Oxidative stress [increased bioavailability of reactive oxygen species (ROS)] plays a role in the endothelial dysfunction and vascular inflammation, which underlie vascular damage in diabetes. Statins are cholesterol-lowering drugs that are vasoprotective in diabetes through unknown mechanisms. We tested the hypothesis that atorvastatin decreases NADPH oxidase (Nox)-derived ROS generation and associated vascular injury in diabetes. Leprdb/Leprdb (db/db) mice, a model of Type 2 diabetes and control Leprdb/Lepr+ (db/+) mice were administered atorvastatin (10 mg/kg per day, 2 weeks). Atorvastatin improved glucose tolerance in db/db mice. Systemic and vascular oxidative stress in db/db mice, characterized by increased plasma TBARS (thiobarbituric acid-reactive substances) levels and exaggerated vascular Nox-derived ROS generation respectively, were inhibited by atorvastatin. Cytosol-to-membrane translocation of the Nox regulatory subunit p47phox and the small GTPase Rac1/2 was increased in vessels from db/db mice compared with db/+ mice, an effect blunted by atorvastatin. The increase in vascular Nox1/2/4 expression and increased phosphorylation of redox-sensitive mitogen-activated protein kinases (MAPKs) was abrogated by atorvastatin in db/db mice. Pro-inflammatory signalling (decreased IκB-α and increased NF-κB p50 expression, increased NF-κB p65 phosphorylation) and associated vascular inflammation [vascular cell adhesion molecule-1 (VCAM-1) expression and vascular monocyte adhesion], which were increased in aortas of db/db mice, were blunted by atorvastatin. Impaired acetylcholine (Ach)- and insulin (INS)-induced vasorelaxation in db/db mice was normalized by atorvastatin. Our results demonstrate that, in diabetic mice, atorvastatin decreases vascular oxidative stress and inflammation and ameliorates vascular injury through processes involving decreased activation of Rac1/2 and Nox. These findings elucidate redox-sensitive and Rac1/2-dependent mechanisms whereby statins protect against vascular injury in diabetes.

CLINICAL PERSPECTIVES

  • Statins, cholesterol-lowering drugs, improve vascular function in diabetes, but exact mechanisms are elusive.

  • We demonstrated that, in a mouse model of Type 2 diabetes (db/db), systemic and vascular oxidative stress, endothelial dysfunction and vascular inflammation were normalized by atorvastatin. These processes were associated with down-regulation of Rac1/2, decreased Nox-derived ROS generation and reduced pro-inflammatory signalling.

  • Our data identify some molecular mechanisms involving Rac1/2-regulated Noxs whereby statins may protect against vascular dysfunction in diabetes. Clinically, statins may have vasoprotective therapeutic potential beyond lipid-lowering in diabetes.

INTRODUCTION

Statins, 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors, inhibit cholesterol biosynthesis and are widely used in the treatment of hypercholesterolaemia [1]. Statins also exhibit actions beyond cholesterol-lowering, the so-called pleiotropic effects, including vasoprotection [2,3]. Recent evidence indicates that statins influence redox-sensitive processes through putative antioxidant properties and by inhibiting generation of reactive oxygen species (ROS) [4,5]. Statins also promote an increase in nitric oxide (NO) production by stimulating endothelial nitric oxide synthase (NOS) activity [6]. As such, statins may be protective in conditions associated with vascular oxidative stress, including hypertension, atherosclerosis and diabetes, possibly by improving endothelial dysfunction and by decreasing vascular inflammation and remodelling [7,8]. Statins have been shown to ameliorate endothelial dysfunction in experimental and clinical diabetes, through unknown mechanisms [911].

Previous studies demonstrated that statins decrease activation of NADPH oxidase (Nox)1, Nox2 and Nox4 (ROS-generating oxidases) in the cardiovascular system and evidence indicates an inhibitory effect of rosuvastatin on vascular Nox4 in diabetes [1214]. The pleiotropic effects of statins are related, in part, to reduced formation of isoprenoids, lipid moieties responsible for post-translational modification of specific signalling proteins. Isoprenoids promote hydrophobic modifications of small GTP-ases, such as Rac1/2, which plays a critical role in the activation of Noxs [15]. Rac1/2 also regulates NOS [16]. Inhibition of hydrophobic modification of Rac1/2 by statins has a significant effect on Nox activation and subsequent ROS generation [15,17,18].

Noxs are the main source of ROS in the vasculature and participate not only in the normal cell function, but also trigger the development of injury in pathological conditions [19,20]. The Nox family is composed of catalytic subunits [Nox1–Nox5, dual oxidase (Duox)1 and Duox2] and the docking subunit p22phox, all present in the cell membrane. The regulatory subunits Nox organizer 1 (Noxo1), Nox activator 1 (Noxa1), p67phox, p47phox and p40phox, are located in the cytosol. Activation of Rac1/2 regulates the translocation and assembly of the Nox subunits in the plasma membrane and is a key event in Nox activation [21,22].

