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Elevated plasma endothelin-1 levels in diabetes mellitus
AJH
2002; 15:967–972
Elevated Plasma Endothelin-1 Levels in Diabetes Mellitus Jochen G. Schneider, Nicole Tilly, Thomas Hierl, Ulrike Sommer, Andreas Hamann, Klaus Dugi, Gudrun Leidig-Bruckner, and Christian Kasperk Background: This study compares plasma endothelin-1 (ET-1) levels in patients with diabetes mellitus or hypertension with healthy controls, and investigates whether ET-1 levels are correlated with glycemic control, metabolic parameters, and vascular complications. Methods: The study population consisted of 103 patients with type 1 diabetes, 124 patients with type 2 diabetes, 35 hypertensive patients without diabetes mellitus, and 99 controls. Results: Plasma ET-1 concentrations were significantly higher in patients with type 1 diabetes (0.28 ⫾ 0.34 fmol/mL, P ⫽ .001), type 2 diabetes (0.31 ⫾ 0.32 fmol/ mL, P ⬍ .0001), and hypertension (0.35 ⫾ 0.26 fmol/mL, P ⬍ .0001) compared to controls (0.08 ⫾ 0.13 fmol/mL). Diabetic patients taking angiotensin converting enzyme (ACE) inhibitors had significantly lower plasma ET-1 levels than patients without (0.22 ⫾ 0.20 fmol/mL v 0.38 ⫾ 0.39 fmol/mL, P ⫽ .029). There were significant associations between ET-1 levels and age (r ⫽ 0.38, P ⬍ .05) and systolic blood pressure (BP) (r ⫽ 0.27, P ⬍ .05) in
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healthy controls. In diabetes we found only nonsignificant associations between ET-1 levels and age or vascular complications and a weak association between plasma ET-1 levels and glycemic control. Conclusions: Patients with diabetes or hypertension have elevated ET-1 levels, but do not exhibit positive correlations between ET-1 levels and BP, which was observed in healthy controls. Increased ET-1 levels do not induce hypertension in diabetes, but were lower in diabetic patients taking ACE inhibitors compared to those without ACE inhibitors. There is no significant association between ET-1 levels and vascular complications. These findings suggest that the plasma ET-1 level is not a marker of endothelial dysfunction but changes in plasma ET-1 levels may precede vascular complications associated with hypertension and diabetes. Am J Hypertens 2002;15: 967–972 © 2002 American Journal of Hypertension, Ltd. Key Words: Endothelins, diabetes mellitus, vascular complications, metabolic control.
ndothelins (ETs) 1 are a family of endogenous peptides produced in a variety of tissues where they play a role as modulators of vascular tone, growth, and hormone production. Endothelins were isolated as potent endogenous vasoconstrictors released by endothelial cells.1 The ET-1 is predominantly produced by endothelial cells, but also by vascular smooth muscle cells, macrophages, and other cells. The ET isoforms 2 and 3 are expressed in a variety of tissues.2 Among the isoforms endothelin-1 (ET-1) comprises most of the circulating ET. Evidence suggests a pathophysiologic role of ET-1 in diseases affecting the cardiovascular system (eg, hypertension, cerebrovascular disease, and heart failure).3 In animal models increased plasma ET-1 levels depress cardiac output and increase vascular resistance.4 Moreover, in patients with heart failure plasma ET-1 levels are two- to
fourfold elevated and correlate with the severity of the disorder.5 Furthermore, both experimental studies and clinical observations support a pathophysiologic role of ET-1 in human coronary atherosclerosis. Increased plasma ET-1 concentrations were observed after myocardial infarction and persistent elevations of ET-1 predict an increased mortality within the subsequent 12 months.6 In humans intravenous injection of exogenous ET-1 induces sustained vasoconstriction resulting in increased systemic blood pressure (BP).1,7 However, elevated ET-1 levels have been found in some but not all studies of patients with essential hypertension.8,9 Hypertension significantly boosts the development of vascular complications in diabetes mellitus.10 Because both hypertension and diabetes mellitus alter endothelial cell structure and function ET-1 may be involved in the development of hypertension and
Received April 17, 2002. First decision May 22, 2002. Accepted July 9, 2002. From the Department of Internal Medicine I, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany. This study was supported by grants from the Deutsche Forschungs-
gemeinschaft DFG (Ka 682/2-3, Ka 682/7-1). Address correspondence and reprint requests to Dr. Christian Kasperk, Department of Internal Medicine I, Endocrinology and Metabolism, University of Heidelberg, Bergheimer Strasse 58, D-69115 Heidelberg, Germany; e-mail: [email protected]
© 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
0895-7061/02/$22.00 PII S0895-7061(02)03060-1
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Table 1. Demographic and metabolic characteristics of the study population and plasma ET-1 levels among different groups
Healthy Number (male/ female) Age (y) ET-1 (fmol/mL) Fasting plasma glucose (mmol/L) HbA1c (%) Plasma creatinine (mol/L) Urinary albumin (mg/L) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Body mass index (kg/m2) Duration of disease (y)
Type 1 Diabetes
Type 2 Diabetes
46/53 46/57 53/71 45.0 ⫾ 16.6 45.3 ⫾ 12.3 63.4 ⫾ 9.9 0.08 ⫾ 0.13 0.28 ⫾ 0.34* 0.31 ⫾ 0.32*† 4.4 ⫾ 0.6 5.1 ⫾ 0.3 68.0 ⫾ 8.8
