The Association of Histological Signs of Plaque Instability with Low eGFR, Higher Neutrophil-to-Lymphocyte Ratio, and Lower Serum MCP-1 Levels in Carotid Endarterectomy Patients-A Single-Center, Prospective Cohort Study

Abstract

Background: Histological signs of carotid atheromatous plaque vulnerability, such as hemorrhage, neovascularization, atherothrombosis, and ulceration, develop against an unstable biological background. Declining renal function contributes to atherosclerotic progression and worsens cardiovascular outcomes. Methods: In a single-center prospective cohort study, we studied 41 endarterectomized patients with severe carotid atherosclerosis. The histological samples were stained with H&E to assess morphology and immunohistochemically labeled with antibodies for CRP and MMP-9 proteins. Complete blood count, the presence of serum biomarkers hsCRP, oxLDL, MCP-1, and MMP-9, and the level of eGFR were determined. Results: Twenty-eight patients with complicated plaques had significantly lower eGFR values: 79.5 (24-110) vs. 94 (69-114) (p = 0.004). Patients with eGFR > 90 mL/min/1.73m² had a higher incidence of intraplaque hemorrhage and histologic complications of any cause (p = 0.012 and p = 0.003). Patients with bleeding and ulceration from the carotid plaque had a higher neutrophil/lymphocyte ratio. Significantly lower levels of MCP-1 were found in the serum of patients with massive inflammatory infiltrate of the carotid plaques, while serum levels of biomarkers like hsCRP, MMP-9, and oxLDL did not show differences in cases with plaque vulnerability. Conclusions: Signs of plaque vulnerability are associated with reduced renal function, a higher neutrophil/lymphocyte ratio, and lower serum levels of MCP-1 in advanced carotid artery stenosis disease.

Keywords: biomarkers; carotid stenosis; eGFR; endarterectomy; intraplaque hemorrhage; plaque vulnerability.

Figure 1

Local synthesis of CRP and MMP-9 by inflammatory cells and vascular smooth muscle cells. Immunohistochemical visualization (3,3′-diaminobenzidine chromogen) of MMP-9 and CRP-labeled cellular and extracellular structures in atherosclerotic plaque. MMP-9 is highly expressed in inflammatory cells (lymphocytes and monocytes) surrounding the lipid core (A), but is also detected in the macrophages in the microenvironment of the lipid core (B). In the plaque, CRP is also synthesized in vascular smooth cells VSMCs, (C) along with macrophages, and various mononuclear elements (D).

Figure 2

The scanned plaque areas corresponding to the CRP-positive signals were considered for analysis (A). Segmentation on anti-CRP/DAB immunolabelled tissue section, followed by annotation of the region of interest. (B–D). The application of the algorithm to measure the thresholded area and calculate the H-score.

Figure 3

The morphological characteristics of plaque vulnerability and plaque complications per H&E stain (representative section): (A) microcalcification (black arrow) or (B) macrocalcification (delimited by black dots—based on nodules of <50 and ≥50 μm); (C) large lipid core (pointed to by blue arrows—cellular detritus predominant in its structure, together with macrophages with foamy cytoplasm and cholesterol crystals); (D) intraplaque inflammatory cell population (delimited by black dashed lines); (E) neovascularisation (gray arrows—the presence of new vessels within the lipid core); and (F) intraplaque hemorrhage (delimited by light brown dashed lines) and fibrous cap damage (ulceration—delimited by green dots).

Figure 4

All-cause (sub-figure (A)) and hemorrhagic (sub-figure (B)) complications in eGFR A, B, and C categories. * p < 0.05, ** p < 0.01.