Open Access

Sirt1 inhibits HG-induced endothelial injury: Role of Mff-based mitochondrial fission and F‑actin homeostasis-mediated cellular migration

  • Authors:
    • Ruijie Qin
    • Lina Zhang
    • Dong Lin
    • Fei Xiao
    • Lixin Guo
  • View Affiliations

  • Published online on: May 8, 2019     https://doi.org/10.3892/ijmm.2019.4185
  • Pages: 89-102
  • Copyright: © Qin et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Although sirtuin 1 (Sirt1) has been found to be involved in diabetic vasculopathy and high glucose (HG)‑mediated endothelial injury, the underlying mechanisms remain to be fully elucidated. The aim of the present study was to investigate the role of Sirt1 in HG‑induced endothelial injury and its potential mechanism. In the present study, it was demonstrated that HG triggers the downregulation of Sirt1 by activating microRNA‑195 in human umbilical vein endothelial cells (HUVECs), as determined by western blot analysis in vivo and in vitro. Furthermore, a lower expression of Sirt1 was correlated with glucose metabolic abnormalities, aortic endothelial dysfunction and endothelial apoptosis as evidenced by western blot analysis and ELISA in mice. By contrast, the loss of Sirt1 evoked mitochondrial fission factor (Mff)‑mediated mitochondrial fission through the c‑Jun N‑terminal kinase (JNK) pathway, which contributes to the apoptosis of HUVECs. In addition, Sirt1 deficiency downregulated the migration of HUVECs through F‑actin dyshomeostasis. Collectively, the results identify Sirt1 as a protective factor, which inhibits the JNK/Mff/mitochondrial fission pathway and sustains F‑actin homeostasis, and has potential implications for novel approaches to diabetic vasculopathy.

Introduction

The incidence of macrovascular complications in diabetic patients is >50% and constitutes a high rate of mortality (1). Hyperglycemia resulting from uncontrolled glucose regulation is mainly considered to form the link between diabetes and diabetic vasculopathy (2). As components of blood barriers, vascular endothelial cells (ECs) are more vulnerable to hyperglycemia damage (3). During the development of diabetes, the impaired EC functioning results in decreased levels of vasodilation, weakened barrier function, and increased reactive oxygen species (ROS) generation and inflammatory activation (4). Therefore, elucidating the mechanisms of endothelial injury is critical to alleviating diabetic vasculopathy.

Mitochondria are not only the focus of cellular energy generation, they are also involved in the regulation of cellular differentiation, proliferation, senility and apoptosis. Several studies have suggested that mitochondrial fission serves an important role in mitochondrial homeostasis (5,6). Furthermore, mitochondrial fission factor (Mff)-mediated mitochondrial fission is considered to be an important facet of diabetic complications (7). Excessive mitochondrial fission leads to cellular energy metabolism disorders, ROS bursts, increased mitochondrial permeability transition pore (mPTP) opening volumes and the activation of mitochondrial-dependent cell apoptotic procedures in response to HG treatment (8). Therefore, maintaining mitochondrial homeostasis by inhibiting mitochondrial fission is necessary to protect endothelial function and survival in diabetic vasculopathy. However, the upstream regulators of mitochondrial fission in HG-induced endothelial injury remain to be elucidated.

Convincing experimental data indicates that Sirt1 serves a critical function in the pathological progression of diabetic vasculopathy by modulating endothelial cellular physiological processes, including metabolism, migration, senescence and apoptosis (9). The activation of Sirt1 by resveratrol ameliorates HG-induced endothelial damage by stabilizing mitochondrial energy metabolism (10). Of note, studies have reported a potential association between Sirt1 and mitochondrial fission (11,12). However, how Sirt1 regulates mitochondrial fission, particularly in HG-induced endothelial injury, remains to be elucidated.

In addition to apoptosis, decreased levels of endothelial cell migration are another key feature of endothelial injury in diabetic vasculopathy (13,14). It is well known that F-actin homeostasis is crucial in the regulation of cellular migration (15,16). However, whether Sirt1 protects cellular migration by regulating F-actin homeostasis remains unclear. Accordingly, the aim of the present study was to examine the role of Sirt1 in repairing diabetic vasculopathy, with a focus on mitochondrial fission and cellular migration.

Materials and methods

Animal procedure and treatment

All experimental procedures described here were conducted in accordance with the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). ApoE−/− mice (male; n=120; supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd; body weight, 18-22 g) were fed a high-fat diet (45% kcal fat, 35% kcal carbohydrates and 20% kcal protein) from 4 weeks of age to the end of the study and housed at 23°C with 40-70% humidity and a 12-h light/dark cycle. 8-week-old apoE−/− mice were intraperitoneally injected with streptozotocin (STZ; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; 50 mg/kg) for five consecutive days. Following this, venous blood was collected from the tails of diabetic mice; blood glucose levels >16.7 mmol/l following a 6-h period of daytime fasting was considered successful model establishment. The diabetic mice (12 weeks old), defined as the diabetic group, were treated with SRT1720 (Selleck Chemicals, Houston, TX, USA; 15 mg/kg/day, defined as the SRT1720 group) for 12 weeks. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg body weight) prior and whole blood was drawn by cardiac puncture or from the eye retroorbital sinus (17). The mice were sacrificed with CO2 using an established CO2 euthanasia method, for endothelial function assessment and histological examination. Briefly, the mice were placed in a chamber and the flow rate of CO2 displacement was 2 l/min (<30% of the chamber volume/min), which was stopped when the mice were no longer breathing and sacrifice was confirmed by cervical dislocation (18,19).

Measurement of biochemical parameters

The blood samples were collected following a 6-h period of fasting. Aortic tissues were homogenized for further biochemical analysis. Glycated hemoglobin (HbA1c) was detected using the in2it A1C system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as described previously (20). The levels of insulin, glucagon, C-peptide, glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were determined using ELISA kits (Beyotime Institute of Biotechnology, Beijing, China).

Measurement of endothelial relaxation function of the thoracic aorta

The measurement of endothelial relaxation functioning was conducted as previously described (3). In brief, the thoracic aorta was immediately dissected and immersed in chilled Krebs-Henseleit solution (Sigma-Aldrich; Merck KGaA) at 37°C and aerated with 95% O2 and 5% CO2 (pH 7.4). The aortic rings were then pre-contracted with U46619 (Sigma-Aldrich; Merck KGaA; 30 nM). Endothelium-dependent and -independent vasodilation were determined using ACh (Selleck Chemicals; 10−9-10−5 M) and SNP (Selleck Chemicals; 10−10-10−6 M), respectively.

Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from the National Infrastructure of Cell Line Resource (Beijing, China; catalog no. 3111C0002000000024). The HUVECs (1×105) were seeded on 6-well plates and cultured in DMEM (high glucose; Gibco; Thermo Fisher Scientific, Inc.) with 10% PBS (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C. To activate mitochondrial fission, FCCP (5 µM, Selleck Chemicals) was used for 2 h at 37°C prior to treatment. To suppress and activate the JNK pathway, SP600125 (SP, 10 µM, Selleck Chemicals) and anisomycin (Ani, 10 µM, Selleck Chemicals) were used 2 h at 37°C prior to treatment, respectively. To inhibit the F-action degradation, Jasplakinolide (2 µM; Selleck Chemicals) was used 2 h before treatment at 37°C.