8.6 ⫾ 3.7 7.0 ⫾ 0.95
70.7 ⫾ 13.3 81.3 ⫾ 54.8
29.8 ⫾ 64.1 30.8 ⫾ 89.4 125 ⫾ 9.4
9.2 ⫾ 3.4 7.4 ⫾ 1.2
126 ⫾ 14.8
103 ⫾ 357 133 ⫾ 18
With ACE Inhibitor (ACEⴙ)
21/14 57/66 61.9 ⫾ 12 51.1 ⫾ 16 0.35 ⫾ 0.26† 0.38 ⫾ 0.39†
31/70 59.4 ⫾ 12 0.22 ⫾ 0.2†‡
5.6 ⫾ 1.5 5.9 ⫾ 0.7
72.2 ⫾ 9.1
71.5 ⫾ 10.8
23.8 ⫾ 3.2
25.4 ⫾ 2.9
8.7 ⫾ 3.6 7.0 ⫾ 1.0
114.3 ⫾ 123.8 79.6 ⫾ 53.9 n.d. 177 ⫾ 29.5
71 ⫾ 4.4
0
Without ACE Inhibitor Hypertensive (ACEⴚ)
39.9 ⫾ 154.17 127 ⫾ 18
8.9 ⫾ 3.3 7.3 ⫾ 1.1 73.3 ⫾ 17.8 42.4 ⫾ 99.9 132 ⫾ 16
104 ⫾ 18
71.2 ⫾ 10
72.2 ⫾ 10.0
28.6 ⫾ 4.2
27.4 ⫾ 5.1
26.2 ⫾ 3.7
28.0 ⫾ 4.0
18.5 ⫾ 12.9 12.4 ⫾ 7.8
9.3 ⫾ 8.7
13.9 ⫾ 10.0
16.3 ⫾ 11.3
ET-1 ⫽ endothelin-1; HbA1c ⫽ glycosylated hemoglobin A1c; ACE ⫽ angiotensin converting enzyme. Data are mean ⫾ SD. Differences in plasma ET-1 levels: * P ⫽ .001; † P ⬍ .0001 v controls; ‡ P ⬍ .05 v ACE (⫺) group.
premature atherosclerosis seen in patients with diabetes mellitus.11,12 Reports on plasma ET-1 levels in patients with diabetes mellitus are conflicting because increased,13,11 unchanged,14 or decreased,15 levels have been reported previously. The goal of the present study was to determine whether plasma ET-1 levels are altered in patients with diabetes mellitus type 1 and type 2 and in hypertensive patients without diabetes mellitus compared to ET-1 levels in healthy subjects. We also studied whether plasma ET-1 levels in diabetic patients are associated with glycemic control or the severity of vascular complications.
Methods Study Population The white study population consisted of 103 patients with type 1 (insulin-dependent), 124 patients with type 2 (non– insulin-dependent) diabetes mellitus, and 35 hypertensive patients without diabetes mellitus. The control group consisted of 99 healthy white volunteers. Informed consent was obtained from all subjects participating in the study. Table 1 summarizes the clinical characteristics of the study population and control group. Study Protocol Venous blood was drawn from the diabetic patients during routine ambulatory visits in the outpatient department. A
spot urine sample was obtained from each patient with diabetes mellitus. Key demographic characteristics were recorded, including age, gender, body mass index (BMI), duration of disease, and history of and present medication. Data about compliance were recorded from the patients’ files. Blood pressure was determined manually in the supine position with a mercury sphygmomanometer after a minimum of 5 min of rest; a mean of three measurements is given. The presence of diabetic retinopathy was assessed by analysis of mydriatic ophthalmoscopic examination. For statistical reasons the group of patients with proliferative retinopathy was compared to those without proliferative changes. Macroangiopathy was diagnosed in subjects with a history of cardiac infarction, coronary artery disease, peripheral arterial occlusive disease, stroke, or in the presence of pathologic ultrasonographic or angiographic examinations. Diabetic neuropathy was diagnosed in subjects with a history of paresthesia or reduced perception of the vibration of a tuning fork when placed on the ankle. Biochemical Analyses For the measurement of ET immunoreactivity blood was collected in ethylenediaminetetra-acetic acid (EDTA)coated polystyrene tubes and centrifuged immediately. The EDTA plasma was stored at ⫺80°C until plasma ET levels were determined by enzyme-linked immunoassay
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(Cat. No. BI-20052; Biomedica, Vienna, Austria). This assay shows 100% cross-reactivity with ET-1 and ET-2, but less than 5% with ET-3. The interassay variance of the plasma ET-1 concentration was 7.0%. It was previously demonstrated that ET-1 comprises most of circulating ETs.16 Thus, for the purpose of this study, results of ET immunoreactivity are referred to as plasma ET-1 concentrations. In ammonium heparinate plasma fasting glucose (FPG), creatinine, cholesterol, and triglycerides were measured by a Beckman LX 20 automatic analyzer (Beckman Instruments, Unterschleissheim, Germany), levels of lowdensity (LDL) and high-density (HDL) lipoproteins were determined after ultracentrifugation. Glycosylated hemoglobin was measured in EDTA blood by the Diamat autoanalyzer (Dade-Behring, Marburg, Germany). Albumin was determined in the morning spot urine samples (Behring Nephelometer II, Marburg, Germany). Statistical Analysis Statistical analyses were performed with SPSS for Windows, release 10.05 (SPSS Inc., Chicago, IL) and SAS software (version 8.1, SAS Institute, Inc., Cary, NC). The ET-1 levels are means ⫾ standard deviation (SD). The relationship between plasma ET-1 concentrations and FPG, glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, and disease duration were first analyzed by inspection of scatterblots. The strength of existing correlations was determined by Pearson’s and Spearman’s correlation coefficients as appropriate and further evaluated by the linear regression model. Retinopathy, neuropathy, and macroangiopathy were entered as dichotomous variables into binary logistic regression analysis. The mean differences in plasma ET-1 concentrations between groups were tested by the linear general model procedure for ANOVA and covariance analysis, adjusting for glycosylated hemoglobin, diastolic and systolic BP, BMI, and age. Statistical significance was accepted at P ⬍ .05.
