Establishment of an interleukin-1β-induced inflammation-activated endothelial cell-smooth muscle cell-mononuclear cell co-culture model and evaluation of the anti-inflammatory effects of tanshinone IIA on atherosclerosis

Abstract

Increasing evidence supports the hypothesis that inflammatory reactions serves an important function in the formation, progression and plaque rupture of atherosclerosis. Interleukin (IL)-1 primarily induces inflammation and is closely associated with the inflammatory environment and the formation of atherosclerosis. The present study aimed to establish an in vitro model for the evaluation of drug efficacy in the intervention of atherosclerosis from the inflammatory perspective, and to observe the anti-inflammatory effects of tanshinone IIA and andrographolide on atherosclerosis. The IL-1β-induced inflammation-activated endothelial cell (EC)-smooth muscle cell (SMC)-mononuclear cell (MC) co-culture model was established, based on the changes in a series of atherosclerosis-associated inflammatory markers secreted by ECs and SMCs. The expression of connexin in ECs, adhesion of MCs and changes in inflammatory signalling molecules were selected as evaluation indices for the inflammatory microenvironment of atherosclerosis. The use of this model revealed that tanshinone IIA exhibited significant efficacy against atherosclerosis and its inflammatory reactions. Inflammatory reactions were regarded as the primary mechanism underlying atherosclerosis. The established model simulated a series of relevant changes in the arterial wall under the inflammatory cytokines with oxidized low-density lipoprotein during the atherosclerotic process. The present study presented a reliable method for the identification of drugs with potential anti-inflammatory activity in atherosclerosis, for investigating the mechanisms of action, considering the improvement of the inflammatory state and the increase in plaque stability observed.

Figure 1

Comparison between EC and SMC monoculture and EC-SMC co-culture following culture for 3 days. (A) Fluorescence microscopy images of cell growth (BCECF-labeled; scale bar=50 µm). (a) EC monoculture, (b) EC in co-culture, (c) SMC monoculture and (d) SMC in co-culture. (B) Concentrations of the atherosclerosis-associated inflammatory markers in the supernatant of the ECs and SMCs. The data are expressed as the mean ± standard deviation of six samples for each condition from a representative experiment (^*P<0.05; ^**P<0.01; ^***P<0.001). EC, endothelial cell; SMC, smooth muscle cell; TNF, tumour necrosis factor; ET, endothelian; NO, nitric oxide; IL, interleukin; MMP, matrix metalloproteinase.

Figure 2

Comparison of the cell growth conditions and atherosclerosis-associated inflammatory markers in the cell supernatant at different time-points of EC-SMC co-culture. (A) Confocal laser scanning microscopy results. (a and e) EC and SMC growth conditions following EC-SMC co-culture for 3 days; (c and f) EC and SMC growth conditions following co-culture for 6 days; (c and g) EC and SMC growth conditions following co-culture for 9 days. (d and h) EC and SMC growth conditions following co-culture for 12 days, respectively (magnification, ×100; scale bar=50 µm). (B) Electron microscopy of the SMC growth conditions following co-culture for 3, 6, 9 and 12 days (magnification, ×1,500). The concentrations of atherosclerosis-associated inflammatory markers in the cell supernatant of the (C) ECs and (D) SMCs following EC-SMC co-culture for 1–9 days, determined using ELISA. The data are expressed as the mean ± standard deviation (^*P<0.05, vs. 1 day). EC, endothelial cell; SMC, smooth muscle cell; TNF, tumour necrosis factor; ET, endothelian; NO, nitric oxide; IL, interleukin; MMP, matrix metalloproteinase.

Figure 3

Comparison of the expression levels of atherosclerosis-associated inflammatory markers in EC-SMC co-culture, with or without MCs, oxLDL and IL-1β. (A) Confocal laser scanning microscopy images of EC-surface adhered MCs (scale bar=50 µm) and the expression of connexin 43 (scale bar=10 µm). (B) Concentrations of atherosclerosis-associated inflammatory markers in the EC supernatant in EC-SMC co-culture, with or without MCs, oxLDL and IL-1βs. The data are expressed as the mean ± standard deviation (^**P<0.01; ^***P<0.001, vs. EC-SMC-MC co-culture with oxLDL and IL-1β groups). EC, endothelial cell; SMC, smooth muscle cell; MC, monocyte; TNF, tumour necrosis factor; ET, endothelian; NO, nitric oxide; IL, interleukin; MMP, matrix metalloproteinase; MCP, monocyte chemoattractant protein; ICAM, intercellular adhesion molecule; TGF, transforming growth factor; oxLDL, oxidative low density lipoprotein; NF, nuclear factor; PPAR, peroxisome proliferator-activated receptor. (C) Expression levels of atherosclerosis-associated inflammatory markers in the SMC supernatant, with or without MCs, oxLDL and IL-1β, measured by enzyme-linked immunosorbent assay. (D) mRNA expression levels of NF-κB and PPARγ in the cells of EC layer were subjected to reverse transcription-quantitative polymerase chain reaction. The data are expressed as the mean ± standard deviation (^*P<0.05; ^**P<0.01, vs. EC-SMC-MC co-culture with oxLDL and IL-1β). EC, endothelial cell; SMC, smooth muscle cell; MC, monocyte; TNF, tumour necrosis factor; ET, endothelian; NO, nitric oxide; IL, interleukin; MMP, matrix metalloproteinase; MCP, monocyte chemoattractant protein; ICAM, intercellular adhesion molecule; TGF, transforming growth factor; oxLDL, oxidative low density lipoprotein; NF, nuclear factor; PPAR, peroxisome proliferator-activated receptor.