Construction of an adenovirus for the overexpression of Sirt1

The Sirt1 adenovirus plasmids (ad-Sirt1) and control adenovirus plasmids (ad-ctrl) were purchased from Vigene Biosciences, Inc. (Rockville, MD, USA). The HUVEC cells (0.5×106) were transfected with 20 nM ad-Sirt1 and ad-ctrl with Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) as described in a previous study (21). The expression of proteins in the transfected cells were determined by western blot analysis.

ROS measurements

The cellular ROS was measured via dihydroethidium (DHE, Invitrogen; Thermo Fisher Scientific, Inc.) staining and was observed through a confocal microscope (Olympus Corporation, Tokyo, Japan). DHE was alternately excited at the wavelengths of 300 and 535 nm following the manufacturer's protocol (22).

Mitochondrial fission analysis, mitochondrial membrane potential (ΔΨm) measurements and mPTP opening

Mitochondrial fission was analyzed according to a previous study (4). The mitochondrion was labelled with Tom20 (1:1,000, Abcam, Cambridge, MA, USA; cat. no. ab56783) at room temperature (25°C) for 1 h and the cell was observed under a confocal microscope. The ΔΨm was measured using the JC-1 kit (Beyotime Institute of Biotechnology) and the mPTP opening was measured as described previously (23). The images were analyzed using ImageJ 1.47 version software (National Institutes of Health).

Cellular viability assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and determination of caspase-9 activity

For the in vivo cellular viability assay, ECs were isolated from the thoracic aorta as described previously (24). Cellular viability was quantitatively measured using an FITC Annexin V Apoptosis Detection kit (BD Biosciences) (25). In brief, the cells were incubated (1×105) with 5 µl of FITC Annexin V and PI for 15 min at room temperature (25°C) in the dark. The samples were then analyzed with a BD FACS-Calibur cytometer (BD Biosciences).

A one-step TUNEL kit (Beyotime Institute of Biotechnology) was used for TUNEL staining, as previously described. Following treatment, the HUVECs were incubated with fluorescein-dUTP (Invitrogen; Thermo Fisher Scientific, Inc.) to stain apoptotic cell nuclei and with DAPI (5 mg/ml) to stain all cell nuclei at room temperature for 3 min. Images of the slides were captured under a confocal microscope with at least five random separate fields. Cellular viability was also measured using an MTT assay, according to the manufacturer's protocol. A caspase 9 activity kit (Beyotime Institute of Biotechnology) was used to measure the activity of caspase 9, according to the manufacturer's protocol (26).

Cell migration and wound healing assay

Following treatment, the cells were seeded in 6-well plates at a density of 0.5×106 cells/well. A wound track was scored in each dish with a pipette head. Debris was removed by rinsing the plates with PBS. Following culture for 24 h, the migration distances were visualized and images were captured (Olympus IX71; Olympus Corporation, Tokyo, Japan). Cell migration was also analyzed using a Transwell chamber assay (24 wells with 8-µm pores and polycarbonate membranes), as previously described (27).

Western blot analysis

Following treatment, the cells were lysed with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) supplemented with phenylmethylsulfonyl fluoride. A bicinchoninic acid protein assay was used to measure the protein concentrations (28). The proteins (50 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature (25°C) and then incubated with primary antibodies overnight at 4°C. Then, the membranes were washed 3 times with PBS and incubated with secondary antibodies at room temperature (25°C) for 1 h. The following antibodies were used: Caspase 3 (1:2,000, CST Biological Reagents Co., Ltd., Shanghai, China; cat. no. 9662), caspase 9 (1:2,000, CST Biological Reagents Co., Ltd., cat. no. 9508), Bcl-2 (1:2,000, CST Biological Reagents Co., Ltd., cat. no. 3498), X-linked inhibitor of apoptosis (x-IAP; 1:1,000, CST Biological Reagents Co., Ltd., cat. no. 2042), phosphorylated (p)-JNK (1:1,000, CST Biological Reagents Co., Ltd., cat. no. 9255), JNK (1:1,000, CST Biological Reagents Co., Ltd., cat. no. 9252), Sirt1 (1:1,000, Abcam, cat. no. ab19A7AB4), dynamin-1-like protein (Drp1; 1:1,000, Abcam, cat. no. ab56788), mitochondrial fission 1 protein (Fis1; 1:1,000, Abcam, cat. no. ab71498), mitofusin-(Mfn)2 (1:1,000, Abcam, cat. no. ab56889), Mfn1 (1:1,000, Abcam, cat. no. ab57602), Mff (1:1,000, CST Biological Reagents Co., Ltd., cat. no. 86668), F-actin (1:1,000, Abcam, cat. no. ab205), G-actin (1:1,000, Abcam, cat. no. ab200046), p-endothelial nitric oxide synthase (eNOS; Ser1117; 1:1,000, Abcam, cat. no. ab184154), eNOS (1:1,000, Abcam, cat. no. ab76198), Bad (1:1,000, Abcam, cat. no. ab32445), Bax (1:2,000, Abcam, cat. no. ab32503), poly (ADP-ribose) polymerase (1:2,000, Abcam, cat. no. ab32064), horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin (Ig)-G (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 7076) and HRP-conjugated anti-rabbit IgG (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 7074). The blots were detected with an enhanced chemiluminescence substrate kit (Thermo Fisher Scientific, Inc.), and band intensity levels were analyzed using Quantity One 4.6 software (Bio-Rad Laboratories, Inc.).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

RT-qPCR was performed according to the manufacturer's instructions. Briefly, total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed with a the TaqMan MicroRNA Reverse Transcription kit (Takara Bio, Inc., Otsu, Japan) at 37°C for 30 min according to the manufacturer's instructions. qPCR was performed using the SYBR-Green RT-PCR kit (Takara Bio, Inc.). The following primers were used for polymerase chain reaction: Sirt1, forward, 5′-GAGAGACGTCTGGTAGATCG-3′ and reverse 5′-GTGCCAGCATGTGTCGTAGT-3′; intercellular adhesion molecule 1 (ICAM-1), forward 5′-GAGACGCAGAGGACCTTAACAG-3′ and reverse 5′-GACGCCGCTCAGAAGAACCA-3′; CRP, forward 5′-GGATGGATTGCACAGCCATT-3′ and reverse 5′-GCGCCGACTCAGAGGTGT-3′; tumor necrosis factor (TNF)α, forward 5′-AGATGGAGCAACCTAAGGTC-3′ and reverse 5′-GCAGACCTCGCTGTTCTAGC-3′; interleukin (IL)6, forward 5′-CAGACTCGCGCCTCTAAGGAGT-3′ and reverse 5′-GATAGCCGATCCGTCGAA-3′; miR-195, forward 5′-UAGCAGCACAGAAAUAUUGGC-3′ and reverse 5′-CAAUAUUUCUGUGCUGCUAUU-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′; and GAPDH, forward 5′-AATGGTGAAGGTCGGTGTG-3′ and reverse 5′-GTGGAGTCATACTGGAACATGTAG-3′. The thermocycling conditions were as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 40 sec, 60°C for 30 sec and 72°C for 30 sec. U6 and GAPDH were selected as internal controls for micro (mi)RNA and mRNA, respectively (23,29). Fold-changes for mRNA expressions were calculated using the 2−ΔΔCq method (30,31).

