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mTOR-mediated autophagy
Neuroscience Letters 712 (2019) 134485
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Research article
SPARC induces phenotypic modulation of human brain vascular smooth muscle cells via AMPK/mTOR-mediated autophagy
T
Tao Lia,b,c, Xianjun Tana,c,d, Shaowei Zhua,c, Weiying Zhonga,c, Bin Huanga,c, Jinhao Sune, ⁎ Feng Lia,c, Yunyan Wanga,c, a
Department of Neurosurgery, Qilu Hospital of Shandong University and Institute of Brain and Brain-Inspired Science, Shandong University, Jinan, China Department of Neurosurgery, The No. 4 People’s Hospital of Jinan, Jinan City, Shandong Province, China c Shandong Key Laboratory of Brain Function Remodeling, China d Department of Neurosurgery, People’s Hospital of Chiping City, Liaocheng City, Shandong Province, China e Key Laboratory for Experimental Teratology of the Ministry of Education and Department of Anatomy, Shandong University, School of Medicine, Jinan, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Secreted protein acidic and rich in cysteine (SPARC) Human Brain Vascular Smooth Muscle Cells (HBVSMCs) Phenotypic modulation Autophagy Intracranial aneurysm
Secreted protein acidic and rich in cysteine (SPARC) was widely expressed in VSMCs of human IAs and could reduce the capability of self-repair. This indicates that SPARC may play a role in the promotion of IAs formation and progression, but the mechanism remains unclear. In this study, we further investigated whether SPARC could induce phenotypic modulation of Human Brain Vascular Smooth Muscle Cells (HBVSMCs) and sought to elucidate the role of SPARC-mediated autophagy involved in it. The results demonstrated that SPARC inhibited the expression of contractile genes in HBVSMCs and induced a synthetic phenotype. More importantly, SPARC significantly up-regulated multiple proteins including autophagy marker microtubule-associated protein light chain 3-II (LC3-II), Beclin-1, and autophagy-related gene 5(ATG5). Furthermore, SPARC could promote p62 degradation. The autophagy inhibitor 3- methyladenine (3-MA) significantly blocked SPARC-induced phenotypic modulation of HBVSMCs. We further sought to elucidate the molecular mechanism involved in SPARCinduced autophagy, and found that SPARC could activate the AMPK/mTOR signaling pathway in HBVSMCs. AMPK could be pharmacologically inhibited by Compound C (CC), which significantly decreased the phosphorylation of AMPK into p-AMPK, increased the phosphorylation of mTOR into p-mTOR, and decreased LC3-II, Beclin-1 and ATG5 levels. This suggested that activated AMPK/ mTOR signaling is related to SPARC-mediated autophagy. These results indicated that SPARC plays a role in the phenotypic modulation of HBVSMCs through autophagy activation by AMPK/mTOR signaling pathway.
1. Introduction Intracranial aneurysms (IAs) are tumor-like protrusions formed by pathological localized expansion of the intracranial arterial wall, with a prevalence of approximately 2%–5% [1,2]. Rupture of IAs is the main cause of subarachnoid hemorrhage with a high morbidity and mortality [3]. So far, the exact pathogenesis of IAs is still not clear. IAs are generally considered to result from a combination of genetics, hemodynamics, and damage of the vascular wall, resulting in the loss of intracranial arterial wall integrity [4,5]. The anatomical basis for promoting the onset of IAs relates to the arterial wall structure. Intracranial arteries have a relatively low amount of smooth muscle in the tunica media and lack elastin in the tunica adventitia. VSMC is characterized
by its high plasticity and contractile function. Under pathological conditions, it can be transformed from a differentiated phenotype with contractile function to a dedifferentiated phenotype with strong proliferation and migration ability. There is increasing evidence that VSMCs play an important role in various vascular disease including IAs [6]. Therefore, VSMCs phenotypic modulation is thought to be involved in the pathogenesis of IA formation, development and rupture [7–9]. Secreted protein acidic and rich in cysteine (SPARC) is a secreted glycoprotein found in the extracellular matrix and is highly expressed in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is genetically transmitted and has a high incidence of IAs [10,11]. SPARC is expressed in various cell types, including osteoblasts, fibroblasts, smooth muscle cells, and endothelial cells. The biological effects of
⁎ Corresponding author at: Department of Neurosurgery, Qilu Hospital of Shandong University and Institute of Brain and Brain-Inspired Science, Shandong University, Jinan, China. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.neulet.2019.134485 Received 7 June 2019; Received in revised form 15 August 2019; Accepted 5 September 2019 Available online 06 September 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
Neuroscience Letters 712 (2019) 134485
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SPARC include tissue remodeling and fibrosis, which have been studied in these cells [12,13]. Our previous study has showed that SPARC is significantly expressed in the tissue of IAs, especially in VSMCs, suggesting that SPARC has a pathogenic effect on the formation of IAs [14]. We also discovered that SPARC can reduce self-healing capacity and promotes damage to the tunica media and internal elastic membrane, so SPARC could promote the formation and development of IAs [15]. However, the specific mechanism by which SPARC participates in the formation and development of IAs is still unclear. The mechanism of SPARC involvement on the phenotypic modulation of HBVSMCs has yet to be identified. Based on the above research, HBVSMCs were obtained to investigate the involvement of SPARC in the pathogenesis of IAs. We sought to elucidate its role in the phenotypic modulation of HBVSMCs and the specifics of the autophagy-related molecular mechanism. The goal of this ongoing work is to provide new insights into the pathogenesis of IAs, with hopes of revealing new therapeutic targets.
2.4. Western blotting(WB)
2. Materials and methods
Since the commonly used method for evaluating autophagy is immunofluorescence staining of LC3-II [16], acidic autophagic vacuoles (AVs) were detected by Cyto-ID Green autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA), according to the manufacturer’s instructions [17,18]. HBVSMCs were placed into a 6-well plate with glass chamber slides. After appropriate treatment, cells were stained with Cyto-ID Green and Hoechst 33342 for 30 min at 37℃. Finally, fluorescence images were obtained using a LSM700 confocal microscope (Carl Zeiss; X800).