Results The mean plasma levels of ET-1 in 103 patients with type 1 and 124 patients with type 2 diabetes mellitus were 0.28 ⫾ 0.34 fmol/mL and 0.31 ⫾ 0.32 fmol/mL, respectively; both values were significantly elevated compared to plasma ET-1 levels in 99 controls (0.08 ⫾ 0.13 fmol/mL, P ⫽ .001 and P ⬍ .0001, respectively). Mean plasma levels of ET-1 in 35 hypertensive patients not suffering from diabetes mellitus were also significantly increased (0.35 ⫾ 0.26 fmol/mL, P ⬍ .0001) compared to healthy controls, but not significantly different from plasma ET-1 levels of patients with type 1 (P ⫽ .8) or type 2 (P ⫽ .6) diabetes (Fig. 1). Adjusting ET-1 values into compared groups for glycosylated hemoglobin, systolic and diastolic BP, age, and BMI did not reveal a relationship of these variables to plasma ET-1 levels. In addition, we found no
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FIG. 1. Plasma endothelin-1 (ET-1) levels (mean ⫾ SEM) in control subjects (n ⫽ 99) are significantly lower than in patients with type 1 diabetes mellitus (n ⫽ 103), type 2 diabetes mellitus (n ⫽ 124), or hypertension (n ⫽ 35).
significant differences in plasma ET-1 levels between genders in the compared groups. Furthermore, plasma ET-1 levels did not significantly correlate with FPG, glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, or disease duration. Plasma ET-1 levels were also not significantly different between patients with diabetic retinopathy, neuropathy, or macroangiopathy compared to those without these complications. However, in patients with type 1 diabetes mellitus suffering from neuropathy and in patients with type 2 diabetes mellitus and macroangiopathy we found a trend toward increased plasma ET-1 levels as compared to those without these complication (0.31 ⫾ 0.38 fmol/mL v 0.22 ⫾ 0.24, P ⫽ .16 and 0.33 ⫾ 0.3 v 0.29 ⫾ 0.36 fmol/mL, P ⫽ .14). In type 1 diabetic patients there was a nonsignificant positive trend between plasma ET-1 levels and age (r ⫽ 0.3, P ⫽ .06). Age was not significantly correlated with plasma ET-1 levels in type 2 diabetes mellitus. Dividing all patients with type 1 and type 2 diabetes mellitus into groups with glycosylated hemoglobin values ⬍8.0% and ⱖ8.0%, there was a nonsignificant trend toward higher plasma ET-1 levels in the group with higher glycosylated hemoglobin (0.35 ⫾ 0.37 fmol/mL v 0.26 ⫾ 0.26 fmol/mL, P ⫽ .13). The mean BP did not differ between patients with type 1 and type 2 diabetes mellitus (Table 1). Thirty-six patients with type 1 and 83 patients with type 2 diabetes were hypertensive and were treated with medication. Angiotensin converting enzyme (ACE) inhibitors were given to 92% of the patients with type 1 diabetes and 80% of the patients with type 2 diabetes. By dividing the diabetic patients into groups treated and not treated with ACE inhibitors, we found significantly decreased plasma ET-1 levels in patients taking ACE inhibitors as compared to patients not taking these drugs (n ⫽ 101, 0.22 ⫾ 0.20 fmol/mL v n ⫽ 123, 0.38 ⫾ 0.39 fmol/mL, P ⫽ .029; Fig. 2). The few patients with diabetes and hypertension taking different BP-lowering drugs had elevated plasma ET-1 levels (n ⫽ 18, 0.36 ⫾ 0.4 fmol/mL). But the difference between these patients and the patients with ACE inhibi-
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FIG. 2. Plasma ET-1 levels (mean ⫾ SEM) in patients with type 1 or type 2 diabetes mellitus taking angiotensin converting enzyme (ACE) inhibitor (Inh.) (n ⫽ 101) are lower as compared to patients with type 1 or type 2 diabetes mellitus without ACE inhibitor (n ⫽ 123). Other abbreviation as in Fig. 1.
tors did not reach statistical significance, probably due to the small number (P ⫽ .12). Comparing the treated and not treated groups, we found a nonsignificant trend between glycosylated hemoglobin and plasma ET-1 level in patients treated with ACE inhibitor (r ⫽ 0.2, P ⫽ .06). We again found no significant correlation between plasma ET-1 levels and glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, disease duration, diabetic retinopathy, neuropathy, or macroangiopathy. After exclusion of type 1 or 2 diabetic patients receiving ACE inhibitors, we still observed no significant correlation between plasma ET-1 levels and FPG, glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, disease duration, diabetic retinopathy, neuropathy, or macroangiopathy. In healthy controls there were significant associations between age and plasma ET-1 level (r ⫽ 0.38, P ⬍ .05) and between systolic BP and plasma ET-1 levels (r ⫽ 0.27, P ⬍ .05). No other relevant correlation between plasma ET-1 levels and the investigated parameters was found in controls.
Discussion Our study clearly demonstrates increased plasma ET-1 levels in patients with diabetes mellitus type 1 or type 2 as compared to healthy controls. Physiologically, ET-1 is primarily secreted abluminally from the endothelial cell layer toward the medial layer of the vessel’s smooth muscle cells,17 indicating that ETs predominantly exert paracrine and autocrine effects and that increased plasma ET-1 levels may indicate early disturbances of endothelial function (leakage of ET-1 into the circulation). These occur before complications (eg, atherosclerotic processes) are clinically present. Due to low plasma levels of circulating ET-1 and its short half-life, ET-1 may be undetectable within a few minutes after blood collection. Therefore, great care was taken in our study to process the blood immediately for ET-1 measurement to prevent ET-1 degradation in the plasma.
