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Up-regulation of 14-3-3β plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation
Biochemical and Biophysical Research Communications xxx (2017) 1e7
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Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation Lishuai Feng, Xu Ma, Jianbo Wang*, Qinghua Tian Department of Interventional Radiology, The Sixth People's Hospital Affiliated to Shanghai Jiaotong University, No 600 Yishan Road, Xuhui District, Shanghai 200233, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2017 Accepted 30 June 2017 Available online xxx
Purpose: The objective of this study was to determine whether diabetes mellitus (DM)-induced upregulation of 14-3-3b (YWHAB) in endothelial cells enhances intimal hyperplasia in carotid arteryinjured DM Sprague-Dawley (SD) rats. Methods: YWHAB expression and rat aortic endothelial cell (RAOEC) vitality were examined using Cell Counting Kit-8 (CCK-8), quantitative reverse transcription PCR (qRT-PCR), and western blot analysis in cells treated with different glucose concentrations (5.6, 10, 15, 25, or 35 mM). For in vivo experiments, a YWHAB small interfering (si) RNA recombinant lentiviral vector (YWHAB-LV) or Mock siRNA recombinant lentiviral vector (Mock-LV) were injected into streptozotocin-induced DM SD rats via the tail vein. YWHAB expression and carotid artery morphology were assessed 7 days post injury using immunofluorescence (IF) and hematoxylin-eosin (HE) staining. The proliferation and migration of Mock-LV and YWHAB-LV-infected RAOECs treated with 25 mM glucose were examined using cell scratch tests and flow cytometry. BCL2-Associated X (BAX) distribution in RAOECs treated with 25 mM glucose was examined using IF staining and western blot analysis. Results: Western blot, qRT-PCR, and CCK-8 analyses demonstrated that both YWHAB expression and cell vitality increased with increasing glucose concentration (p < 0.05). YWHAB IF staining was increased in DM rats compared with the normal group (p < 0.05). HE staining showed that intimal hyperplasia is alleviated in YWHAB-silenced DM rats (p < 0.05). YWHAB silencing suppressed the proliferation and migration of RAOECs treated with 25 mM glucose (p < 0.05). Moreover, western blot analyses and IF staining demonstrated that YWHAB silencing increased the translocation of BAX from the cytoplasm to mitochondria in RAOECs treated with 25 mM glucose (p < 0.05). Conclusions: Our results indicate that hyperglycemia-induced up-regulation of YWHAB in endothelial cell plays a significant role in intimal hyperplasia following carotid artery injury by enhancing endothelial cell proliferation and migration. YWHAB inhibition in hyperglycemic patients may constitute a potential target for therapeutic interventions via restenosis prevention. © 2017 Elsevier Inc. All rights reserved.
Keywords: 14-3-3b Endothelial cells Cell proliferation Diabetes mellitus BAX
1. Introduction Diabetes mellitus (DM) is a serious chronic metabolic disorder resulting from an absolute or relative insufficiency of insulin [1,2]. Approximately 60e80% of DM patients present with hypertension and the vascular complications of DM patients account for approximately 60% of all DM-related deaths [3,4]. Of the various
* Corresponding author. E-mail address: [email protected] (J. Wang).
vascular complications, DM-related excessive proliferation and migration of endothelial cells are thought to underlie the pathogenesis of artery intimal hyperplasia, which may result in pathologies such as atherosclerosis or arterial restenosis following interventional treatment [5,6]. Hyperglycemia in DM is thought to play important roles by increasing the expression of mitogenic growth factors and inflammatory mediators [7,8]; endothelial cells perceive these mechanical signals and then convert them into biological events affecting cell proliferation and apoptosis [9,10]. During this process, vascular complications occur because of excessive endothelial cell
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Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199
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proliferation and migration. For example, vascular endothelial growth factor (VEGF) and its receptors activate pathways leading to endothelial cell proliferation and eventually capillary tube formation [11]. 14-3-3 proteins are phospho-serine/phospho-threonine (pS/T) binding proteins that can be classified into seven isoforms, namely b, g, ε, d, q, h, and z, all of which adopt a similar horseshoe-like structure capable of binding pS/T residues in a sequence specific context [12]. 14-3-3 b, encoded by the YWHAB gene [13], has been identified as a key regulatory component in many vital cellular processes such as signal transduction, protein synthesis, protein folding and degradation, cell cycle, cytoskeleton rearrangement, cellular trafficking, DNA replication, apoptosis, and survival [14]. A number of studies have suggested that expression of the 14-3-3 protein YWHAB might be up-regulated under high glucose concentrations [15,16] and that YWHAB is associated with growth disorders as well as several types of cancer including lung, breast, neck, and brain tumors [17]. Takihara et al. [18] found that overexpression of 14-3-3 b in NIH 3T3 cells could stimulate cell growth and promote tumor formation in nude mice; Clapp et al. [19] identified 14-3-3b/a as a specific inhibitor of apoptosis following the accumulation of reactive oxygen species in human cells; and Zheng et al. [20] confirmed the interaction between 14-3-3 b and Big Mitogen-activated Protein Kinase 1 (BMK1), an important factor that promotes cell proliferation and inhibits cell apoptosis, using yeast two-hybrid analysis. Moreover, Cavet et al. [21] identified 143-3b as a binding protein of p90 Ribosomal S6 Kinase (RSK), whose family members are involved in mitogen-activated cell growth and proliferation, differentiation, and cell survival. However, although an association between diabetes and cardiovascular complications has been identified, to date, no studies have examined the association between high glucose, 14-3-3b expression, and intimal hyperplasia in diabetics following injury. Recently, Haque et al. [15] showed that down-regulation of microRNA (miR)-152 may induce the proliferation of human retinal endothelial cells (hRECs), while Zhao et al. [22] demonstrated that high glucose may suppress the expression of miR-152 in hepatocytes, which may induce the up-regulation of YWHAB [16]. Based these findings, we hypothesized that YWHAB might also be involved in the development of high glucose-induced vascular complications. Therefore, we investigated the role of YWHAB in the proliferation and migration of rat aortic endothelial cells (RAOECs). In addition, to further elucidate the possible mechanism underlying the development of DM-related intimal hyperplasia, we examined the effect of YWHAB on the translocation of BCL2-Associated X (BAX), a proapoptotic member of the Bcl-2 family, which localizes mainly in the cytoplasm but redistributes to the mitochondria in response to apoptotic stimuli by inducing cytochrome C release [23]. 2. Materials and methods 2.1. Rat aortic endothelial cell culturing and infection RAOECs were cultured in 5.6, 10, 15, 25, or 35 mM glucose Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA). Once cells reached at least 70% confluence, they were harvested and subjected to western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analysis or the CCK-8 test. RAOECs treated with 25 mM glucose were infected with either a YWHAB small interfering (si) RNA recombinant lentiviral vector (YWHAB-LV) or corresponding control expression vector (Mock-LV) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The YWHAB-LV and Mock-LV were provided by the laboratory of Prof. Wheeler (Toronto, Canada).