Transfection

The miR-195 mimics (agmir-195, forward 5′-UAGCAGCACAGAAAUAUUGGC-3′ and reverse 5′-CAAUAUUUCUGUGCUGCUAUU-3′; miR-Ctrl, forward 5′-UUCUCCGAACGUGUCACGU-3′ and reverse 5′-ACGUGACACGUUCGGAGAA-3′) and miR-195 inhibitor (antagomir; 5′-GCCAAUAUUUCUGUGCUGCUA-3′) were purchased from GenePharma Co., Ltd. (Shanghai, China). The HUVEC cells (0.5×106) were transfected with 20 nM miR-195 mimic, miR-195 inhibitor and miR negative control (NC) with Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions (32). Transfection was performed for 48 h and the transfection efficiency was evaluated by RT-qPCR.

Immunohistochemistry and immunofluorescence staining

The change in expression of Sirt1 in the thoracic aorta was measured via immunohistochemistry according to a previous study (33,34). Immunofluorescence staining was used to measure cytochrome-c (cyt-c) localization, F/G-actin, and mitochondrial fission. Following treatment, the cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature. Following blocking with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in PBS for 1 h at room temperature, the cells were incubated with primary antibodies for 4 h at room temperature. The secondary antibodies were incubated at room temperature for 1 h in the dark. Images were captured using a laser confocal microscope (TcS SP5; Leica Microsystems, Inc., Buffalo Grove, IL, USA). The primary antibodies, Sirt1 (1:500, Abcam, cat. no. ab19A7AB4), cyt-c (1:500, Abcam, cat. no. ab90529), translocase of outer mitochondrial membrane 20 (1:500, Abcam, cat. no. ab56783) and F-actin (1:500, Abcam, cat. no. ab205) were used. The Alexa Fluor® secondary antibodies, anti-mouse IgG (1:500; cat. no. 4408; green) and anti-rabbit IgG (1:500; cat. no. 4412; green), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). DAPI (5 mg/ml; Sigma-Aldrich; Merck KGaA) was used to stain the nucleus at room temperature for 3 min.

Luciferase activity assay

The wild-type Sirt1 3′-UTR (WT) and mutant Sirt1 3′-UTR (MUT) containing the putative binding site of miR-195 were chemically synthesized and cloned downstream from the firefly luciferase gene in a pGL3-promoter vector (Promega Corporation, Madison, WI, USA). The luciferase plasmids and miR-195 or control miRNA were co-transfected in HUVECs (0.5×106) in DMEM supplemented with 10% FBS using Lipofectamine® 2000 at 37°C according to the manufacturer's instructions. pRL Renilla control reporter vectors (Promega Corporation) were used as an internal control to normalize the values of the experimental reporter gene. Following 48 h of transfection, the intensities were measured with a Luciferase Reporter Assay system (Promega Corporation).

Statistical analysis

All analyses were performed with SPSS 20.0 software (IBM Corp., Armonk, NY, USA). All experiments were repeated three times. Corresponding data are presented as the mean ± standard deviation, and the statistical significance of each variable was estimated by a one-way analysis of variance followed by Tukey's test for post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

Results

Sirt1 attenuates glucose metabolic abnormalities in diabetic mice

First, the expression of Sirt1 in the aorta of diabetic and non-diabetic mice was analyzed via western blot and qPCR analyses (Fig. 1A-C). The results showed that the expression of Sirt1 was significantly reduced at the protein and mRNA levels in the diabetic mice. A previous study showed that the loss of Sirt1 contributed to glucose metabolic abnormalities in diabetics (35). To examine the role of Sirt1 in the glucose metabolic activities observed in diabetic mice, SRT1720, an activator of Sirt1, was used to reactivate Sirt1 in the diabetic mice. SRT1720 reversed the downregulation of Sirt1 in diabetic mice, as indicated by the western blot (Fig. 1A and B), qPCR (Fig. 1C) and immunohistochemical (Fig. 1D and E) analyses. The effects of Sirt1 on the glucose metabolic activities observed in diabetic mice were then evaluated. As is shown in Fig. 1F-K, diabetic mice exhibited higher body weights, fasting blood glucose levels, serum insulin levels, serum-peptide levels, glycosylated hemoglobin A1c (HbA1c) levels and systolic blood pressure (SBP). As expected, the activation of Sirt1 significantly reduced or abrogated the above changes. However, the high levels of glucagon observed in diabetic mice were not altered by SRT1720 (Fig. 1L). These data suggest that the reduced expression of Sirt1 contributes to the glucose metabolic abnormalities observed in diabetic mice.

Sirt1 activation ameliorates aortic endothelial dysfunction in diabetic mice

Endothelial dysfunction is an early marker of chronic HG injury. Endothelial functioning was measured 4 weeks following SRT1720 treatment in mice. As is shown in Fig. 2A, diabetes inhibited the endothelial-dependent vasodilation in response to Ach, which was reversed by SRT1720. By contrast, no significant difference in endothelial-independent vasodilation levels were observed among the four groups. To examine the underlying mechanism involved, fundamental factors, including eNOS phosphorylation (Ser1177), intercellular adhesion molecules, inflammation and oxidative stress, were analyzed. Diabetes decreased the protein levels of p-eNOS (Fig. 2B) and increased the mRNA levels of ICAM1 and VCAM1 (Fig. 2C and D), and this effect was nullified by SRT1720. SRT1720 also decreased the mRNA levels of pro-inflammatory TNF-α, IL-6 and CRP in the diabetic thoracic aortas (Fig. 2E-G). In addition, diabetic mice generated more MDA and consumed more antioxidant factors, including GSH/SOD, and this effect was reversed by SRT1720 (Fig. 2H-J). Together, these data demonstrate that the activation of Sirt1 reduces aortic endothelial dysfunction in diabetic mice.

