Introduction

Cardiovascular system-related diseases are the most common diseases in the elderly population, and one of the major factors associated with the development of these diseases is vascular aging [1]. Vascular aging is closely related to sharp increases in age-dependent diseases of the cardiovascular system and is expected to be a new direction for the prevention and treatment of cardiovascular disease. The main effects of vascular aging include increased vascular stiffness, decreased compliance, and arterial wall thickening [1]. Most studies on vascular aging-related diseases have focused on the vascular intima and media, mainly targeting vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) [2, 3]. However, the importance of the vascular adventitia is increasingly being recognized not only because of its role in supporting blood vessels but also because it has important immune and endocrine functions and plays a key role in the secretion of extracellular matrix components such as collagen, the regulation of vascular stiffness, and the maintenance of vascular elasticity [4,5,6]. Adventitial fibroblasts (AFs) have an important connection to resident medial VSMCs in terms of phenotypic conversion, proliferation, apoptosis, and migratory properties, which contribute to neointima formation and vascular remodeling [5].

Sirtuin 6 (SIRT6), a NAD-dependent protein deacetylase, is a member of the evolutionarily conserved sirtuin family and plays an important role inregulating senescence and lifespan [7,8,9]. SIRT6 participates in various cellular functions, such as DNA repair, genomic stability, transcriptional control, proliferation, differentiation, and metabolism [10]. SIRT6 protects vascular ECs from angiotensin II (Ang II)-induced apoptosis and oxidative stress [11]. SIRT6 also inhibits TNF-α-induced inflammation in vascular AFs through the ROS and Akt signaling pathways [12]. Moreover, SIRT6 shows protective effects against vascular aging [13]. ECs express high levels of SIRT6, and endothelial SIRT6 deficiency accelerates replicative senescence [14].

NF-κB is a key transcription factor involved in the regulation of apoptosis, cell senescence, inflammation, immunity, and aging. Studies have indicated that SIRT6 suppresses certain NF-κB target genes by modifying the chromatin structures of their promoter regions. SIRT6 knockdown in bone marrow stem cells activates NF-κB transcriptional activity and increases the level of acetyl-NF-κB p65 [7]. Subsequently, haploinsufficiency of the NF-κB RELA subunit can extend the lifespan of SIRT6-deficient mice to more than 3 months and attenuate the degenerative phenotypes of SIRT6-deficient animals [15]. SIRT6 mediates these effects via the deacetylation of H3K9-Ac on the promoters of NF-κB target genes, leading to the destabilization of NF-κB on these promoters. In addition, this deacetylation decreases NF-κB-dependent apoptosis and senescence [15]. However, whether SIRT6 and NF-κB participate in adventitia-induced vascular aging remains unknown. Thus, this study aimed to gain a better understanding of the role of SIRT6 in suppressing vascular aging induced by AFs.

Materials and methods

Cell isolation, culture, and identification

AFs were isolated and cultured from the thoracic aortae of 3- to 6-week-old male Sprague-Dawley (SD) rats. The rats were sacrificed, and the full-length aorta was excised from the thoracic cavity and placed in precooled (4 °C) phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin. After the loose connective tissue and collateral vessels were removed, the adventitia was isolated and digested for 20 min by a neutral protease (2 U/ml) at 37 °C. Then, the samples were rapidly cut into small pieces and placed in a 25 cm2 tissue culture flask with DMEM supplemented with 20% FBS and 1% penicillin/streptomycin and incubated at 37 °C with 5% CO2 until the cells reached 70–80% confluence. The isolated AFs were detached by trypsinization and seeded onto new dishes in DMEM containing 10% FBS. The purity of these cultured AFs was 95%, as determined by positive immunocytochemical staining for vimentin and negative staining for desmin and α-smooth muscle actin (α-SMA). Cells that underwent 3–6 population doublings were used in subsequent experiments [7, 16, 17].

All rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The rats were treated ethically. The animal experiment was approved by the Experimental Animal Ethics Committee of Shandong University.

Lentivirus infection

Lentivirus infection was performed as described previously [18].The lentivirus targeting the rat SIRT6 gene sequence 5′-GCCGTCTGGTCATTGTCAA-3′ was synthesized by GeneChem (Shanghai, China). After infection and screening, 8 μg/ml puromycin was added to the cell culture medium to knock down SIRT6.

