A Unique Combination of Nutritionally Active Ingredients Can Prevent Several Key Processes Associated with Atherosclerosis In Vitro

Abstract

Introduction: Atherosclerosis is the underlying cause of cardiovascular disease that leads to more global mortalities each year than any other ailment. Consumption of active food ingredients such as phytosterols, omega-3 polyunsaturated fatty acids and flavanols are known to impart beneficial effects on cardiovascular disease although the combined actions of such agents in atherosclerosis is poorly understood. The aim of this study was to screen a nutritional supplement containing each of these active components for its anti-atherosclerotic effect on macrophages in vitro.

Results: The supplement attenuated the expression of intercellular adhesion molecule-1 and macrophage chemoattractant protein-1 in human and murine macrophages at physiologically relevant doses. The migratory capacity of human monocytes was also hindered, possibly mediated by eicosapentaenoic acid and catechin, while the ability of foam cells to efflux cholesterol was improved. The polarisation of murine macrophages towards a pro-inflammatory phenotype was also attenuated by the supplement.

Conclusion: The formulation was able to hinder multiple key steps of atherosclerosis development in vitro by inhibiting monocyte recruitment, foam cell formation and macrophage polarisation towards an inflammatory phenotype. This is the first time a combination these ingredients has been shown to elicit such effects and supports its further study in preclinical in vivo models.

Fig 1. Physiologically relevant doses of the formulation have no detrimental effect on human macrophage viability or proliferation.

Cell viability (a) or proliferation (b) was assessed in PMA differentiated THP-1 macrophages that were treated with vehicle (vehicle control) or various doses of the complete formulation (+PS) for 24 hours. Data were normalised to the vehicle control that has been arbitrarily assigned as 100%. The data are presented as the mean ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett T3 post-hoc analysis.

Fig 2. Physiologically relevant doses of the formulation can inhibit the IFN-γ induced expression of MCP-1 and ICAM-1 in human macrophages.

Gene transcript levels of MCP-1 (a and c) and ICAM-1 (b, d and e) were assessed in PMA differentiated THP-1 macrophages (a, b, c and d) or HMDM (e) that were either treated with vehicle (vehicle control) or with IFN-γ (250 U/ml) or IFN-γ (250 U/ml) in the presence of the complete formulation (+PS; a, b and e) or with IFN-γ (250 U/ml) in the presence of the formulation lacking PS (-PS; c, d and e) for 3 hours. Gene transcript levels were calculated using the comparative Ct method and normalised to GAPDH levels with values from vehicle treated cells given an arbitrary value of 1. The data are presented as the mean ± SEM from three (a, b, c and d) or four (e) independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett T3 post-hoc analysis on log-transformed data where * p <0.05, ** p <0.01 and *** p <0.001.

Fig 3. A physiologically relevant dose of the formulation can inhibit MCP-1 induced migration of human monocytes.

Cellular migration was assessed using THP-1 monocytes that were treated with vehicle (vehicle control) or treated with MCP-1 (20 ng/ml) alone or with MCP-1 (20 ng/ml) in the presence the complete formulation (+PS) or with MCP-1 (20ng/ml) in the presence the formulation lacking PS (-PS) for 3 hours. Monocyte migration is expressed as a fold-change compared to the proportion of cells that moved from the apical compartment into the basolateral compartment in response to MCP-1 alone that has been arbitrarily set as 1. The data are presented as the mean ± SEM from four independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett T3 post-hoc analysis where ** p <0.01 and *** p <0.001.

Fig 4. A physiologically relevant dose of the formulation can induce cholesterol efflux from human macrophage foam cells.

Cholesterol efflux was assessed in 24 hour [4-¹⁴C]cholesterol loaded PMA-differentiated THP-1 cells in the presence of AcLDL that were treated with ApoA-I (10 μg/ml) in the presence of vehicle or ApoA-I in the presence of the complete formulation (+PS) or ApoA-I in the presence of the formulation lacking PS (-PS) for 24 hours. Data were normalised to the ApoA-I, vehicle treated sample that has been arbitrarily assigned as 1. The data are presented as the mean ± SEM from three independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett T3 post-hoc analysis where ** p <0.01.

Fig 5. A physiologically relevant dose of the formulation can hinder M1 polarisation in murine macrophages.

Gene transcript levels of iNOS (a) and Arg2 (b) were assessed in Raw264.7 murine macrophages that were treated with either vehicle (vehicle control); with IFN-γ (250 U/ml) and LPS (100 ng/ml); with IFN-γ (250 U/ml) and LPS (100 ng/ml) in the presence of the complete formulation (+PS) or with IFN-γ (250 U/ml) and LPS (100 ng/ml) in the presence of the formulation lacking PS (-PS) for 24 hours. Gene transcript levels were calculated using the comparative Ct method and normalised to β-actin levels with the values from IFN-γ and LPS treated cells given an arbitrary value of 1. The data are presented as the mean ± SEM from five independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett T3 post-hoc analysis on square root-transformed data where * p <0.05, ** p <0.01 and *** p <0.001.

Fig 6. Major bioactive components associated with the complete formulation can inhibit MCP-1 induced migration of human monocytes.

Cellular migration was assessed using THP-1 monocytes that were treated with either vehicle (vehicle control) or with MCP-1 (20 ng/ml) or with MCP-1 (20 ng/ml) in the presence of EPA (30 μg/ml), DHA (19.6 μg/ml), (+)-catechin (1.45 μg/ml), stigmasterol (SS, 10 μg/ml), campsterol (CS, 13.9 μg/ml) or β-sitosterol (β-SS, 27.2 μg/ml) for 3 hours. Monocyte migration is expressed as a fold-change compared to the proportion of cells that moved from the apical compartment into the basolateral compartment in response to MCP-1 alone that has been arbitrarily set as 1. The data are presented as the mean ± SEM from four independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett post-hoc analysis on Log transformed data where * p <0.05, ** p <0.01 and *** p <0.001.