Downregulated Sirt1 promotes HG-mediated endothelial cell death

The apoptosis of endothelial cells has been reported to serve a critical role in HG-induced diabetic vasculopathy (36,37). Therefore, the present study measured endothelial cell apoptosis in vivo and in vitro. Annexin V/PI flow cytometry was used to qualify endothelial cell apoptosis in cells extracted from thoracic aortas. Relative to the control group, the diabetic group exhibited clearly elevated ratios of Annexin V+ cells, and this effect was reduced by SRT1720 (Fig. 2K and L). From the in vitro experiments, alterations of Sirt1 were observed in HUVECs prior to and following HG (30 mmol/l) treatment. The expression of Sirt1 was downregulated following HG treatment (Fig. 3A and B), with the lowest expression levels of Sirt1 following 48 h of HG treatment. Therefore, 48 h of HG treatment was used in the following experiments. A gain-of function assay on Sirt1 was performed via adenovirus vector transfection (ad-Sirt1) and the transfection efficiency levels were confirmed via western blotting (Fig. 3C and D). The overexpression of Sirt1 had no influence on cellular viability (Fig. 3E). However, the regaining of Sirt1 reduced the occurrence of HG-induced cellular death (Fig. 3F) as indicated by the expression of cleaved caspase 3 and its substrate, which are indicators of cellular apoptosis (Fig. 3G-I). To further determine whether Sirt1 promotes cellular survival, TUNEL staining was performed (Fig. 3K). The overexpression of Sirt1 also reduced the percentage of TUNEL-positive cells present. These data indicate that Sirt1 is an anti-apoptotic factor of HUVECs under the treatment of HG.

Sirt1 deficiency triggers endothelial death by activating mitochondria-dependent apoptotic pathways

To describe the protective role of Sirt1 in endothelial apoptosis under HG, the present study focused on mitochondria-dependent cellular apoptosis occurring through cellular ROS release, mitochondrial potential collapse, mPTP opening and mitochondrial pro-apoptotic factor leakage. ROS alterations were analyzed through DHE staining. Compared with the control group, HG increased the cellular ROS content levels, which were reduced by the overexpression of Sirt1 (Fig. 4A). HG also induced higher expression levels of Bax, Bad and Caspase 9 but lower expression levels of Bcl-2 and x-IAP, suggesting the activation of mitochondria-related apoptotic pathways (Fig. 4B-G). However, the overexpression of Sirt1 inhibited the pro-apoptotic effects of HG. A JC-1 assay was used to measure the mitochondrial electrochemical potential (ΔΨm) (Fig. 4H and I). HG also impaired ΔΨm, with evidence of decreased levels of red fluorescence and increased levels of green fluorescence. However, the overexpression of Sirt1 reversed the stability of ΔΨm. Furthermore, the rate of mPTP opening was increased by HG but decreased by the overexpression of Sirt1 (Fig. 4J). Following the dissipated ΔΨm and long-lasting mPTP opening, HG increased the leakage of cyt-c from the mitochondria into the cytoplasm, and cyt-c was even observed to migrate into the nucleus (Fig. 4K), whereas an increase in Sirt1 limited cyt-c leakage. These results showed that Sirt1 prevents endothelial apoptosis by inhibiting the mitochondria-dependent apoptotic pathway.

Mitochondrial fission is involved in Sirt1 deficiency-mediated mitochondrial apoptosis under HG treatment

Previous studies have reported that mitochondrial fission serves a critical role in mitochondria-related apoptosis (28,38,39). Therefore, the present study examined whether Sirt1 prevents endothelial apoptosis through the regulation of mitochondrial fission. As is shown in Fig. 5A, HG led to a markedly larger volume of fragmented mitochondria in the Ad-ctrl group. However, upregulating Sirt1 repressed the effects of HG on mitochondrial fragments. Mitochondrial lengths were also recorded (Fig. 5B) to quantify the fission levels, and the results were in accordance with the above findings. These results indicate the role of Sirt1 in activating mitochondrial fission. To examine whether mitochondria are responsible for endothelial apoptosis, FCCP, a mitochondrial fission activator, was used to reactivate mitochondrial fission in Ad-Sirt1 cells (Fig. 5A and B). The protective role of Sirt1 in mPTP opening and cellular apoptosis disappeared following the re-activation of mitochondrial fission in ad-Sirt1 cells (Fig. 5C-E). These results indicate that mitochondrial fission is responsible for Sirt1 deficiency-mediated mitochondrial apoptosis under HG treatment.

Sirt1 regulates mitochondrial fission by activating the JNK/Mff pathway

To determine the means through which Sirt1 regulates mitochondrial fission, changes in Drp1 and in its receptors (Mff, Fis1, Mid49 and Mid51) required to regulate mitochondrial fission were measured. It was first found that HG mainly enhanced the expression of Mff in ad-ctrl cells (Fig. 6A and B) and that the overexpression of Sirt1 reduced the expression of Mff rather than other receptors. Furthermore, HG treatment enhanced the expression of Drp1, which was reversed by the overexpression of Sirt1 (Fig. 6C).

Several studies have confirmed that the activity of Mff is regulated by the JNK pathway (28,40,41). Therefore, the present study examined the JNK pathway to examine the mechanism by which Sirt1 inactivates Mff. Relative to the control group, JNK was activated by HG, as indicated by an increase observed in the expression of p-JNK (Fig. 6D-F). This tendency was reversed by the overexpression of Sirt1. To examine the role of the activation of JNK in Sirt1-induced mitochondrial fission in the present study, SP, an inhibitor of JNK, was added to ad-Ctrl cells to inhibit the JNK pathway. Ani, an activator of the JNK pathway, was also used in ad-Sirt1 cells. Through western botting, it was found that the JNK pathway was triggered by HG treatment or Ani, but was inhibited by the overexpression of Sirt1 or SP (Fig. 6D-F). The expression of Mff and mitochondrial fission were enhanced following HG treatment, but decreased to normal levels upon the overexpression of Sirt1 or SP treatment (Fig. 6D-F). The reactivation of JNK in ad-Sirt1 cells eliminated the inhibition of Mff activation and the protective effects on mitochondrial fission induced by the overexpression of Sirt1 (Fig. 6G and H). This experiment suggests that Sirt1 regulates mitochondrial fission through the JNK pathway.

Overexpression of Sirt1 maintains endothelial migration by sustaining F-actin homeostasis

Endothelial cell mobilization capacities are critical for the repair of endothelial injury. Therefore, whether Sirt1 is involved in cellular migration was measured. First, a Transwell assay was used to investigate associations between HUVEC migration and Sirt1. As is shown in Fig. 7A and B, HG treatment reduced the migration of HUVECs, whereas the overexpression of Sirt1 prevented this change from occurring. These results suggest that Sirt1 is involved in cellular migration. F-actin has been reported to serve a critical role in cellular mobilization. Therefore, the present study examined whether Sir1 regulates cellular migration through F-actin dyshomeostasis. Jasplakinolide, an F-actin depolymerization inhibitor, was used to inhibit F-actin depolymerization in ad-Ctrl cells. As F-actin is composed of G-actin, western blotting was performed to measure the expression of F-actin and G-actin following HG treatment. As is shown in Fig. 7C-F, HG treatment clearly reduced the expression of F-actin and enhanced the expression of G-actin, which was reversed by the overexpression of Sirt1 or jasplakinolide application. Similar results for F-actin were observed by fluorescence (Fig. 7G). Jasplakinolide and the overexpression of Sirt1 significantly enhanced the fluorescence intensity of F-actin, which was reduced by HG. Jasplakinolide also promoted cellular migration under HG treatment, similar to the overexpression of Sirt1 (Fig. 7A and B). These results suggested that Sirt1 regulates cellular migration by inhibiting F-actin depolymerization into G-actin under HG treatment.