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Plasma ET-1 concentrations are known to be elevated under various pathologic conditions.3,8,18,19 However, we did not observe a significant correlation between plasma ET-1 levels and clinical parameters of diabetic disease progression, such as BP, BMI, disease duration, diabetic retinopathy, neuropathy, macroangiopathy, serum creatinine, or albuminuria, which is consistent with the results of a number of smaller studies.14,20 –22 In contrast, other investigators observed significantly elevated plasma ET-1 levels only in type 2 diabetic patients with diabetic retinopathy (n ⫽ 56), and in type 1 diabetic patients with microalbuminuria (n ⫽ 39),23 which may be due to smaller patient numbers. In addition, some reports suggest that elevated plasma ET-1 levels are ameliorated by improvement of metabolic control.24 We found nonsignificant trends toward higher plasma ET-1 levels with higher glycosylated hemoglobin in diabetes, in general, and also in patients treated with ACE inhibitors. The demonstrated absence of an association between plasma ET-1 levels and vascular complications (our data)13,20 does not support a role for ET-1 as a quantitative marker for long-term diabetic vascular complications. We did not observe a significant correlation between plasma ET-1 levels and age in patients with type 1 or type 2 diabetes mellitus. Haak et al11 showed that 60% of patients with type 1 diabetes mellitus and elevated ET-1 levels (higher than the highest value in their controls [2.5 pg/mL]) had diabetes for more than 20 years. In our study no correlation was found between disease duration and ET-1 levels in type 1 or type 2 diabetes, but interestingly, there was a significant association between plasma ET-1 levels and age in the healthy controls and a nonsignificant association between age and plasma ET-1 levels in type 1 diabetic patients. Increased plasma ET-1 levels due to diabetic vasculopathy may obscure the positive correlation between plasma ET-1 levels and age, which in the healthy controls may indicate age-related disturbances of endothelial function. This process might be disturbed in disease such as diabetes because the ET system is already maximally stimulated with abnormal ET-1 levels whose excess secretion could be ameliorated by pharmacologic action. The stimulation of the ET system with increase of ET-1 levels seems to begin at an early state in the metabolic syndrome. Increased plasma ET-1 levels and disturbed nitric oxide production have already been demonstrated in relatives of patients with diabetes, hypertension, or metabolic syndromes.25,26 Endothelin-1 is known to promote sustained increases in BP7 and injected ET-1 dose-dependently increases systemic BP in humans.1 The role of ET-1 in the regulation of BP is demonstrated by the effect of bosentan, a nonselective ET-A/ET-B antagonist, which was shown to lower BP in an animal model and in patients with essential hypertension.27 This reflects at least a close relationship between the ET system and hypertension. Previously, ACE inhibitors were reported to reduce elevated plasma levels
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of ET-1 to normal values in patients with chronic heart failure.28 Mullen et al29 demonstrated an improvement of endothelial dysfunction by treatment with ACE inhibitors assessed noninvasively by high resolution external vascular ultrasound based on brachial artery flow-mediated dilatation, endothelium-dependent stimulus, and response to glyceryl trinitrate, which acts directly on vascular smooth muscle cells. These findings indicate an association between the renin-angiotensin and the ET systems. Angiotensin II activates endothelial angiotensin receptors, which stimulate the production of ET-1.30 This effect can be reversed by reducing angiotensin II levels.31 In our study, patients on ACE inhibitors had significantly lower plasma ET-1 levels than patients without ACE inhibitor, which has already been shown in vitro as well as in patients with hypertension or diabetes.32,33 Thus, the decrease of circulating angiotensin II concentrations by ACE inhibitor treatment may partly explain the decrease in plasma ET-1 levels. However, in our retrospective analysis we did not find a significant effect attributable to the reduced ET-1 plasma concentration. Further prospective studies are needed to clarify a possible additional benefit of ACE inhibitor treatment in patients with diabetes mellitus. In conclusion, elevated plasma ET-1 levels do not induce hypertension in diabetes, because diabetic patients had normal BP in spite of increased plasma ET-1 levels. Increased plasma ET-1 levels are significantly lower in diabetic patients treated with ACE inhibitors. In patients with diabetes or hypertension already exhibiting elevated ET-1 levels, the positive correlation between ET-1 levels and BP seen in healthy controls cannot be shown. In addition, there is no significant association between ET-1 levels and vascular complications in diabetic patients, indicating that under maximal stimulation no further increase is detectable. Therefore, ET-1 is not a quantitative marker for vascular complications but changes in plasma ET-1 levels may precede vascular complications associated with hypertension and diabetes.
