Mol Cell Biochem (2014) 388:75–84 DOI 10.1007/s11010-013-1900-7
DHEA inhibits vascular remodeling following arterial injury: a possible role in suppression of inflammation and oxidative stress derived from vascular smooth muscle cells Jiangbin Chen • Lin Xu • Congxin Huang
Received: 2 May 2013 / Accepted: 15 November 2013 / Published online: 28 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Vascular remodeling is characterized by the aggregation of vascular smooth muscle cells (VSMCs) in intima. Previous studies have demonstrated that dehydroepiandrosterone (DHEA), a steroid hormone, can reverse vascular remodeling. However, it is still far clear that whether and how DHEA participates in the modulation of VSMCs activation and vascular remodeling. VSMCs were obtained from the thoracic aorta of SD rats. Cell proliferation was evaluated by CCK-8 assay and BrdU assay. To measure VSMCs migration activity, a transwell chamber assay was performed. Quantitative real-time RT-PCR and western blot were used to explore the molecular mechanisms. ROS generation by VSMCs was measured by DCF fluorescence. NADPH oxidase activity and SOD activity were measured by the corresponding kits. NF-jB activity was detected by NF-jB luciferase reporter gene assay. A rat carotid artery balloon injury model was built to evaluate the neointimal formation, and plasma PGF2 was measured by ELISA. Our results showed that DHEA significantly inhibited VSMCs proliferation after angiotensin (Ang II) stimulation by down-regulation of NADPH oxidase activity and ERK1/2 phosphorylation. Ang II can increase IL-6 and MCP-1 expression, but DHEA reverses these changes via inhibiting p38-MAPK/NF-jB (p65) signaling pathway. DHEA has no significant effects on VSMCs phenotype transition, but can reduce the neointimal to media area ratio after balloon injury. DHEA can alleviate oxidative stress and inflammation in VSMCs via ERK1/2 and NF-jB
Jiangbin Chen and Lin Xu have contributed equally to this study. J. Chen L. Xu C. Huang (&) Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, People’s Republic of China e-mail: [email protected]
signaling pathway, but has no effect on VSMCs phenotype transition. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury in vivo. Taken together, DHEA might be a promising treatment for vascular injury under pathological condition. Keywords Dehydroepiandrosterone Vascular smooth muscle cells Oxidative stress Inflammation Carotid artery balloon injury
Introduction Endogenous androgens are considered as potential predictors of initiation and development of cardiovascular diseases [1, 2]. Dehydroepiandrosterone (DHEA), one of the most important androgenic hormones, is related to an increased risk of cardiovascular disease [3–6]. Several studies have shown that the serum DHEA level declined in patients with more cardiovascular risk factors and higher burdens of coronary diseases [4–6]. In this case, DHEA has been extensively applied to human for a variety of diseases due to its safety. Some clinic investigations and animal studies have demonstrated that DHEA was a promising therapeutic approach for cardiovascular diseases including atherosclerosis and pulmonary hypertension [7, 8]. Recently, several studies reported that no protective role was found after DHEA supplementation in occlusive vascular diseases [9, 10]. So the benefits of DHEA in clinical use are now challenged. Nowadays it is widely recognized that chronic inflammation and oxidative stress play important roles in the pathogenesis of occlusive vascular diseases [11, 12]. Clinical studies showed a strong correlation between proinflammatory biomarkers and oxidative stress markers in
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atherosclerosis and restenosis [13, 14]. Apart from above, it is a notable feature of vascular remodeling that VSMCs switch from contractile to synthetic phenotype during VSMCs aggregation. The majority of intimal VSMCs after vessel injury are derived from resident medial VSMCs, which undergo a phenotypic modulation and migrate into intima to proliferate and produce extracellular matrix [15, 16]. So all of the three, inflammation, oxidative stress, and phenotypic switch, participate in the vascular remodeling in vascular diseases, but their significance may be different under various conditions. Until now, there is no single study to explore the exact mechanism underlying DHEA reverses vascular remodeling. In present study, we want to illustrate whether if DHEA inhibits inflammation, oxidative stress derived from VSMCs and whether it has the ability to reverse VSMCs phenotype transition after vascular injury. Our study will highlight the potential therapeutic role of DHEA by effectively inhibiting neointimal hyperplasia in vascular diseases.
Methods Primary VSMC culture and DHEA treatment VSMCs were isolated from the thoracic aorta of male SD rats (100–150 g) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing 10 % fetal bovine serum (FBS, Hyclone) according to previous studies [17, 18]. The purity of VSMCs was approximately 90 % assessed by cell immunostaining with anti-smooth muscle a-actin antibody. Only VSMCs between the 3rd and 5th passages were used for experiments. Before treatment, VSMCs were cultured for 24 h in DMEM with 0.5 % FBS. DHEA (Sigma) was prepared in ethanol at the final concentration of 0.1 %, and diluted in media to achieve the desired concentrations before treatment. Cell proliferation assay VSMCs were seeded in 96-well plates at 104 cells per well in 200 ll culture medium. After synchronization, various concentrations of DHEA (0, 10, 50, and 100 lmol/L) were added. After incubation for 60 min, the cells were stimulated with Ang II (1 lmol/L) for another 24 h and then used for evaluation of proliferation by cell counting kit-8 (CCK-8, Dojindo) assay or bromodeoxyuridine (BrdU) assay. According to the CCK-8 manufacturer’s instruction, 20 ll of WST-8 was added into culture medium and the absorbance was measured at 450 nm. Cell viability was assessed by trypan blue exclusion. In this experiment, no significant decrease of cell viability was detected in all investigated groups.
