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Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration Ning Wang, Feng Xu, Saien Lu, Naijin Zhang**, Yingxian Sun* Department of Cardiology, The First Hospital of China Medical University, 155 Nanjing North Street, Heping District, Shenyang, 110001, Liaoning Province, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2020 Accepted 8 February 2020 Available online xxx
Vascular smooth muscle cells (VSMCs) proliferation and migration play a fundamental role during the process of hypertensive angiopathy. Angiotensin-II (Ang-II) is one of the robust phenotype-modulating agents, which changes VSMCs to efficiently proliferate and migrate. The mechanism of the proliferation and migration is not well understood yet. Septin4, as a member of GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes. We approved that Septin4 expression was upregulated in mouse aorta by continuous infusion of Ang-II and in cultured VSMCs treated with Ang-II. Overexpression of Septin4 led to lower level of autophagy and decreased capacity of proliferation and migration. In order to identify the mechanism by which Septin4 interacts with these processes, we blocked autophagy by chloroquine (CQ). After inhibiting the autophagy, the ability of proliferation and migration was further restrained in the Septin4 overexpression VSMCs. In conclusion, our results indicated that during the process of VSMCs proliferation and migration induced by Ang-II, Septin4 modulated autophagy and thus regulated the activity of proliferation and migration. © 2020 Elsevier Inc. All rights reserved.
Keywords: Autophagy Septin4 Hypertensive angiopathy
1. Introduction Although continuous improvement has been made, cardiovascular disease (CVD) is still the most common underlying cause of death in the world [1]. Hypertension accounted for more CVD deaths than any other modifiable CVD risk factor. In the populationbased ARIC (Atherosclerosis Risk in Communities) study, 25% of the cardiovascular events (coronary heart disease, coronary revascularization, stroke, or HF) were attributable to hypertension [2]. Vascular smooth muscle cells (VSMCs) play a fundamental role during the pathophysiologic process of hypertension [3]. Unlike either skeletal or cardiac myocytes that are terminally differentiated, VSMCs preserve remarkable plasticity and may undergo phenotypic alterations in response to changes in local environmental cues. As the major cellular components of blood vessel wall, VSMCs exist in a differentiated contractile phenotype with extremely low rate of proliferation and synthetic function under normal circumstances. In response to some stimuli, VSMCs exhibit
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (N. Zhang), [email protected] (Y. Sun).
aberrant regulation of proliferation, migration, and synthesis of extracellular matrix (ECM) and play a crucial role in the development of hypertension, atherosclerosis and other vascular diseases [4]. The changes in the capacity of proliferation, migration and secretion of VSMCs are attributed to complex and multistep mechanism, which may be induced by a variety of proinflammatory stimuli and hemodynamic alterations. Angiotensin-II (Ang-II) has been approved strong stimuli that leads VSMCs to change from the quiescent contractile state to the active synthetic state and promotes VSMCs proliferation and migration, and ECM deposition. The act of Ang-II upon VSMCs is complicated and the mechanism has been explored widely [5e7]. Among various pathways, autophagy was proposed recent years [8e10]. Septin4 is a subtype of Septins family with GTPase activity, which has multiple splicing variants [11]. It is considered to be an essential component of the cytoskeleton and involved in many important physiological processes, such as cell transport and apoptosis [12]. Recently, our team identified Septin4 as a novel essential factor involved in oxidative stress induced vascular endothelial cells injury by interacting with apoptosis-related protein PARP1 [13]. As to VSMCs which are highly close to endothelial
https://doi.org/10.1016/j.bbrc.2020.02.064 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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cells anatomically and functionally, it is still unclear if Septin4 is involved in the related cardiovascular diseases. Our latest experiments indicated that Septin4 was involved in VSMCs proliferation and migration induced by Ang-II. We found the increased expression of Septin4 by the effect of Ang-II in vivo and in vitro. After treated with Ang-II, the autophagy was activated as well as the promoted capacity of VSMCs proliferation and migration. After overexpressing Septin4 by plasmid transfection and then stimulating by Ang-II, the VSMCs proliferation and migration were inhibited as well as the autophagy level. Furthermore, the inhibition effect was further aggravated by pretreated autophagic inhibitor, chloroquine (CQ). All these results supported the notion that Septin4 was involved in the Ang-II induced VSMCs proliferation and migration through modulating the autophagic activity. 2. Materials and methods 2.1. Animal model C57B6 male mice of 8 weeks age were purchased from Model Organisms Center (Shanghai, China). Through anesthesia by inhaling 2%e3% isoflurane, Osmotic pumps (model 2002; Alzet, USA) filled with Ang-II (Sigma-Aldrich, USA) or 0.9% NaCl as control were implanted subcutaneously through an incision at the back of the mice. The pumping rate for Ang-II was set to 1.5 mg/kg/day. The aortas of the mice were harvested after 14 days implantation for further analysis. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (Shenyang, China). 2.2. Morphological analysis The aorta tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections in 4 mm thickness were cut in series and stained by hematoxylin-eosin (HE) and Masson protocols. Images were analyzed with Image J software version 1.46 (National Institutes of Health, USA).