Cell proliferation assay

For cell proliferation assays, an EdU kit (cat.C0088L; Beyotime Institute of Biotechnology, Shanghai, China) was used to measure cell proliferation in accordance with the manufacturer’s instructions.5-Ethynyl-2′-deoxyuridine (EdU) is similar to thymidine in structure and will insert into DNA during DNA synthesis. EdU can then be labeled with biotin. Then, HRP-streptavidin, TMB, and H2SO4 were added, and the OD450 was measured to determine cell proliferation.

Briefly, AFs were cultured in growth medium in 96-well plates at an initial density of 5 × 103 cells per well and incubated at 37 °C under 5% CO2 for 72 h. The cells were washed with PBS and incubated with 10 μM EdU in the culture medium at room temperature for 2 h. After the addition of the click reaction buffer, HRP-streptavidin solution, TMB, and H2SO4, the resulting absorbance was measured at 450 nm with a spectrophotometer (M5, MD).

Migration assay

The migration of AFs was assessed using a wound healing assay. Briefly, AFs were seeded in the wells of 6-well plates. Twenty-four hours later, the cells were wounded using a 20-μl tip. Photographs were captured at 0, 24, and 48 h under a microscope (IX81, Olympus).

Collagen detection

Rat collagen type I and type III expression was measured using an ELISA kit (Cusabio, China) in accordance with the manufacturer’s instructions. Briefly, 100 μl of supernatant was added to the well and incubated for 2 h at 37 °C. Then, the liquid was removed from each well, and 100 μl of biotinantibody (1×) was added to each well and incubated for 1 hat 37 °C. Each well was aspirated and washed. Then, 100 μl of HRP-avidin (1×) was added to each well and incubated for 1 h at 37 °C. The aspiration/washing process was repeated five times. Next, 90 μl of TMB substrate was added to each well and incubated for 15–30 min at 37 °C. Subsequently, 50 μl of Stop Solution was added to each well. The optical density of each well was measured within 5 min using a microplate reader (M5, MD) at 540 nm.

Western blot (WB) analysis

Cells were lysed in RIPA buffer (Beyotime, China), and the supernatant was collected. The protein concentration was determined using a BCA protein assay kit (Beyotime, China). Approximately 20 μg of protein was separated by SDS-PAGE and wet-transferred onto a PVDF membrane. Nonspecific reactivity was blocked using 1% bovine serum albumin. The blots were incubated with primary antibodies against SIRT6 (1:1000; Abcam), NF-κB p65(1:1000; Cell Signaling Technology), α-SMA (1:1000; Santa Cruz Biotechnology), acetyl-NF-κB p65 (Lys310; 1:1000; Cell Signaling Technology), GAPDH (1:2000; Santa Cruz Biotechnology), and histone H3 (1:1000; Abcam) overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies (1:5000; Cwbio) were used to detect the bound antibodies. The blots were developed using ECL reagent (Cwbio, China) and X-ray film. The protein bands were visualized using a LiDE 100 scanner. A representative immunoblot from three independent experiments with similar results is shown for each WB experiment. Densitometric analysis of the WBs was performed using the AlphaEaseFC-v4.0.0 program.

NF-κB luciferase assay

AFs were cultured in a 24-well plate and cotransfected with pNF-κB-luc and internal control pRL-TK plasmids (Beyotime, China) in serum- and antibiotic-free DMEM. After 6 h, the cells were rinsed with PBS and incubated for an additional 6 h in DMEM containing 10% FBS. Firefly and Renilla luciferase activities were assessed using a luciferase assay kit in accordance with the manufacturer’s protocol (Beyotime, China).

Coimmunoprecipitation (co-IP) assay

A co-IP assay was performed in accordance with a previously described protocol [19] and the instructions of the immunoprecipitation assay kit (Cat. 11719394001, Millipore, USA). Briefly, cells were lysed and solubilized and subsequently sonicated before being clarified in a microcentrifuge. Afterward, 50 μl of sample was collected as the input, and the remaining lysate was subjected to overnight immunoprecipitation with anti-p65 antibodies (ChIP Grade, Abcam) and protein A/G beads. Immunocomplexes were collected by centrifugation and detected by WB analysis.