Sirt1 is negatively regulated by miR-195 via hyperglycemic stimulus

To examine the mechanism by which Sirt1 is downregulated through diabetic vasculopathy, miR-195 was examined. It was found that the expression of miR-195 was significantly enhanced in ad-ctrl cells under HG treatment (Fig. 8A). To elucidate the mechanism by which HG down-regulates Sirt1, a mimic (agmir-195) and inhibitor (antagomir) of miR-195 were used, and the transfection efficiency was evaluated by RT-qPCR analysis (Fig. 8B). As is shown in Fig. 8C, agmir-195 reduced the expression of Sirt1, echoing the results derived following HG treatment. However, inhibitors of miR-195 reversed the downregulated level of Sirt1 under treatment with HG. These results suggest that miR-195 negatively regulates Sirt1 under HG. Furthermore, as shown in Fig. 8D, miR-195 was matched to Sirt1. To examine the direct binding of miR-195 with the 3′UTR of Sirt1, luciferase assays were used by co-transfecting miR-195 mimic with luciferase reporter plasmids with cloned miR-195 binding sites for WT Sirt1-3′UTR or MUT Sirt1-3′UTR (Fig. 8E). Compared with that of the MUT type, luciferase activity was downregulated in cells co-transfected with miR-195 mimics and in the WT Sirt1-3′UTR, suggesting that Sirt1 is a target gene of miR-195. To provide more evidence, the effects of miR-195 on mitochondrial fission, cellular apoptosis and migration were analyzed. It was found that agmir-195 promoted mitochondrial fission, enhanced the expression of caspase-9 and inhibited cellular migration, consistent with the effects of HG treatment (Fig. 8F-I). However, the inhibition of miR-195 inhibited the HG-induced mitochondrial fission, upregulation of caspase-9 and inhibited migration. These results support the hypothesis that HG represses the expression of Sirt1 by upregulating miR-195.

Discussion

Vascular endothelial injury caused by a hyperglycemic environment serves an important role in the occurrence and development of diabetic vasculopathy. Accumulating evidence demonstrates the critical role of Sirt1 in HG-induced endothelial injury. The present study confirmed that i) HG triggers the downregulation of Sirt1 by activating miR-195; ii) reduced expression of Sirt1 contributes to glucose metabolic abnormalities, aortic endothelial dysfunction and EC apoptosis; iii) downregulated Sirt1 triggers EC apoptosis by activating mitochondrial fission; iv) mechanically, the loss of Sirt1 triggers mitochondrial fission by evoking the JNK/Mff pathway; v) Sirt1 deficiency limits EC migration through F-actin dyshomeostasis. These results further enrich current understanding of the molecular pathways of Sirt1-mediated endothelial protection in diabetic vasculopathy.

Sirt1 is related to cellular metabolism, migration, senescence and survival, and is implicated in the pathophysiology of diabetes, neurodegenerative disorders and cardiovascular disease (9,42). In the present study, the activation of Sirt1 with SRT1720 inhibited EC apoptosis and endothelial dysfunction in diabetes. Previous reports have argued that the protective endothelial effect of Sirt1 is closely associated with the regulation of mitochondrial functions by limiting mitochondrial DNA damage and preventing the activation of mitochondrial damaging MMP-9 (11,43,44). Furthermore, mitochondrial dysfunction caused by mitochondrial fission is reported to be involved in the development of diabetic nephropathy (7,45) and diabetic cardiomyopathy (46). These results indicate the involvement of mitochondrial fission in the development of HG-induced endothelial injury and diabetic vasculopathy.

An association between Sirt1 and mitochondrial fission has been illustrated in several studies, which confirms the regulation of Sirt1 on mitochondrial fission. However, how Sirt1 regulates mitochondrial fission in HG-induced endothelial injury remains to be fully elucidated. It has been reported that the JNK pathway is involved in the regulation of mitochondrial fission (28,40,47). In addition, previous studies have confirmed the association between Sirt1 and the JNK pathway (48-50). SIRT1 has been reported to reduce ROS-induced mouse embryonic stem cell apoptosis via the phosphatase and tensin homolog/JNK/Forkhead box O1 pathway (51). Therefore, Sirt1 likely regulates Mff-associated fission via the JNK pathway. The present study confirmed that Sirt1 attenuates mitochondrial fission via the JNK pathway in HG-induced endothelial injury.

Mitochondria are energy metabolism organelles involved in the regulation of ATP generation, ROS production and apoptosis (52-54). It is well known that mitochondrial fission serves an important role in mitochondrial functional regulation. Mechanistically, Drp1, the mitochondrial division executive factor, binds to its corresponding receptor which is located on the mitochondrial outer member, forming a ring on mitochondrial surfaces that contracts and creates a large number of mitochondrial fragments (55,56). In addition, accumulating evidence shows that four proteins, Fis1, Mff, Mid49 and Mid51, are involved in mitochondrial fission (4). As one of the key findings of the present study, it was confirmed that Sirt1 regulates mitochondrial fission mainly by regulating Mff in HG-induced endothelial injury. HG induced the downregulation of Sirt1, leading to the phosphorylated activation of the JNK pathway, which promoted the expression of Mff. Additionally, Sirt1 loss contributed to the upregulation of Drp1. Excessive mitochondrial fission caused mitochondrial depolarization, followed by increased mPTP opening periods and cyt-c leakage into the cytoplasm. As a result of cyt-c release, mitochondria-associated apoptotic pathways were activated, as evidenced by the imbalanced expression of pro- and anti-apoptotic proteins.

The migration capacities of endothelial cells are important in the repair of endothelial injury. Sirt1 is reported to be involved in the regulation of cellular migration capacity, however, the underlying mechanisms involved remain to be fully elucidated. F-actin is a key fiber of cellular migration. Therefore, it was hypothesized that Sirt1 may regulate the cellular migration by regulating F-actin homeostasis. HG treatment reduced the expression of F-actin, enhanced the expression of G-actin and led to impaired migration capacities, which were reversed by the overexpression of Sirt1 and jasplakinolide.

Previous studies have argued that miR-195 is involved in the regulation of Sirt1 (57-59). In human dermal microvascular ECs, Sirt1 is inactivated through the upregulation of miR-195 under HG treatment. The results of the present study also show that miR-195 contributes to the downregulation of Sirt1 under HG treatment in vitro. miR-195 inhibition enhanced the expression of Sirt1, limited mitochondrial fission and cellular apoptosis, and improved migration capacities. However, whether miR-195 contributes to diabetic vasculopathy requires further examination in vivo.

According to previous studies, SRT1720 was used in the present study to reactivate Sirt1 and upregulate the expression of Sirt1 in diabetic mice to examine the role of Sirt1 in diabetic vasculopathy in vivo (30,60,61). However, SRT1720 has been reported not to be a direct activator of SIRT1 (62). Therefore, to provide more accurate evidence, Sirt1 transgenic mice should be used in future studies.

