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Effect of oxysterol-induced apoptosis of vascular smooth muscle cells on experimental hypercholesterolemia.

Perales S, Alejandre MJ, Palomino-Morales R, Torres C, Iglesias J, Linares A - J. Biomed. Biotechnol. (2009)

Bottom Line: Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl-X(L) and Bax and the expression of bcl-2 and bcl-x(L), genes.The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression.These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Smooth muscle cells (SMCs) undergo changes related to proliferation and apoptosis in the physiological remodeling of vessels and in diseases such as atherosclerosis and restenosis. Recent studies also have demonstrated the vascular cell proliferation and programmed cell death contribute to changes in vascular architecture in normal development and in disease. The present study was designed to investigate the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, using an in vivo/in vitro cell model in which SMCs were isolated and culture from chicken exposed to an atherogenic cholesterol-rich diet (SMC-Ch) and/or an antiatherogenic fish oil-rich diet (SMC-Ch-FO). Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl-X(L) and Bax and the expression of bcl-2 and bcl-x(L), genes. The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression. These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis. Our results suggest that the replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of cholesterol-induced changes in the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, making SMCs more resistant to apoptosis.

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Representative Western blots showing expression levels of Bax, Bcl-xl, and Bcl-2 on SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and changes in protein expression after treatment of SMC cultures with 20 μg/mL 25-hydroxycholesterol for 24 hours. Equal amounts of proteins were separated by electrophoresis and processed for immunoblotting. Similar results were obtained in three separate experiments. The ratios were calculated by using O.D protein band reading and normalized against a control (SMC-C) for each protein.
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fig2: Representative Western blots showing expression levels of Bax, Bcl-xl, and Bcl-2 on SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and changes in protein expression after treatment of SMC cultures with 20 μg/mL 25-hydroxycholesterol for 24 hours. Equal amounts of proteins were separated by electrophoresis and processed for immunoblotting. Similar results were obtained in three separate experiments. The ratios were calculated by using O.D protein band reading and normalized against a control (SMC-C) for each protein.

Mentions: Expression of proteins of the Bcl-2 family of oncogenes (bax, bcl-xL, and bcl-2) was studied (Figure 2). Because of the homodimerization and heterodimerization capacity of Bax, Bcl-XL, and Bcl-2, the relationship between expression levels of Bcl-2/Bax and Bcl-XL/Bax determines whether the cells will undergo apoptosis or not after an apoptotic stimulus. Table 3 shows the antiapoptotic/proapoptotic ratios at baseline (after in vivo treatments) and after 25-hydroxycholesterol in vitro treatment. At baseline, expression of Bcl-2 and Bax (Figure 2) was similar among the three culture types, but expression of Bcl-XL was higher in SMC-Ch and SMC-Ch-FO than in SMC-C. Hence, the Bcl-2/Bax ratios were very similar among cultures, whereas the Bcl-XL/Bax (Table 3) was significantly higher in SMC-Ch-FO. The SMC-Ch-FO (Table 3) showed a highly significant increase in the Bcl-XL/Bax ratio but a similar Bcl-2/Bax ratio to that of the control cells, indicating some resistance to apoptosis. Therefore, the replacement of a cholesterol-rich diet with a dish oil-rich diet not only reverses the effects of cholesterol but also protects the SMCs from apoptotic stimuli.


Effect of oxysterol-induced apoptosis of vascular smooth muscle cells on experimental hypercholesterolemia.

Perales S, Alejandre MJ, Palomino-Morales R, Torres C, Iglesias J, Linares A - J. Biomed. Biotechnol. (2009)

Representative Western blots showing expression levels of Bax, Bcl-xl, and Bcl-2 on SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and changes in protein expression after treatment of SMC cultures with 20 μg/mL 25-hydroxycholesterol for 24 hours. Equal amounts of proteins were separated by electrophoresis and processed for immunoblotting. Similar results were obtained in three separate experiments. The ratios were calculated by using O.D protein band reading and normalized against a control (SMC-C) for each protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2734998&req=5

fig2: Representative Western blots showing expression levels of Bax, Bcl-xl, and Bcl-2 on SMC cultures at baseline (SMC-C, SMC-Ch, and SMC-Ch-FO) and changes in protein expression after treatment of SMC cultures with 20 μg/mL 25-hydroxycholesterol for 24 hours. Equal amounts of proteins were separated by electrophoresis and processed for immunoblotting. Similar results were obtained in three separate experiments. The ratios were calculated by using O.D protein band reading and normalized against a control (SMC-C) for each protein.
Mentions: Expression of proteins of the Bcl-2 family of oncogenes (bax, bcl-xL, and bcl-2) was studied (Figure 2). Because of the homodimerization and heterodimerization capacity of Bax, Bcl-XL, and Bcl-2, the relationship between expression levels of Bcl-2/Bax and Bcl-XL/Bax determines whether the cells will undergo apoptosis or not after an apoptotic stimulus. Table 3 shows the antiapoptotic/proapoptotic ratios at baseline (after in vivo treatments) and after 25-hydroxycholesterol in vitro treatment. At baseline, expression of Bcl-2 and Bax (Figure 2) was similar among the three culture types, but expression of Bcl-XL was higher in SMC-Ch and SMC-Ch-FO than in SMC-C. Hence, the Bcl-2/Bax ratios were very similar among cultures, whereas the Bcl-XL/Bax (Table 3) was significantly higher in SMC-Ch-FO. The SMC-Ch-FO (Table 3) showed a highly significant increase in the Bcl-XL/Bax ratio but a similar Bcl-2/Bax ratio to that of the control cells, indicating some resistance to apoptosis. Therefore, the replacement of a cholesterol-rich diet with a dish oil-rich diet not only reverses the effects of cholesterol but also protects the SMCs from apoptotic stimuli.

Bottom Line: Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl-X(L) and Bax and the expression of bcl-2 and bcl-x(L), genes.The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression.These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Smooth muscle cells (SMCs) undergo changes related to proliferation and apoptosis in the physiological remodeling of vessels and in diseases such as atherosclerosis and restenosis. Recent studies also have demonstrated the vascular cell proliferation and programmed cell death contribute to changes in vascular architecture in normal development and in disease. The present study was designed to investigate the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, using an in vivo/in vitro cell model in which SMCs were isolated and culture from chicken exposed to an atherogenic cholesterol-rich diet (SMC-Ch) and/or an antiatherogenic fish oil-rich diet (SMC-Ch-FO). Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl-X(L) and Bax and the expression of bcl-2 and bcl-x(L), genes. The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression. These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis. Our results suggest that the replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of cholesterol-induced changes in the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, making SMCs more resistant to apoptosis.

Show MeSH
Related in: MedlinePlus