Human Alternatively Spliced Tissue Factor Promotes Monocyte-Endothelial Interactions Via Upregulation of Adhesion Molecules In Microvascular Endothelial Cells

Alternatively spliced Tissue Factor (asTF) is a soluble form of Tissue Factor (TF) that lacks the transmembrane domain and exhibits low pro-coagulant activity compared to decrypted full-length TF (Bogdanov et al, Nat Med 2003). Human asTF was recently shown to ligate integrins α6β1 and αVβ3 on endothelial cell surfaces, which triggered neovascularization in vitro, ex vivo, and in vivo in fVIIa- and/or PAR-2 independent fashion (van den Berg et al., PNAS 2009). This principally novel, non-proteolytic biologic activity of asTF was found to activate multiple integrin-linked kinases in human and murine macrovascular endothelial cells (EC), including those comprising the PI3K/Akt pathway. Engagement of PI3K/Akt signaling is known to activate NFkB – a transcription factor involved in upregulation of multiple leukocyte adhesion molecules that play a major role in various disease states, most notably atherogenesis and tumorigenesis. In this study, we sought to determine whether human asTF acts as an agonist on microvascular EC – the endothelial sub-type most relevant to monocyte egress from the circulation.

Primary cardiac and retinal human microvascular endothelial cells (micro-EC) expressed α6, β1, and β3 mRNA. Both micro-EC sub-types rapidly adhered to the recombinant N-terminally His-tagged human asTF: a 20+ fold increase in the number of adherent micro-EC was observed as early as 30 min after the cells were added (n=3,100 ng asTF vs BSA, p<0.0001). In both sub-types of micro-EC, anti-β1 integrin antibody completely blocked adhesion to asTF whereas anti-β3 and/or anti-αvβ3 antibody had no effect, likely due to the known scarcity of non-β1 integrins on the surface of human micro-EC (Wilson et al., IOVS 2003). We then examined the changes in gene expression elicited by asTF in micro-EC using Affymetrix Gene 1.0 ST microarrays. A marked upregulation of several cell adhesion molecules (CAMs) was observed in cardiac and retinal micro-EC including VCAM-1 and E-selectin, which was confirmed at the protein level by western blotting. We proceeded to investigate the functional significance of the upregulation of CAMs by performing adhesion assays using pre-labeled THP-1 cells. Under orbital shear stress conditions, a ∼76% increase in THP-1 adhesion was observed for cardiac micro-EC treated with asTF over control (n=5, p<0.001), and a ∼62% for retinal micro-EC (n=5, p<0.005). Addition of polymyxin B and/or non-charged agarose beads elicited no effect, whereas pre-treatment of asTF samples with Ni-charged beads and heat denaturation eliminated the effect, confirming that the observed findings are asTF-specific and not caused by LPS contamination. In the static assay and the laminar flow chamber assay performed under the flow rates found in postcapillary venules, the increase in THP-1 adhesion was 40% (n=3, p<0.001) and 250% (n=3, p<0.05), respectively, for cardiac micro-EC treated with asTF and 83% (n=3, p<0.001) and 290% (n=3, p<0.05), respectively, for retinal micro-EC. While performing laminar flow chamber assays, we observed relocation of some of the adhered THP-1 cells under the EC monolayer, which indicates that asTF may cause monocyte emigration in the absence of a chemokine gradient. To determine whether monocyte egress through asTF-stimulated micro-EC can also occur under a chemokine gradient, we performed transendothelial migration assays using transwell inserts with MCP-1 placed in the lower chamber. Here, we observed that asTF potentiated THP-1 migration through cardiac micro-EC by ∼206% (n=3, p<0.0001), and through retinal micro-EC – by ∼90% (n=3, p<0.005).

We show for the first time that (i) human asTF ligates β1 integrin(s) on primary human micro-EC, (ii) this leads to transcriptional upregulation of CAMs, and (iii) this upregulation promotes adhesion and transendothelial migration of monocytic cells under physiologically relevant conditions. We note that the concentrations of asTF used here were in the range found in cervical cancer tissue (van den Berg et al., PNAS 2009). Our results expand the potential scope of asTF's contribution to atherogenesis and tumorigenesis. Evaluation of these findings in vivo is highly warranted as monocyte recruitment triggered by asTF may represent a novel nexus in atherosclerotic progression and/or tumor growth.

No relevant conflicts of interest to declare.