Endothelial dysfunction and oxidative stress in type 1 and type 2 diabetic patients without clinical macrovascular complications

Results Decreased GSH levels ( p < 0.05, type 1 and type 2), GSH/GSSG ratio ( p < 0.05 type 1, p < 0.001 type 2) and elevated vWF levels ( p < 0.001, type 1 and type 2) were observed in diabetic patients in comparison with controls. A negative correlation between GSH and vWF ( p < 0.02 and p < 0.001, in type 1 and type 2, respectively) and GSH and BMI ( p < 0.02 in type 1 and type 2) was observed. ET-1 was positively correlated to age ( p < 0.05) and diabetes duration ( p < 0.03) in type 1, while vWF was correlated to systolic blood pressure ( p < 0.05) in type 2 diabetic patients. Urinary VEGF was higher in type 2 ( p < 0.05) in comparison with type 1 diabetic patients and was correlated to glycemia ( p < 0.05) and systolic blood pressure ( p < 0.05). Conclusions These data might indicate that markers of oxidative stress and ED are altered in diabetic patients without clinical macrovascular complications. Keywords Endothelial dysfunction Oxidative stress von Willebrand factor Glutathione redox system 1 Introduction Hyperglycemia is known to promote free radicals production and impairment of the antioxidant systems, such as glutathione reduced form (GSH) and vitamin C [1–6] . Oxidative stress is involved in the pathogenesis of endothelial dysfunction (ED), characterized by increased vascular tone and permeability and by the presence of a prothrombotic and antifibrinolytic status. ED plays a key role in the pathogenesis of atherosclerosis and its presence has been documented in patients with diabetes mellitus [7] . Different markers may be used to assess ED [8] such as endothelin-1 (ET-1), von Willebrand Factor (vWF) and vascular endothelial growth factor (VEGF) levels. Elevated ET-1 and vWF levels [9–12] have been reported in type 1 and type 2 diabetic patients. Some authors evidenced that treatment with vitamin C decreased vWF plasma levels in patients with type 2 diabetes and coronary artery disease, demonstrating a correlation between oxidative stress and ED [13,14] . Furthermore, it has been reported that N -acetyl- l -cysteine treatment increases GSH levels and decreases plasma vascular cell adhesion molecule-1 levels in patients with type 2 diabetes mellitus [15] . Although it is known that ED and oxidative stress are present in subjects with diabetes mellitus it is still debated whether these conditions precede the clinical evidence of macrovascular complications. Thus, in this study we evaluated the relationship among intracellular glutathione redox system and markers of ED (ET-1, vWF, VEGF) in patients with type 1 and type 2 diabetes mellitus without clinical macrovascular complications. 2 Methods The study included 83 consecutive diabetic patients (27 with type 1 and 56 with type 2 diabetes). Attending the Departments of Internal Medicine and of Clinical Sciences, “Sapienza” University of Rome, Italy and 35 control subjects. Patients with poor glycaemic control (HbA1c > 8.5%), a recent acute illness and evident macrovascular complications, evaluated on the basis of clinical history, clinical examination, electrocardiography, echocardiography, carotid and femoral artery ultrasonography, were excluded from the study. Thirty-five healthy subjects, age and sex matched, recruited from hospital staff members represented the control group. This was subdivided into two subgroups: younger for comparison with type 1 and older for comparison with type 2 diabetic patients ( Table 1 ). All subjects were taking no drugs known to interfere with glutathione metabolism or any other antioxidant drugs. This study was approved by the local ethical committee and all participants gave their written informed consent. The screening for microvascular complications was performed in all patients. The presence of renal impairment was evaluated by albumin excretion rate and glomerular filtration rate (GFR) assessment [16] . Albuminuria was measured on timed overnight urine collections by using an enzyme-linked immunosorbent assay (ELISA, Albuwell, Exocell Inc., Philadelphia, USA), and microalbuminuria was diagnosed when albumin excretion rate was >20 μg/min. GFR was calculated using the Cockcroft and Gault equation as follows: K (140 − age) body weight in kg/serum creatinine in μmol/l, with K equal to 1 for men and 0.85 for women. Screening of retinopathy was performed by the ophthalmologist after pupillary dilation, according to scale of the early treatment of diabetic retinopathy (ETDRS) study [17] . Venous blood was drawn in the morning after an overnight fast. Glutathione redox state (GSH, GSSG and GSH/GSSG ratio) was determined in red blood cells from each subject according to an enzymatic method, as previously