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27. Krum H, Viskoper RJ, Lacourciere Y, Budde M, Charlon V: The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. Bosentan Hypertension Investigators. N Engl J Med 1998;338:784 –790. 28. Galatius-Jensen S, Wroblewski H, Emmeluth C, Bie P, Haunso S, Kastrup J: Plasma endothelin in congestive heart failure: effect of the ACE inhibitor, fosinopril. Cardiovasc Res 1996;32:1148 –1154. 29. Mullen MJ, Clarkson P, Donald AE, Thomson H, Thorne SA, Powe AJ, Furuno T, Bull T, Deanfield JE: Effect of enalapril on endothelial function in young insulin-dependent diabetic patients: a randomized, double-blind study. J Am Coll Cardiol 1998;31:1330 – 1335. 30. Barton M, Shaw S, D’Uscio LV, Moreau P, Luscher TF: Angiotensin II increases vascular and renal endothelin-1 and functional
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endothelin converting enzyme activity in vivo: role of ETA receptors for endothelin regulation. Biochem Biophys Res Commun 1997;238:861–865. 31. D’Uscio LV, Shaw S, Barton M, Luscher TF: Losartan but not verapamil inhibits angiotensin II-induced tissue endothelin-1 increase: role of blood pressure and endothelial function. Hypertension 1998;31:1305–1310. 32. Desideri G, Ferri C, Bellini C, De Mattia G, Santucci A: Effects of ACE inhibition on spontaneous and insulin-stimulated endothelin-1 secretion: in vitro and in vivo studies. Diabetes 1997;46:81–86. 33. Ferri C, Laurenti O, Bellini C, Faldetta MR, Properzi G, Santucci A, De Mattia G: Circulating endothelin-1 levels in lean non-insulindependent diabetic patients. Influence of ACE inhibition. Am J Hypertens 1995;8:40 –47.
2002; 15:967–972
Elevated Plasma Endothelin-1 Levels in Diabetes Mellitus Jochen G. Schneider, Nicole Tilly, Thomas Hierl, Ulrike Sommer, Andreas Hamann, Klaus Dugi, Gudrun Leidig-Bruckner, and Christian Kasperk Background: This study compares plasma endothelin-1 (ET-1) levels in patients with diabetes mellitus or hypertension with healthy controls, and investigates whether ET-1 levels are correlated with glycemic control, metabolic parameters, and vascular complications. Methods: The study population consisted of 103 patients with type 1 diabetes, 124 patients with type 2 diabetes, 35 hypertensive patients without diabetes mellitus, and 99 controls. Results: Plasma ET-1 concentrations were significantly higher in patients with type 1 diabetes (0.28 ⫾ 0.34 fmol/mL, P ⫽ .001), type 2 diabetes (0.31 ⫾ 0.32 fmol/ mL, P ⬍ .0001), and hypertension (0.35 ⫾ 0.26 fmol/mL, P ⬍ .0001) compared to controls (0.08 ⫾ 0.13 fmol/mL). Diabetic patients taking angiotensin converting enzyme (ACE) inhibitors had significantly lower plasma ET-1 levels than patients without (0.22 ⫾ 0.20 fmol/mL v 0.38 ⫾ 0.39 fmol/mL, P ⫽ .029). There were significant associations between ET-1 levels and age (r ⫽ 0.38, P ⬍ .05) and systolic blood pressure (BP) (r ⫽ 0.27, P ⬍ .05) in
E
healthy controls. In diabetes we found only nonsignificant associations between ET-1 levels and age or vascular complications and a weak association between plasma ET-1 levels and glycemic control. Conclusions: Patients with diabetes or hypertension have elevated ET-1 levels, but do not exhibit positive correlations between ET-1 levels and BP, which was observed in healthy controls. Increased ET-1 levels do not induce hypertension in diabetes, but were lower in diabetic patients taking ACE inhibitors compared to those without ACE inhibitors. There is no significant association between ET-1 levels and vascular complications. These findings suggest that the plasma ET-1 level is not a marker of endothelial dysfunction but changes in plasma ET-1 levels may precede vascular complications associated with hypertension and diabetes. Am J Hypertens 2002;15: 967–972 © 2002 American Journal of Hypertension, Ltd. Key Words: Endothelins, diabetes mellitus, vascular complications, metabolic control.
ndothelins (ETs) 1 are a family of endogenous peptides produced in a variety of tissues where they play a role as modulators of vascular tone, growth, and hormone production. Endothelins were isolated as potent endogenous vasoconstrictors released by endothelial cells.1 The ET-1 is predominantly produced by endothelial cells, but also by vascular smooth muscle cells, macrophages, and other cells. The ET isoforms 2 and 3 are expressed in a variety of tissues.2 Among the isoforms endothelin-1 (ET-1) comprises most of the circulating ET. Evidence suggests a pathophysiologic role of ET-1 in diseases affecting the cardiovascular system (eg, hypertension, cerebrovascular disease, and heart failure).3 In animal models increased plasma ET-1 levels depress cardiac output and increase vascular resistance.4 Moreover, in patients with heart failure plasma ET-1 levels are two- to
fourfold elevated and correlate with the severity of the disorder.5 Furthermore, both experimental studies and clinical observations support a pathophysiologic role of ET-1 in human coronary atherosclerosis. Increased plasma ET-1 concentrations were observed after myocardial infarction and persistent elevations of ET-1 predict an increased mortality within the subsequent 12 months.6 In humans intravenous injection of exogenous ET-1 induces sustained vasoconstriction resulting in increased systemic blood pressure (BP).1,7 However, elevated ET-1 levels have been found in some but not all studies of patients with essential hypertension.8,9 Hypertension significantly boosts the development of vascular complications in diabetes mellitus.10 Because both hypertension and diabetes mellitus alter endothelial cell structure and function ET-1 may be involved in the development of hypertension and
Received April 17, 2002. First decision May 22, 2002. Accepted July 9, 2002. From the Department of Internal Medicine I, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany. This study was supported by grants from the Deutsche Forschungs-
gemeinschaft DFG (Ka 682/2-3, Ka 682/7-1). Address correspondence and reprint requests to Dr. Christian Kasperk, Department of Internal Medicine I, Endocrinology and Metabolism, University of Heidelberg, Bergheimer Strasse 58, D-69115 Heidelberg, Germany; e-mail: [email protected]
