143 Tissue factor pathway inhibitor regulates angiogenesis independently of tissue factor via inhibition of vascular endothelial growth factor signalling

Abstract

<h3>Introduction</h3> The biological systems of coagulation and angiogenesis show considerable interdependence. Proteases and inhibitors within the tissue factor (TF) pathway of coagulation have emerged as potential regulators of angiogenesis. Tissue factor pathway inhibitor (TFPI), as the primary physiological inhibitor of tissue factor (TF)-mediated coagulation, is ideally situated to modulate the pro-angiogenic effects of TF. However, TFPI may also have effects on angiogenesis independent of its anti-TF ability. <h3>Methods</h3> We determined the effects of altered TFPI expression on the regulation of angiogenesis in vivo using genetically-modified murine models of vascular overexpression (SM22áTFPI strain) and endothelial-specific deletion of the TF-binding domain of TPFI (Tie2TFPI). We then defined the mechanism of these effects in vitro using Human Umbilical Vein Endothelial Cells (HUVECs) overexpressing TFPI or via exogenous addition of TFPI-derived peptides in assays of angiogenesis. <h3>Results</h3> Vascular-directed over-expression of TFPI (SM22áTFPI strain) inhibited angiogenesis in vivo (Abstract 143 figure 1). SM22áTFPI showed significantly impaired recovery from ischaemia in the hindlimb ischaemia model after 3 days (p&lt;0.05, n=5 per group), which persisted throughout the experiment. Survival (until 1-cm tumour dimension) of SM22áTFPI mice vs wild-type control (median survival 14 cf. 10 days) following s.c. B16 melanoma injection (n=7 per group, χ²=4.325, *p&lt;0.05). Endothelial-specific deletion of the TF-binding domain of TFPI failed to reveal a pro-angiogenic phenotype. This led us to suspect that the anti-angiogenic action of TFPI may be independent of TF. Systemic delivery of the murine TFPI carboxyl-terminus (mTFPIct) replicated the effects of endogenous overexpression. In vitro, overexpression of TFPI inhibited endothelial cell tube formation on Matrigel and migration using an injury migration model. Human TFPIct (hTFPIct) inhibited tube formation and migration through inhibition of Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) tyrosine-951 phosphorylation, a key event in migration. hTFPIct did not inhibit VEGF121-induced migration, which lacks the heparin-binding domain of VEGF165. Utilising the chimeric receptor, EGDR, which contains the extracellular domain of epidermal growth factor (EGF) and the intracellular domain of VEGFR2/KDR, a direct effect of TFPIct on the intracellular domain of VEGFR2 was excluded (Abstract 143 figure 2) TFPIct did not block phosphorylation of EGDR when stimulated with EGF. <h3>Conclusion</h3> Angiogeneis is a key biological system in health and disease; enabling cells in a hypoxic environment to stimulate new blood vessel growth. These data demonstrate, both in vivo and in vitro, an inhibitory role for TFPI in angiogenesis that is TF-independent. In addition to it classical role as a TF-antagonist, TFPI, via TFPIct, interferes with the interaction of VEGF165 with the extracellular domain of VEGFR2, thereby limiting angiogenesis.