Elevated H3K4me3 Through MLL2-WDR82 upon Hyperglycemia Causes Jagged Ligand Dependent Notch Activation to Interplay with Differentiation State of Endothelial Cells.

Endothelial-to-mesenchymal transition (EndMT) is a hallmark of diabetes-associated vascular complications. Epigenetic mechanisms emerged as one of the key pathways to regulate diabetes-associated complications. In the current study, we aimed to determine how abrupt changes in histone 3 lysine 4 tri-methylation (H3K4me3) upon hyperglycemia exposure reprograms endothelial cells to undergo EndMT. Through in vitro studies, we first establish that intermittent high-glucose exposure to EC most potently induced partial mesenchyme-like characteristics compared with transient or constant high-glucose-challenged endothelial cells. In addition, glomerular endothelial cells of BTBR Ob/Ob mice also exhibited mesenchymal-like characteristics. Intermittent hyperglycemia-dependent induction of partial mesenchyme-like phenotype of endothelial cells coincided with an increase in H3K4me3 level in both macro- and micro-vascular EC due to selective increase in MLL2 and WDR82 protein of SET1/COMPASS complex. Such an endothelial-specific heightened H3K4me3 level was also detected in intermittent high-glucose-exposed rat aorta and in kidney glomeruli of Ob/Ob mice. Elevated H3K4me3 enriched in the promoter regions of Notch ligands Jagged1 and Jagged2, thus causing abrupt expression of these ligands and concomitant activation of Notch signaling upon intermittent hyperglycemia challenge. Pharmacological inhibition and/or knockdown of MLL2 in cells in vitro or in tissues ex vivo normalized intermittent high-glucose-mediated increase in H3K4me3 level and further reversed Jagged1 and Jagged2 expression, Notch activation and further attenuated acquisition of partial mesenchyme-like phenotype of endothelial cells. In summary, the present study identifies a crucial role of histone methylation in hyperglycemia-dependent reprograming of endothelial cells to undergo mesenchymal transition and indicated that epigenetic pathways contribute to diabetes-associated vascular complications.

Dynamic alterations of H3K4me3 and H3K27me3 at ADAM17 and Jagged-1 gene promoters cause an inflammatory switch of endothelial cells.

Histone protein modifications control the inflammatory state of many immune cells. However, how dynamic alteration in histone methylation causes endothelial inflammation and apoptosis is not clearly understood. To examine this, we explored two contrasting histone methylations; an activating histone H3 lysine 4 trimethylation (H3K4me3) and a repressive histone H3 lysine 27 trimethylation (H3K27me3) in endothelial cells (EC) undergoing inflammation. Through computer-aided reconstruction and 3D printing of the human coronary artery, we developed a unique model where EC were exposed to a pattern of oscillatory/disturbed flow as similar to in vivo conditions. Upon induction of endothelial inflammation, we detected a significant rise in H3K4me3 caused by an increase in the expression of SET1/COMPASS family of H3K4 methyltransferases, including MLL1, MLL2, and SET1B. In contrast, EC undergoing inflammation exhibited truncated H3K27me3 level engendered by EZH2 cytosolic translocation through threonine 367 phosphorylation and an increase in the expression of histone demethylating enzyme JMJD3 and UTX. Additionally, many SET1/COMPASS family of proteins, including MLL1 (C), MLL2, and WDR5, were associated with either UTX or JMJD3 or both and such association was elevated in EC upon exposure to inflammatory stimuli. Dynamic enrichment of H3K4me3 and loss of H3K27me3 at Notch-associated gene promoters caused ADAM17 and Jagged-1 derepression and abrupt Notch activation. Conversely, either reducing H3K4me3 or increasing H3K27me3 in EC undergoing inflammation attenuated Notch activation, endothelial inflammation, and apoptosis. Together, these findings indicate that dynamic chromatin modifications may cause an inflammatory and apoptotic switch of EC and that epigenetic reprogramming can potentially improve outcomes in endothelial inflammation-associated cardiovascular diseases.

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.