Li Li, Peng Gao, Hou-zao Chen, Zhu-qin Zhang, Ting-ting Xu, Yu-yan Jia, Hui-na Zhang, Guan-hua Du, and De-pei Liu

1Beijing Key Laboratory of Drug Target and Screening Research, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

2National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China

NEOINTIMAL thickening, a key event in the pathogenesis of atherosclerosis and restenosis, is the result of the active cellular process involving apoptosis, migration, proliferation, and extracellular matrix modification.1In the vasculature, imbalance between cell proliferation and apoptosis account for the accumulation of vascular smooth muscle cells (VSMCs) in response to arterial injury, a major feature of vascular intimal hyperplasia.2Regression of the thickened arterial wall early in these pathologies could reduce the neointima.3Fas ligand (FasL), a 40-kDa type II membrane protein in the tumor necrosis factor superfamily, triggers apoptosis through cognate binding to its receptor Fas (CD95/Apo-1).2FasL not only promotes apoptosis but also exerts anti-proliferative and anti-inflammatory effects in the course of tissue repair after vascular injury in vivo.1,4,5FasL gene therapy was adopted as an approach for the treatment of vascular smooth muscle proliferative disorders, such as restenosis after coronary angioplasty.6

Sirtuin 1 (SIRT1) is a type of NAD+dependent Class III histone deacetylase and has multiple roles in stress response, genomic stability, tumorigenesis, energy metabolism, cell ageing, and apoptosis.7Accumulating evidence indicates the involvement of SIRT1 in the pathogenesis of various diseases including atherosclerosis and restenosis. Transgenic overexpression of SIRT1 in endothelial cells was reported to retard atherosclerosis.8SIRT1 increases endothelial nitric oxide synthase-derived nitric oxide in endothelial cells9and reduces macrophage foam cell formation by interfering with the nuclear factor-κB signaling pathway.10,11SIRT1 can also retard hyperphosphatemia-induced arterial calcification12and reduce neointima formation following injury by inhibiting VSMC proliferation and migration.13Recently, SIRT1 in VSMCs has been reported to protect against DNA damage, medial degeneration, and atherosclerosis.14

The previous study of our group demonstrated the inhibitory effect of SIRT1 on VSMC proliferation and migration that underlie neointima formation.13In view of the critical role of SIRT1 and FasL in vascular smooth muscle proliferative disorders, we investigated the effect of SIRT1 on FasL expression in flow-restricted mouse carotid arteries and serum-stimulated rat VSMCs, and elucidated the potential mechanisms of the effect.

MATERIALS AND METHODS

Cells and cell cultures

The rat embryonic aorta smooth muscle cell (SMC) lines A10 and A7r5 were purchased from the American Type Culture Collection. Primary rat aortic VSMCs were isolated from the thoracic aorta of male Sprague-Dawley rats by enzymatic digestion. All the cells were cultured in DMEM supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS). VSMCs were maintained at 37°C in a humidified atmosphere containing 5% CO2, and used at passages 3-5.

Carotid artery ligation model

The animal experiment protocols of this study were approved by the Animal Care and Use Committee at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College. SMC-specific human SIRT1 transgenic (Tg) mice were generated as described previously.13Nine- to twelve-week-old male SMC-SIRT1 Tg mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (a flow-restriction model of vascular injury that results in neointima formation, ligation groups, n=9 per genotype) or ligation-excluded operation (sham groups, n=9 per genotype) according to a previously described method.13The carotid arteries were harvested 1 day after ligation.

Adenovirus generation and infection

The replication-defective adenoviral vectors expressing human SIRT1 (Ad-SIRT1) or the control green fluorescent protein (Ad-GFP) were prepared as described previously.8,15Primary rat aortic VSMCs were infected with the above adenoviral vectors for 24 hours at a multiplicity of infection of 100, washed with serum-free DMEM and cultured in serum-free medium without virus for 24 hours followed by treatment with 10% FBS for 1 hour and 4 hours.