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For BrdU incorporation assay, 10 mmol/l BrdU was introduced into the culture medium for cell labeling after Ang II treatment. Then the culture plates were washed and used to colorimetric BrdU cell proliferation assay according to the manufacturer’s instruction (Calbiochem). The cells incubated without BrdU were served as negative control. The absorbance was read at a wavelength of 450 nm with a spectrophotometric plate reader. The mean absorbance for each group was determined by subtracting the mean value of the negative control. Cell migration assay To measure VSMCs migration activity, a transwell chamber (Corning) assay was performed. Briefly, VSMCs (105, overnight serum-starved) were seeded into the upper chamber in serum-free medium with or without DHEA, and the lower chamber was filled with DMEM containing Ang II (1 lmol/L). After incubation for 8 h, all nonmigrant cells were removed from the upper face of the Transwell membrane with a cotton swab and migrant cells were fixed in methanol and stained with 0.5 % crystal violet. Migration was quantified by counting the number of stained cells on the membrane under a microscope. For the wound healing assay, 2.5 9 105 cells were seeded into six-well plates. VSMCs monolayer were serum starved (0.1 % FBS; 24 h) and scratched/wounded using a sterile 200 lL pipette tip. VSMCs were challenged by Ang II (1 lmol/L) with or without DHEA. Photos were taken immediately after wounding (0 h) and at 24 h postwounding. To quantify migration, wound area was calculated at 0 and 24 h time points, and expressed as the relative fold changes compared with those in control group. Quantitative real-time RT-PCR Total RNA was extracted using a commercial RNA isolation kit (Qiagen). A total of 4 lg RNA was used to reverse transcription. Then cDNA was amplified by real-time PCR with SYBR Green PCR master mix (Invitrogen) according to the manufacturer’s instruction. The thermal cycling conditions comprised an initial denaturation step at 94 °C for 10 min, followed by 40 cycles (94 °C for 30 s; 60 °C for 30 s; 72 °C for 60 s). Data were normalized to GAPDH expression by the comparative quantification method (2-DDCt). The sequencespecific primers used to amplify rat genes were synthesized according to a previous study [18]. Western blot analysis VSMCs were washed three times with cold PBS and subsequently lysed in lysis buffer to obtain the nuclear and cytosolic protein according to the manufacturer’s protocol
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(Biovison). Protein concentration was determined using bicinchoninic acid protein assay (Bipec). The proteins were separated by SDS–polyacrylamide gels and transferred to PVDF membranes. For immunoblotting, PVDF membranes were blocked and probed with the corresponding antibodies against phosphorylated p38 MAPK (Cell signaling), IjB (Santa Cruz), p65 (Santa Cruz), SM a-actin (Sigma), SMMHC (Santa Cruz), b-actin (Santa Cruz), GAPDH (Santa Cruz), and H3 (Santa Cruz) overnight at 4 °C. After three washes, the blots were incubated with peroxidase-conjugated secondary antibodies (Pierce) for 1 h at room temperature, and subsequently analyzed by ECL detection system. Reactive oxygen species (ROS) production ROS generation by VSMCs was measured by DCF fluorescence. Briefly, VSMCs were cultured in 6-well plates and serum-starved overnight. After washing with PBS, VSMCs were pre-treated with DHEA (100 lmol/L) for 30 min and then exposed to Ang II (1 lmol/L) for another 60 min. The cells were then incubated with DCF (10 lmol/L, Bipec) for 30 min at room temperature. DCF fluorescence at an excitatory wavelength of 495 nm was recorded on a fluorescence microscopy. Fluorescence intensity was quantified from at least three random fields per well. Measurements of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and superoxide dismutase (SOD) activity VSMCs were treated with 100 lmol/L DHEA for 30 min and then challenged by Ang II (1 lmol/L) for another 60 min. NADPH oxidase activity of whole cell lysates was measured using lucigenin chemiluminescence in the presence of NADPH (100 lmol/L, sigma) as described previously [18]. 50 lg extracted protein was incubated with lucigenin (5 lmol/L, sigma) for 10 min at 37 °C in assay buffer with a final volume of 1 ml. The dynamic tracing of chemiluminescence was recorded for 180 s after addition of NADPH. NADPH oxidase activity was expressed as relative light units/min/mg protein. For SOD activity assay, total SOD activity was determined using the SOD activity assay kit (Biovison) as per the manufacturer’s instructions.
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1 lg of pRL-TK plasmid (Promega) as control using Lipofectamine 2000 (Invitrogen). After transfection for 6 h, cells were pretreated with or without DHEA and then challenged by 1 lmol/L Ang II for additional 12 h. After that, VSMCs were harvested for the dual luciferase assay (Promega). Luciferase activity was normalized to the activity of the internal control. Data were expressed as the fold changes to the luciferase activity in the absence of treatment. Balloon injury of rat carotid artery All the animal protocols were complied strictly with the Institutional Animal Care and Use Committee guidelines. The procedure for balloon injury in rat carotid arteries has been described previously. Briefly, male SD rats (450– 500 g) were anesthetized with pentobarbital (30 mg/kg, ip). The left carotid artery was isolated, and a balloon angioplasty catheter (balloon diameter 1.5 mm, balloon length 20 mm, Medtronic) was introduced through the external carotid arteriotomy incision. After that, the catheter were placed to the aortic arch, then inflated to produce moderate resistance and gradually withdrawn three times. Next, the catheter was removed and the external carotid branch was ligated. The rats for sham-operation underwent the same procedures except balloon insertion. 2 days after surgery, treated rats received intragastric administration with DHEA (3 mg/kg/day) or equal sterile saline. The rats were sacrificed at day 7 or day 28 after balloon injury, and the tissues were harvested for next experiments. Morphometric analysis 28 days after the operation, the injured and control carotid arteries were harvested, fixed in 4 % paraformaldehyde and then embedded in paraffin. For morphologic analysis of neointimal formation, five round cross-sections (4 lm thickness) were cut from the approximate middle of the artery. The intimal and medial cross-sectional areas of the carotid arteries were measured, and the intima/media ratios were calculated. Detection of plasma 8-iso-prostaglandin F2 (8-isoPGF2) in vivo
NF-jB luciferase reporter gene assay The reporter plasmid pGL6-NF-jB-Luc for testing NF-jB transcriptional activity was purchased from Beyotime Institute Biotechnology, China. VSMCs (1 9 105 cells/ well) were plated in 24-well plates before transfection and grew to about 70 % confluence. Cells were then transiently co-transfected with 1 lg of pGL6-NF-jBLuc plasmid and
Blood samples for all animals were obtained from external jugular vein and kept into EDTA anticoagulated tubes 7 days later after balloon injury and centrifugated at 1,5009g for 10 min at 4 °C. The plasma was carefully transferred and stored at -80 °C. The levels of 8-iso-PGF2 were analyzed by a commercial ELISA kit (Abnova Corporation).
