Diabetes mellitus is a leading risk factor for the development of cardiovascular disease.1 Hyperglycemia is the hallmark of diabetes mellitus, and its causal role in the development of microvascular complications is well-established. Its involvement in the development of macrovascular complications is less clear, although there is increasing evidence that poor glycemic control increases the risk for vascular events.
Redox signalling is a key mechanism by which hyperglycemia induces endothelial cell activation.2 High glucose increases the formation of reactive oxygen species (ROS) in endothelial cells.3 In vivo, hyperglycemia induces endothelial cell activation characterized by reduced nitric oxide bioavailibility and impaired vasoreactivity.4,5 Endothelial activation followed by leukocyte adherence and transmigration subsequently drives the pathogenesis of vascular disease.6
The renin-angiotensin system (RAS) may play an important role in vascular complications in diabetes mellitus.7,8 Angiotensin II has considerable pro-oxidant and proinflammatory effects.9,10 Upon binding to its type 1 receptor, angiotensin II activates NADPH oxidase and induces redox signalling in both endothelial cells as well as leucocytes.11 As a result, angiotensin II stimulates the formation of proinflammatory cytokines from the vasculature and mononuclear cells. We therefore postulated that valsartan, an angiotensin II type 1 receptor antagonist, may convey protection against hyperglycemia-induced redox signalling and improve endothelial function and leukocyte activation during hyperglycemia.
Materials and Methods
Eleven healthy, non-smoking, male volunteers were recruited for the study. All individuals were normotensive and had no history of cardiovascular disease, family history of premature atherosclerosis, or type II diabetes mellitus. The subjects did not use any medication. Subjects were asked to refrain from alcohol and caffeine-containing drinks 24 hours before the experimental procedures. The study was approved by the medical ethics review committee of the University Medical Center Utrecht, and written informed consent was obtained from each participant.
An open-label, prospective study was performed. The effects of a hyperglycemic clamp on endothelial function and cytokine release from peripheral blood leukocytes were studied on 2 test days; day 1 (without medication) and day 2 (after 4 weeks of treatment with valsartan). To determine the effects of valsartan after short-term and more sustained hyperglycemia, a 22 hour hyperglycemic clamp was performed. Twenty-two hours instead of twenty-four hours was chosen for logistic reasons and allowed assessment of endothelial function at approximately the same time of the day before and near the end of the clamp. The dosing regimen for valsartan was 80 mg once daily in the first 2 weeks and 160 mg once daily during the latter 2 weeks. During the clamp, liquid potassium chloride (20 mmol) was administered at t = 16 hours. A standard lunch was served after vascular and laboratory assessment at t = 4 hours. A standard evening meal was served at t = 8 hours, after which subjects fasted until vascular and laboratory investigations the next morning (t = 22 hours). No snacks were allowed during the clamp. A time-control experiment was performed in 5 volunteers on a third test day.
A venous catheter was inserted in a large vein at the antecubital fossa and in a more distal vein of the left arm. A hyperglycemic clamp was performed by a variable rate infusion of a 20% glucose solution to achieve and maintain a steady-state plasma glucose concentration of 12 mmol/L. The proximal vein was used for infusion of the glucose solution, the more distal vein for withdrawal of blood. During the first hour of the clamp, 0.5 mL blood samples were withdrawn to measure plasma glucose at 10 minute intervals. Between t = 1 hour and t = 10 hours, plasma glucose was measured at 15 minute intervals, between t = 10 and t = 15 hours at 30 minute intervals, and between t = 15 and t = 22 hours at 1 hour intervals. Insulin was measured at 1 hour intervals during the first 10 hours and every 2 hours during the last 12 hours. For other laboratory investigations, blood samples were collected before the clamp and at t = 4 and t = 22 hours during the clamp.
Plasma glucose was measured immediately using a glucose oxidase method (Yellow Spring Instruments). Plasma for other parameters was obtained by centrifugation at 3000 rpm for 10 min at 4oC and stored at -80o for further analysis. TNF-α and interleukin-6 (IL-6) were determined in duplicate using commercially available ELISA kits (R&D Diagnostics, Abingdon, UK). Insulin was determined by microparticle enzyme immune assay (Axsym, Abbott, Wiesbaden, Germany). Hemoglobin, white blood cell count, creatinin, total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides were analyzed by standard methods.
Assessment of Vascular Function
Ultrasonographic assessment of brachial artery post-ischemic flow-mediated vasodilation (FMD) was performed before initiation of the hyperglycemic clamp and at t = 2, t = 4, and t = 22 hours during the clamp. Upon arrival, patients were asked to rest in a supine position for 15 minutes. Ultrasound measurements were performed at the elbow of the right arm using a vessel wall-movement system (Wall Track System, Pie Medical), which consists of a 10 MHz linear array transducer connected to a data acquisition system and a personal computer. In order to optimize quality of the ultrasound images of the arterial wall, ultrasound gel, a 200 mL water bag as conductive medium, and a fixed probe holder were used. An optimal 2-dimensional B-mode image of the brachial artery was obtained. An M-line perpendicular to the vessel was selected, and the ultrasound system was switched to M-mode. The vessel-movement detector system registered the end-diastolic vessel diameter repeatedly during a period of 12 seconds. The first 3 measurements were averaged to provide a baseline arterial diameter. By inflation of a blood pressure cuff for 5 minutes at a pressure of 200 mm Hg, ischemia was applied to the forearm distal to the location of the transducer. Ultrasonographic measurements continued for 4 minutes after cuff release at 30 second intervals. The widest lumen diameter was taken as a measure for maximal post-ischemic vasodilation. In order to allow the arterial diameter to return to its baseline value, patients rested for 15 minutes. The vasodilatory response to nitroglycerine (400 ug spray), an endothelium-independent vasodilator, was assessed before and during the clamp at t = 22 hours. Brachial artery diameter was assessed at 2, 3, 4, and 5 minutes after nitroglycerin administration, and the widest lumen diameter was used for calculations. FMD and nitroglycerine-induced vasodilation were calculated as the percentage change relative to the baseline diameter.
Ex Vivo Cytokine Release
Blood was collected before and 22 hours after starting the hyperglycemic clamp. Recombinant lipopolysaccharide (LPS) was used as an inflammatory stimulus to induce cytokine release. An LPS dose response curve was generated (0 pg/mL, 0.06 pg/mL, and 0.6 pg/mL) by mixing whole blood with LPS in a sterile plastic tube. Whole blood without LPS was used as a control. After mixing gently, the tubes were placed in a humidified 5% CO2 atmosphere at 37oC. After 4 hours, the samples were centrifuged (420 × g for 10 minutes followed by 1000 × g for 5 minutes), and the supernatants were stored in duplicate at -80oC until further analysis. Concentrations of TNF-α and IL-6 were assessed in all supernatants.
Results are expressed as mean (SEM). The effects of the hyperglycemic clamp on FMD, systemic IL-6 and TNF-α, and LPS-induced cytokine release were analysed by 2-way repeated measures ANOVA. If differences reached statistical significance, a paired t-test was used to assess differences at individual time periods between study day 1 and 2. A paired t-test was used to test the difference in cytokine concentrations before and after incubation. P < 0.05 was considered significant.
All subjects completed the study. Clinical characteristics are shown in Table 1. No changes in systolic or diastolic blood pressure were observed after 4 weeks of treatment with valsartan. Valsartan treatment was well tolerated in all volunteers. Compliance to the study protocol was excellent. On average, 1 tablet per study participant was returned. Blood pressure remained stable during the hyperglycemic clamp, and no difference was observed between the 2 test days (data not shown). On both test days, fasting plasma glucose was 4.7 mmol/L, and similar glucose profiles were achieved during the clamp, with stable glucose concentrations achieved after 30 minutes of glucose infusion (Figure 1a). Glucose infusion rates were similar on both study days (Figure 1b). Insulin concentrations increased during the first 3 hours and after the standard lunch meal (Figure 1c). During vascular assessment at t = 4 and t = 22 hours, insulin concentrations were similar and had been stable for at least 1 hour (Figure 1). There was no significant difference between the insulin concentrations on the 2 study days. No differences in blood pressure or potassium were observed during the time-control and hyperglycemic clamp experiments (data not shown).
Effect of Valsartan on Endothelium-Dependent and Endothelium-Independent Vasodilation During a Hyperglycemic Clamp
The hyperglycemic clamp induced a 40% to 50% reduction in flow-mediated vasodilation compared with baseline (t = 0 hour) on day 1 (P < 0.001; Table 2 and Figure 2). Valsartan (V+) significantly improved flow-mediated vasodilation during the hyperglycemic clamp (P < 0.005; Table 2 and Figure 2). Pre-ischemic brachial artery diameters were not statistically different either before compared with during the hyperglycemic clamp or between study days (Table 2). No reductions in FMD were observed during the time-control experiment (Table 2 and Figure 2).
The hyperglycemic clamp did not affect nitroglycerin-induced vasodilation and, no effect of valsartan on endothelium-independent vasodilation was observed (18.6% ± 4.1% (V−) vs. 19.2% ± 4.6% (V+) before the hyperglycemic clamp; 16.4% ± 4.4% (V−) vs. 15.3% ± 5.4% (V+) at the end of the hyperglycemic clamp).
Effect of Valsartan on Circulating Markers of Inflammation and Endothelial Activation
A significant increase in IL-6 was observed on days 1 and 2 (P < 0.001 for both days) (Table 3). No difference was observed at any time point between the 2 study days. During the time control experiment, a similar increase in IL-6 was present (P < 0.03). TNF-α and soluble VCAM-1 concentrations were not significantly different during the hyperglycemic clamp on both study days or between the study days at similar time points.
Leukocyte Cytokine Release After an Inflammatory Stimulus
A significant reduction in lipopolysaccharide-induced IL-6 and TNF-α release was observed during valsartan treatment both before and at 22 hours during the hyperglycemic clamp (P < 0.05). (Figure 3) The hyperglycemic clamp did not modulate cytokine release after the inflammatory stimulus. No change in white blood cell count was observed during the hyperglycemic clamp or between the test days at any time point. (Table 2).
The main outcome of our study is that, despite the presence of high glucose concentrations, blockade of the angiotensin II type 1 receptor by valsartan significantly improves endothelial function and reduces cytokine release from peripheral blood leukocytes.
Endothelial and leucocyte redox signalling and the associated decreased NO bioavailibility play a key role in the pathophysiology of vascular complications in patients with diabetes mellitus.2 We investigated the role of the angiotensin II type 1 receptor signalling pathway in endothelial dysfunction induced not only by short-term (2 and 4 hours) but also by sustained (22 hours) hyperglycemia. The overnight hyperglycemic clamp was performed in order to learn whether sustained endothelial cell activation would result in a further reduction in vasoreactivity and would induce an exaggerated inflammatory response from leukocytes compared with short-term hyperglycemia. The clamp was well-tolerated, and no electrolyte disturbances or changes in hematocrite that could have influenced the results occurred. A rapid decrease in flow-mediated vasodilation was observed at 2 hours during the hyperglycemic clamp. These data are in line with several other groups showing a reduction in endothelial function after short-term (up to 6 hours) glucose-administration.4,5,12 In contrast, Reed et al reported that hyperglycemia had no effect on endothelial function.13 This discrepancy may be due to (the combination of) a different technique (venous occlusion plethysmography), a different vascular bed (forearm microvessels), and somatostatin infusion during systemic hyperglycemia in their study. We did not observe a further reduction in flow-mediated vasodilation after sustained hyperglycemia, suggesting stable dysfunctional vasoreactivity. Although soluble VCAM-1 is reported to be a marker of endothelial activation, we did not observe an increase in soluble VCAM-1 by hyperglycemia despite the presence of reduced FMD. IL-6 concentrations increased not only on the 2 test days, when a hyperglycemic clamp was performed, but also during the time-control experiment. This may be due to factors related to the experimental procedure and day-time variability.
We and several groups have shown that patients with diabetes mellitus are characterised by a reduction in both endothelium-dependent and endothelium-independent vasodilation.14-16 In the current study, no reduction in endothelium-independent vasodilation was observed after sustained hyperglycemia for 22 hours. These data suggest that factors other than hyperglycemia per se are involved in reduced endothelium-independent vasoreactivity in diabetes mellitus. We show that blockade of the angiotensin II type 1 receptor by valsartan significantly attenuates the impairment of flow-mediated vasodilation induced by hyperglycemia, although FMD did not completely recover to values before the clamp. These results may be due to the fact that activation of the angiotensin II type 1 receptor signalling pathway in humans mediates hyperglycemia-induced endothelial dysfuction.
Recruitment and activation of leucocytes are important drivers of complications in both atherosclerosis and diabetes mellitus.17 Treatment with angiotensin II type 1 receptor blockers reduces proinflammatory cytokines in patients with manifest vascular disease or cardiovascular risk factors.18
Endothelial cell activation is important for leukocyte adherence. After docking to stimulated endothelium, leucocytes are activated by signals from endothelial cells.19 In fact, endothelium-derived proteins, such as adenosine and GM-CSF, induce the production of ROS by neutrophils.20,21 Leukocytes are capable of maintaining the inflammatory insult to the endothelium by releasing inflammatory cytokines, notably TNF-α, that activate endothelial NF-κB.22 Leukocyte NADPH oxidase-generated ROS appears to play a crucial role in this process.22 Redox signalling by hyperglycemia in atherogenesis has been partially attributed to activation of NADPH oxidase.23 The angiotensin II type 1 receptor signalling pathway also shares the activation of protein kinase C with hyperglycemia-induced signalling. We mimicked this pathway of leukocyte activation and cytokine release by stimulating leukocytes ex vivo with lipopolysaccharide, an inflammatory stimulus. Our results show that valsartan significantly reduces the release of TNF-α and IL-6 during both normoglycemia and hyperglycemia to a similar extent. These data extend observations by Dandona et al, who showed lower activation markers in peripheral blood leukocytes after valsartan treatment.24 Whether the reduced release of proinflammatory cytokines by valsartan contributes to the improvement in endothelial function during the hyperglycemic clamp cannot be deduced from our data. However, a reduction in local concentrations of proinflammatory cytokines, released from adherent leukocytes, by angiotensin II type 1 receptor blockade is likely to have a beneficial effect on endothelial function.25 For example, anti-TNF treatment results in improvement of endothelial function in states characterized by vascular inflammation.26
Our study has some limitations. We did not assess blood parameters of blockade of the RAS. However, the marked improvement in endothelial function in combination with the effects on leukocyte release of inflammatory cytokines during valsartan treatment, which confirms and extends previously reported effects of angiotensin II receptor blockers, and the well-established pharmacological profile of valsartan strongly suggest that inhibition of the RAS underlies our results. Whole blood was used in the experiments on cytokine induction from leukocytes; therefore, we were not able to determine the differential cytokine release from mononuclear cells and neutrophils. Although monocytes are considered to be the characteristic atherogenic leucocyte, more recent data show that neutrophils may also be involved in modulation of vascular function.27 In fact, the neutrophil count appears to be predictive for cardiovascular event.28,29 Another consideration is that insulin has been reported to have both adverse and beneficial effects on vascular function. Several studies have shown adverse effects of insulin on vascular smooth muscle,30 endothelial function,31 and release of vasoconstrictors32; however, favourable effects on endothelial cell function and mononuclear cells have also been reported.33-37 We did not administer somatostatin to suppress endogenous insulin secretion because gastrointestinal discomfort is a frequent side effect of prolonged infusion, and this stress factor could affect vascular function. In addition, hyperinsulinemia is a characteristic feature in patients with type 2 diabetes. In our study, similar insulin concentrations were present at 4 and 22 hours during the clamp, when vascular function and laboratory assessment was performed. These concentrations are comparable with post-glucose challenge concentrations in insulin-resistant subjects.
In conclusion, valsartan improves endothelial function and cytokine release from leukocytes during hyperglycemia in the presence of hyperinsulinemia. A pathophysiological link between the effects of hyperglycemia and the RAS on the endothelium and inflammatory cells may contribute to the beneficial effects of inhibitors of the RAS on cardiovascular outcome in patients with diabetes mellitus.
We thank Jos op't Roodt for expert performance of the vascular function tests and Laura Splint for technical laboratory assistance.
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