Mol. Cells 2020; 43(8): 749-762
Published online August 19, 2020
https://doi.org/10.14348/molcells.2020.0085
© The Korean Society for Molecular and Cellular Biology
Correspondence to : yuanby@wust.edu.cn (BYY); zhangtongcun@wust.edu.cn (TCZ)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The migration, dedifferentiation, and proliferation of vascular smooth muscle cells (VSMCs) are responsible for intimal hyperplasia, but the mechanism of this process has not been elucidated. WD repeat domain 1 (WDR1) promotes actin-depolymerizing factor (ADF)/cofilin-mediated depolymerization of actin filaments (F-actin). The role of WDR1 in neointima formation and progression is still unknown. A model of intimal thickening was constructed by ligating the left common carotid artery in Wdr1 deletion mice, and H&E staining showed that Wdr1 deficiency significantly inhibits neointima formation. We also report that STAT3 promotes the proliferation and migration of VSMCs by directly promoting WDR1 transcription. Mechanistically, we clarified that WDR1 promotes the proliferation and migration of VSMCs and neointima formation is regulated by the activation of the JAK2/STAT3/WDR1 axis.
Keywords migration, neointima formation, proliferation, vascular smooth muscle cells, WD repeat domain 1
Vascular smooth muscle cells (VSMCs) are important participants in vascular pathological processes and are the predominant cell type in the arterial wall (Gomez and Owens, 2012; Ni et al., 2019; Owens et al., 2004). Proliferation and migration of VSMCs are key events in intimal hyperplasia (Allahverdian et al., 2018); these events are also prerequisites for diseases such as arteriosclerosis and restenosis after angioplasty (O'Brien et al., 2011; Xie et al., 2017). Under normal physiological conditions, VSMCs in the tunica media remain in a stationary state (Xie et al., 2017). In response to stimuli such as hypoxia and injury, VSMCs undergo a phenotypic transformation and proliferate (Intengan and Schiffrin, 2000; Xie et al., 2011). VSMCs also secrete extracellular matrix components, resulting in the intimal hyperplasia and vascular lumen stenosis that comprise neointima formation (Dzau et al., 2002; Jonasson et al., 1988; Marx and Marks, 2001; Weintraub, 2007).
Under physiological conditions, assembly and depolymerization of actin filaments coincide and maintain a balance, which is known as actin dynamics (Yuan et al., 2018). Cell motility and growth are inseparable from actin dynamics, and this process is regulated by various actin-binding proteins (Cooper and Schafer, 2000). Many factors are involved in depolymerization, such as actin-depolymerizing factor (ADF)/cofilin and WD repeat domain 1 (WDR1) (Bamburg, 1999; Cooper and Schafer, 2000). ADF/cofilin plays a crucial role in actin cleavage and depolymerization (Bravo-Cordero et al., 2013). WDR1 is a major cofactor for ADF/cofilin and promotes ADF/cofilin-mediated depolymerization of actin filaments (F-actin), thereby regulating the balance between actin depolymerization and assembly (Lee and Dominguez, 2010; Xu et al., 2015). WDR1-regulated actin dynamics directly affect cellular processes such as migration, cell-cell junction maintenance, and proliferation (Collazo et al., 2014; Lee et al., 2016). High expression of
Signal transducer and activator of transcription 3 (STAT3) is an important component of the Janus kinase (JAK/STAT) signaling pathway (Qi et al., 2016), which is involved in inflammation, as well as various cellular processes such as division, proliferation, drug resistance, and apoptosis (Fu et al., 2012; Hirano et al., 2000; Jackson and Ceresa, 2017; Limagne et al., 2017; Tripathi et al., 2017; Zulkifli et al., 2017). Binding of an extracellular signaling molecule (e.g., interleukin-6 [IL-6]) to its corresponding receptor on the cell surface induces the phosphorylation of JAK and its downstream transcription factor STATs (e.g., the IL-6/JAK/STAT3 pathway) (Chang et al., 2013; Johnson et al., 2018). Phosphorylated STAT3 forms homologous or heterologous dimers and then translocates to the nucleus (Souissi et al., 2011). STAT3 regulates the expression of downstream genes that are associated with growth, drug resistance, and inflammation, such as STK35, AKT2, and COX-2 (Kou et al., 2011; Qi et al., 2016; Wu et al., 2018). Studies have shown that STAT3 is crucial to neointima formation (Kovacic et al., 2010; Seki et al., 2000). However, the relationship between STAT3 and WDR1 and transcriptional regulation during vascular injury repair is unclear.