Oxidative stress, due to exaggerated Nox-induced ROS formation and decreased NO bioavailability, is a hallmark of diabetic vasculopathy [2325]. We previously demonstrated an important role for Nox1 in accelerated atherosclerosis in diabetes [26]. Considering that the beneficial effects of statins are mediated, in part, by reducing ROS bioavailability, the aim of the present study was to determine whether atorvastatin, a lipid soluble cholesterol-lowering drug of the statin family, decreases ROS-associated vascular dysfunction and inflammation in db/db mice, through Nox-dependent processes. We thus tested the hypothesis that atorvastatin reduces Nox activity and redox signalling in db/db mice. Since statins target small GTPases through post-translational modifications, we also questioned whether changes in Nox activity by atorvastatin are associated with altered activity of vascular Rac1/2 in diabetes.

MATERIALS AND METHODS

Animals

The study was approved by the Animal Ethics Committee of the Ottawa Hospital Research Institute, University of Ottawa. Experiments were conducted in accordance with the guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with Institutional guidelines. Male Leprdb/Lepr+ (db/+) and Leprdb/Leprdb (db/db) mice [B6.BKS(D)-Leprdb/J] were purchased from Jackson Laboratories at the age of 6 weeks. Mice were treated with atorvastatin (10 mg/kg per day atorvastatin; Millipore) for 2 weeks. This dose of atorvastatin was selected as it is a relatively low dose that has been well described for rodents in the literature [27,28]. Atorvastatin was incorporated in the chow (2018 Teklad Global 18% Protein Rodent Diet, Harlan Laboratories) according to the previous food intake assessment (db/+: 0.063 g of atorvastatin/kg of chow; db/db: 0.056 g of atorvastatin/kg of chow). Food intake during the treatment was evaluated daily. Systolic blood pressure (SBP) was measured weekly by tail-cuff plethysmography. At the end of the treatment, mice were killed by isoflurane inhalation and subsequently decapitated.

Plasma biochemistry

Blood was collected immediately prior to killing by cardiac puncture. Plasma glucose, triacylglycerols (triglycerides) and cholesterol were determined by auto-analyser (Beckman Coulter AU5800).

Oral glucose tolerance test

The oral glucose tolerance test (OGTT) was performed to evaluate glucose tolerance in db/db and db/+ mice treated with control or atorvastatin diet. Mice were deprived of food for 6 h. Blood was sampled from the lateral saphenous vein immediately before (baseline, t0) and after (t15, t30, t60, t90, t120 min) administration of 2 g of glucose/kg by oral gavage. Glucose levels were determined using a glucose analyser (Accu-Check, Roche Diagnostics).

Vascular function

Mesenteric vascular beds were isolated from db/db and db/+ mice treated with control or atorvastatin diet. Second-order branches of superior mesenteric artery were dissected and mounted on a wire myograph (DMT, Danish Myo Technology). Vessel segments (2 mm in length) were mounted on 25 μm wires in a vessel bath chamber for isometric tension recording and equilibrated for 30 min in Krebs–Henseleit-modified physiological salt solution (120 mmol/l NaCl, 25 mmol/l NaHCO3, 4.7 mmol/l KCl, 1.18 mmol/l KH2PO4, 1.18 mmol/l MgSO4, 2.5 mmol/l CaCl2, 0.026 mmol/l EDTA and 5.5 mmol/l glucose), at 37°C, continuously bubbled with 95% O2 and 5% CO2, pH 7.4. At the beginning of each experiment, arteries were contracted with 10 μmol/l noradrenaline (norepinephrine; NE) or 90 mmol/l KCl to test for functional integrity. In some experiments, the vascular endothelium was removed by gently rubbing the lumen side of the ring segments. The integrity of the endothelium or its removal was assessed by the presence or absence of relaxation in response to 1 μmol/l acetylcholine (ACh) of NE pre-contracted arteries respectively. Endothelium-dependent relaxation was assessed by the concentration–response curves to ACh (0.1 nmol/l to 10 μmol/l) and insulin (INS; 0.1–10000 ng/dl) in vessels pre-contracted with NE at a concentration to achieve approximately 70% of maximal response. Contractile responses mediated by NE (0.1 nmol/l to 10 μmol/l) were evaluated in endothelium-intact and endothelium-denuded arteries.

Vascular structural and mechanical studies

Second-order branches of superior mesenteric artery (2–3 mm in length) were slipped on to two glass microcannulas, one of which was positioned until vessel walls were parallel, in a pressure myograph (Living Systems). Vessel segments were equilibrated in Krebs–Henseleit-modified physiological salt solution, at 37°C, continuously bubbled with 95% O2 and 5% CO2, pH 7.4, under constant intraluminal pressure (45 mmHg). Vascular structure and mechanical studies were assessed under Ca2+-free conditions to eliminate the effects of myogenic tone. Vessels were perfused for 30 min with Ca2+-free Krebs solution containing 10 mmol/l EGTA. Measurements of media thickness and lumen diameter were taken at stepwise increments of luminal pressure (3–120 mmHg). Vascular, structural and mechanical parameters were calculated as we previously described [29].

