Abstract Body: Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T_H cells (Tregs) and pro-inflammatory T_H17 cells within the aortic wall. The mechanisms regulating CD4⁺T_H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T_H cell phenotype by directly altering key transcription factors in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T_H17 activity within AAA development and to determine key T_H17 cell signaling pathways that are altered
Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1^f/fCD4^cre+) or MLL1 (Mll1^f/fCD4^cre+) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3).
Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4⁺ cells compared to non-aneurysmal control samples. Congruently, CD4⁺T_H cells isolated from both human AAA samples and the murine AngII-induced AAA model displayed a predominance of T_H17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4⁺ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4⁺ T_H cell Notch signaling (Notch1^f/fCD4^cre+) or MLL1 (Mll1^f/fCD4^cre+) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p<0.05) with decreased CD4⁺ T_H17 cell activity and reciprocal increase in Treg expression.
Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.