In this study, we propose a mechanism by which WDR1 regulates the proliferation of VSMCs and pathological intimal thickening. We demonstrated that after vascular injury, WDR1 promotes VSMC proliferation and migration, thereby promoting intimal thickening. After
C57/BL6 male mice (7- to 8-weeks old) were obtained from the Model Animal Research Center of Nanjing University (Yuan et al., 2014).
Primary cultures of murine artery smooth muscle cells (MASMCs) were prepared from arteries. Briefly, aortas were isolated from 8 animals (
Human aortic smooth muscle cells (HASMCs; National Infrastructure of Cell Line Resource, China) were grown in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C in a 5% CO2 incubator. Cells were trypsinized at 80% to 90% confluence for experiments. HASMCs were seeded into 6-well plates, and when the cells reached 60% confluence, angiotensin II (Ang II, 200 nM; Sigma-Aldrich) and PDGF-BB (20 ng/ml; R&D Systems, USA) were added to the medium, and cells were collected at different times after treatment. Some HAVSMC cultures were stimulated with serum. For these cultures, cells were incubated in serum-free DMEM for 12 h and then treated with 10% fetal bovine serum. Cells were collected at various times after treatment.
Small interfering RNA (siRNA) duplexes for silencing
RNA was extracted from HASMCs using TRIzol (Sigma-Aldrich). M-MLV reverse transcriptase (Vazyme Biotech, China) was used to synthesize cDNA. Real-time polymerase chain reaction (PCR) (Real-Time PCR Detection Systems; Bio-Rad, USA) was performed to assess the relative levels of mRNAs. Relative mRNA levels were normalized internally to GAPDH mRNA. Related primers are as follows: GAPDH (F): 5’-CATGTACGTTGCTATCCAGGC-3’; GAPDH (R): 5’-CTCCTTAATGTCACGCACGAT-3’; WDR1 (F): 5’-TTGTCAACTGTGTGCGATTCTC-3’; and WDR1 (R): 5’-GCTGTCGGGACTCCA ACTAA-3’.
Proteins were extracted from HASMCs, MASMCs, and murine artery tissues. Western blotting was performed as described previously (Hu et al., 2018). Primary antibodies included rabbit anti-WDR1 (cat No. 13676-1-AP, 1:1,000; Proteintech, USA), rabbit anti-STAT3 (cat No. 10253-2-AP, 1:1,000; Proteintech), rabbit anti-p-STAT3Tyr705 (cat No. 9415, 1:1,000; Cell Signaling Technology, USA), β-actin (cat No. AP0060, 1:5,000; Bioworld Technology, USA), rabbit anti-JAK2 (cat No. sc390539, 1:500; Santa Cruz Biotechnology, USA), and p-JAK2Tyr1007/1008 (cat No. ab32102, 1:1,000; Abcam, UK). The membrane was washed using TBS-T and incubated with secondary antibody conjugated to horseradish peroxidase in 5% nonfat milk for 1 h at room temperature. Detection was carried out using a chemiluminescence detection kit (cat No.K-12045-D50; Advansta, USA).
Arterial tissue samples were first washed with cold PBS and then fixed with 4% paraformaldehyde at 4°C. The samples were processed by successive incubation in (1) PBS for 20 min; (2) an ethanol series (70%, 85%, 95%, 95%, 100%, and 100%) for 1 h at room temperature; (3) butyl alcohol, three times for 30 min each at room temperature; and (4) fresh paraffin at 65°C, three times for 30 min each. The treated samples were embedded in paraffin, and 5-μm-thick sections were prepared. After the sections were dewaxed and rehydrated, immunofluorescence was performed according to standard protocols. Briefly, sections were incubated with mouse anti-α-SMA (cat No. ab32575, 1:200; Abcam) or rabbit anti-PH3 (cat No. 53348; Cell Signaling Technology) overnight at 4°C. After being washed with PBS three times, sections were incubated with Alexa Fluor 647-conjugated anti-mouse IgG (cat No. 4410S; Cell Signaling Technology) or Alexa Fluor 488-conjugated anti-rabbit IgG (cat No. 4416S; Cell Signaling Technology) for 30 min at room temperature. Slices were then stained with DAPI (1.0 mg/ml; Invitrogen) for 30 min before mounting. Images were captured with an Olympus confocal microscope (Olympus, Japan).