described [18] . The inter-assay and intra-assay variability was less than 10%. The results were expressed as μmol/g Hb. Test-combination for hemoglobin was purchased from Roche GmbH (Mannheim, Germany). GSH, GSSG, 5-sulfosalicylic acid, 5,5′-dithiobis-2-nitrobenzoic acid, triethanolamine, Hystopaque-1077 were purchased from Sigma Chemical Co. (St. Louis, MO), whereas 2-vinylpyridine was purchased from Aldrich (Steinheim, Germany). ET-1 was assayed using an enzyme-linked immunosorbent assay kit (Biomedica Medizinprodukte, GmbH & Co., KG, Wien, Austria) in blood samples collected in EDTA and centrifuged for 15 min at 5 °C and then frozen at −80 °C until assayed. The intra- and inter-assay variability was less than 10%. vWF was measured by enzyme-linked immunosorbent assay using a commercially available kit (American Diagnostica, Inc., Greenwich, CT, USA); the intra- and inter-assay variations of this method were 2.5 and 5%, respectively. HbA1c was assayed with HPLC method (Menarini, Florence, Italy). Because the high variability of plasma VEGF, its concentration was measured in urine samples using a quantitative sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA). The sensitivity of the methods is less than 5.0 pg/ml. VEGF was expressed as the ratio between urine VEGF level (pg/ml) and urine creatinine (mg/ml). The intra- and inter-assay variability was less than 10%. 3 Data analysis Data regarding patient characteristics and biochemical parameters are expressed as mean ± standard error (S.E.M.) or as median (range), as indicated in the text and legends to tables. Mean differences were compared by unpaired Student's t -test or Mann–Whitney U -test for two groups and by one-way ANOVA for more than two groups. The association among the different clinical and biochemical parameters was evaluated with Pearson or Spearman's correlation as appropriate. Univariate and multivariate analyses were performed to correlate, in type 1 diabetic patients, independent variables such as diabetes duration, age, BMI, and vWF with dependent variables such as GSH and ET-1 and in type 2 diabetic patients independent variables such as BMI, fasting plasma glucose, SBP, vWF with dependent variables such as GSH, vWF and VEGF. Adjustments were made for potential confounding factors including gender, age, diabetes duration, and glycemic control (HbA1c). All analyses were performed using SPSS version 11.5 (SPSS, Chicago, IL). A p value <0.05 was considered statistically significant. 4 Results Table 1 shows the clinical characteristics of patients and control groups. The two groups of patients were similar for diabetes duration, but type 2 diabetic patients were older, and with higher BMI, systolic and diastolic blood pressure ( Table 1 ). HbA1c levels and GFR were lower in type 2 in comparison with type 1 diabetic patients ( Table 1 ). 4.1 Microalbuminuria Microalbuminuria was present in two type 1 (7.4%) and 15 (26.8%) type 2 diabetic patients, while diabetic retinopathy was revealed in eight type 1 (29.6%) and 14 type 2 (25%) diabetic patients. 4.2 GSH GSH blood levels were similar in type 1 and type 2 diabetic patients, but significantly reduced in comparison with control subjects ( Table 2 ). The GSH/GSSG ratio was not different in type 1 and type 2 diabetic patients, but it was significantly reduced in type 1 and type 2 diabetic patients in comparison with control subjects ( Table 2 ). 4.3 ET-1 ET-1 values showed a similar behavior in diabetic patients and control subjects. 4.4 vWF vWF levels were not different in type 1 and type 2 diabetic patients, but it was significantly increased in diabetic patients in comparison with control subjects ( Table 2 ). 4.5 VEGF Urinary VEGF concentration was significantly higher in type 2 in comparison with type 1 diabetic patients (80.3 ± 11.0 vs. 45.3 ± 9.0 pg/mg, p < 0.05). 4.6 Correlations in patients with type 1 diabetes In type 1 diabetic patients GSH was negatively correlated with vWF ( r = −0.47, p < 0.02) and BMI ( r = −0.28, p < 0.02), while ET-1 was positively correlated with age ( r = 0.41, p < 0.05) and diabetes duration ( r = 0.43, p < 0.03) ( Table 3 ). After performing multiple regression analysis, the correlation between GSH and vWF was still significant when adjusting for age ( p = 0.04), but it was no more significant after adjusting for gender, diabetes duration and HbA1c. The correlation between GSH and BMI was still significant when adjusting for gender ( p = 0.020) and age ( p = 0.02), but it was no more significant after adjusting for diabetes duration and HbA1c. The correlation between ET-1 and age was still significant when adjusting for gender ( p = 0.0009), diabetes duration ( p = 0.028) and HbA1c ( p = 0.019). The correlation between ET-1 and diabetes duration was no more significant after adjusting for age, while it was still significant when adjusted for gender ( p = 0.008) and HbA1c ( p = 0.005). 