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Reduced beta2-glycoprotein I protects macrophages from ox-LDL-induced foam cell formation and cell apoptosis.

Wang WL, Meng ZX, Zhou SJ, Li CJ, Chen R, Lv L, Ma ZJ, Yu DM, Yu P - Lipids Health Dis (2013)

Bottom Line: Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.05;74.44 ± 5.27 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05).Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein.Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, the Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, 300070, Tianjin, China. yudemintij@126.com.

ABSTRACT

Background: Reduced beta2-glycoprotein I (beta2-GPI) is a free thiol-containing form of beta2-GPI that displays a powerful effect in protecting endothelial cells from oxidative stress-induced cell death. The present study aims to investigate the effect of beta2-GPI or reduced beta2-GPI on ox-LDL-induced foam cell formation and on cell apoptosis and to determine the possible mechanisms.

Methods: The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining and cholesterol measurement were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to detect cell apoptosis. Real-time quantitative PCR was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as CD36, SRB1, ABCA1 and ABCG1. Western blot analysis was used to detect the protein expression of certain apoptosis-related proteins, such as caspase-9, caspase-3, p38 MAPK/p-p38 MAPK and JNK/p-JNK.

Results: Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.05;74.44 ± 5.27 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05). Reduced beta2-GPI decreased the ox-LDL-induced expression of CD36 mRNA and ABCA1 mRNA (p < 0.05), as well as CD36, cleaved caspase-9, cleaved caspase-3, p-p38 MAPK and p-JNK proteins (p < 0.05 or p < 0.01). Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein.

Conclusions: Both beta2-GPI and reduced beta2-GPI inhibit ox-LDL-induced foam cell formation and cell apoptosis, and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake of macrophages by downregulating CD36 as well as protein expression. Reduced beta2-GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38 MAPK and JNK, and the amount of cleaved caspase-3 and caspase-9. Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.

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Intracellular cholesterol content of RAW264.7 macrophage. (A) The content of intracellular TC, FC and CE. (B) The ratio of CE to TC. RAW264.7 macrophages were seeded onto 96-well microtitre plates and then supplemented with ox-LDL alone or along with beta2-GPI or reduced beta2-GPI. HPLC-MS method was used to quantitatively determine the content of free cholesterol. Total protein was detected by the BCA method, and the final cholesterol content was determined using the ratio between the cholesterol concentration and the corresponding protein concentration (μg/mg protein). Ox-LDL increased the content of TC, FC, CE and the value of CE/TC. Beta2-GPI or reduced beta2-GPI decreased TC content in macrophages, whereas latter caused a larger decrease than the former. Note: n = 3, TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester. *: compared with the control group, p < 0.01; ▲: compared with the ox-LDL group, p < 0.05; ▲▲: compared with the ox-LDL group, p < 0.01; #: compared with the beta2-GPI group, p < 0.05.
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Figure 2: Intracellular cholesterol content of RAW264.7 macrophage. (A) The content of intracellular TC, FC and CE. (B) The ratio of CE to TC. RAW264.7 macrophages were seeded onto 96-well microtitre plates and then supplemented with ox-LDL alone or along with beta2-GPI or reduced beta2-GPI. HPLC-MS method was used to quantitatively determine the content of free cholesterol. Total protein was detected by the BCA method, and the final cholesterol content was determined using the ratio between the cholesterol concentration and the corresponding protein concentration (μg/mg protein). Ox-LDL increased the content of TC, FC, CE and the value of CE/TC. Beta2-GPI or reduced beta2-GPI decreased TC content in macrophages, whereas latter caused a larger decrease than the former. Note: n = 3, TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester. *: compared with the control group, p < 0.01; ▲: compared with the ox-LDL group, p < 0.05; ▲▲: compared with the ox-LDL group, p < 0.01; #: compared with the beta2-GPI group, p < 0.05.

Mentions: In the ox-LDL group, the contents of TC, FC, CE and the value of CE/TC were all higher than those contents in the control group (p < 0.01). The CE/TC value exceeded 50%. Compared with the ox-LDL group, the TC content of cells was decreased in both the ox-LDL + beta2-GPI group and in the ox-LDL + reduced beta2-GPI group (p < 0.05), whereas reduced beta2-GPI caused a larger decrease than beta2-GPI (p < 0.05) (Figure 2A,B).


