Chemico-Biological Interactions

Dipeptidyl peptidase-4 inhibition prevents vascular aging in mice under chronic stress: Modulation of oxidative stress and inflammation

Minglong Xin, Xianglan Jin, Xiangdan Cui, Chunzi Jin, Limei Piao, Ying Wan, Shengnan Xu, Shengming Zhang, Xueling Yue, Hailong Wang, Yongshan Nan, Xianwu Cheng
PII: S0009-2797(19)31334-1

Original Article
Dipeptidyl peptidase-4 Inhibition Prevents vascular aging in mice under chronic stress: Modulation of oxidative stress and Inflammation

Minglong Xina, MD; Xianglan Jina, MD; Xiangdan Cuia, MD, PhD; Chunzi Jina, MD, PhD; Limei Piaoa, MD, PhD; Ying Wana, MD; Shengnan Xua, MD; Shengming Zhanga, MD; Xueling Yuea, MS; Hailong Wanga, MD; Yongshan Nana, MD, PhD;
Xianwu Cheng1*, MD, PhD, FAHA
aDepartment of Cardiology and Emergency, Yanbian University Hospital, Yanji, Jilin P.R., 133000, China
Abstract: 207 words; Text:3998 words; Figures: 5; Sup. Figs. S1–S2 Running title: DPP-4-I alleviates stress-related vascular aging

*Corresponding author: Prof. Xian Wu Cheng, Department of Cardiology and Hypertension, Yanbian University Hospital, 1327 Juzijie, Yanji 133000, China. Email: [email protected] or [email protected]

ABSTRACT

Background and aims: Chronic psychosocial stress is a risk factor for cardiovascular disease. In view of the important role of dipeptidyl peptidase-4 (DPP-4) in human pathophysiology, we studied the role of DPP-4 in stress-related vascular aging in mice, focusing on oxidative stress and the inflammatory response.
Methods and Results: Male mice were randomly divided into a non-stress group and an immobilization stress group treated for 2 weeks. Chronic stress accelerates aortic senescence and increases plasma DPP-4 levels. Stress increased the levels of gp91phox, p22phox, p47phox, p67phox, p53, p27, p21, p16INK4A, vascular cell adhesion molecule-1, intracellular adhesion molecule-1, monocyte chemoattractant protein-1, matrix metalloproteinase-2 (MMP-2), MMP-9, cathepsin S (Cat S), and Cat K mRNAs and/or protein in the aorta of the stressed mice and decreased their levels of endothelial nitric oxide synthase and SirTuin1 (SirT1). DPP-4 inhibitors can improve stress-induced targeting molecules and morphological changes. In vitro, the inhibition of DPP-4 also alleviated the changes in the oxidative and inflammatory molecules in response to hydrogen peroxide in human umbilical vein endothelial cells.
Conclusions: DPP-4 inhibition can improve vascular aging in stressed mice, possibly by improving oxidative stress production and vascular inflammation. Our results suggest that DPP-4 may become a new therapeutic target for chronic stress-related vascular aging in metabolic cardiovascular diseases.
Key words: chronic stress, vascular aging, oxidative stress, inflammatory reaction, aorta

Introduction

The first step in aging is vascular endothelial senescence. Vascular aging involves structural and functional changes, and it occurs in different tissues of the vascular wall, including endothelial cells, smooth muscle cells and connective tissue. As an early pathophysiological change, vascular aging plays an important role in the occurrence and development of atherosclerosis. Vascular aging accelerates the aging process of the body. Not only does vascular aging easily develop into atherosclerosis; it also contributes to many diseases that seriously threaten human health and life [1].
Vascular aging progresses into atherogenesis, and the damage to organs should not be underestimated, especially considering vital organs such as the heart, brain and kidney. The incidence and mortality of atherosclerosis-based cardiovascular diseases (ACVDs) are increasing with the aging of the world’s population. It is therefore of great significance to strengthen the research on vascular aging for the prevention and treatment of ACVD. Based on experimental and clinical evidence, chronic psychological stress (CPS) is considered a risk factor for vascular aging and ACVD [2,3]. CPS activates extracellular and intracellular pathways (including the hypothalamus-pituitary-adrenal axis and the sympathetic nervous system) and leads to stress-related ACVD by inducing excessive pathophysiological responses [4–11]. The mechanisms underlying CPS-related vascular aging remain unclear.
Dipeptidyl peptidase-4 (DPP-4) is a serine protease that acts as a membrane-anchored extracellular peptidase, degrading a large number of cytokines, chemokines, hormones and growth factors under physiological and pathological conditions [12]. DPP-4 (also known as CD26) splits dipeptides such as glucagon-like peptide-1 (GLP-1) from the N-terminal of a polypeptide containing proline or alanine. GLP-1 is a highly effective intestinal insulin-stimulating hormone that is secreted by

