Oxidative stress-induced cellular senescence desensitizes cell growth and migration of vascular smooth muscle cells through down-regulation of platelet-derived growth factor receptor-beta

Cobalt dichloride induced VSMC senescence

HIF-1α, a critical transcription factor involved in hypoxia-mediated gene expression, was markedly upregulated at an early time point (6 hr) after treatment with several concentrations (0, 150, and 300 μM) of cobalt dichloride. An increased level of HIF-1α could still be detected at 72 hr post-treatment (Figure 1A). To identify the production of cellular senescence, senescence-associated β-galactosidase (SA-β-gal) activity within cells was detected by cytochemical staining (Figure 1B). Experimental results revealed that 72 hr of treatment with cobalt dichloride dose-dependently induced blue-green staining of a portion of cells, a representative feature of senescent cells. This result suggested that cell senescence was promoted under incubation condition with 300 μM CoCl₂ for 72 hr.

Figure 1. Identification of cellular senescence. A10 cells were incubated with various doses of cobalt dichloride (CoCl₂) for 6 and 72 hr. The protein level of hypoxia-inducible factor (HIF)-1α was analyzed using Western blotting (A). Senescent cells were detected by cytochemical staining of SA-β-gal activity which appeared as a blue-green color (B). * p < 0.05 compared to the group without CoCl₂ treatment.

Cell proliferation and migration were reduced in senescent VSMCs

To understand the influence of CoCl₂-induced cellular senescence on VSMC proliferation and migration, both normal and senescent cells were incubated with PDGF-BB for 24 hr. Data from the cell growth analysis suggested that the growth capacity of senescent cells had obviously decreased compared to normal cells (Figure 2A). Likewise, cell migration of senescent VSMCs was insensitive to PDGF-BB-mediated stimulation according to data from the wound-healing assay (Figure 2B).

Figure 2. Influence of cellular senescence on the proliferative and migratory capacities of A10 cells. Senescence was produced in cells by a 72-hr incubation with 300 μM CoCl₂. After that, platelet-derived growth factor (PDGF)-BB-stimulated cell proliferation and migration were respectively analyzed by an MTT assay (A) and wound-healing analysis (B). * p < 0.05 compared to the normal cell group.

Expression and activation levels of PDGFR-β and its downstream signaling molecules were suppressed in senescent VSMCs

Regulations of the PDGFR-β gene and protein were analyzed in VSMCs treated with various concentrations (0, 150, and 300 μM) of CoCl₂ for 72 hr. Our data indicated that CoCl₂ treatment diminished gene and protein expressions of PDGFR-β in a dose-dependent manner (Figure 3A, 3B). Moreover, the phenomenon of PDGFR-β activation (phosphorylation) induced by 25 ng/ml of PDGF-BB was also obviously attenuated in VSMCs treated with 300 μM CoCl₂, the repression of which was very similar to the inhibitory effect of AG-1295, a PDGFR blocker (Figure 3B). Likewise, activation levels of downstream signaling molecules of PDGFR-β, such as AKT, mTOR, and ERK1/2, were increased after PDGF-BB incubation, which was reversed by adding 25 μM of AG-1295 to normal cells. However, the PDGF-stimulated increases in the activations of AKT, mTOR, and ERK1/2 proteins were obviously reduced in cells incubated with 300 μM CoCl₂ for 72 hr (Figure 3C).

Figure 3. Changes in platelet-derived growth factor (PDGF) receptor (PDGFR)-β-mediated pathways in senescent vascular smooth muscle cells (VSMCs). Cells were treated with different concentrations of CoCl₂ for 72 hr. Expression and activation levels of PDGFR-β were analyzed using an RT-PCR (A) and Western blotting (B), respectively. The phosphorylation levels of the downstream signaling molecules of PDGFR-β were examined after 15 min of stimulation with 25 ng/ml PDGF-BB under serum-free condition (C). * p < 0.05, ** p < 0.01 compared to the group treated with 10% FBS or PDGF-BB alone.