2.2. Cell vitality Once cells reached at least 70% confluence they were incubated with 10 ml Cell Counting Kit-8 (CCK8; Yeasen, Shanghai, China) working solution for 1 h prior to measuring cell optical density (OD) at 450 nm. The OD value represented cell vitality.
2.3. Animal protocols Twenty-four SD male rats (200 ± 20 g), were randomly separated into four groups: uninjured (n ¼ 6), normal (n ¼ 6), diabetes mellitus (DM; n ¼ 6), and DM þ YWHAB silencing group (n ¼ 6). Three rats from each group were used for Hematoxylin-eosin (HE) staining and the other three rats were used for Immunofluorescence (IF) staining. All rats were fed a standard diet ad libitum. Room temperature was maintained at 23e25 C, with 50e60% humidity and an 8 h light period daily. Animals and forage were purchased from the Model Animal Research Centre of Nanjing University (Jiangsu, China). This study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85e23, revised 1996).
2.4. Establishment of the carotid artery injury diabetes mellitus rat model and injection of the lentivirus in vivo Following an intraperitoneal injection of 65 mg/kg streptozotocin to establish the DM rat model, 100 ml of either Mock-LV or (108 TU/ml titer) or YWHAB-LV (108 TU/ml titer) were injected into the tail vein of rats in the DM and DM þ YWHAB silencing groups, respectively. Rats in the normal group served as the negative control. Two weeks post tail lentivirus injection, rats were anesthetized with isofluorane and a 2-French balloon catheter (Edwards Lifesciences, Irvine, CA, USA) was inserted through the left external carotid artery into the common carotid artery and insufflated three times with 2 atm of pressure. Following injury, the external carotid artery was quickly ligated and blood flow was resumed. All DM rats were sacrificed at 7 days post injury.
2.5. Hematoxylin-eosin and immunofluorescence staining Specimens were incubated in 4% paraformaldehyde for 24e48 h and then embedded in paraffin. Paraffin sections (3e5 mm thick) were dewaxed, stained, examined microscopically, and photographed. Images were captured using a fluorescence microscope (Jenoptik, Jena, Germany). YWHAB expression levels were determined using an immunofluorescence assay; 3e5 mm frozen carotid artery sections were cultured for 24 h in complete medium, fixed with 4% paraformaldehyde for 10 min, and permeabilized in 0.01 M phosphate buffered saline (PBS)/0.5% Triton X-100 for 5 min. Intima were then incubated with an anti-YWHAB primary antibody (1:200 dilution; Abcam, Cambridge, UK) for 1 h at room temperature and then with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA). Following washes in PBS, the intima were incubated for 3 min with 0.25 mg/ml 40 ,6diamidino-2-phenylindole (DAPI). A similar protocol was used for the in vivo experiments; briefly, the RAOECs were washed with PBS prior to lysis in RIPA buffer supplemented with protease inhibitors (Roche Applied Sciences, Laval, QC, Canada) and then incubated with a primary antibody against YWHAB or BAX (anti-BAX, 1:200 dilution; Sigma-Aldrich, St. Louis, MO, USA). Mitochondria were visualized with mitochondria-targeted dsRed (Yeasen). Images were captured using a fluorescence microscope (Jenoptik).
Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199
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2.6. Quantitative reverse transcription PCR (qRT-PCR)
2.10. Statistical analyses
Total RNA was extracted using the TRIzol (Invitrogen) and chloroform/isopropanol method. cDNA was generated using the PrimeScript RT Reagent Kit (TAKARA, Dalian, China) and PCR was performed using SYBR Premix Ex Taq (TAKARA) with YWHAB and GAPDH specific primers. PCR data were normalized to GAPDH and presented as 2- DDCt values. The primers used for qRT-PCR are as follows: YWHAB forward: 50 -CAAAGAGTACCGTGAGAAGATCGAG-30 YWHAB reverse: 50 -CGGATGCAACTTCAGAAAGATACC-30 GAPDH forward: 50 -GGCACAGTCAAGGCTGAGAATG-30 GAPDH reverse: 50 -ATGGTGGTGAAGACGCCAGTA-30
Results from at least three independent experiments are presented as means ± SD. Statistical analyses were performed using the 8.0 SAS program. One-way analysis of variance with Student's ttest was applied to compare the statistical significance of the differences between groups. P-values <0.05 were considered statistically significant.
