Effects of Irbesartan on Intracellular Antioxidant Enzyme Expression and Activity in Adolescents and Young Adults With Early Diabetic Angiopathy

All patients gave their informed consent to the study, which was approved by the Ethics Committee of the School of Medicine, University of Chieti, Italy. Subjects ≥18 years of age signed their own consent; for subjects ≤17 years of age, their parents signed consent and the adolescents signed their own assent.

The study group was comprised of 14 adolescents or young adults with type 1 diabetes (ages 14–21 years, duration of diabetes 11–19 years). Of the 14 subjects, 9 had retinopathy (6 with background retinopathy and 3 with preproliferative retinopathy), 5 had persistent microalbuminuria (defined as an albumin excretion rate [AER] >20 μg/min in two of three overnight urinary collections), and 5 had both conditions. The control groups were comprised of 11 type 1 diabetic subjects without angiopathy (ages 12–22 years) and 10 healthy volunteers (ages 16–22 years).

Blood samples were obtained from all participants after a 12-h fast. Each participant performed an overnight urine collection before blood samples were taken. The antioxidant 4-hydroxy-TEMPO (1 mmol/l; Sigma, St. Louis, MO) was added to the urine samples, which were then stored at −20°C until extraction. Blood pressure and AER were measured before and after 3 and 6 months of irbesartan treatment.

Skin fibroblasts were obtained by 5-mm punch biopsies taken under local anesthetic from the anterior surface of the forearm. The fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM; ICN Biochemicals, Thame, U.K.). CuZn superoxide dismutase (CuZnSOD), Mn superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase (GPX) activity and mRNA expression were measured before and after 6 months of treatment with irbesartan (150 mg/day). On both occasions, antioxidant enzyme activity was evaluated ex vivo at different glucose concentrations (5 and 22 mmol/l). Clinical characteristics of the subjects participating in the study are summarized in Table 1.

Arterial blood pressure was measured in all patients and control subjects following the recommendations of the American Heart Association and the American Academy of Pediatrics. The glomerular filtration rate was measured as previously described (7). No patient was taking any other medications at the time of the study.

Fibroblasts were cultured in DMEM (ICN) supplemented with 20% FCS (Life Technologies, Paisley, U.K.), 2 mmol/l glutamine (Sigma, Dorset, U.K.), 50 units/ml penicillin (Life Technologies), and 50 μg/ml streptomycin (Life Technologies). At the fourth passage, cells were cooled gradually and then frozen at −180°C in 10% DMSO in DMEM until used for the experiments. It is well recognized that even long-term cryopreservation does not affect fibroblasts’ functional activities.

All of the experiments were conducted between the sixth and eighth passages with the same batches of medium and FCS. Using these passages is considered to be a suitable method for studying fibroblasts from donor patients (1,2). The purchased medium contained 5 mmol/l glucose, to which mannitol or glucose was added to ensure that the high and normal glucose culture media had the same osmolarity. Cells were cultured in isosmotic normal (5 mmol/l) and high ex vivo glucose (22 mmol/l) concentrations.

Each sample of cells was grown for 12 weeks, with the medium being renewed every 2nd day. For each culture condition (normal or high glucose), 12 80-cm² plastic tissue culture flasks were used: 3 for RNA extraction, 3 for enzyme activity measurement, 3 for the evaluation of cell membrane lipid peroxidation, and 3 to determine cell number.

The medium was aspirated, and the monolayers were washed twice with PBS and detached by treatment with 2.5 ml trypsin-EDTA (Life Technologies) for 4–6 min at 37°C. Trypsin activity was stopped by adding 7 ml of medium containing serum after complete detachment of the cells was verified under the microscope. The cell suspension was passed several times through a fine Pasteur pipette to disaggregate cell clumps and 1 ml was counted in an electronic Coulter counter (ZBI model; Coulter Electronics, Luton, U.K.) equipped with a 100-μm aperture.

The monolayers were rinsed twice with ice-cold PBS, and the cells were harvested with a sterile rubber cell scraper. The cells were sedimented for 4 min at 1,600g and processed for enzyme/protein or mRNA analyses. For enzyme/protein lysates, cells were resuspended in 50 mmol/l potassium phosphate buffer containing 0.5% Triton X-100 and sonicated in an ice-water bath for two 30-s bursts on a Branson sonicator B15 (position 2, continuous setting; Branson Ultrasonic, Danbury, CT) with a 30-s cooling interval. Total protein concentration was determined as previously described (4). For CAT and GPX activities, sonicates were first spun for 5 min at 800g (4°C). The supernatants were assayed according to the procedure of Clairborne (8) for CAT activity and Gunzler and Flohè for GPX activity, as previously described (4).

