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Fish oil supplementation reverses the effect of cholesterol on apoptotic gene expression in smooth muscle cells.

Perales S, Alejandre MJ, Morales RP, Torres C, Linares A - Lipids Health Dis (2010)

Bottom Line: Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions.The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures.Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Sciences, Campus Universitario de Fuentenueva Avenida Severo Ochoa s/n 18071 University of Granada, Spain.

ABSTRACT

Background: Nutritional control of gene regulation guides the transformation of smooth muscle cells (SMC) into foam cells in atherosclerosis. Oxidative stress has been reported in areas of lipid accumulation, activating proliferation genes. Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions. We hypothesized that fish oil consumption may protect against atherosclerotic vascular disease. The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures.

Methods: An in vivo/in vitro cell model was used, culturing SMC isolated from chicks exposed to an atherogenic cholesterol-rich diet with 5% of cholesterol (SMC-Ch) alone or followed by an anti-atherogenic fish oil-rich diet with 10% of menhaden oil (SMC-Ch-FO) and from chicks on standard diet (SMC-C). Cells were exposed to 25-HC, studying apoptosis levels by flow cytometry (Annexin V) and expressions of caspase-3, c-myc, and p53 genes by quantitative real-time reverse transcriptase-polymerase chain reaction.

Results: Exposure to 25-HC produced apoptosis in all three SMC cultures, which was mediated by increases in caspase-3, c-myc, and p53 gene expression. Changes were more marked in SMC-Ch than in SMC-C, indicating that dietary cholesterol makes SMC more susceptible to 25-HC-mediated apoptosis. Expression of p53 gene was elevated in SMC-Ch-FO. This supports the proposition that endogenous levels of p53 protect SMC against apoptosis and possibly against the development of atherosclerosis. Fish oil attenuated the increase in c-myc levels observed in SMC-C and SMC-Ch, possibly through its influence on the expression of antioxidant genes.

Conclusion: Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis.

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Apoptosis induced by 25-Hidroxicholesterol in the cell culture model. Cells were labeled with Annexin V-FITC and PI (propidiun iodide), and apoptosis was assessed by flow cytometry. A, Representative scatter plots of PI (y-axis) versus Annexin V-FITC (x-axis). SMC-C (1), SMC-Ch (2), SMC-Ch-FO (3), SMC-C treated with 20 μg/ml 25-hydroxycholesterol (25-HC) for 24 h (4), SMC-Ch treated with 20 μg/ml 25-HC for 24 h (5), and SMC-Ch-FO treated with 20 μg/ml 25-HC for 24 h (6). Absence of both markers (lower left quadrants) indicates viable cells; PI positive alone (upper left quadrants) indicates cell necrosis, whereas Annexin V staining alone or together with PI (upper right and lower right quadrants) indicates of early- and late-stage apoptosis, respectively. B, Summary data of three experiments showing the % of annexin-V positive cells (upper right and lower right quadrants) in SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and after 24-h treatment in vitro with 20 μg/ml 25-HC for 24 h. *P < 0.05, ** P < 0.01, *** P < 0.001 vs. SMC-C, +P < 0.05, ++P < 0.01, +++ P < 0.001 vs. SMC-C treated with 20 μg/ml 25-HC for 24 h.
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Figure 1: Apoptosis induced by 25-Hidroxicholesterol in the cell culture model. Cells were labeled with Annexin V-FITC and PI (propidiun iodide), and apoptosis was assessed by flow cytometry. A, Representative scatter plots of PI (y-axis) versus Annexin V-FITC (x-axis). SMC-C (1), SMC-Ch (2), SMC-Ch-FO (3), SMC-C treated with 20 μg/ml 25-hydroxycholesterol (25-HC) for 24 h (4), SMC-Ch treated with 20 μg/ml 25-HC for 24 h (5), and SMC-Ch-FO treated with 20 μg/ml 25-HC for 24 h (6). Absence of both markers (lower left quadrants) indicates viable cells; PI positive alone (upper left quadrants) indicates cell necrosis, whereas Annexin V staining alone or together with PI (upper right and lower right quadrants) indicates of early- and late-stage apoptosis, respectively. B, Summary data of three experiments showing the % of annexin-V positive cells (upper right and lower right quadrants) in SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and after 24-h treatment in vitro with 20 μg/ml 25-HC for 24 h. *P < 0.05, ** P < 0.01, *** P < 0.001 vs. SMC-C, +P < 0.05, ++P < 0.01, +++ P < 0.001 vs. SMC-C treated with 20 μg/ml 25-HC for 24 h.

Mentions: The percentage apoptosis was quantified in the three types of SMC cultures (C, Ch, and Ch-FO) after in vivo treatments (baseline) and after in vitro treatments. Figure 1 depicts the flow cytometry results and the percentage of annexin V-FITC-positive cells; the percentages in the upper right and lower left quadrants of the Figure represent the SMC in early and late apoptosis at baseline and after 24-h incubation with 20 μg/ml 25-hydroxycholesterol, respectively. At baseline, the SMC showed a very low level of apoptosis (about 4%)


Fish oil supplementation reverses the effect of cholesterol on apoptotic gene expression in smooth muscle cells.