2.7. Western blot analysis Western blot analysis was performed to detect YWHAB expression and BAX distribution in RAOECs from different groups. RAOEC cellular lysates were isolated from each group using RIPA (Beyotime Biotechnology, Shanghai, China) lysis buffer. Cytoplasmic and mitochondrial fractions were separated from crude cell lysates using the Mitochondria/Cytosol Fractionation Kit (Biovision, Mountain View, CA, USA). Next, total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk in Tris-buffered saline Tween-20 (TBST) for 2 h and then incubated with diluted primary antibodies (anti-GAPDH, CST; antiYWHAB and anti-BAX) overnight at 4 C. The membranes were then washed three times with TBST, incubated for an additional 2 h at 25 C with a FITC-conjugated secondary antibody (SigmaAldrich), and finally washed with TBST. Semi-quantitative gel images were analyzed with an automatic gel imager using Quantity One software (Bio-Rad). GAPDH was used as the internal control.
2.8. Cell wound scratch assay RAOECs were inoculated into 6-well plates (500,000 cells/well) and grown for 24 h to reach at least 70% confluence. Following 24 h of serum withdrawal, three scratches were made in each well using a P-20 pipette. Cells were incubated for 24 h to enable migration and then images were taken of each well to measure the gap change in the scratches.
2.9. Flow cytometric analysis Following infection with either YWHAB-LV or mock-LV for 24 h, 500,000 RAOECs were collected, washed twice with precooled PBS, and resuspended in 100 ml binding buffer (Yeasen). The cells were then stained with Annexin V-FITC/PI (Yeasen), incubated in the dark for 10 min (25 C), resuspended in an additional 400 ml binding buffer, and then each sample was analyzed by flow cytometry. To analyze cell cycle progression, both YWHAB-LV and con-LV infected RAOECs were grown in 6-well plates to sub-confluence for 24 h and then cultured in serum (DMEM supplemented with 0.2% FBS) for 1 day. Cells were then trypsinized, centrifuged (1000 g, 5 min), resuspended and washed twice with 1 ml precooled 1 PBS, and then fixed with 70% ethanol at 4 C overnight. The fixed cells were washed and resuspended in PBS (200 ml), consecutively treated with 20 ml RNase A solution (37 C, 30 min) and 400 ml PI staining solution (4 C, 60 min; Yeasen), and then analyzed by flow cytometry.
3. Results 3.1. YWHAB expression in RAOEC is up-regulated with increasing glucose concentrations In order to maintain high reproducibility with the in vivo experiments performed in this study, our in vitro experiments were performed using RAOEC cell lines. We conducted in vitro studies to determine whether YWHAB expression varied in RAOECs with increasing glucose concentration. YWHAB expression was evaluated following treatment of RAOECs with 5.6, 10, 15, 25, or 35 mM glucose. As shown by qRT-PCR and western blot analyses, YWHAB expression was up-regulated with increasing glucose concentration (Fig. 1A and B). It should be noted that RAOEC vitality also improved with increasing glucose concentration. However, RAOEC vitality significantly decreased at 35 mM because of hyperglycemic toxicity (Fig. 1C). Because of the significant up-regulation of both YWHAB expression and cell vitality compared to 5.6 mM glucose, 25 mM glucose was chosen as the concentration for mimicking DMinduced hyperglycemia and used as the high glucose concentration in subsequent in vitro experiments. These results demonstrate that both YWHAB expression and cell vitality were up-regulated with increasing glucose concentration. 3.2. YWHAB inhibition prevents intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats Because of the high incidence of intimal hyperplasia following interventional treatment in diabetics, we examined whether there is an association between YWHAB up-regulation and intimal hyperplasia. To this end, we established a DM SD rat carotid artery injury model to examine the effect of YWHAB silencing on intimal hyperplasia. Consistent with the in vitro experiment, IF and HE staining showed that intimal YWHAB expression and intimal hyperplasia (neointimal area) were greater in the DM group than in the normal group at Day 7 post-injury (Fig. 2A and B). However, the neointimal area was significantly reduced in the DM þ YWHAB silencing group compared with the DM group (Fig. 2A). These data convincingly demonstrate that YWHAB silencing can indeed attenuate the pathological process of intimal hyperplasia. 3.3. YWHAB inhibition reduces RAOEC proliferation and migration under high glucose concentrations To explore the role of endothelial cells in the development of hyperglycemia-induced intimal hyperplasia following injury, we conducted in vitro experiments. RAOECs were incubated with 25 mM glucose to mimic in vivo hyperglycemia as explained in section 3.1. Following infection with siRNAs (YWHAB-LV or MockLV), RAOEC proliferation and migration were examined by flow cytometry and cell wound scratch assays. The silencing efficiency of YWHAB-LV was evaluated by qRT-PCR and western blot analyses (YWHAB expression in RAOECs treated with 5.6 mM glucose served as the control; Fig. 3D). The results showed that when cultured with 25 mM (high) glucose, the proliferation and migration of RAOECs infected with YWHAB-LV was significantly decreased compared
Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199
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Fig. 1. The effect of glucose concentration on YWHAB expression and RAOEC cell vitality. (A) qRT-PCR and (B) western blot analyses show that YWHAB expression is up-regulated with increasing glucose concentration. (C) CCK-8 analysis shows that RAOEC vitality is also enhanced with increasing glucose concentration. *p < 0.05 vs. 5.6 mmol/L glucose treatment; n ¼ 3.
Fig. 2. Representative image of HE and IF staining of carotid arteries in DM rats. (A) Neointimal area at Day 7 post-injury compared with uninjured group. (B) YWHAB expression (Red), DAPI (Blue). *p < 0.05 vs. Normal group, #p < 0.05 vs. DM group; n ¼ 3. Scale bar ¼ 25 mm.
with the Mock-LV group (Fig. 3A and C). Moreover, a greater number of RAOECs were arrested in the G1 phase in the YWHAB-LV group than in the Mock-LV group (Fig. 3B). These results indicate that YWHAB inhibition reduces RAOEC proliferation and migration under high glucose concentrations.
