R01 – PROJECT SUMMARY/ABSTRACT Solid organ transplant recipients exhibiting HLA donor specific antibodies (DSA) are at risk for graft loss due to chronic antibody-mediated rejection (cAMR) and develop a progressive vascular disease known as transplant vasculopathy (TV). Although cAMR and TV are highly significant clinical problems across different solid organ transplants the mechanisms by which DSAs directed against HLA-I and HLA-II promote cAMR and TV remain incompletely understood. Novel mechanistic targets and therapeutic approaches to prevent and treat cAMR and TV are urgently needed. Our preliminary results show that antibody(Ab)-induced crosslinking of HLA class I (HLA-I) molecules in endothelial cells (ECs) triggers the formation of complexes of HLA-I with integrin β4 (ITGB4) and Toll like receptor 4 (TLR4) which function as HLA-I co-receptors to elicit intracellular signaling in ECs. The HLA-I/ITGB4 complex mediates FAK/Src, PI3K/AKT, mTORC1/2 and ERK but does not lead to exocytosis of Weibel Palade bodies (WPbs), P-selectin externalization or leukocyte recruitment to ECs. Recently, we found that HLA I ligation induces ITGB4-independent signaling via TLR4 and showed that HLA II signaling in ECs also depends on TLR4 as a key co-receptor. Thus, TLR4 emerges as a central co-receptor in HLA-I and HLA-II signaling. However, the mechanism(s) by which TLR4 elicits WPb exocytosis, P-selectin externalization and monocyte recruitment in ECs was unknown. Our preliminary results indicate that the mechanosensitive Ca2+ channel Piezo 1 functions as a transducer of TLR4-induced exocytosis, P-selectin expression and monocyte adherence in ECs thereby exacerbating pro-inflammatory signaling leading to TV. Based on substantial preliminary studies, our central hypothesis is that class I and II HLA DSAs elicit key features of TV by promoting the formation of physical and functional complexes with other membrane receptors and ion channels, including TLR4, ITGB4 and Piezo1 on the surface of ECs of the graft to promote chronic inflammation, recruitment of monocytes and fibrosis. We propose to explore this central hypothesis by pursuing three Specific Aims: 1) Define a central role of TLR4 signaling in mediating HLA-I and HLA-II induced endothelial cell activation, pro- inflammatory signaling cascades, P-selectin externalization and monocyte recruitment, all processes of fundamental importance in the pathogenesis of transplant vasculopathy. 2) Identify a novel function of the Ca2+ channel Piezo 1 in mediating TLR4-induced endothelial cell activation, P-selectin externalization and monocyte recruitment in response to Ab-mediated HLA I and HLA II activation. 3) Determine the impact of genetic deletion or pharmacological inhibition of TLR4 and Piezo1 on cAMR using a novel model of murine heart allograft that develops TV and define the molecular expression of HLA I and II signaling pathways in human cardiac allografts with transplant vasculopathy. We anticipate that ...