Cells were suspended in 100 mmol/l triethanolamine-diethanolamine buffer and homogenized with a Teflon glass Dounce homogenizer. The homogenate was centrifuged at 105,000g for 1 h (4°C), and the supernatant was passed through a small Sephadex G25 (coarse) column to remove low−molecular-weight substances that might interfere with the enzyme assay. An aliquot of the eluate was applied onto a 5.5% polyacrylamide gel to localize superoxide dismutase (SOD) activity, with the exception that no tetramethyl-ethylenediamine was used for staining.

MnSOD activity was determined in mitochondrial fractions prepared by differential centrifugation, as previously described (4). Mitochondria were disrupted by a freeze-thaw procedure in a high ionic-strength buffer (0.25 mmol/l sucrose, 0.12 mol/l KCl, and 10 mmol/l Tris-HCl; pH 7.4). Mitochondrial membranes were removed by sedimentation at 105,000g for 1 h (4°C) and enzyme activity was measured in the supernatant.

Total RNA was prepared according to the procedure of Chirgwin et al. (8a), as previously described (4). Briefly, 10 μg total RNA were electrophoresed on a 1.4% agarose-formaldehyde gel and then transferred to gene screen membranes. The filters were prehybridized in 50 mmol/l Tris-HCl (pH 7.5), 0.1% sodium pyrophosphate, 0.2% Ficoll, 5 mmol/l EDTA, 1% SDS, 2.2% poly(vinylpyrrolidone), 50% formamide, 0.2% BSA, 1 × standard sodium citrate (SSC), and 150 μg/ml denatured salmon sperm DNA at 65°C for 6 h. Blots were hybridized with ³²P-labeled probes for human CuZnSOD, CAT, and MnSOD and bovine GPX to a specific activity of 1 × 10⁶ cpm/ml in hybridization fluid at 65°C overnight (4). The filters were washed at 65°C twice for 15 min with 2 × SSC (0.1%(and twice for 15 min with 0.1 × SSC (0.1%) SDS and then subjected to autoradiography using an intensifying screen at −85°C. Densitometry was performed on an LKB laser scanning densitometer. Hybridization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as an internal control to correct for loading inequalities.

The filters were probed for the four antioxidant enzymes separately and GAPDH was also used separately. The results were normalized against an ideal reference value obtained from healthy individuals at 5 mmol glucose/l ex vivo.

Cells were trypsinized and centrifuged at 250g for 10 min at 4°C. Cell pellets were resuspended in 1 ml cold PBS for assay of thiobarbituric acid−reactive substances and conjugated dienes, as previously described (4).

Urinary 8-isoprostanes prostaglandin F_2α (PGF_2α) excretion rates were measured by previously described radioimmunoassay methods (9). Lipid peroxidation in native LDLs and the plasma lipid peroxide content were evaluated as previously reported (10). Concentrations of monocyte chemoattractant protein 1 (MCP-1) were determined in triplicate by enzyme-linked immunosorbent assay (11). Endothelial dysfunction was evaluated by measuring von Willebrand factor; its propeptide, prothrombin fragment 1 and 2 (F₁₊₂); and tissue-plasminogen activator, as previously described (12,13).

ANOVA was used to test differences among the three groups. For each group of fibroblasts, a paired t test was used to compare the results under conditions of normal versus high ex vivo glucose concentration, and Fisher’s least significant differences test was used to evaluate the differences among the three different groups in the normal or high glucose condition. P <0.05 was considered significant. Data are expressed as means ± SD or as medians (ranges).

In normal ex vivo glucose concentrations, CuZnSOD activity and mRNA expression were not different among the three groups. In high ex vivo glucose conditions, CuZnSOD mRNA and activity increased similarly in all groups (NS by ANOVA).

In normal ex vivo glucose concentrations, MnSOD activity and mRNA expression was not different among the three groups. In high ex vivo glucose conditions, MnSOD did not change in any group.