The total proteins of HBVSMCs were extracted according to the manufacturer’s instructions using Total Protein Extraction Kit (BestBio, Shanghai, China). The samples were separated by electrophoresis using 8%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a PVDF membrane and blocked with 5% defatted milk for 1 h at room temperature. Primary antibodies were added to the membranes at 4 °C overnight, then anti-rabbit IgG HRPconjugated secondary antibodies were added for further incubation. Millipore’s enhanced chemiluminescence (ECL) was utilized for protein detection. The images were analyzed using a Molecular Imager Chemidoc XRS System (Bio-rad Laboratories, Hercules, CA). Protein expression was normalized to GAPDH expression. The WB band abundance was calculated using Imange J software. 2.5. Immunofluorescence staining and confocal imaging
2.1. Materials SPARC was obtained from Genscript (Jiangsu, China). Antibodies against smooth muscle α-actin (SMA), smooth muscle protein 22α (SM22-α), and osteopontin (OPN) were purchased from Proteintech (Rosemont, USA). Antibodies against LC3-II, p62, ATG5, Beclin-1, pAMPK(Thr172)/AMPK, p-mTOR(Ser2448)/mTOR, and anti-rabbit IgG HRP-conjugated secondary antibodies were purchased from Cell Signaling Technologies (San Jose, CA, USA). 3-methyladenine (3-MA), Chloroquine diphosphate (CQ) and Compound C (CC) were obtained from MedChemExpress (New Jersey, USA). Antibodies against GAPDH was purchased from Goodhere Biotechnology (Hangzhou, China). The primers were designed and synthesized by BioSune Biotechnology (Shanghai, China).
2.6. Transmission electron microscopy(TEM) Observation via TEM is the most accurate method for identifying autophagosomes [19,20]. The cultured cells were digested with trypsin an fixed in 4% glutaraldehyde, followed by post-fixation with 1% OsO4 in 0.1 M cacodylate buffer containing 0.1% CaCl2 for 2 h at 4℃. Next, 1% Millipore-filtered uranyl acetate was added to the samples for staining, followed by dehydration in increasing concentrations of alcohols. Finally, the samples were embedded in LX-112 medium (Ladd Research Industries, 21210). An ultra-cut microtome was used to slice the sections after polymerization of resin at 60℃ for 48 h. Sections were stained with 4% uranyl acetate and lead citrate, then observed under JEM-100cxII electron microscope (JEM).
2.2. Cell cultures and SPARC treatment Primary HBVSMCs were obtained from ScienCell Research Laboratories (Cat No.1100, Carlsbad, CA), the 5th passage of which was used in this study. HBVSMCs were cultured in smooth muscle cell medium (SMCM, Cat.No.1101, ScienCell Research Laboratories, Carlsbad, CA) with 10% fetal bovine serum (FBS, Cat.No.0010), 5% smooth muscle cell growth supplement (SMCGS, Cat.No.1152), and 5% penicillin/streptomycin (P/S, Cat. No.0503) in a humidified atmosphere incubator with 5% CO2 at 37℃. Reagents containing various concentrations (0.5, 1 and 2 μg/ml, respectively) of SPARC were added for different time periods (0, 3 h, 6 h, 12 h and 24 h) for cell induction. The HBVSMCs were pre-incubated with 3-MA(10 mM), CQ(10 μM), or CC(10 μM) for 1 h when necessary.
2.7. Statistical analysis Statistical analysis was performed using a statistical software package (SPSS 21.0, Chicago, IL) and GraphPad-Prism5 (GraphPad, CA, USA). Data were presented as mean ± standard deviation (SD) and statistically analyzed by one-way ANOVA. Student’s t-test was used for two-group comparison. Statistical significance was defined by a p value < 0.05.
2.3. RNA extraction and RT-PCR assay 3. Results We performed total RNA extraction from HBVSMCs using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. The appropriate concentration was determined by measuring absorbance at 260 nm using a Nanodrop spectrophotometer (Thermo scientific). The Superscript First-Strand cDNA synthesis system (Invitrogen, CA, USA) was utilized to perform reverse transcriptase reactions. RT-PCR was performed with a One-Step SYBR Prime Script TM RT-PCR Kit II (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The reactions were performed using a LightCycler 2.0 Instrument (Roche). SYBR green was utilized to determine mRNA expression, which was normalized to GAPDH gene expression. The data were analyzed using the 2−△△Ct method and the values were presented by relative quantity.
3.1. SPARC induced phenotypic modulation in HBVSMCs In order to examine the effect of SPARC on the phenotypic regulation of HBVSMCs, we measured the changes in molecular markers of contractile gene (SMA and SM22-α) and synthetic gene (OPN). As shown in Fig. 1A, SPARC treatment for 12 h resulted in a decrease of ˜20% for SMA and SM22-α mRNA expression and a significant increase for OPN expression (1.5-fold). As shown in Fig. 1B and C, compared with control cells, the protein abundance of SMA and SM22-α decreased ˜20% and the OPN protein abundance increased by 2-fold. These results showed that SPARC promotes phenotypic transition of HBVSMCs. 2
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Fig. 1. SPARC induces a contractile to synthetic phenotype switch in HBVSMCs. (A) Quantitative real-time PCR following SPARC (2 μg/ml) stimulation for 12 h. SMA and SM22-α are markers of the contractile phenotype; OPN is associated with a synthetic phenotype. *p < 0.05 and **p < 0.01 vs control, n = 3 per group. (B) WB analysis for contractile and synthetic proteins after SPARC (2 μg/ml) stimulation for 12 h. (C) Quantification of immunoblots from panel (B) (*p < 0.05 and ***p < 0.001 vs control, n = 3 per group).
levels of p-AMPK(Thr172) and increased the levels of p-mTOR(Ser2448). The autophagic protein levels of LC3-II, Beclin-1 and ATG5 decreased and the levels of p62 increased after CC treatment, respectively (Fig. 4E and F). These findings indicated AMPK/mTOR pathway involved in SPARC-induced autophagy in HBVSMCs.
