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Introduction

Diabetes mellitus is a common metabolic condition with signature high blood glucose. It is associated with increased risk of cardiovascular diseases (CVDs), and cardiovascular complication accounts for >80% of diabetic-related mortalities (1). High blood glucose is a major contributor to the development of diabetic cardiovascular complications (2). High glucose (HG) has been indicated to injure vascular endothelial cells and vascular smooth muscle cells (VSMCs), which are associated with the development of CVDs (3–5). It is well established that diabetes-induced vascular dysfunction and remodeling in multiple vascular beds may be related to the activation of the endothelin-1 (ET-1) system (6–8).

ET-1 is a potent vasoconstrictor. The endothelin type A (ETA) and endothelin type B (ETB) receptors are two types of G protein-coupled receptors, and through them, ET-1 induces strong and long-lasting vasoconstriction (9). In healthy arteries, the binding of ET-1 with ETA receptors on VSMCs mediates the main part of the vasoconstriction, while the ETB receptors are predominantly found on the endothelial cells and mediate vasodilatation (9,10). The ETB receptors located on endothelial cells are termed as vasorelaxant ETB receptors. However, under pathogenic conditions, the expression of ETB receptors is upregulated via transcriptional mechanisms on VSMCs and induce vasoconstriction instead, and these are termed vasoconstrictive ETB receptors (11). Previous study has demonstrated that ETB receptors were highly expressed on the vascular smooth muscle layer in a diabetic model (8).

Silent information regulator family protein 1 (Sirt1), a prominent member of a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases, has been reported to affect a wide range of biological functions involving regulation of metabolism, cell survival and organismal lifespan (12). Accumulating studies have demonstrated that Sirt1 serves a protective role in CVDs (13,14), diabetes and its complications (15,16). Diabetes in vivo or HG in vitro may suppress the expression of Sirt1 and its activity in VSMCs (17). Additionally, reduced expression or activity of Sirt1 in VSMCs may contribute to the development of vascular dysfunction and promote vascular aging and CVDs (18). Currently, the underlying molecular mechanisms of this remain unclear.

The present study was designed to determine whether Sirt1 is involved in HG-mediated regulation of ETB receptors in the superior mesenteric arteries (SMA) of rats. The present study may identify novel targets for the mechanism of diabetes-associated ischemic CVDs.

Materials and methods

Chemicals and drugs

Selective ETB receptor agonist sarafotoxin 6c (S6c), inhibitor for extracellular signal-regulated protein kinase 1 and 2 (ERK1/2; U0126) and resveratrol (Res; activator of Sirt1), as well as glucose, were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The inhibitor and Res were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO (vehicle) used in the experiments was 1 µl/ml, which equals the volume of the inhibitor added to the organ culture. The DMSO concentration was the same in all test conditions, and it was used in the organ culture without the inhibitor to serve as a control. S6c was dissolved in 0.9% saline with 0.1% bovine serum albumin (Sigma-Aldrich; Merck KGaA). Glucose was diluted in DMEM just prior to initiation of the experiments.

Tissue preparation and organ culture procedure

A total of 80 male Sprague-Dawley rats (age, 8 weeks; weight, 300–350 g) were obtained from the Laboratory Animal Center of Xi'an Jiaotong University (Xi'an, China). All rats were housed in a temperature-controlled (20±2°C) and humidity-controlled (40–70%) facility on a 12-h light/dark cycle. Rats had free access to food and water. Following euthanasia with CO2, the SMA was gently removed and freed from adhering tissue under a dissecting microscope. The endothelium was denuded by perfusion (4°C) of the vessel for 10 sec with Triton X-100 (0.1%, v/v) followed by another 10 sec with a physiologic buffer solution (NaCl 119 mM, KCl 4.6 mM, NaHCO3 15 mM, NaH2PO4 1.2 mM, MgCl2 1.2 mM, CaCl2 1.5 mM and glucose 5.5 mM). The vessels were then cut into 1–3-mm long cylindrical segments and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air in DMEM [containing L-glutamine (584 mg/l) and normal glucose (5.5 mM)] supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml) (Thermo Fisher Scientific, Inc.) (19,20). To mimic hyperglycemic conditions, the cylindrical segments were exposed for up to 24 h to HG (15 or 25 mM) DMEM (normal DMEM supplemented with HG concentrations) in a CO2 (5%) incubator at 37°C. Other experiments were performed under similar conditions in the absence/presence of inhibitor [U0126 (10 µM)] or Sirt1 activator [Res (10, 50 or 100 µM)], which were added to the medium prior to incubation.