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Fig. 1 DHEA suppressed VSMCs proliferation and migration. VSMCs were pre-incubated with DHEA for 60 min at concentrations varying from 10 to 100 lM and stimulated with Ang II (1 lM) for another 24 h. Quantification of cellular proliferation was performed with CCK-8 test (a) and BrdU incorporation (b) (n = 6). VSMCs
were pre-incubated with DHEA for 60 min at different concentrations and then stimulated with Ang II (1 lM) for 6 h. Quantification of cellular migration was performed with transwell assay (c) (n = 3) and wound assay (d) (n = 3). *P \ 0.05 versus Ang II alone group
Statistical analysis
presence of Ang II was also markedly attenuated after treatment with DHEA (Fig. 1c, d).
All statistical analyses were performed with SPSS 13.0 software. Data were presented as mean ± SEM. All values were analyzed using 1-way ANOVA for multiple comparisons. A P value \ 0.05 was considered to be statistically significant.
Results DHEA attenuates VSMCs proliferation and migration in vitro To examine the effects of DHEA on VSMCs proliferation, VSMCs were incubated with different concentrations of DHEA for 60 min and subsequently treated with Ang II (1 lM) for another 24 h. Pre-treatment with DHEA demonstrated a dose-dependent reduction in Ang II-induced cell growth (Fig. 1a) and BrdU incorporation (Fig. 1b). Additionally, the increase of VSMCs migration in the
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DHEA inhibits oxidative stress-dependent ERK1/2 activation in response to Ang II stimulation VSMCs were pre-treated with 100 lM DHEA for 60 min and then stimulated with 1 lM Ang II for another 60 min. As shown in Fig. 2a, Ang II significantly increased the levels of ROS and such increase was markedly attenuated by DHEA. Consistent with this result, the activity of NADPH oxidase (a major oxidant-producing enzyme) was blunted by DHEA in response to Ang II. No significant alteration of SOD (a key antioxidant enzyme system) activity was observed in DHEA-treated group (Fig. 2b). To explore the molecular mechanisms underlying that DHEA impairs the VSMCs proliferation and migration, we further examined the effects of DHEA on MAPK signaling pathway. We found that the phosphorylation of ERK1/2 and JNK1/2 were significantly increased by 1 lM Ang II stimulation. However, the phosphorylation of ERK1/2 was dramatically blocked by DHEA, whereas the phosphorylation
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Fig. 2 DHEA attenuated oxidative stress and ERK1/2 activation in VSMCs. The same density of VSMCs was pre-treatment with 100 lmol/L DHEA for 60 min and exposed to Ang II (1 lM) for another 60 min. a, b Effect of DHEA on ROS production, NADPH activity and SOD activity. c, d VSMCs were pre-treated with
100 lmol/L DHEA or NAC for 60 min, followed with stimulation of 1 lM Ang II for another 1 h. Protein expression of phosphorylated ERK1/2 and JNK1/2 was examined by western blot. *P \ 0.05 versus control group; #P \ 0.05 versus Ang II group, n = 3
of JNK1/2 was not significantly affected (Fig. 2c). In addition, ERK1/2 phosphorylation was significantly attenuated by pre-incubation with N-acetylcysteine (NAC, 10 mM), a typical antioxidant (Fig. 2d). Our findings suggest that DHEA inhibits oxidative stress-dependent ERK1/2 signaling in response to Ang II.
resulted in a 40 % reduction of Ang II-induced luciferase activation (Fig. 3c).
DHEA suppresses inflammation of VSMCs induced by Ang II To determine whether DHEA can suppress inflammation in VSMCs, we examined the expression of inflammatory mediators MCP-1 and IL-6. Our results showed that DHEA significantly decreased mRNA levels of MCP-1 and IL-6 compared with those in Ang II-treated group (Fig. 3a). Since transcriptional induction of MCP-1 and IL-6 requires p38 MAPK/NF-jB activation, the activity of this signaling pathway was determined. Our results showed that p38 MAPK was significantly phosphorylated by Ang II, while this phosphorylation was blocked by DHEA (Fig. 3b). In addition, DHEA inhibited Ang II-induced degradation of cytoplasmic IjB protein and nuclear translocation of NFjB p65 in VSMCs (Fig. 3b). To further characterize the effect of DHEA on Ang II-mediated NF-jB activity, VSMCs were transfected with NF-jB-Luc plasmid. DHEA
DHEA has no significant effect on VSMCs phenotype transition Previous studies found that VSMCs switching from a differentiated to a dedifferentiated phenotype plays an important role in neointimal hyperplasia via Notch signaling pathway [15]. Here we applied a well-established VSMCs model for phenotypic modulation, in which differentiated VSMCs were induced by serum starvation and dedifferentiated VSMCs were challenged by 10 % serum [19]. Our results suggested that the levels of SM a–actin and SM-MHC were increased after 72 h-serum starvation, and these differentiation markers were reduced by 10 % serum stimulation, which well presents a cell transition from differentiated to dedifferentiated phenotype. Interestingly, as shown in Fig. 4, DHEA has no significant effect on VSMCs phenotype transition markers. In agreement with this effect, no notable alterations were observed on Notch ligand Jagged-1, Notch target genes Hey-1 and Hey-2 mRNA levels, and also Notch 1 intracellular domain protein levels in DHEA-treated group compared to Ang IItreated group (data not shown).
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Fig. 3 DHEA suppressed inflammatory response induced by Ang II. a After pre-treatment with 100 lmol/L DHEA for 60 min, VSMCs were stimulated with 1 lM Ang II for another 3 h. mRNA expression of MCP-1 and IL-6 in VSMCs was analyzed by real-time RT-PCR; b VSMCs pre-treated with 100 lmol/L DHEA for 60 min, followed with stimulation of 1 lM Ang II for another 1 h. Protein expression
of phosphorylated p38 MAPK, IjB and p65 was examined by western blot. *P \ 0.05 versus control group; #P \ 0.05 versus Ang II group, n = 3; c Effect of DHEA on NF-jB activation. After transfection with NF-jB-Luc plasmid, VSMCs were challenged with Ang II for 8 h, and then luciferase activity was determined. *P \ 0.05 versus control group; #P \ 0.05 versus Ang II group, n = 3
Effect of DHEA on neointimal formation in vivo
2.51 ± 0.24, P \ 0.05; n = 5) compared with those in control group (Fig 5c).
As shown in Fig. 5, 8-iso-PGF2, one of oxidative stress markers, was suppressed by DHEA treatment 7 days later after balloon injury, which was companied by reduction of MCP-1 and IL-6 expression. On day 28 after balloon injury, the degree of neointimal formation was evaluated morphologically and quantitatively. Our immunohistochemistry analyses showed that DHEA group had a smaller intima area (0.19 ± 0.027 vs. 0.43 ± 0.059 mm2, P \ 0.05; n = 5) and intima/media ratio (1.19 ± 0.17 vs.