Promoter activity of PDGFR-β was reduced in senescent VSMCs

To further understand the mechanism of downregulation of the PDGFR-β gene in senescent cells, several lengths (0.5, 1.0, 1.5, and 2.0 kb) of PDGFR-β promoter segments were cloned into a luciferase-based reporter vector, pGL4.10[Luc2], to measure their individual promoter activities (Figure 4A, 4B). The analyzed data indicated that the proximal segment ranging -523 to -1 of the PDGFR-β promoter possessed the highest promoter activity among all promoter segments (Figure 4C). Therefore, the constructed reporter vector with the proximal segment (-523 to -1) of the PDGFR-β promoter was further transfected into both normal and senescent cells, and the experimental data demonstrated that promoter activity was significantly reduced in senescent cells compared to that of normal cells (Figure 4D).

Figure 4. Promoter deletion assay of platelet-derived growth factor receptor (PDGFR)-β. Four lengths of the PDGFR-β promoter (A) were individually constructed in a luciferase-based reporter vector to produce four pGL4.10[Luc2]-rPDGFR-β vectors (B). Individual promoter activities were measured in normal cells at 24 hr after transfection with the different pGL4.10[Luc2]-rPDGFR-β plasmids (C). Differences in promoter activities between normal and senescent cells with the PDGFR-β segment (0.5 kb) were measured (D). The transcription start site was defined as +1. ** p < 0.01 compared to the group transfected with the reporter vector with the proximal promoter (0.5 kb) of PDGFR-β. ^‡p < 0.01 compared to the normal cell group.

TF-binding profile analysis of the rat PDGFR-β promoter

To evaluate differences in the binding profiles of 96 TFs on the proximal segment of the PDGFR-β promoter between normal and senescent cells, nuclear extracts isolated from normal or senescent cells were incubated with the proximal segment of the PDGFR-β promoter and a fluorescence-labeled competitive oligonucleotide probe to identify possible TF-DNA interactions using a commercial TF-DNA-binding assay kit. Our results showed that binding levels of 18 TFs obviously decreased (>2-fold difference) in senescent cells compared to those of normal cells (Table 1). Of these, binding levels of four TFs, including NFAT, SATB1, FOXD3, and PPAR, were more than 10-fold lower in senescent cells than that in normal cells.

Bioinformatics prediction of potential TFBSs on the PDGFR-β promoter

To investigate whether these 18 TFs can possibly interact with the proximal segment (-523 to -1) of the PDGFR-β promoter, potential binding sites of these TFs were analyzed and predicted by two TRANSFAC^®-based bioinformatics on-line software programs (PROMO and MatInspector). According to the prediction results, putative binding sites of the TFs, including NFAT, SATB1, FOXD3, PPAR, TFIID, NRF1, Stat5, GR/PR, USF-1, and Pbx1, were predicted to be within the proximal segment (-523 to -1) of the PDGFR-β promoter (Figure 5).

Figure 5. Bioinformatics prediction of putative transcription factor-binding sites (TFBSs) on the platelet-derived growth factor receptor (PDGFR)-β promoter. The putative TFBSs were analyzed on the PDGFR-β promoter region (-529 to -1). The sequence of the PDGFR-β promoter is presented as white text on a black background. The locations of the predicted TFBS are labeled below the PDGFR-β promoter sequence. Transcriptional start site was defined as the +1 position.

Gene expressions of candidate TFs in senescent VSMCs

To clarify whether decreases in TF-binding levels could be attributed to reductions in TF expressions in senescent cells, transcriptional levels of these ten TFs were further measured using a quantitative polymerase chain reaction (qPCR). The experimental data showed that gene expressions of seven TFs, viz., NFATc4, SATB1, PPAR-α, TFIID, PR, STAT5, and Pbx1, were markedly reduced (>2-fold change) in senescent cells compared to those in normal cells (Figure 6).

Figure 6. Quantifications of gene levels of candidate transcription factors. Transcriptional expressions of candidate transcription factors in normal and senescent cells were analyzed by a real-time PCR. Gene expression is shown as the log2 (multiple of change) of the transcription factors after senescence induction.

