A Critical Role of Growth Arrest-Specific Gene 6-Mer Axis in the Pathogenesis of Endothelial Damage Contributing to Thrombotic Microangiopathy Associated with Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) and thrombotic microangiopathy (TMA) are severe complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Host endothelial cells are targets of alloreactive donor cytotoxic T lymphocytes or various proinflammatory cytokines following allo-SCT, and endothelial damage plays an important role in the pathogenic mechanism(s) of TMA associated with GVHD. However, the detailed mechanism(s) of TMA as well as GVHD have not yet been fully elucidated. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family; Tyro3, Axl, and Mer. Gas6 and the TAM receptor tyrosine kinases have been reported to be associated with systemic inflammatory and autoimmune disorders, as well as hemostatic abnormalities. We hypothesized that Gas6-TAM pathway signaling contributed to endothelial dysfunction in the pathogenesis of TMA associated with GVHD. ELISA showed that the serum levels of Gas6 were markedly increased in the all-SCT patients with grades 2 or 3 GVHD at 21-35 days after stem-cell engraftment. The increased serum levels of Gas6 were also correlated with the elevated D-dimer and plasmin-α2 plasmin inhibitor complex values in the allo-SCT patients. Immunostaining showed that the expression of Gas6 and Mer was significantly upregulated in the intestinal acute GVHD lesion. The platelet aggregation test showed that UNC2250 (5 μM), a selective Mer tyrosine kinase inhibitor markedly suppressed increased platelet aggregation caused by exogenous Gas6 and collagen. Using Western blot analysis, exposure of human umbilical vein endothelial cells (ECs) to, Gas6 or 25-50% (vol/vol) serum isolated from patients with GVHD caused the downregulation of thrombomodulin (TM) and plasminogen activator inhibitor type-1 (PAI-1) as well as the upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as assessed by Western blot analyses, which were inhibited by UNC2250 or Mer siRNA. Furthermore, exogenous Gas6 or the addition of serum from patients with GVHD to ECs induced apoptosis of ECs with activation of caspase-3 and poly (ADP-ribose) polymerase, which were inhibited by UNC2250 or Mer siRNA. These results indicate that the Gas6-Mer axis may be crucial for platelet activation, endothelial damage and subsequent hypercoagulation in the pathogenesis of TMA associated with GVHD after allo-SCT. Furthermore, in the murine models of allo-SCT, we observed hepatic GVHD with hepatocellular apoptosis, necrosis and fibrosis and TMA characterized by thrombosis formation in the microvasculature of liver and kidney. The intravenous administration of 3 mg/kg UNC2250 markedly inhibited hepatic GVHD and TMA formations of liver and kidney in murine models of allo-SCT at 21 days after allo-SCT. The present study provides new evidence that the Gas6-Mer axis may be involved in the mechanism(s) underlying the progression of the pathogenesis of TMA associated with GVHD after all-SCT. We suggest that Gas6-Mer axis may be an attractive therapeutic target for treating TMA associated with GVHD caused by endothelial damage.