Histological evaluation

Wistar-Kyoto (WKY) rats were fed a standard diet at The Pharmacological Experimental Animal Center of Shandong University. The rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (300 mg/kg). The aorta was dissected and removed. Some arterial tissues were quickly fixed with 10% formalin, decalcified using 10% EDTA, and embedded in paraffin. Serial sections (5-μm thickness) were stained with hematoxylin and eosin. For collagen staining, the sections were stained with Sirius scarlet and Victoria blue solution and washed with acidified water before being dehydrated. For myofibroblast (MF) staining, the sections were stained with α-SMA (green) and vimentin (red) antibodies and DAPI (blue). Images were taken using a light microscope (CX41, Olympus). The levels of collagen and elastic fibers were quantified by the Image-Pro Plus image analysis system. All sections were reviewed and scored by two independent pathologists who were blinded to the status of the samples.

Statistical analysis

The data are presented as the mean ± SEM and were analyzed by two-tailed Student’s t-tests or one-way ANOVA. Multiple comparisons between groups were performed using the S-N-K method. P values < 0.05 were considered statistically significant. Statistical analysis was conducted with SPSS 17.0 software (SPSS, Inc., Chicago, Illinois).

Results

SIRT6 expression is decreased in aging rat aortae

To identify the relationship between SIRT6 expression and vascular aging, young (16 weeks old) and aged (56 weeks old) WKY rat thoracic aortae were collected to examine wall thickness, collagen staining, and protein expression. Vessel wall thickening and lumen narrowing in the aging rat aorta group were found compared with those in the young rat aorta group (Fig. 1A–D). Sirius scarlet-Victoria blue staining indicated that the collagen area was increased, and the ratio of elastic fiber to collagenous fiber was decreased in the aging rat aorta group (Fig. 1E–G). α-SMA+vimentin+ cells were identified as MFs. The staining data indicated that the aging rat aorta group had more MFs, indicating the transformation of AFs to MFs (Fig. 1H, I), which are involved in vascular remodeling. Moreover, SIRT6 expression was decreased in the adventitia of aging rats compared with that of young rats (Fig. 1J, K). These results suggest that the downregulation of SIRT6 expression in AFs contributes to vascular aging.

Fig. 1
figure 1

SIRT6 expression is decreased in aging rat aortic tissue. Young and aging rat aortic tissues were collected and embedded in paraffin. A–D Serialsections (5-μm thickness) were stained with hematoxylin and eosin (n = 10). Image J was used to determine the lengths. E–G: For collagen staining, the sections were stained with Sirius scarlet-Victoria blue solution (n = 10). Collagen fibers were dyed red, and elastic fibers were dyed blue-green. Image-Pro Plus was used to determine the areas. H and I Representative images of immunohistochemical staining of MFs in young (left) and aging (right) rat aortic tissues. The sections were stained with α-SMA (green) and vimentin (red) antibodies and DAPI (blue). The α-SMA+vimentin+ area was calculated using Image-Pro Plus (n = 5). J and K SIRT6 expression in the adventitia was detected by WB analysis. The results were obtained from representative experiments, and GAPDH was used as a control for normalization. The relative protein levels were calculated by AlphaEase FC and are shown (n = 4).Photographs were taken using an inverted microscope. The data are presented as the means ± SEM. *P ≤ 0.05, **P ≤ 0.01

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SIRT6 knockdown promotes the aging phenotype in AFs

To determine the function of SIRT6 in vascular aging, primary AFs were isolated from rat vascular adventitial tissue. There was almost no α-SMA or desmin expression (data not shown); however, vimentin was strongly expressed (Fig. 2A). These data suggested that the isolated cells were vascular AFs.

Fig. 2
figure 2

SIRT6 knockdown promotes an aging phenotype in AFs. A. AFs were isolated from rat vascular adventitia. The cells were stained with anti-vimentin (green) and DAPI (blue) in an immunofluorescence assay. Bar = 20 μm. AFs were infected with lentivirus. After puromycin screening, AFs with stable SIRT6 knockdown were obtained (B). Then, cell proliferation, collagen secretion, migration, and α-SMA expression were measured using the EdU assay (C), ELISA (D, E), the wound healing assay (F), and WB analysis (B), respectively. G Photographs were taken using an inverted microscope (200×). Bar = 100 μm. The WB results were obtained from three repeated experiments, and GAPDH was used as a control for normalization. The relative protein levels are shown. The data are presented as the means ± SEM. *P ≤ 0.05, **P ≤ 0.01

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A lentivirus targeting SIRT6 mRNA was used to downregulate SIRT6 expression. After puromycin screening, the WB results indicated that SIRT6 expression was markedly downregulated (Fig. 2B). Subsequently, cellular aging phenotypes were analyzed. The EdU assay showed that the cells in the SIRT6-knockdown group grew quickly compared with those in the control group (Fig. 2C). SIRT6 suppression also promoted collagen I secretion and cell migration (Fig. 2D, F, and G). Moreover, α-SMA expression was also upregulated in SIRT6-knockdown cells (Fig. 2B). However, SIRT6 knockdown decreased the secretion of collagen III, which contributes to vascular elasticity (Fig. 2E). Taken together, these results suggest that SIRT6 downregulation induces a vascular adventitial senescence phenotype.

