Ganhua You1,2, Xiangshu Long3,4, Fang Song3,4, Jing Huang3,4, Maobo Tian3,4, Yan Xiao3,4, Shiyan Deng3,4, Qiang Wu3,4. 1. Guizhou University School of Medicine, Guiyang 550025, People's Republic of China. 2. Guizhou Institute for Food and Drug Control, Guiyang 550004, People's Republic of China. 3. Department of Cardiology, Guizhou Provincial People's Hospital, Guiyang 550002, People's Republic of China. 4. Department of Cardiology, People's Hospital of Guizhou University, Guiyang 550002, People's Republic of China.
Abstract
BACKGROUND: Metformin has been shown to inhibit the proliferation and migration of vascular wall cells. However, the mechanism through which metformin acts on atherosclerosis (AS) via the long non-coding RNA taurine up-regulated gene 1 (lncRNA TUG1) is still unknown. Thus, this research investigated the effect of metformin and lncRNA TUG1 on AS. METHODS: First, qRT-PCR was used to detect the expression of lncRNA TUG1 in patients with coronary heart disease (CHD). Then, the correlation between metformin and TUG1 expression in vitro and their effects on proliferation, migration, and autophagy in vascular wall cells were examined. Furthermore, in vivo experiments were performed to verify the anti-AS effect of metformin and TUG1 to provide a new strategy for the prevention and treatment of AS. RESULTS: qRT-PCR results suggested that lncRNA TUG1 expression was robustly upregulated in patients with CHD. In vitro experiments indicated that after metformin administration, the expression of lncRNA TUG1 decreased in a time-dependent manner. Metformin and TUG1 knockdown via small interfering RNA both inhibited proliferation and migration while promoted autophagy via the AMPK/mTOR pathway in vascular wall cells. In vivo experiments with a rat AS model further demonstrated that metformin and sh-TUG1 could inhibit the progression of AS. CONCLUSION: Taken together, our data demonstrate that metformin might function to prevent AS by activating the AMPK/mTOR pathway via lncRNA TUG1.
BACKGROUND: Metformin has been shown to inhibit the proliferation and migration of vascular wall cells. However, the mechanism through which metformin acts on atherosclerosis (AS) via the long non-coding RNA taurine up-regulated gene 1 (lncRNA TUG1) is still unknown. Thus, this research investigated the effect of metformin and lncRNA TUG1 on AS. METHODS: First, qRT-PCR was used to detect the expression of lncRNA TUG1 in patients with coronary heart disease (CHD). Then, the correlation between metformin and TUG1 expression in vitro and their effects on proliferation, migration, and autophagy in vascular wall cells were examined. Furthermore, in vivo experiments were performed to verify the anti-AS effect of metformin and TUG1 to provide a new strategy for the prevention and treatment of AS. RESULTS: qRT-PCR results suggested that lncRNA TUG1 expression was robustly upregulated in patients with CHD. In vitro experiments indicated that after metformin administration, the expression of lncRNA TUG1 decreased in a time-dependent manner. Metformin and TUG1 knockdown via small interfering RNA both inhibited proliferation and migration while promoted autophagy via the AMPK/mTOR pathway in vascular wall cells. In vivo experiments with a rat AS model further demonstrated that metformin and sh-TUG1 could inhibit the progression of AS. CONCLUSION: Taken together, our data demonstrate that metformin might function to prevent AS by activating the AMPK/mTOR pathway via lncRNA TUG1.
Atherosclerosis (AS) is a chronic disease that can be caused by multiple factors. Autophagy has become a new research interest with the increased understanding of the pathogenesis of AS. Studies have shown that autophagy is closely related to cancer, neurodegenerative diseases, and cardiovascular diseases.1–3 The activation of autophagy in vascular wall cells can protect endothelial cells and smooth muscle cells from damage caused by risk factors and contribute to the stability of plaques, whereas the inhibition of autophagy can accelerate apoptosis, necrosis, and aging, thus making plaques more vulnerable.4Long non-coding RNAs (lncRNAs) are a class of RNA molecules with a length of more than 200 nt that do not encode proteins.5,6 Recent studies have suggested that lncRNAs can participate in the onset and development of various types of cardiovascular diseases7,8 including heart failure, myocardial hypertrophy, heart metabolic disease, myocardial infarction, and AS. For example, the lncRNA NEXN-AS1 has been shown to mitigate AS by regulating activity of the actin-binding protein NEXN.9 The lncRNA taurine up-regulated gene 1 (TUG1) is located on chromosome 22q12.2 and has a length of 7.1 kb; it was originally identified in taurine-treated mouse retinal cells.10 Increasing evidence indicates that the dysregulation of TUG1 is involved in the development of a variety of diseases including cancer, ischemic stroke, and diabetes.11–14 However, there is limited knowledge on the function of TUG1 at the molecular level, as well as its exact role in AS.AMP kinase (AMPK) is a key energy sensor that recognizes ATP in cells; it is activated by hepatic protein kinase B1 under conditions of starvation or energy consumption and is a negative regulator of mammalian target of rapamycin (mTOR). The inhibition of mTOR phosphorylation mediated by AMPK phosphorylation can induce autophagy in many different cell types.15,16 Metformin is a widely used antidiabetic drug used to treat patients with type 2 diabetes mellitus. Studies have shown that it can exert protective effects against cardiovascular diseases; specifically, metformin can activate autophagy and provide cardioprotective effects in δ-sarcoglycan-deficient hearts.17 Further, metformin represses cardiac apoptosis through inhibition of the Forkhead box O1 (FoxO1) pathway.18 In addition, clinical trials have shown that metformin has anti-AS properties,19–21 providing data for its potential use for the primary prevention of AS. However, the exact mechanism through which metformin inhibits AS via TUG1, and the mechanism associated with the TUG1-modulated activation of autophagy is currently unknown. Accordingly, the present study aimed to observe the correlation between metformin and TUG1 expression in vitro, as well as their effects on proliferation, migration, and autophagy in vascular wall cells. Then, in vivo experiments were performed to verify the anti-AS effect of metformin and TUG1 to provide a new strategy for the prevention and treatment of AS.
Materials and Methods
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were purchased from the Xiangya Cell Bank of Central South University (Changsha, China). Cells were cultivated in RPMI1640 medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Biological Industries, Beit-Haemek, Israel) and 1% penicillin/streptomycin (Solarbio, Beijing, China) at 37 °C in an atmosphere containing 5% CO2.
Plasmid Construction and Transfection
TUG1 small interfering RNA (si-TUG1 #1, #2), siRNA negative control (si-NC), adeno-associated virus carrying TUG1 shRNA (sh-TUG1), and empty vector (sh-NC) were purchased from GenePharma (Shanghai, China). CRISPR/cas9 single guide RNA with TUG1 overexpression (sg-TUG1 #1, #2, #3) and the corresponding control (sg-NC) were purchased from Syngentech (Beijing, China). The sequences of siRNA/shRNA/sgRNA are listed in . Transfection was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.
Clinical Blood Samples
Thirty-eight individuals (male, 22 cases; female, 16 cases; age, 50–75 years) without coronary heart disease (CHD) were enrolled as healthy controls. In addition, 35 cases (male, 24 cases; female, 11 cases; age, 50–75 years) of patients with CHD diagnosed by coronary angiography in Guizhou Provincial People’s Hospital (Guiyang, China) were enrolled from June 2018 to August 2018. The inclusion criterium was at least one major coronary artery showing more than 80% stenosis for patients with stable angina pectoris. The exclusion criteria were as follows: (1) unstable angina or myocardial infarction; (2) complicated with other organic heart diseases; (3) combined with severe liver disease, kidney diseases, familial hypercholesterolemia, malignant tumors, or inflammatory diseases. The study protocol was approved by the Human Ethics Committee Review Board at the Guizhou Provincial People’s Hospital. Oral informed consent was obtained from each patient (Ethics approval No (2019)068).
Cell Counting Kit-8 Assay
HUVECs were plated in 96-well culture plates (3 × 103 cells/well) either immediately or 24 h after transfection. After treatment with different concentrations of metformin (Solarbio) for 24, 48, or 72 h, 10 μL of cell counting kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was added to each well and cells were incubated for 2 h at 37 °C. The absorbance at 450 nm was measured using a microplate reader (Bio-Tek, Winooski, VT, USA).
5-Ethynyl-2′-Deoxyuridine Assay
Cells were seeded in 96-well culture plates (3 × 103 cells/well) and exposed to media with metformin or transfected with si-TUG1 for 48 h. Thereafter, cells were treated with 5-ethynyl-2′-deoxyuridine (EdU; Ribobio, Guangzhou, China) for 2 h at 37 °C. Then, cells were fixed and exposed to 1× Apollo reaction for 30 min and stained with Hoechst 33,342 for 30 min. Cells were visualized with a fluorescent microscope (100×; Olympus, Tokyo, Japan). The proliferation rate of cells was evaluated based on the proportion of EdU-positive nuclei (red) to blue nuclei.