© 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
0895-7061/02/$22.00 PII S0895-7061(02)03060-1
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Table 1. Demographic and metabolic characteristics of the study population and plasma ET-1 levels among different groups
Healthy Number (male/ female) Age (y) ET-1 (fmol/mL) Fasting plasma glucose (mmol/L) HbA1c (%) Plasma creatinine (mol/L) Urinary albumin (mg/L) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Body mass index (kg/m2) Duration of disease (y)
Type 1 Diabetes
Type 2 Diabetes
46/53 46/57 53/71 45.0 ⫾ 16.6 45.3 ⫾ 12.3 63.4 ⫾ 9.9 0.08 ⫾ 0.13 0.28 ⫾ 0.34* 0.31 ⫾ 0.32*† 4.4 ⫾ 0.6 5.1 ⫾ 0.3 68.0 ⫾ 8.8
8.6 ⫾ 3.7 7.0 ⫾ 0.95
70.7 ⫾ 13.3 81.3 ⫾ 54.8
29.8 ⫾ 64.1 30.8 ⫾ 89.4 125 ⫾ 9.4
9.2 ⫾ 3.4 7.4 ⫾ 1.2
126 ⫾ 14.8
103 ⫾ 357 133 ⫾ 18
With ACE Inhibitor (ACEⴙ)
21/14 57/66 61.9 ⫾ 12 51.1 ⫾ 16 0.35 ⫾ 0.26† 0.38 ⫾ 0.39†
31/70 59.4 ⫾ 12 0.22 ⫾ 0.2†‡
5.6 ⫾ 1.5 5.9 ⫾ 0.7
72.2 ⫾ 9.1
71.5 ⫾ 10.8
23.8 ⫾ 3.2
25.4 ⫾ 2.9
8.7 ⫾ 3.6 7.0 ⫾ 1.0
114.3 ⫾ 123.8 79.6 ⫾ 53.9 n.d. 177 ⫾ 29.5
71 ⫾ 4.4
0
Without ACE Inhibitor Hypertensive (ACEⴚ)
39.9 ⫾ 154.17 127 ⫾ 18
8.9 ⫾ 3.3 7.3 ⫾ 1.1 73.3 ⫾ 17.8 42.4 ⫾ 99.9 132 ⫾ 16
104 ⫾ 18
71.2 ⫾ 10
72.2 ⫾ 10.0
28.6 ⫾ 4.2
27.4 ⫾ 5.1
26.2 ⫾ 3.7
28.0 ⫾ 4.0
18.5 ⫾ 12.9 12.4 ⫾ 7.8
9.3 ⫾ 8.7
13.9 ⫾ 10.0
16.3 ⫾ 11.3
ET-1 ⫽ endothelin-1; HbA1c ⫽ glycosylated hemoglobin A1c; ACE ⫽ angiotensin converting enzyme. Data are mean ⫾ SD. Differences in plasma ET-1 levels: * P ⫽ .001; † P ⬍ .0001 v controls; ‡ P ⬍ .05 v ACE (⫺) group.
premature atherosclerosis seen in patients with diabetes mellitus.11,12 Reports on plasma ET-1 levels in patients with diabetes mellitus are conflicting because increased,13,11 unchanged,14 or decreased,15 levels have been reported previously. The goal of the present study was to determine whether plasma ET-1 levels are altered in patients with diabetes mellitus type 1 and type 2 and in hypertensive patients without diabetes mellitus compared to ET-1 levels in healthy subjects. We also studied whether plasma ET-1 levels in diabetic patients are associated with glycemic control or the severity of vascular complications.
Methods Study Population The white study population consisted of 103 patients with type 1 (insulin-dependent), 124 patients with type 2 (non– insulin-dependent) diabetes mellitus, and 35 hypertensive patients without diabetes mellitus. The control group consisted of 99 healthy white volunteers. Informed consent was obtained from all subjects participating in the study. Table 1 summarizes the clinical characteristics of the study population and control group. Study Protocol Venous blood was drawn from the diabetic patients during routine ambulatory visits in the outpatient department. A
spot urine sample was obtained from each patient with diabetes mellitus. Key demographic characteristics were recorded, including age, gender, body mass index (BMI), duration of disease, and history of and present medication. Data about compliance were recorded from the patients’ files. Blood pressure was determined manually in the supine position with a mercury sphygmomanometer after a minimum of 5 min of rest; a mean of three measurements is given. The presence of diabetic retinopathy was assessed by analysis of mydriatic ophthalmoscopic examination. For statistical reasons the group of patients with proliferative retinopathy was compared to those without proliferative changes. Macroangiopathy was diagnosed in subjects with a history of cardiac infarction, coronary artery disease, peripheral arterial occlusive disease, stroke, or in the presence of pathologic ultrasonographic or angiographic examinations. Diabetic neuropathy was diagnosed in subjects with a history of paresthesia or reduced perception of the vibration of a tuning fork when placed on the ankle. Biochemical Analyses For the measurement of ET immunoreactivity blood was collected in ethylenediaminetetra-acetic acid (EDTA)coated polystyrene tubes and centrifuged immediately. The EDTA plasma was stored at ⫺80°C until plasma ET levels were determined by enzyme-linked immunoassay
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(Cat. No. BI-20052; Biomedica, Vienna, Austria). This assay shows 100% cross-reactivity with ET-1 and ET-2, but less than 5% with ET-3. The interassay variance of the plasma ET-1 concentration was 7.0%. It was previously demonstrated that ET-1 comprises most of circulating ETs.16 Thus, for the purpose of this study, results of ET immunoreactivity are referred to as plasma ET-1 concentrations. In ammonium heparinate plasma fasting glucose (FPG), creatinine, cholesterol, and triglycerides were measured by a Beckman LX 20 automatic analyzer (Beckman Instruments, Unterschleissheim, Germany), levels of lowdensity (LDL) and high-density (HDL) lipoproteins were determined after ultracentrifugation. Glycosylated hemoglobin was measured in EDTA blood by the Diamat autoanalyzer (Dade-Behring, Marburg, Germany). Albumin was determined in the morning spot urine samples (Behring Nephelometer II, Marburg, Germany). Statistical Analysis Statistical analyses were performed with SPSS for Windows, release 10.05 (SPSS Inc., Chicago, IL) and SAS software (version 8.1, SAS Institute, Inc., Cary, NC). The ET-1 levels are means ⫾ standard deviation (SD). The relationship between plasma ET-1 concentrations and FPG, glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, and disease duration were first analyzed by inspection of scatterblots. The strength of existing correlations was determined by Pearson’s and Spearman’s correlation coefficients as appropriate and further evaluated by the linear regression model. Retinopathy, neuropathy, and macroangiopathy were entered as dichotomous variables into binary logistic regression analysis. The mean differences in plasma ET-1 concentrations between groups were tested by the linear general model procedure for ANOVA and covariance analysis, adjusting for glycosylated hemoglobin, diastolic and systolic BP, BMI, and age. Statistical significance was accepted at P ⬍ .05.