intestinal L cells in the presence of nutrients [13]. DPP-4 inhibitors are widely used in the treatment of type 2 diabetes mellitus as anti-hyperglycemic drugs. These drugs inhibit DPP-4, thereby increasing the concentration of GLP-1. Several studies have shown that DPP-4 inhibitors such as sitagliptin and anagliptin have multipotency in the management of ACVDs [14–18]. Clinical studies showed that the serum levels of DPP-4 were increased in patients with coronary artery disease with or without diabetes mellitus [19,20]. Chronic variable pressure can increase DPP-4 levels in plasma and tissues [21,22]. Although many research findings have indicated that DPP-4 plays a key role in the occurrence and development of ACVD, little is known about the role of DPP-4 in CPS-related vascular aging.
In this study, we investigated the hypothesis that increased DPP-4 activity may negatively modulate vascular aging via an enhancement of oxidative production and inflammation in mice that have been subjected to chronic stress.
Methods

Animals

Eight-week-old male BALB/c mice (22–25 g weight) were purchased from the Animal Center of Yanbian University, Jilin, China. All mice were maintained in a 22°C room with a 12-hr light/dark cycle. The mice were given a standard mouse chow and drinking water ad libitum. The experimental protocol was approved by the Animal Care Committee of the Ethics Committee on Animal Research at Yanbian University. All animal laboratory experiments were conducted in accordance with those committees’ guidelines on animal care.

Mouse restraint stress protocols

First, for the evaluation of the impact of chronic stress on vascular aging,

8-week-old male mice (n=16) fed the ordinary diet were randomly assigned to either the non-stress group or the stressed group. Restraint stress was conducted as described [23]. Non-stressed control mice were allowed contact with each other and left undisturbed, whereas the stressed mice were subjected to a 4-hr session in an immobilization stress tube (Cat. 551-BSRR; Natsume Seisakusho, Tokyo) once daily (between 10:00 a.m. and 2:00 p.m.) for 2 weeks.
In separate DPP-4 inhibition experiments, male mice fed the ordinary diet were randomly assigned to one of two groups and given (by oral gavage) vehicle (distilled water, Stress) or the DPP-4 inhibitor anagliptin (30 mg/kg/d, S-Ana, a generous gift from Sanwa Kagaku Pharmaceutical, Mie, Japan; n=15 for each group) twice daily for 2 weeks under continued daily 4-hr immobilization stress.
Sample collections

At the final day of the 2-week stress protocol, all mice were given an intraperitoneal injection with an overdose of urethane (2 mg/kg; Sinopharm Group, Shanghai, China). The mice were then perfused with phosphate-buffered saline (PBS) under physiological pressure, and the heart and aorta were collected. The whole aorta, together with the heart, was isolated from the proximal ascending aorta to the bifurcation of the iliac artery. Following the removal of adventitial adipose tissues, the entire aorta was separated into several parts as follows: The aortic root was fixed with 4% paraformaldehyde for β-galactosidase (β-Gal); others parts (thoracic aorta and abdominal aorta) were stored at −80°C for Western blotting assays or kept in RNAlater Solution for the targeted gene assays.