For immunohistochemistry (IHC), samples were prepared as described above, and sections were incubated with anti-WDR1 primary antibody and detected according to the IHC kit instructions (cat No. KIT-9707; Maixin Biotechnology, China). H&E staining was performed using standard protocols. H&E-stained arterial sections were analyzed by planimetry with ImageJ software.
Cell counting kit 8 (CCK-8) (cat No. A311-01; Vazyme Biotech) was used to detect cell proliferation between 0 h and 72 h according to the manufacturer’s manual. Briefly, after tamoxifen-induced
MASMCs were seeded into 6-well plates for two days after induction with tamoxifen. After serum starvation for 24 h, a scratch was made using a sterile 200 μl pipette tip across each well, creating a cell-free area, based on the technique described by Yuan et al., and then 10 vol% fresh medium was added to the culture. Images were captured with a microscope at 0 h and 12 h. One day after transfection with siRNA, HASMCs were serum-starved for 12 h, and a scratch was made as described above.
The Phanta Super-Fidelity DNA Polymerase (cat No. P515-01; Vazyme Biotech) was used to perform a PCR to clone the murine
The
Similarly,
The integrity and orientation of each insert were confirmed by sequencing.
ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (cat No. 9002S; Cell Signaling Technology). MASMCs were treated with IL-6 (cat No. I9646; Sigma-Aldrich) for 30 min, and the cell lysate was used for immunoprecipitation with an antibody specific for the phosphorylated STAT3 (pSTAT3) protein (cat No. 9415; Cell Signaling Technology). PCR was performed on purified DNA from each sample using specific primers. The primer sequences used are listed below: F: 5’-TTACCCCAGCAGTCTTTGTACCAT-3’ and R: 5’-AGCACTGTCCTAGGGATGTGCTAG-3’.
MASMCs were synchronized and induced with tamoxifen for two days as described above. Cells were fixed overnight in 75% ethanol at 4°C and stained for 10 min at room temperature with propidium iodide (50 mg/ml) and analyzed with a FACSCanto flow cytometer (BD Biosciences, USA). Cell-cycle detection was carried out by propidium iodide staining and fluorescence-activated cell sorting analysis. Each experiment was performed in triplicate.
Mouse carotid arteries were harvested at various times post-injury, and the proteins were extracted with lysis buffer (50 mM Tris-HCl, pH 6.8; 10% glycerol; 1% SDS). Extracted proteins (10 mg) were loaded on 10% SDS-PAGE gels containing 1% gelatin to detect gelatinase activity. After washing in 2.5% Triton X-100, the gels were incubated overnight in buffer (10 mM CaCl2, 0.01% NaN3, and 50 mM Tris-HCl, pH 7.5). The obtained gels were stained with 0.2% Coomassie blue R-250 (Sigma-Aldrich) for 2 h and then destained with 10% acetic acid and 40% methanol.
Comparisons between two groups were performed using unpaired two-tailed Student’s
To investigate the effect of WDR1 on intimal thickening, we constructed a model of vascular injury in mice, and WDR1 levels were measured by western blot on different days post-injury (Figs. 1A and 1B). Furthermore, a gelatin zymography assay indicated that MMP9 activity in the ligation group was significantly increased compared with the sham group, indicating that injury facilitates SMC migration (Fig. 1C). To determine the localization and expression pattern of WDR1 in blood vessels, we performed immunohistochemical staining (Fig. 1D). WDR1 is primarily localized in the smooth muscle cells of blood vessels (Fig. 1D), and its level gradually increases as the number of days post-injury increases. These results suggest that WDR1 plays a key role in the repair of vascular injury.