Results The mean plasma levels of ET-1 in 103 patients with type 1 and 124 patients with type 2 diabetes mellitus were 0.28 ⫾ 0.34 fmol/mL and 0.31 ⫾ 0.32 fmol/mL, respectively; both values were significantly elevated compared to plasma ET-1 levels in 99 controls (0.08 ⫾ 0.13 fmol/mL, P ⫽ .001 and P ⬍ .0001, respectively). Mean plasma levels of ET-1 in 35 hypertensive patients not suffering from diabetes mellitus were also significantly increased (0.35 ⫾ 0.26 fmol/mL, P ⬍ .0001) compared to healthy controls, but not significantly different from plasma ET-1 levels of patients with type 1 (P ⫽ .8) or type 2 (P ⫽ .6) diabetes (Fig. 1). Adjusting ET-1 values into compared groups for glycosylated hemoglobin, systolic and diastolic BP, age, and BMI did not reveal a relationship of these variables to plasma ET-1 levels. In addition, we found no
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FIG. 1. Plasma endothelin-1 (ET-1) levels (mean ⫾ SEM) in control subjects (n ⫽ 99) are significantly lower than in patients with type 1 diabetes mellitus (n ⫽ 103), type 2 diabetes mellitus (n ⫽ 124), or hypertension (n ⫽ 35).
significant differences in plasma ET-1 levels between genders in the compared groups. Furthermore, plasma ET-1 levels did not significantly correlate with FPG, glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, or disease duration. Plasma ET-1 levels were also not significantly different between patients with diabetic retinopathy, neuropathy, or macroangiopathy compared to those without these complications. However, in patients with type 1 diabetes mellitus suffering from neuropathy and in patients with type 2 diabetes mellitus and macroangiopathy we found a trend toward increased plasma ET-1 levels as compared to those without these complication (0.31 ⫾ 0.38 fmol/mL v 0.22 ⫾ 0.24, P ⫽ .16 and 0.33 ⫾ 0.3 v 0.29 ⫾ 0.36 fmol/mL, P ⫽ .14). In type 1 diabetic patients there was a nonsignificant positive trend between plasma ET-1 levels and age (r ⫽ 0.3, P ⫽ .06). Age was not significantly correlated with plasma ET-1 levels in type 2 diabetes mellitus. Dividing all patients with type 1 and type 2 diabetes mellitus into groups with glycosylated hemoglobin values ⬍8.0% and ⱖ8.0%, there was a nonsignificant trend toward higher plasma ET-1 levels in the group with higher glycosylated hemoglobin (0.35 ⫾ 0.37 fmol/mL v 0.26 ⫾ 0.26 fmol/mL, P ⫽ .13). The mean BP did not differ between patients with type 1 and type 2 diabetes mellitus (Table 1). Thirty-six patients with type 1 and 83 patients with type 2 diabetes were hypertensive and were treated with medication. Angiotensin converting enzyme (ACE) inhibitors were given to 92% of the patients with type 1 diabetes and 80% of the patients with type 2 diabetes. By dividing the diabetic patients into groups treated and not treated with ACE inhibitors, we found significantly decreased plasma ET-1 levels in patients taking ACE inhibitors as compared to patients not taking these drugs (n ⫽ 101, 0.22 ⫾ 0.20 fmol/mL v n ⫽ 123, 0.38 ⫾ 0.39 fmol/mL, P ⫽ .029; Fig. 2). The few patients with diabetes and hypertension taking different BP-lowering drugs had elevated plasma ET-1 levels (n ⫽ 18, 0.36 ⫾ 0.4 fmol/mL). But the difference between these patients and the patients with ACE inhibi-
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FIG. 2. Plasma ET-1 levels (mean ⫾ SEM) in patients with type 1 or type 2 diabetes mellitus taking angiotensin converting enzyme (ACE) inhibitor (Inh.) (n ⫽ 101) are lower as compared to patients with type 1 or type 2 diabetes mellitus without ACE inhibitor (n ⫽ 123). Other abbreviation as in Fig. 1.
tors did not reach statistical significance, probably due to the small number (P ⫽ .12). Comparing the treated and not treated groups, we found a nonsignificant trend between glycosylated hemoglobin and plasma ET-1 level in patients treated with ACE inhibitor (r ⫽ 0.2, P ⫽ .06). We again found no significant correlation between plasma ET-1 levels and glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, disease duration, diabetic retinopathy, neuropathy, or macroangiopathy. After exclusion of type 1 or 2 diabetic patients receiving ACE inhibitors, we still observed no significant correlation between plasma ET-1 levels and FPG, glycosylated hemoglobin, plasma creatinine, albuminuria, BP, BMI, disease duration, diabetic retinopathy, neuropathy, or macroangiopathy. In healthy controls there were significant associations between age and plasma ET-1 level (r ⫽ 0.38, P ⬍ .05) and between systolic BP and plasma ET-1 levels (r ⫽ 0.27, P ⬍ .05). No other relevant correlation between plasma ET-1 levels and the investigated parameters was found in controls.