The animal experiments in the present investigation were approved by the Laboratory Animal Administration Committee of Xi'an Jiaotong University and conformed to the Guidelines for Animal Experimentation of Xi'an Jiaotong University (20) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [NIH Publication no. 85–23, revised 2011 (21)].

In vitro pharmacology

The present experiments were performed according to a previously published protocol (20). Briefly, fresh or incubated cylindrical artery segments were immersed in temperature-controlled (37°C) myograph individual baths (Organ Bath Model 700MO; J.P. Trading, Aarhus, Denmark) containing 5 ml physiologic buffer solution. The solution was continuously gassed with 5% CO2 in O2, resulting in a pH of 7.4. The cylindrical arterial segments were mounted for continuous recording of isometric tension with LabChart 7 Pro software (ADInstruments, Hastings, UK). A resting tone of 2 mN was applied to each segment, and the segments were allowed to stabilize at this tension for at least 1.5 h before exposure to a potassium-rich (60 mM K+) buffer solution with the same composition as the standard solution, except that NaCl was replaced by an equimolar concentration of KCl. The potassium-induced contraction was used as a reference for contractile capacity, and the segments were used only if potassium elicited reproducible responses over 1.0 mN. Concentration-response curves for S6c, a selective ETB receptor agonist (10−11–10−7 M), were obtained by cumulative administration of the reagent.

Western blotting

Arterial segment lysate preparation and western blot analysis were performed as previously described (20). Briefly, following organ culture for 24 h, the arterial segments were lysed on ice for 1 h in radioimmuno-precipitation assay buffer [Tris-HCl (pH 8.0) 50 mM, NaCl 150 mM, 1% Triton X-100 (v/v), 1% deoxycholic acid (w/v) and 0.1% sodium dodecyl sulfate] containing 0.5 mM phenylmethylsulfonyl fluoride and protease inhibitors (Roche Diagnostics, Basel, Switzerland). Protein concentration was measured with a BCA protein assay kit (Thermo Fisher Scientific, Inc.). After being denatured by boiling for 5 min in Laemmli loading buffer (Beyotime Institute of Biotechnology, Haimen, China), equal amounts of protein (50 µg) were loaded and separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. To block non-specific binding, the membranes were incubated with 5% bovine serum albumin or non-fat dried milk for 1 h at 37°C. Subsequently, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies contained anti-phospho-p44/42 antibody (1:1,000; 4370; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p44/42 antibody (1:1,000; ab115799), anti-ETB receptor antibody (1:1,000; ab65972), anti-Sirt1 antibody (1:1,000; ab104833) and anti-β-actin antibody (1:1,000; ab8226) (Abcam, Cambridge, MA, USA). After being washed with Tris-buffered saline containing 0.1% Tween-20 (Beyotime Institute of Biotechnology), the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or -rabbit immunoglobulin G (1:1,000; 31430 and 31460; Thermo Fisher Scientific, Inc.) for 1 h at 37°C, followed by enhanced chemiluminescence using a SuperSignal West PicoSubstrate kit (Pierce; Thermo Fisher Scientific, Inc.) and analyzed using the ChemiDoc-it HR 410 imaging system (UVP, LLC, Phoenix, AZ, USA).

Statistical analysis

All data were expressed as the mean ± standard error of the mean. S6c-induced vasoconstriction data were presented as a percentage of contraction induced by 60 mM K+. Two sets of data were compared using the unpaired Student's t-test with Welch's correction or two-way analysis of variance (ANOVA) with Bonferroni post hoc test. One-way ANOVA with Dunnett's post hoc test was applied for comparisons of more than two data sets. Data analysis was performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of HG on ETB receptor-mediated vasoconstriction and ETB receptor protein expression in the SMA

In the fresh SMA ring, S6c induced only negligible contractions (Emax value, 5.12±1.05%). After organ culture alone for 24 h, S6c induced strong contraction of SMA in a concentration-dependent manner, with an Emax value of 35.38±6.79% and a pEC50 value of 6.57±0.33. Culture with different doses of HG (15 and 25 mM) further shifted the S6c-induced concentration-contraction curve of organ culture artery toward the left and significantly increased contractile responses to S6c, with an Emax of 58.46±6.60% and pEC50 of 7.31±0.17 for 15 mM HG, and an Emax of 67.58±8.04% and pEC50 of 7.51±0.15 for 25 mM HG, respectively (P<0.05) (Fig. 1A). This suggests that organ culture enhanced the contraction of the SMA induced by S6c. In addition, HG further enhanced the ETB receptor-mediated contraction of the SMA induced by S6c.

