Intestinal Stem Cells (ISCs) Derived from Menopausal Mice and Implications for Screening Cardioprotective Probiotic Species

Cardiovascular disease (CVD) is the leading cause of death in both men and women; however, morbidity and mortality sharply rise after the onset of menopause in women implicating loss of estrogen as key driver of increased CVD risk. The mechanisms contributing to this shift in CVD risk following menopause are not well understood, therefore limiting our abilities to develop sex-specific therapeutics. Given menopausal women show shifts in their gut microbiomes and our previous work demonstrating strain-specific probiotic cardioprotection, we are investigating the impact of the gut microbiome on CVD susceptibility during menopause. Using our 4-vinylcyclohexene diepoxide model of ovarian failure that uniquely preserves the peri-menopause transition, we generated 3D-cultures of intestinal stem cells (ISCs) derived from pre-, peri-, and menopausal mice. Following protein isolation, samples were used for in-solution, global, unbiased proteomics. We found 2,086 proteins were identified with two or more unique peptides across all groups and treatments, but 398 proteins were found to be significant by ANOVA (p<0.05). Hierarchical clustering and principle component analysis indicated that ISCs cultured from pre- and peri-menopausal mice showed less variance between samples when compared to menopause. Additionally, pairwise comparisons (p<0.05 and |log2 fold change| ≥ 1.5) showed menopausal samples have an overall downregulation (37 and 73 proteins compared to pre- and peri-menopausal cultures, respectively) of proteins that were most enriched for genes associated to metabolic pathways including amino acid biosynthesis, central carbon metabolism, and glycolysis pathway. Interestingly, we found a decreased peptide abundance in menopausal samples for the polymeric immunoglobulin receptor (pIgR). pIgR plays an important role in transcytosis of IgA and IgM across mucosal barriers and found to be involved in tightly regulating populations of gut microbiota. Immunoblotting of freshly isolated ISCs from pre-menopausal and menopausal mice showed that pIgR is significantly decreased in menopausal samples compared to pre-menopause. Furthermore, the gut microbiome and microbial metabolites directly impact gene expression within the mucosal barrier by modulating histone deacetylase (HDAC) activity. Therefore, through immunoblotting we demonstrated that histone H3 acetylation at Lys27 was significantly decreased in ISCs isolated from menopausal mice. These findings validate the use of ISCs isolated from menopausal mice as a model system to elucidate mechanisms of host-microbe interactions in a controlled cell system with implications to derive high-throughput probiotic screening tools. Ralph and Shirley Morgan Cardiovascular Research Award '21-'22, Sarver Heart Center, University of Arizona. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.