Nitric Oxide Mediates Downregulation of Tissue Factor Expression in Primary Human Pericytes through a p38 MAPK Signaling Pathway

Tissue factor (TF) is a high-affinity receptor for FVII/FVIIa that serves as a key initiator of hemostasis and is thought to also play a functional role in angiogenesis. Elevated TF expression has been linked to upregulated angiogenesis in malignant tumors, while reducing TF expression in experimental tumor models results in decreased angiogenesis. Although these data suggest that high TF expression is critical for angiogenesis, we have reported that TF expression declines significantly in pericytes that surround angiogenic vessels at sites of wound healing. This is the only known example of active TF downregulation, suggesting that pericytes regulate their expression of TF by a unique mechanism. Additionally, TF expression increases in response to many mediators, yet none that decrease TF expression have been described. The goal of this study was to characterize TF downregulation in pericytes and identify mediators of this process.

We have previously shown that TF expression in primary cultures of human pericytes is not affected by treatment with various growth factors or inflammatory stimuli, but decreases significantly in response to phorbol 12-myristate 13-acetate (PMA). PMA triggers degradation of TF protein and inhibition of TF mRNA synthesis, both in a Protein Kinase C (PKC)- dependent manner. To identify other signaling molecules in this pathway we used chemical inhibitors to block the activity of signaling molecules downstream of PKC before adding PMA to pericyte cultures. Inhibition of NF-kB, ERK1/2, AKT, JNK, and p38 MAPK did not block degradation of TF protein. However, pericytes that received a p38 inhibitor (SB202190) alone demonstrated significant reduction of TF mRNA. Treatment with SB202190 followed by PMA produced an additive effect on TF mRNA reduction. Western blotting showed that prolonged PMA treatment (>4 hours) produced a sustained decrease in p38 phosphorylation. These data suggest that PMA inhibits p38 activity, and that p38 confers stability to TF mRNA.

We have previously found that basic Fibroblast Growth Factor (bFGF) triggers downregulation of pericyte TF a co-culture system with human microvascular endothelial cells. However, transferring bFGF-conditioned endothelial cell media to pericytes cultured alone failed to reproduce TF loss. bFGF has been shown to stimulate nitric oxide (NO) production, and both bFGF and NO have been linked to angiogenesis. This led us to consider NO as a potential labile mediator of TF downregulation. ±6.1%, p<0.01). However, expression of TF mRNA was not reduced at this time, as it is during culture with PMA. Pericytes treated with DETA NO demonstrated sustained p38 phosphorylation for up to 8 hours. Taken together, these data suggest that DETA NO downregulates TF protein but maintains basal levels of TF mRNA, potentially in a p38-dependent manner.

Based on these data, we hypothesize that the p38 signaling axis is a key component of a unique pathway of TF regulation in pericytes, and that endothelial nitric oxide contributes to downregulation of pericyte TFin vivo at sites of physiologic angiogenesis.