China) reagent mixture (10:1; 110 ml/well) and incubated for another 90 min. The optical density value was determined at a wavelength of 450 nm by microplate spectrophotometer (Tecan Infinite F50; Tecan Group, Ltd.).The same steps were repeatedly conducted in the Flag-control and Flag-Septin4 HASMCs with or without Ang-II interference. When autophagy inhibitor was required according to experimental design, 20 mM CQ was added to DMEM at 1:1000 and incubated for 10 h before optical analysis.
2.6. Cell migration assay A total of 300 ml cell suspension (8 103 cells) was added to the upper chambers of a transwell culture plate (Corning, USA) upon a 24-well plate. Ang-II at concentration gradient as 0 M, 108 M, 107 M, 106 M, 105 M were added in the cell suspension. To the bottom chambers, 600 ml of medium containing 10% FBS was added.After 48 h, cells on the upper surface of the polycarbonate membrane were gently removed. After fixed in methanol for 30 min, remaining cells on the polycarbonate membrane were stained with Giemsa solution for 30 min, then washed and observed under a microscope. The same steps were repeatedly conducted in the Flag-control and Flag-Septin4 HASMCs with or without Ang-II interference. When autophagic inhibitor was required according to experimental design, 20 mM CQ was added to DMEM at 1:1000 and incubated for 10 h before cell harvest.
2.7. Western blot assay After tissue harvesting or cell incubating as described above, cells were lysed in lysis buffer and centrifuged at 12000 rpm/min for 20 min at 4 C. The supernatants were collected. Protein concentration was calculated by Coomassie Brilliant Blue Statistical analysis. Equal content of protein was fractionated by SDS-PAGE followed by transferring to PVDF membrane and interacted with the primary and secondary antibodies. Data were normalized to the GAPHD content of the same sample quantified by using Image J software version 1.46 (National Institutes of Health, USA).
2.3. Plasmid, antibodies and agent Plasmid encoding the full-length human Septin4 (GeneChem, China) was cloned to Flag tagged destination vectors. Antibodies against rabbit MMP-2, PCNA, P62, LC3, Col-I were purchased from Proteintech, USA. Antibodies against Flag and GAPDH were purchased from Cell Signaling Technology, USA. Antibodies against Septin4 were from Abcam, USA.
2.8. Statistical analysis Results were presented as mean ± standard deviation.Statistics was analyzed by SPSS software 22.0 version. Significant differences between groups were determined by t-test or ANOVA. A P-value < 0.05 was considered statistically significant.
2.4. Cell culture and transfection 3. Results Human aortic smooth muscle cells (HASMCs cellline, BeNa Culture Collection, China) were cultured in a humidified incubator at 37 C with 5% CO2 and in Dulbecco’s modified eagle medium (DMEM) (HyClone, USA) with 10% fetal bovine serum (FBS) (HyClone, USA). Ang-II was used to stimulate proliferation and migration. CQ (Sigma-Aldrich, USA) was used as autophagy inhibitor. Cell transfection was performed by Lipofectamine 3000 (Invitrogen, USA) based on the manufacturer’s instructions. 2.5. Cell viability assay 104 cells/well were subcultured in a 96-well plate. Then Ang-II at concentration gradient as 0 M, 108 M, 107 M, 106 M, 105 M were added and incubated for 48 h. The primary DMEM was replaced by DMEM/CCK-8 (Beyotime Institute of Biotechnology,
3.1. Ang-II increased Septin4 expression, the intima-media thickness and collagen content in mice aortas We performed HE and Masson staining to demonstrate the morphological changes of the aorta. After 14 days of continuous subcutaneous infusion of Ang-II, the intima-media thickness of the mice aortas was dramatically increased as shown in Fig. 1. Besides, the collagen content was elevated represented as the blue area by masson staining, which was secreted by synthetic VSMCs. It indicated the vascular fibrosis level under the influence of Ang-II. Through analysis of separated aortic tissue, Septin4 protein expression was upregulated. These results indicated that Septin4 was involved in the process of the intimal-medial hyperplasia and matrix secretion induced by Ang-II in vivo.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 1. Ang-II increased Septin4 expression, the intima-media thickness and collagen content in mice aortas. (A) the intima-media thickness by HE staining. (B) Ang-II increased the intima-media thickness. (C) vascular fibrosis level by Masson staining. The blue area represented the vascular fibrosis. (D) Ang-II increased the fibrosis level. (E) Septin4 protein level in mice aortas by Western blot. (F) Ang-II increased the Septin4 protein level.***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.2. With the increase of Ang-II concentration, Septin4 expression was increased, as well as the capacity of VSMCs proliferation and migration As shown in Fig. 2, with the increase of Ang-II concentration, the migration cell number via filter membrane was increased accordingly. Similarly, cell viability increased with the increase of Ang-II concentration, which was detected by CCK8 method. The higher concentration of Ang-II was added, the higher expression of Septin4 was identified by Western blot assay. These findings suggested that Septin4 played a role during the boost of VSMCs proliferative and migratory capacity stimulated by Ang-II in vitro. 3.3. Stimulation effects of Ang-II on proliferation, migration and autophagy were alleviated by Septin4 overexpression For further investigating the effect of Septin4 upon VSMCs proliferation and migration stimulated by Ang-II, we overexpressed Septin4 in VSMCs by plasmid transfection method and results were illustrated in Fig. 3. Firstly, stimulated by different concentration of
