Circulating CCL20 as a New Biomarker of Abdominal Aortic Aneurysm

Abstract

Autoimmunity appears to play a role in abdominal aortic aneurysm (AAA) pathology. Although the chemokine CCL20 has been involved in autoimmune diseases, its relationship with the pathogenesis of AAA is unclear. We investigated CCL20 expression in AAA and evaluated it as a potential biomarker for AAA. CCL20 was measured in plasma of AAA patients (n = 96), atherosclerotic disease (AD) patients (n = 28) and controls (n = 45). AAA presence was associated with higher plasma levels of CCL20 after adjustments for confounders in the linear regression analysis. Diagnostic performance of plasma CCL20 was assessed by ROC curve analysis, AUC 0.768 (CI:0.678-0.858; p<0.001). Classification and regression tree analysis classified patients into two CCL20 plasma level groups. The high-CCL20 group had a higher number of AAA than the low-CCL20 group (91% vs 54.3%, p< 0.001). mRNA of CCL20 and its receptor CCR6 were higher in AAA (n = 89) than in control aortas (n = 17, p<0.001). A positive correlation was found between both mRNA in controls (R = 0674; p = 0.003), but not in AAA. Immunohistochemistry showed that CCR6 and CCL20 colocalized in the media and endothelial cells. Infiltrating leukocytes immunostained for both proteins but only colocalized in some of them. Our data shows that CCL20 is increased in AAA and circulating CCL20 is a high sensitive biomarker of AAA.

Figure 1

CCL20 plasma concentration in healthy subjects (n = 45), abdominal aortic aneurysm patients (AAA, n = 96) and patients with atherosclerotic disease and without AAA (AD, n = 28). No group fit normal distribution; ns, means no significant differences (p > 0.05).

Figure 2

(A) Receiver-operating-characteristic (ROC) curve for CCL20 plasma levels in abdominal aortic aneurysm patients (AAA, n = 96). The area under the curve was 0.768 (CI 0.678–0.858; p < 0.001). (B) CART analysis classification regarding CCL20 plasma levels as independent variable and using AAA condition as the dependent variable; including all patients in the statistics. (C) Calculated probability from the equation Logit P = 0.901 + 2.006 ∗ log10 [CCL20] obtained in the Logistic Regression Analysis, considering CCL20 plasma concentration ([CCL20]) as independent variable and occurrence of AAA as the dependent dichotomous variable ([CCL20] coefficient p < 0.001).

Figure 3

(A) CCL20 plasma concentration in abdominal aortic aneurysm patients stratified by MD (AAA) and patients with atherosclerotic disease without AAA (AD). No group fit normal distribution. (B) Statistical correlation of between CCL20 and sELAF in AAA samples.

Figure 4

(A) CCL20 and its receptor (CCR6) transcription levels in normal aorta from healthy donors (control, n = 17) and abdominal aortic aneurysm lesions (AAA, n = 89). No group fit normal distribution. (B) Statistical correlation between CCL20 and its receptor transcription levels in normal aortas (control) and AAA samples.

Figure 5

(A) Representative immunohistochemistry images of CCL20 and its receptor (CCR6) in normal aorta (NA) and in AAA samples. Arrows show some immunostained cells. (B) Representative immunofluorescent double staining for CCL20 and CCR6 in normal aorta (NA) and in AAA samples. Arrows show double immunostained cells. Bars are 50 µm; L, indicates the light of microvessels; I, indicates leukocyte infiltration areas; and M, indicates media layer.