| Background and ObjectiveAtherosclerosis (AS) is the most common and important cardiovascular disease, and is a critical inducement of cardiopathy, myocardial infarction and peripheral vascular diseases. It causes great harm to human health. AS mainly manifests as lipid accumulation at the vascular endarterium leading to atheromatous plaque formation, arterial wall thickening, harden, becoming inflexible and artery stenosis. More seriously, plaque ruption would cause thrombus formation and further give rise to acute coronary syndromes. There are various explanations about the pathogenesis of AS, including lipid infiltration theory, thrombus formation theory and smooth muscle cell clones theory and so on. But the concrete pathomechanism of AS is still not clear.Vascular endothelial cell (VEC), locating at the interior surface of vascular wall, plays important roles in cardiovascular system. It acts as an effective permeable barrier between circulating blood and tissues. Meanwhile, these cells also participated in the regulation of cell cholesterol, lipid homeostasis, signal transduction, immunity, inflammation and haemostasis. Dysfunction of endothelium is a critical inducer of AS and other cardiovascular diseases. Recently, more and more study concentrate on the importance of endothelial cells in atherosclerosis and gradually forms a new AS pathomechanism theory, called endothelial injury reaction theory, and endothelial cells become a novel AS therapeutic target.The mammalian target of rapamycin (mTOR), also known as FK506binding proteinl2-rapamycin associated protein1A, is a serine/threonine protein kinase of the phosphatidylinositol-3-OH kinase (PI(3)K)-related family. mTOR forms two different complexes:mTOR complex1(mTORC1) and mTOR complex2(mTORC2), and is a critical regulator in response to upstream cellular signals, such as growth factors, energy, stress and nutrients, and participated in controlling cell growth, proliferation and metabolism. Dysregulation of mTOR signaling induces a variety of human diseases including Alzheimer’s disease, cancer, diabetes and its cardiovascular complications.Thus,mTOR has become a new target for the treatment of diseases and themTOR-targeted therapies are currently being evaluated in clinical trials. mTOR inhibitors are already approved as anticancer agents.Recently, the application of chemical genetics has provided a novel method to discover new key regulators in physiological and pathological process and to develop novel drug for the treatment of diseases. We previously found that a novel y-butyrolactone derivative,3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BD0), inhibited LPS and chloroquine induced autophagy injury in human umbilical vein endothelial cells (HUVECs) and performed as a good endothelial protective agent. Further work identified3BDO to be an mTORC1activator. But it is still unknown that whether3BDO participated in oxidized low density lipoprotein (oxLDL)-induced endothelial injury by activating mTORC1, and whether3BDO exerted a role in protecting endothelial cell and inhibiting atherosclerosis.To answer these scientific questions mentioned above, this study investigated the effect of3BDO on endothelial autophagy and the molecular mechanism of atherosclerosis by using HUVECs and apoE-/-mice as animal model, and further explored the role of mTORC1in endothelial function and atherosclerosis. Our research will provide new evidence for VEC autophagy regulation and provide new potential compound and target for AS treatment.Results1. The mechanism how3BDO activated mTORCl and inhibited oxLDL-induced endothelial autophagy 1.13BD0activated mTORCl and inhibited oxLDL-induced increase of FLJ11812and ATG13in HUVECsFirst, we observed the effect of3BDO on oxLDL-induced mTORC1activity decrease in HUVECs, by detecting the phosphorylation of p70S6K and4E-binding protein1(4EBP1), two substrates of mTORC1. Western blot results showed that oxLDL inhibited the phosphorylation of p70S6K and4E-binding protein1(4EBP1) and3BD0treatment eliminated oxLDL-induced inhibition of p70S6K and4EBP1phosphorylation.1.23BDO inhibited oxLDL-induced FLJ11812production in HUVECsIn our recent work, we found that long noncoding RNA FLJ11812was a new factor downstream mTORCl and mTORC1inhibits FLJ11812production via TIA-1. RT-PCR result showed that oxLDL promoted FLJ11812production and3BDO treatment inhibited oxLDL-induced FLJ11812increase.1.33BDO inhibited oxLDL-increased ATG13protein level and contributed to ATG13phosphorylation in HUVECsFLJ11812is a long noncoding RNA which participated in translation regulation by combining with miRNA. Previously, we found that FLJ11812directly bound with miR-4459, and participated in the regulation of miR-4459targeted ATG13expression. Western blot result showed that oxLDL up regulated ATG13protein level.3BDO treatment inhibited oxLDL-induced increase of ATG13. The activation of mTOR directly promoted ATG13phosphorylation. Our result showed that oxLDL treatment inhibited the phosphorylation of ATG13and3BDO addition increased the phosphorylation level of ATG13.1.43BDO inhibited oxLDL-induced autophagy in endothelial cells in HUVECsATG13is necessary for autophagy initiation. The decrease of ATG13protein level