SIRT6 knockdown facilitates Ang II-mediated promotion of vascular aging

Ang II plays a critical role in inducing vascular senescence [20]. However, whether Ang II participates in SIRT6-related vascular aging remains to be elucidated. Ang II promoted proliferation, collagen I secretion, migration, and α-SMA expression in rat AFs (Fig. 3A, B, D, and E) and decreased SIRT6 expression and collagen III secretion (Fig. 3C, E). Lentivirus-mediated SIRT6 downregulation promoted proliferation, collagen I secretion, migration, and α-SMA expression in rat AFs compared with those in the vector control group (Fig. 3F, G, I). SIRT6 knockdown also decreased collagen III secretion (Fig. 3H). Moreover, SIRT6 suppression strengthened the effect of Ang II on the proliferation, collagen secretion, and migration of rat AFs (Fig. 3F–I). Taken together, these data indicated that lentivirus-mediated SIRT6 suppression strengthened the effect of Ang II on the induction of vascular aging.

Fig. 3
figure 3

SIRT6 knockdown facilitates the effect of Ang II on the induction of vascular aging. AFs were treated with 100 nM Ang II for 48 h. Then, proliferation (A), collagen secretion (B, C), migration (D), and protein expression (E) were analyzed. SIRT6-knockdown AFs were also used to examine proliferation (F), collagen secretion (G, H), and migration (I) and compared with those of vector control AFs. The WB results were obtained from three repeated experiments, and GAPDH was used as a control for normalization. The relative protein levels are shown. The data are presented as the means ± SEM. *P ≤ 0.05, **P ≤ 0.01

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Ang II induces vascular aging by activating the NF-κB signaling pathway

The NF-κB signaling pathway plays an important role in vascular aging. Ang II and the NF-κB pathway also contribute to malignant hypertensive nephrosclerosis [21]. However, whether Ang II induces vascular aging through this pathway in the rat vascular adventitia remains to be elucidated. Therefore, AFs were treated with Ang II, and the vascular aging phenotype was analyzed. The data indicated that Ang II could induce proliferation, collagen I secretion, and migration in AFs (Fig. 4A–C). Moreover, collagen III secretion was suppressed by Ang II treatment (Fig. 4D). To identify the role of the NF-κB signaling pathway in this process, the NF-κB pathway inhibitor BAY 11–7082 was used [22]. BAY 11–7082markedly reduced the effect of Ang II on the proliferation, collagen secretion, and migration of AFs (Fig. 4A–D). Mechanistically, Ang II promoted the translocation of p65 from the cytoplasm to the nucleus (Fig. 4E). BAY 11–7082 reduced the effect of Ang II on the induction of p65nuclear translocation (Fig. 4E). Taken together, these data indicate that Ang II activates the NF-κB signaling pathway, leading to an aging phenotype in the rat vascular adventitia.

Fig. 4
figure 4

Ang II activates the NF-κB signaling pathway, leading to vascular aging. AFs were pretreated with the NF-κB pathway inhibitor BAY 11–7082 (1 µM) before being treated with 100 nM Ang II for 48 h. Then, AFs and supernatants were collected to examine proliferation (A), collagen secretion (B, C), and migration (D). AF nuclei were isolated, and nuclear p65 expression was analyzed by WB analysis (E). The WB results were obtained from three repeated experiments, and histone H3 was used as a control for normalization. The relative protein levels are shown. The data are presented as the means ± SEM. *P ≤ 0.05, **P ≤ 0.01

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SIRT6 knockdown contributes to Ang II-induced vascular aging in rats by activating the NF-κB signaling pathway

