SIRT1 and FOXO Mediate Contractile Differentiation of Vascular Smooth Muscle Cells under Cyclic Stretch

Vascular smooth muscle cells (VSMCs) in the media of arterial wall are exposed to mechanical cyclic stretch in vivo, which is caused by lumen pressure [1]. It has been shown that physiological cyclic stretch inhibits VSMC migration, proliferation, and apoptosis and maintains the contractile differentiation of VSMCs, which contributes to vascular homeostasis [2]. However, the underlying mechanism/signal transduction pathways remain unclear.

VSMCs changes from a contractile state to a synthetic state, which plays important roles in vascular remodeling during hypertension and atherosclerosis [3,4]. In mature vessels, VSMCs mostly have a differentiated contractile state with highly expressed contractile markers, including smooth muscle myosin heavy chain (SM-MHC), SM α-actin, SM22α, calponin, and smoothelin, which are associated with the contractile function of VSMCs [5]. VSMCs with differentiated contractile state have low proliferation ratio and migration ability [6]. However, in response to pathological stimulus, such as injury, hypertension and atherosclerosis, VSMCs de-differentiated from a contractile to a synthetic state, which plays a critical role in arterial remodeling [7].

It has been shown that various intracellular signaling pathways are involved in stretch-induced VSMC functions. For example, Rho family GTPases, mitogen-activated protein kinases (MAPKs) and PI3K/Akt initiate the differentiation, migration, and proliferation of VSMCs [8,9]. However, the molecular mechanism by which mechanical cyclic stretch modulates differentiation of VSMCs remains to be further elucidated.

SIRT1 is an important NAD⁺-dependent class III histone deacetylase, and it has been proved to play protective roles in vascular homeostasis [10]. SIRT1 modulates the differentiated response of VSMCs during development and following injury [11,12]. Pre-B-cell colony-enhancing factor (PBEF) regulates SIRT1 activity by increasing intracellular NAD⁺ via the salvage pathway and promotes VSMC differentiation into a contractile state [13]. In addition, SIRT1 appears to control the cellular responses by regulating the expression and activity of the Forkhead transcription factor (FOXO) family, such as FOXO3a and FOXO4 [14,15], which have been widely reported to be important molecules in modulating the functions of VSMCs.

Yang et al. reported that FOXO3a regulates the gene transcription of myocardin, which participates in VSMC phenotypic modulation [16]. FOXO3a and its downstream gene, apoptotic protease activating factor 1, play important roles in VSMC survival during vessel remodeling and atherogenesis [17]. FOXO4 and mTORC1 are involved in adiponectin-induced VSMC differentiation [18].

Although these studies have revealed that SIRT1, FOXO3a and FOXO4 play important roles in the functional regulation of VSMCs, the effects of SIRT1, FOXO3a and FOXO4 on the differentiation of VSMCs, especially in responsive to mechanical stretch, has not yet been clarified. The objective of this study was to determine whether the SIRT1/FOXO pathway is involved in the cyclic stretch-induced differentiation of VSMCs and to detect the molecular mechanisms that are involved in this process. These results elucidate the biomechanical mechanism that underlies vascular homeostasis and vascular remodeling.

The animal care and experimental protocols conformed to the Animal Management Rules of China (Documentation 55, 2001, Ministry of Health, China), and the study was approved by the Animal Research Committee of Shanghai Jiao Tong University.

Because Sirt1 homozygous knockout (Sirt1^-/-) mice have a low perinatal survival rate, Sirt1 heterozygous knockout (Sirt1^+/-) mice, provided as a gift from Michael W. McBurney at the Center for Cancer Therapeutics, Ottawa Hospital Research Institute, Canada, were used in our studies [19,20]. Mice were maintained under a specific pathogen-free condition in the vivarium facility of Shanghai Jiao Tong University with a 12 hours light/dark cycle (lights were turned on at 6:00 am). The offspring were genotyped from DNA obtained by a tail clip at weaning [19,20]. Male Sirt1^+/- mice, 18-20 g in weight, were used for further analysis.