3.4. YWHAB inhibition promotes the translocation of BAX from cytoplasm to mitochondria As a number of 14-3-3 family isoforms bind to BAX and the distribution of BAX, a pro-apoptotic member of the Bcl-2 family, is
Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199
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Fig. 3. The effect of YWHAB silencing on the proliferation and migration of RAOECs treated with 25 mM glucose. Flow cytometry shows that (A) cell vitality was lower and (B) the G1 arrest rate was higher in the YWHAB-LV group compared with the Mock-LV group. (C) The cell scratch assay shows that the wound closure rate was lower in the YWHAB-LV group compared with the Mock-LV group. (D) Western blot analyses demonstrating YWHAB silencing efficiency. YWHAB expression in 5.6 mM glucose-treated RAOECs served as the control group. *p < 0.05 vs. Mock-LV group, #p < 0.05 vs. control group; n ¼ 3.
related to the regular apoptotic process of cells, we next examined BAX distribution under different glucose concentrations and the effect of YWHAB silencing on BAX distribution in RAOECs. Although mostly localized to the cytoplasm, distribution of BAX to the mitochondria is essential for maintaining the normal apoptosis process of cells by inducing cytochrome C release. Western blot analyses and IF staining showed that BAX expression was significantly decreased in the mitochondria of RAOECs treated with 25 mM glucose compared to 5.6 mM glucose. However, YWHAB silencing promoted BAX translocation to the mitochondria (Fig. 4A and B). These results indicate that high glucose-induced up-regulation of YWHAB inhibited the regular apoptosis process by sequestering BAX in the cytoplasm and suggest that YWHAB silencing may play a role in preventing excessive RAOEC proliferation and migration under high glucose conditions. 4. Discussion Hyperglycemia is considered the main factor underlying the development of vascular complications in diabetes [24]; hyperglycemia triggers a cascade of metabolic and biochemical changes occurring in this pathology. Excessive proliferation and migration of endothelial cells are important features in the pathogenesis of intimal hyperplasia, which could become a greater risk factor for arterial restenosis and atherosclerosis following injury or angioplasty [25,26]. Several studies have identified a connection between hyperglycemia and excessive endothelial cell proliferation. Wang et al. showed that scutellarin treatment could inhibit high glucose-induced proliferation in hRECs [27]; Chen et al. found that
30 mM glucose significantly promoted the migration and proliferation of endothelial cells, which was blocked by 1 mg/ml adrenomedullin [26]; and Yuan et al. [28] showed that heparanase knockdown in high glucose-treated cells could decrease migration and proliferation in human retinal vascular endothelial cells (HRECs). In addition, Li et al. [29] found that excessive proliferation of RAOECs could be inhibited by miR-98 via regulation of cyclin D2. Similarly, in this study, we demonstrated that 25 mM glucose significantly promoted RAOEC proliferation compared with 5.6 mM glucose treatment, thus confirming the role of glucose concentration in the growth status of endothelial cells. In addition, we found that YWHAB is up-regulated with increasing glucose concentration. YWHAB is a member of the highly conserved 14-3-3 family, which mediate signal transduction by binding to phosphoserinecontaining proteins [14]. Wang et al. [30] identified the binding between YWAHB and Wee1 and the following effect on the biochemical activity of Wee1. In addition, Uchida et al. [31] found that the binding between CDC25B and YWHAB also plays a role in regulating the subcellular redistribution of CDC25B; the interaction between YWHAB and CDC25B promotes the translocation of CDC25B from the nucleus to the cytoplasm. Although several studies have already demonstrated that YWHAB plays a role in the development and progression of tumors [21], the relationship between the effect of hyperglycemia on YWHAB expression and intimal hyperplasia following injury remains elusive. Regular apoptosis plays a critical role in the maintenance of tissue homeostasis. The regulation of mitochondrial membrane integrity and the release of cytochrome C from mitochondria are important processes during apoptosis and are controlled by the Bcl-
Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199
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Fig. 4. The effect of YWHAB silencing on BAX distribution in RAOECs incubated with 25 mM glucose. Cytoplasmic (Cyto) and mitochondrial (Mito) fractions were purified from RAOEC whole-cell lysates and BAX expression was detected by (A) western blot analyses. BAX expression in RAOECs treated with 5.6 mM glucose served as the control (Con). (B) The distribution of BAX (Green), mitochondria (Mito-dsRED; Red), and DAPI (Blue) were visualized by IF staining. *p < 0.05 vs. Con; n ¼ 3. Scale bar ¼ 10 mm.
