Using In Silico Methods to Examine Atherosclerosis in the ApoE KO Mouse: a Basic Science Meta-Analysis

by Rachel Xiang

Mentor: Iris Jaffe, Cell, Molecular, and Developmental Biology, TUMS; Funding source: Nathan Gantcher Student Summer Scholars Fund

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Atherosclerosis is a chronic inflammatory disease where lipid-rich plaques form in blood vessels. Atherosclerosis is a potent risk factor for more severe forms of cardiovascular disease (CVD), such as heart and stroke, which occur when plaques rupture, disrupting blood flow. Highly inflamed plaques with large lipid cores are more vulnerable to rupture, implicating mechanisms that regulate lipid metabolism and inflammation as integral to outcomes of atherosclerosis. As CVD remains the leading cause of death worldwide, there is a critical need to study the mechanism by which atherosclerosis develops.

Animal models are commonly used to study atherosclerosis, and the ApoE KO mouse is a popular preclinical model of atherosclerosis. Since its development in 1995, over 6,000 studies have been published using the ApoE KO model system. Most have examined the effect of a single gene perturbation on atherosclerosis. However, rather than considering each gene target as a unique contributor to atherosclerosis, it’s more likely that these genes are connected and may act together. With this in mind, we hypothesized that a systematic analysis of all genes that have been experimentally implicated in atherosclerosis in the ApoE KO mouse would allow us to identify common regulators and pathways.

This summer, we looked for patterns across genes that have been experimentally shown to affect atherosclerosis in 10% of all published ApoE KO mouse atherosclerosis studies. We collected data from studies describing directional changes in plaque size, lipid content, and inflammation that resulted from a single gene perturbation. The analysis was performed using a bioinformatics software called Ingenuity Pathway Analysis (IPA), which looks for patterns in gene expression data and offers predictions of pathway enrichment and relevant regulatory networks.

Common upstream regulators and pathways:

Three significant upstream regulators of plaque size were identified, all of which are known to interact with COX-2, an enzyme that mediates inflammatory responses in macrophages. COX-2 was predicted to be activated, implicating COX-2 as a potential master regulator of gene changes associated with increased plaque size.

However, selective COX-2 inhibitors are associated with increased cardiovascular risk, which does not align with our findings of COX-2 as a regulator of plaque size increase. One possible reconciliation of this discrepancy is that COX-2 may not lie as upstream as we had thought, and that it is merely activated as a response to other changes associated with increased plaque size and composition. Overall, further investigation is needed to understand COX-2’s role in atherosclerosis.

Significantly enriched pathways were also identified. LXR/RXR signaling, which is involved in lipid metabolism and macrophage inflammatory responses, was inhibited. TREM1 signaling, which stimulates a number of inflammatory responses, was activated.

Sex differences:

In humans, CVD develops differently in men and women, underscoring the importance of examining biological mechanisms that might explain these sex differences.

Certain pathways were differentially regulated in males and females. Estrogen receptor (ESR) signaling was inhibited in females but activated in males with atherosclerosis. ESR signaling may have an atheroprotective effect in females, as inhibition of ESR signaling was associated with larger plaques. This finding is consistent with the observation that the risk of atherosclerosis increases in post-menopausal women, after estrogen production stops.

PTEN signaling was also oppositely regulated by sex. PTEN is a tumor suppressor that is well characterized in cancer, but less so in CVD. PTEN has been shown to regulate proliferation, migration, and survival in vascular smooth muscle cells, and aberrant vascular smooth muscle cell proliferation contributes to plaque development. Here, our findings suggest a novel role for PTEN signaling as a contributor to sex differences in CVD outcomes.

Further exploration of sex-dependent mechanisms may allow for the identification of sex-specific drug targets.

Differences in early and late stages of atherosclerosis:
The comparison of early vs. late atherosclerosis has important clinical implications. Typically, patients are unlikely to seek treatment during early stages of atherosclerosis, meaning pathways and functions enriched in late atherosclerosis may be more effective drug targets. Functions related to inflammation and cell movement were activated early, and functions related to lipid metabolism and cell survival were inhibited late. Our comparison of pathways and functions associated with early and late atherosclerosis proposes a timeline for different processes involved.

Overall, meta-analyses are common in clinical research but, until now, have not yet been implemented in basic science research. Systematic analyses of basic science studies may prove to be a powerful tool in identifying relevant pathways and regulators of atherosclerosis, and a better understanding of these mechanisms could result in a broader array of treatments designed to inhibit this process.