VSMCs were harvested from rat thoracic aorta by an explanted technique [21]. Briefly, the media layer of thoracic aorta was isolated surgically and minced into small pieces that were plated onto 25 cm² culture flasks in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin, and incubated at 37°C with humidified 5% CO₂. VSMCs displayed the typical spindle-shaped morphology and ‘‘hill-and-valley'' pattern of growth, and they were characterized by immunohistochemical staining for smooth muscle-specific α-actin (Sigma). Cells between passages 4-8 were used.

Rat VSMCs were seeded on type I collagen-coated [21] flexible silicone membrane (Flexercell International, USA) at an initial density of 3 × 10⁵ cells per well (9.32 cm²). After seeding for 24 hours, the cells were starved with DMEM (0% FBS) for 24 hours to arrest growth and synchronize the cells. VSMCs were then subjected to cyclic stretch that was produced by a computer-controlled vacuum (FX-4000T Strain Unit, Flexercell International, USA) as previously described [21]. The following mechanical parameters were applied: stretch magnitude of 10%, frequency of 1.25 Hz, and duration of 24 hours. VSMCs cultured under the same conditions, but no mechanical stretch applied was used as the static control.

After cyclic stretch application, the attached VSMCs were fixed in 4% paraformaldehyde at room temperature for 20 minutes, permeabilized in 0.5% Triton X-100 on ice for 5 minutes, and blocked in 1% BSA at room temperature for 30 minutes. Then cells were incubated with SIRT1 antibody (1:100, Abcam) at 4°C over night. All samples were washed with PBS for 30 minutes and then incubated with an Alexa Fluor 488-conjugated secondary antibody (Cell Signaling Technology, 1:1000) and DAPI for 1 hour at room temperature. The samples were examined with a laser scanning confocal microscope (Olympus, LV1000).

Rat VSMCs were seeded at a density of 2.0×10⁵ cells per well in six-well plates and grown in DMEM with 10% FBS. After seeding for 24 hours, VSMCs were starved with DMEM (0% FBS) for 24 hours. Resveratrol (Sigma) was added to the DMEM culture medium at a concentration of 1 µM, 10 µM, 25 µM, 50 µM and 100 µM for 24 hours. Alcohol, which was the vehicle of resveratrol, was used as the control.

The mRNA sequence of rat SIRT1 (NM_001107627) was acquired from NCBI GenBank. Small interfering RNAs (siRNAs) targeting rat SIRT1 were designed and synthesized by GenePharma Biological Company (Shanghai, P.R. China). The sequences of siRNA for SIRT1 were 5'-GAU UUA UUA CCA GAA ACA ATT-3' and 5'-UUG UUU CUG GUA AUA AAU CTT-3'. The sequences of NC were 5'-UUC UCC GAA CGU GUC ACG UTT-3' and 5'-ACG UGA CAC GUU CGG AGA ATT-3'. Rat VSMCs were seeded at a density of 2.0 × 10⁵ cells per well in six-well plates and grown in DMEM with 10% FBS. After seeding for 24 hours, the cells were transfected with 100 nmol siRNA and 5 µL Lipofectamine™ 2000 (Invitrogen) for 6 hours according to the manufacturer's instruction. Non-silencing siRNA that does not recognize any known genes that are homologous to rat genes was synthesized as a negative control (NC).

pcDNA3-hSIRT1-FLAG mammalian expression plasmid was a kind gift from Dr. Fuyuki Ishikawa (Kyoto University, Graduate School of Biostudies). FLAG-FOXO4 (Addgene plasmid ID 17549) and pcDNA3 flag FKHRL1 AAA (Addgene plasmid ID 10709) plasmid were obtained from Addgene USA. Rat VSMCs were seeded at a density of 2.0 × 10⁵ cells per well in six-well plates, and they were transfected with X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's instructions. The transfection efficiency was confirmed by western blot. pcDNA3.1 empty vector was used as the control.