To investigate the potential effects of WDR1 in vascular biology and pathophysiology, we used the murine model of vascular injury for
The response of WDR1 to various stimuli associated with vascular injury was investigated
Although WDR1 levels are increasing during neointima formation, it is unclear how WDR1 affects the process. Therefore, we performed CAL in
Knockdown of
We investigated STAT3 activation by western blot in carotid artery tissue after ligation (Fig. 5A). After the injury, the levels of pSTAT3 (activated STAT3) increase and reach the highest level at 7 days post-ligation. The levels of WDR1 display a trend similar to those of pSTAT3 (Fig. 5A). These results suggest that STAT3 may regulate WDR1 to affect neointimal thickening. To test this hypothesis, we exposed cells to IL-6 and Ang II to simulate the release of cytokines following vascular injury. The proliferation and migration of SMCs are improved in response to IL-6 and Ang II (data not shown). As expected, pSTAT3 and WDR1 are elevated. In addition, we silenced
To study the interaction between WDR1 and STAT3, we used siRNA to decrease
Based on the results obtained above, we hypothesized that STAT3 binds directly to the
WDR1-mediated SMCs migration and proliferation require the activation of STAT3. To elucidate additional details about this result, we conducted
Intimal thickening caused by abnormal proliferation of SMCs is associated with a variety of diseases (Xie et al., 2017). The proliferation and migration of SMCs caused by endogenous and exogenous factors are crucial steps in the repair of vascular injury (O'Brien et al., 2011; Zhang et al., 2014). However, our understanding of the underlying mechanisms for these responses in SMCs is poor.
WDR1 acts as a cofactor of cofilin for actin depolymerization and plays a vital role in cellular processes, such as proliferation and migration (Yuan et al., 2018). However, the function of WDR1 in SMCs is not understood. Therefore, we constructed
The process of vascular remodeling after vascular injury is divided into four stages (Schwartz et al., 1995; Seki et al., 2000). The first stage occurs within a few days of injury when the proliferation of the medial VSMCs is maximal. The second stage involves the migration of SMCs to the intima, starting between day 4 and 5. The third stage involves the continuous proliferation of SMCs in the neointima. During the fourth stage, extracellular matrix components are secreted and deposited. Inflammation, cytokine production, and VSMC proliferation are key aspects of vascular repair, and inflammation, and cytokines are active in the first phase of vascular remodeling. Unfortunately, endometrial remodeling elicits a series of negative results. Decreased lumen diameter causes thrombosis, which in turn leads to serious cardiovascular diseases such as myocardial and cerebral infarctions.
JAK and STAT proteins transmit intracellular signals in a variety of cell types in response to various cytokines (Li et al., 2019). Previous reports have shown that JAK2/STAT3 is responsive to angiotensin II type 1 receptor and is activated after tyrosine phosphorylation in rat smooth muscle cells (Marrero et al., 1997; Seki et al., 2000; Watanabe et al., 2004). Activated STAT3 translocates to the nucleus after homologous or heterologous dimer formation and binds to cis-inducing elements in the nucleus, leading to transcriptional activation of early growth response genes (Li et al., 2019). When SMCs were treated with the JAK2-specific inhibitor AG490, proliferation was significantly inhibited, suggesting that the JAK2/STAT3 pathway is vital to smooth muscle cell proliferation.
In this study, we investigated the role of the STAT3-WDR1 axis in neointima formation after vascular injury. JAK2/STAT3 was induced in the tunica media and intima, beginning at the first stage and peaking in the second and third stages. We found that levels of STAT3 and WDR1 increased synergistically in a certain period of time during arterial injury repair. Therefore, we hypothesized that STAT3 regulates the transcription of
We successfully combined
This work was supported by the National Natural Science Foundation of China (No. 31701266).
J.S.H. and B.Y.Y. conceived and designed the study. J.S.H., S.J.P., M.R.X., X.H., Z.Y.L., and R.A. performed the experiments. J.S.H. wrote the paper. J.S.H., B.Y.Y., S.J.P., M.R.X., and T.C.Z. reviewed and edited the manuscript. All authors read and approved the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(8): 749-762
Published online August 31, 2020 https://doi.org/10.14348/molcells.2020.0085
Copyright © The Korean Society for Molecular and Cellular Biology.
