2025 Theses Doctoral

Comprehensive Multimodal Profiling of Atherosclerosis Reveals Bhlhe40 as a Potential Regulator of Vascular Smooth Muscle Cell Phenotypic Modulation

Ibikunle, Chinyere Onyinyechi

Background:
Atherosclerosis is an inflammatory disease of the arterial vessels characterized by the accumulation of lipids and cells such as vascular smooth muscle cells (VSMCs), endothelial cells, fibroblasts, and immune cells, which form a plaque or lesion that obstructs proper blood flow. Studies have shown that the high lipid and inflammatory micro-environment within atherosclerotic plaques drive the phenotypic modulation of VSMCs from their contractile state to multiple modulated states such as fibroblast-like, myofibroblast-like, osteoblast-like, and macrophage-like states; as well as their uptake of lipids to form VSMC foam cells. While these modulated states have been well recognized in the field, the transcriptomic profile and molecular drivers underlying VSMC phenotypic modulation in atherosclerosis progression remain under characterized.

Methods:
In this study, we aimed to understand how VSMCs transition to macrophage-like and lipid-rich foam cells during atherosclerosis progression and the molecular factors regulating this process. We first performed a multiomics study using single-cell RNA and CITE-seq, on a lineage tracing atherosclerotic Ldlr-/- mouse model fed with a high-fat diet at multiple time points (each time point reflective of disease progression). This allowed for the identification of VSMCs and their derived cell types in atherosclerotic lesions in vivo. Our multiomics study and immunohistochemical staining were also extended to human atherosclerotic lesions to study the transcriptomic profile and differentially expressed proteins unique to VSMCs, their subtypes, and phenotypically modulated states during disease progression, including VSMC-macrophage-like cells and VSMC-foam cells.

Furthermore, to understand the modulation of contractile VSMCs to VSMC foam cells, we conducted bulk RNA sequencing of atherosclerotic plaques from conditional VSMC-lineage tracing Ldlr-/- mouse models maintained on a high-fat diet. Using LipidTox staining, VSMCs and VSMC-derived foam cells were identified and isolated using flow cytometry at key time points representing late and advanced stages of disease progression and further sequenced to characterize their genomic profile and identify a panel of key transcriptional regulators upregulated in late to advanced stages of atherosclerosis progression. Further re-analysis of our single-cell data on the expression pattern of these transcription regulators showed their expression within VSMC subpopulations at the single-cell level, with the transcription factor Bhlhe40 showing the highest specificity of being differentially expressed in modulated VSMC clusters. To understand the functional role of Bhlhe40, we performed an siRNA knockdown of Bhlhe40 in primary mouse VSMCs under atherosclerotic stress induced by TNFα and MBD-Cholesterol.

Results:
Our single-cell analysis revealed that VSMC-derived macrophage-like cells are a rare cell population even in advanced stages of atherosclerosis, while a substantial number of VSMC-lineage foam cells existed in mouse atherosclerotic lesions. Histological studies utilizing immunohistochemistry and in situ hybridization provided additional critical insights into the localization of rare VSMC-derived macrophage-like cells within atherosclerotic lesions. Notably, these studies further showed that in both mice and humans, VSMC-derived macrophage-like cells are very few compared to their myeloid-derived counterparts in lesions.

In contrast, lipid staining combined with bulk RNA sequencing analyses demonstrated that approximately 70% of foam cells in advanced lesions are derived from VSMCs. These VSMC-derived foam cells showed enrichment in gene pathways associated with proliferation, migration, and cancer-related transcription factors, supporting the hypothesis that VSMC-derived cells in atherosclerosis exhibit characteristics akin to tumor biology.

Our findings also reveal that the expression of the Basic Helix-Loop-Helix Family Member E40, Bhlhe40 (also known as Dec1), a pro-inflammatory transcription factor, is activated in VSMC-derived foam cells during atherosclerosis progression and enriched in modulated and proliferative VSMC-derived cells measured by scRNA-seq of atherosclerotic lesions in mice and humans. Upstream regulator analysis suggests that Bhlhe40 may play a pivotal role in driving the differentiation of VSMCs into foam cells. Furthermore, functional in vitro studies in mouse primary VSMCs showed that siRNA Bhlhe40 knockdown inhibits VSMC phenotypic modulation and lipid uptake during atherosclerotic stress induced in vitro.

Conclusions:
These findings advance our understanding of VSMC phenotypic modulation in atherosclerosis and identify Bhlhe40 as a regulator of VSMC foam cell formation during development and progression of atherosclerosis. Elucidating the molecular and transcriptomic landscape of VSMC-modulated cells will significantly contribute to the field and address key gaps in the current understanding of atherosclerosis while offering potential pathways for innovative therapeutic strategies.