Ang-II, cells expressing Flag-Septin4 exhibited lower viability compared with the Flag-control expressing group. Transwell migration assay showed that the migratory ability of Flag-Septin4 expressing cells reduced significantly. Additionally, we examined PCNA protein level as the proliferation indicator [14], col-1 [15] as the matrix secretory indicator and MMP-2 [16] as the migratory indicator. As shown in Fig. 3, PCNA, col-1, MMP-2 protein were significantly increased after being stimulated by Ang-II in VSMCs but relatively decreased in Flag-Septin4 expressing cells. Furthermore, we detected the autophagy activity by examining the formation of p62 and LC3-II [17]. Overexpressing Septin4 in VSMCs elevated the p62 protein level shown in Fig. 3. As a widely used autophagic flux marker, p62 is degraded by itself autophagy and therefore its quantities accumulates when autophagy is inhibited. LC3-II is another commonly used indicator which correlates with the number of autophagosomes, It was shown that Ang-II dramatically increased the LC3-II amount in cells expressing Flagcontrol and the positive effect was significantly impaired in FlagSeptin4 cells. These results suggested that the promotion effect of Ang-II on proliferation, migration and autophagy was alleviated by
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 2. With the increase of Ang-II concentration, Septin4 expression was increased, as well as the capacity of VSMCs proliferation and migration. (A, B) Ang-II increased migratory cells in a concentration-dependant manner by transwell method. (C) Ang-II increased cells viability in a concentration-dependant manner by CCK8. (D, E) Ang-II increased Septin4 expression in a concentration-dependant manner by western blot assay. ***p < 0.001; **p < 0.01.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 3. Stimulation effects of Ang-II on proliferation, migration and autophagy were alleviated by Septin4 overexpression. (A) Stimulation effect of Ang-II on cells viability was alleviated by Septin4 overexpression by CCK8 method. (B, C) Stimulation effect of Ang-II on cells migration was alleviated by Septin4 overexpression by transwell assay. Ang-II was treated at the concentration of 106M. ###p < 0.001 by ANOVA. (D, E) Stimulation effects of Ang-II on PCNA, col-1 and MMP-2 protein levels were alleviated by Septin4 overexpression by western blot. #p < 0.05 by ANOVA. (F, G) Under the condition of Ang-II, Septin4 overexpression increased p62 and decreased LC3-II amount by western blot, which indicated suppressed autophagic flux. #p < 0.05 by ANOVA.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 4. Ang-II stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor. (A) Stimulation effect of Ang-II on cell viability was further alleviated in Septin4 overexpressing cells treated with CQ. #p < 0.05 by ANOVA. (B,C) Stimulation effect of Ang-II on cell migration was further alleviated in Septin4 overexpressing cells treated with CQ. #p < 0.05 by ANOVA. (D, E) Stimulation effects of Ang-II on PCNA, col-1 and MMP2 protein levels were alleviated in Septin4 overexpressing cells treated with CQ. #p < 0.05 by ANOVA. (F, G) Higher protein level of p62 and LC3-II was detected in Septin4 overexpressing cells treated with CQ, which indicated higher degree of inhibition of autophagic activity. #p < 0.05 by ANOVA.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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overexpressing Septin4. 3.4. Ang-II stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor In order to clarify the mechanism of Septin4 on VSMCs proliferation and migration, we blocked autophagy flux with CQ. CQ acts in the final step of autophagy which blocks the autophagosome to fuse with the lysosome and thus makes itelf an effective autophagic inhibitor [18]. Here, we designed six groups as follows, Flag-control, Flag-control þ Ang-II, Flag-control þ Ang-II þ CQ, Flag-Septin4, Flag- Septin4þAng-II, Flag-Septin4þAng-II þ CQ and results were listed in Fig. 4. Fig. 4A showed that without Ang-II, Flag-Septin4 cells viability was decreased after CQ treatment. Generally, after treated with Ang-II, Flag-control, Flag-Septin4 and Flag-Septin4þ CQ cells viability was increased separately, but the increase effect was weakened in the latter two groups. Especially, the increasing trend in Flag-Septin4 cells was further restrained after CQ treatment. Similar finding was seen in transwell assay (Fig. 4C). CQ attenuated the positive effect of Ang-II on migratory ability in FlagSeptin4 expressing cells. By western blot tests, PCNA, col-1 and MMP-2 expression were decreased in CQ treated Flag-Septin4 cells. Fig. 4F showed the p62 and LC3-II accumulation after CQ treatment with and without Ang-II. Thus, it indicated that CQ further inhibited autophagic activity in Septin4 overexpressing group compared with control.These results suggested that Ang-II stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor, and autophagy mediated the effect of Septin4 upon the VSMCs proliferation and migration which were promoted by Ang-II. 4. Discussion Our results firstly reported that Septin4 was involved in VSMCs proliferation and migration in response to Ang-II via regulating autophagy. We identified the elevated level of Septin4 protein expression both in mice aortas and in cultured VSMCs after stimulated by Ang-II. It has been reported that Ang-II promotes VSMCs proliferation, migration and the autophagic activity [8,19], and the notion got supported by our data. Furthermore, our results showed that overexpressing Septin4 attenuated the Ang-II influence on VSMCs. Following Ang-II stimulation, Septin4 limited the increase of cell viability and prevented cells passing through the filter membrane. Autophagy was considered as one of the mechanisms of VSMC phenotype switching according to the published literature [8,10], which facilitated cell proliferation and migration. Overexpressing Septin4 significantly upregulated p62 and downregulated LC3-II which indicated that Septin4 attenuated the promotion effect of Ang-II upon autophagic activity. To further investigate the inner