Overall, the results of the present study illustrate the protective mechanism of Sirt1 in diabetic vasculopathy, which limits endothelial apoptosis by inhibiting JNK/Mff/mitochondrial fission and enhances endothelial migration by stabilizing F-actin homeostasis. The mechanisms underlying the downregulation of Sirt1 induced by HG were also examined. HG repressed the expression of Sirt1 by upregulating miR-195. Further investigations are required to examine the clinical value of Sirt1 in diabetic vasculopathy.

Funding

The present study was supported by a grant from the National Natural Science Foundation of China (grant no. BJ-2014-066).

Availability of data and materials

The data and materials used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

RQ, LZ and DL conceived and designed the experiments, performed the experiments, and contributed reagents/materials/analysis tools. FX and LG analyzed the data. RQ and LG wrote the paper. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Experimental Animal Ethics Committee of Beijing Vital River Laboratory Animal Technology (Beijing, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

References

1 

Booth GL, Kapral MK, Fung K and Tu JV: Recent trends in cardiovascular complications among men and women with and without diabetes. Diabetes Care. 29:32–37. 2006. View Article : Google Scholar

2 

Brownlee M: The pathobiology of diabetic complications: A unifying mechanism. Diabetes. 54:1615–1625. 2005. View Article : Google Scholar

3 

Lu J, Xiang G, Liu M, Mei W, Xiang L and Dong J: Irisin protects against endothelial injury and ameliorates atherosclerosis in apolipoprotein E-Null diabetic mice. Atherosclerosis. 243:438–448. 2015. View Article : Google Scholar

4 

Zhou H, Wang S, Zhu P, Hu S, Chen Y and Ren J: Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission. Redox Biol. 15:335–346. 2018. View Article : Google Scholar :

5 

Bohuszewicz O and Low HH: Structure of a mitochondrial fission dynamin in the closed conformation. Nat Struct Mol Biol. 25:722–731. 2018. View Article : Google Scholar : PubMed/NCBI

6 

Mansouri A, Gattolliat CH and Asselah T: Mitochondrial dysfunction and signaling in chronic liver diseases. Gastroenterology. 155:629–647. 2018. View Article : Google Scholar : PubMed/NCBI

7 

Sheng J, Li H, Dai Q, Lu C, Xu M, Zhang J and Feng J: NR4A1 promotes diabetic nephropathy by activating mff-mediated mitochondrial fission and suppressing parkin-mediated mitophagy. Cell Physiol Biochem. 48:1675–1693. 2018. View Article : Google Scholar : PubMed/NCBI

8 

Liu WJ, Jiang HF, Rehman FU, Zhang JW, Chang Y, Jing L and Zhang JZ: Lycium barbarum polysaccharides decrease hyperglycemia-aggravated ischemic brain injury through maintaining mitochondrial fission and fusion balance. Int J Biol Sci. 13:901–910. 2017. View Article : Google Scholar : PubMed/NCBI

9 

Wils J, Favre J and Bellien J: Modulating putative endothelial progenitor cells for the treatment of endothelial dysfunction and cardiovascular complications in diabetes. Pharmacol Ther. 170:98–115. 2017. View Article : Google Scholar

10 

Fang WJ, Wang CJ, He Y, Zhou YL, Peng XD and Liu SK: Resveratrol alleviates diabetic cardiomyopathy in rats by improving mitochondrial function through PGC-1alpha deacetylation. Acta Pharmacol Sin. 39:59–73. 2018. View Article : Google Scholar

11 

Ding M, Feng N, Tang D, Feng J, Li Z, Jia M, Liu Z, Gu X, Wang Y, Fu F and Pei J: Melatonin prevents Drp1-mediated mitochondrial fission in diabetic hearts through SIRT1-PGC1alpha pathway. J Pineal Res. 65:e124912018. View Article : Google Scholar

12 

Ou X, Lee MR, Huang X, Messina-Graham S and Broxmeyer HE: Sirt1 positively regulates autophagy and mitochondria function in embryonic stem cells under oxidative stress. Stem Cells. 32:1183–1194. 2014. View Article : Google Scholar : PubMed/NCBI

13 

Shi Y, Lv X, Liu Y, et al: Elevating ATP-binding cassette transporter G1 improves re-endothelialization function of endothelial progenitor cells via Lyn/Akt/eNOS in diabetic mice. FASEB J. Jun 12–2018.Epub ahead of print. View Article : Google Scholar

14 

Wang W, Yang C, Wang XY, Zhou LY, Lao GJ, Liu D, Wang C, Hu MD, Zeng TT, Yan L and Ren M: MicroRNA-129 and - 335 promote diabetic wound healing by inhibiting sp1-mediated mmp-9 expression. Diabetes. 67:1627–1638. 2018. View Article : Google Scholar : PubMed/NCBI

15 

Ma TJ, Zhang ZW, Lu YL, Zhang YY, Tao DC, Liu YQ and Ma YX: CLOCK and BMAL1 stabilize and activate RHOA to promote F-actin formation in cancer cells. Exp Mol Med. 50:1302018. View Article : Google Scholar : PubMed/NCBI

16 

Kilian P, Campbell S, Bilodeau L, Guimond MO, Roberge C, Gallo-Payet N and Payet MD: Angiotensin II type 2 receptor stimulation increases the rate of NG10815 cell migration via actin depolymerization. Endocrinology. 149:2923–2933. 2008. View Article : Google Scholar : PubMed/NCBI

17 

Chang CH, Hsu CC, Lee AS, Wang SW, Lin KT, Chang WL, Peng HC, Huang WC and Chung CH: 4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFkappaB suppression. BMC Complement Altern Med. 18:1082018. View Article : Google Scholar

18 

Li Y, Wang H, Zhang R, Zhang G, Yang Y and Liu Z: Leukemia growth is inhibited by benzoxime without causing any harmful effect in rats bearing RBL-1 xenotransplants. Oncol Lett. 17:1934–1938. 2019.PubMed/NCBI

19 

Cavariani MM, de Mello Santos T, Pereira DN, de Almeida Chuffa LG, Felipe Pinheiro PF, Scarano WR and Domeniconi RF: Maternal protein restriction differentially alters the expression of AQP1, AQP9 and VEGFr-2 in the epididymis of rat offspring. Int J Mol Sci. 20:E4692019. View Article : Google Scholar : PubMed/NCBI

20 

Xu Y, Bai L, Chen X, Li Y, Qin Y, Meng X and Zhang Q: 6-Shogaol ameliorates diabetic nephropathy through anti-inflammatory, hyperlipidemic, anti-oxidative activity in db/db mice. Biomed Pharmacother. 97:633–641. 2018. View Article : Google Scholar

21 

Liu Y and Zheng Y: Bach1 siRNA attenuates bleomycin-induced pulmonary fibrosis by modulating oxidative stress in mice. Int J Mol Med. 39:91–100. 2017. View Article : Google Scholar