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Discussion The results from our study demonstrate that DHEA protects against VSMCs proliferation in vivo. The protective role of DHEA in VSMCs proliferation is mediated by direct interruption of ROS-dependent ERK1/2 signaling and p38 MAPK/NF-jB signaling pathway. Moreover, daily DHEA
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Fig. 4 Effect of DHEA on VSMCs phenotype-transition. Differentiated VSMCs were induced by serum starvation for 72 h after 24-h synchronization and then challenged by 10 % serum for another 24 h. Protein expression of VSMCs markers (SMa-actin and SM-MHC) were examined by western blot. *P \ 0.05 versus non-serum group, n = 3
Fig. 5 Effects of DHEA on neointimal formation in injured carotid arteries. a Levels of plasma 8-iso-PGF2 were detected by ELISA (n = 6); b mRNA expressions of IL-6 and MCP-1 were analyzed by real-time RT-PCR (n = 3, each group contained three arteries); c Representative histologic sections of hematoxylin eosin-stained
carotid arteries. Scale bar represents 100 lm. Quantitative analysis of intimal area and ratio of intima to media were calculated 28 days after balloon injury (n = 6). *P \ 0.05 versus control group, #P \ 0.05 versus injury group
feeding markedly reduced intima-media thickening concomitant with inhibition of oxidative stress and inflammation after carotid balloon injury in rat. These findings support the concept that DHEA could be an effective therapeutical candidate against VSMC proliferation and vascular hyperplasia. The mechanism underlying that DHEA has anti-proliferative effects remains largely unclear. Until now, it’s been
reported that DHEA regulated VSMCs proliferation through Skp2 signaling pathway and redox regulation [20, 21]. Another research revealed that DHEA inhibited vascular remodeling and reduced neointima formation after vascular injury via its effects on VSMCs phenotypic modulation, functions, and apoptosis by up-regulating p16(INK4a)/activating PPARalpha [22]. DHEA has multiple biologic effects on cardiovascular diseases, and our
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Fig. 6 Schematic diagram of the anti-proliferative mechanisms of DHEA in VSMCs. DHEA can suppress MAPK activation, and then prevent the degradation of cytoplasmic IkB and reduced nuclear level of p65. MAPK/p65 signaling pathway plays an important role in induction of inflammation and oxidative stress. Through these mechanisms, DHEA prevents the activation of MAPK/p65 and the terminal events in proliferation and migration
study highlight its inhibitory effects on oxidative stress and inflammation in VSMCs. There is increasing evidence for the involvement of oxidative stress in several steps leading to the development of vascular diseases [11]. Some extracellular signals, such as growth factors or inflammatory stimuli, induce ROS and subsequently activate ERK1/ 2 which is an important regulator of VSMCs growth [23–25]. In present study, we investigated whether the antiproliferative effect of DHEA is mediated by inhibition of ROS generation. Intriguingly, the results indicated that inhibition of ROS in vitro and in vivo was a key mechanism for the anti-proliferative activity of DHEA. The potential mechanism might be associated with the decreased activity of NADPH oxidase. In accordance with our finding, several researchers also found that DHEA prevented oxidative stress in endothelial cell, myocardial cell, and cancer cells though NADPH oxidase pathway [26–28]. Although ROS plays a key role in mediating vessel hyperplasia, the precise molecular targets through which DHEA regulates VSMCs growth signaling are not well defined. To explore the molecular mechanisms, we examined the effects of DHEA on MAPK signaling pathway, and demonstrated that DHEA markedly blocked ERK1/2 activation in response to Ang II in vitro. Importantly, the classical antioxidant NAC simulated the effects of DHEA on the activation of ERK1/2. This result suggests that the inhibitory effect of DHEA on ERK1/2 signaling may be mainly via inhibition of ROS.
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Evidences showed that up-regulation of inflammatory genes played a major role in the pathology of vascular diseases [29, 30]. The inflammatory gene expression is primarily regulated by transcription factor NF-jB [31, 32]. Activated NF-jB has been detected in atherosclerotic plaques [33] and in cultured VSMCs stimulated by cytokines, such as Ang II [34] and thrombin [32]. Since DHEA inhibited the expression of these molecules in VSMCs, we investigated a potential interference of DHEA to NF-jB activation. NF-jB family consists of five members and these subunits form homodimers or heterodimers [35]. The most common transcription form of NF-jB is composed of a p50 DNA-binding subunit attaching by a p65 transactivation subunit [35]. The present study found that DHEA inhibited Ang II-induced NF-jB activity, and this result indicated that DHEA was the transcription inhibitor of NFjB component p65. Previous studies suggest that p38 MAPK participates in the regulation of NF-jB transcriptional activity [36, 37]. We also showed that DHEA inhibited the phosphorylation of p38 MAPK. This inducible negative regulation of p38 MAPK activity may help to clarify the inhibitory role of DHEA on NF-jB activation induced by Ang II in VSMCs. Human autopsy studies suggest an important role for VSMCs in the pathogenesis of occlusive arterial diseases. Several growth factors including Ang II have been implicated in neointimal growth and restenosis in injured arteries [38, 39]. Initial studies have confirmed that Ang II and its receptors exerted hypertrophic effects on VSMC biology via different signaling pathways [40, 41]. Besides these traditional hypertrophic stimuli, VSMCs switch from a contractile to a synthetic phenotype under the stimulation of other polypeptide growth factors during the aggregation of VSMCs within the intima, such as platelet-derived growth factor and serum [42, 43]. Thus the pathological state of the arteries is probably determined by the synergetic effects of the two different classes of stimulation. In our study, DHEA was found to have an obvious effect on inhibition of VSMCs proliferation and migration induced by Ang II, but not on VSMCs phenotype transition. From these results, we suspected that DHEA might be more sensitive to hypertrophic stimuli than phenotype-transition factors. Since occlusive vessel diseases require efficient and individualized treatment approaches, it is possible that DHEA has more powerful impact on vascular pathophysiology triggered by hypertrophic factors than phenotypetransition factors. Taken together, our findings suggest that DHEA can alleviate inflammation and oxidative stress in VSMCs. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury (See Fig. 6). Therefore, DHEA might be a promising therapy for vascular injury.
Mol Cell Biochem (2014) 388:75–84 Acknowledgments This work was supported by a grant from Fundamental Research Funds for the Central Universities (No. 302274023).