Analysis of the relationship between age and in-stent restenosis

To understand possible relationships between age and in-stent restenosis, clinical records of coronary artery disease patients with vascular stenting were collected and divided into younger (<45 years old; n=8) and older (>60 years old; n=36) groups. The data indicated that proportions of restenosis were 75.0% and 55.6% in the younger and older groups, respectively (Table 2). Moreover, younger men with vascular stenting had a relatively higher proportion of restenosis compared to that in older men (75.0% vs. 60.0%). In the older group, the proportion of restenosis in men was relatively higher than that in women (60.0% vs. 45.5%). However, Pearson’s correlation test showed that no significant correlation (r = -0.057, p = 0.725) existed between age and the vascular status (with or without restenosis) in overall patients who had been followed-up 6 months after vascular stenting. Similarly, there was also no statistical correlation when analyzing different gender groups (r = -0.064, p = 0.735 for males; r = 0.176, p = 0.606 for females).

Chemicals and reagents

Dulbecco’s modified Eagle medium (DMEM; #12800-017) was purchased from Gibco (Rockville, MD, USA). Hyclone™ Antibiotic Antimycotic solution (#SV30079.01) and fetal bovine serum (FBS; FetalClone III™, #SH30109.03) were procured from GE Healthcare Life Sciences (Chicago, IL, USA). Primary antibodies against phospho-AKT (#GTX50128), PDGFR-β (#GTX61115), phospho-PDGFR-β (#GTX61797), and HIF-1α (#GTX127309), as well as horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse (#GTX213112-01) and rabbit (#GTX213110-01) immunoglobulin G (IgG) were purchased from GeneTex (Irvine, CA, USA). Antibodies detecting β-actin (#66009-1-Ig), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2; #05-797R), and phospho-mammalian target of rapamycin (mTOR; #ab109268) were respectively obtained from Proteintech (Rosemont, IL, USA), Millipore (Bedford, MA, USA), and Abcam (Cambridge, MA, USA). Recombinant human PDGF-BB (#100-14B) and AG-1295 (#14529), a PDGFR-β inhibitor, were respectively bought from PeproTech (Rocky Hill, NJ, USA) and Cayman Chemical (Ann Arbor, MI, USA). Cobalt dichloride (#C8661) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

A10 cells (#60082), a cell line of rat thoracic aorta smooth muscle, were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and maintained in DMEM supplemented with 10% (v/v) FBS and a 100x-diluted antibiotic antimycotic solution. Cells were kept at 37°C in a humidified incubator with 5% (v/v) CO₂ and 95% (v/v) air. The culture medium was refreshed every 2~3 days.

Induction and identification of cellular senescence

Cellular senescence of A10 cells was induced by 72 hr of incubation with cobalt dichloride at a concentration of 150 or 300 μM, and was identified by detecting intracellular senescence-associated beta-galactosidase (SA-β-gal) activity according to previous reports [57, 58]. Briefly, cells were washed twice with 1× phosphate-buffered saline (PBS), fixed with a 2% (v/v) glutaraldehyde-PBS solution for 5 min at room temperature, washed three times in PBS for 1 min each, and then stained with a staining solution containing 40 mM of citric acid/Na phosphate buffer (pH 6.0), 5 mM of K₄[Fe(CN)₆]·3H₂O, 5 mM K₃[Fe(CN)₆], 150 mM sodium chloride, 2 mM magnesium chloride, and 1 mg/mL X-gal. After overnight incubation at 37°C, cells were washed twice with PBS for 30 s each, rinsed with methanol, and air-dried. Subsequently, cells were photographed using ToupView™ image acquisition software (ToupTek Photonics, Zhejiang, China) at 40× magnification under an inverted microscope (#Eclipse TS100; Nikon, Melville, NY, USA) equipped with a digital camera (#E3ISPM06300KPA-IP106300A; Suzhou Vision Photonics, Jiangsu, China).

