The immunogenicity of graft microvascular endothelial cells (ECs) has immense biological significance in the modulation of the local intragraft microenvironment. EC may either support Teffector activity and rejection or local immunoregulation. Studies have shown that endothelial Tie2 and its ligands, Angpts (1/2), are crucial in the maintenance of vascular stability and endothelial quiescence. The treatment of graft recipients with agonistic ligand mimetics (e.g. Angpt1) or inhibitors of pathological Angpt2 improves graft survival. However, the mechanisms whereby the Tie2-receptor prolongs survival is not known. We hypothesize that Tie2 expression on intragraft EC modulates the local environment to support immunoregulation. We screened an FDA drug library of 650 drugs, and identified simvastatin (Simva) as a potent regulator of Angpt2, and we observed that it induces Tie2 expression. The Simva-mediated (0.1-30micM) increase in Tie2 expression was validated at the mRNA and protein level in human ECs (2.4 fold, n=5) and in primary mouse heart (MHEC) and kidney (MKEC) ECs (5.8 fold, n=6, p<0.01). We employed a proteomic screen using SWATH-MS, and identified a Tie2-regulated protein-protein network that promotes endothelial cell as well as lymphocyte activation. Furthermore, we observed a dramatic increase in the expression of the co-inhibitory molecules PD-L1 (2.6 fold; n=6, p<0.01) and LGALS9 (~2.8 fold; n=6, p<0.001) in Simva treated EC. To test the interplay among Tie2 signals and PD-L1 expression, by using si-RNA (25nM) we silenced Tie2 (~75%) in murine ECs and find that the silencing of cells is associated with a significant (n=6, p<0.01) reduction in PD-L1 mRNA and protein expression. Furthermore, Tie2 siRNA transfected MHECs failed to respond to Simva with the induction of PD-L1 and LGALS9 (p<0.001). To understand the functional consequences of Simva treatment of ECs, we performed transcostimulation assays where human EC (+/-10 micM Simva) were co-cultured with human CD4+ T cells and observed Simva treatment of EC reduced ~50% (n=3, p<0.001) proliferation as compared to control ECs. Collectively, these data suggest that intragraft endothelial Tie2 may function to regulate the expression of immunoregulatory molecules on EC, which in turn supports a pro-tolerogenic microenvironment. Our findings also suggest that simvastatin may be therapeutic to augment local intragraft immunoregulation and pro-tolerogenic immunosuppressive regimens to prolong graft survival.

CITATION INFORMATION: Ghosh C., Agur T., Mak A-.L., Pal S., Wedel J., David B. Novel Function of TIE2 in Immunoregulation Am J Transplant. 2017;17 (suppl 3).

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