The C Loop of Fifth Epidermal Growth Factor-like Domain of Thrombomodulin Exerts Cytoprotective and Angiogenic Effects in Vitro and In Vivo

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerted cytoprotective and pro-angiogenic function in vitro and in vivo via G-protein coupled receptor 15 (GPR15). TME5 comprises three S-S bonds which divides TME5 into three loops: A (TME5A), B (TME5B) and C (TME5C). The present study aimed to identify the minimum structure of TME5 that produces favorable effects in vascular endothelial cells. The bromodeoxyuridine (BrdU) incorporation and in vivo Matrigel plug assays found that TME5C but not TME5A or TME5B stimulated proliferation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis, respectively. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs. Western blot analysis found that exposure of HUVECs to TME5C increased levels of anti-apoptotic myeloid cell leukemia-1 (Mcl-1) protein in association with activation of signal transduction pathways including extracellular signal-regulated kinase (ERK), AKT and mitogen-activated protein kinase p38. Importantly, TME5C did not affect thrombin clotting time in vitro. The cytoprotective function of TME5C was mediated by cell surface expressed GPR15, as TME5C was not able to protect vascular endothelial cells isolated from GPR15 knockout mice. Strikingly, TME5C successfully ameliorated hematopoietic stem cell transplantation (HSCT)-induced sinusoidal obstruction syndrome (SOS) in a murine model in association with counteracting reduction of sinusoidal ECs numbers, loss of integrity of sinusoidal wall and sinusoidal ECs detachment. Taken together, cytoprotective and the pro-angiogenetic functions of TM are preserved in TME5C. Use of TME5C may be a promising treatment strategy to prevent or treat lethal complications such as SOS whose pathogenesis is based on the endothelial insults.