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DHEA inhibits vascular remodeling following arterial injury: a possible role in suppression of inflammation and oxidative stress derived from vascular smooth muscle cells Jiangbin Chen • Lin Xu • Congxin Huang
Received: 2 May 2013 / Accepted: 15 November 2013 / Published online: 28 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Vascular remodeling is characterized by the aggregation of vascular smooth muscle cells (VSMCs) in intima. Previous studies have demonstrated that dehydroepiandrosterone (DHEA), a steroid hormone, can reverse vascular remodeling. However, it is still far clear that whether and how DHEA participates in the modulation of VSMCs activation and vascular remodeling. VSMCs were obtained from the thoracic aorta of SD rats. Cell proliferation was evaluated by CCK-8 assay and BrdU assay. To measure VSMCs migration activity, a transwell chamber assay was performed. Quantitative real-time RT-PCR and western blot were used to explore the molecular mechanisms. ROS generation by VSMCs was measured by DCF fluorescence. NADPH oxidase activity and SOD activity were measured by the corresponding kits. NF-jB activity was detected by NF-jB luciferase reporter gene assay. A rat carotid artery balloon injury model was built to evaluate the neointimal formation, and plasma PGF2 was measured by ELISA. Our results showed that DHEA significantly inhibited VSMCs proliferation after angiotensin (Ang II) stimulation by down-regulation of NADPH oxidase activity and ERK1/2 phosphorylation. Ang II can increase IL-6 and MCP-1 expression, but DHEA reverses these changes via inhibiting p38-MAPK/NF-jB (p65) signaling pathway. DHEA has no significant effects on VSMCs phenotype transition, but can reduce the neointimal to media area ratio after balloon injury. DHEA can alleviate oxidative stress and inflammation in VSMCs via ERK1/2 and NF-jB
Jiangbin Chen and Lin Xu have contributed equally to this study. J. Chen L. Xu C. Huang (&) Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, People’s Republic of China e-mail: [email protected]
signaling pathway, but has no effect on VSMCs phenotype transition. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury in vivo. Taken together, DHEA might be a promising treatment for vascular injury under pathological condition. Keywords Dehydroepiandrosterone Vascular smooth muscle cells Oxidative stress Inflammation Carotid artery balloon injury
Introduction Endogenous androgens are considered as potential predictors of initiation and development of cardiovascular diseases [1, 2]. Dehydroepiandrosterone (DHEA), one of the most important androgenic hormones, is related to an increased risk of cardiovascular disease [3–6]. Several studies have shown that the serum DHEA level declined in patients with more cardiovascular risk factors and higher burdens of coronary diseases [4–6]. In this case, DHEA has been extensively applied to human for a variety of diseases due to its safety. Some clinic investigations and animal studies have demonstrated that DHEA was a promising therapeutic approach for cardiovascular diseases including atherosclerosis and pulmonary hypertension [7, 8]. Recently, several studies reported that no protective role was found after DHEA supplementation in occlusive vascular diseases [9, 10]. So the benefits of DHEA in clinical use are now challenged. Nowadays it is widely recognized that chronic inflammation and oxidative stress play important roles in the pathogenesis of occlusive vascular diseases [11, 12]. Clinical studies showed a strong correlation between proinflammatory biomarkers and oxidative stress markers in
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atherosclerosis and restenosis [13, 14]. Apart from above, it is a notable feature of vascular remodeling that VSMCs switch from contractile to synthetic phenotype during VSMCs aggregation. The majority of intimal VSMCs after vessel injury are derived from resident medial VSMCs, which undergo a phenotypic modulation and migrate into intima to proliferate and produce extracellular matrix [15, 16]. So all of the three, inflammation, oxidative stress, and phenotypic switch, participate in the vascular remodeling in vascular diseases, but their significance may be different under various conditions. Until now, there is no single study to explore the exact mechanism underlying DHEA reverses vascular remodeling. In present study, we want to illustrate whether if DHEA inhibits inflammation, oxidative stress derived from VSMCs and whether it has the ability to reverse VSMCs phenotype transition after vascular injury. Our study will highlight the potential therapeutic role of DHEA by effectively inhibiting neointimal hyperplasia in vascular diseases.
Methods Primary VSMC culture and DHEA treatment VSMCs were isolated from the thoracic aorta of male SD rats (100–150 g) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing 10 % fetal bovine serum (FBS, Hyclone) according to previous studies [17, 18]. The purity of VSMCs was approximately 90 % assessed by cell immunostaining with anti-smooth muscle a-actin antibody. Only VSMCs between the 3rd and 5th passages were used for experiments. Before treatment, VSMCs were cultured for 24 h in DMEM with 0.5 % FBS. DHEA (Sigma) was prepared in ethanol at the final concentration of 0.1 %, and diluted in media to achieve the desired concentrations before treatment. Cell proliferation assay VSMCs were seeded in 96-well plates at 104 cells per well in 200 ll culture medium. After synchronization, various concentrations of DHEA (0, 10, 50, and 100 lmol/L) were added. After incubation for 60 min, the cells were stimulated with Ang II (1 lmol/L) for another 24 h and then used for evaluation of proliferation by cell counting kit-8 (CCK-8, Dojindo) assay or bromodeoxyuridine (BrdU) assay. According to the CCK-8 manufacturer’s instruction, 20 ll of WST-8 was added into culture medium and the absorbance was measured at 450 nm. Cell viability was assessed by trypan blue exclusion. In this experiment, no significant decrease of cell viability was detected in all investigated groups.