4.7 Correlations in patients with type 2 diabetes In type 2 diabetic patients, GSH was negatively correlated with vWF ( r = −0.54, p < 0.001) and BMI ( r = −0.52, p < 0.02). In the same patient group, vWF was positively correlated with systolic blood pressure ( r = 0.26, p < 0.05) and VEGF with fasting plasma glucose ( r = 0.44, p < 0.05) and systolic blood pressure ( r = 0.57, p < 0.05), Table 3 . The correlation between GSH and vWF was still significant after adjusting for gender ( p = 0.001), age ( p = 0.001), diabetes duration ( p = 0.001) and HbA1c ( p = 0.001). The correlation between GSH and BMI was still significant after adjusting for gender ( p = 0.046), age ( p = 0.042), diabetes duration ( p = 0.042), but it was no more significant when adjusting for HbA1c. The correlation between vWF and systolic blood pressure was still significant when adjusting for gender ( p = 0.04), age ( p = 0.036), diabetes duration ( p = 0.035) and HbA1c ( p = 0.038). The correlation found between VEGF and fasting plasma glucose ( p < 0.05) was no more significant after adjusting for gender, age, diabetes duration and HbA1c. The correlation between VEGF and systolic blood pressure ( p < 0.05) was still significant after adjusting for gender ( p = 0.04) and HbA1c ( p = 0.036), while it was no more significant after adjusting for age and diabetes duration. 4.8 Markers of endothelial dysfunction and diabetic complications When considering all diabetic patients, those with microalbuminuria had decreased GSH levels and increased urinary VEGF levels in comparison with the normoalbuminuric group (5.2 ± 0.3 vs. 6.2 ± 0.2 μmol/g Hb, p < 0.05; and 99.0 ± 27.6 vs. 58.1 ± 6.8 pg/mg, p < 0.05; means ± S.E.M.). Furthermore, urinary VEGF levels were increased, but not significantly, in patients with diabetic retinopathy (78.0 ± 20.0 vs. 65.7 ± 8.9 pg/mg, mean ± S.E.M.) and hypertension (90.2 ± 16.5 vs. 58.1 ± 8.8 pg/mg, mean ± S.E.M.) in comparison with patients without these complications and in smokers (67.8 ± 13.0 vs. 54.2 ± 13.7 pg/mg, mean ± S.E.M.) compared with non-smokers. In type 2 diabetic patients no correlation was found between diabetes and hypertension therapy and the different parameters studied. 4.9 Markers of endothelial dysfunction and diabetic treatment With respect to diabetes therapy, no differences in GSH, GSSG, GSH/GSSG, vWF, VEGF and ET-1 levels were found between patients treated and untreated with metformin. 5 Discussion In this study the presence of oxidative stress and ED in patients with type 1 and type 2 diabetes, without clinically evident macrovascular complications, has been demonstrated. It is known that hyperglycemia is responsible for an increased production of reactive oxygen species (ROS) through glucose auto oxidation and/or non-enzymatic glycation, following an impairment of the antioxidant defense system. The ROS, especially superoxide anion and hydrogen peroxide, are important signaling molecules of endothelial cells damage [18,19] . Previous reports have demonstrated that ROS participate in the modulation of endothelial function and overexpression of inflammatory cytokines [20] . In this study a decreased GSH and GSH/GSSG ratio was found in patients with type 1 and type 2 diabetes, suggesting the presence of a damage at the antioxidant defense system level. ED represents a major cause of arterial wall damage and in this work increased levels of vWF in diabetic patients, without clinical evidence of macrovascular complications have been found [2,3] . This finding could indicate the presence of an activation and/or damage of endothelial cells even in this condition. Moreover, the hypothesis of a link between oxidative stress and ED has been supported by the finding, in this study, of a negative correlation between vWF and GSH in the two groups of diabetic patients. Since GSH is the major intracellular antioxidant, it is possible that the impairment of GSH redox system in diabetes makes the endothelial cells more susceptible to ROS injury with consequent ED. Interestingly, multiple regression analysis showed a difference between patients with type 1 and type 2 diabetes. In type 1 diabetic patients the negative correlation between GSH and vWF was dependent on diabetes duration and glycemic control as measured by HbA1c. In patients with type 2 diabetes, this relationship between oxidative stress and endothelial dysfunction was confirmed after adjustment for confounding variables (gender, age, BMI, diabetes duration, HbA1c). It has been demonstrated that in insulin resistance states, such as obesity and type 2 diabetes, an impairment of insulin-mediated vasodilatation and ED are present [21] . Moreover, it