3.2. SPARC induced autophagy activation in HBVSMCs Autophagy is a catabolic lysosome-mediated process responsible for the degradation and recycling of damaged or dysfunctional cytoplasmic components and intracellular organelles. In order to determine whether treatment with SPARC could induced autophagy, the protein levels of LC3-II, p62, Beclin-1 and ATG5 of HBVSMCs were measured after SPARC treatment. As shown in Fig. 2A and B, LC3-II formation after SPARC treatment increased in a time-dependent manner. After 12 h following treatment, LC3-II formation showed a significant increase, suggesting that SPARC promotes autophagy. Consistent with this hypothesis, there was an increase in expression of Beclin-1 and ATG5 and a decrease in expression of p62. As shown in Fig. 2C and D, the protein levels of LC3-II, Beclin-1 and ATG5 in HBVSMCs increased and p62 decreased in a dose-dependent manner of SPARC, which indicated that SPARC could induce autophagy activation. The concentration of 2 μg/ ml of SPARC could induce a significant increase in autophagy. So, 2 μg/ ml of SPARC was chosen for the following experiment concentration and 12 h post-treatment with SPARC was selected as the appropriate detection time.
3.5. SPARC induced autophagy flux increasing of HBVSMCs Due to the transient formation of LC3-II and its rapid degradation into lysosomes, an increase in LC3-II could also paradoxically indicate a decrease in autophagy. Therefore, to more accurately assess autophagic flux, HBVSMCs were respectively pretreated with 3-MA(10 mM) and CQ(10 μM) for 1 h before treated with SPARC(2 μg/ml). SPARC could induce a significant increase in LC3-II, Beclin-1 and ATG5 expression and a decrease in p62 expression. These effects were partly blocked by 3-MA, which suppressed the early stage of autophagosome formation, so SPARC indeed plays a role in the stimulation of autophagic flux in cultured HBVSMCs (Fig. 5A and B). Compared to the control, CQ could significantly increase the expression of LC3-II, Beclin-1, ATG5 and p62 expression (Fig. 5A and B), which reflects autophagosome accumulation because of autophagic flux inhibition. These results indicated that SPARC mediated HBVSMCs autophagy flux in vitro.
3.3. SPARC induced ultrastructural changes and extensive vacuolization in HBVSMCs
3.6. SPARC-induced autophagy involved in HBVSMCs phenotypic switching Autophagosome formation is considered as the “gold-standard” to document autophagy. We utilized Cyto-ID Green autophagy detection kit to visualize autophagosome formation via confocal microscopy. There was a significant increase in autophagic vacuoles(AVs) after HBVSMCs treated with SPARC(2 μg/ml) for 12 h, and this increase could be blocked by 3-MA (Fig. 3A and B). TEM was used for the analysis of ultrastructural changes in autophagosome formation. As shown in Fig. 3C and D, there was an approximate 1-fold increase in number of AVs within HBVSMCs treated with SPARC (2 μg/ml) for 12 h, and 3-MA could block this increase. Taken together, these structural changes further supported our immunological and fluorescence-imaging data, which suggested that SPARC was a robust inducer of the autophagic program.
Next, in order to elucidate whether autophagy plays a role in phenotypic switching of HBVSMCs, the HBVSMCs was pretreated with autophagy inhibitor 3-MA before SPARC treatment. As shown in Fig. 6A and B, 3-MA pretreatment could partially block SPARC-induced losses of SMA and SM22-α. Simultaneously, inhibition of autophagy with 3MA could block the SPARC-mediated increase of OPN. Collectively, these results indicated that autophagy is important for HBVSMC phenotype, and 3-MA is particularly effective in preventing conversion to the synthetic phenotype. 4. Discussion In this study, we found that SPARC could induce autophagy in HBVSMCs phenotypic modulation by activation of the AMPK/mTOR signaling pathway. We determined that SPARC, which promotes the phenotypic development of synthetic HBVSMCs, was a strong inducer of autophagy. In particular, 3-MA could partly block the SPARC-induced degradation of SMA and SM22-α. These findings demonstrated that SPARC-mediated autophagy takes part in the regulation of HBVSMC phenotype and function. Recent studies have shown that phenotypic modulation of VSMCs is closely related to IAs [9,21]. Therefore, it is essential to identify the specific pathways involved in these processes. Until now, SPARC involvement in VSMCs phenotypic modulation has remained undefined. In the current study, we explored the various impacts of SPARC on phenotypic modulation of HBVSMCs
3.4. SPARC induced autophagy activation through AMPK/mTOR signaling pathway Autophagy activation in mammalian cells usually involves inhibition of the mammalian target of rapamycin (mTOR), a downstream target of the AMPK pathway. To explore the potential molecular mechanisms by which SPARC induces autophagy, we detected the AMPK/ mTOR signaling pathway in this experiment. As demonstrated in Fig. 4A and B, there was a dose-dependent increase in p-AMPK(Thr172) protein levels upon SPARC treatment, and a dose-dependent decrease in p-mTOR(Ser2448). Then, HBVSMCs were incubated with the AMPK inhibitor CC(10 μM). As shown in Fig. 4C and D, CC reduced the protein 3
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Fig. 2. SPARC induces autophagy in HBVSMCs. (A) Time-dependent expression of autophagy-related proteins (LC3Ⅱ, p62, Beclin-1 and ATG5) of HBVSMCs treated with SPARC (2 μg/ml) were detected by WB. (B) Quantification of immunoblots from panel (A). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control, n = 3 per group. (C) Representative WB immunoblots of autophagy-related proteins in HBVSMCs treated with different concentrations of SPARC (0, 0.5, 1 and 2 μg/ml) for 12 h. (D) Quantification of immunoblots from panel (C). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control, n = 3 per group.
factors could trigger phenotypic alternation of VSMCs [25]. Autophagy plays an important role in the phenotypic alternation of VSMCs, and previous studies have also shown that platelet-derived growth factor (PDGF)-BB [26] and shear stress [27] could promote VSMCs phenotypic transformation by activating autophagy. Autophagy, a process that maintains energy balance of cells, plays a vital role in the physiological and pathophysiological processes in the vasculature, and has been shown to be involved in the development of various cardiovascular and cerebrovascular diseases including atherosclerosis [28], restenosis [29] and IAs [30] et al. In our study, enhanced autophagic activity was observed in the cultured HBVSMCs exposed to SPARC. SPARC- induced autophagy both occured in a time-dependent manner and a dose-dependent manner. Inhibition of autophagy could partly block SPARCinduced HBVSMCs phenotypic transition. These results demonstrated the causal role of autophagy induced by SPARC in the HBVSMCs phenotypic conversion.
