Co-culture models of endothelial cells, macrophages, and vascular smooth muscle cells for the study of the natural history of atherosclerosis

Abstract

Background This work aims to present a fast, affordable, and reproducible three-cell co-culture system that could represent the different cellular mechanisms of atherosclerosis, extending from atherogenesis to pathological intimal thickening. Methods and results We built four culture models: (i) Culture model #1 (representing normal arterial intima), where human coronary artery endothelial cells were added on top of Matrigel-coated collagen type I matrix, (ii) Culture model #2 (representing atherogenesis), which demonstrated the subendothelial accumulation and oxidative modification of low-density lipoproteins (LDL), (iii) Culture model #3 (representing intimal xanthomas), which demonstrated the monocyte adhesion to the endothelial cell monolayer, transmigration into the subendothelial space, and transformation to lipid-laden macrophages, (iv) Culture model #4 (representing pathological intimal thickening), which incorporated multiple layers of human coronary artery smooth muscle cells within the matrix. Coupling this model with different shear stress conditions revealed the effect of low shear stress on the oxidative modification of LDL and the upregulation of pro-inflammatory molecules and matrix-degrading enzymes. Using electron microscopy, immunofluorescence confocal microscopy, protein and mRNA quantification assays, we showed that the behaviors exhibited by the endothelial cells, macrophages and vascular smooth muscle cells in these models were very similar to those exhibited by these cell types in nascent and intermediate atherosclerotic plaques in humans. The preparation time of the cultures was 24 hours. Conclusion We present three-cell co-culture models of human atherosclerosis. These models have the potential to allow cost- and time-effective investigations of the mechanobiology of atherosclerosis and new anti-atherosclerotic drug therapies.

Modeling Foam Cell Formation in A Hydrogel‐Based 3D‐Intimal Model: A Study of The Role of Multi‐Diseases During Early Atherosclerosis

“…Previous findings supported such LDL transfusion and retention in the hydrogel construct. [39,51] However, those studies employed higher concentrations of LDL (4 to 40 times higher) with more prolonged incubation in their systems than ours. Although the transportation of native LDL in the subendothelial space was not depicted in our model, it is likely to pass through the intercellular junctions under high glucose or infection stimulation.…”

“…A recent study (published in January 2023), however, considered the role of vascular smooth muscle cells (VSMCs) in atherosclerosis by developing a collagen‐based coculture model with vascular and inflammatory cells to study the effects of shear stress on native LDL oxidation and pro‐inflammatory molecules released during atherosclerosis. [ 39 ] The model embedded VSMCs in the collagen construct to represent pathological intimal thickening. However, the major limitation of this model is that the total preparation time of this coculture system is only 24 h. The study seeded the HUVEC on the top of the collagen construct for 2 h and then immediately added native LDL and THP‐1 monocytes to observe LDL diffusion and monocyte migration.…”

Vascular smooth muscle cells enhance immune/vascular interplay in a 3-cell model of vascular inflammation

“…1 and 2 ). More recent 3-cell models of atherosclerosis rely on the use of non-differentiated THP1 monocytes 21 , so it is questionable whether the levels of inflammation induced in these systems are fully representative of immune/vascular interplay in native atheroma.…”

Role of triggering receptor expressed on myeloid cells-1 in the mechanotransduction signaling pathways that link low shear stress with inflammation

“…S2 . The substrate of the matrix was consisted of collagen type I mixed with layers of human coronary artery smooth muscle cells (HCASMCs) 3 . The collagen matrix was coated with a single layer of Matrigel (Corning Life Sciences, Durham, NC, USA) representing the basic membrane.…”