Wound Healing Assay
Cells with or without transfection were cultured in 6-well culture plates. After reaching 90% confluence, the cells were scratched with a 200-µL pipette tip, washed with PBS, and cultured in low-serum 1640 medium. Wounds were observed under an inverted microscope (40×, Olympus) and photographed at different time points. ImageJ software (NIH, Bethesda, MD, USA) was used to measure the wound areas.
mRFP-GFP-LC3 Staining
Cells were cultured on confocal culture dishes and transfected with control vector or the mRFP-GFP-LC3 lentiviral vector (2.65 × 108 PFU/mL, SyngenTech) for 48 h. Then, the cells were incubated with metformin or transfected with si-TUG1 for 24 h and fixed with 4% paraformaldehyde. The expression of monomeric red fluorescence protein (mRFP) and green fluorescence protein (GFP) was viewed under a laser scanning confocal microscope (630×, Carl Zeiss, Oberkochen, Germany).
Quantitative Real-Time Reverse Transcriptase PCR
RNA from peripheral blood according to the instructions of the kit (Bioteke, Beijing, China). Total RNA from cells/tissues was isolated with TRIzol reagent (Invitrogen), For quantitative reverse transcriptase PCR (qRT-PCR), 1000 ng total RNA was reverse-transcribed into first-strand cDNA using a PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan). RNA expression was examined by qRT-PCR using a Two Step SYBR PrimeScript RT-PCR Kit (Takara) with the Illumina P05775 system (Illumina, San Diego, CA, USA). The primer sequences used are listed as follows: lncRNA TUG1 (human): forward 5′-CCACTTTGTCACAAGAGAAGGC-3′, reverse 5′-CACAAATTCCCATCATTCCC-3′; lncRNA TUG1 (rat): forward 5′-TGCTGAAGTTGTTTGCCTGC-3′, reverse 5′-TCCTTGGTGGAATTGGGCAC-3′; GAPDH (human): forward 5′-TCACCATCTTCCCAGGAGCGAG-3′, reverse 5′-TGTCGCTGTTGAAGTCAGAG-3′; GAPDH (rat): forward 5′-GGTGAAGGTCGGTGTGAACG-3′ and reverse 5′-CCACTTTGTCACAAGAGAAGGC-3′. Relative gene expression was calculated using the 2−ΔΔCt method with GAPDH used as a normalization control.22
Western Blotting
Cells were lysed with RIPA buffer (Solarbio) supplemented with a complete protease inhibitor cocktail (KangChen, Shanghai, China) for 30 min on ice. Then, the cell debris was collected and centrifuged at 12,000 g for 15 min. The protein concentration was detected using the bicinchoninic acid (BCA) Protein assay kit (Beyotime, Beijing, China). Equal concentrations of protein extracts were separated by 8–12% SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked with 5% nonfat milk for 2 h and incubated with primary antibodies against LC3II, P62, AMPK, mTOR, and ATG3 (1:1000, Abcam, Cambridge, UK), p-AMPK (Thr172) and p-mTOR (Ser2448) (1:1000, Cell Signal Technology, Danvers, MA, USA), and GAPDH (1:1000, Goodhere, Inc., Hangzhou, China) at 4 °C overnight. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (1:5000; Cell Signaling Technology, Danvers, MA, USA) for 1.5 h at room temperature. An enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) was used to visualize the blots, and protein bands were quantified using ImageJ software.
Laboratory Animals and Grouping
A total of 70 male Wistar rats weighing 170 ± 10 g were purchased from Changsha Tianqin Biotechnology Co., Ltd (License number: SCXK (Xiang) 2014–0011, Changsha, China). All animal experiments were approved by the Experimental Animal Ethics Committee of Guizhou Provincial People’s Hospital.The rats were randomly divided into seven groups with 10 rats in each group as follows: control group (no AS), AS model group, sh-NC group, sh-TUG1 group, metformin group, metformin + sh-TUG1 group, and atorvastatin positive control group. Rats in the control group were fed a normal diet, whereas all other rats were fed a high-fat diet and vitamin D3 to induce AS (the composition of the rat diet is listed in ) and was in accordance with the literature.23,24,25 Food was provided adlibitum. After allowing the rats to acclimatize for 30 days, 40 μL sh-TUG1 (1.1 × 1013 vector genomes/mL) or empty vector was injected through the sublingual vein of rats in the corresponding group. Furthermore, in the metformin group, rats were given 100 mg/kg/day metformin (Bristol-Myers Squibb, New York, USA) via intragastrical administration for 30 days. Rats in the atorvastatin positive control group were given 2.1 mg/kg/day atorvastatin (Jialin Pharmaceutical, Beijing, China) via intragastrical administration for 30 days. Except those of the control group, the other rats were maintained on a high-fat diet, and all rats were then sacrificed for further data analysis. All animal studies were approved by the Ethics Committee of Guizhou Provincial of People’s Hospital, and conformed to the Guide for Care and Use of Laboratory Animals by the National Institutes of Health (NIH).
Hematoxylin and Eosin Staining
To observe the changes in the aortic root tissue morphology, hematoxylin and eosin staining (H&E) was performed as follows. Tissues fixed in 4% paraformaldehyde were placed in decreasing concentrations of alcohol, cleared with xylene, dipped in wax, embedded in wax blocks, and sliced into 5-μm sections. Briefly, the sections were dewaxed, stained with hematoxylin followed by 1% hydrochloric acid alcohol, stained with eosin solution, dehydrated with a gradient alcohol series, and cleared with xylene. The slides were then observed using a microscope (40×, Olympus) to identify pathological changes.
Immunohistochemistry
Paraffin-embedded artery tissue sections were deparaffinized in xylene and rehydrated through a graded series of ethanol solutions (100–70%). Sections were heated in EDTA (Zsgb-bio, Beijing, China) for 15 min and incubated for 10 min in 3% H2O2. Thereafter, sections were blocked with 10% goat serum (Bioss, Beijing, China) for 30 min at 37 °C. The slides were then incubated separately with primary antibodies against LC3 or p62 (1:100, Proteintech, Wuhan, China) at 4 °C overnight. The next day, the IgG-HRP conjugated secondary antibody (Bioss) was added and samples were incubated for 1 h at room temperature, followed by the addition of 3, 3′-diaminobenzidine (DAB, Zsgb-bio) and hematoxylin counterstain. Tissues were then microscopically observed and photographed (200×, Olympus). DAB staining was analyzed by two pathologists as previously described.26
Statistical Analysis
Data were calculated as the mean from at least three independent experiments. Numerical data are presented as the mean ± SD. All statistical analyses were performed using GraphPad 7.0 software (GraphPad Software, Inc., La Jolla, CA, USA). When data obeyed normal distribution, Student’s t test was used to analyze difference between two groups, analysis of variance (ANOVA) was performed for three or more groups. When data did not obey normal distribution, Mann–Whitney was used to analyze difference between two groups. Kruskal–Wallis was used in animal experiment. A value of P < 0.05 was considered statistically significant.
Results
Expression Levels of TUG1 in Patients with CHD and in Cells Incubated with Metformin
To investigate the correlation between lncRNA TUG1 and AS, qRT-PCR was used to detect TUG1 expression in the peripheral blood of healthy controls and patients with CHD. The results suggested that TUG1 expression was robustly upregulated in patients with CHD, compared with that in healthy controls (Figure 1A). Then, cells were incubated with metformin (10 mmol/L) for 24, 48, or 72 h. As shown in Figure 1B, the expression of TUG1 was downregulated in a time-dependent manner.
Figure 1
Expression levels of TUG1 in patients with coronary heart disease (CHD) and in cells incubated with metformin.
Notes: (A) qRT-PCR was used to detect TUG1 expression in the peripheral blood of healthy controls (n = 38) and patients with CHD (n = 35). (B) Expression levels of TUG1 after HUVECs were incubated with metformin for 24, 48, or 72 h. Data are presented as the mean ± SD. **p < 0.01 vs the control group.
Expression levels of TUG1 in patients with coronary heart disease (CHD) and in cells incubated with metformin.Notes: (A) qRT-PCR was used to detect TUG1 expression in the peripheral blood of healthy controls (n = 38) and patients with CHD (n = 35). (B) Expression levels of TUG1 after HUVECs were incubated with metformin for 24, 48, or 72 h. Data are presented as the mean ± SD. **p < 0.01 vs the control group.Abbreviations:
TUG1, taurine up-regulated gene 1; CHD, coronary heart disease; qRT-PCR, quantitative reverse transcription PCR; HUVECs, human umbilical vein endothelial cells; SD, standard deviation.
