Soluble tissue factor has unique angiogenic activities that selectively promote migration and differentiation but not proliferation of endothelial cells

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Abstract

The level of circulating tissue factor (TF) is up-regulated in human angiogenesis-related malignancies. However, whether circulating TF has angiogenic activities has not been determined. Soluble TF (sTF) is the main domain of circulating TF. Here, using cell migration, wound healing, and tubule formation assays, human recombinant sTF was found to significantly promote the migration and differentiation of endothelial cells. The stress fiber formation and rearrangement induced by sTF observed through immunofluorescence microscope may be responsible for the stimulatory migration effect of sTF. Nevertheless, sTF had no effects on endothelial cell proliferation. Interestingly, sTF can be internalized by endothelial cells, which implies a novel mechanism for sTF in angiogenesis. These results suggest that sTF has unique angiogenic activities and may serve as a potential therapeutic target to treat diseases associated with angiogenesis such as cancer and rheumatoid arthritis.

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Materials and methods

Human recombined sTF from E. coli. Coding region for human sTF (TF1-219, residues 1–219) was amplified by PCR using TF cDNA. sTF, constructed and expressed in E. coli BL21/DE3, was purified and refolded thereafter. The clotting activity of refolded sTF was analyzed and verified after reconstitution with phospholipids as described previously [9].

Cell culture. Primary HUVECs were isolated from freshly delivered umbilical cords and grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen)

sTF stimulates the migration of endothelial cells

Angiogenesis involves cell migration, proliferation, and tubule formation [6]. The mobilization of endothelial cells at the microvascular level is a prerequisite for the neovascular occurrence [8]. Thus, we first set out to determine the migratory effect of sTF on HMECs and HUVECs using Millicell separate culture plate inserts. After 6 h, the migration of HMECs on collagen-precoated transwell filter in response to the increasing concentrations of sTF was significantly stimulated over that in the

Discussion

TF, a transmembrane receptor protein, is the primary initiator of coagulation and also participates in tumor angiogenesis, growth, and metastasis [2]. Importantly, elevated levels of circulating TF were discovered in the blood of patients with angiogenesis-related tumor, hypercoagulability, and vascular diseases. For example, circulating TF and membrane TF expressed by tumor cells positively correlates with tumorigenesis and angiogenesis in human colorectal carcinoma [4]. Besides, it was

Acknowledgments

This study was supported in part by the General Programs of National Natural Science Foundation of China (Nos. 30670419, 30771083), and the State Key Development Program for Basic Research of China (No. 2006CB910305).

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