In normal ex vivo glucose concentrations, CAT and GPX activity and mRNA expression were not different among the three groups. In high ex vivo glucose conditions, CAT and GPX mRNA (P < 0.001) and activity (P < 0.001) were significantly different among the groups by ANOVA (Tables 2 and 3). In high-glucose conditions before irbesartan treatment, CAT and GPX mRNA expression and protein activity were significantly higher in control subjects and diabetic subjects without angiopathy versus angiopathic diabetic subjects (Table 2 and 3); there was no difference among the control subjects and those without diabetic angiopathy.

High ex vivo glucose concentrations significantly increased lipid peroxidation in every group of cells. Higher levels were found in cells of adolescents and young adults with diabetic angiopathy (P < 0.001).

Treatment with irbesartan (150 mg/day) lasted for 6 months. No adverse events were evident in any patient. Irbesartan ameliorated the antioxidant enzymatic activity in both normal and hyperglycemic conditions (Fig. 1). mRNA expression was also increased after treatment with irbesartan (Fig. 2).

Irbesartan reduced lipid peroxidation in cells of adolescents and young adults with diabetic angiopathy. Serum markers of oxidant status and MCP-1 levels were decreased after the 6-month treatment (Fig. 3); urinary excretion of 8-isoprostanes PGF_2α was also reduced by inhibition of the ANG-II receptor (Fig. 3). Endothelial dysfunction was significantly improved after treatment with irbesartan (Fig. 4).

The urinary AER was reduced after treatment with irbesartan (median 72 [range 28–135] vs. 32 [10–54] μg/min; P < 0.01). Mean blood pressure was also reduced by irbesartan (97.5 [90–103] vs. 93.1 [88–96] mmHg; P < 0.05).

No relation between HbA_1c (A1C) values and markers of oxidative stress was found. No significant difference in A1C was observed after 6 months of treatment with irbesartan in patients with diabetic microangiopathy.

Our findings indicate that exposure to high ex vivo glucose concentrations induced an increase in mRNA levels and the biological activity of CuZnSOD, CAT, and GPX in fibroblasts from control subjects and adolescents without diabetic angiopathy; in contrast, the levels and activity of only CuZnSOD were increased in fibroblasts from diabetic adolescents with angiopathy. This finding may have important consequences with regard to glucose-induced oxidative stress injury to cells; in fact, glucose-induced oxidative stress has been demonstrated to damage several cells, including endothelial and mesangial cells (1).

Both CuZnSOD, which is located primarily in the cytoplasm, and MnSOD, a structurally distinct protein located in the mitochondria, catalyze the reaction O₂⁻ + O₂⁻ + 2H⁺ = O₂ + H₂O₂ (1). H₂O₂ is converted to H₂O in peroxisomes by the antioxidant enzyme CAT and in the cytoplasm by GPX (14). These antioxidant enzymes protect the cell from oxidative stress, but the threshold of protection can vary dramatically as a function of the activity and balance of these enzymes (15). CAT and GPX are far more efficient than CuZnSOD in protecting fibroblasts against oxidative stress (15). However, in several instances, cells with increased levels of CuZnSOD are hypersensitive to oxidative stress rather than protected from it (15). This occurs because CuZnSOD increases the formation of H₂O₂, which, if not efficiently converted to H₂O by an adequate level of CAT and GPX, may be detrimental to the cell (15). It is therefore not surprising that an increase in CuZnSOD is generally accompanied by a concomitant increase in CAT and GPX (15). We confirmed this phenomenon in fibroblasts derived from control subjects and young diabetic patients without microvascular complications in the presence of high ex vivo glucose concentrations. In the fibroblasts of our subjects with childhood-onset diabetes and angiopathy, a high glucose concentration induced a significant increase only in CuZnSOD, with no change in the activity of CAT and GPX. These results confirm previous data obtained in adult type 1 diabetic patients with macroproteinuria and overt nephropathy (2) and suggest that cells of type 1 diabetic adolescents and young adults with incipient angiopathy are not able to adjust their antioxidant mechanisms when high ex vivo glucose−induced oxidative stress is produced; consequently, they are more susceptible to oxidative stress. Alternatively, it could be argued that in the absence of the ability to increase CAT and GPX, the cells may “decide” not to enhance CuZnSOD and MnSOD; in that situation, the mechanism could simply be switched off. However, this event should also be operative in diabetic patients without angiopathy and control subjects.