Metformin Suppresses Proliferation and Migration while Induces Autophagy in HUVECs
First, HUVECs were incubated with metformin at different concentrations (0, 2, 5, 10, 15, or 20 mmol/L) for 24, 48, or 72 h. The CCK-8 assay results indicated that metformin had a concentration-and time-dependent inhibitory effect on cell growth in HUVECs (Figure 2A). A concentration of 10 mmol/L metformin effectively inhibited cell proliferation without causing changes in cell morphology; thus, 10 mmol/L was the drug concentration used for subsequent experiments. Furthermore, the EdU assay showed that cells incubated with metformin for 48 h showed significantly supressed proliferation ability compared with that of the control group (Figure 2B). Then, we performed a wound healing assay, the results showed that metformin could reduce HUVEC migration in a time-dependent manner (Figure 2C). To validate the autophagy-inducing effects of metformin on HUVECs, we transfected the cells with mRFP-GFP-LC3 lentivirus to observe LC3 dots. As shown in Figure 2D, the numbers of both yellow and red dots were markedly increased in metformin-treated HUVECs compared with those in the control group. Lastly, the cells were treated with metformin at different time points (0, 3, 6, 12, 24, 36 h), we found that it could downregulate the expression of p62 and increase the ratio of LC3II/LC3I (Figure 2E). Moreover, the changes in p62 expression and the LC3II/LC3I ratio were most prominent at 24 h; thus, we chose 24 h as the incubation time for subsequent Western blotting experiments.
Figure 2
Metformin (Met) suppresses proliferation and migration while induces autophagy in HUVECs.
Notes: (A) The CCK-8 and (B) EdU assays were performed to measure the proliferation of HUVECs treated with or without Met. (C) A wound healing assay was performed to analyze the effect of Met on the migration of HUVECs. (D) mRFP-GFP-LC3 staining was used to observe the induction of autophagosomes and autolysosomes by Met. (E) Western blotting was used to analyze the levels of p62 and LC3 at different time points after cells were treated with Met. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group.
Abbreviations: Met, Metformin; HUVECs, human umbilical vein endothelial cells; CCK-8, cell counting kit-8; EdU, 5-ethynyl-2′-deoxyuridine; SD, standard deviation.
Metformin (Met) suppresses proliferation and migration while induces autophagy in HUVECs.Notes: (A) The CCK-8 and (B) EdU assays were performed to measure the proliferation of HUVECs treated with or without Met. (C) A wound healing assay was performed to analyze the effect of Met on the migration of HUVECs. (D) mRFP-GFP-LC3 staining was used to observe the induction of autophagosomes and autolysosomes by Met. (E) Western blotting was used to analyze the levels of p62 and LC3 at different time points after cells were treated with Met. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group.Abbreviations: Met, Metformin; HUVECs, human umbilical vein endothelial cells; CCK-8, cell counting kit-8; EdU, 5-ethynyl-2′-deoxyuridine; SD, standard deviation.
si-TUG1 Suppresses Proliferation and Migration while Induces Autophagy in HUVECs
Next, HUVECs were transfected with si-TUG1(#1, #2) to knock down the expression of TUG1. Following transfection, TUG1 expression decreased sharply in the si-TUG1 groups compared with that in the si-NC group (Figure 3A). Similar to the results of metformin treatment, we found that knockdown of TUG1 expression by siRNA inhibited proliferation (Figure 3B, C and F) and migration (Figure 3D and G), while promoted autophagy (Figure 3E, H and I) in HUVECs.
Figure 3
si-TUG1 suppresses proliferation and migration while induces autophagy in HUVECs.
Notes: (A) Transfection efficiency of si-TUG1 (#1, #2). (B) The CCK-8 and (C and F) EdU assays were performed to determine the proliferation of HUVECs treated with or without si-TUG1. (D and G) A wound healing assay was performed to detect the effect of si-TUG1 on the migration ability of HUVECs. (E and H) mRFP-GFP-LC3 staining was used to observe the induction of autophagosomes and autolysosomes by si-TUG1. (I) Western blotting was used to analyze the levels of p62 and LC3 induced by si-TUG1. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the si-NC group.
Abbreviations: siRNA, small interfering RNA; TUG1, taurine up-regulated gene 1; HUVECs, human umbilical vein endothelial cells; CCK-8, cell counting kit-8; EdU, 5-ethynyl-2′-deoxyuridine; SD, standard deviation; NC, normal control.
si-TUG1 suppresses proliferation and migration while induces autophagy in HUVECs.Notes: (A) Transfection efficiency of si-TUG1 (#1, #2). (B) The CCK-8 and (C and F) EdU assays were performed to determine the proliferation of HUVECs treated with or without si-TUG1. (D and G) A wound healing assay was performed to detect the effect of si-TUG1 on the migration ability of HUVECs. (E and H) mRFP-GFP-LC3 staining was used to observe the induction of autophagosomes and autolysosomes by si-TUG1. (I) Western blotting was used to analyze the levels of p62 and LC3 induced by si-TUG1. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the si-NC group.Abbreviations: siRNA, small interfering RNA; TUG1, taurine up-regulated gene 1; HUVECs, human umbilical vein endothelial cells; CCK-8, cell counting kit-8; EdU, 5-ethynyl-2′-deoxyuridine; SD, standard deviation; NC, normal control.
Metformin Activates the AMPK/mTOR Pathway in HUVECs via lncRNA TUG1
First, the transfection efficiency of TUG1 overexpression (OE #1, #2, #3) was measured by qRT-PCR (Figure 4A). OE #1 resulted in the greatest overexpression efficiency and was thus selected for subsequent experiments. Second, we found that metformin could increase the expression of p-AMPK/AMPK, ATG3, and LC3II/LC3I and decrease the expression of p-mTOR/mTOR and p62 (Figure 4B–G). In addition, the effects of metformin on the AMPK/mTOR pathway were partially reversed by OE #1 transfection. Third, CCK-8 and wound healing assays were conducted (Figure 4H and I); here, OE #1 obviously reversed the metformin-induced effects on proliferation and migration.
Figure 4
Metformin (Met) activates the AMPK/mTOR pathway in HUVECs via lncRNA TUG1.
Notes: (A) Transfection efficiency of sg-TUG1 (OE #1,#2,#3). (C) Western blotting was used to detect (B) p-AMPK/AMPK, (D) p-mTOR/mTOR, (E) ATG3, (F) p62, and (G) LC3II/LC3I protein levels after cells were incubated with Met or transfected with OE#1. (H) CCK-8 and (I) wound healing assays were performed to determine the cell viability and migration ability, respectively, after cells were incubated with Met or transfected with OE #1. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group.
Abbreviations: Met, metformin; HUVECs, human umbilical vein endothelial cells; lncRNA, long non-coding RNA; TUG1, taurine up-regulated gene 1; sg-TUG1, single guide taurine up-regulated gene 1; OE, overexpression; CCK-8, cell counting kit-8; SD, standard deviation.
Metformin (Met) activates the AMPK/mTOR pathway in HUVECs via lncRNA TUG1.Notes: (A) Transfection efficiency of sg-TUG1 (OE #1,#2,#3). (C) Western blotting was used to detect (B) p-AMPK/AMPK, (D) p-mTOR/mTOR, (E) ATG3, (F) p62, and (G) LC3II/LC3I protein levels after cells were incubated with Met or transfected with OE#1. (H) CCK-8 and (I) wound healing assays were performed to determine the cell viability and migration ability, respectively, after cells were incubated with Met or transfected with OE #1. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group.Abbreviations: Met, metformin; HUVECs, human umbilical vein endothelial cells; lncRNA, long non-coding RNA; TUG1, taurine up-regulated gene 1; sg-TUG1, single guide taurine up-regulated gene 1; OE, overexpression; CCK-8, cell counting kit-8; SD, standard deviation.
si-TUG1 Induces Autophagy in HUVECs via the AMPK/mTOR Pathway
Western blotting was then used to detect the protein levels of AMPK, p-AMPK, mTOR, p-mTOR, ATG3, p62, and LC3 after cells were transfected with si-TUG1 (#2, 50 nm). si-TUG1 (#2) resulted in better knockdown efficiency and was thus selected for subsequent experiments. As expected, the expression of p-AMPK/AMPK, ATG3, and LC3II/LC3I was upregulated, whereas that of p-mTOR/mTOR and p62 was downregulated by si-TUG1 (Figure 5A–F). However, the effect of si-TUG1 on the AMPK/mTOR pathway was reversed by treatment with Compound C (C.C, 50 μmol/L, an AMPK inhibitor), which suggested that TUG1 knockdown could activate the AMPK/mTOR pathway in HUVECs. In addition, as shown in Figure 5G and H, C.C reversed the effects on proliferation and migration induced by si-TUG1. Figure 5I shows the technical rote of metformin and lncRNA TUG1 in AS.
