Atherosclerosis Cell Models for Research

Atherosclerosis is the underlying cause of most cardiovascular diseases (CVD), including myocardial infarction, stroke, and peripheral artery disease. According to the World Health Organization (WHO), CVDs are the leading cause of death globally, accounting for an estimated 17.9 million deaths each year (WHO, 2021). Atherosclerosis is a chronic inflammatory disease characterized by the buildup of plaques in arterial walls, leading to luminal narrowing and thrombosis. Major risk factors include hyperlipidemia, hypertension, smoking, diabetes, and obesity. The 5-year survival after a major cardiovascular event varies; for example, after a first myocardial infarction, the 5-year survival is approximately 80% in high-income countries, but lower in low- and middle-income settings (NCI, SEER data). The economic burden is substantial, with direct and indirect costs exceeding billions annually.

Atherosclerosis is ideal for mechanistic studies due to its complex pathophysiology involving multiple cell types (endothelial cells, smooth muscle cells, macrophages, T cells) and lipid metabolism. Public datasets, such as those from the Genotype-Tissue Expression (GTEx) project and the Framingham Heart Study, provide rich genetic and expression data. Open questions include the precise molecular triggers of plaque rupture, the role of specific genetic variants in disease susceptibility, and the development of targeted therapies. Gene-edited cell models enable precise dissection of these mechanisms.

Atherosclerosis involves several interconnected pathways:

1. Endothelial dysfunction: Injury to the endothelium leads to increased permeability and expression of adhesion molecules (e.g., VCAM-1, ICAM-1).

2. Lipid retention and modification: Low-density lipoprotein (LDL) particles infiltrate the intima and undergo oxidation, triggering inflammation.

3. Monocyte recruitment and foam cell formation: Monocytes adhere to activated endothelium, migrate into the intima, differentiate into macrophages, and engulf oxidized LDL to become foam cells.

4. Smooth muscle cell proliferation and matrix deposition: Smooth muscle cells migrate from the media to the intima, proliferate, and produce extracellular matrix, forming a fibrous cap.

5. Inflammation and plaque progression: Inflammatory cytokines (e.g., TNF-α, IL-6) perpetuate the cycle, leading to plaque growth and instability.

While atherosclerosis is not a cancer, genetic variations significantly influence risk. Key genes include:

Data from ClinVar, NCBI Gene, and population studies.

Key signaling networks involved in atherosclerosis include:

• • Inflammatory signaling: NF-κB pathway activated by cytokines and oxidized lipids, leading to expression of adhesion molecules and inflammatory genes.
• • Lipid metabolism: LXR and SREBP pathways regulate cholesterol efflux and synthesis; dysregulation promotes foam cell formation.
• • MAPK/ERK pathway: Mediates smooth muscle cell proliferation and migration in response to growth factors.
• • PI3K/AKT pathway: Promotes cell survival and proliferation; also involved in insulin signaling and endothelial function.
• • Notch signaling: Regulates cell fate decisions in vascular development and inflammation.

These networks are targets for therapeutic intervention.

Common cell lines used in atherosclerosis research:

Organoids: 3D vascular organoids derived from induced pluripotent stem cells (iPSCs) can recapitulate vessel structure and function, offering a more physiologically relevant model for studying atherosclerosis.

Animal models for atherosclerosis include:

• • ApoE knockout mice: Spontaneously develop atherosclerotic lesions on a high-fat diet; widely used.
• • LDLR knockout mice: Develop hypercholesterolemia and atherosclerosis; useful for studying LDL metabolism.
• • PCSK9 gain-of-function mice: Mimic familial hypercholesterolemia.
• • Rabbits on high-cholesterol diet: Develop lesions similar to human plaques.
• • Porcine models: Larger animals with similar cardiovascular physiology.

These models are essential for in vivo validation but have limitations in recapitulating human disease.

CRISPR-based gene editing enables the creation of isogenic cell lines with precise genetic modifications. For atherosclerosis research, examples include:

• • LDLR knockout cell lines (e.g., in HepG2 or HUVEC) to study LDL uptake and cholesterol metabolism.
• • PCSK9 knock-in cell lines with gain-of-function mutations to investigate LDL receptor degradation.
• • APOE knockout macrophages (e.g., THP-1) to study lipid accumulation and inflammation.
• • NOS3 knockout endothelial cells to model endothelial dysfunction.

These sequence-verified models are commercially available and accelerate research by providing consistent, reproducible systems. They are essential for functional genomics and drug discovery.

Gene-edited cell lines are used to validate the role of specific genes in atherosclerosis. For example:

• • Knocking out LDLR in hepatocytes confirms its role in LDL uptake.
• • Introducing PCSK9 gain-of-function mutations demonstrates its effect on LDLR degradation.
• • Silencing inflammatory genes (e.g., IL-6) in macrophages reveals their contribution to foam cell formation.

These models allow precise loss- and gain-of-function studies.

Isogenic cell line pairs (e.g., wild-type vs. LDLR knockout) are used to screen for compounds that modulate lipid metabolism. For example:

• • Screening for PCSK9 inhibitors using cells with PCSK9 overexpression.
• • Testing anti-inflammatory drugs in TNF-α-stimulated endothelial cells.
• • Assessing drug resistance in smooth muscle cells with specific mutations.

These models improve the predictive power of in vitro screens.

CRISPR-based synthetic lethality screens can identify novel therapeutic targets. For example:

• • In macrophages, knocking out genes involved in cholesterol efflux (e.g., ABCA1) can identify compensatory pathways.
• • Genome-wide CRISPR screens in endothelial cells can reveal genes that protect against oxidative stress.

These approaches lead to biomarker discovery and new drug targets.