Mouse thoracic aortas were lysed with homogenizers, and cultured rat VSMCs were lysed for 5 minutes in lysis buffer at 4°C. Respective nuclear and cytoplasmic proteins from VSMCs were extracted by using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, China). Protein concentration was determined by an Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). 30 µg proteins (per lane) were subjected to electrophoretic separation by 10% SDS-PAGE and transferred to nitrocellulose membranes (Hybond, Amersham). Western blots were performed using antibodies directed against SIRT1 (1:300, Santa Cruz Biotechnology), FOXO4 (1:300, Cell Signaling Technologies), FOXO3a (1:500, Cell Signaling Technologies), SM22α (1:800, Abcam), smooth muscle α-actin (1:1000, Sigma), calponin (1:500, Sigma) and GAPDH (1:1000, Santa Cruz Biotechnology). After incubation with alkaline phosphatase-conjugated secondary antibodies (Jackson Immunoresearch), the signals were visualized by nitroblue tetrazolium-bromochloroindolyl phosphate (Bio Basic, Inc.), and quantified using Quantity One software (Bio-Rad).

The potential diseases and functions of FOXO3a or FOXO4 in cardiovascular system and the possible networks among FOXO3a, FOXO4 and SIRT1 were obtained by Ingenuity Pathway Analysis (IPA) software (http://www.ingenuity.com/products/ipa). The significance values for analyses were calculated by comparing the molecule that participates in a given function or pathway relative to the total number of occurrences in all functional/pathway annotations stored in the Ingenuity Pathways Knowledge Database Literature.

Each experiment was performed at least in triplicate, and all values are expressed as mean ± SD. Oneway ANOVA was used to compare the difference between the two groups followed by Fisher's t-test for multiple comparisons. Values of P < 0.05 were accepted as statistically significant.

Compared to the static control, 10% cyclic stretch significantly increased the expression level of SIRT1 in VSMCs (Fig. 1A). The expression of SIRT1 in nucleus and cytoplasm are both significantly increased (Fig. 1B, 1C, 1D), and the increase in the nucleus was more remarkable than that in the cytoplasm (Fig. 1B, 1C).

Our results revealed that a 10% mechanical stretch significantly increased the expression level of the transcription factor FOXO3a but decreased the expression level of FOXO4 (Fig. 2A, 2B). The expression levels of contractile VSMC markers, including α-actin, SM22α and calponin were all increased by the application of mechanical stretch (Fig. 2C) compared to the static control. Microscopic photograph revealed that after cyclic-stretch application, VSMCs were elongated and almost perpendicular aligned to the stretch direction (Fig. 2D).

These in vitro results revealed that a physiological stretch that is 10% cyclic stretch [21], maintains the VSMC contractile differentiation.

To further study the role of SIRT1, FOXO3a and FOXO4 in response to mechanical stretch, resveratrol, an activator of SIRT1 [22] was used to stimulate SIRT1 in VSMCs under static conditions. The results showed that VSMCs treated with resveratrol (1 µM) did not have a significant effect on the expression of SIRT1, whereas 10 µM, 25 µM, 50 µM and 100 µM Resveratrol all significantly increased the expression of SIRT1 (Fig. 3A). Furthermore, resveratrol (25 µM) treatment significantly increased the expression of FOXO3a (Fig. 3B), decreased the expression of FOXO4 (Fig. 3C), and increased contractile VSMC markers (Fig. 3D), indicating that resveratrol treatment increased the expression of FOXO3a and contractile protein markers but decreased the expression of FOXO4.

SIRT1 target siRNA transfection in VSMCs decreased the expression of SIRT1 (Fig. 4A), which repressed FOXO3a expression (Fig. 4B) but elevated FOXO4 expression (Fig. 4C). The expression of contractile markers was decreased by SIRT1 target siRNA transfection (Fig. 4D).

Furthermore, when VSMCs were transfected with SIRT1 siRNA and then subjected to a 10% cyclic stretch, SIRT1 siRNA decreased the expression of SIRT1 (Fig. 4E) and repressed the expression of FOXO3a (Fig. 4F), whereas the expression of FOXO4 was increased in this condition (Fig. 4G). The expression of contractile markers was decreased in this group (Fig. 4H).

Transfection with the SIRT1 overexpression plasmid revealed that the up regulated SIRT1 (Fig. 5A) increased the expression of FOXO3a (Fig. 5B) and contractile VSMC markers, i.e., α-actin, SM22α and calponin (Fig. 5D), but decreased the expression of FOXO4 (Fig. 5C).