Whether SIRT6 suppresses the NF-κB signaling pathway to reduce Ang II-induced vascular aging remains unknown. To clarify this issue, we treated SIRT6-knockdown cells with Ang II, BAY 11–7082, or a combination of BAY 11–7082 and Ang II. Our data indicated that compared with the vector control, SIRT6 downregulation contributed to Ang II-induced rat vascular adventitial aging and affected AF proliferation, collagen secretion, migration, and α-SMA expression (Fig. 5A–E). Moreover, BAY 11–7082 reduced the effect of SIRT6 suppression on the Ang II-induced vascular aging phenotype (Fig. 5A–E). These data suggest that SIRT6 exerts its effects by inhibiting the NF-κB pathway. To further elucidate the functional relationship between SIRT6and the NF-κB pathway, we examined the expression of acetyl-NF-κB p65 (Lys310) and NF-κB transcriptional activity. SIRT6 suppression increased the expression of acetyl-NF-κB p65 (Lys310) and promoted NF-κB transcriptional activity in SIRT6-knockdown AFs compared with vector control AFs (Fig. 5F, G). However, BAY 11–7082 reduced the effect of SIRT6 knockdown on the NF-κB pathway via acetyl-NF-κB p65 (Lys310) expression and NF-κB transcriptional activity (Fig. 5F, G). To determine whether SIRT6 physically interacts with the NF-κB p65 subunit, AF lysates were immunoprecipitated with anti-p65 antibodies. WB analysis of the immunoprecipitates (IPs) revealed that p65 physically associated with SIRT6 (Fig. 5H). Taken together, these results suggest that SIRT6 knockdown contributes to Ang II-induced rat vascular aging by activating the NF-κB signaling pathway.

Fig. 5
figure 5

SIRT6 knockdown facilitates Ang II-induced rat vascular aging by activating the NF-κB signaling pathway. SIRT6-knockdown and vector control AFs were pretreated with BAY 11–7082 (1 µM) before being treated with 100 nM Ang II for 48 h. Then, AFs and supernatants were collected to examine proliferation (A), collagen secretion (B, C), migration (D), and α-SMA expression (E). SIRT6-knockdown and vector control AFs were collected or transfected with pNF-κB-luc or the internal control plasmid pRL-TK to examine the expression of acetyl-NF-κB p65 (Lys310) (F) and NF-κB transcriptional activity (G). AF lysates were immunoprecipitated with anti-p65 antibodies. WB analysis was used to measure SIRT6 in the IP complexes (H). The WB results were obtained from three repeated experiments, and GAPDH was used as a control for normalization. The relative protein levels are shown. The data are presented as the means ± SEM. *P ≤ 0.05, **P ≤ 0.01

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Discussion

In the 21st century, the aging of the world population has become a heavy burden for developed and developing countries. With increasing age, the morphology and function of the cardiovascular system undergo a series of aging-associated changes. The characteristic remodeling of the vascular structure and function associated with aging is called vascular aging. Vascular aging includes adventitial remodeling, such as thickening of the adventitia, fibroblast proliferation, and the accumulation and disorder of collagen fibers. In this study, Sirius scarlet-Victoria blue double staining showed that in aged rats, the arterial wall was significantly thickened; the red collagen fibers had severely proliferated, especially in the middle layer; the multilayered elastic fiber plates were destroyed and exhibited a disordered structure; and the ratio of elastic fiber to collagen fiber was decreased. Moreover, in young rats, the morphology of the arterial wall was normal, the structure of the blue-green elastic plate was clear, and the distribution of red collagen fibers was normal (Fig. 1E–G).Cell and rat model experiments were performed, and the use of the two different strains of rats for direct comparisons is a limitation of the present study. However, we do not think this affects the conclusion of this study because WKY and SD rats have been used to research vascular aging (WKY rats [23, 24], SD rats [25, 26]).

According to the traditional concept, vascular injury is mainly characterized by medial proliferation and the migration of VSMCs. Therefore, research on vascular remodeling has focused on ECs and VSMCs, which are mainly associated with endothelial injury, repair, and hypertrophy or the proliferation of smooth muscle cells in the middle layer, ignoring adventitial and vascular fibroblasts. Recent studies have shown that the adventitia plays a more important role in vascular function than merely supporting and nourishing blood vessels [27]. The role of the adventitia and AFs in the development of vascular remodeling has attracted increasing attention. The adventitia is considered the most sensitive region to injury factors, followed by the intima and media. It is not only an important participant in vascular remodeling but also the initiating and “leading factor” of intimal and medial injury and can even initiate vascular injury [28]. In atherosclerosis, hypertension, and vascular injury, AF sare activated and differentiated into MFs, which can migrate and proliferate in the media and neointima, accompanied by an increase in collagen and elastic fiber synthesis, which exacerbates vascular remodeling. A number of follow-up studies confirmed [29,30,–31] that AFs are activated earlier than VSMCs. During the development of lesions, the proliferative activity of AFs was significantly stronger than that of middle VSMCs, and the expression of α-SMA was positive, which indicated that AFs transformed into MFs [32]. MFs show stronger proliferation, migration, collagen synthesis, and secretion than AFs, resulting in wall thickening, lumen stenosis, and pathological vascular remodeling [5, 33]. Given that vimentin is a marker of fibroblasts andα-SMA is a marker of VSMCs, an immunofluorescence double-labeling method was used to identify vascular wall cells: VSMCs only express α-SMA, while MFs coexpress vimentin and α-SMA. Our results showed that the number of MFs in the vascular wall of aged rats was significantly increased (Fig. 1H, I).Therefore, we focused on AFs as our research model.