2 family [32]. In our study, YWHAB silencing increased the distribution of BAX in mitochondria when RAOECs were incubated with 25 mM glucose. Studies examining the association between YWHAB and other apoptosis factors, such as BMK1, RSK, and CHOP, are required to elucidate the role of YWHAB up-regulation in the development of diabetic vascular complications [33]. In conclusion, this study shows that under DM conditions, excess YWHAB plays a role in intimal hyperplasia following carotid artery injury by promoting endothelial cell proliferation and migration via sequestration of BAX from the mitochondria. These findings identify YWHAB as a diagnostic biomarker and YWHAB silencing as a therapeutic target for intimal hyperplasia-related restenosis or atherosclerosis in diabetics. Conflicts of interest None. Acknowledgments This work was financially supported by the Shanghai Shen Kang Hospital Development Center Suburb of Three Hospital Capacity-
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Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation Lishuai Feng, Xu Ma, Jianbo Wang*, Qinghua Tian Department of Interventional Radiology, The Sixth People's Hospital Affiliated to Shanghai Jiaotong University, No 600 Yishan Road, Xuhui District, Shanghai 200233, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2017 Accepted 30 June 2017 Available online xxx
Purpose: The objective of this study was to determine whether diabetes mellitus (DM)-induced upregulation of 14-3-3b (YWHAB) in endothelial cells enhances intimal hyperplasia in carotid arteryinjured DM Sprague-Dawley (SD) rats. Methods: YWHAB expression and rat aortic endothelial cell (RAOEC) vitality were examined using Cell Counting Kit-8 (CCK-8), quantitative reverse transcription PCR (qRT-PCR), and western blot analysis in cells treated with different glucose concentrations (5.6, 10, 15, 25, or 35 mM). For in vivo experiments, a YWHAB small interfering (si) RNA recombinant lentiviral vector (YWHAB-LV) or Mock siRNA recombinant lentiviral vector (Mock-LV) were injected into streptozotocin-induced DM SD rats via the tail vein. YWHAB expression and carotid artery morphology were assessed 7 days post injury using immunofluorescence (IF) and hematoxylin-eosin (HE) staining. The proliferation and migration of Mock-LV and YWHAB-LV-infected RAOECs treated with 25 mM glucose were examined using cell scratch tests and flow cytometry. BCL2-Associated X (BAX) distribution in RAOECs treated with 25 mM glucose was examined using IF staining and western blot analysis. Results: Western blot, qRT-PCR, and CCK-8 analyses demonstrated that both YWHAB expression and cell vitality increased with increasing glucose concentration (p < 0.05). YWHAB IF staining was increased in DM rats compared with the normal group (p < 0.05). HE staining showed that intimal hyperplasia is alleviated in YWHAB-silenced DM rats (p < 0.05). YWHAB silencing suppressed the proliferation and migration of RAOECs treated with 25 mM glucose (p < 0.05). Moreover, western blot analyses and IF staining demonstrated that YWHAB silencing increased the translocation of BAX from the cytoplasm to mitochondria in RAOECs treated with 25 mM glucose (p < 0.05). Conclusions: Our results indicate that hyperglycemia-induced up-regulation of YWHAB in endothelial cell plays a significant role in intimal hyperplasia following carotid artery injury by enhancing endothelial cell proliferation and migration. YWHAB inhibition in hyperglycemic patients may constitute a potential target for therapeutic interventions via restenosis prevention. © 2017 Elsevier Inc. All rights reserved.
Keywords: 14-3-3b Endothelial cells Cell proliferation Diabetes mellitus BAX
1. Introduction Diabetes mellitus (DM) is a serious chronic metabolic disorder resulting from an absolute or relative insufficiency of insulin [1,2]. Approximately 60e80% of DM patients present with hypertension and the vascular complications of DM patients account for approximately 60% of all DM-related deaths [3,4]. Of the various
* Corresponding author. E-mail address: [email protected] (J. Wang).
vascular complications, DM-related excessive proliferation and migration of endothelial cells are thought to underlie the pathogenesis of artery intimal hyperplasia, which may result in pathologies such as atherosclerosis or arterial restenosis following interventional treatment [5,6]. Hyperglycemia in DM is thought to play important roles by increasing the expression of mitogenic growth factors and inflammatory mediators [7,8]; endothelial cells perceive these mechanical signals and then convert them into biological events affecting cell proliferation and apoptosis [9,10]. During this process, vascular complications occur because of excessive endothelial cell
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Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199
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proliferation and migration. For example, vascular endothelial growth factor (VEGF) and its receptors activate pathways leading to endothelial cell proliferation and eventually capillary tube formation [11]. 14-3-3 proteins are phospho-serine/phospho-threonine (pS/T) binding proteins that can be classified into seven isoforms, namely b, g, ε, d, q, h, and z, all of which adopt a similar horseshoe-like structure capable of binding pS/T residues in a sequence specific context [12]. 14-3-3 b, encoded by the YWHAB gene [13], has been identified as a key regulatory component in many vital cellular processes such as signal transduction, protein synthesis, protein folding and degradation, cell cycle, cytoskeleton rearrangement, cellular trafficking, DNA replication, apoptosis, and survival [14]. A number of studies have suggested that expression of the 14-3-3 protein YWHAB might be up-regulated under high glucose concentrations [15,16] and that YWHAB is associated with growth disorders as well as several types of cancer including lung, breast, neck, and brain tumors [17]. Takihara et al. [18] found that overexpression of 14-3-3 b in NIH 3T3 cells could stimulate cell growth and promote tumor formation in nude mice; Clapp et al. [19] identified 14-3-3b/a as a specific inhibitor of apoptosis following the accumulation of reactive oxygen species in human cells; and Zheng et al. [20] confirmed the interaction between 14-3-3 b and Big Mitogen-activated Protein Kinase 1 (BMK1), an important factor that promotes cell proliferation and inhibits cell apoptosis, using yeast two-hybrid analysis. Moreover, Cavet et al. [21] identified 143-3b as a binding protein of p90 Ribosomal S6 Kinase (RSK), whose family members are involved in mitogen-activated cell growth and proliferation, differentiation, and cell survival. However, although an association between diabetes and cardiovascular complications has been identified, to date, no studies have examined the association between high glucose, 14-3-3b expression, and intimal hyperplasia in diabetics following injury. Recently, Haque et al. [15] showed that down-regulation of microRNA (miR)-152 may induce the proliferation of human retinal endothelial cells (hRECs), while Zhao et al. [22] demonstrated that high glucose may suppress the expression of miR-152 in hepatocytes, which may induce the up-regulation of YWHAB [16]. Based these findings, we hypothesized that YWHAB might also be involved in the development of high glucose-induced vascular complications. Therefore, we investigated the role of YWHAB in the proliferation and migration of rat aortic endothelial cells (RAOECs). In addition, to further elucidate the possible mechanism underlying the development of DM-related intimal hyperplasia, we examined the effect of YWHAB on the translocation of BCL2-Associated X (BAX), a proapoptotic member of the Bcl-2 family, which localizes mainly in the cytoplasm but redistributes to the mitochondria in response to apoptotic stimuli by inducing cytochrome C release [23]. 2. Materials and methods 2.1. Rat aortic endothelial cell culturing and infection RAOECs were cultured in 5.6, 10, 15, 25, or 35 mM glucose Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA). Once cells reached at least 70% confluence, they were harvested and subjected to western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analysis or the CCK-8 test. RAOECs treated with 25 mM glucose were infected with either a YWHAB small interfering (si) RNA recombinant lentiviral vector (YWHAB-LV) or corresponding control expression vector (Mock-LV) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The YWHAB-LV and Mock-LV were provided by the laboratory of Prof. Wheeler (Toronto, Canada).
2.2. Cell vitality Once cells reached at least 70% confluence they were incubated with 10 ml Cell Counting Kit-8 (CCK8; Yeasen, Shanghai, China) working solution for 1 h prior to measuring cell optical density (OD) at 450 nm. The OD value represented cell vitality.
2.3. Animal protocols Twenty-four SD male rats (200 ± 20 g), were randomly separated into four groups: uninjured (n ¼ 6), normal (n ¼ 6), diabetes mellitus (DM; n ¼ 6), and DM þ YWHAB silencing group (n ¼ 6). Three rats from each group were used for Hematoxylin-eosin (HE) staining and the other three rats were used for Immunofluorescence (IF) staining. All rats were fed a standard diet ad libitum. Room temperature was maintained at 23e25 C, with 50e60% humidity and an 8 h light period daily. Animals and forage were purchased from the Model Animal Research Centre of Nanjing University (Jiangsu, China). This study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85e23, revised 1996).
2.4. Establishment of the carotid artery injury diabetes mellitus rat model and injection of the lentivirus in vivo Following an intraperitoneal injection of 65 mg/kg streptozotocin to establish the DM rat model, 100 ml of either Mock-LV or (108 TU/ml titer) or YWHAB-LV (108 TU/ml titer) were injected into the tail vein of rats in the DM and DM þ YWHAB silencing groups, respectively. Rats in the normal group served as the negative control. Two weeks post tail lentivirus injection, rats were anesthetized with isofluorane and a 2-French balloon catheter (Edwards Lifesciences, Irvine, CA, USA) was inserted through the left external carotid artery into the common carotid artery and insufflated three times with 2 atm of pressure. Following injury, the external carotid artery was quickly ligated and blood flow was resumed. All DM rats were sacrificed at 7 days post injury.
2.5. Hematoxylin-eosin and immunofluorescence staining Specimens were incubated in 4% paraformaldehyde for 24e48 h and then embedded in paraffin. Paraffin sections (3e5 mm thick) were dewaxed, stained, examined microscopically, and photographed. Images were captured using a fluorescence microscope (Jenoptik, Jena, Germany). YWHAB expression levels were determined using an immunofluorescence assay; 3e5 mm frozen carotid artery sections were cultured for 24 h in complete medium, fixed with 4% paraformaldehyde for 10 min, and permeabilized in 0.01 M phosphate buffered saline (PBS)/0.5% Triton X-100 for 5 min. Intima were then incubated with an anti-YWHAB primary antibody (1:200 dilution; Abcam, Cambridge, UK) for 1 h at room temperature and then with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA). Following washes in PBS, the intima were incubated for 3 min with 0.25 mg/ml 40 ,6diamidino-2-phenylindole (DAPI). A similar protocol was used for the in vivo experiments; briefly, the RAOECs were washed with PBS prior to lysis in RIPA buffer supplemented with protease inhibitors (Roche Applied Sciences, Laval, QC, Canada) and then incubated with a primary antibody against YWHAB or BAX (anti-BAX, 1:200 dilution; Sigma-Aldrich, St. Louis, MO, USA). Mitochondria were visualized with mitochondria-targeted dsRed (Yeasen). Images were captured using a fluorescence microscope (Jenoptik).
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2.6. Quantitative reverse transcription PCR (qRT-PCR)
2.10. Statistical analyses
Total RNA was extracted using the TRIzol (Invitrogen) and chloroform/isopropanol method. cDNA was generated using the PrimeScript RT Reagent Kit (TAKARA, Dalian, China) and PCR was performed using SYBR Premix Ex Taq (TAKARA) with YWHAB and GAPDH specific primers. PCR data were normalized to GAPDH and presented as 2- DDCt values. The primers used for qRT-PCR are as follows: YWHAB forward: 50 -CAAAGAGTACCGTGAGAAGATCGAG-30 YWHAB reverse: 50 -CGGATGCAACTTCAGAAAGATACC-30 GAPDH forward: 50 -GGCACAGTCAAGGCTGAGAATG-30 GAPDH reverse: 50 -ATGGTGGTGAAGACGCCAGTA-30
Results from at least three independent experiments are presented as means ± SD. Statistical analyses were performed using the 8.0 SAS program. One-way analysis of variance with Student's ttest was applied to compare the statistical significance of the differences between groups. P-values <0.05 were considered statistically significant.
