VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis

Objective: VEGF (vascular endothelial growth factor) plays a critical role in physiological and pathological angiogenesis. Endothelial 3D podosomes (3DPs) are a type of F-actin-rich membrane microdomain, predominantly found in endothelial tip cells controlled by VEGF signaling during sprouting angiogenesis, such as occurs in retinal vasculature development. The molecular mechanisms governing 3DP formation have not been completely elucidated. Approach and Results: By using in vitro cell models and in vivo mouse models, we study the role of TM (thrombomodulin) in VEGF-induced endothelial 3DPs. Here, we report that VEGF can induce the expression of TM via ROCK2 (Rho-associated coiled-coil kinase 2). Furthermore, ROCK2 can catalyze the phosphorylated activation of ezrin to promote the association of the cytoplasmic domain of TM with F-actin in 3DPs and thereby promote the formation of 3DPs. We used endothelial cells transfected with different TM mutants as models to verify the role of TM domains in 3DPs and angiogenic activity. TM expression in endothelial cells augments angiogenic activity, a response that is dependent on the interaction of the cytoplasmic tail of TM with ezrin, and the integrity of the lectin-like domain of TM. Thus, as compared with wild-type counterparts, mice lacking the lectin-like domain of TM exhibit reduced neovascularization of granulation tissues during cutaneous wound healing and less retinal neovascularization in a model of oxygen-induced retinopathy. Conclusions: VEGF-ROCK2-ezrin-TM-F-actin axis promotes the formation of the lipid raft membrane-associated complex configuration, 3DP, which plays a critical role in mediating tube formation and cell migration of endothelial cells in sprouting angiogenesis.