mechanism of VSMCs plasticity and autophagic activity changes, we conducted pharmacologic inhibition of autophagy by CQ. CQ further inhibited the positive effect of Ang-II upon cells viability and migratory number in Septin4-Flag expressing cells. These results indicated that autophagy mediated the effect of Septin4 upon the VSMCs proliferation and migration which were promoted by Ang-II. In conclusion, our results implied that Septin4 played a role during the Ang-II induced transformation of VSMCs proliferative and migratory properties and the mechanism may be dependent of regulating autophagic activity. Autophagy is the major intracellular process for lysosomemediated dynamic recycling system [20]. The purpose of autophagy is to eliminate the damaged cytoplasmic material and produce new building blocks and energy for cellular renovation and homeostasis. Thus, it is important in metabolic adaption, intracellular quality control, and renovation during development and
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differentiation. Autophagy is a critical determinant in the changes of VSMCs proliferative and migratory properties [21]. Effective autophagy in VSMCs has been shown to promote the conversion of contractile VSMCs to a synthetic phenotype which is characterized by enhanced capacity of proliferation and migration. Treatment of VSMCs with PDGF induces autophagy resulted in the removal of contractile proteins through a 50 adenosine monophosphateactivated protein kinase (AMPK)-independent and mammalian target of rapamycin (mTOR)-independent mechanism [10]. Another interesting finding was that secreted Sonic hedgehog (Shh) increased the expression of the synthetic phenotype markers and promoted autophagy in an Akt-dependent manner [22]. Conversely, the pharmacological inhibition of autophagy by 3methyladenine (3-MA) prevents PDGF or Shh induced VSMCs proliferation [10,22]. Generally, these findings demonstrated that autophagy was indispensable during the process of VSMCs plasticity changes. Autophagy is activated in VSMCs by various stimuli and stressors including reactive oxygen and lipid species, cytokines, growth factors and metabolic stress. Treatment of VSMCs with angiotensin II induces autophagy in a dose- and time-dependent manner via activation of AT1 receptor and NADPH oxidase [19]. Our result confirmed the pro-proliferative and migratory effect of autophagy on VSMCs induced by Ang-II and identified a new modulator-Septin4 as an autophagy inhibitor. Septins have been traditionally studied for their role in cytokinesis and their filament-forming abilities. With further research, more functions were discovered including membrane dynamics, cytoskeletal reorganization, vesicle trafficking, and tumorigenesis [12]. The human Septin4 gene encodes two major protein isoforms, Sept4_i1 (H5/PNUTL2) and Sept4_i2/ARTS. ARTS locates in mitochondria and induces apoptosis by interacting with inhibitors of apoptosis proteins (IAPs) which has been the best-studied family of caspase inhibitors. Despite of pro-apoptotic function, it is unclear if Septin4 plays a role in autophagic activity. We firstly proposed the notion that Septin4 manipulated VSMCs autophagy and thus prevented them from transforming to a proliferative and migratory state induced by Ang-II. Therefore, Septin4 may be a new potential therapeutic target in related cardiovascular diseases. As Septin4 was newly identified as a modulator for autophagy involved in Ang-II induced VSMCs proliferation and migration, there were plenty of details remained unclear. For example, more efforts may be made to investigate the specific molecular pathways through which Septin4 manipulates autophagy. As a goldenstandard for autophagic activity, electron microscope may be required for further mechanism exploration. In conclusion, our study indicated that Septin4 was involved in the VSMCs switching to a proliferative and migratory state stimulated by Ang-II through modulating autophagic activity. And it provided a potential target for therapeutic strategy in related cardiovascular diseases. Funding This work was supported by National Natural Science Foundation of China (Grant No. 81670231, 81970211). Declaration of competing interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper. References [1] E.J. Benjamin, M.J. Blaha, S.E. Chiuve, et al., Heart disease and stroke statistics-
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration Ning Wang, Feng Xu, Saien Lu, Naijin Zhang**, Yingxian Sun* Department of Cardiology, The First Hospital of China Medical University, 155 Nanjing North Street, Heping District, Shenyang, 110001, Liaoning Province, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2020 Accepted 8 February 2020 Available online xxx
Vascular smooth muscle cells (VSMCs) proliferation and migration play a fundamental role during the process of hypertensive angiopathy. Angiotensin-II (Ang-II) is one of the robust phenotype-modulating agents, which changes VSMCs to efficiently proliferate and migrate. The mechanism of the proliferation and migration is not well understood yet. Septin4, as a member of GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes. We approved that Septin4 expression was upregulated in mouse aorta by continuous infusion of Ang-II and in cultured VSMCs treated with Ang-II. Overexpression of Septin4 led to lower level of autophagy and decreased capacity of proliferation and migration. In order to identify the mechanism by which Septin4 interacts with these processes, we blocked autophagy by chloroquine (CQ). After inhibiting the autophagy, the ability of proliferation and migration was further restrained in the Septin4 overexpression VSMCs. In conclusion, our results indicated that during the process of VSMCs proliferation and migration induced by Ang-II, Septin4 modulated autophagy and thus regulated the activity of proliferation and migration. © 2020 Elsevier Inc. All rights reserved.