Perales S, Alejandre MJ, Morales RP, Torres C, Linares A - Lipids Health Dis (2010)

Apoptosis induced by 25-Hidroxicholesterol in the cell culture model. Cells were labeled with Annexin V-FITC and PI (propidiun iodide), and apoptosis was assessed by flow cytometry. A, Representative scatter plots of PI (y-axis) versus Annexin V-FITC (x-axis). SMC-C (1), SMC-Ch (2), SMC-Ch-FO (3), SMC-C treated with 20 μg/ml 25-hydroxycholesterol (25-HC) for 24 h (4), SMC-Ch treated with 20 μg/ml 25-HC for 24 h (5), and SMC-Ch-FO treated with 20 μg/ml 25-HC for 24 h (6). Absence of both markers (lower left quadrants) indicates viable cells; PI positive alone (upper left quadrants) indicates cell necrosis, whereas Annexin V staining alone or together with PI (upper right and lower right quadrants) indicates of early- and late-stage apoptosis, respectively. B, Summary data of three experiments showing the % of annexin-V positive cells (upper right and lower right quadrants) in SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and after 24-h treatment in vitro with 20 μg/ml 25-HC for 24 h. *P < 0.05, ** P < 0.01, *** P < 0.001 vs. SMC-C, +P < 0.05, ++P < 0.01, +++ P < 0.001 vs. SMC-C treated with 20 μg/ml 25-HC for 24 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914009&req=5

Figure 1: Apoptosis induced by 25-Hidroxicholesterol in the cell culture model. Cells were labeled with Annexin V-FITC and PI (propidiun iodide), and apoptosis was assessed by flow cytometry. A, Representative scatter plots of PI (y-axis) versus Annexin V-FITC (x-axis). SMC-C (1), SMC-Ch (2), SMC-Ch-FO (3), SMC-C treated with 20 μg/ml 25-hydroxycholesterol (25-HC) for 24 h (4), SMC-Ch treated with 20 μg/ml 25-HC for 24 h (5), and SMC-Ch-FO treated with 20 μg/ml 25-HC for 24 h (6). Absence of both markers (lower left quadrants) indicates viable cells; PI positive alone (upper left quadrants) indicates cell necrosis, whereas Annexin V staining alone or together with PI (upper right and lower right quadrants) indicates of early- and late-stage apoptosis, respectively. B, Summary data of three experiments showing the % of annexin-V positive cells (upper right and lower right quadrants) in SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and after 24-h treatment in vitro with 20 μg/ml 25-HC for 24 h. *P < 0.05, ** P < 0.01, *** P < 0.001 vs. SMC-C, +P < 0.05, ++P < 0.01, +++ P < 0.001 vs. SMC-C treated with 20 μg/ml 25-HC for 24 h.
Mentions: The percentage apoptosis was quantified in the three types of SMC cultures (C, Ch, and Ch-FO) after in vivo treatments (baseline) and after in vitro treatments. Figure 1 depicts the flow cytometry results and the percentage of annexin V-FITC-positive cells; the percentages in the upper right and lower left quadrants of the Figure represent the SMC in early and late apoptosis at baseline and after 24-h incubation with 20 μg/ml 25-hydroxycholesterol, respectively. At baseline, the SMC showed a very low level of apoptosis (about 4%)

Bottom Line: Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions.The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures.Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Sciences, Campus Universitario de Fuentenueva Avenida Severo Ochoa s/n 18071 University of Granada, Spain.

ABSTRACT

Background: Nutritional control of gene regulation guides the transformation of smooth muscle cells (SMC) into foam cells in atherosclerosis. Oxidative stress has been reported in areas of lipid accumulation, activating proliferation genes. Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions. We hypothesized that fish oil consumption may protect against atherosclerotic vascular disease. The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures.

Methods: An in vivo/in vitro cell model was used, culturing SMC isolated from chicks exposed to an atherogenic cholesterol-rich diet with 5% of cholesterol (SMC-Ch) alone or followed by an anti-atherogenic fish oil-rich diet with 10% of menhaden oil (SMC-Ch-FO) and from chicks on standard diet (SMC-C). Cells were exposed to 25-HC, studying apoptosis levels by flow cytometry (Annexin V) and expressions of caspase-3, c-myc, and p53 genes by quantitative real-time reverse transcriptase-polymerase chain reaction.

Results: Exposure to 25-HC produced apoptosis in all three SMC cultures, which was mediated by increases in caspase-3, c-myc, and p53 gene expression. Changes were more marked in SMC-Ch than in SMC-C, indicating that dietary cholesterol makes SMC more susceptible to 25-HC-mediated apoptosis. Expression of p53 gene was elevated in SMC-Ch-FO. This supports the proposition that endogenous levels of p53 protect SMC against apoptosis and possibly against the development of atherosclerosis. Fish oil attenuated the increase in c-myc levels observed in SMC-C and SMC-Ch, possibly through its influence on the expression of antioxidant genes.

Conclusion: Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis.

Show MeSH
Related in: MedlinePlus