JiSheng Hu1,2 , ShangJing Pi1,2
, MingRui Xiong1
, ZhongYing Liu1
, Xia Huang1
, Ran An1
, TongCun Zhang1,*
, and BaiYin Yuan1, *
1Institute of Biology and Medicine, College of Life Science and Health, Wuhan University of Science and Technology, Hubei 430081, China, 2These authors contributed equally to this work.
Correspondence to:yuanby@wust.edu.cn (BYY); zhangtongcun@wust.edu.cn (TCZ)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The migration, dedifferentiation, and proliferation of vascular smooth muscle cells (VSMCs) are responsible for intimal hyperplasia, but the mechanism of this process has not been elucidated. WD repeat domain 1 (WDR1) promotes actin-depolymerizing factor (ADF)/cofilin-mediated depolymerization of actin filaments (F-actin). The role of WDR1 in neointima formation and progression is still unknown. A model of intimal thickening was constructed by ligating the left common carotid artery in Wdr1 deletion mice, and H&E staining showed that Wdr1 deficiency significantly inhibits neointima formation. We also report that STAT3 promotes the proliferation and migration of VSMCs by directly promoting WDR1 transcription. Mechanistically, we clarified that WDR1 promotes the proliferation and migration of VSMCs and neointima formation is regulated by the activation of the JAK2/STAT3/WDR1 axis.
Keywords: migration, neointima formation, proliferation, vascular smooth muscle cells, WD repeat domain 1
Vascular smooth muscle cells (VSMCs) are important participants in vascular pathological processes and are the predominant cell type in the arterial wall (Gomez and Owens, 2012; Ni et al., 2019; Owens et al., 2004). Proliferation and migration of VSMCs are key events in intimal hyperplasia (Allahverdian et al., 2018); these events are also prerequisites for diseases such as arteriosclerosis and restenosis after angioplasty (O'Brien et al., 2011; Xie et al., 2017). Under normal physiological conditions, VSMCs in the tunica media remain in a stationary state (Xie et al., 2017). In response to stimuli such as hypoxia and injury, VSMCs undergo a phenotypic transformation and proliferate (Intengan and Schiffrin, 2000; Xie et al., 2011). VSMCs also secrete extracellular matrix components, resulting in the intimal hyperplasia and vascular lumen stenosis that comprise neointima formation (Dzau et al., 2002; Jonasson et al., 1988; Marx and Marks, 2001; Weintraub, 2007).
Under physiological conditions, assembly and depolymerization of actin filaments coincide and maintain a balance, which is known as actin dynamics (Yuan et al., 2018). Cell motility and growth are inseparable from actin dynamics, and this process is regulated by various actin-binding proteins (Cooper and Schafer, 2000). Many factors are involved in depolymerization, such as actin-depolymerizing factor (ADF)/cofilin and WD repeat domain 1 (WDR1) (Bamburg, 1999; Cooper and Schafer, 2000). ADF/cofilin plays a crucial role in actin cleavage and depolymerization (Bravo-Cordero et al., 2013). WDR1 is a major cofactor for ADF/cofilin and promotes ADF/cofilin-mediated depolymerization of actin filaments (F-actin), thereby regulating the balance between actin depolymerization and assembly (Lee and Dominguez, 2010; Xu et al., 2015). WDR1-regulated actin dynamics directly affect cellular processes such as migration, cell-cell junction maintenance, and proliferation (Collazo et al., 2014; Lee et al., 2016). High expression of
Signal transducer and activator of transcription 3 (STAT3) is an important component of the Janus kinase (JAK/STAT) signaling pathway (Qi et al., 2016), which is involved in inflammation, as well as various cellular processes such as division, proliferation, drug resistance, and apoptosis (Fu et al., 2012; Hirano et al., 2000; Jackson and Ceresa, 2017; Limagne et al., 2017; Tripathi et al., 2017; Zulkifli et al., 2017). Binding of an extracellular signaling molecule (e.g., interleukin-6 [IL-6]) to its corresponding receptor on the cell surface induces the phosphorylation of JAK and its downstream transcription factor STATs (e.g., the IL-6/JAK/STAT3 pathway) (Chang et al., 2013; Johnson et al., 2018). Phosphorylated STAT3 forms homologous or heterologous dimers and then translocates to the nucleus (Souissi et al., 2011). STAT3 regulates the expression of downstream genes that are associated with growth, drug resistance, and inflammation, such as STK35, AKT2, and COX-2 (Kou et al., 2011; Qi et al., 2016; Wu et al., 2018). Studies have shown that STAT3 is crucial to neointima formation (Kovacic et al., 2010; Seki et al., 2000). However, the relationship between STAT3 and WDR1 and transcriptional regulation during vascular injury repair is unclear.