Figure 5
si-TUG1 induces autophagy in HUVECs via the AMPK/mTOR pathway.
Notes: (A) Western blotting was used to detect (B) p-AMPK/AMPK, (C) p-mTOR/mTOR, (D) ATG3, (E) p62, and (F) LC3II/LC3I protein levels after cells were transfected with si-TUG1 or incubated with Compound C (C,C). (G) CCK-8 and (H) wound healing assay were performed to measure the cell viability and migration ability, respectively, after cells were transfected with si-TUG1 or incubated with C,C. (I) The technical rote of metformin and lncRNA TUG1 in atherosclerosis (AS). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group.
Abbreviations: siRNA, small interfering RNA; TUG1, taurine up-regulated gene 1; HUVECs, human umbilical vein endothelial cells; CCK-8, cell counting kit-8; lncRNA, long non-coding RNA; SD, standard deviation.
si-TUG1 induces autophagy in HUVECs via the AMPK/mTOR pathway.Notes: (A) Western blotting was used to detect (B) p-AMPK/AMPK, (C) p-mTOR/mTOR, (D) ATG3, (E) p62, and (F) LC3II/LC3I protein levels after cells were transfected with si-TUG1 or incubated with Compound C (C,C). (G) CCK-8 and (H) wound healing assay were performed to measure the cell viability and migration ability, respectively, after cells were transfected with si-TUG1 or incubated with C,C. (I) The technical rote of metformin and lncRNA TUG1 in atherosclerosis (AS). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group.Abbreviations: siRNA, small interfering RNA; TUG1, taurine up-regulated gene 1; HUVECs, human umbilical vein endothelial cells; CCK-8, cell counting kit-8; lncRNA, long non-coding RNA; SD, standard deviation.
Anti-AS Effects of Metformin and lncRNA TUG1 in vivo
Finally, we assessed the effects of metformin and lncRNA TUG1 in the AS rat model. Histopathological changes were observed through H&E staining, and aortic root lesion sizes were significantly decreased in the metformin and sh-TUG1 groups compared with those in the AS model or empty vector group, demonstrating that metformin and sh-TUG1 have protective effects on high-fat diet-induced AS injury (Figure 6A and C). Immunohistochemistry was then used to detect the expression of autophagy-related proteins. Compared with that in the control group, the expression of p62 increased, whereas that of LC3 decreased, in the model and sh-NC groups. Furthermore, compared with levels in the model or sh-NC group, p62 was downregulated, whereas LC3 was upregulated, in the metformin, sh-TUG1, metformin + sh-TUG1, and atorvastatin groups (Figure 6B and D). Through qRT-PCR, we demonstrated that compared with that in the control group, the expression of TUG1 was increased in the model group and sh-NC group. Additionally, compared with those in the model or sh-NC group, the levels of TUG1 were decreased in the metformin, sh-TUG1, and metformin + sh-TUG1 groups (Figure 6E). Western blotting indicated that compared with that in the control group, the ratio of p-mTOR/mTOR was increased in the model or sh-NC group. Further, compared with that in the model or sh-NC group, the ratio of p-AMPK/AMPK was increased, whereas that of p-mTOR/mTOR was decreased, in the metformin, sh-TUG1, metformin + sh-TUG1, and atorvastatin groups (Figure 6F).
Figure 6
Anti-atherosclerosis (AS) effects of metformin and lncRNA TUG1 in vivo.
Notes: (A and C) The histopathological changes in the aortic root of rats in each group were observed by H&E staining. (B and D) Immunohistochemistry was performed to detect the expression of autophagy-related proteins p62 and LC3. (E) The expression of TUG1 in each group was measured by qRT-PCR. (F)The expression levels of proteins related to the AMPK/mTOR pathway in each group were measured by Western blotting. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group. #p < 0.05, ##p < 0.01 vs the model group. &p < 0.05, &&p < 0.01 vs the sh-NC group.
Abbreviations: lncRNA, long non-coding RNA; TUG1, taurine up-regulated gene 1; H&E staining, hematoxylin and eosin staining; qRT-PCR, quantitative reverse transcription PCR; SD, standard deviation; NC, normal control.
Anti-atherosclerosis (AS) effects of metformin and lncRNA TUG1 in vivo.Notes: (A and C) The histopathological changes in the aortic root of rats in each group were observed by H&E staining. (B and D) Immunohistochemistry was performed to detect the expression of autophagy-related proteins p62 and LC3. (E) The expression of TUG1 in each group was measured by qRT-PCR. (F)The expression levels of proteins related to the AMPK/mTOR pathway in each group were measured by Western blotting. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 vs the control group. #p < 0.05, ##p < 0.01 vs the model group. &p < 0.05, &&p < 0.01 vs the sh-NC group.Abbreviations: lncRNA, long non-coding RNA; TUG1, taurine up-regulated gene 1; H&E staining, hematoxylin and eosin staining; qRT-PCR, quantitative reverse transcription PCR; SD, standard deviation; NC, normal control.
Discussion
Atherosclerotic diseases such as CHD remain the leading cause of death worldwide; the lifetime risk of CHD is 67% in humans over 55 years of age.27–29 Studies have shown that lncRNAs are vital regulatory factors in the progression of AS.30–32 Thus, defining their functions might help to identify novel diagnostic and therapeutic targets for AS. In this study, we found that lncRNA TUG1 was significantly upregulated in the peripheral blood of patients with CHD compared with healthy individuals. This indicates that lncRNA TUG1 may promote CHD progression.Autophagy is the process through which cytoplasmic components such as proteins and organelles that need to be degraded are encapsulated and eventually transported to the lysosomes for degradation. Studies have shown that autophagy inhibits the progression of AS, whereas defective autophagy in vascular wall cells enhances its progression.33,34 In this study, we found that metformin and si-TUG1 reduced the proliferation and migration of HUVECs. There is a strong connection between autophagy and vascular wall cell proliferation/migration in AS. Research has suggested that autophagy defects in vascular smooth muscle cells might induce cell proliferation and migration, leading to the acceleration of AS progression.35 However, the upregulation of autophagy in vascular wall cells is known to reduce proliferation and inhibit fibrosis.36Markers involved in autophagy are classified as autophagy-related genes (ATGs), and approximately 30 ATGs are known to participate in different stages of autophagy,37–39 including Beclin-1, ATG3, ATG5, ATG7, ATG8, and ATG12.40 During autophagy, LC3I is converted to LC3II and p62 enters the autophagosome to be degraded; thus, the LC3II/I ratio and p62 are commonly used as indicators of autophagy.41,42 To explore the effect of TUG1 on autophagy, we measured the levels of autophagy-related proteins and several major signaling pathways. We found that the expression of p-mTOR/mTOR and p62 was reduced when TUG1 was knocked down, whereas that of p-AMPK/AMPK and LC3II/LC3I was elevated, which was accompanied by an increase in ATG3 expression. C.C, which acts through the specific inhibition of p-AMPK in the AMPK/mTOR pathway, is recognized as an autophagy inhibitor. We found that the changes in protein expression induced by si-TUG1 could be reversed by C.C; at the same time, C.C successfully reversed the changes in proliferation and migration mediated by si-TUG1. These data suggest that si-TUG1 activates autophagy via the AMPK/mTOR pathway to suppress proliferation and migration.It has been reported that metformin can be used to treat diseases through the regulation of lncRNA expression.43,44 Because metformin was found to attenuate TUG1 expression in a time-dependent manner in the present study, we further investigated its correlation with TUG1 expression. The results indicated that metformin activates autophagy via lncRNA TUG1 in HUVECs.Finally, we established an AS rat model. As expected, immunohistochemistry and Western blotting in the metformin and sh-TUG1 groups were consistent with the results of our in vitro experiments. Interestingly, immunohistochemistry showed an increase in the expression of p62 and a decrease in that of LC3 in the AS model group compared with levels in the control group, indicating that autophagy was somewhat disrupted in rats of the AS model group. Furthermore, we found that atorvastatin treatment activated the AMPK/mTOR pathway; however, to the best of our knowledge, the role of atorvastatin in preventing AS via the AMPK/mTOR pathway has not yet been reported.
Conclusion
In summary, we provide clear evidence that metformin attenuates the progression of AS via lncRNA TUG1. This study not only expands our understanding of the role of metformin in preventing AS, but also provides new perspectives into its molecular mechanisms. TUG1 is also suggested to be potential a therapeutic target for the prevention of AS.