and the related molecular mechanism in vitro. VSMCs have contractile functions in normal mature vessels. When subjected to various stimuli, VSMC can change from a contractile phenotype to a synthetic phenotype [9,22]. In our research, after exposure to SPARC in vitro, cultured HBVSMCs would dedifferentiate from a contractile state to a synthetic state, confirmed by decreased expression of contractile genes (SMA and SM22-α) and increased expression of synthetic gene (OPN). The protein levels of OPN significantly increased in the tissue of IAs, especially in unruptured IAs [23], it could also mediate cells death through enhanced autophagy in abdominal aortic aneurysms [24]. These results indicated the significance of SPARC involvement on phenotypic modulation of HBVSMCs, but the specific mechanisms have not been completely elucidated. Previous studies have shown that numerous stimuli including injury, blood flow shear stress, cytokine production and biochemical 4
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Fig. 3. SPARC increases autophagosome formation and promotes extensive vacuolization. (A) Representative confocal imaging of HBVSMCs stained with Cyto-ID Green autophagy detection kit treated with or without SPARC and 3-MA. (B) Quantification of AVs in images from panel(A) (> 20 cells counted per field in each group, ***p < 0.001 vs the indicated groups, n = 3 per group. (C) Representative transmission electron micrographs of cells treated with or without SPARC and 3-MA. (D) Quantification of AVs in images from panel (C) (*p < 0.05 vs the indicated groups, n = 3 per group).
Next, we explored the SPARC-mediated autophagy flux increased in HBVSMCs. The increased levels of autophagosomes could be caused by excessive activation of autophagy or dysfunctional autophagic flux [31,32]. We discovered that SPARC promoted a vigorous form of autophagy characterized by increased LC3-II expression, augmented autophagic flux, and the formation of autophagosomes and AVs. An increased number of autophagosomes in steady-state could result from an increase of autophagy, a block in downstream lysosomal processing of these autophagosomes, or both. It is important to note that the improved autophagic flux from SPARC occurs due to the increased delivery of the autophagosome to the autophagolysosome, as well as the activation of autophagosome initiation or formation. The following two findings are supportive of this. Firstly, the lysosome inhibitor CQ blocked the autophagic flux and 3-MA alleviated the increase of autophagy in HBVSMCs treated with SPARC. Secondly, SPARC regulated the activation of mTOR and AMPK, indicating the possible involvement of classical autophagy upstream signaling in HBVSMCs. Then, we investigated the potential molecular mechanisms
underlying that SPARC-induced autophagy in HBVSMCs. The AMPK/ mTOR signaling pathway is a classical pathway in the complex signal network that mediates autophagy [33]. We analyzed the effectiveness of AMPK/mTOR signaling activation in the up-regulation of SPARCinduced autophagy. Previous studies have shown that SPARC could interact with AMPK, the activation of AMPK would increases SPARC expression, while inhibition of endogenous AMPK expression would reduce SPARC expression [34]. The data from our study demonstrated a decrease ratio of p-mTOR/mTOR and an increase ratio of p-AMPK/ AMPK in HBVSMCs in a dose-dependent manner. To further vertify above results, CC was introduced for pretreatment. We found that pretreatment with CC was able to significantly block the increase expression of p-AMPK and the decrease expression of p-mTOR mediated by SPARC. Furthermore, CC administration markedly reduced the effect of autophagy activation in HBVSMCs suffering from SPARC, with a lower LC3-II, Beclin-1, ATG5 and a higher p62 protein level. All in all, these results further suggest that AMPK/mTOR signaling pathway plays a role in SPARC-induced autophagy activation. 5
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Fig. 4. SPARC targets the AMPK/mTOR pathway in HBVSMCs. (A) Representative WB immunoblots of p-AMPK(Thr172), AMPK, p-mTOR(Ser2448) and mTOR in HBVSMCs treated with different concentrations of SPARC for 12 h. (B) Quantification of immunoblots from panel (A). Each phosphor protein was normalized to its non-phosphorylated total protein band intensity, *p < 0.05 and ***p < 0.001 vs control, n = 3 per group. (C) Representative WB immunoblots of p-AMPK(Thr172), AMPK, p-mTOR(Ser2448), and mTOR in HBVSMCs treated without or with SPARC and CC. Each phosphor protein was normalized to its non-phosphorylated total protein band intensity too. (D) Quantification of immunoblots from panel (C). Each phosphor protein was normalized to its non-phosphorylated total protein band intensity, *p < 0.05 and ***p < 0.001 vs the indicated groups, n = 3 per group. (E) Representative WB immunoblots of autophagy-related proteins (LC3Ⅱ, p62, Beclin-1 and ATG5) in HBVSMCs treated without or with SPARC and CC. (F) Quantification of immunoblots from panel (E). *p < 0.05, **p < 0.01 and ***p < 0.001 vs the indicated groups, n = 3 per group.
5. Conclusions
mediated autophagy and SPARC-induced phenotypic modulation in HBVSMCs. We hope that the information obtained from this study can contribute to the development of new targets for early diagnosis and pharmacologic treatment options for IAs.
In conclusion, the results from our study provide new insights into the autophagic mechanisms by which SPARC induces phenotypic modulation of HBVSMCs from contractile to synthetic state. Furthermore, there is a strong correlation between AMPK/mTOR6
Neuroscience Letters 712 (2019) 134485
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Fig. 5. SPARC can mediate autophagy flux increase of HBVSMCs. (A) Representative WB immunoblots of autophagy-related proteins (LC3Ⅱ, p62, Beclin-1 and ATG5) in HBVSMCs treated without or with SPARC, 3-MA or CQ. (B) Quantification of immunoblots from panel (A). *p < 0.05, **p < 0.01 and ***p < 0.001 vs the indicated groups, n = 3 per group.
Author’s contribution
a potential conflict of interest.
TL and XT carried out the experiments and wrote the manuscript. SZ analyzed the data. WZ and FL prepared the figures and tables. JS and BH provided technical support and revised the paper. YW were responsible for experimental design and contributed some reagents.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China [No. 81171172 and No.81701160] and Shandong Provincial Science and Development planning Program of China [No. 2016GSF201230].
Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as
Fig. 6. Inhibition of autophagy prevents SPARC-induced phenotypic changes. (A) Representative WB immunoblots of contractile (SMA and SM22-α) and synthetic (OPN) proteins in HBVSMCs treated without or with SPARC, 3-MA. (B) Quantification of immunoblots in panel (A). *p < 0.05, **p < 0.01 and ***p < 0.001 vs the indicated groups, n = 3 per group. 7
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
SPARC induces phenotypic modulation of human brain vascular smooth muscle cells via AMPK/mTOR-mediated autophagy
T
Tao Lia,b,c, Xianjun Tana,c,d, Shaowei Zhua,c, Weiying Zhonga,c, Bin Huanga,c, Jinhao Sune, ⁎ Feng Lia,c, Yunyan Wanga,c, a
Department of Neurosurgery, Qilu Hospital of Shandong University and Institute of Brain and Brain-Inspired Science, Shandong University, Jinan, China Department of Neurosurgery, The No. 4 People’s Hospital of Jinan, Jinan City, Shandong Province, China c Shandong Key Laboratory of Brain Function Remodeling, China d Department of Neurosurgery, People’s Hospital of Chiping City, Liaocheng City, Shandong Province, China e Key Laboratory for Experimental Teratology of the Ministry of Education and Department of Anatomy, Shandong University, School of Medicine, Jinan, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Secreted protein acidic and rich in cysteine (SPARC) Human Brain Vascular Smooth Muscle Cells (HBVSMCs) Phenotypic modulation Autophagy Intracranial aneurysm
Secreted protein acidic and rich in cysteine (SPARC) was widely expressed in VSMCs of human IAs and could reduce the capability of self-repair. This indicates that SPARC may play a role in the promotion of IAs formation and progression, but the mechanism remains unclear. In this study, we further investigated whether SPARC could induce phenotypic modulation of Human Brain Vascular Smooth Muscle Cells (HBVSMCs) and sought to elucidate the role of SPARC-mediated autophagy involved in it. The results demonstrated that SPARC inhibited the expression of contractile genes in HBVSMCs and induced a synthetic phenotype. More importantly, SPARC significantly up-regulated multiple proteins including autophagy marker microtubule-associated protein light chain 3-II (LC3-II), Beclin-1, and autophagy-related gene 5(ATG5). Furthermore, SPARC could promote p62 degradation. The autophagy inhibitor 3- methyladenine (3-MA) significantly blocked SPARC-induced phenotypic modulation of HBVSMCs. We further sought to elucidate the molecular mechanism involved in SPARCinduced autophagy, and found that SPARC could activate the AMPK/mTOR signaling pathway in HBVSMCs. AMPK could be pharmacologically inhibited by Compound C (CC), which significantly decreased the phosphorylation of AMPK into p-AMPK, increased the phosphorylation of mTOR into p-mTOR, and decreased LC3-II, Beclin-1 and ATG5 levels. This suggested that activated AMPK/ mTOR signaling is related to SPARC-mediated autophagy. These results indicated that SPARC plays a role in the phenotypic modulation of HBVSMCs through autophagy activation by AMPK/mTOR signaling pathway.
1. Introduction Intracranial aneurysms (IAs) are tumor-like protrusions formed by pathological localized expansion of the intracranial arterial wall, with a prevalence of approximately 2%–5% [1,2]. Rupture of IAs is the main cause of subarachnoid hemorrhage with a high morbidity and mortality [3]. So far, the exact pathogenesis of IAs is still not clear. IAs are generally considered to result from a combination of genetics, hemodynamics, and damage of the vascular wall, resulting in the loss of intracranial arterial wall integrity [4,5]. The anatomical basis for promoting the onset of IAs relates to the arterial wall structure. Intracranial arteries have a relatively low amount of smooth muscle in the tunica media and lack elastin in the tunica adventitia. VSMC is characterized
by its high plasticity and contractile function. Under pathological conditions, it can be transformed from a differentiated phenotype with contractile function to a dedifferentiated phenotype with strong proliferation and migration ability. There is increasing evidence that VSMCs play an important role in various vascular disease including IAs [6]. Therefore, VSMCs phenotypic modulation is thought to be involved in the pathogenesis of IA formation, development and rupture [7–9]. Secreted protein acidic and rich in cysteine (SPARC) is a secreted glycoprotein found in the extracellular matrix and is highly expressed in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is genetically transmitted and has a high incidence of IAs [10,11]. SPARC is expressed in various cell types, including osteoblasts, fibroblasts, smooth muscle cells, and endothelial cells. The biological effects of
⁎ Corresponding author at: Department of Neurosurgery, Qilu Hospital of Shandong University and Institute of Brain and Brain-Inspired Science, Shandong University, Jinan, China. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.neulet.2019.134485 Received 7 June 2019; Received in revised form 15 August 2019; Accepted 5 September 2019 Available online 06 September 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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SPARC include tissue remodeling and fibrosis, which have been studied in these cells [12,13]. Our previous study has showed that SPARC is significantly expressed in the tissue of IAs, especially in VSMCs, suggesting that SPARC has a pathogenic effect on the formation of IAs [14]. We also discovered that SPARC can reduce self-healing capacity and promotes damage to the tunica media and internal elastic membrane, so SPARC could promote the formation and development of IAs [15]. However, the specific mechanism by which SPARC participates in the formation and development of IAs is still unclear. The mechanism of SPARC involvement on the phenotypic modulation of HBVSMCs has yet to be identified. Based on the above research, HBVSMCs were obtained to investigate the involvement of SPARC in the pathogenesis of IAs. We sought to elucidate its role in the phenotypic modulation of HBVSMCs and the specifics of the autophagy-related molecular mechanism. The goal of this ongoing work is to provide new insights into the pathogenesis of IAs, with hopes of revealing new therapeutic targets.
2.4. Western blotting(WB)
2. Materials and methods
Since the commonly used method for evaluating autophagy is immunofluorescence staining of LC3-II [16], acidic autophagic vacuoles (AVs) were detected by Cyto-ID Green autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA), according to the manufacturer’s instructions [17,18]. HBVSMCs were placed into a 6-well plate with glass chamber slides. After appropriate treatment, cells were stained with Cyto-ID Green and Hoechst 33342 for 30 min at 37℃. Finally, fluorescence images were obtained using a LSM700 confocal microscope (Carl Zeiss; X800).