Figure 1 HG increases the receptor-mediated contractile response and ETB receptor protein expression. (A) After the rat SMA rings were cultured with dif-ferent doses of HG (15 or 25 mM) for 24 h, the contractile response curves induced by sarafotoxin 6c were measured and presented as a percentage of 60 mM K+-induced contraction. Data are presented as the mean ± SEM (n=6–8 artery ring segments). (B) ETB receptor protein expression levels were detected after 24 h of organ culture with HG (25 mM) in SMA without endothelium. Data are presented as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. culture; #P<0.05 and ##P<0.01 as indicated. HG, high glucose; ETB, endothelin type B; SMA, superior mesenteric artery; SEM, standard error of the mean.

There were no significant differences in the K+-induced contraction among the groups, and incubation with control in the concentration used did not affect the contractile response to S6c (data not shown).

The SMA segments were cultured for 24 h in the presence or absence of HG (25 mM). ETB receptor protein expression in vascular smooth muscles were assessed using western blotting. The results demonstrated that there were low levels of ETB receptor protein in fresh SMA segments. Organ culture alone induced an increase in the ETB receptor protein expression level compared to the fresh group; however, this did not achieve statistical significance. Furthermore, HG (25 mM) significantly elevated protein expression levels of ETB receptors in cultured SMA segments, compared with the culture alone group (P<0.05) (Fig. 1B).

Activating Sirt1 with Res blocks the HG-increased protein expression of ETB receptors and receptor-mediated vasoconstriction in the SMA

Culture with HG and different doses of Res (10, 50 or 100 µM) inhibited the HG-enhanced contractile responses to S6c, and decreased the Emax from 67.03±9.27% in HG-cultured artery to 33.56±9.87% (10 µM; P<0.05), 21.95±4.56% (50 µM; P<0.01) and 4.32±0.63% (100 µM; P<0.01) (Fig. 2A).

Figure 2 Activating Sirt1 with Res blocks the HG-increased receptor-mediated contractile response and ETB receptor expression. (A) After the rat SMA rings were cultured with HG (25 mM) in the presence or absence of different doses of Res (10, 50 or 100 µM) for 24 h, the contractile response curves induced by sarafotoxin 6c were measured and presented as a percentage of 60 mM K+-induced contraction. Data are presented as the mean ± SEM (n=6–8 artery ring segments). (B) ETB receptor protein expression levels were detected after 24 h of organ culture with or without HG (25 mM) in the presence or absence of Res (100 µM) in SMA. Data are presented as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. HG ± DMSO; ##P<0.01 as indicated. Sirt1, silent information regulator family protein 1; Res, resveratrol; HG, high glucose; ETB, endothelin type B; SMA, superior mesenteric artery; SEM, standard error of the mean; DMSO, dimethyl sulfoxide.

The ETB receptor protein expression in the SMA following organ culture with Res (100 µM) was examined using western blotting. Compared with the control group (culture alone), HG significantly upregulated the levels of ETB receptors (P<0.01) (Fig. 2B). However, Res (100 µM) significantly inhibited the HG-induced elevation of the ETB receptor protein levels (P<0.01) (Fig. 2B).

These findings suggest that HG-increased protein expression of ETB receptors and receptor-mediated contractile function were related to Sirt1 signaling pathways. Sirt1 activator may effectively inhibit HG-induced upregulation of ETB receptor protein expression and receptor-mediated contractile function.

ERK1/2 signaling pathway is involved in the upregulation of ETB receptor protein expression and receptor-mediated vasoconstriction induced by HG in the organ culture SMA

As demonstrated in Fig. 3, culture with HG and the inhibitor for ERK1/2 (U0126) significantly attenuated the HG-enhanced contractile responses to S6c, and decreased the Emax from 66.89±8.36% in HG-cultured artery to 16.44±5.51% (P<0.01) (Fig. 3A).

Figure 3 The ERK1/2 signaling pathway is involved in the upregulation of ETB receptor protein expression and receptor-mediated vasoconstriction induced by HG. (A) After the rat SMA segments were cultured with HG (25 mM) in the presence or absence of ERK1/2 inhibitor (U0126) for 24 h, the contractile response curves induced by sarafotoxin 6c were measured and presented as a percentage of 60 mM K+-induced contraction. Data are presented as the mean ± SEM (n=6–8 artery ring segments). (B) ETB receptor protein expression levels were detected after 24 h of organ culture with or without HG (25 mM) in the presence or absence of ERK1/2 inhibitor (U0126) in SMA. Data are presented as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. HG ± DMSO; ##P<0.01 as indicated. ERK1/2, extracellular signal-regulated protein kinase 1/2; HG, high glucose; ETB, endothelin type B; SMA, superior mesenteric artery; SEM, standard error of the mean; DMSO, dimethyl sulfoxide.

The level of phosphorylated ERK1/2 (p-ERK1/2) and ETB receptor protein expression in the SMA were examined following culture with or without HG (25 mM) in the presence of or absence of U0126. As demonstrated in Fig. 4, the level of p-ERK1/2 was low in the control group following organ culture for 24 h. Compared with the control group, HG (25 mM) significantly upregulated the levels of p-ERK1/2 (P<0.05) (Fig. 4B). However, the inhibitor for ERK1/2 (U0126) significantly reduced the increases in ETB receptor protein expression induced by HG (P<0.01) (Fig. 3B). Additionally, U0126 almost completely abolished the HG-induced upregulation of ETB receptor protein expression in SMA segments (Fig. 3B). These results suggest that HG activated the ERK1/2 signaling pathway, which resulted in upregulation of ETB receptor expression and receptor-mediated contractile function.

Figure 4 Activating Sirt1 with Res inhibits the HG-induced activation of the ERK1/2 signaling pathways. After the superior mesenteric artery rings were cultured with or without HG (25 mM) in the presence or absence of Res (100 µM) for 24 h, the protein expression levels of (A) Sirt1 and (B) p-ERK1/2 were detected. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05 and **P<0.01 as indicated. Sirt1, silent information regulator family protein 1; Res, resveratrol; HG, high glucose; ERK1/2, extracellular signal-regulated protein kinase 1/2; p-, phosphorylated.

Activating Sirt1 with Res inhibits the HG-induced activation of ERK1/2 signaling pathways

In order to further study the relationship between Sirt1 and ERK1/2 signaling pathways in HG-cultured rat SMA, Res (100 µM) was used for activating Sirt1, and then the level of p-ERK1/2 was detected using western blotting. The results demonstrated that the level of Sirt1 was relatively high in SMA segments following organ culture for 24 h. However, culture with HG significantly decreased Sirt1 protein expression compared with the level in culture alone (P<0.01) (Fig. 4A). Res significantly reversed the decrease in Sirt1 protein expression caused by HG (P<0.01) (Fig. 4A). In addition, Res also inhibited the increase in the level of p-ERK1/2 induced by HG in SMA segments (P<0.01) (Fig. 4B). These results indicate that the activation of Sirt1 could inhibit the HG-induced activation of ERK1/2 signaling pathways. Therefore, in the present study, HG upregulated ETB receptor expression and the receptor-mediated contractile response by downregulating the level of Sirt1 and activating the ERK1/2 signaling pathway in SMA (Fig. 5).

Figure 5 Schematic illustration of the signaling pathway involved in the effect of HG on ETB receptor expression and the receptor-mediated contractile response in rat SMA. HG downregulates the level of Sirt1 and activates the ERK1/2 signaling pathway, and subsequently induces the upregulation of ETB receptor expression and the receptor-mediated contractile response in rat SMA. HG, high glucose; ETB, endothelin type B; SMA, superior mesenteric artery; Sirt1, silent information regulator family protein 1; ERK1/2, extracellular signal-regulated protein kinase 1/2; p-, phosphorylated.

Discussion

The present study investigated the effect of HG on ETB receptor protein expression in VSMCs and its mechanisms. The results suggested that HG enhanced receptor-mediated contractile responses to S6c and increased the ETB receptor protein expression in rat SMA. Meanwhile, the level of Sirt1 was downregulated and the ERK1/2 signaling pathway was activated in the HG-cultured rat SMA. Additionally, the activation of ERK1/2 signaling pathways could be inhibited by Sirt1 activator. This suggested that HG decreased the level of Sirt1 protein and activated the ERK1/2 signaling pathway, which resulted in upregulation of ETB receptor protein expression and receptor-mediated vasoconstriction in rat SMA.

Phenotypic modification of VSMCs may occur in physiological and pathophysiological settings, which is the basis of VSMC vasomotion and proliferation (22). Study has demonstrated that ETB receptors were involved in phenotypic modification of VSMCs (11). Organ culture provides a model for exploring the mechanisms involved in upregulation of vascular smooth muscle ETB receptors (11). In the present study, organ culture of the SMA was also used as a model to investigate the effect of HG on ETB receptor expression in rat SMA. The present results demonstrated that HG significantly upregulated ETB receptor protein expression and the receptor-mediated vasoconstriction induced by S6c in the organ culture SMA. In endothelial cells, ETB receptors mediate vasodilatation via release of nitric oxide and prostacyclin (23). However, ETB receptors induce vasoconstriction in VSMCs, which may be related to the phosphatidylinositol 4,5-bisphosphate system (24). In addition, in the present study, the level of Sirt1 in HG-cultured rat SMA was significantly decreased. This suggested that Sirt1 may be involved in the process of HG increasing ETB receptor expression and the receptor-mediated vasoconstriction in the organ culture SMA.

Sirt1 has been implicated in the process of aging, metabolism and tolerance to oxidative stress (25). In recent years, Sirt1 has been implicated in the pathogenesis of CVDs (26). Additionally, studies have recently identified that Sirt1 could improve VSMC functions (27) and serve a protective role in CVDs (13,14). Sirt1 deficiency in VSMCs could lead to vascular dysfunction and promote CVDs (18). A study by Badi et al (28) reported that downregulation of Sirt1 by miR-34a in VSMCs promotes senescence and inflammation. Other researchers also agreed that the loss of endogenous Sirt1 protein in human VSMCs directly contributes to the induction of cellular senescence and deficits of cellular function, including an impaired stress response, and reduced capacity for cell migration and proliferation (29). However, diabetes induction in vivo and HG concentrations in vitro significantly downregulated the level of Sirt1 protein in rat VSMCs (17). This finding was also demonstrated in the present study. Sirt1 is a NAD-dependent deacetylase, and it requires NAD for its enzymatic activity (30). Previous study demonstrated that inhibition of NAD biosynthesis may be one of the major mechanisms involved in the downregulation of Sirt1 induced by HG or hyperglycemia (17). In the present study, activating Sirt1 with Res significantly inhibited HG-induced upregulation of ETB receptor expression and receptor-mediated vasoconstriction in the organ culture SMA. This suggested that the enhancement of receptor-mediated vasoconstriction and the increase in ETB receptor protein expression was related to the downregulation of Sirt1 in HG-cultured rat SMA.

Additionally, the present study demonstrated that the level of p-ERK1/2 was significantly increased in HG-cultured rat SMA segments. ERK1/2 is one of three main signaling pathways of mitogen-activated protein kinases, and it has received attention for its involvement in the regulation of ETB receptors in VSMCs (20). Risk factors for CVDs, including cigarette smoke particles (31), minimally modified low-density lipoprotein (32,33), low-density lipoprotein (19) or homocysteine (20), could induce ETB receptors to be highly expressed on VSMCs through the ERK1/2 signaling pathway. Notably, when a specific ERK1/2 inhibitor (U0126) was used to block activation of ERK1/2 in the present study, the HG-induced upregulation of ETB receptor protein expression and receptor-mediated vasoconstriction in the organ culture SMA were blocked distinctly. This suggested that HG upregulated receptor-mediated vasoconstriction and ETB receptor expression by activating ERK1/2 signaling pathways.

A previous study has demonstrated that Sirt1 overexpression in VSMCs could significantly inhibit angiotensin II-induced VSMC hypertrophy by suppressing phosphorylation of ERK1/2 (34). This suggested that Sirt1 could regulate the activity of the ERK1/2 pathway. In the present study, activating Sirt1 with Res significantly blocked the upregulation of p-ERK induced by HG in the organ culture SMA. This result was in accordance with a previous study, which demonstrated that Sirt1 activator (Res) inhibited ERK1/2 phosphorylation in VSMCs (35). In other cells, Sirt1 activator partly reversed the ultraviolet B-induced damage on human retinal pigment epithelial cells by inhibiting AKT and ERK phosphorylation (36). Conversely, researchers reported that Sirt1 deletion led to enhanced pro-inflammatory signaling as demonstrated by increased signal transducer and activator of transcription and ERK phosphorylation (37).

In conclusion, the present results indicate that HG upregulated ETB receptor expression through the Sirt1-ERK1/2 signaling pathways in SMA. The upregulation of ETB receptor expression could enhance receptor-mediated vascular contractile responses and result in CVDs. The present study may provide novel therapeutic targets for the prevention and treatment of vasospasm and diabetes-associated CVDs.

Acknowledgments

The present study was supported in part by the Natural Science Foundation of Shaanxi Province (grant no. 2014PT013) and the Science and Technological Project of Shaanxi Province (grant no. 2016JQ8043).

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References