2.7. Western blot analysis Western blot analysis was performed to detect YWHAB expression and BAX distribution in RAOECs from different groups. RAOEC cellular lysates were isolated from each group using RIPA (Beyotime Biotechnology, Shanghai, China) lysis buffer. Cytoplasmic and mitochondrial fractions were separated from crude cell lysates using the Mitochondria/Cytosol Fractionation Kit (Biovision, Mountain View, CA, USA). Next, total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk in Tris-buffered saline Tween-20 (TBST) for 2 h and then incubated with diluted primary antibodies (anti-GAPDH, CST; antiYWHAB and anti-BAX) overnight at 4 C. The membranes were then washed three times with TBST, incubated for an additional 2 h at 25 C with a FITC-conjugated secondary antibody (SigmaAldrich), and finally washed with TBST. Semi-quantitative gel images were analyzed with an automatic gel imager using Quantity One software (Bio-Rad). GAPDH was used as the internal control.
2.8. Cell wound scratch assay RAOECs were inoculated into 6-well plates (500,000 cells/well) and grown for 24 h to reach at least 70% confluence. Following 24 h of serum withdrawal, three scratches were made in each well using a P-20 pipette. Cells were incubated for 24 h to enable migration and then images were taken of each well to measure the gap change in the scratches.
2.9. Flow cytometric analysis Following infection with either YWHAB-LV or mock-LV for 24 h, 500,000 RAOECs were collected, washed twice with precooled PBS, and resuspended in 100 ml binding buffer (Yeasen). The cells were then stained with Annexin V-FITC/PI (Yeasen), incubated in the dark for 10 min (25 C), resuspended in an additional 400 ml binding buffer, and then each sample was analyzed by flow cytometry. To analyze cell cycle progression, both YWHAB-LV and con-LV infected RAOECs were grown in 6-well plates to sub-confluence for 24 h and then cultured in serum (DMEM supplemented with 0.2% FBS) for 1 day. Cells were then trypsinized, centrifuged (1000 g, 5 min), resuspended and washed twice with 1 ml precooled 1 PBS, and then fixed with 70% ethanol at 4 C overnight. The fixed cells were washed and resuspended in PBS (200 ml), consecutively treated with 20 ml RNase A solution (37 C, 30 min) and 400 ml PI staining solution (4 C, 60 min; Yeasen), and then analyzed by flow cytometry.
3. Results 3.1. YWHAB expression in RAOEC is up-regulated with increasing glucose concentrations In order to maintain high reproducibility with the in vivo experiments performed in this study, our in vitro experiments were performed using RAOEC cell lines. We conducted in vitro studies to determine whether YWHAB expression varied in RAOECs with increasing glucose concentration. YWHAB expression was evaluated following treatment of RAOECs with 5.6, 10, 15, 25, or 35 mM glucose. As shown by qRT-PCR and western blot analyses, YWHAB expression was up-regulated with increasing glucose concentration (Fig. 1A and B). It should be noted that RAOEC vitality also improved with increasing glucose concentration. However, RAOEC vitality significantly decreased at 35 mM because of hyperglycemic toxicity (Fig. 1C). Because of the significant up-regulation of both YWHAB expression and cell vitality compared to 5.6 mM glucose, 25 mM glucose was chosen as the concentration for mimicking DMinduced hyperglycemia and used as the high glucose concentration in subsequent in vitro experiments. These results demonstrate that both YWHAB expression and cell vitality were up-regulated with increasing glucose concentration. 3.2. YWHAB inhibition prevents intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats Because of the high incidence of intimal hyperplasia following interventional treatment in diabetics, we examined whether there is an association between YWHAB up-regulation and intimal hyperplasia. To this end, we established a DM SD rat carotid artery injury model to examine the effect of YWHAB silencing on intimal hyperplasia. Consistent with the in vitro experiment, IF and HE staining showed that intimal YWHAB expression and intimal hyperplasia (neointimal area) were greater in the DM group than in the normal group at Day 7 post-injury (Fig. 2A and B). However, the neointimal area was significantly reduced in the DM þ YWHAB silencing group compared with the DM group (Fig. 2A). These data convincingly demonstrate that YWHAB silencing can indeed attenuate the pathological process of intimal hyperplasia. 3.3. YWHAB inhibition reduces RAOEC proliferation and migration under high glucose concentrations To explore the role of endothelial cells in the development of hyperglycemia-induced intimal hyperplasia following injury, we conducted in vitro experiments. RAOECs were incubated with 25 mM glucose to mimic in vivo hyperglycemia as explained in section 3.1. Following infection with siRNAs (YWHAB-LV or MockLV), RAOEC proliferation and migration were examined by flow cytometry and cell wound scratch assays. The silencing efficiency of YWHAB-LV was evaluated by qRT-PCR and western blot analyses (YWHAB expression in RAOECs treated with 5.6 mM glucose served as the control; Fig. 3D). The results showed that when cultured with 25 mM (high) glucose, the proliferation and migration of RAOECs infected with YWHAB-LV was significantly decreased compared
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Fig. 1. The effect of glucose concentration on YWHAB expression and RAOEC cell vitality. (A) qRT-PCR and (B) western blot analyses show that YWHAB expression is up-regulated with increasing glucose concentration. (C) CCK-8 analysis shows that RAOEC vitality is also enhanced with increasing glucose concentration. *p < 0.05 vs. 5.6 mmol/L glucose treatment; n ¼ 3.
Fig. 2. Representative image of HE and IF staining of carotid arteries in DM rats. (A) Neointimal area at Day 7 post-injury compared with uninjured group. (B) YWHAB expression (Red), DAPI (Blue). *p < 0.05 vs. Normal group, #p < 0.05 vs. DM group; n ¼ 3. Scale bar ¼ 25 mm.
with the Mock-LV group (Fig. 3A and C). Moreover, a greater number of RAOECs were arrested in the G1 phase in the YWHAB-LV group than in the Mock-LV group (Fig. 3B). These results indicate that YWHAB inhibition reduces RAOEC proliferation and migration under high glucose concentrations.
