Introduction.β-Carotene-rich diet has been shown to be inversely associated with the risk of coronary heart disease. However, clinical trials using synthetic all-trans-β-carotene failed to demonstrate a beneficial effect. We therefore sought to study the effect of natural source ofβ-carotene, the algaDunaliella, containing both all-trans and 9-cis-β-carotene on atherosclerosis. In a previous study we showed that 9-cis-β-carotene-rich powder of the algaDunaliellainhibits early atherogenesis in low-density lipoprotein receptor knockout mice.Aims. The aims of the current work were to study whether diet enriched withDunaliellapowder would inhibit the progression of established atherosclerosis in old male apoE-deficient mice and to compare the effect ofDunaliellaon lipid profile and atherosclerosis in a low-versus high-fat diet fed mice.Methods. In the first experiment, young mice (12 weeks old) were allocated into 3 groups: (1) low-fat diet; (2) low-fat diet + Dunaliellapowder (8%); (3) low-fat diet + β-carotene-deficientDunaliella. In the second experiment, old mice (7 months old) with established atherosclerotic lesions were allocated into 4 groups: (1) low-fat diet; (2) low-fat diet + Dunaliella; (3) high fat-diet; (4) high-fat diet + Dunaliella.Results. In young mice fed a low-fat diet, a trend toward lower atherosclerotic lesion area in the aortic sinus was found in theDunaliellagroup compared with the control group. In old mice with established atherosclerotic lesion,Dunaliellainhibited significantly plasma cholesterol elevation and atherosclerosis progression in mice fed a high-fat diet.Conclusion. The results of this study suggest that a diet containing natural carotenoids, rich in 9-cis-β-carotene, has the potential to inhibit atherosclerosis progression, particularly in high-fat diet regime.

Vitamin A is involved in regulation of glucose concentrations, lipid metabolism, and inflammation, which are major risk factors for atherogenesis. However, the effect of vitamin A deficiency on atherogenesis has not been investigated. Therefore, the objective of the current study was to examine whether vitamin A deficiency accelerates atherogenesis in apolipoprotein E-deficient mice (apoE^−/−). ApoE^−/−mice were allocated into the following groups: control, fed vitamin A-containing chow diet; BC, fed chow diet fortified withDunaliellapowder containingβc isomers; VAD, fed vitamin A-deficient diet; and VAD-BC group, fed vitamin A-deficient diet fortified with aDunaliellapowder. Following 15 weeks of treatment, liver retinol concentration had decreased significantly in the VAD group to about 30% that of control group. Vitamin A-deficient diet significantly increased both plasma cholesterol concentrations and the atherosclerotic lesion area at the aortic sinus (+61%) compared to the control group. Dietaryβc fortification inhibited the elevation in plasma cholesterol and retarded atherogenesis in mice fed the vitamin A-deficient diet. The results imply that dietary vitamin A deficiency should be examined as a risk factor for atherosclerosis and that dietaryβc, as a sole source of retinoids, can compensate for vitamin A deficiency.

Cholesterol efflux from macrophages is a key process in reverse cholesterol transport and, therefore, might inhibit atherogenesis. 9-cis-β-carotene (9-cis-βc) is a precursor for 9-cis-retinoic-acid (9-cis-RA), which regulates macrophage cholesterol efflux. Our objective was to assess whether 9-cis-βc increases macrophage cholesterol efflux and induces the expression of cholesterol transporters. Enrichment of a mouse diet with βc from the alga Dunaliella led to βc accumulation in peritoneal macrophages. 9-cis-βc increased the mRNA levels of CYP26B1, an enzyme that regulates RA cellular levels, indicating the formation of RA from βc in RAW264.7 macrophages. Furthermore, 9-cis-βc, as well as all-trans-βc, significantly increased cholesterol efflux to high-density lipoprotein (HDL) by 50% in RAW264.7 macrophages. Likewise, food fortification with 9-cis-βc augmented cholesterol efflux from macrophages ex vivo. 9-cis-βc increased both the mRNA and protein levels of ABCA1 and apolipoprotein E (APOE) and the mRNA level of ABCG1. Our study shows, for the first time, that 9-cis-βc from the diet accumulates in peritoneal macrophages and increases cholesterol efflux to HDL. These effects might be ascribed to transcriptional induction of ABCA1, ABCG1, and APOE. These results highlight the beneficial effect of βc in inhibition of atherosclerosis by improving cholesterol efflux from macrophages.

Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15’-monooxygenase (BCMO1) is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis.