 30 and 180-240 minutes post-incubation with TF-positive, but not TF-deficient microparticles. However, only the second peak was concurrent with high TF activity as determined by a chromogenic thrombin-generation assay. Annexin V-labelling of HDBEC showed phosphatidylserine exposure following 90 minutes incubation with microparticles, which explains the high TF activity associated with the second antigen peak. Analysis of TF mRNA levels revealed no de novo expression of TF mRNA in response to microparticles, and pre-incubation of cells with cycloheximide did not prevent the appearance of TF. However, blocking endocytosis with a dynamin inhibitor prolonged the disappearance and prevented the reappearance of TF antigen on the cell surface. Incubation of HDBEC with microparticles containing TF-GFP revealed the early co-localisation of TF with Rab4 and Rab5, followed by co-localisation with the late endosomal/trans-Golgi network marker Rab9, and the recycling endosome marker Rab11. siRNA-mediated suppression of Rab11 reduced the reappearance of TF on the cell surface. These data suggest a mechanism by which TF-containing microparticles are internalised by endothelial cells and the TF moiety recycled to the cell surface. Together with the exposure of phosphatidylserine, this is capable of inducing a substantial increase in the procoagulant potential of the surface of endothelial cells.

Pressure-reducing mattresses.

A clinical evaluation of eight base foam pressure-reducing mattresses was undertaken at Addenbrooke's NHS Trust, Cambridge. Data were collected on the medical and nutritional status, skin condition, medication, weight and Waterlow score for each patient, together with ratings on mattress comfort. At the beginning and end of the study, mattresses were assessed for interface pressures and the general condition of each mattress and its cover was evaluated.

Intranuclear binding of [3H]dihydrotestosterone by cultured human fibroblasts.

The uptake of [1,2,4,5,6,7-3H]dihydrotestosterone into whole cells and nuclei has been assessed in fibroblasts grown from genital skin of 10 controls and 9 subjects with hereditary male pseudohermaphroditism due to androgen resistance. The cells were exposed to hypotonic buffer and ruptured by passage through a 25-gauge needle, and the nuclei were purified by sedimentation through 2.1 M sucrose. Uptake of the hormone into nuclei reached an apparent plateau in 45 min, was saturable at 1 nM dihydrotestosterone, and was not detectable in the presence of excess nonradioactive hormones. Over a wide range of uptake by intact cells from control subjects and from subjects with androgen resistance due to 5alpha-reductase deficiency or receptor deficiency, nuclear uptake averaged about half of the total cell uptake. Furthermore, in cells from two unrelated 46,XY phenotypic females with androgen resistance but normal 5alpha-reductase activity and normal whole cell dihydrotestosterone binding, uptake into the nucleus was also normal. In these subjects, the defect in androgen action must be at some terminal phase of androgen action.