Mechanisms of AMR Mediated By DSA: Activation of Endothelial Notch Pathway Triggers M1/M2 Polarization in Human Cardiac Transplants.

Abstract

Notch signaling is a major pathway in cell fate decisions and it is considered as an important player in vascular homeostasis through the control of cell differentiation, proliferation and apoptosis. However, the contribution of the Notch pathway to vascular injury and endothelial dysfunction in transplantation remains unclear. This study investigated the contribution of Notch receptors and ligands to vascular injury associated with donor specific antibody (DSA)-mediated rejection (AMR) in human cardiac allografts.Regulation of Notch receptors (Notch1, 2, 3, 4) and ligands (Jagged1, Dll4) was analyzed by quantitative PCR and by immunohistochemistry in cardiac biopsies from patients with stable graft or non failing heart (n=13) or with AMR (n=8). The impact of Notch signaling activity was further investigated by modulating Dll4 in cellular co-culture models using endothelial cells (EC) isolated from donor transplants and monocytes. Our results show that AMR is characterized by an up-regulation in Notch1, -2, Dll4 and Jagged1 transcripts (respectively, 3.7-, 4.7-, 4.5- and 2.1-fold increase versus controls, p<0.01) and a drastic down-regulation of Notch3 and of the endothelial Notch4 (5.1-fold decrease versus controls, p<0.01). A Notch4/Dll4 imbalance, at endothelial level, upon AMR was confirmed by immunohistochemistry on biopsies and also observed in cellular models. We also found that overexpression of Dll4 in human EC strongly induces Notch activity in monocytes, reflected by increased Hes1 expression. Moreover, Dll4 enhances IL-1β, IL-6, IL-8 and TNF mRNA expression in monocytes and induces a phenotypic switch of monocyte markers.Overall, our findings provide the first evidence that impaired Notch activity in graft EC is a key event associated with AMR triggering EC dysfunction and monocytes/macrophages polarization.