22 

Ekim Kocabey A, Kost L, Gehlhar M, Rodel G and Gey U: Mitochondrial sco proteins are involved in oxidative stress defense. Redox Biol. 21:1010792018. View Article : Google Scholar : PubMed/NCBI

23 

Zhu P, Hu S, Jin Q, Li D, Tian F, Toan S, Li Y, Zhou H and Chen Y: Ripk3 promotes ER stress-induced necroptosis in cardiac IR injury: A mechanism involving calcium overload/XO/ROS/mPTP pathway. Redox Biol. 16:157–168. 2018. View Article : Google Scholar : PubMed/NCBI

24 

Liang W, Buluc M, van Breemen C and Wang X: Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells. Cell Calcium. 36:431–443. 2004. View Article : Google Scholar : PubMed/NCBI

25 

Pickart L and Thaler MM: Growth-modulating human plasma tripeptide: Relationship between molecular structure and DNA synthesis in hepatoma cells. FEBS Lett. 104:119–122. 1979. View Article : Google Scholar : PubMed/NCBI

26 

Shi C, Cai Y, Li Y, Li Y, Hu N, Ma S, Hu S, Zhu P, Wang W and Zhou H: Yap promotes hepatocellular carcinoma metastasis and mobilization via governing cofilin/F-actin/lamellipodium axis by regulation of JNK/Bnip3/SERCA/CaMKII pathways. Redox Biol. 14:59–71. 2018. View Article : Google Scholar

27 

Zhou H, Zhu P, Guo J, Hu N, Wang S, Li D, Hu S, Ren J, Cao F and Chen Y: Ripk3 induces mitochondrial apoptosis via inhibition of FUNDC1 mitophagy in cardiac IR injury. Redox Biol. 13:498–507. 2017. View Article : Google Scholar : PubMed/NCBI

28 

Jin Q, Li R, Hu N, Xin T, Zhu P, Hu S, Ma S, Zhu H, Ren J and Zhou H: DUSP1 alleviates cardiac ischemia/reperfusion injury by suppressing the Mff-required mitochondrial fission and Bnip3-related mitophagy via the JNK pathways. Redox Biol. 14:576–587. 2018. View Article : Google Scholar

29 

Shuang Y, Li C, Zhou X, Huang Y and Zhang L: MicroRNA-195 inhibits growth and invasion of laryngeal carcinoma cells by directly targeting DCUN1D1. Oncol Rep. 38:2155–2165. 2017. View Article : Google Scholar : PubMed/NCBI

30 

Ren Y, Du C, Shi Y, Wei J, Wu H and Cui H: The Sirt1 activator, SRT1720, attenuates renal fibrosis by inhibiting CTGF and oxidative stress. Int J Mol Med. 39:1317–1324. 2017. View Article : Google Scholar : PubMed/NCBI

31 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar

32 

Zhao WJ, Zhang HF and Su JY: Downregulation of microRNA-195 promotes angiogenesis induced by cerebral infarction via targeting VEGFA. Mol Med Rep. 16:5434–5440. 2017. View Article : Google Scholar : PubMed/NCBI

33 

Zhou H, Wang J, Zhu P, Hu S and Ren J: Ripk3 regulates cardiac microvascular reperfusion injury: The role of IP3R-dependent calcium overload, XO-mediated oxidative stress and F-action/filopodia-based cellular migration. Cell Signal. 45:12–22. 2018. View Article : Google Scholar : PubMed/NCBI

34 

Dai Y, Zhang J, Xiang J, Li Y, Wu D and Xu J: Calcitriol inhibits ROS-NLRP3-IL-1beta signaling axis via activation of Nrf2-antioxidant signaling in hyperosmotic stress stimulated human corneal epithelial cells. Redox Biol. 21:1010932018. View Article : Google Scholar

35 

Park SJ, Ahmad F, Um JH, Brown AL, Xu X, Kang H, Ke H, Feng X, Ryall J, Philp A, et al: Specific Sirt1 activator-mediated improvement in glucose homeostasis requires Sirt1-independent activation of AMPK. EBioMedicine. 18:128–138. 2017. View Article : Google Scholar : PubMed/NCBI

36 

Liu J, Jiang C, Ma X and Wang J: Notoginsenoside Fc attenuates high glucose-induced vascular endothelial cell injury via upregulation of PPAR-gamma in diabetic spraguedawley rats. Vascul Pharmacol. 109:27–35. 2018. View Article : Google Scholar : PubMed/NCBI

37 

Kojima H, Otani A, Oishi A, Makiyama Y, Nakagawa S and Yoshimura N: Granulocyte colony-stimulating factor attenuates oxidative stress-induced apoptosis in vascular endothelial cells and exhibits functional and morphologic protective effect in oxygen-induced retinopathy. Blood. 117:1091–1100. 2011. View Article : Google Scholar

38 

Zhou H, Hu S, Jin Q, Shi C, Zhang Y, Zhu P, Ma Q, Tian F and Chen Y: Mff-dependent mitochondrial fission contributes to the pathogenesis of cardiac microvasculature ischemia/reperfusion injury via induction of mROS-mediated cardiolipin oxidation and HK2/VDAC1 disassociation-involved mPTP opening. J Am Heart Assoc. 6:e0053282017. View Article : Google Scholar : PubMed/NCBI

39 

Wang Y, Subramanian M, Yurdagul A Jr, Barbosa-Lorenzi VC, Cai B, de Juan-Sanz J, Ryan TA, Nomura M, Maxfield FR and Tabas I: Mitochondrial fission promotes the continued clearance of apoptotic cells by macrophages. Cell. 171:331–345.e22. 2017. View Article : Google Scholar : PubMed/NCBI

40 

Xu P, Zhang G, Sha L and Hou S: DUSP1 alleviates cerebral ischaemia reperfusion injury via inactivating JNK-Mff pathways and repressing mitochondrial fission. Life Sci. 210:251–262. 2018. View Article : Google Scholar : PubMed/NCBI

41 

Ji K, Lin K, Wang Y, Du L, Xu C, He N, Wang J, Liu Y and Liu Q: TAZ inhibition promotes IL-2-induced apoptosis of hepatocellular carcinoma cells by activating the JNK/F-actin/mitochondrial fission pathway. Cancer Cell Int. 18:1172018. View Article : Google Scholar : PubMed/NCBI

42 

Hubbard BP and Sinclair DA: Small molecule SIRT1 activators for the treatment of aging and age-related diseases. Trends Pharmacol Sci. 35:146–154. 2014. View Article : Google Scholar : PubMed/NCBI

43 

Zhang Q, Deng Q, Zhang J, Ke J, Zhu Y, Wen RW, Ye Z, Peng H, Su ZZ, Wang C and Lou T: Activation of the Nrf2-ARE pathway ameliorates hyperglycemia-mediated mitochondrial dysfunction in podocytes partly through Sirt1. Cell Physiol Biochem. 48:1–15. 2018. View Article : Google Scholar : PubMed/NCBI