Figure 3

Comparison of the expression levels of atherosclerosis-associated inflammatory markers in EC-SMC co-culture, with or without MCs, oxLDL and IL-1β. (A) Confocal laser scanning microscopy images of EC-surface adhered MCs (scale bar=50 µm) and the expression of connexin 43 (scale bar=10 µm). (B) Concentrations of atherosclerosis-associated inflammatory markers in the EC supernatant in EC-SMC co-culture, with or without MCs, oxLDL and IL-1βs. The data are expressed as the mean ± standard deviation (^**P<0.01; ^***P<0.001, vs. EC-SMC-MC co-culture with oxLDL and IL-1β groups). EC, endothelial cell; SMC, smooth muscle cell; MC, monocyte; TNF, tumour necrosis factor; ET, endothelian; NO, nitric oxide; IL, interleukin; MMP, matrix metalloproteinase; MCP, monocyte chemoattractant protein; ICAM, intercellular adhesion molecule; TGF, transforming growth factor; oxLDL, oxidative low density lipoprotein; NF, nuclear factor; PPAR, peroxisome proliferator-activated receptor. (C) Expression levels of atherosclerosis-associated inflammatory markers in the SMC supernatant, with or without MCs, oxLDL and IL-1β, measured by enzyme-linked immunosorbent assay. (D) mRNA expression levels of NF-κB and PPARγ in the cells of EC layer were subjected to reverse transcription-quantitative polymerase chain reaction. The data are expressed as the mean ± standard deviation (^*P<0.05; ^**P<0.01, vs. EC-SMC-MC co-culture with oxLDL and IL-1β). EC, endothelial cell; SMC, smooth muscle cell; MC, monocyte; TNF, tumour necrosis factor; ET, endothelian; NO, nitric oxide; IL, interleukin; MMP, matrix metalloproteinase; MCP, monocyte chemoattractant protein; ICAM, intercellular adhesion molecule; TGF, transforming growth factor; oxLDL, oxidative low density lipoprotein; NF, nuclear factor; PPAR, peroxisome proliferator-activated receptor.

Figure 4

Effects of atorvastatin and indomethacin on atherosclerosis-associated inflammatory markers in the IL-1β-induced inflammation-activated EC-SMC-MC co-culture model. (A) EC-surface adhered MC count and the expression of Connexin 43. Concentrations of atherosclerosis-associated inflammatory markers in the (B) EC and (C) SMC supernatant were measured by ELISA. (D) The mRNA expression levels of NF-κB and PPARγ in cells from the EC layer were subjected to reverse transcription quantitative polymerase chain reaction. The data are expressed as the mean ± standard deviation (^*P<0.05, ^**P<0.01, ^***P<0.001, vs. Model). E, endothelial cell; S, smooth muscle cell; M, monocyte; O, oxidated low density lipoprotein; I, interleukin-1β induced; TNF, tumour necrosis factor; IL, interleukin; MMP, matrix metalloproteinase; MCP, monocyte chemoattractant protein; TGF, transforming growth factor; NF, nuclear factor; PPAR, peroxisome proliferator-activated receptor.

Figure 5

Effects of tanshinone IIA and andrographolide on atherosclerosis-associated inflammatory markers in the IL-1β-induced inflammation-activated EC-SMC-MC co-culture model. (A) EC-surface adhered MC count and the expression of Connexin 43. Concentrations of atherosclerosis-associated inflammatory markers in the (B) EC and (C) SMC supernatant were measured by ELISA. (D) The mRNA expression levels of NF-κB and PPARγ in cells from the EC layer were subjected to reverse transcription quantitative polymerase chain reaction. The data are expressed as the mean ± standard deviation (^*P<0.05, ^**P<0.01, ^***P<0.001, vs. Model). EC, endothelial cell; SMC, smooth muscle cell; MC, monocyte; TNF, tumour necrosis factor; IL, interleukin; MMP, matrix metalloproteinase; MCP, monocyte chemoattractant protein; TGF, transforming growth factor; NF, nuclear factor; PPAR, peroxisome proliferator-activated receptor.