Abstract Th0054: Investigating the Role of Zhx2 in Macrophage Cell Death in the Context of Atherosclerosis

Background: Atherosclerosis is a chronic inflammatory artery disease, causing about 50% of deaths in Western societies. It results from lipid accumulation and inflammation, leading to atherosclerotic cardiovascular disease (ASCVD) like heart attacks and strokes. In our previous studies, we observed that BALB/cJ mice with a naturally occurring null mutation (KO) in the Zhx2 gene, on a hyperlipidemic Ldlr−/− background, exhibited up to a 10-fold reduction in atherosclerotic lesion size compared to isogenic mice with the wild-type (WT) allele. This reduction was associated with primarily increased macrophage apoptosis in the atherosclerotic lesions. Objective: To uncover the underlying mechanism of lesional macrophage apoptosis in Zhx2-null mice, we performed single-cell analysis on the aortae of Zhx2 WT and KO mice fed an atherogenic diet. The data were analyzed and validated using RNA and protein-based assays. Methods: Eight to ten-week-old Zhx2 WT and KO BALB/cJ female mice were fed an atherogenic diet (1% cholesterol) for 14 weeks. Aortae from four mice per group were pooled, enzymatically digested, and analyzed through single-cell analysis. The data were then examined to identify signature genes and pathways in aortic macrophages from WT and KO mice. Apoptosis was confirmed by measuring Bax and Bcl2 expression in BMDMs as well. Western blot analysis was also performed on BMDMs to validate the findings from the single-cell analysis. Results: In the abstract graphic. Conclusions: Single-cell analysis reveals significant downregulation of the PI3K-Akt pathway, along with elevated apoptosis-related gene expression in KO mice, which was validated by qPCR analysis of BAX and Bcl2. Gene enrichment analysis identifies spp1 (protein: osteopontin) as a key gene involved in the PI3K-Akt pathway. Further examination of the OPN/PI3K pathway at the protein level, using Western blot analysis in bone marrow-derived macrophages, showed downregulation of OPN and phosphorylated PI3K and mTOR, along with increased Caspase-9 expression in KO BMDMs. These findings suggest that Zhx2 promotes macrophage survival in atherosclerosis through the OPN/PI3K pathway.