Nonproteolytic Properties of Murine Alternatively Spliced Tissue Factor: Implications for Integrin-Mediated Signaling in Murine Models

Richard C Godby,Yascha W Van Den Berg,David Y Hui,Ramprasad Srinivasan,Henri H Versteeg,Evgeny Ozhegov,Robert Sturm,Wolfram Ruf,Bruce J Aronow,Vladimir Y Bogdanov,Stephen F Konieczny

doi:10.2119/molmed.2011.00416

Abstract

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.

Highlights

Tissue factor (TF), an integral membrane glycoprotein, serves as an enzymatic cofactor of the serine protease FVII/FVIIa and acts as the principle physiologic trigger of the blood coagu-lation cascade
We examined the expression patterns of murine alternatively spliced tissue factor (masTF) and murine full-length tissue factor (mflTF) in pancreatic tissue of genetically modified mice that spontaneously develop preneoplastic lesions (PanINs) and pancreatic ductal adenocarcinoma (PDAC); as shown in Figure 1A, the intensity of staining for masTF as well as mflTF increased as lesions progressed from early PanINs to high-grade PDAC phenotype, indicating that masTF is likely to contribute to pancreatic tumor growth in the murine setting
Compared to mflTF, staining for masTF in PanINs and PDAC tissue appeared to be somewhat more diffuse; to ascertain whether masTF is present in the extracellular stromal compartment, immunofluorescence studies were performed and, as shown in Figure 1B, masTF was found in abundance in tumor cells as well as extracellular stroma, whereas mflTF was exclusively cell associated

Summary

Introduction

Tissue factor (TF), an integral membrane glycoprotein, serves as an enzymatic cofactor of the serine protease FVII/FVIIa and acts as the principle physiologic trigger of the blood coagu-lation cascade. Tissue factor (TF), an integral membrane glycoprotein, serves as an enzymatic cofactor of the serine protease FVII/FVIIa and acts as the principle physiologic trigger of the blood coagu-. Aside from its role in the maintenance of normal hemostasis and its involvement in a variety of thrombotic disorders, TF is known to affect angiogenesis via protease activated receptor-2 signaling and interactions with α3β1 and α6β1 integrins [1]. The term “angiogenesis” collectively refers to the processes that result in the formation of new vasculature from preexisting blood vessels. Angiogenesis depends on a delicate interplay between the endothelium and pericytes [2,3]. Tip cells migrate from the existing vessel and are followed by stalk cells, which divide and form a lumen, creating a capillary [4].

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