Measurement of plasma lipid peroxidation products

Plasma lipid peroxidation was determined by quantifying thiobarbituric acid-reactive substances (TBARS) [30]. Plasma samples were mixed with 2% butylated hydroxytoluene and quintanilla reagent (26 mmol/l thiobarbituric acid and 15% trichloroacetic acid). The mixture was boiled for 15 min. Subsequently, the mixture was cooled (4°C) and centrifuged at 3000 g for 10 min. TBARS formed in each of the samples was assessed by measuring absorbance of the supernatant at 535 nm with an absorbance microplate reader (BioTek ELx808). In parallel, MDA (malondialdehyde) standards were diluted in a range of 0–6 nmol/ml. TBARS were calculated by plotting the obtained absorbance against an MDA concentration standard curve.

Lucigenin-enhanced chemiluminescence

Vascular ROS generation was measured by a luminescence assay with lucigenin as the electron acceptor and NADPH as the substrate. Aortic segments from db/db and db/+ mice treated with control or atorvastatin diet were homogenized in assay buffer (50 mmol/l KH2PO4, 1 mmol/l EGTA and 150 mmol/l sucrose, pH 7.4) with a glass-to-glass homogenizer. The assay was performed with 100 μl of sample, 1.25 μl of lucigenin (5 μmol/l), 25 μl of NADPH (0.1 mmol/l) and assay buffer to a total volume of 250 μl. Luminescence was measured for 30 cycles of 18 s each by a luminometer (Lumistar Galaxy, BMG Labtechnologies). Basal readings were obtained prior to the addition of NADPH to the assay. The reaction was started by the addition of the substrate. Basal and buffer blank values were subtracted from the NADPH-derived luminescence. Superoxide production was expressed as relative luminescence unit (RLU)/μg of protein.

Western blotting

Total protein was extracted from aortas. Frozen tissues were homogenized in 50 mmol/l Tris/HCl (pH 7.4) lysis buffer (containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.1% SDS, 2 mmol/l Na3VO4, 1 mmol/l PMSF, 1 μg/ml pepstatin A, 1 μg/ml leupeptin and 1 μg/ml aprotinin). Total protein extracts were cleared by centrifugation at 12000 g for 10 min and pellet was discarded. Proteins from homogenates of vascular tissues (20 μg) were separated by electrophoresis on a polyacrylamide gel (10%) and transferred on to a nitrocellulose membrane. Non-specific binding sites were blocked with 5% skim milk or 1% BSA in Tris-buffered saline solution with Tween for 1 h at 24°C. Membranes were then incubated with specific antibodies overnight at 4°C. Antibodies were as follows: anti-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), anti-extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) MAPK (Thr202/Tyr204), anti-c-Jun N-terminal kinase (JNK) MAPK (Thr183/Tyr185), anti-nuclear factor κB (NF-κB) p65 (Ser536) (Cell Signaling); anti-Nox1 (Sigma); anti-Nox2, anti-Nox4, anti-vascular cell adhesion molecule-1 (VCAM-1), anti-NF-κB p50, anti-inhibitor of κB-α (IκB-α; Santa Cruz Biotechnology). Antibody to β-actin (Sigma) was used as internal housekeeping control. After incubation with secondary antibodies, signals were revealed with chemiluminescence, visualized by autoradiography and quantified densitometrically.

Cytosol and membrane fractionation

Mesenteric arterial beds were homogenized in 50 mM Tris/HCl, pH 7.4, lysis buffer containing 5 mmol/l EGTA and 2 mmol/l EDTA, 0.1 mmol/l PMSF, 0.2 mmol/l Na3VO4, 1 μmol/l pepstatin A, 1 μmol/l leupeptin and 1 μmol/l aprotinin. Homogenates were centrifuged at 100000 g for 1 h at 4°C. The supernatant (cytosolic fraction) was collected. The pellet, containing the particulate fraction, was re-suspended in lysis buffer containing 1% Triton X-100 and centrifuged at 10000 g for 10 min at 4°C. The resultant supernatant was collected (membrane-enriched fraction). Protein analysis was performed by Western blotting as described above using anti-p47phox (1:500 dilution, Santa Cruz) and anti-Rac1/2 (1:1000 dilution, Cell Signaling) antibodies. Antibody to β-actin (Sigma) was used as internal housekeeping control. Results are expressed as membrane to cytosol ratio of protein content in the cell fractions as an index of translocation and activation.

Vascular inflammatory response: macrophage adhesion

The adhesion assay was performed in a 24-well plate coated with 4% agarose. Aortic rings (5 mm) were cleaned and opened longitudinally. The vascular segments were positioned endothelium-side up in the solid agarose surface (one segment/well), fixed with sharp pointed pins and kept in F12 medium at 37°C. Murine-derived J774A.1 monocyte/macrophage cell line was obtained from the American Type Culture Collection. J774A.1 adherent cells were cultured in Dulbecco's modified Eagle's medium and 10% heat-inactivated FBS. For cell fluorescent labelling, macrophages (105 cells/ml) were suspended in 1% BSA supplemented PBS containing 1 μmol/l calcein-AM (Molecular Probes) and incubated for 20 min at 37°C. Labelled macrophages were washed twice with PBS and suspended in Hanks’ buffered salt solution. Fluorescence-labelled cells (105 cells/well) were then added to the wells containing the vascular segments and were allowed to adhere for 30 min at 37°C in 5% CO2. Non-adherent cells were removed by gently washing with pre-warmed Hanks’ buffered salt solution. The number of adherent cells was counted using fluorescence microscopy. Four fields were evaluated per segment. Imaging was acquired with an Axiovert Imaging System.