is known that an oxidative stress may be involved in the pathogenesis of the insulin resistance [22] . In type 2 diabetic patients included in this study, the correlation between oxidative stress and ED is independent of glycemic control and diabetes duration, thus it can be hypothesized that ED might be correlated to some component of the insulin resistance syndrome. On the other hand, the presence of a correlation between GSH and BMI, dependent of metabolic control both in type 1 and type 2 diabetic patients has been demonstrated. Visceral obesity is a component of the metabolic syndrome and frequently associated to insulin resistance and it is characterized by the production by adipose tissue of inflammatory molecules. It is possible to postulate that the presence of obesity and poor metabolic control in type 2 diabetic patients is a necessary condition to impair the antioxidant system. On the other hand in patients with type 2 diabetes a correlation was found between vWF and systolic blood pressure, and the correlation was independent of different confounding factors such as age, diabetes duration and metabolic control. These data confirm the hypothesis of a link between ED and insulin resistance, being hypertension frequently associated to insulin resistance syndrome. Increased levels of plasma ET-1 have been evidenced in patients with diabetes mellitus. It is known that this molecule influences vascular tone and endothelial function [23–26] . In this study, a correlation between ET-1 and age was found, in accordance with previous reports that have demonstrated a similar behavior in the general population [27] . In the patients with type 1 diabetes, this correlation was independent of metabolic control and diabetes duration and no correlation was found between ET-1 and GSH, suggesting that different mechanisms are involved in the alteration of these parameters. Moreover, the loss of a significant correlation between ET-1 and the duration of the disease, when adjusted for age, in the same group of patients, may indicate that aging plays a major role in the increase of this molecule. In this study increased urinary VEGF levels were found in type 2 in comparison with type 1 diabetic patients. Honkanen et al. demonstrated that urinary VEGF value was not different in patients with type 1 diabetes with nephropathy and healthy subjects, while Lenz et al. found a positive correlation between urinary VEGF and microalbuminuria in patients with type 2 diabetes [28–30] . In accordance with Lenz et al. report, an increase of urinary VEGF in microalbuminuric type 2 diabetic patients was observed in this study. VEGF increases vascular permeability and its higher concentration in urine from microalbuminuric diabetic patients may indicate the presence of a generalized endothelial dysfunction in these patients. Moreover, a correlation between VEGF and systolic blood pressure was evidenced in the same group. It is known that hypertensive type 2 diabetic patients show urinary VEGF levels higher than normotensive diabetic patients [16] . The correlation between VEGF and systolic blood pressure, here found, was independent of metabolic control, supporting the hypothesis that many factors concur to the development of the endothelial dysfunction in diabetes and that the increased permeability at vascular level, represented by increased albumin and VEGF urine excretion, is frequently associated to hypertension and to an increased cardiovascular risk. In patients with microalbuminuria, decreased GSH levels, in comparison with normoalbuminuric patients, were observed. This finding is in accordance with several reports that support the role of oxidative stress in the pathogenesis of renal complication [31,32] . In conclusion, this study indicates that oxidative stress and ED are present in patients with type 1 and type 2 diabetes mellitus without clinically evident macrovascular complications. Furthermore, the correlation between ED and oxidative stress in patients with type 2 diabetes without clinical vascular complications support the hypothesis that the impairment of intracellular antioxidant system and ED are frequently associated in diabetes mellitus. While in type 1 diabetic patients this correlation seems to be dependent of metabolic control, in patients with type 2 diabetes other factors (i.e. metabolic syndrome components) could be involved in the development of oxidative stress and ED. Conflict of interest The authors confirm to have no relationship such as: consultancies, stock ownership or other equity interests, patents received and/or pending, or any commercial relationship which might be in any way considered related to a submitted article. References [1] A. Fortuno G.S. 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