Keywords: Autophagy Septin4 Hypertensive angiopathy
1. Introduction Although continuous improvement has been made, cardiovascular disease (CVD) is still the most common underlying cause of death in the world [1]. Hypertension accounted for more CVD deaths than any other modifiable CVD risk factor. In the populationbased ARIC (Atherosclerosis Risk in Communities) study, 25% of the cardiovascular events (coronary heart disease, coronary revascularization, stroke, or HF) were attributable to hypertension [2]. Vascular smooth muscle cells (VSMCs) play a fundamental role during the pathophysiologic process of hypertension [3]. Unlike either skeletal or cardiac myocytes that are terminally differentiated, VSMCs preserve remarkable plasticity and may undergo phenotypic alterations in response to changes in local environmental cues. As the major cellular components of blood vessel wall, VSMCs exist in a differentiated contractile phenotype with extremely low rate of proliferation and synthetic function under normal circumstances. In response to some stimuli, VSMCs exhibit
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (N. Zhang), [email protected] (Y. Sun).
aberrant regulation of proliferation, migration, and synthesis of extracellular matrix (ECM) and play a crucial role in the development of hypertension, atherosclerosis and other vascular diseases [4]. The changes in the capacity of proliferation, migration and secretion of VSMCs are attributed to complex and multistep mechanism, which may be induced by a variety of proinflammatory stimuli and hemodynamic alterations. Angiotensin-II (Ang-II) has been approved strong stimuli that leads VSMCs to change from the quiescent contractile state to the active synthetic state and promotes VSMCs proliferation and migration, and ECM deposition. The act of Ang-II upon VSMCs is complicated and the mechanism has been explored widely [5e7]. Among various pathways, autophagy was proposed recent years [8e10]. Septin4 is a subtype of Septins family with GTPase activity, which has multiple splicing variants [11]. It is considered to be an essential component of the cytoskeleton and involved in many important physiological processes, such as cell transport and apoptosis [12]. Recently, our team identified Septin4 as a novel essential factor involved in oxidative stress induced vascular endothelial cells injury by interacting with apoptosis-related protein PARP1 [13]. As to VSMCs which are highly close to endothelial
https://doi.org/10.1016/j.bbrc.2020.02.064 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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cells anatomically and functionally, it is still unclear if Septin4 is involved in the related cardiovascular diseases. Our latest experiments indicated that Septin4 was involved in VSMCs proliferation and migration induced by Ang-II. We found the increased expression of Septin4 by the effect of Ang-II in vivo and in vitro. After treated with Ang-II, the autophagy was activated as well as the promoted capacity of VSMCs proliferation and migration. After overexpressing Septin4 by plasmid transfection and then stimulating by Ang-II, the VSMCs proliferation and migration were inhibited as well as the autophagy level. Furthermore, the inhibition effect was further aggravated by pretreated autophagic inhibitor, chloroquine (CQ). All these results supported the notion that Septin4 was involved in the Ang-II induced VSMCs proliferation and migration through modulating the autophagic activity. 2. Materials and methods 2.1. Animal model C57B6 male mice of 8 weeks age were purchased from Model Organisms Center (Shanghai, China). Through anesthesia by inhaling 2%e3% isoflurane, Osmotic pumps (model 2002; Alzet, USA) filled with Ang-II (Sigma-Aldrich, USA) or 0.9% NaCl as control were implanted subcutaneously through an incision at the back of the mice. The pumping rate for Ang-II was set to 1.5 mg/kg/day. The aortas of the mice were harvested after 14 days implantation for further analysis. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (Shenyang, China). 2.2. Morphological analysis The aorta tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections in 4 mm thickness were cut in series and stained by hematoxylin-eosin (HE) and Masson protocols. Images were analyzed with Image J software version 1.46 (National Institutes of Health, USA).