Data analysis

Results are presented as means±S.E.M. Comparisons were performed by one-way ANOVA followed by the Bonferroni test or the Newman–Keuls test, when appropriate. P<0.05 was considered statistically significant.

RESULTS

Metabolic parameters in db/+ and db/db mice treated with control or atorvastatin diet

Treatment with atorvastatin did not affect body weight or food intake in db/+ and db/db mice, compared with control counterparts (Supplementary Figure S1). Table 1 shows that atorvastatin decreased plasma cholesterol and triacylglycerol levels in db/db mice compared with control-diet-treated db/db mice. Non-fasting db/db mice treated with atorvastatin displayed slightly reduced, but non-significant, plasma glucose levels as compared with db/db mice on control diet. Glucose tolerance was determined by OGTT (Figure 1). No differences were observed in the fasting blood glucose levels at baseline between db/db mice receiving atorvastatin or control diet. Overall, db/db mice receiving control diet displayed impaired glucose tolerance as compared with db/+ mice. Atorvastatin-treated db/db mice showed a decrease in blood glucose compared with control-diet-treated db/db mice at 60, 90 and 120 min after the glucose challenge. The statin treatment had no effect on fasting blood glucose levels at baseline or glucose tolerance in db/+ mice.

Table 1
Plasma biochemistry

Biochemistry analysis of plasma from db/+ and db/db treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Results are means±S.E.M. of 12 mice in each experimental group.*P<0.05 compared with db/+ control diet; **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Group
Parameterdb/+db/dbdb/+ +atorvastatindb/db +atorvastatin
Cholesterol (mmol/l) 2.37±0.6 3.16±0.4* 2.25±0.3 2.53±0.2** 
Triacylglycerols (mmol/l) 0.93±0.35 2.14±0.9* 1.07±0.1 1.28±0.5** 
Glucose (mmol/l) 11.79±2.1 32.05±13.9* 11.89±3.0 27.58±7.1** 
Group
Parameterdb/+db/dbdb/+ +atorvastatindb/db +atorvastatin
Cholesterol (mmol/l) 2.37±0.6 3.16±0.4* 2.25±0.3 2.53±0.2** 
Triacylglycerols (mmol/l) 0.93±0.35 2.14±0.9* 1.07±0.1 1.28±0.5** 
Glucose (mmol/l) 11.79±2.1 32.05±13.9* 11.89±3.0 27.58±7.1** 

Atorvastatin improves glucose tolerance in db/db mice

Figure 1
Atorvastatin improves glucose tolerance in db/db mice

OGTT was performed in db/+ and db/db mice treated with control or atorvastatin (10 mg/kg per day) diet (2 weeks). After 6 h fasting, baseline blood glucose was measured. Mice received 2 mg/kg glucose by gavage and blood samples were collected at 15, 30, 60, 90 and 120 min after the challenge. Results are means±S.E.M. of 12 mice in each experimental group. Line graphs, time course of blood glucose after the challenge. Bar graph, area under the curve (AUC) in the plot of blood glucose concentration against time. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Figure 1
Atorvastatin improves glucose tolerance in db/db mice

OGTT was performed in db/+ and db/db mice treated with control or atorvastatin (10 mg/kg per day) diet (2 weeks). After 6 h fasting, baseline blood glucose was measured. Mice received 2 mg/kg glucose by gavage and blood samples were collected at 15, 30, 60, 90 and 120 min after the challenge. Results are means±S.E.M. of 12 mice in each experimental group. Line graphs, time course of blood glucose after the challenge. Bar graph, area under the curve (AUC) in the plot of blood glucose concentration against time. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Systolic blood pressure in db/+ and db/db mice treated with control or atorvastatin diet

Diabetic db/db mice and db/+ control mice had similar SBP levels (Supplementary Figure S2). Atorvastatin did not affect the SBP in either db/+ or db/db mice.

Status of oxidative stress in arteries from db/+ and db/db mice

The potential antioxidant effect of atorvastatin was evaluated in db/db and db/+ mice. Figure 2(A) demonstrates that plasma TBARS levels were significantly higher in db/db mice compared with db/+ and this increase was inhibited by atorvastatin treatment. NADPH-dependent superoxide anion generation was measured in aortic homogenates from db/+ and db/db mice. Figure 2(B) shows that lucigenin-derived luminescence was significantly higher in arteries from db/db mice compared with db/+. Atorvastatin reduced the increase in superoxide anion generation in db/db mice. Translocation of the Nox cytosolic subunit p47phox and the small GTPase Rac1/2 from the cytosol to the cell membrane was evaluated as an index of the oxidase activation. Expression of p47phox (Figure 2C) and Rac1/2 (Figure 2D) in membrane-enriched fractions was increased in mesenteric arteries from db/db mice. This effect was abrogated by atorvastatin.