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For BrdU incorporation assay, 10 mmol/l BrdU was introduced into the culture medium for cell labeling after Ang II treatment. Then the culture plates were washed and used to colorimetric BrdU cell proliferation assay according to the manufacturer’s instruction (Calbiochem). The cells incubated without BrdU were served as negative control. The absorbance was read at a wavelength of 450 nm with a spectrophotometric plate reader. The mean absorbance for each group was determined by subtracting the mean value of the negative control. Cell migration assay To measure VSMCs migration activity, a transwell chamber (Corning) assay was performed. Briefly, VSMCs (105, overnight serum-starved) were seeded into the upper chamber in serum-free medium with or without DHEA, and the lower chamber was filled with DMEM containing Ang II (1 lmol/L). After incubation for 8 h, all nonmigrant cells were removed from the upper face of the Transwell membrane with a cotton swab and migrant cells were fixed in methanol and stained with 0.5 % crystal violet. Migration was quantified by counting the number of stained cells on the membrane under a microscope. For the wound healing assay, 2.5 9 105 cells were seeded into six-well plates. VSMCs monolayer were serum starved (0.1 % FBS; 24 h) and scratched/wounded using a sterile 200 lL pipette tip. VSMCs were challenged by Ang II (1 lmol/L) with or without DHEA. Photos were taken immediately after wounding (0 h) and at 24 h postwounding. To quantify migration, wound area was calculated at 0 and 24 h time points, and expressed as the relative fold changes compared with those in control group. Quantitative real-time RT-PCR Total RNA was extracted using a commercial RNA isolation kit (Qiagen). A total of 4 lg RNA was used to reverse transcription. Then cDNA was amplified by real-time PCR with SYBR Green PCR master mix (Invitrogen) according to the manufacturer’s instruction. The thermal cycling conditions comprised an initial denaturation step at 94 °C for 10 min, followed by 40 cycles (94 °C for 30 s; 60 °C for 30 s; 72 °C for 60 s). Data were normalized to GAPDH expression by the comparative quantification method (2-DDCt). The sequencespecific primers used to amplify rat genes were synthesized according to a previous study [18]. Western blot analysis VSMCs were washed three times with cold PBS and subsequently lysed in lysis buffer to obtain the nuclear and cytosolic protein according to the manufacturer’s protocol
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(Biovison). Protein concentration was determined using bicinchoninic acid protein assay (Bipec). The proteins were separated by SDS–polyacrylamide gels and transferred to PVDF membranes. For immunoblotting, PVDF membranes were blocked and probed with the corresponding antibodies against phosphorylated p38 MAPK (Cell signaling), IjB (Santa Cruz), p65 (Santa Cruz), SM a-actin (Sigma), SMMHC (Santa Cruz), b-actin (Santa Cruz), GAPDH (Santa Cruz), and H3 (Santa Cruz) overnight at 4 °C. After three washes, the blots were incubated with peroxidase-conjugated secondary antibodies (Pierce) for 1 h at room temperature, and subsequently analyzed by ECL detection system. Reactive oxygen species (ROS) production ROS generation by VSMCs was measured by DCF fluorescence. Briefly, VSMCs were cultured in 6-well plates and serum-starved overnight. After washing with PBS, VSMCs were pre-treated with DHEA (100 lmol/L) for 30 min and then exposed to Ang II (1 lmol/L) for another 60 min. The cells were then incubated with DCF (10 lmol/L, Bipec) for 30 min at room temperature. DCF fluorescence at an excitatory wavelength of 495 nm was recorded on a fluorescence microscopy. Fluorescence intensity was quantified from at least three random fields per well. Measurements of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and superoxide dismutase (SOD) activity VSMCs were treated with 100 lmol/L DHEA for 30 min and then challenged by Ang II (1 lmol/L) for another 60 min. NADPH oxidase activity of whole cell lysates was measured using lucigenin chemiluminescence in the presence of NADPH (100 lmol/L, sigma) as described previously [18]. 50 lg extracted protein was incubated with lucigenin (5 lmol/L, sigma) for 10 min at 37 °C in assay buffer with a final volume of 1 ml. The dynamic tracing of chemiluminescence was recorded for 180 s after addition of NADPH. NADPH oxidase activity was expressed as relative light units/min/mg protein. For SOD activity assay, total SOD activity was determined using the SOD activity assay kit (Biovison) as per the manufacturer’s instructions.
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1 lg of pRL-TK plasmid (Promega) as control using Lipofectamine 2000 (Invitrogen). After transfection for 6 h, cells were pretreated with or without DHEA and then challenged by 1 lmol/L Ang II for additional 12 h. After that, VSMCs were harvested for the dual luciferase assay (Promega). Luciferase activity was normalized to the activity of the internal control. Data were expressed as the fold changes to the luciferase activity in the absence of treatment. Balloon injury of rat carotid artery All the animal protocols were complied strictly with the Institutional Animal Care and Use Committee guidelines. The procedure for balloon injury in rat carotid arteries has been described previously. Briefly, male SD rats (450– 500 g) were anesthetized with pentobarbital (30 mg/kg, ip). The left carotid artery was isolated, and a balloon angioplasty catheter (balloon diameter 1.5 mm, balloon length 20 mm, Medtronic) was introduced through the external carotid arteriotomy incision. After that, the catheter were placed to the aortic arch, then inflated to produce moderate resistance and gradually withdrawn three times. Next, the catheter was removed and the external carotid branch was ligated. The rats for sham-operation underwent the same procedures except balloon insertion. 2 days after surgery, treated rats received intragastric administration with DHEA (3 mg/kg/day) or equal sterile saline. The rats were sacrificed at day 7 or day 28 after balloon injury, and the tissues were harvested for next experiments. Morphometric analysis 28 days after the operation, the injured and control carotid arteries were harvested, fixed in 4 % paraformaldehyde and then embedded in paraffin. For morphologic analysis of neointimal formation, five round cross-sections (4 lm thickness) were cut from the approximate middle of the artery. The intimal and medial cross-sectional areas of the carotid arteries were measured, and the intima/media ratios were calculated. Detection of plasma 8-iso-prostaglandin F2 (8-isoPGF2) in vivo
NF-jB luciferase reporter gene assay The reporter plasmid pGL6-NF-jB-Luc for testing NF-jB transcriptional activity was purchased from Beyotime Institute Biotechnology, China. VSMCs (1 9 105 cells/ well) were plated in 24-well plates before transfection and grew to about 70 % confluence. Cells were then transiently co-transfected with 1 lg of pGL6-NF-jBLuc plasmid and
Blood samples for all animals were obtained from external jugular vein and kept into EDTA anticoagulated tubes 7 days later after balloon injury and centrifugated at 1,5009g for 10 min at 4 °C. The plasma was carefully transferred and stored at -80 °C. The levels of 8-iso-PGF2 were analyzed by a commercial ELISA kit (Abnova Corporation).
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Fig. 1 DHEA suppressed VSMCs proliferation and migration. VSMCs were pre-incubated with DHEA for 60 min at concentrations varying from 10 to 100 lM and stimulated with Ang II (1 lM) for another 24 h. Quantification of cellular proliferation was performed with CCK-8 test (a) and BrdU incorporation (b) (n = 6). VSMCs
were pre-incubated with DHEA for 60 min at different concentrations and then stimulated with Ang II (1 lM) for 6 h. Quantification of cellular migration was performed with transwell assay (c) (n = 3) and wound assay (d) (n = 3). *P \ 0.05 versus Ang II alone group
Statistical analysis
presence of Ang II was also markedly attenuated after treatment with DHEA (Fig. 1c, d).
