Ox-LDL increases OX40L in endothelial cells through a LOX-1-dependent mechanism

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis, and it can stimulate the expression of a variety of inflammatory signals. As a new and highly sensitive inflammation index, OX40L may be a key to understanding the mechanisms that regulate interactions between cells within the vessel wall and inflammatory mediators during the development of atherosclerosis. To investigate whether Ox-LDL regulates OX40L expression through an oxidized LDL-1 receptor (LOX-1)-mediated mechanism, we investigated the effect of different concentrations of Ox-LDL (50, 100, 150 µg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells, and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression.

Atherosclerosis; Ox-LDL; LOX-1; OX40/OX40L; Polyinosinic acid

Authorship

Q. Dong

Department of Cardiology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, ChinaChongqing Medical UniversityChinaChongqing, ChinaDepartment of Cardiology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China

R. Xiang

D.Y. Zhang

S. Qin

Correspondence: S. Qin, Department of Cardiology, The First Affiliated Hospital, Chongqing Medical University, 1 Youyi Road, Chongqing 400016, China. +86-23-8901-1429. E-mail: clb436@126.com

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Tables (2)

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Figure 1
HUVEC shape by inverted microscopy. A, Control HUVECs showing normal adherent growth with a spindle or pentagonal shape. B, HUVECs incubated with Ox-LDL (150 µg/mL) for 24 h. The cells are shrunken, with an increased rate of cell apoptosis indicated by an increased formation of apoptotic bodies. HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein.

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Figure 2
A, Cell cycle of normal HUVECs. B, HUVECs incubated with Ox-LDL (100 µg/mL) for 24 h. HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein.

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Figure 3
A, Apoptosis rates of normal HUVECs.B, HUVECs incubated with Ox-LDL (100 µg/mL) for 24 h. Rates increased from 5.26% in control to 15.69% in the Ox-LDL-treated cells. HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; PI: propidium iodide.

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Figure 4
A, OX40L expression levels in HUVECs treated with 1) no Ox-LDL (untreated control); 2) 100 µg/mL Ox-LDL, and 3) 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. B, The expression of OX40L in group 2 was increased compared with group 1, while OX40L protein expression in group 3 was decreased compared with group 2 (n=4; *P<0.05, t-test). HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

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Figure 5
Change in OX40L expression with A, no Ox-LDL;B, 100 µg/mL Ox-LDL; C, 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. The cells were observed by laser confocal microscope (bar=75 μm). Green fluorescence indicates OX40L protein. Red fluorescence indicates nucleolus. Red and green fluorescence show the location and volume of OX40L protein. D, Average fluorescence value of 8 randomly selected cells. OX40L was expressed on HUVEC membranes. OX40L protein expression in group B was increased compared to group A, while the expression of OX40L in group C was decreased compared to group B (n=4; *P<0.05, t-test). HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

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Figure 6
A, Quantification cycles of OX40L mRNA.B, OX40L expression with no Ox-LDL (Control), 100 µg/mL Ox-LDL, and 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. OX40L mRNA expression in Ox-LDL was increased compared to control while OX40L mRNA expression in Ox-LDL+Poly 1 was decreased compared to Ox-LDL (n=4; *P<0.05, t-test). Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

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Figure 7
A, LOX-1 expression levels in HUVECs treated with 1) no Ox-LDL (untreated control); 2) 100 µg/mL Ox-LDL, and 3) 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. B, The expression of LOX-1 in group 2 was increased compared to group 1. LOX-1 protein expression in group 3 was decreased compared to group 2 (n=4; *P<0.05, t-test). LOX-1: oxidized low-density lipoprotein-1 receptor; HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

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Figure 1 HUVEC shape by inverted microscopy. A, Control HUVECs showing normal adherent growth with a spindle or pentagonal shape. B, HUVECs incubated with Ox-LDL (150 µg/mL) for 24 h. The cells are shrunken, with an increased rate of cell apoptosis indicated by an increased formation of apoptotic bodies. HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein.

Figure 2 A, Cell cycle of normal HUVECs. B, HUVECs incubated with Ox-LDL (100 µg/mL) for 24 h. HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein.

Figure 3 A, Apoptosis rates of normal HUVECs.B, HUVECs incubated with Ox-LDL (100 µg/mL) for 24 h. Rates increased from 5.26% in control to 15.69% in the Ox-LDL-treated cells. HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; PI: propidium iodide.

Figure 4 A, OX40L expression levels in HUVECs treated with 1) no Ox-LDL (untreated control); 2) 100 µg/mL Ox-LDL, and 3) 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. B, The expression of OX40L in group 2 was increased compared with group 1, while OX40L protein expression in group 3 was decreased compared with group 2 (n=4; *P<0.05, t-test). HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

Figure 5 Change in OX40L expression with A, no Ox-LDL;B, 100 µg/mL Ox-LDL; C, 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. The cells were observed by laser confocal microscope (bar=75 μm). Green fluorescence indicates OX40L protein. Red fluorescence indicates nucleolus. Red and green fluorescence show the location and volume of OX40L protein. D, Average fluorescence value of 8 randomly selected cells. OX40L was expressed on HUVEC membranes. OX40L protein expression in group B was increased compared to group A, while the expression of OX40L in group C was decreased compared to group B (n=4; *P<0.05, t-test). HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

Figure 6 A, Quantification cycles of OX40L mRNA.B, OX40L expression with no Ox-LDL (Control), 100 µg/mL Ox-LDL, and 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. OX40L mRNA expression in Ox-LDL was increased compared to control while OX40L mRNA expression in Ox-LDL+Poly 1 was decreased compared to Ox-LDL (n=4; *P<0.05, t-test). Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

Figure 7 A, LOX-1 expression levels in HUVECs treated with 1) no Ox-LDL (untreated control); 2) 100 µg/mL Ox-LDL, and 3) 250 μg/μL Poly I plus 100 µg/mL Ox-LDL. B, The expression of LOX-1 in group 2 was increased compared to group 1. LOX-1 protein expression in group 3 was decreased compared to group 2 (n=4; *P<0.05, t-test). LOX-1: oxidized low-density lipoprotein-1 receptor; HUVECs: human umbilical vein endothelial cells; Ox-LDL: oxidative low-density lipoprotein; Poly I: polyinosinic acid.

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Ox-LDL increases OX40L in endothelial cells through a LOX-1-dependent mechanism

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[1] Correspondence: S. Qin, Department of Cardiology, The First Affiliated Hospital, Chongqing Medical University, 1 Youyi Road, Chongqing 400016, China. +86-23-8901-1429. E-mail: clb436@126.com