Atorvastatin reduced vascular oxidative stress in db/db mice

Figure 2
Atorvastatin reduced vascular oxidative stress in db/db mice

Plasma, aorta and mesenteric arteries were obtained from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). ROS status was assessed by plasma TBARS levels (A) and lucigenin-enhanced chemiluminescence (B). Translocation of p47phox (C) and Rac1/2 (D) was assessed by the protein expression in membrane and cytosolic fractions isolated from mesenteric arterial bed homogenates. Results are means±S.E.M. of 6–8 mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Figure 2
Atorvastatin reduced vascular oxidative stress in db/db mice

Plasma, aorta and mesenteric arteries were obtained from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). ROS status was assessed by plasma TBARS levels (A) and lucigenin-enhanced chemiluminescence (B). Translocation of p47phox (C) and Rac1/2 (D) was assessed by the protein expression in membrane and cytosolic fractions isolated from mesenteric arterial bed homogenates. Results are means±S.E.M. of 6–8 mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Expression of Nox isoforms in arteries from db/+ and db/db mice

Since enhanced oxidative stress was observed in the vasculature of db/db mice, we evaluated the protein expression of the Nox isoforms in aortas from of db/+ and db/db mice. Figure 3 demonstrates that the protein expression of Nox1, 2 and 4 was up-regulated in aortas from db/db mice compared with the db/+ group. Atorvastatin reduced expression of the catalytic subunit of the oxidase isoforms in the vasculature of diabetic mice.

Effects of atorvastatin on Nox protein expression in the vasculature of db/m and db/db mice

Figure 3
Effects of atorvastatin on Nox protein expression in the vasculature of db/m and db/db mice

Protein levels of Nox1 (A), Nox2 (B) and Nox4 (C) were evaluated in mesenteric arteries isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblots of Nox1, Nox2, Nox4 and β-actin. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Figure 3
Effects of atorvastatin on Nox protein expression in the vasculature of db/m and db/db mice

Protein levels of Nox1 (A), Nox2 (B) and Nox4 (C) were evaluated in mesenteric arteries isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblots of Nox1, Nox2, Nox4 and β-actin. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Effects of atorvastatin treatment on MAPK phosphorylation in arteries from db/+ and db/db mice

The effect of atorvastatin on the phosphorylation levels of MAPKs, the downstream signalling targets of ROS, is demonstrated in Figure 4. In the aorta, p38 MAPK phosphorylation (Figure 4A) was increased in db/db mice compared with db/+ mice. Similar results were observed for ERK1/2 MAPK (Figure 4B) and JNK MAPK (Figure 4C). Atorvastatin inhibited the increase in MAPK phosphorylation observed in the db/db mice.

Increased MAPK phosphorylation is inhibited by atorvastatin in the vasculature of db/db mice

Figure 4
Increased MAPK phosphorylation is inhibited by atorvastatin in the vasculature of db/db mice

Phosphorylation levels of p38 MAPK (A), ERK1/2 MAPK (B) and JNK MAPK (C) were evaluated in mesenteric arteries isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblots of p38 MAPK (Thr180/Tyr182), ERK1/2 MAPK (Thr202/Tyr204), JNK MAPK (Thr183/Tyr185) and β-actin. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Figure 4
Increased MAPK phosphorylation is inhibited by atorvastatin in the vasculature of db/db mice

Phosphorylation levels of p38 MAPK (A), ERK1/2 MAPK (B) and JNK MAPK (C) were evaluated in mesenteric arteries isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblots of p38 MAPK (Thr180/Tyr182), ERK1/2 MAPK (Thr202/Tyr204), JNK MAPK (Thr183/Tyr185) and β-actin. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Pro-inflammatory responses in arteries from db/+ and db/db mice treated with atorvastatin

The redox-sensitive NF-κB family of transcription factors regulates multiple cellular processes including inflammation. Increased phosphorylation levels of the NF-κB p65 subunit were observed in aortas from db/db mice (Figure 5A). Aortas from db/db mice exhibited increased protein expression of the NF-κB subunit p50 (Figure 5B) with reduced expression of the regulatory protein IκB-α compared with db/+ mice (Figure 5C). These effects were normalized by atorvastatin.

Atorvastatin affects NF-κB proteins phosphorylation and expression in the vasculature of db/db mice

Figure 5
Atorvastatin affects NF-κB proteins phosphorylation and expression in the vasculature of db/db mice

Phosphorylation of NF-κB p65 (A) and the expression of NF-κB p50 (B) and IκB-α (C) were evaluated in mesenteric arteries isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblots of NF-κB p65 (Ser536), NF-κB p50, IκB-α and β-actin. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Figure 5
Atorvastatin affects NF-κB proteins phosphorylation and expression in the vasculature of db/db mice

Phosphorylation of NF-κB p65 (A) and the expression of NF-κB p50 (B) and IκB-α (C) were evaluated in mesenteric arteries isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblots of NF-κB p65 (Ser536), NF-κB p50, IκB-α and β-actin. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

One of the initial steps in vascular inflammation is expression of adhesion molecules such as VCAM-1. VCAM-1 content was increased in the vasculature of db/db mice versus controls (Figure 6A). Figure 6(B) shows adherent green fluorescent macrophages in aortic segments. Increased numbers of adherent macrophages were observed in aortic segments of db/db mice compared with db/+ mice. Atorvastatin reduced vascular inflammatory responses as evidenced by decreased VCAM-1 expression and macrophage adhesion in db/db mice.

