549Monocytes activate the non-canonical Wnt5a pathway in microvascular endothelial cells and induce tissue factor expression and tube formation

Purpose: Atherosclerosis is characterized by lipid accumulation, inflammation and neovascularization. Advanced atherosclerotic plaques can be destabilized and become vulnerable to rupture by microcapillary formation and inflammatory cell infiltration; and can lead to thrombosis and acute coronary syndromes. We have demonstrated in previous studies that increased tissue factor (TF) expression, that it is induced in microvascular endothelial cells (mECs), triggers angiogenesis; however, the signals that induce TF expression in mECs are barely understood. Here, we hypothesize that monocytes, that are strongly involved in atherosclerotic plaque inflammation, can interact with mECs and induce TF expression and, consequently, neovessel formation. Methods: The crosstalk between monocytes and mEC was studied in vitro by using both wound repair and matrigel assays. Angiogenesis was evaluated through tube length, nodule number and angiotube-covered area in the mEC cultured with MCM (isolated monocyte-derived conditioned medium). TF and Wnt5a genes were silenced through small interfering RNA (siRNA) in mEC and monocytes, TF expression was measured by PCR and immunoblotting and TF promoter was cloned into luciferase reporter vector pGL3. Finally, matrigel plugs seeded with monocytes and mECs were implanted in nude mice to test in vivo angiogenesis. Results: MCM enhanced wound repair, neovessel formation and expression of TF in in vitro assays. Wnt5a was identified as a protein released by monocytes that promoted TF expression and angiogenesis. Wnt5a protein increased up to 7 fold both TF expression in mEC and neotube formation. Furthermore, Wnt5a upregulated the receptor FZD5 in mEC and induced Ca2+ intracellular secretion and increased the transcription factor NF-κB for TF expression. In vivo, monocytes-mEC plugs showed significantly higher angiogenesis. Conclusions: This study demonstrate that monocytes crosstalk with mEC through Wnt5a secreted monocyte activates the non-canonical Wnt-pathway in mEC by interacting with FZD5 and triggering TF expression and inducing angiogenesis.