These results suggest that SIRT1, whose expression was changed by mechanical stretch, has opposite effects on the expression of FOXO3a and FOXO4, which could subsequently regulate VSMC differentiation.

To further detect the changed expression of FOXO3a and FOXO4 upon VSMC differentiation, FOXO3a or FOXO4 overexpression plasmids were transfected into VSMCs under static condition. The data indicated that overexpressed FOXO3a (Fig. 6A) increased the expression of α-actin, SM22α and calponin (Fig. 6C), while overexpressed FOXO4 (Fig. 6B) decreased the expression of α-actin, SM22α and calponin (Fig. 6D). These results suggested that the altered expression of FOXO3a and FOXO4 plays different roles in VSMC differentiation.

Because Sirt1 homozygous knockout mice have a low perinatal survival rate [19,20], Sirt1 heterozygous knockout (SIRT1 ^+/-) mice were used to decrease the expression of SIRT1 and to further validate our findings in vivo. Compared with the wild type (SIRT ^+/+) mice (male), the expression of SIRT1 in thoracic aorta from SIRT1 ^+/- mice (male) was repressed (Fig. 7A). The expression of FOXO3a was repressed (Fig. 7B), but the expression of FOXO4 was significantly increased in VSMCs (Fig. 7C). The expression of contractile VSMC markers was also repressed (Fig. 7D) compared with that in the wild type mice.

Our results demonstrated that cyclic stretch increased the expression of SIRT1, FOXO3a and contractile VSMC markers, but decreased the expression of FOXO4. These increases were repressed by decreasing SIRT1 with SIRT1 siRNA transfection, whereas increasing SIRT1 by resveratrol or transfection of the SIRT1-overexpression plasmid increased the expression of the contractile markers. The results demonstrated that the cyclic stretch modulated VSMC differentiation was affected by the SIRT1-FOXO pathway (Fig. 8A) .

Blood vessels are subjected to mechanical stretch due to the pulsatile nature of blood flow, and mechanical stretch profoundly influences the orientation, morphology, migration, proliferation, apoptosis and phenotype of VSMCs [23,24]. Pathologically increased cyclic stretch contributes to vascular remodeling during aging and hypertension [25,26], whereas the physiological level of cyclic stretch is essential for maintaining the vascular wall structure and inhibiting the growth factor-stimulated proliferation of VSMCs [27]. Our present results showed that physiological cyclic stretch (10%-1.25 Hz) significantly increased the expression of VSMC contractile markers. The similar physiological responses of VSMCs are also revealed by culture of whole vascular tissue [26,28]. Therefore, physiological cyclic stretch is a protective stimulus to maintain the contractile differentiation of VSMCs and to maintain vascular homeostasis.

SIRT1, as an important class III histone deacetylase, participates in many cell functions, i.e., senescence, proliferation, differentiation, and apoptosis [2,29,30]. It has been shown that mammalian SIRT1 is involved in chromatin remodeling, gene silencing, DNA damage response [31], and caloric restriction [32]. Activation of SIRT1 induced by caloric restriction significantly attenuates age-related vascular oxidative stress and inflammation, and improves endothelial homeostasis [33,34]. Moderate overexpression of SIRT1 in the heart of transgenic mice attenuated age-dependent increases in cardiac hypertrophy, apoptosis/fibrosis, cardiac dysfunction, and the expression of senescence markers [35]. A recent study revealed that in VSMCs, SIRT1 protects against oxidized low-density lipoprotein-induced DNA damage and senescence and inhibits medial degeneration and atherosclerosis [36]. The results from these studies suggest a protective effect of SIRT1 on the cardiovascular system.

The new findings of our present study revealed that SIRT1 is a crucial mechano-responsive molecule. Kim et al. recently revealed that the expression of SIRT1 is regulated by microRNA-34a [37], which had been proved to be an important mechano-sensitive molecule in our previous work [38]. Interestingly, our unpublished data revealed that physiological cyclic stretch decreases the expression of microRNA-34a in VSMCs. Whether the decreased expression of microRNA-34a can target on SIRT1 and regulate its expression in VSMCs will be examined in the future.