The total proteins of HBVSMCs were extracted according to the manufacturer’s instructions using Total Protein Extraction Kit (BestBio, Shanghai, China). The samples were separated by electrophoresis using 8%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a PVDF membrane and blocked with 5% defatted milk for 1 h at room temperature. Primary antibodies were added to the membranes at 4 °C overnight, then anti-rabbit IgG HRPconjugated secondary antibodies were added for further incubation. Millipore’s enhanced chemiluminescence (ECL) was utilized for protein detection. The images were analyzed using a Molecular Imager Chemidoc XRS System (Bio-rad Laboratories, Hercules, CA). Protein expression was normalized to GAPDH expression. The WB band abundance was calculated using Imange J software. 2.5. Immunofluorescence staining and confocal imaging
2.1. Materials SPARC was obtained from Genscript (Jiangsu, China). Antibodies against smooth muscle α-actin (SMA), smooth muscle protein 22α (SM22-α), and osteopontin (OPN) were purchased from Proteintech (Rosemont, USA). Antibodies against LC3-II, p62, ATG5, Beclin-1, pAMPK(Thr172)/AMPK, p-mTOR(Ser2448)/mTOR, and anti-rabbit IgG HRP-conjugated secondary antibodies were purchased from Cell Signaling Technologies (San Jose, CA, USA). 3-methyladenine (3-MA), Chloroquine diphosphate (CQ) and Compound C (CC) were obtained from MedChemExpress (New Jersey, USA). Antibodies against GAPDH was purchased from Goodhere Biotechnology (Hangzhou, China). The primers were designed and synthesized by BioSune Biotechnology (Shanghai, China).
2.6. Transmission electron microscopy(TEM) Observation via TEM is the most accurate method for identifying autophagosomes [19,20]. The cultured cells were digested with trypsin an fixed in 4% glutaraldehyde, followed by post-fixation with 1% OsO4 in 0.1 M cacodylate buffer containing 0.1% CaCl2 for 2 h at 4℃. Next, 1% Millipore-filtered uranyl acetate was added to the samples for staining, followed by dehydration in increasing concentrations of alcohols. Finally, the samples were embedded in LX-112 medium (Ladd Research Industries, 21210). An ultra-cut microtome was used to slice the sections after polymerization of resin at 60℃ for 48 h. Sections were stained with 4% uranyl acetate and lead citrate, then observed under JEM-100cxII electron microscope (JEM).
2.2. Cell cultures and SPARC treatment Primary HBVSMCs were obtained from ScienCell Research Laboratories (Cat No.1100, Carlsbad, CA), the 5th passage of which was used in this study. HBVSMCs were cultured in smooth muscle cell medium (SMCM, Cat.No.1101, ScienCell Research Laboratories, Carlsbad, CA) with 10% fetal bovine serum (FBS, Cat.No.0010), 5% smooth muscle cell growth supplement (SMCGS, Cat.No.1152), and 5% penicillin/streptomycin (P/S, Cat. No.0503) in a humidified atmosphere incubator with 5% CO2 at 37℃. Reagents containing various concentrations (0.5, 1 and 2 μg/ml, respectively) of SPARC were added for different time periods (0, 3 h, 6 h, 12 h and 24 h) for cell induction. The HBVSMCs were pre-incubated with 3-MA(10 mM), CQ(10 μM), or CC(10 μM) for 1 h when necessary.
2.7. Statistical analysis Statistical analysis was performed using a statistical software package (SPSS 21.0, Chicago, IL) and GraphPad-Prism5 (GraphPad, CA, USA). Data were presented as mean ± standard deviation (SD) and statistically analyzed by one-way ANOVA. Student’s t-test was used for two-group comparison. Statistical significance was defined by a p value < 0.05.
2.3. RNA extraction and RT-PCR assay 3. Results We performed total RNA extraction from HBVSMCs using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. The appropriate concentration was determined by measuring absorbance at 260 nm using a Nanodrop spectrophotometer (Thermo scientific). The Superscript First-Strand cDNA synthesis system (Invitrogen, CA, USA) was utilized to perform reverse transcriptase reactions. RT-PCR was performed with a One-Step SYBR Prime Script TM RT-PCR Kit II (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The reactions were performed using a LightCycler 2.0 Instrument (Roche). SYBR green was utilized to determine mRNA expression, which was normalized to GAPDH gene expression. The data were analyzed using the 2−△△Ct method and the values were presented by relative quantity.
3.1. SPARC induced phenotypic modulation in HBVSMCs In order to examine the effect of SPARC on the phenotypic regulation of HBVSMCs, we measured the changes in molecular markers of contractile gene (SMA and SM22-α) and synthetic gene (OPN). As shown in Fig. 1A, SPARC treatment for 12 h resulted in a decrease of ˜20% for SMA and SM22-α mRNA expression and a significant increase for OPN expression (1.5-fold). As shown in Fig. 1B and C, compared with control cells, the protein abundance of SMA and SM22-α decreased ˜20% and the OPN protein abundance increased by 2-fold. These results showed that SPARC promotes phenotypic transition of HBVSMCs. 2
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Fig. 1. SPARC induces a contractile to synthetic phenotype switch in HBVSMCs. (A) Quantitative real-time PCR following SPARC (2 μg/ml) stimulation for 12 h. SMA and SM22-α are markers of the contractile phenotype; OPN is associated with a synthetic phenotype. *p < 0.05 and **p < 0.01 vs control, n = 3 per group. (B) WB analysis for contractile and synthetic proteins after SPARC (2 μg/ml) stimulation for 12 h. (C) Quantification of immunoblots from panel (B) (*p < 0.05 and ***p < 0.001 vs control, n = 3 per group).
levels of p-AMPK(Thr172) and increased the levels of p-mTOR(Ser2448). The autophagic protein levels of LC3-II, Beclin-1 and ATG5 decreased and the levels of p62 increased after CC treatment, respectively (Fig. 4E and F). These findings indicated AMPK/mTOR pathway involved in SPARC-induced autophagy in HBVSMCs.
3.2. SPARC induced autophagy activation in HBVSMCs Autophagy is a catabolic lysosome-mediated process responsible for the degradation and recycling of damaged or dysfunctional cytoplasmic components and intracellular organelles. In order to determine whether treatment with SPARC could induced autophagy, the protein levels of LC3-II, p62, Beclin-1 and ATG5 of HBVSMCs were measured after SPARC treatment. As shown in Fig. 2A and B, LC3-II formation after SPARC treatment increased in a time-dependent manner. After 12 h following treatment, LC3-II formation showed a significant increase, suggesting that SPARC promotes autophagy. Consistent with this hypothesis, there was an increase in expression of Beclin-1 and ATG5 and a decrease in expression of p62. As shown in Fig. 2C and D, the protein levels of LC3-II, Beclin-1 and ATG5 in HBVSMCs increased and p62 decreased in a dose-dependent manner of SPARC, which indicated that SPARC could induce autophagy activation. The concentration of 2 μg/ ml of SPARC could induce a significant increase in autophagy. So, 2 μg/ ml of SPARC was chosen for the following experiment concentration and 12 h post-treatment with SPARC was selected as the appropriate detection time.