Atorvastatin reduces VCAM-1 expression and macrophages adhesion in aortas from db/db mice

Figure 6
Atorvastatin reduces VCAM-1 expression and macrophages adhesion in aortas from db/db mice

Aortas were isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). VCAM-1 expression in aortic homogenates (A) and the number of adherent fluorescent macrophages to aortic segments (B) were evaluated. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Figure 6
Atorvastatin reduces VCAM-1 expression and macrophages adhesion in aortas from db/db mice

Aortas were isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). VCAM-1 expression in aortic homogenates (A) and the number of adherent fluorescent macrophages to aortic segments (B) were evaluated. Results are presented as means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Effect of atorvastatin on vascular function in arteries from db/+ and db/db mice

Figure 7 and Tables 2 and 3 show the effects of atorvastatin on vascular function of db/+ and db/db mice. Endothelium-intact mesenteric arteries from db/db mice were more sensitive to NE compared with those from db/+ mice, as evidenced by the leftward shift in the concentration–response curve to the agonist. Atorvastatin abolished the increased sensitivity to NE in arteries from db/db mice (Figure 7A). No differences in the response to NE were observed in endothelium-denuded arteries from db/+ and db/db mice (Figure 7B). Relaxation in response to ACh was significantly reduced in arteries from db/db mice, effects that were restored by atorvastatin (Figure 7C). Maximum relaxation in response to INS was decreased in mesenteric arteries from db/db mice compared with those from db/+ mice. Atorvastatin improved the endothelium-dependent relaxation in response to INS in arteries from db/db mice (Figure 7D). Atorvastatin did not affect the endothelium-dependent relaxant responses in arteries from db/+ mice.

Atorvastatin decreases the sensitivity to NE and improves endothelium dependent relaxation in small mesenteric arteries from db/db mice

Figure 7
Atorvastatin decreases the sensitivity to NE and improves endothelium dependent relaxation in small mesenteric arteries from db/db mice

Resistance mesenteric arteries were isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks) and mounted in a wire myograph. Concentration–response curves to NE (0.1 nmol/l to 10 μmol/l) were performed in endothelium-intact (A) or endothelium-denuded (B) arteries. Endothelium-intact arteries were pre-contracted with NE (10−6 mol/l) and concentration–response curves to (C) ACh (1 nmol/l to 10 μmol/l) and (D) INS (0.1–10000 ng/dl) were performed. Results are mean±S.E.M. of 5–8 mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Figure 7
Atorvastatin decreases the sensitivity to NE and improves endothelium dependent relaxation in small mesenteric arteries from db/db mice

Resistance mesenteric arteries were isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks) and mounted in a wire myograph. Concentration–response curves to NE (0.1 nmol/l to 10 μmol/l) were performed in endothelium-intact (A) or endothelium-denuded (B) arteries. Endothelium-intact arteries were pre-contracted with NE (10−6 mol/l) and concentration–response curves to (C) ACh (1 nmol/l to 10 μmol/l) and (D) INS (0.1–10000 ng/dl) were performed. Results are mean±S.E.M. of 5–8 mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Table 2
Vascular reactivity to NE, ACh and INS: maximum effect

Effects of atorvastatin treatment (10 mg/kg per day for 2 weeks) on vascular responses [maximum effect (Emax) of second order mesenteric arteries)] to NE, ACh and INS in db/+ and db/db mice. Concentration–effect curves were performed in endothelium-intact (+E) and endothelium-denuded (−E) mesenteric rings. Results are means±S.E.M. of seven mice in each experimental group. Emax values for NE are presented as a percentage of KCl (60 mM) responses, whereas ACh and INS are presented in relation of pre-contraction of 0.1 μM NE. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Group (Emax)
Agonistdb/+db/dbdb/+ +atorvastatindb/db +atorvastatin
NE (+E) 101.4±13.3 110.0±10.9 119.3±12.53 97.3±14.80 
NE (-E) 91.1±10.8 98.7±11.8 91.5±9.0 89.8±16.42 
ACh 85.2±5.6 48.0±5.7* 87.9±5.8 79.6±11.3** 
INS 68.1±5.4 42.2±5.2* 77.2±8.2 65.9±7.8** 
Group (Emax)
Agonistdb/+db/dbdb/+ +atorvastatindb/db +atorvastatin
NE (+E) 101.4±13.3 110.0±10.9 119.3±12.53 97.3±14.80 
NE (-E) 91.1±10.8 98.7±11.8 91.5±9.0 89.8±16.42 
ACh 85.2±5.6 48.0±5.7* 87.9±5.8 79.6±11.3** 
INS 68.1±5.4 42.2±5.2* 77.2±8.2 65.9±7.8** 
Table 3
Vascular reactivity to NE, ACh and INS: pD2

Effects of atorvastatin treatment (10 mg/kg per day for 2 weeks) on vascular responses [pD2 (negative logarithm of the EC50 of second-order mesenteric arteries)] to NE, ACh and INS in db/+ and db/db mice. Concentration–effect curves were performed in endothelium-intact (+E) and endothelium-denuded (−E) mesenteric rings. Results are means±S.E.M. of seven mice in each experimental group. *P<0.05 compared with db/+ control diet, **P<0.05 db/db atorvastatin diet compared with db/db control diet.

Group (pD2)
Agonistdb/+db/dbdb/+ +atorvastatindb/db +atorvastatin
NE (+E) 7.53±0.3 8.55±0.3* 6.78±0.5 7.41±0.1** 
NE (−E) 7.25±0.3 7.66±0.3 7.53±0.2 7.69±0.2 
ACh 7.36±0.2 8.66±0.9* 7.73±0.2 7.23±0.2** 
INS 6.72±0.2 7.32±0.5* 6.61±0.1 7.17±0.2 
Group (pD2)
Agonistdb/+db/dbdb/+ +atorvastatindb/db +atorvastatin
NE (+E) 7.53±0.3 8.55±0.3* 6.78±0.5 7.41±0.1** 
NE (−E) 7.25±0.3 7.66±0.3 7.53±0.2 7.69±0.2 
ACh 7.36±0.2 8.66±0.9* 7.73±0.2 7.23±0.2** 
INS 6.72±0.2 7.32±0.5* 6.61±0.1 7.17±0.2 

Vascular morphology and mechanics in arteries from db/+ and db/db mice

Mesenteric arteries from db/+ and db/db mice presented similar wall thickness, lumen diameter and cross-sectional area in response to stepwise increments of intraluminal pressure (Supplementary Figures S3A–S3C). No differences were observed in the stress–strain relationship curve between arteries from db/+ and db/db mice (Supplementary Figure S3D). Atorvastatin did not affect morphological parameters and mechanical properties in all experimental groups.

DISCUSSION

Major findings in the present study demonstrate that INS resistance, endothelial dysfunction and vascular inflammation in diabetic db/db mice are ameliorated by atorvastatin through processes associated with reduced vascular oxidative stress and decreased pro-inflammatory signalling. These phenomena were associated with decreased activation of Rac1/2, down-regulation of Nox isoforms and decreased generation of ROS. Our data provide some novel insights involving Rac1/2-regulated Noxs whereby statins, such as atorvastatin, protect against vasculopathy in diabetes. Statins as adjuvant therapy in the management of diabetes may have beneficial metabolic and vascular effects beyond lipid lowering.

The major morbidities associated with diabetes are cardiovascular disease and nephropathy due in large part to vascular injury [31]. In our study, db/db mice exhibited significant endothelial dysfunction and vascular inflammation as evidenced by reduced ACh- and INS-induced endothelium-dependent vasorelaxation and increased monocyte adhesion with augmented VCAM-1 expression. Vascular dysfunction has been described in patients with diabetes [32,33] and in experimental models of Type 1 diabetes (OVE26 mice and streptozotocin-induced diabetes) [34,35] and Type 2 diabetes (db/db, fat-fed) [36,37]. Our findings support others that have shown improved endothelium-dependent NO-mediated vasorelaxation by another statin, rosuvastatin, in experimental diabetes and are in line with clinical studies demonstrating that statins improve flow-mediated vasodilation in patients with Type 2 diabetes [12,38,39]. Although these vascular effects have been attributed to mechanisms independent of lipid-lowering effects [12], the metabolic and lipid profiles of db/db mice in our study were improved by atorvastatin. Accordingly, we cannot exclude the possibility that some vasoprotection occurs through lipid lowering.

Exact mechanisms whereby statins affect directly on vascular function still remain elusive. However, growing evidence indicates that these drugs influence signalling pathways involved in the generation of ROS and NO [40]. Statins inhibit HMG-CoA reductase [1], which normally catalyses the biosynthesis of mevalonate, the main intermediate fatty acid in cholesterol biosynthesis. Mevalonate is also the primary precursor of lipid isoprenoid intermediates, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are essential for the prenylation and activation of small GTPases [15,41], such as Rac1/2. Hence, statins have the capacity to inhibit Rac1/2 activation, which could affect Rac-dependent signalling, including Nox-ROS pathways [42]. db/db mice exhibited increased activation of Rac1/2 as evidenced by increased cytosol-to-membrane translocation, with associated increased p47phox translocation and activation of Noxs. Increased Rac1/2 activation has also been demonstrated in streptozotocin-induced diabetic rats [43] and we showed that vascular and renal Nox-mediated ROS generation is amplified in streptozotocin-induced and db/db diabetic mice [26,44]. Of the Nox isoforms, we previously demonstrated an important role for Nox1 and Nox4 in cardiovascular and renal injury in diabetic mice [26,42]. Other studies have implied a role for Nox2 [45,46]. In the present study, we found increased expression of all three Nox isoforms in db/db vessels and as such cannot identify which Nox is primarily responsible for the increased ROS generation in our model. However, since Nox1 and Nox2 have an obligatory need for p47phox, which was also activated in db/db mice, these Noxs may be particularly important, similar to what was previously reported.