Authors: Sebastian Cremer; Katharina M Michalik; Ariane Fischer; Larissa Pfisterer; Nicolas Jaé; Carla Winter; Reinier A Boon; Marion Muhly-Reinholz; David John; Shizuka Uchida; Christian Weber; Wolfgang Poller; Stefan Günther; Thomas Braun; Daniel Y Li; Lars Maegdefessel; Ljubica Perisic Matic; Ulf Hedin; Oliver Soehnlein; Andreas Zeiher; Stefanie Dimmeler Journal: Circulation Date: 2019-03-05 Impact factor: 29.690
Authors: Andrea Natali; Lorenzo Nesti; Elena Venturi; Angela C Shore; Faisel Khan; Kim Gooding; Phillip E Gates; Helen C Looker; Fiona Dove; Isabel Goncalves; Margaretha Persson; Jan Nilsson Journal: Diabetes Obes Metab Date: 2018-10-02 Impact factor: 6.577
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Gad Galili; Inmaculada Galindo; Maria F Galindo; Giovanna Galliciotti; Lorenzo Galluzzi; Luca Galluzzi; Vincent Galy; Noor Gammoh; Sam Gandy; Anand K Ganesan; Swamynathan Ganesan; Ian G Ganley; Monique Gannagé; Fen-Biao Gao; Feng Gao; Jian-Xin Gao; Lorena García Nannig; Eleonora García Véscovi; Marina Garcia-Macía; Carmen Garcia-Ruiz; Abhishek D Garg; Pramod Kumar Garg; Ricardo Gargini; Nils Christian Gassen; Damián Gatica; Evelina Gatti; Julie Gavard; Evripidis Gavathiotis; Liang Ge; Pengfei Ge; Shengfang Ge; Po-Wu Gean; Vania Gelmetti; Armando A Genazzani; Jiefei Geng; Pascal Genschik; Lisa Gerner; Jason E Gestwicki; David A Gewirtz; Saeid Ghavami; Eric Ghigo; Debabrata Ghosh; Anna Maria Giammarioli; Francesca Giampieri; Claudia Giampietri; Alexandra Giatromanolaki; Derrick J Gibbings; Lara Gibellini; Spencer B Gibson; Vanessa Ginet; Antonio Giordano; Flaviano Giorgini; Elisa Giovannetti; Stephen E Girardin; Suzana Gispert; Sandy Giuliano; Candece L Gladson; Alvaro Glavic; Martin Gleave; 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Jörg Höhfeld; Marina K Holz; Yonggeun Hong; David A Hood; Jeroen Jm Hoozemans; Thorsten Hoppe; Chin Hsu; Chin-Yuan Hsu; Li-Chung Hsu; Dong Hu; Guochang Hu; Hong-Ming Hu; Hongbo Hu; Ming Chang Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Ya Hua; Canhua Huang; Huey-Lan Huang; Kuo-How Huang; Kuo-Yang Huang; Shile Huang; Shiqian Huang; Wei-Pang Huang; Yi-Ran Huang; Yong Huang; Yunfei Huang; Tobias B Huber; Patricia Huebbe; Won-Ki Huh; Juha J Hulmi; Gang Min Hur; James H Hurley; Zvenyslava Husak; Sabah Na Hussain; Salik Hussain; Jung Jin Hwang; Seungmin Hwang; Thomas Is Hwang; Atsuhiro Ichihara; Yuzuru Imai; Carol Imbriano; Megumi Inomata; Takeshi Into; Valentina Iovane; Juan L Iovanna; Renato V Iozzo; Nancy Y Ip; Javier E Irazoqui; Pablo Iribarren; Yoshitaka Isaka; Aleksandra J Isakovic; Harry Ischiropoulos; Jeffrey S Isenberg; Mohammad Ishaq; Hiroyuki Ishida; Isao Ishii; Jane E Ishmael; Ciro Isidoro; Ken-Ichi Isobe; Erika Isono; Shohreh Issazadeh-Navikas; Koji Itahana; Eisuke Itakura; Andrei I Ivanov; 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Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Bertrand Kaeffer; Katarina Kågedal; Alon Kahana; Shingo Kajimura; Or Kakhlon; Manjula Kalia; Dhan V Kalvakolanu; Yoshiaki Kamada; Konstantinos Kambas; Vitaliy O Kaminskyy; Harm H Kampinga; Mustapha Kandouz; Chanhee Kang; Rui Kang; Tae-Cheon Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Marc Kantorow; Maria Kaparakis-Liaskos; Orsolya Kapuy; Vassiliki Karantza; Md Razaul Karim; Parimal Karmakar; Arthur Kaser; Susmita Kaushik; Thomas Kawula; A Murat Kaynar; Po-Yuan Ke; Zun-Ji Ke; John H Kehrl; Kate E Keller; Jongsook Kim Kemper; Anne K Kenworthy; Oliver Kepp; Andreas Kern; Santosh Kesari; David Kessel; Robin Ketteler; Isis do Carmo Kettelhut; Bilon Khambu; Muzamil Majid Khan; Vinoth Km Khandelwal; Sangeeta Khare; Juliann G Kiang; Amy A Kiger; Akio Kihara; Arianna L Kim; Cheol Hyeon Kim; Deok Ryong Kim; Do-Hyung Kim; Eung Kweon Kim; Hye Young Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; Jin Hyoung Kim; Kwang Woon Kim; Michael D Kim; Moon-Moo Kim; Peter K Kim; Seong Who Kim; Soo-Youl Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Jason S King; Karla Kirkegaard; Vladimir Kirkin; Lorrie A Kirshenbaum; Shuji Kishi; Yasuo Kitajima; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Rudolf A Kley; Walter T Klimecki; Michael Klinkenberg; Jochen Klucken; Helene Knævelsrud; Erwin Knecht; Laura Knuppertz; Jiunn-Liang Ko; Satoru Kobayashi; Jan C Koch; Christelle Koechlin-Ramonatxo; Ulrich Koenig; Young Ho Koh; Katja Köhler; Sepp D Kohlwein; Masato Koike; Masaaki Komatsu; Eiki Kominami; Dexin Kong; Hee Jeong Kong; Eumorphia G Konstantakou; Benjamin T Kopp; Tamas Korcsmaros; Laura Korhonen; Viktor I Korolchuk; Nadya V Koshkina; Yanjun Kou; Michael I Koukourakis; Constantinos Koumenis; Attila L Kovács; Tibor Kovács; Werner J Kovacs; Daisuke Koya; Claudine Kraft; Dimitri Krainc; Helmut Kramer; Tamara Kravic-Stevovic; Wilhelm Krek; Carole Kretz-Remy; Roswitha Krick; Malathi Krishnamurthy; Janos Kriston-Vizi; Guido Kroemer; Michael C Kruer; Rejko Kruger; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Christian Kuhn; Addanki Pratap Kumar; Anuj Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Rakesh Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Atsushi Kuno; Sheng-Han Kuo; Jeff Kuret; Tino Kurz; Terry Kwok; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert R La Spada; Frank Lafont; Tim Lahm; Aparna Lakkaraju; Truong Lam; Trond Lamark; Steve Lancel; Terry H Landowski; Darius J R Lane; Jon D Lane; Cinzia Lanzi; Pierre Lapaquette; Louis R Lapierre; Jocelyn Laporte; Johanna Laukkarinen; Gordon W Laurie; Sergio Lavandero; Lena Lavie; Matthew J LaVoie; Betty Yuen Kwan Law; Helen Ka-Wai Law; Kelsey B Law; Robert Layfield; Pedro A Lazo; Laurent Le Cam; Karine G Le Roch; Hervé Le Stunff; Vijittra Leardkamolkarn; Marc Lecuit; Byung-Hoon Lee; Che-Hsin Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Hsinyu Lee; Jae Keun Lee; Jongdae Lee; Ju-Hyun Lee; Jun Hee Lee; Michael Lee; Myung-Shik Lee; Patty J Lee; Sam W Lee; Seung-Jae Lee; Shiow-Ju Lee; Stella Y Lee; Sug Hyung Lee; Sung Sik Lee; Sung-Joon Lee; Sunhee Lee; Ying-Ray Lee; Yong J Lee; Young H Lee; Christiaan Leeuwenburgh; Sylvain Lefort; Renaud Legouis; Jinzhi Lei; Qun-Ying