and the increase of ATG13phosphorylation both inhibited autophagy. We further observed whether3BDO inhibited oxLDL-induced autophagy in HUVECs. The results of LC3puncta, LC3-Ⅱ level and p62accumulation analysis showed that3BDO significantly inhibited oxLDL-induced autophagy.1.53BDO inhibited oxLDL-induced upregulation of inflammatory factors in HUVECs OxLDL-caused endothelial injury leads to the upregualtion of a series of inflammatory factors, such as adherence factors, interleukins and so on. Our result showed that3BD0obviously inhibited oxLDL-induced expression of ICAM-1and VCAM-1. Elisa analysis result showed that3BDO significantly decreased oxLDL-induced secretion of IL-6and IL-8.2. The effect of3BDO on atherosclerosis development and plaque stability in apoE-/-mice2.13BDO decreased the lipid accumulation in the whole aortas and aortic roots in apoE-/-miceApoE-/-mice were fed an atherogenic diet for12weeks and then divided into3groups (n=6mice/group) for treatment:control (DMSO), low-dose3BD0(50mg/kg/d;3BDO-LD) and high-dose3BDO (100mg/kg/d;3BDO-HD). The control,3BDO-LD and3BDO-HD groups were treated respectively for8weeks. A series of indexes of atherosclerosis were detected. Oil Red O staining of the whole aortas and aortic roots both revealed that3BDO significantly decreased the lipid deposition.2.23BDO restricted atherosclerosis development and stabilized established atherosclerotic lesion in apoE-/-miceWe next further investigated the size and phenotype of the atherosclerotic lesion in aortic root of apoE-/-mice. H&E staining of aortic root showed that the atherosclerotic lesion areas in the3BDO treatment groups were significantly decreased than the control group. The phenotype of atherosclerotic lesion showed3BDO promoted the stability of plaque, with increased smooth muscle cells, reduced macrophage cells and MMP2/9activity. The level of smooth muscle cells and macrophages was respectively determined by a-smooth muscle actin and lysosome-associated membrane protein2(lamp-2) immunofluorescence staining. All these results indicated that3BDO treatment stabilized established atherosclerotic lesions in apoE-/-mice.2.33BDO inhibited the atherosclerotic inflammatory response in apoE-/-miceFurthermore, we investigated the effect of3BDO on inflammatory response which is an important index of plaque stability. Compared with control group,3BDO treatment significantly decreased the level of IL-6and IL-8in the serum. 2.43BD0did not affect the body weight of apoE-/-miceDuring the injection period, we measured the body weight of the mice every week. Result showed that compared to the control group,3BD0did not cause any change on the body weight of apoE-/-mice, which excluded the toxity of3BD0.These data suggested that3BD0inhibited the atherosclerosis development and further alleviated the inflammatory response.3. The mechanism how3BDO promoted plaque stability and inhibited AS development in apoE-/-mice3.13BDO did not affect the lipid metabolism of apoE-/-miceFirst, we evaluated the serum lipid level of apoE-/-mice. Result showed that3BD0did not change the serum lipid level which indicated that3BD0did not partipaited in lipid metabolism.3.23BDO activated mTORC1in plaque endothelium of apoE-/-miceThe mTORC1activity in plaque endothelium of apoE-/-mice was detected by immunofluorescence staining. Result showed that, compared to control group,3BD0treatment increased the levels of p-p70S6K in plaque endothelium of apoE-/-mice,which indicated that3BDO promoted plaque endothelium mTORC1activity.3.33BDO decreased the level of ATG13in plaque endothelium of apoE-/-miceThe protein level of ATG13was evaluated by immunofluorescence staining. Results showed that3BDO treatment decreased the protein level of ATG13in plaque endothelium of apoE-/-mice.3.43BDO selectively inhibited oxLDL-triggered autophagy in vascular endothelium of apoE-/-miceEn face staining of LC3patches in vascular endothelium of apoE-/-mice showed that compared to control group,3BD0treatment obviously decreased the amount of LC3patches, which revealed that3BDO inhibited endothelium autophagy in apoE-/-mice.Besides,3BDO did not affect oxLDL-induced autophagy level in macrophage cells and smooth muscle cells, which indicated the cell specifity of3BD0.3.53BDO inhibited oxLDL-triggered apoptosis in plaque endothelium of apoE-/-mice TUNEL staining in plaque endothelium of apoE-/-mice showed that compared to control group,3BD0treatment obviously inhibited endothelium apoptosis.Our result indicated that3BD0inhibited AS development by protecting against endothelium injury.3BD0activated endothelial mTORCl and inhibited endothelial apoptosis induced by autophagy injury.ConclusionOur results revealed that in endothelial cells, oxLDL decreased the mTORCl activity and contributed to the development of atherosclerosis. First, inhibition of mTORC1by oxLDL contributed to the downstream FLJ11812increase. FLJ11812, with decoy activity for miR-4459and by sequestering miR-4459, regulates the level of miR-4459targeted gene ATG13, thus promoted autophagy injury and endothelium apoptosis. As an mTORC1activator,3BDO inhibited oxLDL-induced mTORCl acvitity decrease and further restrained atherosclerosis development and promoted plaque stability. |
