TLR2 Promotes Vascular Smooth Muscle Cell Chondrogenic Differentiation and Consequent Calcification via the Concerted Actions of Osteoprotegerin Suppression and IL-6–Mediated RANKL Induction

Abstract

Objective— Vascular smooth muscle cell (VSMC) transformation to an osteochondrogenic phenotype is an initial step toward arterial calcification, which is highly correlated with cardiovascular disease–related morbidity and mortality. TLR2 (Toll-like receptor 2) plays a pathogenic role in the development of vascular diseases, but its regulation in calcification of arteries and VSMCs remains unclear. We postulate that TLR2-mediated inflammation participates in mediating atherosclerotic arterial calcification and VSMC calcification. Approach and Results— We found that ApoE −/− Tlr2 −/− genotype in mice suppressed high-fat diet–induced atherosclerotic plaques formation during initiation but progressively lost its preventative capacity, compared with ApoE −/− mice. However, TLR2 deficiency prohibited high-fat diet–induced advanced atherosclerotic calcification, chondrogenic metaplasia, and OPG (osteoprotegerin) downregulation in the calcified lesions. Incubation of VSMCs in a calcifying medium revealed that TLR2 agonists significantly increased VSMC calcification and chondrogenic differentiation. Furthermore, TLR2 deficiency suppressed TLR2 agonist–mediated VSMC chondrogenic differentiation and consequent calcification, which were triggered via the concerted actions of IL (interleukin)-6–mediated RANKL (receptor activator of nuclear factor κB ligand) induction and OPG suppression. Inhibition experiments with pharmacological inhibitors demonstrated that IL-6–mediated RANKL induction is signaled by p38 and ERK1/2 (extracellular signal-regulated kinase 1/2) pathways, whereas the OPG is suppressed via NF-κB (nuclear factor κB) dependent signaling mediated by ERK1/2. Conclusions— We concluded that on ligand binding, TLR2 activates p38 and ERK1/2 signaling to selectively modulate the upregulation of IL-6–mediated RANKL and downregulation of OPG. These signaling pathways act in concert to induce chondrogenic transdifferentiation of VSMCs, which in turn leads to vascular calcification during the pathogenesis of atherosclerosis.

Vanadium Derivative Exposure Promotes Functional Alterations of VSMCs and Consequent Atherosclerosis via ROS/p38/NF-κB-Mediated IL-6 Production

“…Notably, these NaVO 3 -induced atherosclerotic pathologies in ApoE −/− mice were prevented by NAC treatment, indicating that ROS is required for NaVO 3 -driven atherosclerosis. Our results further show that VOSO 4 and NaVO 3 promote phenotypic transitions of VSMCs from the quiescent contractile state to the active synthetic state and VSMC proliferation and migration, which has been implicated in deleterious consequences of atherosclerosis [3,25,26,29]. The present study provides experimental evidence that vanadium salts, VOSO 4 and NaVO 3 , drive VSMC pathological responses by inducing ROS production and thereby activating p38/NF-κB signaling.…”

“…The decrease in VSMC differentiation marker proteins such as SM22α in atherosclerotic lesions is a common characteristic of atherosclerosis [3,29], we determined whether intranasal administration of NaVO 3 significantly decreased SM22α. A significant decrease in the immunopositive areas for SM22α in the atherosclerotic lesions ( Figure 1F) was found to be highly associated with enhanced neointimal formation in the atherosclerotic lesion of NaVO 3 -exposed mice ( Figure 1G).…”

The Role of Osteoprotegerin and Its Ligands in Vascular Function

“…Neutrophils produce OPG and IL-17. IL-17 increases the recruitment of neutrophils at the site of inflammation and influences the production of various proinflammatory mediators [15,16]. A recent study reported a significant elevation of circulating OPG in septic patients with different levels of severity and in those who progressed to acute kidney injury; OPG thus appears to be a reliable biomarker [17].…”

The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification

“…We mainly focused on the relationship between miR-34a and the prototypical pro-inflammatory SASP component IL6, which is canonically upregulated in cells that have undergone senescence, increases with age and plays a causal role in inflammaging and age-related diseases [8,30,32,33]. Besides, IL6 is one of the most abundant cytokines produced by senescent VSMCs and exerts pro-calcification activity [8,34,35]. We found that miR-34a and IL6 levels are upregulated and positively correlate in aortas of old mice and with HASMCs donors' age and senescence (Figure 1).…”