Plasma DPP-4 level analysis

The DPP-4 levels of the mice were evaluated by using the DPP-4 Glo Protease

Assay (Promega, Madison, WI) with an aminoluciferin substrate as described [23]. In brief, the luminogenic substrate containing the Gly-Pro sequence was cleaved by DPP-4. After the DPP-4 cleavage, the substrate is released, resulting in the luciferase reaction and the production of light. For the blood DPP4 activity assays, the plasma was isolated using VENOJectII vacuum blood collection tubes containing anticoagulants without serine protease inhibitor (Terumo, Tokyo) and then diluted in
0.1 mM Tris-HCl buffer (pH 8.0) by 30-fold.

Equal amounts of protein (200 µg/25 µL) and diluted plasma (25 µL) were subjected to a DPP4 Glo assay (Promega) in the presence or absence of the DPP4 inhibitor anagliptin (20 µmol/L). Human recombinant DPP4 (Sigma-Aldrich, St. Louis, MO) was used to drive a standard curve. The luminescence intensity was calculated using a POWERSCAN4 (BioTek Instruments, Winooski, VT) as described [19]. The anagliptin-sensitive value (i.e., the absence value minus the presence value as an absolute value of the DPP4 activity) in relative light units per mL of plasma was calculated with the standard curve to represent the DPP4 level (ng/mL) [24].

Gene expression assay

Total RNA was isolated from the lysates of the aortic tissues with the use of the RNeasy Fibrous Tissue Mini-Kit (Qiagen, Hilden, Germany) and was subjected to reverse transcription [25]. The quantitative real-time polymerase reaction chain (RT-PCR) analysis was performed with primers specific for p22phox, p47phox, gp91phox, p67phox, endothelial nitric oxide synthase (eNOS), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), metalloproteinase-9 (MMP-9), MMP-2, tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, cathepsin S (CatS), and CatK with the use of an ABI 7300 PCR System (Applied Biosystems, Foster City, CA) [22]. The

expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured in parallel to that of the genes of interest and was used as an internal standard for the quantitative comparison of mRNA levels. The primer sequences are listed in Supplementary Table S1.

Senescence-associated β-Gal staining

We performed β-Gal staining as described [26]. The aorta specimens were fixed for 1 hr in the fixative solution (Cat. K320-250, Senescence Detection kit; Wako Pure Chemical Industries, Osaka, Japan). After being washed twice with PBS, the aortas were incubated with staining solution containing X-Gal (40 mg/mL) at 37°C for the senescence-associated β-Gal activity assay according to the manufacturer’s protocol. The vessels were then incised longitudinally along the artery. Positive areas of β-Gal staining in the aortas were calculated using a BZ-X700 microscope and BZ-X analyzer (Keyence, Osaka, Japan).

Western blot analysis

The total protein was isolated with lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% NP-40 or Triton-100, and fresh 1× protease inhibitor cocktail; pH 7.4). The protein concentration was examined with a BCA protein assay kit (Solarbio Life Sciences, Beijing). The same amounts of protein (40 µg) were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were incubated overnight with primary antibodies against p16INK4A (10883-1-AP, Proteintech, Rosemont, IL; 1:1,000), p21 (ab109199, abcam, Cambridge, MA; 1:1,000), p22phox (sc-271968, Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000), p27 (#2552), p53 (#2524), p-AktSer473 (#4060), SIRT1 (#8469,

Cell Signaling Technology, Boston, MA; 1:1,000), GAPDH (E-AB-20072, Elabscience Biotechnology, Wuhan, China; 1:2,000), gp9phox (611415), eNOS (610296, BD Transduction Laboratories, San Jose, CA; 1:1,000) and then incubated with the related secondary antibodies at a 1:2,000–5,000 dilution. The SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA) was used for the evaluation of the targeted proteins. The levels of the targeted proteins quantified by Western blots were normalized by loading GAPDH levels.

Endothelial cell culture and experiments

Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA) and cultured in endothelial basal medium-2 (EBM-2) plus 10% fetal bovine serum (FBS) and endothelial growth medium-2 (EGM-2) and SingleQuotes (Clonetics) in a humidified atmosphere of 5% CO2 and 95% air. After being cultured in serum-free EBM-2 for 12 hr, the HUVECs were used for the following experiments.
(1) First, the cells were cultured in the presence or absence of H2O2 (0, 200, 400 µmol/L) in EBM-2 medium for 24 hr and then subjected to a Western blot analysis for the examination of eNOS expression. (2) Second, the cells pretreated with anagliptin (10, 30 µmol/L) at the indicated concentrations for 30 min were cultured in the presence or absence of H2O2 (400 µmol/L) in EBM-2 medium for 24 hr and then subjected to cellular assays (Western blot/RT-PCR). (3) Third, in order to explore the mechanism underlying the induction of eNOS/p-Akt expression by DPP-4 inhibitors under oxidative stress, we pretreated HUVECs with or without two inhibitors, i.e., the phosphatidylinositol-3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002, 20 µmol/L) and the

janus kinase (JAK)/STAT3 pathway inhibitor tyrphostin (AG490; 5 µmol/L; all from APExBIO Technology, Houston, TX). The cells were then cultured in the presence or absence of anagliptin (30 µmol/L)/H2O2 (400 µmol/L) for 24 hr and subjected to Western blotting for the examination of eNOS/p-Akt expression. For all cell culture assays, at least three independent experiments were performed in triplicate.
Statistical analyses

The results are expressed as means ± standard error of the mean (SEM). Student’s t-test (for comparisons between two groups) or a one-way analysis of variance (ANOVA; for comparisons of three or more groups) followed by Tukey’s post-hoc test were performed. All parameter calculations were conducted by two observers blinded to the treatment of the mice. After the status of the data distribution was determined, the data were subjected to the statistical analyses. A p-value <0.05 was considered significant.

Results

Effects of chronic stress on the body weight, plasma DPP-4 level, and aortic aging
Compared to the non-stressed group of mice, the body weights of the mice and the amounts of subcutaneous and inguinal adipose were reduced by stress (Fig. 1A,B). The stressed mice had increased levels of plasma DPP-4 compared to the control mice (Fig. 1C). The β-Gal staining revealed that the stressed aortas had increased senescence-associated expressions of β-galactosidase activity (Fig. 1D,E), indicating that chronic stress accelerated vascular aging, accompanied by increased levels of plasma DPP-4.

The impact of the stress on oxidative stress, inflammation, and proteolysis

As the second step in studying the relationship between stress and oxidative stress and inflammation, we analyzed the expression of oxidative stress- and inflammation-related genes in the aorta of mice after 14 days of immobilized stress. The quantitative PCR data showed that the levels of oxidative stress-related genes (p22phox, p47phox, p67phox, gp91phox) and inflammatory genes (ICAM-1, VCAM-1 and MCP-1) were higher in the stressed mice compared to the non-stress mice (Fig. 2A,B), whereas the levels of eNOS were lower than those of the control (non-stress) mice (Fig. 2C). We also observed that the levels of proteolysis-related genes (CatS, CatK, MMP-2, MMP-9, TIMP-1, and TIMP-2) in the stressed mice were higher than those in the non-stress mice (Fig. 2D–F). The Western blotting analysis showed that the stress decreased the expressions of p-eNOSSer1177 and SirT1 protein in the aorta, whereas the stress increased the expressions of p16INK4A, p21, p22phox, p27, p53, gp91phox, p-AktSer473 protein (Suppl. Fig. S1A–C).

DPP-4 inhibition prevented stress-related oxidative stress production and inflammation
Compared to the mice that received stress alone, the DPP-4 inhibitor anagliptin decreased the weights of the subcutaneous and inguinal adipose and the plasma DPP-4 levels and vascular senescence in the mice (Fig. 3A–C). As shown in Figure 4A,B, the levels of target genes (ICAM-1, VCAM-1, MCP-1, P22phox, P47phox, p67phox, and gp91phox) were lower in the aortas of the S-Ana mice compared to the levels in the stress-alone mice. Anagliptin also decreased the levels of CatS, CatK, MMP-2, MMP-9, TIMP-1, and TIMP-2 genes and increased the levels of eNOS genes

in the mouse aorta (Fig. 4C–F). As expected, the targeted protein changes (p16INK4A, p21, p22phox, p27, p53, gp91phox, p-AktSer473, eNOS, and SirT1) were sensitive to DPP-4 inhibition (Fig. 3D–F).

Effects of oxidative stress on endothelial cells

In vitro, HUVECs were exposed to H2O2 for 24 hr, and eNOS protein was detected. The decrease of eNOS expression in HUVECs by H2O2 was dose-dependent (Suppl. Fig. S2A,B). As shown in Figure 5A, H2O2 treatment (400 µmol/L) markedly decreased the eNOS gene expression in the HUVECs. Conversely, H2O2 increased the levels of NADPH oxidase subunits (gp91phox, p22phox), inflammatory chemokines (ICAM-1, VCAM-1 and MCP-1), and proteolytic enzymes (MMP-2, MMP-9, CatS and CatK) (Fig. 5A,B).
Effects of DPP-4 inhibitor pretreatment on the oxidative stress of endothelial cells
To further study the effects of DPP-4 inhibitor in vitro, we pretreated HUVECs with different concentrations of anagliptin under oxidative stress conditions. Cell detection was performed 24 hr later. The decrease in the eNOS mRNA expression induced by H2O2 was ameliorated by DPP-4 inhibition in a dose-dependent manner (Fig. 5A). DPP-4 inhibitor also dramatically inhibited the expressions of gp91phox, p22phox, VACM-1, ICAM-1, MCP-1, CatS, and CatK genes (Fig. 5A,B). As expected, the expression of eNOS protein was also improved, and the expressions of p16INK4A and p21 protein were inhibited (Fig. 5C,D).
We next used both the phosphatidylinositol 3-kinase inhibitor (PI3K) LY294002 and the janus kinase/signal transducer and activator of transcription 3

(Jak3/Stat) inhibitor AG490 to explore the mechanisms underlying DPP-4 inhibition-mediated improvement of eNOS expression in HUVECs. Pretreatment with LY294002 diminished the beneficial effects of anagliptin on eNOS expression and AktSer473 phosphorylation in HUVECs (Suppl. Fig. S2C,D). By comparison, AG490 had little effect on either of these targeted molecule changes (Suppl. Fig. S2C,D).
Discussion
We investigated the role of DPP-4 in vascular senescence in mice under chronic stress, with a special focus on oxidative stress and inflammation in vivo and in vitro. Our experiments revealed the significant finding that chronic stress increased the plasma DPP-4 levels in the mice. This increase raised the expressions of oxidative stress- and inflammation-related genes in the aortas, thereby accelerating vascular aging. This change also resulted in an increase in the expressions of proteolysis-related targeted MMP and cathepsin family members and vascular senescence-related targeted molecules, producing a harmful vascular metabolic change. DPP4 inhibition ameliorated the chronic stress-related aging under our experimental conditions. In the in vitro experiments, the DPP-4 inhibitor mitigated the oxidative stress-induced eNOS expression by activating the PI3K/Akt signaling pathway.
NADPH oxidase, which produces peroxides, is present in phagocytes, neuroepithelial cells, vascular smooth muscle cells, and endothelial cells. It consists of a membrane-bound yellow cytochrome containing two subunits, gp91phox and p22phox, as well as the cytoplasmic proteins p47phox and p67phox. Genetic and pharmacological interventions targeting NADPH oxidase subunits have been shown to be able to improve the development of diet-induced atherosclerosis [27]. In the