SIRT1 has been proved to play critical roles in vascular homeostasis and remodeling [22,39]. Here, using SIRT1 activator-resveratrol, SIRT1 overexpression plasmid, specific siRNA transfection, and knockout mice, we revealed the protective role of SIRT1 in maintaining the normal contractile differentiation of VSMCs. Furthermore, physiological cyclic stretch increased SIRT1 expression, which subsequently modulated the expression of FOXO3a and FOXO4 and then the differentiation of VSMCs.

It has been revealed that SIRT1 deacetylates FOXOs, thus attenuating cell apoptosis and cell-cycle arrest [40]. Acetylated FOXO3a was deacetylated by SIRT1 in an NAD-dependent manner [40]. Treatment with nicotinamide, a SIRT1 inhibitor, results in the deacetylation of FOXO3 in 293T cells [41,42]. SIRT1 can also participate in FOXOs-induced cellular functions via modulating the expression of FOXOs. Our results showed that the expression of FOXO4 was decreased, while the expression of FOXO3a was increased by SIRT1. It has also been reported by other studies that other than deacetylation, the expression of FOXOs is also regulated by SIRT1 in several types of cells [14,15]. Recent research demonstrated that host cell factor 1 (HCF-1) participates in the SIRT1-regulated expression of FOXOs in worms [15].

FOXOs, as important transcriptional regulators, play important roles in the maintenance of vascular homeostasis and vascular stability [43]. FOXO3a is a negative transcription factor of CYR61 which is crucial for VSMC proliferation and neointimal hyperplasia [44]. Here we revealed a protective role of increased FOXO3a in maintaining the differentiation of VSMCs, but a pathological role of increased FOXO4. Liu et al. also reported that FOXO4 inhibits contractile genes expression in VSMCs [45]. IPA bioinformatics analysis suggested that FOXO3a and FOXO4 participates in various cellular functions in cardiovascular system, including cell cycle, cell death and survival, cellular assembly and organization, cellular growth and proliferation, cellular development, cell morphology, and et al. (Table 1, 2). Since contractile differentiated VSMCs shows a less intent to proliferate, migrate, and synthesize extracellular matrix (ECM), which contribute to maintain vascular homeostasis [2], the roles of FOXO3a and FOXO4 on VSMC functions, such as proliferation, migration, apoptosis and synthesize, need further demonstration.

Although our present research suggested that SIRT1 may be the upstream molecule of FOXO3a and FOXO4 in stretch-induced VSMC phenotypic modulation, the underlying mechanism in this process is complex and requires further study. Using IPA bioinformatics software, the potential relationships among SIRT1, FOXO3a and FOXO4 in mammal cells were revealed (Fig. 8B). It suggested that many transcription factors may participate in this regulating network, including cyclin-dependent kinase inhibitor 2B (CDKN2B), cyclin G2 (CCNG2), host cell factor C1 (HCFC1), host cell factor C2 (HCFC2), SIRT2, forkhead box G1 (FOXG1), and ovo-like zinc finger 1 (OVOL1). However, the effects of these transcription factors on the expression of FOXO3a and FOXO4 modulated by SIRT1 in VSMCs are still unclear, which need to be demonstrated in the future.

In conclusion, our studies suggest that the up regulation of SIRT1 by physiological cyclic stretch induced contractile differentiation of VSMCs, and the opposite variation of FOXO3a and FOXO4 are involved in this process. These results define a novel role of SIRT1 as an important regulator to maintain vascular homeostasis, and the results also raise the possibility that the activation of SIRT1 could be a useful strategy for treating pathological vascular remodeling in hypertension and other vascular diseases.

This research was supported by grants from the National Natural Science Foundation of China, Nos. 11232010, 10972120, 11229202 and 11172176.

We thank Dr. Fuyuki Ishikawa at the Graduate School of Biostudies, Kyoto University for the kind gift of the pcDNA3-hSIRT1-FLAG plasmid. We thank Dr. Domenico Accili at Addgene for providing the FLAG-FOXO4 plasmid and Dr. William Sellers at Addgene for providing the pcDNA3 flag FKHRL1 AAA. We also thank Michael W. McBurney at the Center for Cancer Therapeutics, Ottawa Hospital Research Institute, Canada for the kind gift of the Sirt1 transgenic mice.

None declared.