In atorvastatin-treated db/db mice, Nox activation was attenuated as evidenced by reduced NADPH-induced generation of ROS, down-regulation of Nox isoforms, reduced p47phox translocation and decreased levels of TBARS. These effects may relate to Rac1/2 inhibition since atorvastatin blocked translocation of Rac from the cytosol to the membrane. Rac1/2 normally regulates Noxs by promoting translocation of the p47phox/p67phox/p40phox cytosolic complex to the Nox/p22phox complex in the cell membrane and is important in the initial steps of the electron transfer reaction for superoxide anion generation. Hence, statins probably blunt Nox activity, at least in part, through inhibitory effects on Rac1/2 and possibly by modulating assembly of oxidase subunits. Statins have been shown to regulate Rac activity through various mechanisms including stimulation of small GTP-binding protein GDP dissociation stimulator (SmgGDS), which negatively regulates Rac1/2, and by inhibiting post-translational modifications [47]. Our findings, in the present paper, expand those observations and provide a putative molecular mechanism whereby statins interfere with Rac-regulated Nox-derived ROS in diabetes, which probably reduces oxidative stress, normalizes endothelial function and prevents vascular inflammation in db/db mice. These processes probably involve reduced pro-inflammatory signalling through MAPKs and NF-κB [4850], pathways that were down-regulated by treatment in diabetic mice. Such effects may relate to blunted Rac1/2 and Nox activation. It is also possible that atorvastatin improved vasorelaxation through NOS/NO-dependent pathways, since statins have been shown to stimulate activity of NOS with increased NO generation [51,52].

Statin treatment had a positive effect on the metabolic profile and INS sensitivity in db/db mice, which could also have an effect on improved vascular status in these mice, as previously suggested [53]. In our experiments, atorvastatin decreased vascular sensitivity to NE and improved endothelium-dependent vasorelaxation in db/db mice. These phenomena were associated with reduced vascular oxidative damage and inflammation and may have been due, at least in part, to changes in INS sensitivity in vascular cells.

In support of our results in db/db mice others have shown in experimental rat models of diabetes [53,54] and in patients with diabetes [55] that atorvastatin improves INS sensitivity. However, data from some clinical studies reported that statins actually worsen diabetes and that they promote new onset diabetes and INS resistance. Reasons for these conflicting data are unclear, but it should be stressed that studies reporting pro-diabetic actions of statins were primarily retrospective analyses of large clinical trials that demonstrated associations between statin treatment and development of diabetes [5659]. In such associative studies, causality cannot be established and in none of those investigations was a direct negative effect of statins on INS sensitivity actually demonstrated. Further in depth investigations are required to determine whether statins directly influence INS metabolism in diabetes.

In conclusion, we elucidate some molecular mechanisms whereby atorvastatin protects against vascular damage in diabetes. We also demonstrate that statin treatment improves INS sensitivity, which in its own right could positively influence vascular status. Taken together, our data suggest that statins may have important vasoprotective effects in diabetes beyond lipid lowering.

Abbreviations

     
  • ACh

    acetylcholine

  •  
  • Duox

    dual oxidase

  •  
  • ERK

    extracellular-signal-regulated protein kinase

  •  
  • HMG-CoA

    3-hydroxy-3-methylglutaryl CoA

  •  
  • INS

    insulin

  •  
  • IκB-α

    inhibitor of κB-α

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MDA

    malondialdehyde

  •  
  • NE

    noradrenaline (norepinephrine)

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NO

    nitric oxide

  •  
  • NOS

    nitric oxide synthase

  •  
  • Nox

    NADPH oxidase

  •  
  • OGTT

    oral glucose tolerance test

  •  
  • ROS

    reactive oxygen species

  •  
  • SBP

    systolic blood pressure

  •  
  • TBARS

    thiobarbituric acid-reactive substances

  •  
  • VCAM-1

    vascular cell adhesion molecule-1

AUTHOR CONTRIBUTION

The study was conceived by Rhian Touyz and Rita Tostes, and developed by Thiago Bruder-Nascimento and Glaucia Callera. Thiago Bruder-Nascimento conducted the studies with help from Glaucia Callera and Ying He. The paper was written by Thiago Bruder-Nascimento, Glaucia Callera, Rita Tostes and Rhian Touyz, with contributions from Augusto Montezano, Tayze Antunes and Aurelie Nguyen Dinh Cat.

FUNDING

This work was supported by the São Paulo Research Foundation (FAPESP) [grant numbers 2010/52214-6 (to R.C.T); 2011/01785-6; 2011/22035-5 (to T.B.-N.)]; the Agence Universitaire de la Francophonie [grant number 58145FT103]; the Juvenile Diabetes Research Foundation [grant number 4-2010-528]; the Canadian Institutes of Health Research [grant number 44018]; and the British Heart Foundation [grant number RG/13/7/30099].

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Supplementary data