Inhibition of c-Jun N-terminal kinase attenuates low shear stress-induced atherogenesis in apolipoprotein E-deficient mice

Abstract

Atherosclerosis begins as local inflammation of arterial walls at sites of disturbed flow, such as vessel curvatures and bifurcations with low shear stress. c-Jun NH₂-terminal kinase (JNK) is a major regulator of flow-dependent gene expression in endothelial cells in atherosclerosis. However, little is known about the in vivo role of JNK in low shear stress in atherosclerosis. We aimed to observe the effect of JNK on low shear stress-induced atherogenesis in apolipoprotein E-deficient (ApoE(-/-)) mice and investigate the potential mechanism in human umbilical vein endothelial cells (HUVECs). We divided 84 male ApoE(-/-) mice into two groups for treatment with normal saline (NS) (n = 42) and JNK inhibitor SP600125 (JNK-I) (n = 42). Perivascular shear stress modifiers were placed around the right carotid arteries, and plaque formation was studied at low shear stress regions. The left carotid arteries without modifiers represented undisturbed shear stress as a control. The NS group showed atherosclerotic lesions in arterial regions with low shear stress, whereas the JNK-I group showed almost no atherosclerotic lesions. Corresponding to the expression of proatherogenic vascular cell adhesion molecule 1 (VCAM-1), phospho-JNK (p-JNK) level was higher in low shear stress regions with NS than with JNK-I inhibitor. In HUVECs under low shear stress, siRNA knockdown and SP600125 inhibition of JNK attenuated nuclear factor (NF)-κB activity and VCAM-1 expression. Furthermore, siRNA knockdown of platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31) reduced p-JNK and VCAM-1 levels after low shear stress stimulation. JNK may play a critical role in low shear stress-induced atherogenesis by a PECAM-1-dependent mechanosensory pathway and modulating NF-κB activity and VCAM-1 expression.

Figure 1

Morphology of carotid artery arteriosclerotic plaque and the efficiency of SP600125 to inhibit JNK at 10 wks after cast placement in ApoE^−/− mice. (A) Representative images from low and undisturbed flow regions in two groups (hematoxylin and eosin staining; 200×). Bar = 200 μm. (B) Quantification of the intima-media ratio obtained by measuring the area confined by the external elastic lamina of the instrumented carotid artery in NS and JNK-I mice. (C) Western blot analysis of JNK protein expression (including p-JNK and t-JNK) for two groups of mice. (D) The activity of JNK (ratio of p-JNK to t-JNK) in different groups. All data are mean ± SD of sections from 15 different animals. undist, undisturbed shear stress; low, low shear stress. ^▴P < 0.05 versus undist regions in NS group. *P < 0.05 versus low regions in NS group.

Figure 2

Relative VCAM-1 protein expression in different carotid artery regions. (A) Immuno-fluorescence analyses of VCAM-1 expression within vascular endothelium including primary and secondary antibody controls. Representative images acquired by laser scanning confocal microscopy (400×) showed vWF staining (green) of intact endothelium, VCAM-1 staining (red) and DAPI staining (blue) for cell nuclei. Bar = 100 μm; arrows denote the ECs. (B) Relative quantification of VCAM-1 expression in low and undisturbed (undist) regions by measuring the integrated optical density (IOD) of positive red area normalized to undisturbed flow region of the NS group. Western blot analysis of VCAM-1 in different regions (C) and quantification of protein expression of VCAM-1 relative to β-actin (D). Data are mean ± SD from six animals from low flow regions from experiments performed in triplicate. *P < 0.05 versus low regions in NS the group. ^▴P < 0.05 versus undisturbed regions in the NS group.

Figure 3

Relative levels of VCAM-1 with low shear stress (4 dyn/cm²) at various times in HUVECs. (A) RT-PCR results of VCAM-1 mRNA expression relative to β-actin. (B) Western blot analysis of VCAM-1 and β-actin protein expression in different groups. (C) Quantification of VCAM-1 protein expression. Control represents static (0 h). *P < 0.05 versus control.

Figure 4

Western blot and immunofluorescence analysis of the active JNK (p-JNK) and NF-κB (p-p65) expression induced by low shear stress for 12 h in HUVECs. (A) Western blot analysis of JNK protein expression (p-JNK and t-JNK) and (B) active NF-κB (p-p65) of control and low shear stress groups. (C) Quantification of activity of JNK (p-JNK/t-JNK) before and after low shear stress stimulation. (D) Quantification of p-p65 relative to β-actin before and after low shear stress stimulation. (E) Representative immunofluorescence images by laser scanning microscopy showing p-JNK staining (green) of HUVECs before and after low shear stress for 12 h. (F) Representative immunofluorescence images showing p-p65 staining (green) of HUVECs. DAPI blue staining of cell nuclei is shown. Bar = 20 μm. (G) Quantification of relative p-JNK level by measuring the positive green area of two groups. (H) Quantification of p-p65 relative level to control. All relative expressions were normalized to the control group. *P < 0.05 versus control.

Figure 5

The efficiency of siRNA in knockdown of CD31 and JNK in HUVECs. (A) Western blot image of CD31 expression and β-actin with or without si-CD31 or low shear stress pretreatment. (B) Relative quantitation of CD31 normalized to control of static group in different groups. (C) Western blot image of t-JNK expression after si-CD31 or low shear stress treatments. (D) Quantitation of relative JNK level normalized to control of static group. Each data point was obtained from triplicate experiments. ^▴P < 0.05 versus static control. ^•P < 0.05 versus low shear stress control.

Figure 6

Activities of JNK (p-JNK) detected by Western blot and immunofluorescence analysis in HUVECs transfected with or without si-CD31 and si-Neg before and after low shear stress treatment. (A) Western blot analysis of JNK expression (p-JNK and t-JNK) in HUVECs with different stimulations. (B) Representative immunofluorescence images showing p-JNK staining (green). DAPI blue staining of cell nuclei is shown. Bar = 20 μm. (C) Quantitation of JNK (p-JNK/t-JNK) in different groups by Western blot analysis. (D) Quantitation of relative p-JNK level (IOD) normalized to control of static group. *P < 0.05 versus static group. ^▴P < 0.05 versus control of static group. ^•;P < 0.05 versus control of low shear stress group.

Figure 7

Activity of NF-κB (p-p65) and VCAM-1 expression detected by immunofluorescence and Western blot analysis with or without downregulation of JNK before and after low shear stress treatment. (A) Representative immunofluorescence images of p-p65 expression staining (green) in HUVECs with different stimulations. DAPI blue staining of cell nuclei. Bar = 20 μm. (B) Activity of p-p65 in different groups detected by immunofluorescence (IOD) was normalized to control of static group. (C) Western blot analysis of p-p65 and VCAM-1 expression of different groups. Quantitation of p-p65 level (D) and VCAM-1 level relative to β-actin (E). SP, SP600125; si-Neg was control for si-JNK. *P < 0.05 versus static group. ^•P < 0.05 versus control within low group.