Cell growth analysis by a 3-[4,5-dimethyl thiazol-2-yl]- 2,5-diphenyl tetrazolium bromide (MTT) assay

These experiments were carried out according to our previous study [59]. Briefly, cells (10⁴ cells/well) were seeded on a 96-well plate for 24 hr of adaptation. After that, the culture medium was refreshed with serum-free medium with or without 25 ng/mL PDGF-BB. After 24 hr of incubation, cells were treated with 5 mg/mL of MTT for 2 hr. Subsequently, cells were washed with PBS, and then 100 μL of dimethyl sulfoxide (DMSO) was added to each well. Absorbance values at 570 nm were determined for each well using 650 nm as the reference wavelength. Cell growth is presented as the ratio (multiple of change) of the absorbance of cells treated with PDGF-BB to that without PDGF-BB treatment.

Wound-healing assay

This experimental procedure was according to a method described in our previous study [60]. Briefly, cells were seeded on 12-well plates (5 × 10⁵ cells/well). After 24 hr of serum-free starvation, a pipette tip was used to create an original cell-free region on the confluent cell monolayer, which was then photographed at 0 hr under a microscope at 40× magnification. After that, cells were treated with 6 ng/mL of PDGF-BB. At 16 hr after treatment, the cell number within the original cell-free region was counted on photographic images using the particle analysis function of Image J analytical software (National Institutes of Health (NIH), Bethesda, MD, USA) according to the official web-based manual (https://imagej.net/Particle_Analysis). The extent of cell migration is shown as the multiple of change of migrating cell numbers compared to the normal cell group.

Immunoblotting

Immunoblot experiments were carried out as described previously [59]. Harvested cells were lysed using lysis buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% (w/v) sodium dodecylsulfate (SDS), 5 mM ethylenediaminetetraacetic acid (EDTA; pH 8.0), 1 mM dithiothreitol (DTT), 1% (v/v) Triton X-100, 20% (v/v) glycerol, a proteinase inhibitor cocktail (#4693159001; cOmplete™; Roche, Basel, Switzerland), and a phosphatase inhibitor cocktail (#04906837001; PhosSTOP™; Roche). Cell lysates were further centrifuged at 13,000 ×g and 4 °C for 10 min to collect the supernatants for SDS-polyacrylamide gel electrophoresis (PAGE). Protein concentrations were measured with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Aliquots containing 30 μg of protein were electrophoresed using 10% slab SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Immun-bot^®; Bio-Rad). After blocking non-specific binding sites with 5% (w/v) non-fat milk at room temperature for 1 h, the membrane was incubated with primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies at 4°C for 3 hr. Substrates were visualized using a T-Pro LumiLong Plus Chemiluminescent Substrate Kit (T-Pro Biotechnology, New Taipei City, Taiwan). The luminescence signal was acquired by the Azure C300 imaging System (Azure Biosystems, Dublin, CA, USA) and quantified using AzureSpot software (v14.0; Azure Biosystems). Results for each experiment were normalized to the band density of β-actin. The relative protein expression of the group without CoCl₂ treatment was defined as 100%.

Analysis of gene expressions

The procedures examining gene expressions were conducted according to our previous study [61]. Briefly, 1 mL of 3-Zol™ reagent (#2001; MDBio, Taipei, Taiwan) was added to harvested cells, which were then vortexed for 30 s and incubated on ice for 5 min. After that, 0.2 mL of chloroform was added to the cell lysate, vortex-mixed for 15 s, and incubated at room temperature for 3 min. After centrifugation at 12,000 ×g for 15 min, the aqueous phase was transferred to a clean tube, precipitated with 0.5 mL of isopropanol, and centrifuged at 12,000 ×g for 15 min. The pellet was then washed with 1 ml of 75% (v/v) cold ethanol prepared with 0.1% (v/v) diethyl pyrocarbonate (DEPC)-treated water, centrifuged at 12,000 ×g at 4°C for 15 min, dried for 20 min at room temperature, re-suspended in 50 μL DEPC-treated water, and stored at -80°C.

Complementary (c)DNA synthesis was carried out using the ReverTra Ace set (#PU-TRT-200; TOYOBO, Osaka, Japan) according to the manufacturer’s manual. Briefly, 1 μg of total RNA was supplemented in a total reaction volume of 20 μL with 1× reverse-transcription buffer, 1 mM dNTPs, 0.5 nM oligo(dT)20, 0.5 units of an RNase inhibitor, and 5 units of ReverTra Ace (reverse transcriptase). After incubation for 20 min at 42°C, the mixture was incubated for 5 min at 99°C to denature the products. Finally, the cDNA product was stored at -80°C.

