Knockdown of mTOR by lentivirus-mediated RNA interference suppresses atherosclerosis and stabilizes plaques via a decrease of macrophages by autophagy in apolipoprotein E-deficient mice

Abstract

Atherosclerotic plaque destabilization and rupture leads to acute coronary syndromes which cause serious damage to human health worldwide. However, there is currently a lack of efficient therapeutic methods. Mammalian target of rapamycin (mTOR) has been suggested to be involved in the development of atherosclerotic plaques and serves as a therapeutic target. The present study was performed to determine whether RNA interference (RNAi) of mTOR in vivo by LV‑mediated small hairpin RNA (shRNA) was capable of inhibiting the progression of atherosclerotic plaques. LV‑mediated shRNA against mTOR (LV‑shmTOR) was designed and obtained. Male apolipoprotein E‑deficient mice were fed a high‑fat diet and a constrictive collar was placed around the right carotid arteries of these mice to induce plaque formation. Eight weeks after surgery, mice were randomly divided into the mTOR RNA interference (LV‑shmTOR) group, receiving treatment with LV‑mTOR‑shRNA; the LV‑shCON group, receiving treatment with LV‑non‑specific‑shRNA; and the control group, receiving treatment with phosphate‑buffered saline. Following transfection, the mice were sacrificed to evaluate the effects of mTOR expression silencing on atherosclerosis. Transfection of LV‑mTOR‑shRNA markedly inhibited the mRNA and protein expression levels. Knockdown of mTOR ameliorated dysregulated blood lipid metabolism and stabilized aortic atherosclerotic plaques by decreasing the plaque area and increasing the fibrous cap and cap‑to‑core ratio. Furthermore, macrophages were decreased by silencing mTOR in atherosclerotic plaques. In addition, western blot analysis revealed that the knockdown of mTOR increased autophagy‑related protein 13 (Atg13) dephosphorylation and light chain 3‑I/light chain 3‑II (LC3‑I/LC3‑II) ratios, both of which were associated with a high activity of autophagy, suggesting an increase of autophagy in atherosclerotic plaques. Moreover, genes including matrix metalloproteinase 2, monocyte chemoattractant protein 1 and tissue factor, which promote plaque instability, were downregulated by silencing mTOR. These results demonstrate that LV‑mediated mTOR silencing by RNAi treatment induces macrophage autophagy and is a potential strategy for the treatment of atherosclerotic plaques.

An activator of mTOR inhibits oxLDL-induced autophagy and apoptosis in vascular endothelial cells and restricts atherosclerosis in apolipoprotein E-/- mice

“…Previous studies reported that inhibition of mTOR by sirolimus or everolimus decreased the number of macrophages by inducing autophagy and suppressng atherosclerosis1011. Recent research also demonstrated that suppression of mTOR in an atherosclerotic mouse model by left ventricular-mediated RNAi inhibited atherosclerosis and stabilized plaques via a decrease of macrophages by autophagy28. Compared to macrophage activity, the elimination and injury of ECs is detrimental for atherosclerotic plaque stability.…”

Plasma MicroRNA-100 Is Associated With Coronary Plaque Vulnerability

“…21 A recent study showed that knockdown of mTOR ameliorated dysregulated blood lipid metabolism and stabilized aortic atherosclerotic plaques by decreasing plaque area and increasing the size of fibrous cap and cap-to-core ratio via a decrease in the number of macrophages by autophagy in apolipoprotein E-deficient mice. 22 Another recent study showed that increased mTOR activity upregulated sterol regulatory element-binding protein-2-mediated cholesterol uptake through the phosphorylated retinoblastoma tumor suppressor protein. 23 In accordance with these studies, a recent clinical study also showed that mRNA expression of mTOR and other pro-inflammatory mediators, including tumor necrosis factor-α and monocyte chemotactic protein-1, was significantly higher in mononuclear cells extracted from carotid endarterectomy specimens compared with peripheral mononuclear cells.…”

7‐Ketocholesterol‐induced lysosomal dysfunction exacerbates vascular smooth muscle cell calcification via oxidative stress

“…Furthermore, induction of ALP in macrophages via knockdown of mTOR suppressed atherosclerosis and stabilized plaques (Wang et al . ). In TGF‐β‐induced calcification, atorvastatin decreased Ca deposition in VSMCs through induction of ALP (Liu et al .…”