Downregulation Of Tissue Factor Expression In Human Pericytes In An Endothelial Cell Co-Culture Model
Authors: Snyder Stacey A. , Hoffman Maureane , Monroe Dougald , Lasater Samantha L. 
Published: October 2019

Abstract

Introduction
Tissue factor (TF) is a protein expressed by perivascular cells including pericytes (PCs) where it is responsible for initiation of blood coagulation. Many conditions have been associated with increased cellular TF expression, including inflammation, malignant transformation and tumor angiogenesis. However, our previous studies have shown that TF expression in PCs paradoxically decreases during wound healing.{McDonald:2008fy} To date, no studies have demonstrated the mechanism responsible for the downregulation of TF expression in any cell type. Because angiogenesis is a critical occurrence during wound healing, pro-angiogenic cytokines are likely candidates responsible for the downregulation of TF expression in PCs. Therefore, several well-characterized pro-angiogenic cytokines were studied for their effects on TF expression and activity in PCs.

Materials and Methods
PCs (PromoCell, Heidielberg, Germany) were treated directly with each of five individual angiogenic factors: angiopoietin 2 (ANG2, 1-1000ng/mL), basic fibroblast growth factor (bFGF, 0.1-10 ng/mL), platelet derived growth factor (PDGF, 1-1000ng/mL), transforming growth factor beta (TGFB 0.2-200 ng/mL), and vascular endothelial growth factor (VEGF, 1-1000ng/mL). Serum rich media was used for all experiments and all treatments lasted 24 hours. PCs were assessed for TF activity using a coagulation factor X (FX) activation assay. In addition to direct treatment, PCs were assayed for TF protein expression following treatment in a co-culture model. Briefly, PCs were plated in a standard tissue culture treated plate. Human Dermal Microvessel Endothelial Cells (ECs) were placed inside a 0.4um polyester membrane transwell insert, which was placed into the well containing the PCs. This allowed for communication via soluble factors through the membrane, but not direct contact between the two cell types. Co-cultured ECs were treated with angiogenic factors as described above. Additionally, ECs cells were cultured for 24 hours with bFGF and the conditioned media was collected, cleared of cells, and applied to cultured pericytes. Following treatment PCs were lysed and whole cell lysate was separated by polyacrylamide gel electrophoresis and Western blot (WB). WB membranes were probed with anti-TF antibody for total protein determination. Actin was used as a loading control for all blots.

Results
Following direct stimulation for 24 hours, none of the angiogenic growth factors assayed produced a reduction in TF activity or antigen, even at supraphysiological concentrations. Following treatment of ECs in a co-culture model neither ANG2, PDGF, TGFB, nor VEGF produced a change in TF expression in pericytes. However, treatment of ECs with bFGF for 24 hours produced a marked decrease in total TF protein expression in co-cultured PCs. Four independent experiments confirmed the decreased expression of TF under these conditions. In contrast, bFGF conditioned EC media applied to PCs for 24 hours resulted in no change in TF protein expression.

Conclusions
The observation that bFGF stimulation of co-cultured ECs, but not direct stimulation, causes a decrease in TF protein expression in PCs suggests that a soluble mediator is responsible for the effect. The finding that a decrease in TF expression was not observed in PCs treated with bFGF-conditioned ECs media suggests that a labile mediator may be responsible.

Disclosures:
Hoffman: Novo Nordisk: Consultancy, Research Funding; CSL-Behring: Consultancy, Research Funding; Boehringer Ingelheim: Research Funding.