In this study, we propose a mechanism by which WDR1 regulates the proliferation of VSMCs and pathological intimal thickening. We demonstrated that after vascular injury, WDR1 promotes VSMC proliferation and migration, thereby promoting intimal thickening. After
C57/BL6 male mice (7- to 8-weeks old) were obtained from the Model Animal Research Center of Nanjing University (Yuan et al., 2014).
Primary cultures of murine artery smooth muscle cells (MASMCs) were prepared from arteries. Briefly, aortas were isolated from 8 animals (
Human aortic smooth muscle cells (HASMCs; National Infrastructure of Cell Line Resource, China) were grown in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C in a 5% CO2 incubator. Cells were trypsinized at 80% to 90% confluence for experiments. HASMCs were seeded into 6-well plates, and when the cells reached 60% confluence, angiotensin II (Ang II, 200 nM; Sigma-Aldrich) and PDGF-BB (20 ng/ml; R&D Systems, USA) were added to the medium, and cells were collected at different times after treatment. Some HAVSMC cultures were stimulated with serum. For these cultures, cells were incubated in serum-free DMEM for 12 h and then treated with 10% fetal bovine serum. Cells were collected at various times after treatment.
Small interfering RNA (siRNA) duplexes for silencing
RNA was extracted from HASMCs using TRIzol (Sigma-Aldrich). M-MLV reverse transcriptase (Vazyme Biotech, China) was used to synthesize cDNA. Real-time polymerase chain reaction (PCR) (Real-Time PCR Detection Systems; Bio-Rad, USA) was performed to assess the relative levels of mRNAs. Relative mRNA levels were normalized internally to GAPDH mRNA. Related primers are as follows: GAPDH (F): 5’-CATGTACGTTGCTATCCAGGC-3’; GAPDH (R): 5’-CTCCTTAATGTCACGCACGAT-3’; WDR1 (F): 5’-TTGTCAACTGTGTGCGATTCTC-3’; and WDR1 (R): 5’-GCTGTCGGGACTCCA ACTAA-3’.
Proteins were extracted from HASMCs, MASMCs, and murine artery tissues. Western blotting was performed as described previously (Hu et al., 2018). Primary antibodies included rabbit anti-WDR1 (cat No. 13676-1-AP, 1:1,000; Proteintech, USA), rabbit anti-STAT3 (cat No. 10253-2-AP, 1:1,000; Proteintech), rabbit anti-p-STAT3Tyr705 (cat No. 9415, 1:1,000; Cell Signaling Technology, USA), β-actin (cat No. AP0060, 1:5,000; Bioworld Technology, USA), rabbit anti-JAK2 (cat No. sc390539, 1:500; Santa Cruz Biotechnology, USA), and p-JAK2Tyr1007/1008 (cat No. ab32102, 1:1,000; Abcam, UK). The membrane was washed using TBS-T and incubated with secondary antibody conjugated to horseradish peroxidase in 5% nonfat milk for 1 h at room temperature. Detection was carried out using a chemiluminescence detection kit (cat No.K-12045-D50; Advansta, USA).
Arterial tissue samples were first washed with cold PBS and then fixed with 4% paraformaldehyde at 4°C. The samples were processed by successive incubation in (1) PBS for 20 min; (2) an ethanol series (70%, 85%, 95%, 95%, 100%, and 100%) for 1 h at room temperature; (3) butyl alcohol, three times for 30 min each at room temperature; and (4) fresh paraffin at 65°C, three times for 30 min each. The treated samples were embedded in paraffin, and 5-μm-thick sections were prepared. After the sections were dewaxed and rehydrated, immunofluorescence was performed according to standard protocols. Briefly, sections were incubated with mouse anti-α-SMA (cat No. ab32575, 1:200; Abcam) or rabbit anti-PH3 (cat No. 53348; Cell Signaling Technology) overnight at 4°C. After being washed with PBS three times, sections were incubated with Alexa Fluor 647-conjugated anti-mouse IgG (cat No. 4410S; Cell Signaling Technology) or Alexa Fluor 488-conjugated anti-rabbit IgG (cat No. 4416S; Cell Signaling Technology) for 30 min at room temperature. Slices were then stained with DAPI (1.0 mg/ml; Invitrogen) for 30 min before mounting. Images were captured with an Olympus confocal microscope (Olympus, Japan).
