Graft microvascular endothelial cells (EC) represent a critical interface between the recipient and the donor. Stable/quiescent EC support immunoregulation in part via the expression of cell surface and secreted anti-inflammatory molecules. The EC receptor tyrosine kinase Tie2 and its canonical ligand Angiopoietin (Angpt)-1 is critical in the maintenance of endothelial quiescence. In contrast, Angpt2 acts as a context-dependent agonist/antagonist. We hypothesize that signals elicited by Tie2 facilitate an intragraft microenvironment that fosters tolerance and/or long-term graft survival. We used an established FDA drug library (650 drugs) to screen EC, and identified HMG-CoAreductase inhibitors (statins) as potent repressors of Angpt2 mRNA and protein production. In the present study, we evaluated the potential of statins to regulate Tie2 and EC stability. We isolated primary EC from C57/BL6 mouse heart and kidney (MHEC and MKEC respectively), and treated cultures with Simvastatin (Simva) (0-30[micro]M), and evaluated Angpt2 and Tie2 mRNA and protein expression by RT-PCR, ELISA, WB and FACS. IncuCyte cell imaging was also used to evaluate the live-time effect of Simva on cell morphology. We found a marked phenotypic change in ECs within 4hrs of Simva (10mM) treatment (vs controls) including cobblestoning and increased junctional VE-Cadherin, reflective of a quiescent morphology. Further, Simva-treated ECs (24hrs) had reduced levels of Angpt-2 (2 and 1.5-fold) and increased (2.5 and 3.4 fold) levels of Tie2 mRNA in MHEC and MKEC respectively. Moreover, when Simva-pretreated (10[micro]M) EC were challenged with TNFα (0-100units/ml), inducible expression of Angpt-2 was significantly inhibited (P<0.0001). In addition, we found that Simva-treated EC had notable expression of PD-L1 (1.6 fold);and also siRNA knockdown of Tie2 reduced its level of expression. In pilot in vivo analyses, we also find that Simva treatment (1-2mg/kg) reduces Angpt-2/Angpt-1 mRNA expression ratios. Collectively, these data for the first time identify the HMG-CoA reductase inhibitor family agents as potential therapeutics to target the stability and immunogenicity of microvascular EC within the graft, independent of cholesterol/lipid homeostasis. Statin dosing to target this axis should be considered in future tolerance induction clinical trials.

CITATION INFORMATION: Ghosh C, Mak A, McGourty M, Parikh S, Chen X, Goldberg S, Wedel J, Briscoe D. TIE'ing Statins to Microvascular Stability and Endothelial Cell Immunogenicity. Am J Transplant. 2017;17 (suppl 3).

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