Of the SIRT family members, SIRT6 is located on heterochromatin in the nucleus and is mainly involved in DNA repair, chromatin compaction, telomerase function, and genomic stability, indicating thatSIRT6is at the forefront of antiaging [34]. SIRT6 protects ECs from premature aging by maintaining their ability to replicate and form blood vessels in vitro [35]. Recent evidence shows that SIRT6 has a protective effect on vascular endothelial dysfunction by inhibiting apoptosis and oxidative stress induced by Ang II [11, 13, 36]. SIRT6 overexpression protects ECs from apoptosis and oxidative stress induced by oxygen and glucose deprivation and reperfusion [37]. According to the latest research results on the sirtuin family and the particularly important role of SIRT6 in delaying aging, this study established a rat model of different ages by collecting vascular adventitia and fibroblasts as the breakthrough point. During cell culture, the endogenous expression of SIRT6 in AFs and the changes in SIRT6 expression in MFs induced by SIRT6 were examined for the first time.SIRT6-mediated improvements in blood vessels has been discussed from many aspects to investigate the importance of SIRT6 in delaying physiological and pathological vascular aging.

In this study, we isolated and cultured rat vascular AFs and found that SIRT6 played an important role in rat vascular aging. This is the first study connecting SIRT6 and the adventitia in the context of vascular senescence. Vascular senescence includes adventitial remodeling, such as thickening of the adventitia, fibroblast proliferation, and the accumulation and disorder of collagen fibers. Type I collagen acts as a scaffold and is related to the tensile strength of the vascular wall. Type III collagen determines the diameter and elasticity of the collagen fibers and is related to the degree to which the vasculature can expand. Lentivirus-mediated SIRT6 knockdown promoted AF proliferation, collagen I secretion, and migration (Fig. 2B–D, F). SIRT6 knockdown also decreased collagen III secretion (Fig. 2E). These findings are consistent with the vascular aging phenotype. In vascular aging, AFs transform into MFs, which are involved in vascular remodeling and the expression of α-SMA [32]. SIRT6 knockdown also increased α-SMA expression (Fig. 2B). Our findings are consistent with those of previous studies showing that SIRT6 inhibits cardiac fibroblast differentiation into MFs, which prevents Ang II-induced cardiac fibrosis and pathological cardiac remodeling [38]. Ang II treatment downregulates SIRT6 expression [39, 40]. Our data also suggested that Ang II suppressed SIRT6 expression, and SIRT6 knockdown facilitated the effect of Ang II on the induction of vascular aging (Fig. 3E–I).

Ang II plays an important role in inducing vascular remodeling. Age-induced NF-κB signaling contributes to cardiovascular risks [41]. Our data indicated that Ang II could activate the NF-κB signaling pathway, leading to vascular aging (Fig. 3A–D). Deficiency in mammalian SIRT6 leads to a shortened lifespan and an aging-like phenotype in mice [15]. SIRT6 could bind to p65 physically, and its downregulation increased acetyl-NF-κB p65 expression and NF-κB transcriptional activity and facilitated NF-κB signaling, leading to an aging phenotype (Fig. 5). The mechanism may also involve SIRT6-induced deacetylation at the NF-κB-related gene promoter region, which remains to be studied in our future research.

Conclusions

Taken together, our data indicated that SIRT6 knockdown could lead to vascular aging. The data from aging rat aortic tissue also support our hypothesis. In the aging rat group, the vascular wall was thickened, and collagen fibers were increased. Moreover, AFs were activated, and these cells transformed into MFs. SIRT6 expression was downregulated compared with that in the young rat group. Therefore, SIRT6 maybe a biomarker of vascular aging, and activating SIRT6 maybe a therapeutic strategy to delay vascular aging.