Functional analyses of TET2 in vivo and in vitro reveal endothelial-specific roles in interferon responses that are dysregulated in hyperglycaemia

Abstract Background Endothelial dysfunction represents a critical early event in the development of vascular disease but its aetiology remains unknown. However, risk factors for cardiovascular disease, including hyperglycaemia, correlate with altered DNA methylation patterns both in vivo and in vitro. Ten-Eleven-Translocation 2 (TET2) catalyses successive oxidation of 5-methylcytosine to facilitate DNA demethylation. Loss-of-function mutations of TET2 in haematopoietic cells are associated with cardiovascular disease, and decreased TET2 protein stability is observed in diabetic patient peripheral blood mononuclear cells. However, the contribution of TET2 to endothelial function and the extent to which dysregulated TET2 activity might participate in endothelial dysfunction in diabetes remain incompletely understood. Methods Responses of wild-type and TET2fl/fl Cdh5-CreERT2 mouse aortae to vasoconstrictors and vasodilators were measured in an organ bath. Targets of TET2-mediated transcriptional regulation were identified by RNA sequencing of lung CD31 + cells from these mice, and by qPCR following silencing of TET2 in human umbilical vein endothelial cells (HUVEC). Comparisons were made between genes differentially expressed upon TET2 ablation or exposure to hyperglycaemia in vivo. Specific gene loci associated with altered TET activity following interferon gamma (IFNγ) treatment were assessed by hydroxymethylated DNA immunoprecipitation sequencing. Results Endothelial-specific deletion of TET2 did not alter responses of aortic rings to acetylcholine or phenylephrine. The absence of TET2 in murine lung CD31 + cells dysregulated pathways including response to IFNγ and leukocyte activation, bearing significant resemblance to pathways dysregulated by exposure to hyperglycaemia. The IFNγ-induced transcription of CXCL9 and CXCL10 in HUVEC correlated with a striking gain of 5hmC deposition at an enhancer region between CXCL9 and CXCL10 gene loci. Silencing of TET2 or high glucose administration acted similarly to reduce this IFNγ-induced expression. Conclusions TET2 transcriptionally regulates IFNγ responses and leukocyte activation pathways in endothelial cells. TET2 is likely to regulate the IFNγ-induced expression of CXCL9 and CXCL10 via its catalytic activity, causing demethylation of an intergenic enhancer region. Decreased TET2 activity may contribute to the altered expression of a subset of genes dysregulated by hyperglycaemic exposure. These findings may provide insight into the epigenetic and transcriptional reprogramming induced by hyperglycaemia, leading to endothelial dysfunction and vascular disease.