Reduced beta2-glycoprotein I protects macrophages from ox-LDL-induced foam cell formation and cell apoptosis.

Wang WL, Meng ZX, Zhou SJ, Li CJ, Chen R, Lv L, Ma ZJ, Yu DM, Yu P - Lipids Health Dis (2013)

Intracellular cholesterol content of RAW264.7 macrophage. (A) The content of intracellular TC, FC and CE. (B) The ratio of CE to TC. RAW264.7 macrophages were seeded onto 96-well microtitre plates and then supplemented with ox-LDL alone or along with beta2-GPI or reduced beta2-GPI. HPLC-MS method was used to quantitatively determine the content of free cholesterol. Total protein was detected by the BCA method, and the final cholesterol content was determined using the ratio between the cholesterol concentration and the corresponding protein concentration (μg/mg protein). Ox-LDL increased the content of TC, FC, CE and the value of CE/TC. Beta2-GPI or reduced beta2-GPI decreased TC content in macrophages, whereas latter caused a larger decrease than the former. Note: n = 3, TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester. *: compared with the control group, p < 0.01; ▲: compared with the ox-LDL group, p < 0.05; ▲▲: compared with the ox-LDL group, p < 0.01; #: compared with the beta2-GPI group, p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842777&req=5

Figure 2: Intracellular cholesterol content of RAW264.7 macrophage. (A) The content of intracellular TC, FC and CE. (B) The ratio of CE to TC. RAW264.7 macrophages were seeded onto 96-well microtitre plates and then supplemented with ox-LDL alone or along with beta2-GPI or reduced beta2-GPI. HPLC-MS method was used to quantitatively determine the content of free cholesterol. Total protein was detected by the BCA method, and the final cholesterol content was determined using the ratio between the cholesterol concentration and the corresponding protein concentration (μg/mg protein). Ox-LDL increased the content of TC, FC, CE and the value of CE/TC. Beta2-GPI or reduced beta2-GPI decreased TC content in macrophages, whereas latter caused a larger decrease than the former. Note: n = 3, TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester. *: compared with the control group, p < 0.01; ▲: compared with the ox-LDL group, p < 0.05; ▲▲: compared with the ox-LDL group, p < 0.01; #: compared with the beta2-GPI group, p < 0.05.
Mentions: In the ox-LDL group, the contents of TC, FC, CE and the value of CE/TC were all higher than those contents in the control group (p < 0.01). The CE/TC value exceeded 50%. Compared with the ox-LDL group, the TC content of cells was decreased in both the ox-LDL + beta2-GPI group and in the ox-LDL + reduced beta2-GPI group (p < 0.05), whereas reduced beta2-GPI caused a larger decrease than beta2-GPI (p < 0.05) (Figure 2A,B).

Bottom Line: Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.05;74.44 ± 5.27 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05).Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein.Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, the Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, 300070, Tianjin, China. yudemintij@126.com.

ABSTRACT

Background: Reduced beta2-glycoprotein I (beta2-GPI) is a free thiol-containing form of beta2-GPI that displays a powerful effect in protecting endothelial cells from oxidative stress-induced cell death. The present study aims to investigate the effect of beta2-GPI or reduced beta2-GPI on ox-LDL-induced foam cell formation and on cell apoptosis and to determine the possible mechanisms.

Methods: The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining and cholesterol measurement were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to detect cell apoptosis. Real-time quantitative PCR was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as CD36, SRB1, ABCA1 and ABCG1. Western blot analysis was used to detect the protein expression of certain apoptosis-related proteins, such as caspase-9, caspase-3, p38 MAPK/p-p38 MAPK and JNK/p-JNK.

Results: Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.05;74.44 ± 5.27 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05). Reduced beta2-GPI decreased the ox-LDL-induced expression of CD36 mRNA and ABCA1 mRNA (p < 0.05), as well as CD36, cleaved caspase-9, cleaved caspase-3, p-p38 MAPK and p-JNK proteins (p < 0.05 or p < 0.01). Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein.

Conclusions: Both beta2-GPI and reduced beta2-GPI inhibit ox-LDL-induced foam cell formation and cell apoptosis, and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake of macrophages by downregulating CD36 as well as protein expression. Reduced beta2-GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38 MAPK and JNK, and the amount of cleaved caspase-3 and caspase-9. Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.

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