Lei; David A Leib; Gil Leibowitz; Istvan Lekli; Stéphane D Lemaire; John J Lemasters; Marius K Lemberg; Antoinette Lemoine; Shuilong Leng; Guido Lenz; Paola Lenzi; Lilach O Lerman; Daniele Lettieri Barbato; Julia I-Ju Leu; Hing Y Leung; Beth Levine; Patrick A Lewis; Frank Lezoualc'h; Chi Li; Faqiang Li; Feng-Jun Li; Jun Li; Ke Li; Lian Li; Min Li; Min Li; Qiang Li; Rui Li; Sheng Li; Wei Li; Wei Li; Xiaotao Li; Yumin Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Yulin Liao; Joana Liberal; Pawel P Liberski; Pearl Lie; Andrew P Lieberman; Hyunjung Jade Lim; Kah-Leong Lim; Kyu Lim; Raquel T Lima; Chang-Shen Lin; Chiou-Feng Lin; Fang Lin; Fangming Lin; Fu-Cheng Lin; Kui Lin; Kwang-Huei Lin; Pei-Hui Lin; Tianwei Lin; Wan-Wan Lin; Yee-Shin Lin; Yong Lin; Rafael Linden; Dan Lindholm; Lisa M Lindqvist; Paul Lingor; Andreas Linkermann; Lance A Liotta; Marta M Lipinski; Vitor A Lira; Michael P Lisanti; Paloma B Liton; Bo Liu; Chong Liu; Chun-Feng Liu; Fei Liu; Hung-Jen Liu; Jianxun Liu; Jing-Jing Liu; Jing-Lan Liu; Ke Liu; Leyuan Liu; Liang Liu; Quentin Liu; Rong-Yu Liu; Shiming Liu; Shuwen Liu; Wei Liu; Xian-De Liu; Xiangguo Liu; Xiao-Hong Liu; Xinfeng Liu; Xu Liu; Xueqin Liu; Yang Liu; Yule Liu; Zexian Liu; Zhe Liu; Juan P Liuzzi; Gérard Lizard; Mila Ljujic; Irfan J Lodhi; Susan E Logue; Bal L Lokeshwar; Yun Chau Long; Sagar Lonial; Benjamin Loos; Carlos López-Otín; Cristina López-Vicario; Mar Lorente; Philip L Lorenzi; Péter Lõrincz; Marek Los; Michael T Lotze; Penny E Lovat; Binfeng Lu; Bo Lu; Jiahong Lu; Qing Lu; She-Min Lu; Shuyan Lu; Yingying Lu; Frédéric Luciano; Shirley Luckhart; John Milton Lucocq; Paula Ludovico; Aurelia Lugea; Nicholas W Lukacs; Julian J Lum; Anders H Lund; Honglin Luo; Jia Luo; Shouqing Luo; Claudio Luparello; Timothy Lyons; Jianjie Ma; Yi Ma; Yong Ma; Zhenyi Ma; Juliano Machado; Glaucia M Machado-Santelli; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; John D MacMicking; Lee Ann MacMillan-Crow; Frank Madeo; Muniswamy Madesh; Julio Madrigal-Matute; Akiko Maeda; Tatsuya Maeda; Gustavo Maegawa; Emilia Maellaro; Hannelore Maes; Marta Magariños; Kenneth Maiese; Tapas K Maiti; Luigi Maiuri; Maria Chiara Maiuri; Carl G Maki; Roland Malli; Walter Malorni; Alina Maloyan; Fathia Mami-Chouaib; Na Man; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Serge N Manié; Claudia Manzoni; Kai Mao; Zixu Mao; Zong-Wan Mao; Philippe Marambaud; Anna Maria Marconi; Zvonimir Marelja; Gabriella Marfe; Marta Margeta; Eva Margittai; Muriel Mari; Francesca V Mariani; Concepcio Marin; Sara Marinelli; Guillermo Mariño; Ivanka Markovic; Rebecca Marquez; Alberto M Martelli; Sascha Martens; Katie R Martin; Seamus J Martin; Shaun Martin; Miguel A Martin-Acebes; Paloma Martín-Sanz; Camille Martinand-Mari; Wim Martinet; Jennifer Martinez; Nuria Martinez-Lopez; Ubaldo Martinez-Outschoorn; Moisés Martínez-Velázquez; Marta Martinez-Vicente; Waleska Kerllen Martins; Hirosato Mashima; James A Mastrianni; Giuseppe Matarese; Paola Matarrese; Roberto Mateo; Satoaki Matoba; Naomichi Matsumoto; Takehiko Matsushita; Akira Matsuura; Takeshi Matsuzawa; Mark P Mattson; Soledad Matus; Norma Maugeri; Caroline Mauvezin; Andreas Mayer; Dusica Maysinger; Guillermo D Mazzolini; Mary Kate McBrayer; Kimberly McCall; Craig McCormick; Gerald M McInerney; Skye C McIver; Sharon McKenna; John J McMahon; Iain A McNeish; Fatima Mechta-Grigoriou; Jan Paul Medema; Diego L Medina; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Yide Mei; Ute-Christiane Meier; Alfred J Meijer; Alicia Meléndez; Gerry Melino; Sonia Melino; Edesio Jose Tenorio de Melo; Maria A Mena; Marc D Meneghini; Javier A Menendez; Regina Menezes; Liesu Meng; Ling-Hua Meng; Songshu Meng; Rossella Menghini; A Sue Menko; Rubem Fs Menna-Barreto; Manoj B Menon; Marco A Meraz-Ríos; Giuseppe Merla; Luciano Merlini; Angelica M Merlot; Andreas Meryk; Stefania Meschini; Joel N Meyer; Man-Tian Mi; Chao-Yu Miao; Lucia Micale; Simon Michaeli; Carine Michiels; Anna Rita Migliaccio; Anastasia Susie Mihailidou; Dalibor Mijaljica; Katsuhiko Mikoshiba; Enrico Milan; Leonor Miller-Fleming; Gordon B Mills; Ian G Mills; Georgia Minakaki; Berge A Minassian; Xiu-Fen Ming; Farida Minibayeva; Elena A Minina; Justine D Mintern; Saverio Minucci; Antonio Miranda-Vizuete; Claire H Mitchell; Shigeki Miyamoto; Keisuke Miyazawa; Noboru Mizushima; Katarzyna Mnich; Baharia Mograbi; Simin Mohseni; Luis Ferreira Moita; Marco Molinari; Maurizio Molinari; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Marco Mongillo; Martha M Monick; Serena Montagnaro; Craig Montell; Darren J Moore; Michael N Moore; Rodrigo Mora-Rodriguez; Paula I Moreira; Etienne Morel; Maria Beatrice Morelli; Sandra Moreno; Michael J Morgan; Arnaud Moris; Yuji Moriyasu; Janna L Morrison; Lynda A Morrison; Eugenia Morselli; Jorge Moscat; Pope L Moseley; Serge Mostowy; Elisa Motori; Denis Mottet; Jeremy C Mottram; Charbel E-H Moussa; Vassiliki E Mpakou; Hasan Mukhtar; Jean M Mulcahy Levy; Sylviane Muller; Raquel Muñoz-Moreno; Cristina Muñoz-Pinedo; Christian Münz; Maureen E Murphy; James T Murray; Aditya Murthy; Indira U Mysorekar; Ivan R Nabi; Massimo Nabissi; Gustavo A Nader; Yukitoshi Nagahara; Yoshitaka Nagai; Kazuhiro Nagata; Anika Nagelkerke; Péter Nagy; Samisubbu R Naidu; Sreejayan Nair; Hiroyasu Nakano; Hitoshi Nakatogawa; Meera Nanjundan; Gennaro Napolitano; Naweed I Naqvi; Roberta Nardacci; Derek P Narendra; Masashi Narita; Anna Chiara Nascimbeni; Ramesh Natarajan; Luiz C Navegantes; Steffan T Nawrocki; Taras Y Nazarko; Volodymyr Y Nazarko; Thomas Neill; Luca M Neri; Mihai G Netea; Romana T Netea-Maier; Bruno M Neves; Paul A Ney; Ioannis P Nezis; Hang Tt Nguyen; Huu Phuc Nguyen; Anne-Sophie Nicot; Hilde Nilsen; Per Nilsson; Mikio Nishimura; Ichizo Nishino; Mireia Niso-Santano; Hua Niu; Ralph A Nixon; Vincent Co Njar; Takeshi Noda; Angelika A Noegel; Elsie Magdalena Nolte; Erik Norberg; Koenraad K Norga; Sakineh Kazemi Noureini; Shoji Notomi; Lucia Notterpek; Karin Nowikovsky; Nobuyuki Nukina; Thorsten Nürnberger; Valerie B O'Donnell; Tracey O'Donovan; Peter J O'Dwyer; Ina Oehme; Clara L Oeste; Michinaga