China) reagent mixture (10:1; 110 ml/well) and incubated for another 90 min. The optical density value was determined at a wavelength of 450 nm by microplate spectrophotometer (Tecan Infinite F50; Tecan Group, Ltd.).The same steps were repeatedly conducted in the Flag-control and Flag-Septin4 HASMCs with or without Ang-II interference. When autophagy inhibitor was required according to experimental design, 20 mM CQ was added to DMEM at 1:1000 and incubated for 10 h before optical analysis.
2.6. Cell migration assay A total of 300 ml cell suspension (8 103 cells) was added to the upper chambers of a transwell culture plate (Corning, USA) upon a 24-well plate. Ang-II at concentration gradient as 0 M, 108 M, 107 M, 106 M, 105 M were added in the cell suspension. To the bottom chambers, 600 ml of medium containing 10% FBS was added.After 48 h, cells on the upper surface of the polycarbonate membrane were gently removed. After fixed in methanol for 30 min, remaining cells on the polycarbonate membrane were stained with Giemsa solution for 30 min, then washed and observed under a microscope. The same steps were repeatedly conducted in the Flag-control and Flag-Septin4 HASMCs with or without Ang-II interference. When autophagic inhibitor was required according to experimental design, 20 mM CQ was added to DMEM at 1:1000 and incubated for 10 h before cell harvest.
2.7. Western blot assay After tissue harvesting or cell incubating as described above, cells were lysed in lysis buffer and centrifuged at 12000 rpm/min for 20 min at 4 C. The supernatants were collected. Protein concentration was calculated by Coomassie Brilliant Blue Statistical analysis. Equal content of protein was fractionated by SDS-PAGE followed by transferring to PVDF membrane and interacted with the primary and secondary antibodies. Data were normalized to the GAPHD content of the same sample quantified by using Image J software version 1.46 (National Institutes of Health, USA).
2.3. Plasmid, antibodies and agent Plasmid encoding the full-length human Septin4 (GeneChem, China) was cloned to Flag tagged destination vectors. Antibodies against rabbit MMP-2, PCNA, P62, LC3, Col-I were purchased from Proteintech, USA. Antibodies against Flag and GAPDH were purchased from Cell Signaling Technology, USA. Antibodies against Septin4 were from Abcam, USA.
2.8. Statistical analysis Results were presented as mean ± standard deviation.Statistics was analyzed by SPSS software 22.0 version. Significant differences between groups were determined by t-test or ANOVA. A P-value < 0.05 was considered statistically significant.
2.4. Cell culture and transfection 3. Results Human aortic smooth muscle cells (HASMCs cellline, BeNa Culture Collection, China) were cultured in a humidified incubator at 37 C with 5% CO2 and in Dulbecco’s modified eagle medium (DMEM) (HyClone, USA) with 10% fetal bovine serum (FBS) (HyClone, USA). Ang-II was used to stimulate proliferation and migration. CQ (Sigma-Aldrich, USA) was used as autophagy inhibitor. Cell transfection was performed by Lipofectamine 3000 (Invitrogen, USA) based on the manufacturer’s instructions. 2.5. Cell viability assay 104 cells/well were subcultured in a 96-well plate. Then Ang-II at concentration gradient as 0 M, 108 M, 107 M, 106 M, 105 M were added and incubated for 48 h. The primary DMEM was replaced by DMEM/CCK-8 (Beyotime Institute of Biotechnology,
3.1. Ang-II increased Septin4 expression, the intima-media thickness and collagen content in mice aortas We performed HE and Masson staining to demonstrate the morphological changes of the aorta. After 14 days of continuous subcutaneous infusion of Ang-II, the intima-media thickness of the mice aortas was dramatically increased as shown in Fig. 1. Besides, the collagen content was elevated represented as the blue area by masson staining, which was secreted by synthetic VSMCs. It indicated the vascular fibrosis level under the influence of Ang-II. Through analysis of separated aortic tissue, Septin4 protein expression was upregulated. These results indicated that Septin4 was involved in the process of the intimal-medial hyperplasia and matrix secretion induced by Ang-II in vivo.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 1. Ang-II increased Septin4 expression, the intima-media thickness and collagen content in mice aortas. (A) the intima-media thickness by HE staining. (B) Ang-II increased the intima-media thickness. (C) vascular fibrosis level by Masson staining. The blue area represented the vascular fibrosis. (D) Ang-II increased the fibrosis level. (E) Septin4 protein level in mice aortas by Western blot. (F) Ang-II increased the Septin4 protein level.