Fig 1.
Graphical illustration of the co-culture models and study design.
HCAECs: human coronary artery endothelial cells; HCASMCs: human coronary artery smooth muscle cells; LDL: low-density lipoproteins.
Fig 2.
Culture model #1 representing normal arterial intima.
A: Schematic illustration: Normal HCAECs cultured on top of Matrigel-coated collagen matrix, B and C: Scanning electron microscopic images showing the ECs monolayer with gap junctions and the underlying fibers of collagen matrix, D and E: Transmission electron microscopic images showing vertical section of the cultures F and G: 3D image of the culture by immunofluorescence confocal microscopy showing the endothelial cell monolayer (blue) connected by VE-cadherin (red) at the gap junctions (ECs: endothelial cells; gj: gap junctions).
Fig 3.
Co-culture model #2 representing atherogenesis.
A: Schematic illustration: Addition LDL particles on the monolayer of the HCAECs cultured on top of Matrigel-coated collagen matrix, B: Scanning electron microscopic images, C and D: Transmission electron microscopic images. Note the presence of lipid droplets within the ECs cytoplasm, E, F and G: 3D image of the co-culture by non-immunofluorescence confocal microscopy, showing the transportation of the LDL particles (red) from the medium to the subendothelial space through the pre-stained HCAECs monolayer (green and blue), H: Presence of oxidized LDL in the medium in a native LDL concentration-dependent manner (EC: endothelial cells; gj: gap junctions; Mit: mitochondria; L: low-density lipoproteins; LS: lysosome). *p<0.05, **p< 0.01.
Fig 4.
Co-culture model #3 representing intimal xanthomas.
A: Schematic illustration: Addition of native LDL and monocytes to the monolayer of HCAECs cultured on top of Matrigel-coated collagen matrix, B-D: Scanning electron microscopic images showing the characteristic membrane changes of monocytes adhering on the ECs (B, C), and transmigrating through the gap junctions into the subendothelial space (D), E and F: Magnified transmission electron microscopic images showing the characteristic appearance of subendothelial macrophage with eccentric nucleus, villous plasma projections (white asterisks) and lipid droplets, G-I: Non-immunofluorescence confocal microscopy with pre-stained monocytes (green). Note the presence of LDL-laden macrophages in the matrix (I), J: Presence of oxidized LDL in the medium in a native LDL concentration-dependent manner (EC: endothelial cells; gj: gap junctions; L: low-density lipoproteins; Mφ: macrophages; N: nucleus). **p< 0.01.
Fig 5.
Co-culture model #4 representing pathological intimal thickening.
A: Schematic illustration: Addition of LDL and monocytes on top of HCAECs cultured on Matrigel-coated matrix containing layers of HCASMCs, B and C: Scanning electron microscopic images showing the transportation stages of monocytes: Monocytes adhering on the ECs (B), transmigrating monocytes through gap junctions (C), and transmigrant monocytes (white asterisks in B), D-F: Transmission electron microscopic images showing monocytes adhering on the ECs (D), and layers of spindle-shaped VSMCs in the collagen matrix (E) with characteristic surface connections with neighboring collagen fibers (black arrows in F), G-I: 3D images of the co-culture by non- immunofluorescence confocal microscopy showing the monolayer of ECs on top of the matrix, LDL (red) and pre-stained monocytes (green) on top of the ECs, LDL-laden macrophages in the matrix (merged green and red), and pre-stained spindle-shaped smooth muscle cells (green), J: High magnification of immunofluorescence confocal microscopy showing α-smooth muscle actin stained VSMCs (green), K: Immunoblotting of cell-specific markers in HCAECs and HCASMCs, isolated from the co-cultures by magnetic beads. Note the expression of CD31 predominantly by the HCAECs and α-smooth muscle actin predominantly by HCASMCs, L: Presence of oxidized LDL in the medium and matrix in a native LDL concentration-dependent manner (EC: endothelial cells; N: nucleus; L: low-density lipoprotein; gj: gap junctions; Mφ: macrophages; VSMC: vascular smooth muscle cells; SMA: α-smooth muscle actin). ***p<0.001.
Fig 6.
Differentiation of monocytes into lipid-laden macrophage cells in the collagen matrix.
A: 3D confocal immunofluorescence image of monocytes mixed within collagen matrix and cultured for 24 h, showing their transformation into CD68-positive macrophages (green), B: 3D confocal immunofluorescence image of monocytes cultured within EC-plated collagen matrix followed by the addition of Dil-labelled LDL (red). Note the transformation of monocytes into LDL-laden, CD68-positive macrophages (white arrow; merged green and red; Mφ: macrophages; LDL: low-density lipoproteins), C: 3D confocal non-immunofluorescence (monocytes and SMCs were pre-stained with calcein AM) image of monocytes/macrophages and SMCs cultured within EC-plated collagen matrix followed by the addition of Dil-labelled ox-LDL (red). Note the co-localization of ox-LDL with SMCs and monocytes/macrophages (M: monocytes; Mφ: macrophages; SMC: smooth muscle cells.
Table 1.
Comparison of preparation time of three-cell co-culture models of atherosclerosis.
Fig 7.
Effects of shear stress and native LDL on the release of oxidized LDL, expression of pro-inflammatory molecules, and matrix-degrading enzymes.
A: Low shear stress significantly increased the expression of oxidized LDL in the co-culture medium of model #4, compared to high shear stress or static controls, B-F: Similarly, expression of MCP-1, IL-1ß, IL-6, cathepsin L, and MMP-1 was increased in cultures exposed to low shear stress, compared to high shear stress or static controls (white bars). Notably, addition of LDL in the medium significantly augmented the expression of oxidized LDL, MCP-1, IL-1ß, IL-6, cathepsin L, and MMP-1 (black bars; LDL: low-density lipoproteins; MCP-1: monocyte chemoattractant protein 1; IL: interleukin; MMP: matrix metalloproteinases) *p<0.05; **p<0.01; ***p<0.001.
Fig 8.
Effects of low shear stress and native LDL on oxidized-LDL and expression of pro-inflammatory molecules and matrix-degrading enzymes.
Dose-response effect of native (non-oxidized) LDL on the quantities of oxidized LDL (A), MCP-1, IL-1ß, IL-6, IL-8, MMP-1 and -9, and cathepsins L and S (B-I), released into the medium of co-culture model #4 (white bars). Note that the pro-atherosclerotic effect of native LDL is significantly augmented by exposing the co-cultures to low shear stress (5±3 dynes/cm2) for 1 hour, prior to the addition of native LDL (black bars; ox-LDL: oxidized low-density lipoproteins, MCP: monocyte chemoattractant protein; IL: interleukin; MMP: matrix metalloproteinases) * p<0.05; **p < 0.01; ***p<0.001.
Table 2.
Qualitative comparison of two- and three-cell co-culture models of atherosclerosis.