Ogawa; Besim Ogretmen; Yuji Ogura; Young J Oh; Masaki Ohmuraya; Takayuki Ohshima; Rani Ojha; Koji Okamoto; Toshiro Okazaki; F Javier Oliver; Karin Ollinger; Stefan Olsson; Daniel P Orban; Paulina Ordonez; Idil Orhon; Laszlo Orosz; Eyleen J O'Rourke; Helena Orozco; Angel L Ortega; Elena Ortona; Laura D Osellame; Junko Oshima; Shigeru Oshima; Heinz D Osiewacz; Takanobu Otomo; Kinya Otsu; Jing-Hsiung James Ou; Tiago F Outeiro; Dong-Yun Ouyang; Hongjiao Ouyang; Michael Overholtzer; Michelle A Ozbun; P Hande Ozdinler; Bulent Ozpolat; Consiglia Pacelli; Paolo Paganetti; Guylène Page; Gilles Pages; Ugo Pagnini; Beata Pajak; Stephen C Pak; Karolina Pakos-Zebrucka; Nazzy Pakpour; Zdena Palková; Francesca Palladino; Kathrin Pallauf; Nicolas Pallet; Marta Palmieri; Søren R Paludan; Camilla Palumbo; Silvia Palumbo; Olatz Pampliega; Hongming Pan; Wei Pan; Theocharis Panaretakis; Aseem Pandey; Areti Pantazopoulou; Zuzana Papackova; Daniela L Papademetrio; Issidora Papassideri; Alessio Papini; Nirmala Parajuli; Julian Pardo; Vrajesh V Parekh; Giancarlo Parenti; Jong-In Park; Junsoo Park; Ohkmae K Park; Roy Parker; Rosanna Parlato; Jan B Parys; Katherine R Parzych; Jean-Max Pasquet; Benoit Pasquier; Kishore Bs Pasumarthi; Daniel Patschan; Cam Patterson; Sophie Pattingre; Scott Pattison; Arnim Pause; Hermann Pavenstädt; Flaminia Pavone; Zully Pedrozo; Fernando J Peña; Miguel A Peñalva; Mario Pende; Jianxin Peng; Fabio Penna; Josef M Penninger; Anna Pensalfini; Salvatore Pepe; Gustavo Js Pereira; Paulo C Pereira; Verónica Pérez-de la Cruz; María Esther Pérez-Pérez; Diego Pérez-Rodríguez; Dolores Pérez-Sala; Celine Perier; Andras Perl; David H Perlmutter; Ida Perrotta; Shazib Pervaiz; Maija Pesonen; Jeffrey E Pessin; Godefridus J Peters; Morten Petersen; Irina Petrache; Basil J Petrof; Goran Petrovski; James M Phang; Mauro Piacentini; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Federico Pietrocola; Felipe X Pimentel-Muiños; Mario Pinar; Benjamin Pineda; Ronit Pinkas-Kramarski; Marcello Pinti; Paolo Pinton; Bilal Piperdi; James M Piret; Leonidas C Platanias; Harald W Platta; Edward D Plowey; Stefanie Pöggeler; Marc Poirot; Peter Polčic; Angelo Poletti; Audrey H Poon; Hana Popelka; Blagovesta Popova; Izabela Poprawa; Shibu M Poulose; Joanna Poulton; Scott K Powers; Ted Powers; Mercedes Pozuelo-Rubio; Krisna Prak; Reinhild Prange; Mark Prescott; Muriel Priault; Sharon Prince; Richard L Proia; Tassula Proikas-Cezanne; Holger Prokisch; Vasilis J Promponas; Karin Przyklenk; Rosa Puertollano; Subbiah Pugazhenthi; Luigi Puglielli; Aurora Pujol; Julien Puyal; Dohun Pyeon; Xin Qi; Wen-Bin Qian; Zheng-Hong Qin; Yu Qiu; Ziwei Qu; Joe Quadrilatero; Frederick Quinn; Nina Raben; Hannah Rabinowich; Flavia Radogna; Michael J Ragusa; Mohamed Rahmani; Komal Raina; Sasanka Ramanadham; Rajagopal Ramesh; Abdelhaq Rami; Sarron Randall-Demllo; Felix Randow; Hai Rao; V Ashutosh Rao; Blake B Rasmussen; Tobias M Rasse; Edward A Ratovitski; Pierre-Emmanuel Rautou; Swapan K Ray; Babak Razani; Bruce H Reed; Fulvio Reggiori; Markus Rehm; Andreas S Reichert; Theo Rein; David J Reiner; Eric Reits; Jun Ren; Xingcong Ren; Maurizio Renna; Jane Eb Reusch; Jose L Revuelta; Leticia Reyes; Alireza R Rezaie; Robert I Richards; Des R Richardson; Clémence Richetta; Michael A Riehle; Bertrand H Rihn; Yasuko Rikihisa; Brigit E Riley; Gerald Rimbach; Maria Rita Rippo; Konstantinos Ritis; Federica Rizzi; Elizete Rizzo; Peter J Roach; Jeffrey Robbins; Michel Roberge; Gabriela Roca; Maria Carmela Roccheri; Sonia Rocha; Cecilia Mp Rodrigues; Clara I Rodríguez; Santiago Rodriguez de Cordoba; Natalia Rodriguez-Muela; Jeroen Roelofs; Vladimir V Rogov; Troy T Rohn; Bärbel Rohrer; Davide Romanelli; Luigina Romani; Patricia Silvia Romano; M Isabel G Roncero; Jose Luis Rosa; Alicia Rosello; Kirill V Rosen; Philip Rosenstiel; Magdalena Rost-Roszkowska; Kevin A Roth; Gael Roué; Mustapha Rouis; Kasper M Rouschop; Daniel T Ruan; Diego Ruano; David C Rubinsztein; Edmund B Rucker; Assaf Rudich; Emil Rudolf; Ruediger Rudolf; Markus A Ruegg; Carmen Ruiz-Roldan; Avnika Ashok Ruparelia; Paola Rusmini; David W Russ; Gian Luigi Russo; Giuseppe Russo; Rossella Russo; Tor Erik Rusten; Victoria Ryabovol; Kevin M Ryan; Stefan W Ryter; David M Sabatini; Michael Sacher; Carsten Sachse; Michael N Sack; Junichi Sadoshima; Paul Saftig; Ronit Sagi-Eisenberg; Sumit Sahni; Pothana Saikumar; Tsunenori Saito; Tatsuya Saitoh; Koichi Sakakura; Machiko Sakoh-Nakatogawa; Yasuhito Sakuraba; María Salazar-Roa; Paolo Salomoni; Ashok K Saluja; Paul M Salvaterra; Rosa Salvioli; Afshin Samali; Anthony Mj Sanchez; José A Sánchez-Alcázar; Ricardo Sanchez-Prieto; Marco Sandri; Miguel A Sanjuan; Stefano Santaguida; Laura Santambrogio; Giorgio Santoni; Claudia Nunes Dos Santos; Shweta Saran; Marco Sardiello; Graeme Sargent; Pallabi Sarkar; Sovan Sarkar; Maria Rosa Sarrias; Minnie M Sarwal; Chihiro Sasakawa; Motoko Sasaki; Miklos Sass; Ken Sato; Miyuki Sato; Joseph Satriano; Niramol Savaraj; Svetlana Saveljeva; Liliana Schaefer; Ulrich E Schaible; Michael Scharl; Hermann M Schatzl; Randy Schekman; Wiep Scheper; Alfonso Schiavi; Hyman M Schipper; Hana Schmeisser; Jens Schmidt; Ingo Schmitz; Bianca E Schneider; E Marion Schneider; Jaime L Schneider; Eric A Schon; Miriam J Schönenberger; Axel H Schönthal; Daniel F Schorderet; Bernd Schröder; Sebastian Schuck; Ryan J Schulze; Melanie Schwarten; Thomas L Schwarz; Sebastiano Sciarretta; Kathleen Scotto; A Ivana Scovassi; Robert A Screaton; Mark Screen; Hugo Seca; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Jose M Seguí-Simarro; Juan Segura-Aguilar; Ekihiro Seki; Christian Sell; Iban Seiliez; 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Keiji Tanaka; Masaki Tanaka; Daolin Tang; Dingzhong Tang; Guomei Tang; Isei Tanida; Kunikazu Tanji; Bakhos A Tannous; Jose A Tapia; Inmaculada Tasset-Cuevas; Marc Tatar; Iman Tavassoly; Nektarios Tavernarakis; Allen Taylor; Graham S Taylor; Gregory A Taylor; J Paul Taylor; Mark J Taylor; Elena V Tchetina; Andrew R Tee; Fatima Teixeira-Clerc; Sucheta Telang; Tewin Tencomnao; Ba-Bie Teng; Ru-Jeng Teng; Faraj