3.5. SPARC induced autophagy flux increasing of HBVSMCs Due to the transient formation of LC3-II and its rapid degradation into lysosomes, an increase in LC3-II could also paradoxically indicate a decrease in autophagy. Therefore, to more accurately assess autophagic flux, HBVSMCs were respectively pretreated with 3-MA(10 mM) and CQ(10 μM) for 1 h before treated with SPARC(2 μg/ml). SPARC could induce a significant increase in LC3-II, Beclin-1 and ATG5 expression and a decrease in p62 expression. These effects were partly blocked by 3-MA, which suppressed the early stage of autophagosome formation, so SPARC indeed plays a role in the stimulation of autophagic flux in cultured HBVSMCs (Fig. 5A and B). Compared to the control, CQ could significantly increase the expression of LC3-II, Beclin-1, ATG5 and p62 expression (Fig. 5A and B), which reflects autophagosome accumulation because of autophagic flux inhibition. These results indicated that SPARC mediated HBVSMCs autophagy flux in vitro.
3.3. SPARC induced ultrastructural changes and extensive vacuolization in HBVSMCs
3.6. SPARC-induced autophagy involved in HBVSMCs phenotypic switching Autophagosome formation is considered as the “gold-standard” to document autophagy. We utilized Cyto-ID Green autophagy detection kit to visualize autophagosome formation via confocal microscopy. There was a significant increase in autophagic vacuoles(AVs) after HBVSMCs treated with SPARC(2 μg/ml) for 12 h, and this increase could be blocked by 3-MA (Fig. 3A and B). TEM was used for the analysis of ultrastructural changes in autophagosome formation. As shown in Fig. 3C and D, there was an approximate 1-fold increase in number of AVs within HBVSMCs treated with SPARC (2 μg/ml) for 12 h, and 3-MA could block this increase. Taken together, these structural changes further supported our immunological and fluorescence-imaging data, which suggested that SPARC was a robust inducer of the autophagic program.
Next, in order to elucidate whether autophagy plays a role in phenotypic switching of HBVSMCs, the HBVSMCs was pretreated with autophagy inhibitor 3-MA before SPARC treatment. As shown in Fig. 6A and B, 3-MA pretreatment could partially block SPARC-induced losses of SMA and SM22-α. Simultaneously, inhibition of autophagy with 3MA could block the SPARC-mediated increase of OPN. Collectively, these results indicated that autophagy is important for HBVSMC phenotype, and 3-MA is particularly effective in preventing conversion to the synthetic phenotype. 4. Discussion In this study, we found that SPARC could induce autophagy in HBVSMCs phenotypic modulation by activation of the AMPK/mTOR signaling pathway. We determined that SPARC, which promotes the phenotypic development of synthetic HBVSMCs, was a strong inducer of autophagy. In particular, 3-MA could partly block the SPARC-induced degradation of SMA and SM22-α. These findings demonstrated that SPARC-mediated autophagy takes part in the regulation of HBVSMC phenotype and function. Recent studies have shown that phenotypic modulation of VSMCs is closely related to IAs [9,21]. Therefore, it is essential to identify the specific pathways involved in these processes. Until now, SPARC involvement in VSMCs phenotypic modulation has remained undefined. In the current study, we explored the various impacts of SPARC on phenotypic modulation of HBVSMCs
3.4. SPARC induced autophagy activation through AMPK/mTOR signaling pathway Autophagy activation in mammalian cells usually involves inhibition of the mammalian target of rapamycin (mTOR), a downstream target of the AMPK pathway. To explore the potential molecular mechanisms by which SPARC induces autophagy, we detected the AMPK/ mTOR signaling pathway in this experiment. As demonstrated in Fig. 4A and B, there was a dose-dependent increase in p-AMPK(Thr172) protein levels upon SPARC treatment, and a dose-dependent decrease in p-mTOR(Ser2448). Then, HBVSMCs were incubated with the AMPK inhibitor CC(10 μM). As shown in Fig. 4C and D, CC reduced the protein 3
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Fig. 2. SPARC induces autophagy in HBVSMCs. (A) Time-dependent expression of autophagy-related proteins (LC3Ⅱ, p62, Beclin-1 and ATG5) of HBVSMCs treated with SPARC (2 μg/ml) were detected by WB. (B) Quantification of immunoblots from panel (A). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control, n = 3 per group. (C) Representative WB immunoblots of autophagy-related proteins in HBVSMCs treated with different concentrations of SPARC (0, 0.5, 1 and 2 μg/ml) for 12 h. (D) Quantification of immunoblots from panel (C). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control, n = 3 per group.
factors could trigger phenotypic alternation of VSMCs [25]. Autophagy plays an important role in the phenotypic alternation of VSMCs, and previous studies have also shown that platelet-derived growth factor (PDGF)-BB [26] and shear stress [27] could promote VSMCs phenotypic transformation by activating autophagy. Autophagy, a process that maintains energy balance of cells, plays a vital role in the physiological and pathophysiological processes in the vasculature, and has been shown to be involved in the development of various cardiovascular and cerebrovascular diseases including atherosclerosis [28], restenosis [29] and IAs [30] et al. In our study, enhanced autophagic activity was observed in the cultured HBVSMCs exposed to SPARC. SPARC- induced autophagy both occured in a time-dependent manner and a dose-dependent manner. Inhibition of autophagy could partly block SPARCinduced HBVSMCs phenotypic transition. These results demonstrated the causal role of autophagy induced by SPARC in the HBVSMCs phenotypic conversion.