Discussion Our study clearly demonstrates increased plasma ET-1 levels in patients with diabetes mellitus type 1 or type 2 as compared to healthy controls. Physiologically, ET-1 is primarily secreted abluminally from the endothelial cell layer toward the medial layer of the vessel’s smooth muscle cells,17 indicating that ETs predominantly exert paracrine and autocrine effects and that increased plasma ET-1 levels may indicate early disturbances of endothelial function (leakage of ET-1 into the circulation). These occur before complications (eg, atherosclerotic processes) are clinically present. Due to low plasma levels of circulating ET-1 and its short half-life, ET-1 may be undetectable within a few minutes after blood collection. Therefore, great care was taken in our study to process the blood immediately for ET-1 measurement to prevent ET-1 degradation in the plasma.
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Plasma ET-1 concentrations are known to be elevated under various pathologic conditions.3,8,18,19 However, we did not observe a significant correlation between plasma ET-1 levels and clinical parameters of diabetic disease progression, such as BP, BMI, disease duration, diabetic retinopathy, neuropathy, macroangiopathy, serum creatinine, or albuminuria, which is consistent with the results of a number of smaller studies.14,20 –22 In contrast, other investigators observed significantly elevated plasma ET-1 levels only in type 2 diabetic patients with diabetic retinopathy (n ⫽ 56), and in type 1 diabetic patients with microalbuminuria (n ⫽ 39),23 which may be due to smaller patient numbers. In addition, some reports suggest that elevated plasma ET-1 levels are ameliorated by improvement of metabolic control.24 We found nonsignificant trends toward higher plasma ET-1 levels with higher glycosylated hemoglobin in diabetes, in general, and also in patients treated with ACE inhibitors. The demonstrated absence of an association between plasma ET-1 levels and vascular complications (our data)13,20 does not support a role for ET-1 as a quantitative marker for long-term diabetic vascular complications. We did not observe a significant correlation between plasma ET-1 levels and age in patients with type 1 or type 2 diabetes mellitus. Haak et al11 showed that 60% of patients with type 1 diabetes mellitus and elevated ET-1 levels (higher than the highest value in their controls [2.5 pg/mL]) had diabetes for more than 20 years. In our study no correlation was found between disease duration and ET-1 levels in type 1 or type 2 diabetes, but interestingly, there was a significant association between plasma ET-1 levels and age in the healthy controls and a nonsignificant association between age and plasma ET-1 levels in type 1 diabetic patients. Increased plasma ET-1 levels due to diabetic vasculopathy may obscure the positive correlation between plasma ET-1 levels and age, which in the healthy controls may indicate age-related disturbances of endothelial function. This process might be disturbed in disease such as diabetes because the ET system is already maximally stimulated with abnormal ET-1 levels whose excess secretion could be ameliorated by pharmacologic action. The stimulation of the ET system with increase of ET-1 levels seems to begin at an early state in the metabolic syndrome. Increased plasma ET-1 levels and disturbed nitric oxide production have already been demonstrated in relatives of patients with diabetes, hypertension, or metabolic syndromes.25,26 Endothelin-1 is known to promote sustained increases in BP7 and injected ET-1 dose-dependently increases systemic BP in humans.1 The role of ET-1 in the regulation of BP is demonstrated by the effect of bosentan, a nonselective ET-A/ET-B antagonist, which was shown to lower BP in an animal model and in patients with essential hypertension.27 This reflects at least a close relationship between the ET system and hypertension. Previously, ACE inhibitors were reported to reduce elevated plasma levels
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of ET-1 to normal values in patients with chronic heart failure.28 Mullen et al29 demonstrated an improvement of endothelial dysfunction by treatment with ACE inhibitors assessed noninvasively by high resolution external vascular ultrasound based on brachial artery flow-mediated dilatation, endothelium-dependent stimulus, and response to glyceryl trinitrate, which acts directly on vascular smooth muscle cells. These findings indicate an association between the renin-angiotensin and the ET systems. Angiotensin II activates endothelial angiotensin receptors, which stimulate the production of ET-1.30 This effect can be reversed by reducing angiotensin II levels.31 In our study, patients on ACE inhibitors had significantly lower plasma ET-1 levels than patients without ACE inhibitor, which has already been shown in vitro as well as in patients with hypertension or diabetes.32,33 Thus, the decrease of circulating angiotensin II concentrations by ACE inhibitor treatment may partly explain the decrease in plasma ET-1 levels. However, in our retrospective analysis we did not find a significant effect attributable to the reduced ET-1 plasma concentration. Further prospective studies are needed to clarify a possible additional benefit of ACE inhibitor treatment in patients with diabetes mellitus. In conclusion, elevated plasma ET-1 levels do not induce hypertension in diabetes, because diabetic patients had normal BP in spite of increased plasma ET-1 levels. Increased plasma ET-1 levels are significantly lower in diabetic patients treated with ACE inhibitors. In patients with diabetes or hypertension already exhibiting elevated ET-1 levels, the positive correlation between ET-1 levels and BP seen in healthy controls cannot be shown. In addition, there is no significant association between ET-1 levels and vascular complications in diabetic patients, indicating that under maximal stimulation no further increase is detectable. Therefore, ET-1 is not a quantitative marker for vascular complications but changes in plasma ET-1 levels may precede vascular complications associated with hypertension and diabetes.
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