1439PRAS40 attenuates mTOR-dependent endothelial inflammation and atherosclerosis
Authors: Zhang K , Schecker J , Woeltje K , Krull A , Stangl V , Stangl K , Katus H , Voelkers M , Althoff T F 
Published: October 2019

Abstract

Introduction
Endothelial inflammation plays a pivotal role in atherosclerosis. Many inflammatory and metabolic signals converge upon mechanistic target of rapamycin (mTOR), and inhibition of mTOR has been shown to reduce atherosclerosis. However, clinical use of mTOR-inhibitors is limited by serious adverse effects, of which insulin resistance and dyslipidemia are particularly troubling in the context of atherosclerosis. In that respect, targeting PRAS40, an endogenous modulator of mTOR complex 1 (mTORC1) with highly cell type-specific effects on mTOR signaling, may be a more promising approach. In fact, we have previously demonstrated that, in contrast to conventional mTOR inhibitors, PRAS40 gene therapy substantially improves metabolic profile in obese mice. However, the function of PRAS40 in endothelial cells and its role in atherosclerosis have never been investigated.

Methods and results
To define the impact of PRAS40 on endothelial mTORC1-signaling in this context, cultured human umbilical vein endothelial cells (HUVECs) were exposed to the atherogenic cytokine TNFα. TNFα induced mTOR signaling as evidenced by increased phosphorylation of S6 kinase and ribosomal S6 protein. Interestingly, this effect was strongly augmented upon siRNA-mediated knock-down of PRAS40, indicating a negative regulation of mTORC1 by PRAS40 in endothelial cells. Moreover, PRAS40-knockdown promoted TNFα-induced inflammatory signaling as reflected by increased proliferative activity, upregulation of atherogenic markers like CCL2 and VCAM-1, as well as enhanced monocyte recruitment in the THP-1 adhesion assay. In contrast, PRAS40-overexpression blocked TNFα-induced activation of mTORC1 and consistently suppressed all of these measures of inflammatory activation. All effects of PRAS40-overexpression could be reproduced by the mTORC1 inhibitors rapamycin and torin1. Thus, our in vitro studies suggest that in endothelial cells PRAS40 exerts anti-atherogenic effects by negative regulation of mTORC1. To validate these findings in vivo in the context of atherosclerosis we created transgenic mice with tamoxifen-inducible endothelium-specific PRAS40-deficiency (EC-PRAS40-KO). These mice were exposed to a model of accelerated atherosclerosis based on western diet and partial carotid ligation: Four weeks after partial carotid ligation, neointimal and atherosclerotic lesion formation was strongly enhanced in EC-PRAS40-KO mice. Moreover, mTORC1 activity as well as CCL2 and VCAM-1 expression were markedly increased compared to control mice.

Conclusion
Our data indicate that PRAS40 suppresses atherosclerosis via inhibition of mTORC1-mediated inflammatory signaling in endothelial cells. In conjunction with its favourable effects on metabolic homeostasis, the overall therapeutic profile of PRAS40-treatment appears to be beneficial compared to conventional mTOR-inhibitors. Taken together PRAS40 may qualify as a promising therapeutic target for the treatment of atherosclerosis.

Acknowledgement/Funding
German Federal Ministry of Education and Research, DZHK (German Centre for Cardiovascular Research)