Restoration of 5-methoxytryptophan protects against atherosclerotic chondrogenesis and calcification in ApoE\u2212/\u2212 mice fed high fat diet

Guan-Lin Lee,Jing-Yiing Wu,Cheng-Chin Kuo,Kenneth K. Wu,Tsai-Lien Liao

doi:10.1186/s12929-021-00771-1

Abstract

BackgroundToll-like receptor-2 (TLR2) promotes vascular smooth muscle cell (VSMC) transdifferentiation to chondrocytes and calcification in a p38 MAPK-dependent manner. Vascular 5-methoxytryptophan (5-MTP) is a newly identified factor with anti-inflammatory actions. As 5-MTP targets p38 MAPK for its actions, we postulated that 5-MTP protects against vascular chondrogenesis and calcification.MethodsHigh-fat diet-induced advanced atherosclerosis in mice were performed to investigate the effect of 5-MTP on atherosclerotic lesions and calcification. VSMCs were used to determine the role of 5-MTP in VSMC chondrogenic differentiation and calcification. Alizarin red S and Alcian blue staining were used to measure VSMC calcification and chondrogenic differentiation, respectively.Results5-MTP was detected in aortic tissues of ApoE−/− mice fed control chow. It was reduced in ApoE−/− mice fed high-fat diet (HFD), but was restored in ApoE−/−Tlr2−/− mice, suggesting that HFD reduces vascular 5-MTP production via TLR2. Intraperitoneal injection of 5-MTP or its analog into ApoE−/− mice fed HFD reduced aortic atherosclerotic lesions and calcification which was accompanied by reduction of chondrogenesis and calcium deposition. Pam3CSK4 (Pam3), ligand of TLR2, induced SMC phenotypic switch to chondrocytes. Pretreatment with 5-MTP preserved SMC contractile proteins and blocked Pam3-induced chondrocyte differentiation and calcification. 5-MTP inhibited HFD-induced p38 MAPK activation in vivo and Pam3-induced p38 MAPK activation in SMCs. 5-MTP suppressed HFD-induced CREB activation in aortic tissues and Pam3-induced CREB and NF-κB activation in SMCs.ConclusionsThese findings suggest that 5-MTP is a vascular arsenal against atherosclerosis and calcification by inhibiting TLR2–mediated SMC phenotypic switch to chondrocytes and the consequent calcification. 5-MTP exerts these effects by blocking p38 MAPK activation and inhibiting CREB and NF-κB transactivation activity.

Highlights

Toll-like receptor-2 (TLR2) promotes vascular smooth muscle cell (VSMC) transdifferentiation to chondrocytes and calcification in a p38 MAPK-dependent manner
The results show that vascular production of 5-MTP was suppressed by HFDinduced atherosclerosis in ApoE−/− mice, which was restored by genetic deletion of TLR2. 5-MTP administration reduced chondrogenesis and calcification in HFDinduced atherosclerosis in ApoE−/− mice and inhibited VSMC phenotypic switch to calcified chondrocytes induced by TLR2 and TLR4 activation. 5-MTP inhibits
High fat diet reduces vascular and plasma 5‐MTP via toll‐like receptor 2 We previously reported that vascular endothelial cells (EC) produce 5-MTP and secrete it into extracellular milieu via Golgi vesicle trafficking

Summary

Introduction

Toll-like receptor-2 (TLR2) promotes vascular smooth muscle cell (VSMC) transdifferentiation to chondrocytes and calcification in a p38 MAPK-dependent manner. There is accumulating evidence that pro-inflammatory cytokines drive vascular smooth muscle cell (VSMC) migration and promote its phenotypic switch from contractile to osteochondrogenic phenotype which lead to vascular calcification [7,8,9,10]. We reported that toll like receptor 2 (TLR2) activation induces vascular SMC transdifferentiation into chondrocytes with calcium deposition [10]. Endogenous TLR2 activation plays a crucial role in aortic chondrogenesis and calcification in ApoE−/− mice fed with high fat diet (HFD) as atheromatous calcification is abrogated in HFD-fed ApoE−/−Tlr2−/− double-knockout mice [10]. TLR2 activation promotes atheromatous calcification via p38 MAPK signaling pathway which leads to enhanced IL-6 expression [10]. IL-6 was shown to drive VSMC migration and VSMC transdifferentiation into chondrocytes and calcium deposition [10,11,12]

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