Trimethylamine-N-Oxide Promotes Vascular Calcification Through Activation of NLRP3 (Nucleotide-Binding Domain, Leucine-Rich-Containing Family, Pyrin Domain-Containing-3) Inflammasome and NF-κB (Nuclear Factor κB) Signals

Abstract

Objectives: Vascular calcification is highly prevalent in patients with chronic kidney disease. Increased plasma trimethylamine N-oxide (TMAO), a gut microbiota-dependent product, concentrations are found in patients undergoing hemodialysis. However, a clear mechanistic link between TMAO and vascular calcification is not yet established. In this study, we investigate whether TMAO participates in the progression of vascular calcification using in vitro, ex vivo, and in vivo models. Approach and Results: Alizarin red staining revealed that TMAO promoted calcium/phosphate-induced calcification of rat and human vascular smooth muscle cells in a dose-dependent manner, and this was confirmed by calcium content assay. Similarly, TMAO upregulated the expression of bone-related molecules including Runx2 (Runt-related transcription factor 2) and BMP2 (bone morphogenetic protein-2), suggesting that TMAO promoted osteogenic differentiation of vascular smooth muscle cells. In addition, ex vivo study also showed the positive regulatory effect of TMAO on vascular calcification. Furthermore, we found that TMAO accelerated vascular calcification in rats with chronic kidney disease, as indicated by Mico-computed tomography analysis, alizarin red staining and calcium content assay. By contrast, reducing TMAO levels by antibiotics attenuated vascular calcification in chronic kidney disease rats. Interestingly, TMAO activated NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome and NF-κB (nuclear factor κB) signals during vascular calcification. Inhibition of NLRP3 inflammasome and NF-κB signals attenuated TMAO-induced vascular smooth muscle cell calcification. Conclusions: This study for the first time demonstrates that TMAO promotes vascular calcification through activation of NLRP3 inflammasome and NF-κB signals, suggesting the potential link between gut microbial metabolism and vascular calcification. Reducing the levels of TMAO could become a potential treatment strategy for vascular calcification in chronic kidney disease.

NAFLD and increased risk of cardiovascular disease: clinical associations, pathophysiological mechanisms and pharmacological implications

“…PERK is a receptor for TMAO, and its binding results in PERK activation and induction of the transcription factor FoxO1, a key factor in metabolic disorders [101]. Interestingly, TMAO may directly activate pro-inflammatory pathways as it up-regulates NLRP3 and nuclear factor (NF)-κB and thereby promotes vascular calcification [102]. Thus, TMAO reflects a crucial microbiota-derived biomarker of atherosclerosis and potentially of NAFLD-associated CVD.…”

Vascular Calcification—New Insights into Its Mechanism

“…Phosphate-regulated intracellular signaling includes Wnt/β-catenin, protein kinase B (PKB or Akt), nuclear factor-kappa B (NF-κB), and serum-and glucocorticoid-inducible kinase 1 (SGK1) [57,[73][74][75][76]. The role of the inflammasome is also important [77,78]. In addition, oxidative stress, caused by an imbalance of antioxidants and reactive oxygen species (ROS), is related to osteoinductive signals and apoptosis, and may be regulated by the Gas6/Axl pathway, Akt, AMP-activated protein kinase (AMPK), etc., in which phosphate directly regulates apoptosis [77,79,80].…”

“…The role of the inflammasome is also important [77,78]. In addition, oxidative stress, caused by an imbalance of antioxidants and reactive oxygen species (ROS), is related to osteoinductive signals and apoptosis, and may be regulated by the Gas6/Axl pathway, Akt, AMP-activated protein kinase (AMPK), etc., in which phosphate directly regulates apoptosis [77,79,80].…”

Identification of Novel Biomarkers and Pathways for Coronary Artery Calcification in Nondiabetic Patients on Hemodialysis Using Metabolomic Profiling

“…[26][27][28] These translocated metabolites and toxins may then contribute to chronic inflammation and ultimately to cardiovascular disease. [29][30][31][32] Animal studies have shown that bile acids may have direct effects on cardiovascular calcification via farnesoid X receptor, a bile acid nuclear receptor. 33,34 In our study, bile acid synthesis was one of the top 3 pathways identified by the pathway enrichment analyses, but its pathway impact was low compared to that of arginine/proline metabolism and urea cycle (0.01 versus 0.21 and 0.23), and the association between the bile acid synthesis pathway and CAC status was not significant.…”