Recombinant Thrombomodulin Exerts Anti-autophagic Action in Endothelial Cells and Provides Anti-atherosclerosis Effect in Apolipoprotein E Deficient Mice

Po-Sheng Chen,Yi-Heng Li,Kuan-Chieh Wang,Chawn-Yau Luo,Ting-Hsing Chao,Kuan-Chieh Wang,Hua-Lin Wu,Guey-Yueh Shi,Shi-Ya Tseng,Hsing-Chun Chung

doi:10.1038/s41598-017-03443-z

Abstract

Stress-induced alteration in endothelial cells (ECs) integrity precedes the development of atherosclerosis. Previous studies showed that the soluble recombinant thrombomodulin (rTM) not only increases ECs proliferation but also exerts anti-apoptotic activity in ECs. However, the functional significance of soluble rTM on autophagy-related apoptosis in ECs is still undetermined. Implicating a cytoprotective role for rTM in persistent serum starvation (SS)-induced autophagy in cultured ECs, we found that treatment of rTM decreased the expression of SS-induced autophagy-related proteins, ATG5 and LC3, and the formation of autophagosomes through activation of AKT/mTOR pathway. In addition, treatment of rTM decreased SS-induced EC apoptosis, but this effect of rTM could not be recapitulated by co-treatment with a potent autophagy inducer, rapamycin and in ECs with ATG5 knockdown. In human atherosclerosis specimens, expression of autophagy markers, ATG13 and LC3, were more abundant in aortic intimal ECs with severe atherosclerosis than those without atherosclerosis. Moreover, compared to saline treatment group, administration of rTM reduced LC3 and ATG13 expression, intimal EC apoptosis, and atherosclerotic lesion severity in the aorta of apolipoprotein E deficient mice. In conclusion, treatment with rTM suppressed stress-induced autophagy overactivation in ECs, provided ECs protective effects, and decreased atherosclerosis in apolipoprotein E deficient mice.

Highlights

Consisting autophagy related proteins (ATGs) that initiate autophagosome formation[6]
These results demonstrated that recombinant TM (rTM) treatment suppressed stress-induced autophagy overactivation in endothelial cells (ECs)
The major findings of this study were: (1) rTM suppressed stress-induced autophagy overactivation in ECs; (2) autophagy modulated ECs cell behavior; (3) autophagy was activated in aortic ECs in human atherosclerosis and apoE deficient mice; (4) rTM treatment suppressed aortic ECs autophagy and decreased atherosclerosis in apoE deficient mice and (5) rTM effect on autophagy was achieved through mTOR activation via the fibroblast growth factor receptor 1 (FGFR1)/ FGFR substrate 2α (FRS2α)-mediated PI3K/AKT pathway

Summary

Introduction

Consisting autophagy related proteins (ATGs) that initiate autophagosome formation[6]. Later studies showed that soluble recombinant TM (rTM) confers direct ECs protective effect by decreasing stress-induced ECs apoptosis and increasing ECs proliferation[11, 12]. Our previous studies demonstrated that the cytoprotective effects of rTM are independent of its thrombin inhibition or APC producing functions, and works by direct binding of rTM to fibroblast growth factor receptor 1 (FGFR1) on ECs with subsequent PI3K/AKT pathway activation[12,13,14] which is important in inhibition of autophagy. We hypothesized that: (1) rTM is an autophagy inhibitor by activating PI3K/AKT/mTOR complex 1 signaling pathway; (2) rTM suppresses stress-induced autophagy overactivation in ECs to provide ECs protective effects; and (3) rTM treatment may suppress overactivated autophagy in ECs during the process of atherosclerosis formation and decrease atherosclerosis severity in animal atherosclerosis model.

Methods

Results

Conclusion