Abstract 288: Nitric Oxide Mediates Active Downregulation of Tissue Factor Expression in Human Pericytes

Abstract

Tissue Factor (TF) is a transmembrane protein that not only expresses pro-coagulant enzymatic activity, but also mediates signal transduction by several second messenger pathways. It plays an obvious role in hemostasis and thrombosis, as well as modulating angiogenesis, inflammation, immune responses and progression of malignancy and metastasis. TF is normally expressed by a limited subset of cell types. It can also be induced on other cells by a plethora of mediators, mostly linked to inflammation and immune responses. By contrast, TF has only been shown to be actively downregulated in a single circumstance: we have reported that constitutive expression of TF by pericytes around dermal vessels disappears during angiogenesis associated with wound healing. This suggests that downregulation of pericyte TF may play a functional role in the process of angiogenesis. We previously reported that TF protein in cultured human pericytes is significantly reduced 8 hours after treatment with phorbol 12-myristate 13-acetate (PMA) (94.71% reduction + 2.18%, p<0.001). This loss of TF protein is facilitated by reduction of TF mRNA. Downregulation of TF protein and mRNA depends on Protein Kinase C. The goal of the current study was to identify a physiologic mediator of pericyte TF downregulation. Treatment of human pericytes with growth factors and cytokines involved in wound healing did not change TF expression. Co-culture of human pericytes with human microvascular endothelial cells in transwells in the presence of basic fibroblast growth factor (bFGF) resulted in downregulation of pericyte TF. However, conditioned media from endothelial cells grown in the presence of bFGF did not trigger a decrease of TF in pericytes. bFGF has been shown to stimulate NO production, and both bFGF and NO have been linked to angiogenesis. This led us to consider nitric oxide (NO) as a potential labile mediator of TF downregulation. TF protein was reduced 8 hours after treatment with DETA/NO (44% reduction + 6.1%, p<0.01). However, expression of TF mRNA was not reduced at this time, as it is during culture with PMA. Our working hypothesis is that TF downregulation in pericytes is partially mediated by endothelial cell-derived NO, but is also mediated by additional PKC-dependent pathways.