Inflammatory biomarkers in atherosclerosis: pentraxin 3 can become a novel marker of plaque vulnerability

Abstract

Inflammation is crucially involved in the development of carotid plaques. We examined the relationship between plaque vulnerability and inflammatory biomarkers using intraoperative blood and tissue specimens. We examined 58 patients with carotid stenosis. Following carotid plaque magnetic resonance imaging, 41 patients underwent carotid artery stenting (CAS) and 17 underwent carotid endarterectomy (CEA). Blood samples were obtained from the femoral artery (systemic) and common carotid artery immediately before and after CAS (local). Seventeen resected CEA tissue samples were embedded in paraffin, and histopathological and immunohistochemical analyses for IL-6, IL-10, E-selectin, adiponectin, and pentraxin 3 (PTX3) were performed. Serum levels of IL-6, IL-1β, IL-10, TNFα, E-selectin, VCAM-1, adiponectin, hs-CRP, and PTX3 were measured by multiplex bead array system and ELISA. CAS-treated patients were classified as stable plaques (n = 21) and vulnerable plaques (n = 20). The vulnerable group showed upregulation of the proinflammatory cytokines (IL-6 and TNFα), endothelial activation markers (E-selectin and VCAM-1), and inflammation markers (hs-CRP and PTX3) and downregulation of the anti-inflammatory markers (adiponectin and IL-10). PTX3 levels in both systemic and intracarotid samples before and after CAS were higher in the vulnerable group than in the stable group. Immunohistochemical analysis demonstrated that IL-6 was localized to inflammatory cells in the vulnerable plaques, and PTX3 was observed in the endothelial and perivascular cells. Our findings reveal that carotid plaque vulnerability is modulated by the upregulation and downregulation of proinflammatory and anti-inflammatory factors, respectively. PTX3 may thus be a potential predictive marker of plaque vulnerability.

Figure 1. Systemic and local levels of proinflammatory and anti- inflammatory markers.

Serum levels of inflammatory markers in systemic, pre-, and post-procedural local samples were measured by ELISA. Results are expressed as mean ± IQRs. Differences between the samples are not significant, except for those indicated by *P<0.05.

Figure 2. Comparison of proinflammatory and anti- inflammatory markers for plaque vulnerability.

Serum levels of proinflammatory and anti-inflammatory markers from vulnerable and stable plaques in systemic, pre-, and post-procedural local samples were measured by ELISA. Results are expressed as mean ± IQRs. Differences between the samples are not significant, except for those indicated by *P<0.05.

Figure 3. Comparison of systemic and control samples.

Serum levels of proinflammatory and anti-inflammatory markers in systemic samples of patients with vulnerable and stable plaques, and controls were measured by ELISA. Results are expressed as mean ± IQRs. Differences between the samples are not significant, except for those indicated by *P<0.05.

Figure 4. Immunochemical staining of atherosclerotic carotid endarterectomy specimens and emboli captured by distal protection devices.

Serial sections were stained with hematoxylin and eosin (HE) (A, B, C, Q, R), anti-IL-6 (D, E, F), anti-PTX3 (G, H, I), and anti-E-selectin (J, K, L) antibodies. Sections from CEA (A to L), quantification of immunohistochemistry analysis of IL-6, PTX3, and E-selectin staining of sections from CEA (M, N, O), and embolic debris from CAS captured by the distal filter device (P, Q, R) are shown. HE staining showed inflammatory cell infiltrations in the vulnerable plaques (A, B, Q) and cholesterin crystals in the stable plaques (C, R). IL-6 staining is observed in the vulnerable plaques with inflammatory cell infiltration (D, E) but not in the stable plaques (F). PTX3 expression is observed in endothelial and perivascular cells and in the basement membrane in the vulnerable plaques (G, H) but not in the stable plaques (I). Immunohistochemistry revealed E-selectin expression in endothelial cells in the vulnerable plaques (J, K) but not in the stable plaques (L). The second line on the left of the vulnerable plaques (B, E, H, K) shows enlargement of the cropped areas. Scale bar, 100 µm.

Figure 5. Receiver-operating curve of PTX3 levels for predicting vulnerable plaque.

The area under the curve of PTX3 levels were obtained from all patients.

Figure 6. Proinflammatory and anti-inflammatory biomarkers in vulnerable plaques.

Decreased adiponectin levels induce endothelial cell dysfunction with E-selectin release, with subsequent low IL-10 levels and high TNFα levels. Impaired endothelial cells release E-selectin, PTX3, and TNFα. Macrophages produce PTX3, TNFα, and IL-6.