44 

Mishra M, Duraisamy AJ and Kowluru RA: Sirt1: A guardian of the development of diabetic retinopathy. Diabetes. 67:745–754. 2018. View Article : Google Scholar : PubMed/NCBI

45 

Ayanga BA, Badal SS, Wang Y, Galvan DL, Chang BH, Schumacker PT and Danesh FR: Dynamin-related protein 1 deficiency improves mitochondrial fitness and protects against progression of diabetic nephropathy. J Am Soc Nephrol. 27:2733–2747. 2016. View Article : Google Scholar : PubMed/NCBI

46 

Galloway CA and Yoon Y: Mitochondrial dynamics in diabetic cardiomyopathy. Antioxid Redox Signal. 22:1545–1562. 2015. View Article : Google Scholar : PubMed/NCBI

47 

Sheng J, Li H, Dai Q, Lu C, Xu M, Zhang J and Feng J: DUSP1 recuses diabetic nephropathy via repressing JNK-Mff-mitochondrial fission pathways. J Cell Physiol. 234:3043–3057. 2019. View Article : Google Scholar

48 

Becatti M, Barygina V, Mannucci A, Emmi G, Prisco D, Lotti T, Fiorillo C and Taddei N: Sirt1 protects against oxidative stress-induced apoptosis in fibroblasts from psoriatic patients: A new insight into the pathogenetic mechanisms of psoriasis. Int J Mol Sci. 19:E15722018. View Article : Google Scholar : PubMed/NCBI

49 

Zhang L, Bao D, Li P, Lu Z, Pang L, Chen Z, Guo H, Gao Z and Jin Q: Particle-induced SIRT1 downregulation promotes osteoclastogenesis and osteolysis through ER stress regulation. Biomed Pharmacother. 104:300–306. 2018. View Article : Google Scholar : PubMed/NCBI

50 

Suzuki M, Bandoski C and Bartlett JD: Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling. Free Radic Biol Med. 89:369–378. 2015. View Article : Google Scholar : PubMed/NCBI

51 

Chae HD and Broxmeyer HE: SIRT1 deficiency downregulates PTEN/JNK/FOXO1 pathway to block reactive oxygen species-induced apoptosis in mouse embryonic stem cells. Stem Cells Dev. 20:1277–1285. 2011. View Article : Google Scholar :

52 

Yamamoto K, Imamura H and Ando J: Shear stress augments mitochondrial ATP generation that triggers ATP release and Ca2+ signaling in vascular endothelial cells. Am J Physiol Heart Circ Physiol. 315:H1477–H1485. 2018. View Article : Google Scholar

53 

Li X, Fang P, Li Y, Kuo YM, Andrews AJ, Nanayakkara G, Johnson C, Fu H, Shan H, Du F, et al: Mitochondrial reactive oxygen species mediate lysophosphatidylcholine-induced endothelial cell activation. Arterioscler Thromb Vasc Biol. 36:1090–1100. 2016. View Article : Google Scholar : PubMed/NCBI

54 

Placido AI, Pereira CM, Correira SC, Carvalho C, Oliveira CR and Moreira PI: Phosphatase 2a inhibition affects endoplasmic reticulum and mitochondria homeostasis via cytoskeletal alterations in brain endothelial cells. Mol Neurobiol. 5:154–168. 2017. View Article : Google Scholar

55 

Park SJ, Lee H, Jo DS, Jo YK, Shin JH, Kim HB, Seo HM, Rubinsztein DC, Koh JY, Lee EK and Cho DH: Heterogeneous nuclear ribonucleoprotein A1 post-transcriptionally regulates Drp1 expression in neuroblastoma cells. Biochim Biophys Acta. 1849:1423–1431. 2015. View Article : Google Scholar : PubMed/NCBI

56 

Kashatus DF: Restraining the divider: A drp1-phospholipid interaction inhibits drp1 activity and shifts the balance from mitochondrial fission to fusion. Mol Cell. 63:913–915. 2016. View Article : Google Scholar : PubMed/NCBI

57 

Zheng D, Yu Y, Li M, Wang G, Chen R, Fan GC, Martin C, Xiong S and Peng T: Inhibition of MicroRNA 195 prevents apoptosis and multiple-organ injury in mouse models of sepsis. J Infect Dis. 213:1661–1670. 2016. View Article : Google Scholar :

58 

Mortuza R, Feng B and Chakrabarti S: miR-195 regulates sirt1-mediated changes in diabetic retinopathy. Diabetologia. 57:1037–1046. 2014. View Article : Google Scholar : PubMed/NCBI

59 

Zhu H, Yang Y, Wang Y, Li J, Schiller PW and Peng T: MicroRNA- 195 p romotes palmitate-induced apoptosis in cardiomyocytes by down-regulating sirt1. Cardiovasc Res. 92:75–84. 2011. View Article : Google Scholar : PubMed/NCBI

60 

Khader A, Yang WL, Hansen LW, Rajayer SR, Prince JM, Nicastro JM, Coppa GF and Wang P: SRT1720, a sirtuin 1 activator, attenuates organ injury and inflammation in sepsis. J Surg Res. 219:288–295. 2017. View Article : Google Scholar : PubMed/NCBI

61 

Guo S, Liao H, Liu J, Liu J, Tang F, He Z, Li Y and Yang Q: Resveratrol activated sonic hedgehog signaling to enhance viability of NIH3T3 cells in vitro via regulation of sirt1. Cell Physiol Biochem. 50:1346–1360. 2018. View Article : Google Scholar : PubMed/NCBI

62 

Pacholec M, Bleasdale JE, Chrunyk B, Cunningham D, Flynn D, Garofalo RS, Griffith D, Griffor M, Loulakis P, Pabst B, et al: SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J Biol Chem. 285:8340–8351. 2010. View Article : Google Scholar : PubMed/NCBI

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July-2019
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Qin R, Zhang L, Lin D, Xiao F and Guo L: Sirt1 inhibits HG-induced endothelial injury: Role of Mff-based mitochondrial fission and F‑actin homeostasis-mediated cellular migration. Int J Mol Med 44: 89-102, 2019
APA
Qin, R., Zhang, L., Lin, D., Xiao, F., & Guo, L. (2019). Sirt1 inhibits HG-induced endothelial injury: Role of Mff-based mitochondrial fission and F‑actin homeostasis-mediated cellular migration. International Journal of Molecular Medicine, 44, 89-102. https://doi.org/10.3892/ijmm.2019.4185
MLA
Qin, R., Zhang, L., Lin, D., Xiao, F., Guo, L."Sirt1 inhibits HG-induced endothelial injury: Role of Mff-based mitochondrial fission and F‑actin homeostasis-mediated cellular migration". International Journal of Molecular Medicine 44.1 (2019): 89-102.
Chicago
Qin, R., Zhang, L., Lin, D., Xiao, F., Guo, L."Sirt1 inhibits HG-induced endothelial injury: Role of Mff-based mitochondrial fission and F‑actin homeostasis-mediated cellular migration". International Journal of Molecular Medicine 44, no. 1 (2019): 89-102. https://doi.org/10.3892/ijmm.2019.4185