Plasmid construction and luciferase reporter assay

The plasmid containing the coding sequence of human SIRT1 (WT-SIRT1/pcDNA3.1-SIRT1) and the mutant with the point mutation (CAT to TAT) at codon 363 (SIRT1H363Y) were kind gifts from Professor Fuyuki Ishikawa (Kyoto University, Japan). The base substitution that replaces a conserved histidine residue at position 363 into a tyrosine residue in SIRT1H363Y leads to the inactivation of deacetylase activity.16-18The plasmid containing the coding sequence of full-length mouse GATA-6 (pCMVTag3b-GATA-6) was kindly provided by Professor Patricia A. Mericko (University of Pennsylvania, USA). For the luciferase assay, a 2.1 kb of rat FasL promoter was amplified with polymerase chain reaction (PCR) from rat genome and cloned into pGL3-basic (FasL-Luc). A10 cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) with the above plasmids and luciferase assays were performed with commercial kit (Promega, Madison, WI, USA).

Western blotting

Cellular and mouse tissue proteins were extracted using radioimmunoprecipitation assay buffer. The proteins were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto polyvinylidene fluoride membranes. The membranes were blocked in Tris-buffered saline containing 5% non-fat milk for 2 hours. Anti-SIRT1 antibody (anti-mSirt1, Millipore, Temecula, CA, USA； anti-hSIRT1, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-FasL antibody (Santa Cruz Biotechno- logy Inc.), or anti-β-actin antibody (loading control, Sigma-Aldrich, St. Louis, MO, USA) were added overnight at 4°C. A horse radish peroxidase-conjugated secondary antibody was used for the electrochemiluminescence detection. Band intensities were analyzed by densitometry.

Reverse transcription-polymerase chain reaction (RT-PCR)

TRIzol reagent (Invitrogen) was used to extract the total RNA in cells according to the manufacturer's instructions, and reversely transcribed to cDNA as template for PCR. The primers used are as follows: human SIRT1 (hSIRT1), 5'-CTTCAGGTCAAGGGATGGTAT-3' (forward),5'-GCGTGTC- TATGTTCTGGGTAT-3' (reverse)； rat FasL,195'-GTCCTGTCC- TTGACACTTCAGTCTCC-3' (forward), 5'-TCAACTTCTTCTCC- TCCATTAGCACC-3' (reverse)； rat β-actin (internal control), 5'- GAGAGGGAAATCGTGCGTGAC-3' (forward), 5'-TAGAG- CCACCAATCCACACAGAG-3' (reverse). The fragment length of amplified products was determined using an agarose gel electrophoresis.

DNA analysis by flow cytometry

Cellular apoptosis was monitored by measuring hypodiploid DNA content. Adenovirus-infected VSMCs were cultured in serum-free DMEM for 24 hours, followed by stimulation with 10% FBS for 4 hours. Attached and floating VSMCs were harvested and fixed in cold 70% ethanol and then resuspended in staining buffer consisting of RNase and propidium iodide. DNA content was analyzed by flow cytometry using a FACStar flow cytometer (Becton Dickinson, Mountain View, CA, USA). Gating was set to exclude debris and cellular aggregates, and 10 000 events were counted for each analysis.

Statistical analysis

All statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Data were presented as means±SEM. Differences among groups were determined with two-way analysis of variance, followed by Bonferroni post hoc test. Comparisons between two groups were performed with unpaired t-test. P＜0.05 was considered statistically significant.

RESULTS

Endogenous SIRT1 expression in vascular smooth muscle

Western blotting showed that SIRT1 was expressed in both cultured rat aortic VSMCs (Fig. 1A) and arteries of WT mouse (Fig. 1B).

SIRT1 up-regulated FasL expression in injured carotid artery

Western blotting results showed efficient human SIRT1 overexpression in the carotid arteries of SMC-SIRT1 Tg mice. FasL protein level significantly increased in response to SIRT1 overexpression in injured carotid artery 1 day after ligation (Fig. 2).