For immunohistochemistry (IHC), samples were prepared as described above, and sections were incubated with anti-WDR1 primary antibody and detected according to the IHC kit instructions (cat No. KIT-9707; Maixin Biotechnology, China). H&E staining was performed using standard protocols. H&E-stained arterial sections were analyzed by planimetry with ImageJ software.
Cell counting kit 8 (CCK-8) (cat No. A311-01; Vazyme Biotech) was used to detect cell proliferation between 0 h and 72 h according to the manufacturer’s manual. Briefly, after tamoxifen-induced
MASMCs were seeded into 6-well plates for two days after induction with tamoxifen. After serum starvation for 24 h, a scratch was made using a sterile 200 μl pipette tip across each well, creating a cell-free area, based on the technique described by Yuan et al., and then 10 vol% fresh medium was added to the culture. Images were captured with a microscope at 0 h and 12 h. One day after transfection with siRNA, HASMCs were serum-starved for 12 h, and a scratch was made as described above.
The Phanta Super-Fidelity DNA Polymerase (cat No. P515-01; Vazyme Biotech) was used to perform a PCR to clone the murine
The
Similarly,
The integrity and orientation of each insert were confirmed by sequencing.
ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (cat No. 9002S; Cell Signaling Technology). MASMCs were treated with IL-6 (cat No. I9646; Sigma-Aldrich) for 30 min, and the cell lysate was used for immunoprecipitation with an antibody specific for the phosphorylated STAT3 (pSTAT3) protein (cat No. 9415; Cell Signaling Technology). PCR was performed on purified DNA from each sample using specific primers. The primer sequences used are listed below: F: 5’-TTACCCCAGCAGTCTTTGTACCAT-3’ and R: 5’-AGCACTGTCCTAGGGATGTGCTAG-3’.
MASMCs were synchronized and induced with tamoxifen for two days as described above. Cells were fixed overnight in 75% ethanol at 4°C and stained for 10 min at room temperature with propidium iodide (50 mg/ml) and analyzed with a FACSCanto flow cytometer (BD Biosciences, USA). Cell-cycle detection was carried out by propidium iodide staining and fluorescence-activated cell sorting analysis. Each experiment was performed in triplicate.
Mouse carotid arteries were harvested at various times post-injury, and the proteins were extracted with lysis buffer (50 mM Tris-HCl, pH 6.8; 10% glycerol; 1% SDS). Extracted proteins (10 mg) were loaded on 10% SDS-PAGE gels containing 1% gelatin to detect gelatinase activity. After washing in 2.5% Triton X-100, the gels were incubated overnight in buffer (10 mM CaCl2, 0.01% NaN3, and 50 mM Tris-HCl, pH 7.5). The obtained gels were stained with 0.2% Coomassie blue R-250 (Sigma-Aldrich) for 2 h and then destained with 10% acetic acid and 40% methanol.
Comparisons between two groups were performed using unpaired two-tailed Student’s
To investigate the effect of WDR1 on intimal thickening, we constructed a model of vascular injury in mice, and WDR1 levels were measured by western blot on different days post-injury (Figs. 1A and 1B). Furthermore, a gelatin zymography assay indicated that MMP9 activity in the ligation group was significantly increased compared with the sham group, indicating that injury facilitates SMC migration (Fig. 1C). To determine the localization and expression pattern of WDR1 in blood vessels, we performed immunohistochemical staining (Fig. 1D). WDR1 is primarily localized in the smooth muscle cells of blood vessels (Fig. 1D), and its level gradually increases as the number of days post-injury increases. These results suggest that WDR1 plays a key role in the repair of vascular injury.
