Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation

Epigenetic mechanisms have emerged as one of the key pathways promoting diabetes-associated complications. Herein, we explored the role of enhancer of zeste homolog 2 (EZH2) and its product histone 3 lysine 27 trimethylation (H3K27me3) in high glucose-mediated endothelial inflammation. To examine this, we treated cultured primary endothelial cells (EC) with different treatment conditions—namely, constant or intermittent or transient high glucose. Intermittent high glucose maximally induced endothelial inflammation by upregulating transcript and/or protein-level expression of ICAM1 and P-selectin and downregulating eNOS, KLF2, and KLF4 protein levels. We next investigated the underlining epigenetic mechanisms responsible for intermittent hyperglycemia-dependent endothelial inflammation. Compared with other high glucose treatment groups, intermittent high glucose-exposed EC exhibited an increased level of H3K27me3 caused by reduction in EZH2 threonine 367 phosphorylation and nuclear retention of EZH2. Intermittent high glucose also promoted polycomb repressive complex-2 (PRC2) assembly and EZH2′s recruitment to histone H3. Abrupt enrichment of H3K27me3 on KLF2 and KLF4 gene promoters caused repression of these genes, further supporting endothelial inflammation. In contrast, reducing H3K27me3 through small molecule and/or siRNA-mediated inhibition of EZH2 rescued KLF2 level and inhibited endothelial inflammation in intermittent high glucose-challenged cultured EC and isolated rat aorta. These findings indicate that abrupt chromatin modifications cause high glucose-dependent inflammatory switch of EC.

Intermittent High Glucose Exacerbates A-FABP Activation and Inflammatory Response through TLR4-JNK Signaling in THP-1 Cells

Background. Glucose fluctuation confers additional risks on diabetes-related vascular diseases, but the underlying mechanisms are unknown. Macrophage activation mediated by TLR4-JNK signaling plays an important role during the progress of diabetes. In the present study, we hypothesize that glucose fluctuation results in macrophage inflammation through TLR4-JNK signaling pathways. Methods. THP-1 cells were treated with normal glucose (5 mM), constant high glucose (25 mM), and intermittent high glucose (rotation per 6 h in 5 mM or 25 mM) for 24 h. The mRNA and protein expression levels of TLR4, p-JNK, and adipocyte fatty acid-binding protein (A-FABP) were determined, and the proinflammatory cytokines TNF-α and IL-1β were quantified. Results. In constant high glucose, TLR4 expression and JNK phosphorylation levels increased, and this effect was more pronounced in intermittent high glucose. Accordingly, the expression of A-FABP and the release of the proinflammatory cytokines TNF-α and IL-1β also increased in response to constant high glucose, an effect that also was more evident in intermittent high glucose. The inhibition of p-JNK by SP600125 did not attenuate TLR4 expression, but totally inhibited both A-FABP expression and the production of the proinflammatory cytokines TNF-α and IL-1β in both constant and intermittent high glucose. Conclusions. Intermittent high glucose potentiates A-FABP activation and inflammatory responses via TLR4/p-JNK signaling in THP-1 cells. These findings suggest a more detrimental impact of glucose fluctuation on macrophage inflammation in diabetes-related vascular diseases than thus far generally assumed.