All statistical analyses were performed with SPSS 13.0 software. Data were presented as mean ± SEM. All values were analyzed using 1-way ANOVA for multiple comparisons. A P value \ 0.05 was considered to be statistically significant.
Results DHEA attenuates VSMCs proliferation and migration in vitro To examine the effects of DHEA on VSMCs proliferation, VSMCs were incubated with different concentrations of DHEA for 60 min and subsequently treated with Ang II (1 lM) for another 24 h. Pre-treatment with DHEA demonstrated a dose-dependent reduction in Ang II-induced cell growth (Fig. 1a) and BrdU incorporation (Fig. 1b). Additionally, the increase of VSMCs migration in the
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DHEA inhibits oxidative stress-dependent ERK1/2 activation in response to Ang II stimulation VSMCs were pre-treated with 100 lM DHEA for 60 min and then stimulated with 1 lM Ang II for another 60 min. As shown in Fig. 2a, Ang II significantly increased the levels of ROS and such increase was markedly attenuated by DHEA. Consistent with this result, the activity of NADPH oxidase (a major oxidant-producing enzyme) was blunted by DHEA in response to Ang II. No significant alteration of SOD (a key antioxidant enzyme system) activity was observed in DHEA-treated group (Fig. 2b). To explore the molecular mechanisms underlying that DHEA impairs the VSMCs proliferation and migration, we further examined the effects of DHEA on MAPK signaling pathway. We found that the phosphorylation of ERK1/2 and JNK1/2 were significantly increased by 1 lM Ang II stimulation. However, the phosphorylation of ERK1/2 was dramatically blocked by DHEA, whereas the phosphorylation
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Fig. 2 DHEA attenuated oxidative stress and ERK1/2 activation in VSMCs. The same density of VSMCs was pre-treatment with 100 lmol/L DHEA for 60 min and exposed to Ang II (1 lM) for another 60 min. a, b Effect of DHEA on ROS production, NADPH activity and SOD activity. c, d VSMCs were pre-treated with
100 lmol/L DHEA or NAC for 60 min, followed with stimulation of 1 lM Ang II for another 1 h. Protein expression of phosphorylated ERK1/2 and JNK1/2 was examined by western blot. *P \ 0.05 versus control group; #P \ 0.05 versus Ang II group, n = 3
of JNK1/2 was not significantly affected (Fig. 2c). In addition, ERK1/2 phosphorylation was significantly attenuated by pre-incubation with N-acetylcysteine (NAC, 10 mM), a typical antioxidant (Fig. 2d). Our findings suggest that DHEA inhibits oxidative stress-dependent ERK1/2 signaling in response to Ang II.
resulted in a 40 % reduction of Ang II-induced luciferase activation (Fig. 3c).
DHEA suppresses inflammation of VSMCs induced by Ang II To determine whether DHEA can suppress inflammation in VSMCs, we examined the expression of inflammatory mediators MCP-1 and IL-6. Our results showed that DHEA significantly decreased mRNA levels of MCP-1 and IL-6 compared with those in Ang II-treated group (Fig. 3a). Since transcriptional induction of MCP-1 and IL-6 requires p38 MAPK/NF-jB activation, the activity of this signaling pathway was determined. Our results showed that p38 MAPK was significantly phosphorylated by Ang II, while this phosphorylation was blocked by DHEA (Fig. 3b). In addition, DHEA inhibited Ang II-induced degradation of cytoplasmic IjB protein and nuclear translocation of NFjB p65 in VSMCs (Fig. 3b). To further characterize the effect of DHEA on Ang II-mediated NF-jB activity, VSMCs were transfected with NF-jB-Luc plasmid. DHEA
DHEA has no significant effect on VSMCs phenotype transition Previous studies found that VSMCs switching from a differentiated to a dedifferentiated phenotype plays an important role in neointimal hyperplasia via Notch signaling pathway [15]. Here we applied a well-established VSMCs model for phenotypic modulation, in which differentiated VSMCs were induced by serum starvation and dedifferentiated VSMCs were challenged by 10 % serum [19]. Our results suggested that the levels of SM a–actin and SM-MHC were increased after 72 h-serum starvation, and these differentiation markers were reduced by 10 % serum stimulation, which well presents a cell transition from differentiated to dedifferentiated phenotype. Interestingly, as shown in Fig. 4, DHEA has no significant effect on VSMCs phenotype transition markers. In agreement with this effect, no notable alterations were observed on Notch ligand Jagged-1, Notch target genes Hey-1 and Hey-2 mRNA levels, and also Notch 1 intracellular domain protein levels in DHEA-treated group compared to Ang IItreated group (data not shown).
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Fig. 3 DHEA suppressed inflammatory response induced by Ang II. a After pre-treatment with 100 lmol/L DHEA for 60 min, VSMCs were stimulated with 1 lM Ang II for another 3 h. mRNA expression of MCP-1 and IL-6 in VSMCs was analyzed by real-time RT-PCR; b VSMCs pre-treated with 100 lmol/L DHEA for 60 min, followed with stimulation of 1 lM Ang II for another 1 h. Protein expression
of phosphorylated p38 MAPK, IjB and p65 was examined by western blot. *P \ 0.05 versus control group; #P \ 0.05 versus Ang II group, n = 3; c Effect of DHEA on NF-jB activation. After transfection with NF-jB-Luc plasmid, VSMCs were challenged with Ang II for 8 h, and then luciferase activity was determined. *P \ 0.05 versus control group; #P \ 0.05 versus Ang II group, n = 3
Effect of DHEA on neointimal formation in vivo
2.51 ± 0.24, P \ 0.05; n = 5) compared with those in control group (Fig 5c).
As shown in Fig. 5, 8-iso-PGF2, one of oxidative stress markers, was suppressed by DHEA treatment 7 days later after balloon injury, which was companied by reduction of MCP-1 and IL-6 expression. On day 28 after balloon injury, the degree of neointimal formation was evaluated morphologically and quantitatively. Our immunohistochemistry analyses showed that DHEA group had a smaller intima area (0.19 ± 0.027 vs. 0.43 ± 0.059 mm2, P \ 0.05; n = 5) and intima/media ratio (1.19 ± 0.17 vs.