and the related molecular mechanism in vitro. VSMCs have contractile functions in normal mature vessels. When subjected to various stimuli, VSMC can change from a contractile phenotype to a synthetic phenotype [9,22]. In our research, after exposure to SPARC in vitro, cultured HBVSMCs would dedifferentiate from a contractile state to a synthetic state, confirmed by decreased expression of contractile genes (SMA and SM22-α) and increased expression of synthetic gene (OPN). The protein levels of OPN significantly increased in the tissue of IAs, especially in unruptured IAs [23], it could also mediate cells death through enhanced autophagy in abdominal aortic aneurysms [24]. These results indicated the significance of SPARC involvement on phenotypic modulation of HBVSMCs, but the specific mechanisms have not been completely elucidated. Previous studies have shown that numerous stimuli including injury, blood flow shear stress, cytokine production and biochemical 4
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Fig. 3. SPARC increases autophagosome formation and promotes extensive vacuolization. (A) Representative confocal imaging of HBVSMCs stained with Cyto-ID Green autophagy detection kit treated with or without SPARC and 3-MA. (B) Quantification of AVs in images from panel(A) (> 20 cells counted per field in each group, ***p < 0.001 vs the indicated groups, n = 3 per group. (C) Representative transmission electron micrographs of cells treated with or without SPARC and 3-MA. (D) Quantification of AVs in images from panel (C) (*p < 0.05 vs the indicated groups, n = 3 per group).
Next, we explored the SPARC-mediated autophagy flux increased in HBVSMCs. The increased levels of autophagosomes could be caused by excessive activation of autophagy or dysfunctional autophagic flux [31,32]. We discovered that SPARC promoted a vigorous form of autophagy characterized by increased LC3-II expression, augmented autophagic flux, and the formation of autophagosomes and AVs. An increased number of autophagosomes in steady-state could result from an increase of autophagy, a block in downstream lysosomal processing of these autophagosomes, or both. It is important to note that the improved autophagic flux from SPARC occurs due to the increased delivery of the autophagosome to the autophagolysosome, as well as the activation of autophagosome initiation or formation. The following two findings are supportive of this. Firstly, the lysosome inhibitor CQ blocked the autophagic flux and 3-MA alleviated the increase of autophagy in HBVSMCs treated with SPARC. Secondly, SPARC regulated the activation of mTOR and AMPK, indicating the possible involvement of classical autophagy upstream signaling in HBVSMCs. Then, we investigated the potential molecular mechanisms
underlying that SPARC-induced autophagy in HBVSMCs. The AMPK/ mTOR signaling pathway is a classical pathway in the complex signal network that mediates autophagy [33]. We analyzed the effectiveness of AMPK/mTOR signaling activation in the up-regulation of SPARCinduced autophagy. Previous studies have shown that SPARC could interact with AMPK, the activation of AMPK would increases SPARC expression, while inhibition of endogenous AMPK expression would reduce SPARC expression [34]. The data from our study demonstrated a decrease ratio of p-mTOR/mTOR and an increase ratio of p-AMPK/ AMPK in HBVSMCs in a dose-dependent manner. To further vertify above results, CC was introduced for pretreatment. We found that pretreatment with CC was able to significantly block the increase expression of p-AMPK and the decrease expression of p-mTOR mediated by SPARC. Furthermore, CC administration markedly reduced the effect of autophagy activation in HBVSMCs suffering from SPARC, with a lower LC3-II, Beclin-1, ATG5 and a higher p62 protein level. All in all, these results further suggest that AMPK/mTOR signaling pathway plays a role in SPARC-induced autophagy activation. 5
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Fig. 4. SPARC targets the AMPK/mTOR pathway in HBVSMCs. (A) Representative WB immunoblots of p-AMPK(Thr172), AMPK, p-mTOR(Ser2448) and mTOR in HBVSMCs treated with different concentrations of SPARC for 12 h. (B) Quantification of immunoblots from panel (A). Each phosphor protein was normalized to its non-phosphorylated total protein band intensity, *p < 0.05 and ***p < 0.001 vs control, n = 3 per group. (C) Representative WB immunoblots of p-AMPK(Thr172), AMPK, p-mTOR(Ser2448), and mTOR in HBVSMCs treated without or with SPARC and CC. Each phosphor protein was normalized to its non-phosphorylated total protein band intensity too. (D) Quantification of immunoblots from panel (C). Each phosphor protein was normalized to its non-phosphorylated total protein band intensity, *p < 0.05 and ***p < 0.001 vs the indicated groups, n = 3 per group. (E) Representative WB immunoblots of autophagy-related proteins (LC3Ⅱ, p62, Beclin-1 and ATG5) in HBVSMCs treated without or with SPARC and CC. (F) Quantification of immunoblots from panel (E). *p < 0.05, **p < 0.01 and ***p < 0.001 vs the indicated groups, n = 3 per group.
5. Conclusions
mediated autophagy and SPARC-induced phenotypic modulation in HBVSMCs. We hope that the information obtained from this study can contribute to the development of new targets for early diagnosis and pharmacologic treatment options for IAs.
In conclusion, the results from our study provide new insights into the autophagic mechanisms by which SPARC induces phenotypic modulation of HBVSMCs from contractile to synthetic state. Furthermore, there is a strong correlation between AMPK/mTOR6
Neuroscience Letters 712 (2019) 134485
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Fig. 5. SPARC can mediate autophagy flux increase of HBVSMCs. (A) Representative WB immunoblots of autophagy-related proteins (LC3Ⅱ, p62, Beclin-1 and ATG5) in HBVSMCs treated without or with SPARC, 3-MA or CQ. (B) Quantification of immunoblots from panel (A). *p < 0.05, **p < 0.01 and ***p < 0.001 vs the indicated groups, n = 3 per group.
Author’s contribution
a potential conflict of interest.
TL and XT carried out the experiments and wrote the manuscript. SZ analyzed the data. WZ and FL prepared the figures and tables. JS and BH provided technical support and revised the paper. YW were responsible for experimental design and contributed some reagents.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China [No. 81171172 and No.81701160] and Shandong Provincial Science and Development planning Program of China [No. 2016GSF201230].
Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as
Fig. 6. Inhibition of autophagy prevents SPARC-induced phenotypic changes. (A) Representative WB immunoblots of contractile (SMA and SM22-α) and synthetic (OPN) proteins in HBVSMCs treated without or with SPARC, 3-MA. (B) Quantification of immunoblots in panel (A). *p < 0.05, **p < 0.01 and ***p < 0.001 vs the indicated groups, n = 3 per group. 7
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