Terro; Gianluca Tettamanti; Arianne L Theiss; Anne E Theron; Kelly Jean Thomas; Marcos P Thomé; Paul G Thomes; Andrew Thorburn; Jeremy Thorner; Thomas Thum; Michael Thumm; Teresa Lm Thurston; Ling Tian; Andreas Till; Jenny Pan-Yun Ting; Vladimir I Titorenko; Lilach Toker; Stefano Toldo; Sharon A Tooze; Ivan Topisirovic; Maria Lyngaas Torgersen; Liliana Torosantucci; Alicia Torriglia; Maria Rosaria Torrisi; Cathy Tournier; Roberto Towns; Vladimir Trajkovic; Leonardo H Travassos; Gemma Triola; Durga Nand Tripathi; Daniela Trisciuoglio; Rodrigo Troncoso; Ioannis P Trougakos; Anita C Truttmann; Kuen-Jer Tsai; Mario P Tschan; Yi-Hsin Tseng; Takayuki Tsukuba; Allan Tsung; Andrey S Tsvetkov; Shuiping Tu; Hsing-Yu Tuan; Marco Tucci; David A Tumbarello; Boris Turk; Vito Turk; Robin Fb Turner; Anders A Tveita; Suresh C Tyagi; Makoto Ubukata; Yasuo Uchiyama; Andrej Udelnow; Takashi Ueno; Midori Umekawa; Rika Umemiya-Shirafuji; Benjamin R Underwood; Christian Ungermann; Rodrigo P Ureshino; Ryo Ushioda; Vladimir N Uversky; Néstor L Uzcátegui; Thomas Vaccari; Maria I Vaccaro; Libuše Váchová; Helin Vakifahmetoglu-Norberg; Rut Valdor; Enza Maria Valente; Francois Vallette; Angela M Valverde; Greet Van den Berghe; Ludo Van Den Bosch; Gijs R van den Brink; F Gisou van der Goot; Ida J van der Klei; Luc Jw van der Laan; Wouter G van Doorn; Marjolein van Egmond; Kenneth L van Golen; Luc Van Kaer; Menno van Lookeren Campagne; Peter Vandenabeele; Wim Vandenberghe; Ilse Vanhorebeek; Isabel Varela-Nieto; M Helena Vasconcelos; Radovan Vasko; Demetrios G Vavvas; Ignacio Vega-Naredo; Guillermo Velasco; Athanassios D Velentzas; Panagiotis D Velentzas; Tibor Vellai; Edo Vellenga; Mikkel Holm Vendelbo; Kartik Venkatachalam; Natascia Ventura; Salvador Ventura; Patrícia St Veras; Mireille Verdier; Beata G Vertessy; Andrea Viale; Michel Vidal; Helena L A Vieira; Richard D Vierstra; Nadarajah Vigneswaran; Neeraj Vij; Miquel Vila; Margarita Villar; Victor H Villar; Joan Villarroya; Cécile Vindis; Giampietro Viola; Maria Teresa Viscomi; Giovanni Vitale; Dan T Vogl; Olga V Voitsekhovskaja; Clarissa von Haefen; Karin von Schwarzenberg; Daniel E Voth; Valérie Vouret-Craviari; Kristina Vuori; Jatin M Vyas; Christian Waeber; Cheryl Lyn Walker; Mark J Walker; Jochen Walter; Lei Wan; Xiangbo Wan; Bo Wang; Caihong Wang; Chao-Yung Wang; Chengshu Wang; Chenran Wang; Chuangui Wang; Dong Wang; Fen Wang; Fuxin Wang; Guanghui Wang; Hai-Jie Wang; Haichao Wang; Hong-Gang Wang; Hongmin Wang; Horng-Dar Wang; Jing Wang; Junjun Wang; Mei Wang; Mei-Qing Wang; Pei-Yu Wang; Peng Wang; Richard C Wang; Shuo Wang; Ting-Fang Wang; Xian Wang; Xiao-Jia Wang; Xiao-Wei Wang; Xin Wang; Xuejun Wang; Yan Wang; Yanming Wang; Ying Wang; Ying-Jan Wang; Yipeng Wang; Yu Wang; Yu Tian Wang; Yuqing Wang; Zhi-Nong Wang; Pablo Wappner; Carl Ward; Diane McVey Ward; Gary Warnes; Hirotaka Watada; Yoshihisa Watanabe; Kei Watase; Timothy E Weaver; Colin D Weekes; Jiwu Wei; Thomas Weide; Conrad C Weihl; Günther Weindl; Simone Nardin Weis; Longping Wen; Xin Wen; Yunfei Wen; Benedikt Westermann; Cornelia M Weyand; Anthony R White; Eileen White; J Lindsay Whitton; Alexander J Whitworth; Joëlle Wiels; Franziska Wild; Manon E Wildenberg; Tom Wileman; Deepti Srinivas Wilkinson; Simon Wilkinson; Dieter Willbold; Chris Williams; Katherine Williams; Peter R Williamson; Konstanze F Winklhofer; Steven S Witkin; Stephanie E Wohlgemuth; Thomas Wollert; Ernst J Wolvetang; Esther Wong; G William Wong; Richard W Wong; Vincent Kam Wai Wong; Elizabeth A Woodcock; Karen L Wright; Chunlai Wu; Defeng Wu; Gen Sheng Wu; Jian Wu; Junfang Wu; Mian Wu; Min Wu; Shengzhou Wu; William Kk Wu; Yaohua Wu; Zhenlong Wu; Cristina Pr Xavier; Ramnik J Xavier; Gui-Xian Xia; Tian Xia; Weiliang Xia; Yong Xia; Hengyi Xiao; Jian Xiao; Shi Xiao; Wuhan Xiao; Chuan-Ming Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Yuyan Xiong; Chuanshan Xu; Congfeng Xu; Feng Xu; Haoxing Xu; Hongwei Xu; Jian Xu; Jianzhen Xu; Jinxian Xu; Liang Xu; Xiaolei Xu; Yangqing Xu; Ye Xu; Zhi-Xiang Xu; Ziheng Xu; Yu Xue; Takahiro Yamada; Ai Yamamoto; Koji Yamanaka; Shunhei Yamashina; Shigeko Yamashiro; Bing Yan; Bo Yan; Xianghua Yan; Zhen Yan; Yasuo Yanagi; Dun-Sheng Yang; Jin-Ming Yang; Liu Yang; Minghua Yang; Pei-Ming Yang; Peixin Yang; Qian Yang; Wannian Yang; Wei Yuan Yang; Xuesong Yang; Yi Yang; Ying Yang; Zhifen Yang; Zhihong Yang; Meng-Chao Yao; Pamela J Yao; Xiaofeng Yao; Zhenyu Yao; Zhiyuan Yao; Linda S Yasui; Mingxiang Ye; Barry Yedvobnick; Behzad Yeganeh; Elizabeth S Yeh; Patricia L Yeyati; Fan Yi; Long Yi; Xiao-Ming Yin; Calvin K Yip; Yeong-Min Yoo; Young Hyun Yoo; Seung-Yong Yoon; Ken-Ichi Yoshida; Tamotsu Yoshimori; Ken H Young; Huixin Yu; Jane J Yu; Jin-Tai Yu; Jun Yu; Li Yu; W Haung Yu; Xiao-Fang Yu; Zhengping Yu; Junying Yuan; Zhi-Min Yuan; Beatrice Yjt Yue; Jianbo Yue; Zhenyu Yue; David N Zacks; Eldad Zacksenhaus; Nadia Zaffaroni; Tania Zaglia; Zahra Zakeri; Vincent Zecchini; Jinsheng Zeng; Min Zeng; Qi Zeng; Antonis S Zervos; Donna D Zhang; Fan Zhang; Guo Zhang; Guo-Chang Zhang; Hao Zhang; Hong Zhang; Hong Zhang; Hongbing Zhang; Jian Zhang; Jian Zhang; Jiangwei Zhang; Jianhua Zhang; Jing-Pu Zhang; Li Zhang; Lin Zhang; Lin Zhang; Long Zhang; Ming-Yong Zhang; Xiangnan Zhang; Xu Dong Zhang; Yan Zhang; Yang Zhang; Yanjin Zhang; Yingmei Zhang; Yunjiao Zhang; Mei Zhao; Wei-Li Zhao; Xiaonan Zhao; Yan G Zhao; Ying Zhao; Yongchao Zhao; Yu-Xia Zhao; Zhendong Zhao; Zhizhuang J Zhao; Dexian Zheng; Xi-Long Zheng; Xiaoxiang Zheng; Boris Zhivotovsky; Qing Zhong; Guang-Zhou Zhou; Guofei Zhou; Huiping Zhou; Shu-Feng Zhou; Xu-Jie Zhou; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Wenhua Zhu; Xiao-Feng Zhu; Yuhua Zhu; Shi-Mei Zhuang; Xiaohong Zhuang; Elio Ziparo; Christos E Zois; Teresa Zoladek; Wei-Xing Zong; Antonio Zorzano; Susu M Zughaier Journal: Autophagy Date: 2016 Impact factor: 16.016
Authors: Anastasia V Poznyak; Vasily N Sukhorukov; Alexander Zhuravlev; Nikolay A Orekhov; Vladislav Kalmykov; Alexander N Orekhov Journal: Int J Mol Sci Date: 2022-01-21 Impact factor: 5.923
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