present investigation we observed that the expressions of gp91 phox, p22 phox, p67 phox and p47 phox mRNA and the expression of gp91phox and p22phox in aortic tissue were significantly increased under chronic stress.
Beta-galactosidase and p53 are markers related to aging [28]; p53 is the upstream regulator of p21 and p16INK4A [29]. Our observations from the β-Gal staining and the expressions of p16INK4A, p53 and p21 proteins in the Western blotting assay indicate that chronic stress accelerated the process of vascular senescence. This notion was further supported by the in vitro experiments of cultured HUVECs revealing that oxidative stress increased the expressions of p16INK4A and p21 proteins. We therefore speculated that the enhancement of oxidative stress may contribute to the aggravation of vascular aging via the modulation of p53-mediated p16INK4A/p21 signaling activation in mice under our experimental conditions.
eNOS is beneficial to various vascular diseases [30,31]. DPP-4 inhibitors can increase the phosphorylation of eNOS in the heart and muscle of mice [32–34]. The decrease of eNOS activity in aging endothelial cells is due to the decrease in eNOS protein expression and the Akt-mediated phosphorylation of eNOS [35]. β-Gal staining of the aorta in the present study’s stress group showed that the aging of blood vessels was clear, whereas DPP-4 inhibition alleviated it. The levels of eNOS mRNA and p-Akt protein in the aortas of the stressed mice were significantly decreased; these changes were reversed by DPP-4 inhibitor treatment. In vitro, DPP-4 inhibition prevented the harmful changes in those molecules in HUVECs. Moreover, LY294002 abolished the anagliptin-mediated beneficial effects on the eNOS expression and p-Akt phosphorylation in cultured HUVECs, but AG490 had no effect. Collectively, our results demonstrated that the DPP-4 inhibition-mediated vascular protective effect is not due to Jak3/Stat signaling pathway activation but rather to PI3K signaling

pathway activation.

The functional integrity of the vascular system, including membrane transport and barrier functions, depends on normal cellular energy metabolism. The nicotinamide adenine dinucleotide (NAD+)-dependent pre-survival enzyme SIRT1 (SirTuin1) regulates mitochondrial function in the vascular system and controls mitochondrial biogenesis, mitochondrial reactive oxygen species (ROS) production and cellular energy metabolism [36–38], and it removes damaged mitochondria [39] through autophagy. Oxidative stress can increase mitochondrial damage [40]. SIRT1 has been shown to be involved in the regulation of cardiovascular function. The overexpression of SIRT1 has been shown to inhibit oxidative stress-induced endothelial aging, and the inhibition of SIRT1 can lead to premature aging [41]. SIRT1 also has anti-inflammatory effects, and a decrease in SIRT1 activity may lead to vascular inflammation during aging. [39,42]. The biological activity of SIRT1 can alleviate vasculitis in aged mice [43]. At the same time, chronic stress can enhance the inflammatory process in vascular tissue [44,45]. The expression of SIRT1 protein in the aorta of the mice in the present study’s stress group was decreased; the expression of SIRT1 protein was increased in the S-Ana mice, and the aging of blood vessels improved. We also observed that the expression of inflammation-related factors (MCP-1, ICAM-1, VCAM-1) in the aorta of the stressed mice was increased whereas that of the S-Ana mice was decreased. We therefore favor the hypothesis that a stress-mediated decrease in SIRT1 had a salutary effect on the vasculature by promoting vascular inflammation, mitochondrial damage, and dysfunction in the mice under our experimental conditions.
It is well known that the synthesis and degradation of the extracellular matrix is critical to the maintenance of vascular integrity. The degradation of the extracellular

matrix is largely dependent on the members of the MMP family, especially MMP-2 and MMP-9 [45–47]. Genetic and pharmacological inhibitions of MMP-2 and MMP-9 prevented vascular remodeling and neointimal hyperplasia after injury [48]. In addition to these MMPs, cathepsin family members, especially CatS and CatK, are also involved in elastolysis and collagen dissolution activities during the onset and progression of atherosclerosis, and they participate in vascular wall matrix degradation [25,49]. We observed that chronic stress increased the expressions of proteolytic enzyme genes (MMP-2, MMP-9, TIMP-1, TIMP-2, CatS and CatK) in the aorta of mice. Similarly, HUVECs treated with H2O2 also showed the enhanced expression of protein hydrolase mRNA in vitro. Anagliptin mitigated the expressions of the targeted enzymes in response to oxidative stress both in vivo and in vitro. Thus, the vasculo-protective actions of the DPP-4 inhibitor were mediated, at least in part, through the reduction of MMP-2/-9 and CatS/K-mediated proteolysis.
In conclusion, chronic stress increased the plasma concentration of DPP-4 in mice, resulting in oxidation production, inflammation and protein hydrolysis, leading to vascular senescence. A pharmacological intervention targeted toward DPP-4 activity ameliorated the chronic stress-related vascular aging associated with the modulation of oxidative stress production, inflammatory over-actions and proteolytic activity. This is the first study to demonstrate the beneficial action of anagliptin in chronic psychological stress (CPS)-related vascular aging, providing a potential pharmacological treatment alternative.

