Evidence from osteoprotegerine-RANKL system in explanted human carotid plaques and correlation with increased arterial stiffness

Background: Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF)-related family and part of the OPG/receptor activator of NF-k B ligand (RANKL)/ receptor activator of NF- k B (RANK) triad. The effects of OPG in the development of atheroma is dual: on one hand,OPGis capable to reduce the inflammatory process by binding RANKL, and therefore blocking NF- kB-mediated inflammation; on the other hand, OPG can reduce the differentiation of mature osteoclast, allowing the calcification of atherosclerotic plaques. Furthermore, the production of OPG in endothelial cells may reflect endothelial dysfunction and arterial stiffness. However, the most of knowledge in this context is currently provided from serum levels of OPG and this accounts for its low specificity. Wewanted to investigate whether the expression ofOPGandRANKL, inthecarotid atherosclerotic wall, relates to a) carotid stiffness and b) cerebrovascular events (CVE). Methods: An ultrasound examination (UE) of carotid arteries was performed, through an Esaote My Lab 70 system, on a cohort of 22 patients (18 male, 72.3+8.4 yrs) who underwent thromboendarteriectomy. Physician that performed UEs was blinded from patient clinical history. Pulse wave velocity (PWV) and b index were measured as parameters of arterial stiffness. Ultrasonographic carotid plaques morphology was confirmed by a blinded pathologist. A carotid specimen comprehensive of both plaque and plaque-free wall was analyzed in order to investigate by Western blot the expression of OPG and RANKL. Results: Nine (41%) out of 22 patients, previously had a CVE and 55% of them also showed a history of ischemic cardiomyopathy. The UE revealed that all patients with CVE had fibrous/hypoechoic plaques, in contrast those without CVE showed fibrocalcific/ hyperechoic plaques. Moreover, an increased arterial stiffness was identified in the entire cohort (PWV= 10.8+1.9 m/s, b= 19.2+5.8). Specimens’ analysis revealed an enhanced expression of OPG particularly in fibrocalcific/hyperechoic plaques (7.94+1 integrated intensity) rather than in fibrous/hypoechoic plaques (5.9+1.1 integrated intensity, p=0.006). In addition, a negative correlation between CVE and OPG expression (rho =- 0.67, p= 0.008) and between PWV and RANKL values (r= -0.71, p= 0.04) was identified in overall cohort. Conclusion: OPG expression is increased in patients with advanced atherosclerosis, particularly in those with fibrocalcific plaques but no CVE; the correlation between RANKL and PWV validates the role of arterial stiffness in the atherosclerotic process in elderly patients.