SIRT1 up-regulated FasL expression in serum-stimu- lated VSMCs

Transduction of VSMCs with Ad-SIRT1 led to expression of GFP in ＞90% of the cells 48 hours after infection (Fig. 3A). Expression of human SIRT1 mRNA and protein in the infected cells was verified by RT-PCR and Western blotting (Fig. 3B and 3C). The mRNA and protein levels of FasL increased along with SIRT1 overexpression in VSMCs treated with 10% FBS for 1 hour and 4 hours, respectively (Fig. 3B and 3C).

Figure 1. Western blotting results of endogenous Sirtuin 1 (SIRT1) expression in primary rat vascular smooth muscle cells (VSMCs), cell lines of A10 and A7r5 (A) and arteries of wild-type (WT) mouse (B). β-actin was used as the loading control.

Figure 2. Effect of SIRT1 overexpression on Fas ligand (FasL) protein level in vivo. Western blotting shows pooled samples of carotid arteries from WT and smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) mice 1 day after ligation or the sham operation. For the sham group, the entire operation was performed excluding the ligation. Quantitative analyses of Western blotting results (normalized to the expression level of β-actin) are presented in the histogram. *P＜0.001 compared with ligated WT mice. Data are represented as means±SEM (the experiment was repeated thrice with 3 mice in each pool).

Implication of GATA-6 in the transcriptional regulation of FasL by SIRT1

Transcriptional factor GATA-6 co-transfecting in the luciferase assay increased the promoter activity of FasL, and the induction of the FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (Fig. 4). In contrast, SIRT1H363Y significantly relieved the enhanced GATA-6 transcriptional activation of FasL promoter by WT-SIRT1 (Fig. 4).

Apoptosis detection result of VSMCs with SIRT1 overexpression

As shown in Figure 5, infection of VSMCs with the adenoviral vector expressing SIRT1 or control GFP led to little cell death in serum-free medium. Furthermore, no significant difference of cell death was observed between the two groups in serum-free medium or treated with 10% FBS for 4 hours.

DISCUSSION

Figure 3. Effect of SIRT1 overexpression on FasL protein level in VSMCs. Rat aortic VSMCs were infected with adenoviral vectors expressing SIRT1 (Ad-SIRT1) or those expressing green fluorescent protein (Ad-GFP) for 24 hours, followed by serum starvation for 24 hours, then exposed to 10% fetal bovine serum (FBS) for 1 hour or 4 hours. A. fluorescence microscopic images of adenovirus-infected VSMCs, representative of 3 independent experiments； B. effect of SIRT1 overexpression on FasL mRNA level in quiescent VSMCs treated with 10% FBS for 1 hour； C. effect of SIRT1 overexpression on FasL protein level in quiescent VSMCs treated with 10% FBS for 4 hours. *P＜0.05, **P＜0.001 compared with Ad-GFP-infected VSMCs treated with 10% FBS. Data are presented as means±SEM (n=3).

Figure 4. Effects of GATA-6, WT-SIRT1, and SIRT1H363Y (containing a CAT to TAT base substitution at position 363) on the rat FasL promoter activity. A10 cells were transfected with individual test plasmids in the absence or presence of WT-SIRT1/SIRT1H363Y while co-transfecting with GATA-6. PcDNA3.1 was transfected as a control. The relative luciferase activity, after normalizing to pcDNA3.1 activity, was compared among each group. *P＜0.001 compared with pcDNA3.1 control； #P＜0.001 compared with pcDNA3.1+GATA-6 control； †P ＜ 0.001 compared with WT-SIRT1+GATA-6 group. Data are presented as means±SEM (n=3).

Figure 5. Flow cytometry result showing the hypodiploid DNA content in VSMCs infected with adenoviral vectors expressing green fluorescent protein (Ad-GFP) or human SIRT1 (Ad-SIRT1) following serum starvation for 24 hours and then serum stimulation for 4 hours. Data are presented as means±SEM (n=3).