Conflicts of interest

The authors declare that they have no conflicts of interest to disclose with respect to this manuscript.
Financial Support
This work was supported in part by grants from the National Natural Science Foundation of China (nos. 81560240, 81460082, 81660240, and 81770485) and a grant from the Young Investigator Foundation Cathepsin Inhibitor 1 of Yanbian University (no. 2016-30).
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Figure Legends

Fig. 1. Two weeks of the immobilized stress increased the plasma levels of DPP-4 and the aortic vascular aging. A: Body weights in the experimental groups at days 0 and 14 after chronic stress. B: Representative photos show the abdominal subcutaneous fat and inguinal fat. C: The levels of plasma DPP-4 in the two experimental groups at 2 weeks of stress. D: β-Gal+ staining of the aortic root in mice of the non-stress and stress groups. E: The β-Gal+ staining area was calculated in the aortic roots. Scale bars: 50 µm. Results are mean ± SEM (n=6–8). *p<0.05, **p<0.001 vs. the non-stressed group by one-way ANOVA followed by Tukey post hoc tests or Student’s t-test.

Fig. 2. Stress increased the expressions of the oxidative stress-, inflammation- and proteolysis-related genes in the aortas of the stressed mice. A–F: Quantitative PCR data show the levels of p22phox, p47phox, p67phox, gp91phox, MCP-1, VCAM-1, and ICAM-1, eNOS, CatK and CatS, MMP-2 and MMP-9, TIMP-1 and TIMP-2 mRNAs.
Results are mean ± SEM (n=6–8). *p<0.05, **p<0.001 vs. non-stressed group by one-way ANOVA followed by Tukey post hoc tests.

Fig. 3. Anagliptin alleviated vascular aging and the levels of the related proteins in mice exposed to chronic stress for 2 weeks. A: The levels of DPP-4 in plasma during 2 weeks of stress. B: β-Gal+ staining of aortic root in the non-stress and stress groups. C: The β-Gal+ staining area was calculated in the aortic roots. D–F: Representative Western blots and quantitative data show the levels of eNOS, SirT1, p-AktSer473, p22phox, gp91phox, p53, p27, p22phox, p21 and p16INK4A proteins in the aortas of both experimental groups. Results are mean ± SEM (n=4). **p<0.001 vs. non-stressed group by Student’s t-test or one-way ANOVA followed by Tukey post hoc tests.

Fig. 4. DPP-4 inhibition reduced the levels of the oxidative stress-, inflammation- and proteolysis-related genes in stressed mice. A–F: PCR results showing the levels of all

targeted genes (eNOS, p22phox, p47phox, p67phox, gp91phox, MCP-1, VCAM-1, and ICAM-1, MMP-2 and MMP-9, TIMP-1 and TIMP-2, CatK and CatS). Results are mean ± SEM (n=4–8). *p<0.05, **p<0.001 vs. the non-stressed group by one-way ANOVA followed by Tukey post hoc tests or Student’s t-test.

Fig. 5. The effects of anagliptin on the targeted genes in HUVECs in response to oxidative stress. After being cultured in serum-free EBM-2 for 12 hr, the HUVECs pretreated with anagliptin (10, 30 µmol/L) at the indicated concentrations for 30 min were cultured in the presence or absence of H2O2 (400 µmol/L) in EBM-2 medium for 24 hr. A,B: PCR results showing the levels of all targeted genes (eNOS, p22phox, gp91phox, MCP-1, VCAM-1, and ICAM-1, MMP-2, CatK and CatS). C,D: The expression of eNOS, p-AktSer473, p16INK4A and p21 in cells. The results are mean ± SEM (n=4–9). *p<0.05, **p<0.01 vs. corresponding controls by one-way ANOVA followed by Tukey post hoc tests.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: