Telmisartan Shows an Equivalent Effect of Vitamin C in Further Improving Endothelial Dysfunction After Glycemia Normalization in Type 1 Diabetes

Experiments were performed in 36 type 1 diabetic patients. Twelve age- and BMI-matched healthy volunteers served as control subjects (supplemental Table 1 [available at http://dx.doi.org/10.2337/dc07-0318]). Diabetic subjects were divided into three subgroups and matched for age, sex, BMI, metabolic control, and duration of the disease (supplemental Table 1). None of the selected patients were hypertensive, microalbuminuric, or on drug treatment (excluding insulin). Lipid levels were also normal. The study was approved by the local ethics committee, and all subjects gave written informed consent.

All experiments were performed in a quiet, temperature-controlled room (24–25°C) after an overnight fast. Subjects were not allowed to smoke, drink (except for water), or eat for at least 10 h before the experiment. They were studied in the supine position and remained in bed during the experiments. On the morning of the experiments, diabetic patients omitted their insulin injection. In the diabetic patients, a catheter was inserted into an antecubital vein for the infusion of one of the three treatments, described as follows.

Insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) and/or 5% glucose was infused to keep blood glucose levels between 4 and 6 mmol/l for 24 h. The amount of insulin infused was based on the individual daily insulin dose. Blood glucose levels were determined every 5 min with adjustment of the intravenous insulin infusion until steady-state glucose levels were between 4 and 6 mmol/l. At the steady state, venous glucose samples were drawn every 30 min. After 12 h, vitamin C infusion, at the rate of 3 mg/min (15), was also started and continued for the remaining 12 h.

Vitamin C, at the rate of 3 mg/min (15), was infused for 24 h. After 12 h, insulin and/or 5% glucose was infused, aiming to maintain blood glucose levels between 4 and 6 mmol/l (as described above) for the remaining 12 h.

Insulin and/or 5% glucose (to keep blood glucose levels between 4 and 6 mmol/l) and vitamin C were infused at the rate of 3 mg/min (15) for 24 h.

After the first phase of the study, telmisartan (40 mg/day, six patients from each group) or placebo (six patients from each group) treatment was started for 1 month. At the end of the treatment period, the protocols described above were repeated.

In all experiments of the first and second phase, glycemia, endothelial function (flow-mediated dilation [FMD]), and nitrotyrosine plasma level were evaluated at baseline and after 12 and 24 h.

Cholesterol and triglycerides were measured enzymatically (Roche Diagnostics, Basel, Switzerland). HDL cholesterol was estimated after precipitation of apolipoprotein B with phosphotungstate/magnesium (16). LDL cholesterol was calculated after lipoprotein separation (17). Plasma glucose was measured by the glucose-oxidase method, and A1C was measured by high-performance liquid chromatography.

Nitrotyrosine plasma concentration was assayed by enzyme-linked immunosorbent assay, as previously described (18).

Endothelial function was evaluated measuring the FMD of the brachial artery, as previously reported (19). At the end of each test, subjects laid quietly for 15 min. Then, sublingual nitroglycerin (0.3 mg) was administered, and 3 min later, the last measurements were performed. Response to nitroglycerin was used as a measure of endothelium independent vasodilation. All studies were performed in a quiet and temperature-controlled room (22–23°C).

As in previous studies (6,20), the Kolmogorov-Smirnov algorithm was used to determine whether each variable had a normal distribution. Comparisons of baseline data among the groups were performed with unpaired Student's t test. Paired Student's t test was used to compare the various parameters before and after each treatment. Changes in variables during the tests were assessed by two-way repeated-measures ANOVA. If differences reached statistical significance, post hoc analyses with a two-tailed paired t test were used to assess differences at individual time periods in the study with Bonferroni's correction for multiple comparisons. Statistical significance was defined as P < 0.05.

Baseline nitrotyrosine was increased in diabetic patients, whereas FMD was reduced (supplemental Table 1).

As previously reported (12), in protocols A and C, glycemia was almost normalized after 12 and 24 h (P < 0.001 vs. baseline) (supplemental Figs. 1 and 3), whereas in protocol B, glycemia was normal at 24 h (P < 0.001 vs. baseline) (supplemental Fig. 2). After 12 h, nitrotyrosine plasma levels significantly decreased in all protocols (P < 0.01 vs. baseline) (supplemental Figs. 1–3). However, protocol C decreased more significantly compared with protocol A and B (P < 0.01 vs. A and P < 0.05 vs. B), and protocol B decreased more significantly compared with protocol A (P < 0.05). After 12 h, FMD increased (P < 0.01 vs. baseline) (supplemental Figs. 1–3), again more significantly in treatment C compared with the two others (P < 0.01 vs. A and P < 0.05 vs. B). FMD also increaesd more in protocol B compared with protocol A (P < 0.05).

After 24 h, the plasma levels of nitrotyrosine were significantly decreased compared with the plasma levels after 12 h in protocols A and B (P < 0.01) but not in C. After 24 h, there were no differences among the plasma levels of nitrotyrosine reached in each of the three protocols.

FMD was significantly increased compared with the FMD levels after 12 h in protocols A and B (P < 0.01) but not in C. After 24 h for FMD, there were also no differences among the three protocols. Endothelium independent vasodilation did not change during any of the tests (data not shown).

One month of treatment with telmisartan significantly improved baseline FMD and reduced nitrotyrosine plasma levels (supplemental Table 2).

In protocol A, when glycemia was normalized after 12 h, FMD was also almost normalized (P < 0.01 vs. baseline, P < 0.05 vs. placebo) (supplemental Fig. 1), whereas nitrotyrosine was significantly reduced (P < 0.01 vs. baseline, P < 0.05 vs. placebo) (supplemental Fig. 1) in the group treated with telmisartan. Adding vitamin C did not change the results.

Similarly, in protocol B, the infusion of vitamin C had no impact on both FMD and the level of nitrotyrosine plasma, whereas the normalization of glycemia after 24 h normalized FMD (P < 0.01 vs. baseline, P < 0.05 vs. placebo) (supplemental Fig. 2) and reduced nitrotyrosine plasma levels (P < 0.01 vs. baseline, P < 0.05 vs. placebo) (supplemental Fig. 2).

In protocol C, the normalization of FMD (P < 0.01 vs. baseline, P < 0.05 vs. placebo at both 12 and 24 h) (supplemental Fig. 3) and the reduction of nitrotyrosine plasma levels (P < 0.01 vs. baseline, P < 0.05 vs. placebo at both 12 and 24 h) (supplemental Fig. 3) were achieved after 12 h and persisted for 24 h. Variation of the endothelial independent vasodilation was not found during all of the experiments.

This study confirms that in type 1 diabetic patients, only the simultaneous normalization of glycemia and the use of an antioxidant, such as vitamin C, can normalize endothelial dysfunction (12). At the same time, the possibility of improving endothelial function in type 1 diabetes, as already reported for type 2 diabetic patients using an AT-1 receptor blocker (in this case telmisartan), is also reported (20). However, for the first time, we are able to show that telmisartan in combination with the normalization of glycemia may lead to the near normalization of endothelial function. Furthermore, it is also shown that adding vitamin C does not influence the effect of treatment with telmisartan.

The role of oxidative stress in this phenomenon appears to be crucial: in the first phase of the study, when endothelial function is still altered after 12 h of normalization of glycemia or 12 h of vitamin C treatment, nitrotyrosine (a good marker of peroxynitrite and nitrosative stress) is still increased, whereas when endothelial function is normalized by combining glycemic control with vitamin C, nitrotyrosine is also normalized. It has already been reported that telmisartan reduces free radical production and oxidative stress (21,22). In this study, we show that telmisartan influences the generation of oxidative stress in diabetes. In the second phase of the study and as already reported for type 2 diabetic patients (23), chronic treatment with an AT-1 blocker decreases nitrotyrosine plasma levels, whereas the effect of telmisartan on nitrotyrosine plasma levels in all the three protocols is equivalent to that of vitamin C. Interestingly, when vitamin C is added during the protocols in the patients already on telmisartan treatment, it does not influence the results. Together, these data suggest that telmisartan reduces oxidative stress and achieves the same results of a well-established antioxidant such as vitamin C. This is particularly interesting, considering that hyperglycemia-induced protein kinase C overexpression mediated the activation of the NADPH oxidase and may cause endothelial nitric oxide synthase uncoupling (24), which can, in turn, favor peroxynitrite generation, which actually causes the increase in nitrotyrosine and is able to oxidize biopterin 4 (the endothelial nitric oxide synthase cofactor) to the biopterin 3 radical (25). In this case, vitamin C acts as a biopterin 3 radical scavenger rather than an antioxidant, which reacts with superoxide (25). Because telmisartan prevents NADPH activation, this property may help explain our results (25).

Our data suggest that hyperglycemia convincingly induces endothelial dysfunction through the generation of oxidative stress because the administration of vitamin C (in protocol B) in the presence of hyperglycemia restores endothelial function and reduces nitrotyrosine plasma levels. Additionally, the normalization of glycemia (in protocol A) is accompanied by an improvement of both endothelial function and nitrotyrosine. Therefore, considering the results as a whole, it seems reasonable that telmisartan improves endothelial function because it reduces oxidative stress.

Particularly for protocol A, a possible role for insulin in determining the improvement of endothelial dysfunction might be supposed more than for the reduction of hyperglycemia. However, very recently, Ellger et al. (26) demonstrated in an animal model that the reduction of glucose toxicity, more than the action of insulin, improves endothelial function.

As reported above, previous studies have shown that in type 1 diabetic patients, endothelial dysfunction persists even with normalization of glycemia (8,9), which suggests that long-lasting hyperglycemia can induce several permanent alterations in endothelial cells and lead to permanent endothelial dysfunction. Our data suggest that this permanent alteration induces the persistence of the endothelial dysfunction through the generation of oxidative stress because by adding vitamin C or telmisartan to insulin, endothelial function is almost normalized. Interestingly, these in vivo data are consistent with in vitro and animal studies. In the endothelial cells of cultures and in the retina of diabetic rats, an overproduction of free radicals persists, even after the normalization of glucose, and is accompanied by a prolongation of the induction of protein kinase C β, NADPH oxidase, Bax, collagen, and fibronectin, in addition to nitrotyrosine (27), suggesting that oxidative stress may be involved in this effect. In animals, evidence is more consistent. The effect of the reinstitution of good glucose control on hyperglycemia-induced increased oxidative and nitrative stress has been evaluated in the retina of rats maintained in poor glucose control before initiation of good control (28). In diabetic rats, 2 or 6 months of poor control (GHb >11.0%) was followed by 7 months of good control (GHb <5.5%). The reinstitution of good control after 2 months of poor control inhibited elevations of retinal lipid peroxides and nitric oxide levels by ∼50% but failed to have any beneficial effects on nitrotyrosine formation. However, the reversal of hyperglycemia after 6 months of poor control had no significant effect on retinal oxidative stress and nitric oxide levels. In the same rats, inducible nitric oxide synthase expression and nitrotyrosine levels remained elevated by >80% compared with normal rats or rats kept in good control for the duration (28). In a similar study, caspase-3 activity in diabetic rats kept in poor control for 13 months was 175% that in normal rats (29). The reinstitution of good glycemic control after 2 months of poor control partially normalized the hyperglycemia-induced activation of caspase-3 (to 140% of normal values), whereas the reinstitution of good control after 6 months of poor control had no significant effect on the activation of caspase-3. In the same study, nuclear factor-κB (NF-κB) activity was 2.5-fold higher in diabetic rats kept in poor control than in normal rats. The reinstitution of good control after 2 months of poor control partially reversed this increase, but good control after 6 months of poor control had no effect. Initiation of good control soon after the induction of diabetes in rats prevented activation of retinal caspase-3 and NF-κB (29). Similar results are available for the kidney. Diabetic rats were maintained in good glycemic control (GHb = 5%) soon or 6 months after the induction of hyperglycemia and were killed 13 months after the induction of diabetes (30). For rats in which good control was initiated shortly after the induction of diabetes, oxidative stress (as measured by the levels of lipid peroxides, 8-hydroxy-2′-deoxyguanosine, and reduced glutathione) and nitric oxide in urine and the renal cortex were not different from that observed in normal control rats; however, when the reinstitution of good control was delayed for 6 months after the induction of diabetes, oxidative stress and nitric oxide levels remained elevated in both urine and the renal cortex (30).

These data suggest that hyperglycemia-induced oxidative stress and nitric oxide, as well as activation of apoptosis and NF-κB, can be prevented if good glycemic control is initiated very early but are not easily reversed if poor glycemic control is maintained for longer durations. Therefore, these findings suggest a persistence of the hyperglycemia-induced damage in such organs, even after its normalization. Because in vitro evidence shows that almost the same pathways are activated by hyperglycemia in endothelial cells (32), it is reasonable that the cardiovascular system receives the same imprinting in the retina and kidney.

The finding that only the simultaneous control of glycemia and oxidative stress can normalize endothelial function in type 1 diabetic patients is clearly relevant. This evidence seems to suggest the existence of two different pathways working in the generation of endothelial dysfunction in type 1 diabetes: one directly related to hyperglycemia and one not. The explanation of this second pathway may be, at the moment, only speculative. It is well recognized that hyperglycemia, both chronic or acute, can induce endothelial dysfunction through the generation of oxidative stress and is convincingly generated at the level of mitochondria (11,32). However, it is also well recognized that chronic hyperglycemia inducing the formation of advanced end products and chronic hyperglycemia is thought to alter mitochondrial function through the glycation of mitochondrial proteins (33). This premise is important because a recent study, for the first time, described a direct relationship among the formation of intracellular advanced end products on mitochondrial proteins, the decline in mitochondrial function, and the excess formation of reactive species (33). Additionally, mitochondrial respiratory chain proteins that underwent glycation were prone to produce more ·O₂⁻, independently from the level of hyperglycemia. Therefore, a possible explanation for the previously described evidence and for our data are that two pathways are simultaneously working: one due to the actual level of glycemia and another to the long-lasting damage induced in the endothelial cells by chronic hyperglycemia (possibly through nonenzymatic glycation of mitochondria). This hypothesis has recently been reviewed (34).

It is also surprising that vitamin C or telmisartan alone cannot normalize endothelial function if oxidative stress is the key convergent effector of both hyperglycemia and of the long-lasting damage (possibly through nonenzymatic glycation of mitochondria). However, it may be hypothesized that when hyperglycemia is present, it induces the generation of free radicals, which can only partly be counterbalanced by the antioxidant treatment. The persistence of increased levels of nitrotyrosine in protocol B or after long-term telmisartan treatment supports this hypothesis. Conversely, when hyperglycemia is normalized, the possible presence of a second pathway that still produces free radicals may also explain the incomplete action of vitamin C or telmisartan.

Regardless, although the molecular basis for our findings is not clear, we believe that the clinical impact of our finding is important. In particular, the evidence that telmisartan has the same beneficial effect of vitamin C in improving the long-lasting effect of hyperglycemia, in our opinion, deserves attention. In fact, the effectiveness of a chronic long-term treatment with vitamin C is still an open debate (35), whereas telmisartan and other AT-1 receptor blockers are already widely used for the prevention of diabetes complications, particularly nephropathy (36). It seems reasonable to suggest that an AT-1 receptor blocker is a much better form of therapy than vitamin C because a recent study indicates that vitamin C intake increases mortality in postmenopausal women with type 2 diabetes (37).

Moreover, the persistence of endothelial dysfunction, which is a strong predictor of a cardiovascular event (4), even when glycemia is normalized, may contribute to explain the recent results from the Epidemiology of Diabetes Interventions and Complications Study/Diabetes Control and Complications Trial (EDIC/DCCT), showing, for the first time, the influence of the early glycemic control on the progression to macrovascular events (3). Evidence in our study that nitrotyrosine is still elevated even in the presence of near-normal glycemia is also relevant because nitrotyrosine has also been shown to be an independent predictor of CVD (38). Furthermore, our data seem to confirm that early and continuous aggressive treatment of glycemia is important to avoid future complications. Incidentally, in our opinion, the recent findings showing the existence of a continuum between the values of glycemia, endothelial dysfunction, and the risk of a cardiovascular event, even in nondiabetic patients, supports this concept (39,40).

The use of AT-1 blockers for the prevention of cardiovascular complications, particularly in diabetes, is still an open debate (41). Our study showing, for the first time, that a normalization of both endothelial dysfunction and oxidative stress can be achieved in type 1 diabetic patients combining the near normalization of glycemia and telmisartan treatment adds credibility to the use of these compounds. Future long-term controlled trials may help in understanding the possible clinical impact of this finding on the prognosis of CVD in type 1 diabetic patients.