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Discussion The results from our study demonstrate that DHEA protects against VSMCs proliferation in vivo. The protective role of DHEA in VSMCs proliferation is mediated by direct interruption of ROS-dependent ERK1/2 signaling and p38 MAPK/NF-jB signaling pathway. Moreover, daily DHEA
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Fig. 4 Effect of DHEA on VSMCs phenotype-transition. Differentiated VSMCs were induced by serum starvation for 72 h after 24-h synchronization and then challenged by 10 % serum for another 24 h. Protein expression of VSMCs markers (SMa-actin and SM-MHC) were examined by western blot. *P \ 0.05 versus non-serum group, n = 3
Fig. 5 Effects of DHEA on neointimal formation in injured carotid arteries. a Levels of plasma 8-iso-PGF2 were detected by ELISA (n = 6); b mRNA expressions of IL-6 and MCP-1 were analyzed by real-time RT-PCR (n = 3, each group contained three arteries); c Representative histologic sections of hematoxylin eosin-stained
carotid arteries. Scale bar represents 100 lm. Quantitative analysis of intimal area and ratio of intima to media were calculated 28 days after balloon injury (n = 6). *P \ 0.05 versus control group, #P \ 0.05 versus injury group
feeding markedly reduced intima-media thickening concomitant with inhibition of oxidative stress and inflammation after carotid balloon injury in rat. These findings support the concept that DHEA could be an effective therapeutical candidate against VSMC proliferation and vascular hyperplasia. The mechanism underlying that DHEA has anti-proliferative effects remains largely unclear. Until now, it’s been
reported that DHEA regulated VSMCs proliferation through Skp2 signaling pathway and redox regulation [20, 21]. Another research revealed that DHEA inhibited vascular remodeling and reduced neointima formation after vascular injury via its effects on VSMCs phenotypic modulation, functions, and apoptosis by up-regulating p16(INK4a)/activating PPARalpha [22]. DHEA has multiple biologic effects on cardiovascular diseases, and our
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Fig. 6 Schematic diagram of the anti-proliferative mechanisms of DHEA in VSMCs. DHEA can suppress MAPK activation, and then prevent the degradation of cytoplasmic IkB and reduced nuclear level of p65. MAPK/p65 signaling pathway plays an important role in induction of inflammation and oxidative stress. Through these mechanisms, DHEA prevents the activation of MAPK/p65 and the terminal events in proliferation and migration
study highlight its inhibitory effects on oxidative stress and inflammation in VSMCs. There is increasing evidence for the involvement of oxidative stress in several steps leading to the development of vascular diseases [11]. Some extracellular signals, such as growth factors or inflammatory stimuli, induce ROS and subsequently activate ERK1/ 2 which is an important regulator of VSMCs growth [23–25]. In present study, we investigated whether the antiproliferative effect of DHEA is mediated by inhibition of ROS generation. Intriguingly, the results indicated that inhibition of ROS in vitro and in vivo was a key mechanism for the anti-proliferative activity of DHEA. The potential mechanism might be associated with the decreased activity of NADPH oxidase. In accordance with our finding, several researchers also found that DHEA prevented oxidative stress in endothelial cell, myocardial cell, and cancer cells though NADPH oxidase pathway [26–28]. Although ROS plays a key role in mediating vessel hyperplasia, the precise molecular targets through which DHEA regulates VSMCs growth signaling are not well defined. To explore the molecular mechanisms, we examined the effects of DHEA on MAPK signaling pathway, and demonstrated that DHEA markedly blocked ERK1/2 activation in response to Ang II in vitro. Importantly, the classical antioxidant NAC simulated the effects of DHEA on the activation of ERK1/2. This result suggests that the inhibitory effect of DHEA on ERK1/2 signaling may be mainly via inhibition of ROS.
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Evidences showed that up-regulation of inflammatory genes played a major role in the pathology of vascular diseases [29, 30]. The inflammatory gene expression is primarily regulated by transcription factor NF-jB [31, 32]. Activated NF-jB has been detected in atherosclerotic plaques [33] and in cultured VSMCs stimulated by cytokines, such as Ang II [34] and thrombin [32]. Since DHEA inhibited the expression of these molecules in VSMCs, we investigated a potential interference of DHEA to NF-jB activation. NF-jB family consists of five members and these subunits form homodimers or heterodimers [35]. The most common transcription form of NF-jB is composed of a p50 DNA-binding subunit attaching by a p65 transactivation subunit [35]. The present study found that DHEA inhibited Ang II-induced NF-jB activity, and this result indicated that DHEA was the transcription inhibitor of NFjB component p65. Previous studies suggest that p38 MAPK participates in the regulation of NF-jB transcriptional activity [36, 37]. We also showed that DHEA inhibited the phosphorylation of p38 MAPK. This inducible negative regulation of p38 MAPK activity may help to clarify the inhibitory role of DHEA on NF-jB activation induced by Ang II in VSMCs. Human autopsy studies suggest an important role for VSMCs in the pathogenesis of occlusive arterial diseases. Several growth factors including Ang II have been implicated in neointimal growth and restenosis in injured arteries [38, 39]. Initial studies have confirmed that Ang II and its receptors exerted hypertrophic effects on VSMC biology via different signaling pathways [40, 41]. Besides these traditional hypertrophic stimuli, VSMCs switch from a contractile to a synthetic phenotype under the stimulation of other polypeptide growth factors during the aggregation of VSMCs within the intima, such as platelet-derived growth factor and serum [42, 43]. Thus the pathological state of the arteries is probably determined by the synergetic effects of the two different classes of stimulation. In our study, DHEA was found to have an obvious effect on inhibition of VSMCs proliferation and migration induced by Ang II, but not on VSMCs phenotype transition. From these results, we suspected that DHEA might be more sensitive to hypertrophic stimuli than phenotype-transition factors. Since occlusive vessel diseases require efficient and individualized treatment approaches, it is possible that DHEA has more powerful impact on vascular pathophysiology triggered by hypertrophic factors than phenotypetransition factors. Taken together, our findings suggest that DHEA can alleviate inflammation and oxidative stress in VSMCs. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury (See Fig. 6). Therefore, DHEA might be a promising therapy for vascular injury.
Mol Cell Biochem (2014) 388:75–84 Acknowledgments This work was supported by a grant from Fundamental Research Funds for the Central Universities (No. 302274023).
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