In this study, we found that SIRT1, a longevity gene, was expressed in both cultured VSMCs and mouse arteries. Forced expression of SIRT1 increased FasL expression both in flow-restricted mouse carotid arteries and VSMCs treated with serum. Moreover, SIRT1 facilitated the activation of FasL promoter by GATA-6. In contrast, expression of SIRT1H363Y, a catalytically inactive SIRT1 protein, relieved the enhanced GATA-6 transcriptional activation of FasL promoter by SIRT1. These findings suggest that SIRT1 might participate in the regulation of FasL expression at the transcriptional level.

FasL is a death factor that induces apoptosis in Fas-bearing cells. FasL gene transfer to the vessel wall was reported to inhibit intimal hyperplasia after balloon injury via pro-apoptotic and anti-inflammatory effects.1,4,20A previous study by our group has demonstrated that SIRT1 inhibits neointima formation after injury by reducing VSMC proliferation and migration.13In the present study, FasL expression was found up-regulated by SIRT1 both in vitro and in vivo, suggesting that FasL might be involved in the repressing effect of SIRT1 on neointima formation. Given that no notable cell death or significant difference of apoptosis was observed between SIRT1 overexpression group and control both in vivo13and in vitro, the pro-apoptotic effect of FasL may not contribute to the neointima formation suppression by SIRT1. Activation of Fas/FasL also exerts anti-inflammatory effects that are independent of apoptosis.1Therefore, other effects exerted by FasL may contribute to the inhibitory effect of SIRT1 on neointima formation, though the effects still need to be further investigated.

Besides the post-translational regulation,21FasL gene expression is also controlled by distinct protein-DNA interactions at the FasL promoter.3A number of transcriptional factors have been characterized to regulate FasL promoter activity in some certain cell types, including NFAT, NF-κB, AP-1, Sp1, Ets-1, Egr-1/2/3, IRF-1, c-Myc, FKHRL1, and c-Jun.3GATA-6, the major GATA factor expressed in cultured VSMCs, maintains the differentiated VSMC phenotype following vascular injury.22Like FasL and SIRT1, forced expression of GATA-6 in injured carotid arteries inhibits neointima formation.22Moreover, two potential GATA binding elements in the FasL promoter have been identified and GATA-6 was demonstrated to regulate FasL transcription through one (-298TTATCA-303) of them.23In the present study, SIRT1 was found to enhance the activation of FasL promoter by GATA-6. FasL was up-regulated by SIRT1 in both flow-restricted mouse carotid arteries and serum-stimulated rat VSMCs. Given that GATA-6 is transiently expressed in balloon-injured rat carotid arteries23and transiently up-regulated in serum-stimulated rat VSMCs,24GATA-6 might contribute to the transcriptional regulation of FasL by SIRT1 both in vivo and in vitro. The results of this study also showed that SIRT1H363Y abolished the enhanced GATA-6 transcriptional activation of FasL promoter by SIRT1, suggesting that the deacetylase activity of SIRT1 may be involved in the transcriptional regulation of FasL by SIRT1. Trichostatin A, a potent histone deacetylase inhibitor, was reported to abolish angiotension II (Ang II)-mediated GATA-6 binding to the authentic FasL promoter.23In our previous study, SIRT1 was found to interact with GATA-6 in vitro and inhibit GATA-6-induced Nox1 expression in VSMCs.25Hence, whether SIRT1 can deacetylate GATA-6 to mediate the transcriptional regulation of FasL still needs to be determined in the future. Considering that there are a number of transcriptional factors involved in neointima formation and regulation of FasL promoter activity, further work will be necessary to demonstrate detailed mechanism in the regulatory process of FasL expression by SIRT1.

ACKNOWLEDGEMENTS

We would like to thank Rui-feng Yang, Ran Zhang, Guo-wei Zhao, and Shuang Zhou for their technical assistance in this study.

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