To investigate the potential effects of WDR1 in vascular biology and pathophysiology, we used the murine model of vascular injury for
The response of WDR1 to various stimuli associated with vascular injury was investigated
Although WDR1 levels are increasing during neointima formation, it is unclear how WDR1 affects the process. Therefore, we performed CAL in
Knockdown of
We investigated STAT3 activation by western blot in carotid artery tissue after ligation (Fig. 5A). After the injury, the levels of pSTAT3 (activated STAT3) increase and reach the highest level at 7 days post-ligation. The levels of WDR1 display a trend similar to those of pSTAT3 (Fig. 5A). These results suggest that STAT3 may regulate WDR1 to affect neointimal thickening. To test this hypothesis, we exposed cells to IL-6 and Ang II to simulate the release of cytokines following vascular injury. The proliferation and migration of SMCs are improved in response to IL-6 and Ang II (data not shown). As expected, pSTAT3 and WDR1 are elevated. In addition, we silenced
To study the interaction between WDR1 and STAT3, we used siRNA to decrease
Based on the results obtained above, we hypothesized that STAT3 binds directly to the
WDR1-mediated SMCs migration and proliferation require the activation of STAT3. To elucidate additional details about this result, we conducted
Intimal thickening caused by abnormal proliferation of SMCs is associated with a variety of diseases (Xie et al., 2017). The proliferation and migration of SMCs caused by endogenous and exogenous factors are crucial steps in the repair of vascular injury (O'Brien et al., 2011; Zhang et al., 2014). However, our understanding of the underlying mechanisms for these responses in SMCs is poor.
WDR1 acts as a cofactor of cofilin for actin depolymerization and plays a vital role in cellular processes, such as proliferation and migration (Yuan et al., 2018). However, the function of WDR1 in SMCs is not understood. Therefore, we constructed
The process of vascular remodeling after vascular injury is divided into four stages (Schwartz et al., 1995; Seki et al., 2000). The first stage occurs within a few days of injury when the proliferation of the medial VSMCs is maximal. The second stage involves the migration of SMCs to the intima, starting between day 4 and 5. The third stage involves the continuous proliferation of SMCs in the neointima. During the fourth stage, extracellular matrix components are secreted and deposited. Inflammation, cytokine production, and VSMC proliferation are key aspects of vascular repair, and inflammation, and cytokines are active in the first phase of vascular remodeling. Unfortunately, endometrial remodeling elicits a series of negative results. Decreased lumen diameter causes thrombosis, which in turn leads to serious cardiovascular diseases such as myocardial and cerebral infarctions.
JAK and STAT proteins transmit intracellular signals in a variety of cell types in response to various cytokines (Li et al., 2019). Previous reports have shown that JAK2/STAT3 is responsive to angiotensin II type 1 receptor and is activated after tyrosine phosphorylation in rat smooth muscle cells (Marrero et al., 1997; Seki et al., 2000; Watanabe et al., 2004). Activated STAT3 translocates to the nucleus after homologous or heterologous dimer formation and binds to cis-inducing elements in the nucleus, leading to transcriptional activation of early growth response genes (Li et al., 2019). When SMCs were treated with the JAK2-specific inhibitor AG490, proliferation was significantly inhibited, suggesting that the JAK2/STAT3 pathway is vital to smooth muscle cell proliferation.
In this study, we investigated the role of the STAT3-WDR1 axis in neointima formation after vascular injury. JAK2/STAT3 was induced in the tunica media and intima, beginning at the first stage and peaking in the second and third stages. We found that levels of STAT3 and WDR1 increased synergistically in a certain period of time during arterial injury repair. Therefore, we hypothesized that STAT3 regulates the transcription of
We successfully combined
This work was supported by the National Natural Science Foundation of China (No. 31701266).
J.S.H. and B.Y.Y. conceived and designed the study. J.S.H., S.J.P., M.R.X., X.H., Z.Y.L., and R.A. performed the experiments. J.S.H. wrote the paper. J.S.H., B.Y.Y., S.J.P., M.R.X., and T.C.Z. reviewed and edited the manuscript. All authors read and approved the manuscript.
The authors have no potential conflicts of interest to disclose.
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