Introduction: Altered hemodynamic forces and lowered shear stress, which mainly occur at arterial bifurcations and curvatures, predispose these areas for the development of atherosclerotic lesions. An extracellular layer on the endothelium, the glycocalyx, has been shown to be involved in endothelial sensing of these mechanical forces. To determine, how changes in structure and functionality of the endothelial glycocalyx might be related to atherosclerosis progression, we investigated two of its main components, the membrane bound glypican-4 (GPC4) and the associated heparan sulfate (HS), and their expression pattern in human carotid plaque specimens.

Methods: 51 plaque specimens were collected from patients undergoing carotid endarterectomy. Based on plaque cross-sections, specimens plaques were classified into vulnerable (25), stable (21) and initial (5) plaque sections, according to established parameters, such as neovascularization, fibrous cap thickness and size of the necrotic core. Surface expression of CD68, GPC4 and HS was visualized by immunohistochemistry. Microscopic images (x150 magnification) were obtained at the plaque shoulders (PS) and the fibrous cap (FC) region. Numbers of infiltrated CD68 positive macrophages in the shoulder regions were assessed to detect the PS that expressed a higher level of inflammation, which in further analysis was compared do the PS showing a lower degree of inflammation. Subsequently, the endothelial glycocalyx was characterized by determining the percentage of endothelium expressing GPC4 or HS, respectively and compared between the FC and the two PS. Further parameters of plaque vulnerability were analyzed, measuring FC thickness at the thinnest site, the degree of stenosis and necrotic core area.

Results: In vulnerable plaque sections a significantly lower percentage of endothelial GPC4 expression was observed in the more-inflamed compared to the less-inflamed plaque shoulder (p=0.01), while no such differences were observed in stable or initial plaque sections. HS showed a similar expression pattern compared to GPC4, but the differences between the two plaque shoulders were less pronounced and not significant (p=0.11). Comparing different stages of plaque progression (vulnerable vs. stable vs. initial), vulnerable plaque sections showed significantly lower endothelial GPC4 (p=0.03) and HS (p=0.01) expression in the inflamed PS, while highest expression of both molecules were found in initial plaques. Analyses of the FC showed no significant differences in GPC4 or HS expression between the three plaque types. Investigation of further plaque parameters showed significant differences regarding FC thickness (p<0.001), with thinnest FC in vulnerable plaque sections. Furthermore we identified a significantly higher proportional area of the necrotic core related to the whole plaque (p<0.001), with decreasing necrotic core sizes from vulnerable via stable to initial plaque sections.

Conclusion: Plaque inflammation seems to correlate with degradation of the glyococalix, suggesting that the absence of an intact gylcocaliyx may be an important trigger for plaque progession and coronary artery disease events.