Shear stress-induced changes in atherosclerotic plaque composition are modulated by chemokines

Abstract

We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-gamma-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.

Figure 1. Representative H&E-stained cross-sections of murine carotid arteries at different time points after cast placement in mice on a Western diet.

(A) Sections from the OSS region, LSS region, and undisturbed region (control) 1, 3, and 9 weeks after cast placement. The arrows indicate accumulations of macrophages in fatty streaks (original magnification, ×200). (B) Intima/media ratio in the control regions (gray bars), the LSS regions (white bars), and the OSS regions (black bars) at the 3 different time points. *P < 0.05 versus control; ^#P < 0.05 versus LSS.

Figure 2. Validation of the expression levels of the upregulated chemokines.

MCP-1 (A), GRO-α/KC (B), IP-10 (C), and fractalkine (D) expression levels were measured by QPCR using pooled nonamplified cDNA samples (5 animals per pool; n = 3) at different time points (1, 3, and 9 weeks) and different locations (LSS, control, and OSS regions). *P < 0.05 versus control; ^#P < 0.05 versus LSS. (E) Placement of a nonconstrictive, control cast that does not alter the laminar shear stress did not induce changes in gene expression.

Figure 3. Confirmation of gene expression results of selected chemokines by immunohistological analysis of the protein levels in carotid arteries at different time points after cast placement in mice on a Western diet.

(A) Representative cross-sections of murine carotid arteries stained for fractalkine 9 weeks after cast placement and MCP-1 and IP-10 1 week after cast placement, in the control, LSS, and OSS regions (original magnification, ×200). Chemokine expression is represented by a red signal. Green autofluorescence is from the elastic laminae and demarcates the intimal and medial areas. (B) Immunohistological quantification of the chemokine signals of the LSS, OSS, and control regions is shown. n = 5. *P < 0.05 versus control; ^#P < 0.05 versus LSS.

Figure 4. Accumulation of CX3CR1⁺ cells in the different shear stress–induced mature atherosclerotic lesions 9 weeks after cast placement.

(A) Representative cross-sections of murine carotid arteries stained for CX3CR1 (red signal) and nuclei (blue signal). Arrows indicate CX3CR1⁺ cells. Asterisks indicate the position of the lumen, while the white dotted lines demarcate the boundaries between lumen and intima. (B) Immunohistological quantification of the chemokine/receptor signal in the LSS and the OSS region. n = 5. ^#P < 0.05 versus LSS.

Figure 5. CX3CR1-expressing cells located in the advanced lesions are mainly monocytes and macrophages.

Representative cross-sections of murine carotid arteries stained for CX3CR1 and CD11b (top row), CD68 (middle row), and VSMC α-actin (bottom row). In the right column, overlays show the colocalization signal in yellow. Asterisks indicate the position of the lumen, while the white dotted lines demarcate the boundaries between lumen and intima. Double staining for the identification of CX3CR1-expressing cell types was repeated in 4 different experiments.

Figure 6. Fractalkine function was inhibited during cast-induced atherogenesis in apoE^–/– mice by administration of a neutralizing antibody from week 6 until week 9.

Representative cross-sections are shown stained for: lesion morphology by H&E (A), macrophages (B), lipids (C), VSMCs (D), collagen (E), and fractalkine receptor (CX3CR1) (F). Asterisks indicate the position of the lumen, while the white dotted lines demarcate the boundaries between lumen and intima. Bar diagrams represent the lesion area and quantification of the (immuno)histological analysis of the different plaque components. The percentages of positive area at 9 weeks in the intima in the LSS and the OSS regions are indicated, comparing the control antibody group (–) with the anti-fractalkine antibody group (+). n = 8. *P < 0.05 versus control antibody group.

Figure 7. The effect of fractalkine inhibition on cap thickness and the size of the necrotic core in the LSS and OSS lesions.

Quantification of cap thickness (left) and the necrotic core (right) relative to total intimal area of the LSS and the OSS regions, comparing the control antibody group with the anti-fractalkine antibody group. n = 8. *P < 0.05 versus LSS without antibody treatment.

Figure 8. The effect of fractalkine inhibition on T cells (A) and mast cells infiltration (B) and IL-6 and c-reactive protein (CRP) expression (C) in the LSS and OSS lesions.

Left panels: representative cross-sections taken from the LSS lesions from apoE^–/– mice 9 weeks after cast placement with administration of rabbit IgG control antibodies or with the neutralizing antibody against fractalkine. Cross-sections were stained for CD3⁺ T cells (A) and mast cells using toluidine blue (B) (original magnification, ×200). CD3⁺ T cells and mast cells are indicated by arrows. Asterisks indicate the intimal area. Right panels: The number of positive cells counted in the intima (CD3⁺ T cells) and adventitia (mast cells) in the LSS and OSS lesions, with administration of rabbit IgG control antibodies or with the neutralizing antibody against fractalkine. n = 6. *P < 0.05 versus control antibody group. (C) Expression levels of CRP and IL-6 in carotid arteries at 9 weeks after cast placement in mice on a Western diet in response to fractalkine inhibition. Expression profiles were measured by QPCR using amplified RNA samples (10 animals per pool; n = 3). In the control antibody group, IL-6 and CRP levels in the LSS region were significantly upregulated versus the control and OSS regions. *P < 0.05 versus anti-fractalkine antibody group.

Figure 9. Fractalkine function was inhibited during atherosclerosis development in the BCAs in apoE^–/– mice on a Western diet by administration of a neutralizing antibody from week 6 until week 9.

Representative cross-sections are shown stained for: collagen (upper panels), lipids (middle panels), and macrophages (lower panels). Asterisks indicate the position of the atherosclerotic lesions. Bar diagrams represent quantification of the (immuno-)histological analysis of the different plaque components. The percentages of positive area at time point 9 weeks in the intima of the BCAs are indicated, comparing the control antibody group with the fractalkine antibody group. n = 6. *P < 0.05 versus control antibody group.