A traditional polymerase chain reaction (PCR) was conducted using the OnePCR™ HotStar system (#SM206-0100; GeneDireX, Miaoli, Taiwan) in a thermocycler (Labcycler™; SensoQuest, Gottingen, Germany). Briefly, the reaction mixture contained 1 μg cDNA, 0.2 μM primer (Table 3), and 25 μL of the OnePCR™ HotStar reagent in a total volume of 50 μL. After hot-start activation for 5 min at 94°C, 20~35 cycles were carried out, each consisting of 30 s at 94°C, 1 min at 60°C, and 2 min at 72 °C. PCR products were electrophoresed on a 2% agarose gel in 0.5× Tris-borate-EDTA (TBE) running buffer at 100 V for 1 h. DNA bands were visualized with a fluorescent dye (Novel Juice™; GeneDireX, Taichung, Taiwan). The band signals were acquired and quantified with the Azure C300 imaging System and AzureSpot software (Azure Biosystems), respectively. The band intensity of PDGFR-β was normalized to that of the internal control (GAPDH), and the relative gene expression of the group without CoCl₂ treatment was defined as 100%.

A real-time PCR was performed using the Smart Quant Green Master Mix with dUTP and ROX (#SA-SQGR-V2-1ml; Protech, Taipei, Taiwan) in an ABI Prism 7300 sequence detector (Applied Biosystems, Foster City, CA, USA) according to the procedure of our previous study with minor modifications [61]. The reaction mixture contained 1 μg cDNA, 2 μL of each primer (10 μM), and 10 μL of the Master Mix in a total volume of 20 μL. After hot-start activation for 15 min at 95°C, 40 cycles were carried out, each consisting of 15 s at 95°C, 15 s at 59°C, and 30 s at 72°C. The primer pairs used for the real-time PCR are listed in Table 3. The relative transcript expression was calculated using the equation 2^–ΔΔCt, and results are presented as multiples of change relative to the control group.

Plasmid construction and promoter activity assay

The sequence of the potential promoter region (ranging -2001 to -1) of the rat PDGFR-β was identified by aligning sequences of PDGFR-β messenger (m)RNA (#XM_006254789) and a chromosome 18 fragment (#NC_005117) obtained from the GenBank genetic sequence database. Four promoter segments of different lengths, of 2001, 1526, 1021, and 523 bp, were individually amplified by a PCR using appropriate primer pairs with restriction enzyme recognition sequences (Table 3). Amplified promoter segments were further cloned into the pGL4.10[Luc2] plasmid (#E6651; Promega, Madison, WI, USA) using digestion of the restriction enzymes, XhoI (#R6161; Promega) and BglII (#R6081; Promega), to construct four complete reporter plasmids, pGL4.10[Luc2]-rPDGFR-β, with different PDGFR-β promoter regions. The promoter sequence within the recombinant plasmids was verified with Sanger sequencing conducted by Mission Biotech (Taipei, Taiwan).

Cells seeded on 6-well plates (at ca. 80% cell confluence) were transfected with the four individual pGL4.10[Luc2]-rPDGFR-β plasmids using the TransIT^®-LT1 transfection reagent (#2300; Mirus Bio LLC, Madison, WI, USA) according to the procedure in the manufacturer’s manual. In brief, 2.5 μg of plasmid DNA was mixed with 250 μL serum-free culture medium and then mixed with 7.5 μL of transfection reagent for a 30-min incubation at room temperature to form a stable complex of plasmid DNA/transfection reagent. After that, medium within the wells was refreshed with 2.5 mL of culture medium, and the mixture of plasmid DNA/transfection reagent complex was added drop-wise to different areas of the wells. After a 24-h incubation for cell transfection, PDGFR-β promoter activity was measured with a commercial luciferase assay system kit (#E4030; Promega) in accordance with the manufacturer’s instructions. Briefly, cells were washed twice with PBS, and then 200 μL of 1× Reporter lysis buffer was added to each well. After a 15-min incubation at -20°C, cell lysates were harvested, vortexed for 15 s, and then centrifuged at 13,000 rpm for 5 min at 4°C. After that, 20 μL of supernatant was mixed with 100 μL of luciferase assay reagent. The luminescent signal (or relative light units; RLU) of cell supernatants was detected with a luminometer (Varioskan™ Flash; Thermo Scientific, Waltham, MA, USA) with the following parameters: a 2-s measurement delay followed by a 10-s measurement read. The RLU value was further normalized to the protein concentration from the same cell supernatant. Finally, the normalized RLU value of the pGL4.10[Luc2]-rPDGFR-β plasmid with the 0.5-kb promoter segment was defined as 100%.