3.4. YWHAB inhibition promotes the translocation of BAX from cytoplasm to mitochondria As a number of 14-3-3 family isoforms bind to BAX and the distribution of BAX, a pro-apoptotic member of the Bcl-2 family, is
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Fig. 3. The effect of YWHAB silencing on the proliferation and migration of RAOECs treated with 25 mM glucose. Flow cytometry shows that (A) cell vitality was lower and (B) the G1 arrest rate was higher in the YWHAB-LV group compared with the Mock-LV group. (C) The cell scratch assay shows that the wound closure rate was lower in the YWHAB-LV group compared with the Mock-LV group. (D) Western blot analyses demonstrating YWHAB silencing efficiency. YWHAB expression in 5.6 mM glucose-treated RAOECs served as the control group. *p < 0.05 vs. Mock-LV group, #p < 0.05 vs. control group; n ¼ 3.
related to the regular apoptotic process of cells, we next examined BAX distribution under different glucose concentrations and the effect of YWHAB silencing on BAX distribution in RAOECs. Although mostly localized to the cytoplasm, distribution of BAX to the mitochondria is essential for maintaining the normal apoptosis process of cells by inducing cytochrome C release. Western blot analyses and IF staining showed that BAX expression was significantly decreased in the mitochondria of RAOECs treated with 25 mM glucose compared to 5.6 mM glucose. However, YWHAB silencing promoted BAX translocation to the mitochondria (Fig. 4A and B). These results indicate that high glucose-induced up-regulation of YWHAB inhibited the regular apoptosis process by sequestering BAX in the cytoplasm and suggest that YWHAB silencing may play a role in preventing excessive RAOEC proliferation and migration under high glucose conditions. 4. Discussion Hyperglycemia is considered the main factor underlying the development of vascular complications in diabetes [24]; hyperglycemia triggers a cascade of metabolic and biochemical changes occurring in this pathology. Excessive proliferation and migration of endothelial cells are important features in the pathogenesis of intimal hyperplasia, which could become a greater risk factor for arterial restenosis and atherosclerosis following injury or angioplasty [25,26]. Several studies have identified a connection between hyperglycemia and excessive endothelial cell proliferation. Wang et al. showed that scutellarin treatment could inhibit high glucose-induced proliferation in hRECs [27]; Chen et al. found that
30 mM glucose significantly promoted the migration and proliferation of endothelial cells, which was blocked by 1 mg/ml adrenomedullin [26]; and Yuan et al. [28] showed that heparanase knockdown in high glucose-treated cells could decrease migration and proliferation in human retinal vascular endothelial cells (HRECs). In addition, Li et al. [29] found that excessive proliferation of RAOECs could be inhibited by miR-98 via regulation of cyclin D2. Similarly, in this study, we demonstrated that 25 mM glucose significantly promoted RAOEC proliferation compared with 5.6 mM glucose treatment, thus confirming the role of glucose concentration in the growth status of endothelial cells. In addition, we found that YWHAB is up-regulated with increasing glucose concentration. YWHAB is a member of the highly conserved 14-3-3 family, which mediate signal transduction by binding to phosphoserinecontaining proteins [14]. Wang et al. [30] identified the binding between YWAHB and Wee1 and the following effect on the biochemical activity of Wee1. In addition, Uchida et al. [31] found that the binding between CDC25B and YWHAB also plays a role in regulating the subcellular redistribution of CDC25B; the interaction between YWHAB and CDC25B promotes the translocation of CDC25B from the nucleus to the cytoplasm. Although several studies have already demonstrated that YWHAB plays a role in the development and progression of tumors [21], the relationship between the effect of hyperglycemia on YWHAB expression and intimal hyperplasia following injury remains elusive. Regular apoptosis plays a critical role in the maintenance of tissue homeostasis. The regulation of mitochondrial membrane integrity and the release of cytochrome C from mitochondria are important processes during apoptosis and are controlled by the Bcl-
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Fig. 4. The effect of YWHAB silencing on BAX distribution in RAOECs incubated with 25 mM glucose. Cytoplasmic (Cyto) and mitochondrial (Mito) fractions were purified from RAOEC whole-cell lysates and BAX expression was detected by (A) western blot analyses. BAX expression in RAOECs treated with 5.6 mM glucose served as the control (Con). (B) The distribution of BAX (Green), mitochondria (Mito-dsRED; Red), and DAPI (Blue) were visualized by IF staining. *p < 0.05 vs. Con; n ¼ 3. Scale bar ¼ 10 mm.
2 family [32]. In our study, YWHAB silencing increased the distribution of BAX in mitochondria when RAOECs were incubated with 25 mM glucose. Studies examining the association between YWHAB and other apoptosis factors, such as BMK1, RSK, and CHOP, are required to elucidate the role of YWHAB up-regulation in the development of diabetic vascular complications [33]. In conclusion, this study shows that under DM conditions, excess YWHAB plays a role in intimal hyperplasia following carotid artery injury by promoting endothelial cell proliferation and migration via sequestration of BAX from the mitochondria. These findings identify YWHAB as a diagnostic biomarker and YWHAB silencing as a therapeutic target for intimal hyperplasia-related restenosis or atherosclerosis in diabetics. Conflicts of interest None. Acknowledgments This work was financially supported by the Shanghai Shen Kang Hospital Development Center Suburb of Three Hospital Capacity-
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Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199
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Please cite this article in press as: L. Feng, et al., Up-regulation of 14-3-3b plays a role in intimal hyperplasia following carotid artery injury in diabetic Sprague Dawley rats by promoting endothelial cell migration and proliferation, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.199