High glucose concentrations in vitro and hyperglycemia in vivo are well-known stimuli for the production of free radicals and the generation of oxidative stress, with a consequent increase in the expression and activity of antioxidant enzymes (1), which act as a defense system against cell damage (14). The observation that despite hyperglycemia, only a portion of type 1 diabetic patients will progress to diabetic microangiopathy might indicate that there is individual diversity in cell response to high ex vivo glucose concentrations. It is therefore of great relevance that a disturbance in the mechanisms of protection from oxidative stress was found only in the cells of adolescents with angiopathy. By contrast, in adolescents and young adults with long-term type 1 diabetes but no angiopathy, a group that appears to be protected from vascular complications, the defense mechanisms against high glucose-induced oxidative stress were intact, as they were in our nondiabetic control subjects.

The novel finding of this study is that treatment with the selective ANG-II receptor antagonist irbesartan (150 mg/day) was able to substantially improve antioxidant enzyme production and activity in young patients showing early signs of diabetic angiopathy. Irbesartan was effective in decreasing markers of oxidant status and MCP-1 levels. Urinary excretion of 8-isoprostanes PGF_2α was also reduced and endothelial dysfunction was significantly improved after 6-month treatment with irbesartan.

Considerable evidence suggests that the intrarenal renin-angiotensin system (RAS) plays an important role in the development of diabetic nephropathy (16). Blockade of the RAS with either ACE inhibitors or ANG-II receptor antagonists delays the progression of renal injury associated with diabetes (17). In addition, inhibition of ACE by lisinopril may decrease retinopathy progression in normotensive patients with type 1 diabetes (18) and losartan (an angiotensin II receptor antagonist) may also be effective in preventing diabetic retinopathy (19).

Irbesartan is a newly approved ANG-II receptor antagonist with higher bioavailability, lower plasma protein binding, and a longer half-life than other similar drugs (e.g., losartan, valsartan). It selectively binds to the ANG-II receptor subtype 1, thereby inhibiting the activity of ANG-II. Recent studies in rats with streptozocin-induced diabetes have clearly demonstrated that irbesartan exerts a renal protective effect, reducing proteinuria and albuminuria and preventing renal hyperfiltration (20). Specifically, these recent results suggest that the renoprotective effect of irbesartan may be related to its inhibition of renal hypertrophy and expression of growth factors such as transforming growth factor-β₁ and connective tissue growth factor (20). More recently, a new ANG-II receptor blocker (l-158,809) has demonstrated a role in attenuating overexpression of vascular endothelial growth factor in diabetic podocytes (21). Taken together, these data provide robust evidence that ANG-II may be as dangerous as hyperglycemia in inducing vascular and renal damage in diabetic individuals (22). In a recent study, irbesartan was shown to be capable of reducing the incidence of congestive heart failure in patients with type 2 diabetes and overt nephropathy (23).

This is the first study to give solid evidence that irbesartan (150 mg/day) is able to significantly improve intracellular antioxidant enzyme production and activity in skin fibroblasts of adolescents and young adults with childhood-onset type 1 diabetes. This finding is not surprising because it is now well recognized that ANG-II and activated angiotensin I receptors produce intracellular oxidative stress; furthermore, recent data have shown that hyperglycemia is able to directly modulate cellular angiotensin production (24). Indeed, ACE inhibitors and ANG-II receptor antagonists have been demonstrated to act as causal antioxidants; it has been suggested that this property may account for their beneficial effect on diabetic complications (8).

The finding that irbesartan was able to significantly reduce serum markers of oxidative stress and improve endothelial function in young patients with diabetic angiopathy gives further support to the role of ANG-II in the progression of vascular damage in diabetes. Irbesartan has beneficial effects not only on blood pressure and microalbuminuria, but also on antioxidant capacity.

In conclusion, this study confirms that exposure to high ex vivo glucose concentrations induces an antioxidant defense mechanism in skin fibroblasts of normal young subjects and that a failure of this defensive mechanism is present in fibroblasts of young patients with childhood-onset diabetes and early signs of diabetic retinopathy and nephropathy. Treatment with irbesartan, at a dosage of 150 mg/day, significantly improves these cellular antioxidant mechanisms, reduces serum markers of oxidative stress, and ameliorates endothelial dysfunction.

This study was supported by a grant from the Italian Ministry of Research and a generous donation by Fondazione Carlo Erba, Italy.

We acknowledge the editorial assistance provided by Antonella Bascelli. We also thank the Regional Juvenile Diabetes Association and in particular Doriana D’Alimonte D’Attilio for support in childhood diabetes care and research.