Analysis of a TF-binding array

Nuclear proteins were extracted using the Nuclear Extraction Kit (#SK-0001; Signosis, Santa Clara, CA, USA) according to the manufacturer’s instructions. A rat PDGFR-β promoter segment (523 bp; -523 to -1) was amplified using a PCR, and PCR clean-up was conducted using a Plus DNA clean/Extraction kit (#DP034P; GMbiolab, Taichung, Taiwan). After that, binding profiles of 96 TFs on the rat’s PDGFR-β promoter segment were examined using Promoter-Binding TF Profiling Plate Array II (#FA-2002; Signosis). Briefly, 8 μg of the nuclear extract, 0.3 μM of the PDGFR-β promoter segment, and a competitive biotin-labeled TF oligo probe were well mixed and then incubated in the TF binding buffer mixed solution for 30 min at room temperature. Subsequently, the TF-bound oligo probes were separated using a membrane-based spin column (Isolation column) and then hybridized with complementary sequences labeled in different wells of a 96-well plate. After that, the captured oligo probes were detected with streptavidin-HRP and a chemiluminescent substrate. Luminescent signals expressed as relative light unit (RLU) were quantified with a luminometer, and RLU values of <200 were considered background. Final data are presented as the ratio of the RLU value in normal cells to that in senescent cells.

Bioinformatics prediction of binding motifs

The rat PDGFR-β promoter sequence (ranging -523 to -1) was analyzed using web-based bioinformatics tools (PROMO and MatInspector^®) for predicting putative TF-binding sites (TFBSs) defined in the TRANSFAC^® database [62–64]. In the promoter analysis with PROMO (ALGGEN, Barcelona, Spain), parameters were set up by restricting both factor species and site species as animals with a maximum matrix dissimilarity rate of ≤5%. In addition, putative TFBSs within this promoter segment were also analyzed by MatInspector (Genomatix AG, Munich, Germany) using default parameters.

Collection of clinical data

Clinical studies (#201405019 and #N201603042) were approved by the Taipei Medical University (TMU) Joint Institutional Review Board (TMU-JIRB) and were performed from June 2015 to June 2017. In total, 104 patients were recruited from TMU Hospital, Wan-Fang Hospital, and Shuang-Ho Hospital in Taiwan. Inclusion criteria were patients (≥30 years old) who had received vascular stenting, and patients suffering from diabetes, hepatitis, uremia, cancer, hemophilia, or autoimmune diseases were excluded. Restenosis was defined as >50% diameter stenosis within the vascular lesion with vascular stenting, and the vascular status (with or without restenosis) was diagnosed at the first follow-up visit (6 months after surgery). Finally, the medical records of 44 eligible patients were collected to analyze the relationship between age and vascular status (with or without restenosis). At the end of the clinical studies, 60 patients who had been diagnosed with diabetes, a common comorbidity of restenosis, or who had withdrawn from the study were excluded.

Statistical analysis

All data are presented as the mean ± standard deviation (SD). The experiments were run in triplicate. Statistical significance among multiple groups was evaluated by a one-way analysis of variance (ANOVA). Pearson's correlation test was used to analyze the correlation between age and vascular status (with or without restenosis) of patients who had been followed-up 6 months after vascular stenting. A value of p < 0.05 was regarded as statistically significant.