High Phosphate-Induced Calcification of Vascular Smooth Muscle Cells is Associated with the TLR4/NF-κb Signaling Pathway

Abstract

Background/Aims: Hyperphosphatemia is one of the most notable features of chronic kidney disease (CKD). Numerous epidemiological and clinical studies have found that high serum phosphate concentrations are associated with calcification in the coronary arteries. However, the mechanisms underlying the vascular calcification induced by high phosphate have not been understood fully. Methods: Vascular smooth muscle cells (VSMCs) were cultured in highphosphate media to induce vascular calcification, which was detected by Alizarin red S staining. Gene expression and protein levels of differentiation markers were determined by real-time RT-PCR and western blotting, respectively. Protein levels of phosphorylated NF-κB and TLR4 were detected by western blotting, and the role of NF-κB/TLR4 was further confirmed by using an NF-κB inhibitor or TLR4 siRNA. Results: Our results showed that high-phosphate media induced obvious calcification of VSMCs. Simultaneously, VSMC differentiation was confirmed by the increased expression of bone morphogenetic protein-2 and Runt-related transcription factor 2 and decreased expression of the VSMC-specific marker SM22α, which was accompanied by the increased expression of inflammatory cytokines. Moreover, a significant upregulation of TLR4 and phosphorylated NF-κB was also detected in VSMCs with high-phosphate media. In contrast, VSMC calcification and the increased expression of inflammatory cytokines were markedly attenuated by pretreatment with TLR4 siRNA and pyrrolidine dithiocarbamic acid, an NF-κB inhibitor. Conclusion: These data suggest that high-phosphate conditions directly induce vascular calcification via the activation of TLR4/NF-κB signaling in VSMCs. Moreover, inhibition of the TLR4/NF-κB signaling pathway might be a key intervention to prevent vascular calcification in patients with CKD.

Indoxyl sulfate‐induced calcification of vascular smooth muscle cells via the PI3K/Akt/NF‐κB signaling pathway

“…NF‐κB is a key transcription factor involved in diverse biological processes, which is recognized as a pivotal regulator of cell proliferation, differentiation, apoptosis, inflammation and so forth (Almaden et al, ; Viatour et al, ; Weng & Koh, ). Recent studies have demonstrated that NF‐κB has profound functions on the pathogenesis of VC by regulating the VSMC osteogenic transformation (Zhang et al, ; Zhao et al, ). Thus, we speculate that NF‐κB may also play a critical role in the IS‐induced VC.…”

“…It has been identified as a central regulator of gene expression associated with cell proliferation, differentiation, immunity and inflammation (Almaden et al, ; De Silva, Silva, Anderson, Bhagat, & Klein, ; Viatour, Merville, Bours, & Chariot, ). Recent studies have shown that activation of NF‐κB is expressed at a higher level in calcified vessels and inhibition of NF‐κB eliminates the Pi or TNF‐induced VSMC calcification in vitro (He et al, ; Zhang et al, ; Zhao et al, ; Zhao et al, ), suggesting that NF‐κB may also play a crucial role in the pathological of VC in CKD patients. Recently, numerous studies have reported that IS stimulated the activation of NF‐κB in various cells involved in the vessel senescence, endothelium dysfunction, and neuroinflammation (Adelibieke, Shimizu, Muteliefu, Bolati, & Niwa, ; Adesso et al, ; Shen et al, ).…”

Lower Serum Irisin Levels Are Associated with Increased Vascular Calcification in Hemodialysis Patients

“…Now, it is believed that vascular calcification involves a tightly regulated transformation of vascular smooth muscle cells to an osteo/chondrocytic cell that expresses Runt-related transcription factor 2 (Runx2) and produces matrix vesicles under hyperphosphate circumstance [26][27][28][29]. Recently, Chen [30], et al reviewed that high mobility group box 1 have a series of pro-calcification effects, such as promoting vascular smooth muscle osteo/chondrogenic differentiation, apoptosis and release of calcific extracellular vesicles, inflammation, oxidative stress and autophagy signaling, these indicated that inhibition of high mobility group box 1 may be a potential therapy for attenuating vascular calcification.…”

Effects of oral activated charcoal on hyperphosphatemia and vascular calcification in Chinese patients with stage 3–4 chronic kidney disease

“…These matrix vesicles provide sites for further calcification. High-phosphorus stimulation increases the secretion of matrix vesicles, thereby aggravating vascular calcification [22, 23]. …”