***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.2. With the increase of Ang-II concentration, Septin4 expression was increased, as well as the capacity of VSMCs proliferation and migration As shown in Fig. 2, with the increase of Ang-II concentration, the migration cell number via filter membrane was increased accordingly. Similarly, cell viability increased with the increase of Ang-II concentration, which was detected by CCK8 method. The higher concentration of Ang-II was added, the higher expression of Septin4 was identified by Western blot assay. These findings suggested that Septin4 played a role during the boost of VSMCs proliferative and migratory capacity stimulated by Ang-II in vitro. 3.3. Stimulation effects of Ang-II on proliferation, migration and autophagy were alleviated by Septin4 overexpression For further investigating the effect of Septin4 upon VSMCs proliferation and migration stimulated by Ang-II, we overexpressed Septin4 in VSMCs by plasmid transfection method and results were illustrated in Fig. 3. Firstly, stimulated by different concentration of
Ang-II, cells expressing Flag-Septin4 exhibited lower viability compared with the Flag-control expressing group. Transwell migration assay showed that the migratory ability of Flag-Septin4 expressing cells reduced significantly. Additionally, we examined PCNA protein level as the proliferation indicator [14], col-1 [15] as the matrix secretory indicator and MMP-2 [16] as the migratory indicator. As shown in Fig. 3, PCNA, col-1, MMP-2 protein were significantly increased after being stimulated by Ang-II in VSMCs but relatively decreased in Flag-Septin4 expressing cells. Furthermore, we detected the autophagy activity by examining the formation of p62 and LC3-II [17]. Overexpressing Septin4 in VSMCs elevated the p62 protein level shown in Fig. 3. As a widely used autophagic flux marker, p62 is degraded by itself autophagy and therefore its quantities accumulates when autophagy is inhibited. LC3-II is another commonly used indicator which correlates with the number of autophagosomes, It was shown that Ang-II dramatically increased the LC3-II amount in cells expressing Flagcontrol and the positive effect was significantly impaired in FlagSeptin4 cells. These results suggested that the promotion effect of Ang-II on proliferation, migration and autophagy was alleviated by
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 2. With the increase of Ang-II concentration, Septin4 expression was increased, as well as the capacity of VSMCs proliferation and migration. (A, B) Ang-II increased migratory cells in a concentration-dependant manner by transwell method. (C) Ang-II increased cells viability in a concentration-dependant manner by CCK8. (D, E) Ang-II increased Septin4 expression in a concentration-dependant manner by western blot assay. ***p < 0.001; **p < 0.01.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 3. Stimulation effects of Ang-II on proliferation, migration and autophagy were alleviated by Septin4 overexpression. (A) Stimulation effect of Ang-II on cells viability was alleviated by Septin4 overexpression by CCK8 method. (B, C) Stimulation effect of Ang-II on cells migration was alleviated by Septin4 overexpression by transwell assay. Ang-II was treated at the concentration of 106M. ###p < 0.001 by ANOVA. (D, E) Stimulation effects of Ang-II on PCNA, col-1 and MMP-2 protein levels were alleviated by Septin4 overexpression by western blot. #p < 0.05 by ANOVA. (F, G) Under the condition of Ang-II, Septin4 overexpression increased p62 and decreased LC3-II amount by western blot, which indicated suppressed autophagic flux. #p < 0.05 by ANOVA.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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Fig. 4. Ang-II stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor. (A) Stimulation effect of Ang-II on cell viability was further alleviated in Septin4 overexpressing cells treated with CQ. #p < 0.05 by ANOVA. (B,C) Stimulation effect of Ang-II on cell migration was further alleviated in Septin4 overexpressing cells treated with CQ. #p < 0.05 by ANOVA. (D, E) Stimulation effects of Ang-II on PCNA, col-1 and MMP2 protein levels were alleviated in Septin4 overexpressing cells treated with CQ. #p < 0.05 by ANOVA. (F, G) Higher protein level of p62 and LC3-II was detected in Septin4 overexpressing cells treated with CQ, which indicated higher degree of inhibition of autophagic activity. #p < 0.05 by ANOVA.
Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064
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overexpressing Septin4. 3.4. Ang-II stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor In order to clarify the mechanism of Septin4 on VSMCs proliferation and migration, we blocked autophagy flux with CQ. CQ acts in the final step of autophagy which blocks the autophagosome to fuse with the lysosome and thus makes itelf an effective autophagic inhibitor [18]. Here, we designed six groups as follows, Flag-control, Flag-control þ Ang-II, Flag-control þ Ang-II þ CQ, Flag-Septin4, Flag- Septin4þAng-II, Flag-Septin4þAng-II þ CQ and results were listed in Fig. 4. Fig. 4A showed that without Ang-II, Flag-Septin4 cells viability was decreased after CQ treatment. Generally, after treated with Ang-II, Flag-control, Flag-Septin4 and Flag-Septin4þ CQ cells viability was increased separately, but the increase effect was weakened in the latter two groups. Especially, the increasing trend in Flag-Septin4 cells was further restrained after CQ treatment. Similar finding was seen in transwell assay (Fig. 4C). CQ attenuated the positive effect of Ang-II on migratory ability in FlagSeptin4 expressing cells. By western blot tests, PCNA, col-1 and MMP-2 expression were decreased in CQ treated Flag-Septin4 cells. Fig. 4F showed the p62 and LC3-II accumulation after CQ treatment with and without Ang-II. Thus, it indicated that CQ further inhibited autophagic activity in Septin4 overexpressing group compared with control.These results suggested that Ang-II stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor, and autophagy mediated the effect of Septin4 upon the VSMCs proliferation and migration which were promoted by Ang-II. 4. Discussion Our results firstly reported that Septin4 was involved in VSMCs proliferation and migration in response to Ang-II via regulating autophagy. We identified the elevated level of Septin4 protein expression both in mice aortas and in cultured VSMCs after stimulated by Ang-II. It has been reported that Ang-II promotes VSMCs proliferation, migration and the autophagic activity [8,19], and the notion got supported by our data. Furthermore, our results showed that overexpressing Septin4 attenuated the Ang-II influence on VSMCs. Following Ang-II stimulation, Septin4 limited the increase of cell viability and prevented cells passing through the filter membrane. Autophagy was considered as one of the mechanisms of VSMC phenotype switching according to the published literature [8,10], which facilitated cell proliferation and migration. Overexpressing Septin4 significantly upregulated p62 and downregulated LC3-II which indicated that Septin4 attenuated the promotion effect of Ang-II upon autophagic activity. To further investigate the inner mechanism of VSMCs plasticity and autophagic activity changes, we conducted pharmacologic inhibition of autophagy by CQ. CQ further inhibited the positive effect of Ang-II upon cells viability and migratory number in Septin4-Flag expressing cells. These results indicated that autophagy mediated the effect of Septin4 upon the VSMCs proliferation and migration which were promoted by Ang-II. In conclusion, our results implied that Septin4 played a role during the Ang-II induced transformation of VSMCs proliferative and migratory properties and the mechanism may be dependent of regulating autophagic activity. Autophagy is the major intracellular process for lysosomemediated dynamic recycling system [20]. The purpose of autophagy is to eliminate the damaged cytoplasmic material and produce new building blocks and energy for cellular renovation and homeostasis. Thus, it is important in metabolic adaption, intracellular quality control, and renovation during development and
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differentiation. Autophagy is a critical determinant in the changes of VSMCs proliferative and migratory properties [21]. Effective autophagy in VSMCs has been shown to promote the conversion of contractile VSMCs to a synthetic phenotype which is characterized by enhanced capacity of proliferation and migration. Treatment of VSMCs with PDGF induces autophagy resulted in the removal of contractile proteins through a 50 adenosine monophosphateactivated protein kinase (AMPK)-independent and mammalian target of rapamycin (mTOR)-independent mechanism [10]. Another interesting finding was that secreted Sonic hedgehog (Shh) increased the expression of the synthetic phenotype markers and promoted autophagy in an Akt-dependent manner [22]. Conversely, the pharmacological inhibition of autophagy by 3methyladenine (3-MA) prevents PDGF or Shh induced VSMCs proliferation [10,22]. Generally, these findings demonstrated that autophagy was indispensable during the process of VSMCs plasticity changes. Autophagy is activated in VSMCs by various stimuli and stressors including reactive oxygen and lipid species, cytokines, growth factors and metabolic stress. Treatment of VSMCs with angiotensin II induces autophagy in a dose- and time-dependent manner via activation of AT1 receptor and NADPH oxidase [19]. Our result confirmed the pro-proliferative and migratory effect of autophagy on VSMCs induced by Ang-II and identified a new modulator-Septin4 as an autophagy inhibitor. Septins have been traditionally studied for their role in cytokinesis and their filament-forming abilities. With further research, more functions were discovered including membrane dynamics, cytoskeletal reorganization, vesicle trafficking, and tumorigenesis [12]. The human Septin4 gene encodes two major protein isoforms, Sept4_i1 (H5/PNUTL2) and Sept4_i2/ARTS. ARTS locates in mitochondria and induces apoptosis by interacting with inhibitors of apoptosis proteins (IAPs) which has been the best-studied family of caspase inhibitors. Despite of pro-apoptotic function, it is unclear if Septin4 plays a role in autophagic activity. We firstly proposed the notion that Septin4 manipulated VSMCs autophagy and thus prevented them from transforming to a proliferative and migratory state induced by Ang-II. Therefore, Septin4 may be a new potential therapeutic target in related cardiovascular diseases. As Septin4 was newly identified as a modulator for autophagy involved in Ang-II induced VSMCs proliferation and migration, there were plenty of details remained unclear. For example, more efforts may be made to investigate the specific molecular pathways through which Septin4 manipulates autophagy. As a goldenstandard for autophagic activity, electron microscope may be required for further mechanism exploration. In conclusion, our study indicated that Septin4 was involved in the VSMCs switching to a proliferative and migratory state stimulated by Ang-II through modulating autophagic activity. And it provided a potential target for therapeutic strategy in related cardiovascular diseases. Funding This work was supported by National Natural Science Foundation of China (Grant No. 81670231, 81970211). Declaration of competing interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper. References [1] E.J. Benjamin, M.J. Blaha, S.E. Chiuve, et al., Heart disease and stroke statistics-
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Please cite this article as: N. Wang et al., Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.064