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Department of Obstetrics and Gynecology, University of Glasgow, and Vascular Assessment Unit, Glasgow Caledonian University (C.H.), Glasgow, United Kingdom G3 8SJ
Address all correspondence and requests for reprints to: Dr. M. A. Lumsden, University Department of Obstetrics and Gynecology, Queen Mother’s Hospital, Glasgow, United Kingdom G3 8SJ. E-mail: malumsden{at}clinmed.gla.ac.uk
Abstract
Diabetes is associated with vascular dysfunction, which may be due in part to altered vascular responses to endogenous peptides such as endothelin-1. These altered responses may also contribute to the decreased maternal peripheral resistance in pregnancy. The aim of this study was to examine the effect of diabetes on the vasoconstrictor response to endothelin-1 in pregnant women.
Small arteries were isolated from nine healthy pregnant, seven type 1 diabetic pregnant women, and five healthy nonpregnant women. Contraction curves were performed on a wire myograph for noradrenaline (1 nM to 30 µM) and endothelin-1 (1 pM to 0.3 µM). Maximum responses and sensitivity were compared by t test.
No differences in maximum response to noradrenaline or potassium were seen among the three groups. The maximum response to endothelin-1 was significantly increased in pregnancy (P < 0.05), whereas endothelin-1 sensitivity was reduced in the diabetic compared with the nondiabetic pregnant women (P < 0.05).
Pregnant women have an increased maximum vasoconstriction response to endothelin-1 compared with nonpregnant women, whereas diabetic pregnant women demonstrate reduced sensitivity to endothelin-1. These observations suggest that endothelin-1 may play a role in maintaining peripheral vascular tone in normal pregnancy, and the decreased sensitivity seen in pregnant women with diabetes may reflect abnormal vascular reactivity.
DIABETES MELLITUS IS the most common endocrine disorder in the world, and a risk factor in the development of both macro- and microvascular disease (1), increasing the incidence of ischemic heart disease, stroke, retinopathy, and nephropathy (2, 3). Women with diabetes also have an increased risk of adverse pregnancy outcome, such as preeclampsia, macrosomia, and stillbirth, contributing to both maternal and fetal morbidity and mortality (4). Vascular and, in particular, endothelial dysfunction is thought to underlie many of these complications, with the greatest effect at the level of the resistance vasculature where alterations in vessel reactivity can significantly alter blood flow and tissue perfusion (5, 6). Hyperglycemia, although not the only factor involved in the development of endothelial dysfunction, plays an important role in the pathogenesis of diabetic complications, possibly through the generation of oxygen-derived free radicals, which results in endothelial damage, or increased mechanical stress (7).
Endothelin-1 (ET-1) is one of the most potent endogenous vasoconstrictors known. It is preferentially and predominantly released from the vascular endothelium and has a major role in the regulation of vascular tone and systemic blood pressure. ET-1 has binding affinities to two different G-coupled protein receptors, ETA and ETB, the latter of which has two subtypes, ETB1 and ETB2 (8). After release from the vascular endothelium, ET-1 binds to the endothelial ETB1 receptors and mediates vasorelaxation through nitric oxide release (9). This is followed by stimulation of both ETA and ETB2 receptors found on the vascular smooth muscle cell, resulting in a slower, but more sustained, vasoconstriction (10).
In pregnancy, ET-1 is thought to be important in the modulation of placental blood flow (11) and in normal fetal development (12). Plasma levels have been shown to remain constant throughout pregnancy, with similar levels seen in nonpregnant women. However, in pregnant women with diabetes, plasma ET-1 levels are doubled compared with those in nondiabetic pregnant women (13). It is possible that the elevated plasma ET-1 levels seen in diabetic patients may be one of the factors contributing to the development of the vascular complications of the disease.
Impaired endothelial function is present in patients with type 1 diabetes and women with gestational diabetes and is thought to be due to abnormal glucose homeostasis (5, 7, 14, 15). Changes in the response of vascular tissue to ET-1 may result from alterations in ET-1 receptor expression/density or uptake characteristics and may be seen as a consequence of pathology. This study aims to investigate the effect of type 1 diabetes on the vasoconstrictor response to ET-1 in pregnant women. We hypothesize that isolated arteries obtained from pregnant women with diabetes will demonstrate differences in both maximum response and sensitivity when exposed to ET-1 compared with nondiabetic pregnant women.
Subjects and Methods
Subjects
Local ethical committee approval was granted, and the study was
carried out in accordance with the Declaration of Helsinki (1989).
Written informed consent was obtained before cesarean section in nine
nondiabetic pregnant women and seven diabetic pregnant women at term,
and before elective laparotomy in five nondiabetic nonpregnant women.
All women were normotensive and matched for maternal and gestational
age. Diabetic patients were well controlled on insulin and did not
require significant manipulation of their regimens with advancing
gestation. Further patient characteristics, including blood pressures,
fetal weights, hemoglobin A1c levels, and disease
duration, are summarized in Table 1
.
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Experimental protocols
Biopsy specimens of sc fat were obtained from the laparotomy
wound edge and transported to the laboratory in 0.9% sodium chloride
solution in ice. Small arteries (
280 µm) were isolated and mounted
onto a wire myograph (Danish Myotech, Aarhus, Denmark).
Isometric tension was measured (Myodaq/Myodata 2.01, Danish Myotech, Aarhus, Denmark) using standard methodology (16). The vessels were allowed to equilibrate for 1 h in physiological saline solution (PSS) at 37 ± 0.1 C. They were exposed to 95% O2/5% CO2 to achieve a pH of 7.4 at 37 C. After this period of equilibration, the vessels underwent a normalization process, which determined the passive tension characteristics of each individual vessel. This allowed the internal circumference of each vessel to be set at a value 90% of that it would have if relaxed in vivo under a transmural pressure of 100 mm Hg. This value has been shown to be the internal circumference at which the vessels will produce a maximum contractile response (16). Before commencing experimentation, vessel viability (contractile and endothelial function) was confirmed by contraction to a high potassium chloride solution (123 mM KCl in PSS) and relaxation to acetylcholine [3 µM in vessels preconstricted with 10 µM noradrenaline (NA)]. Finally, to provide a measure of maximal vasoconstriction, the vessels were constricted with NA (10 µM) in KCl/PSS, allowed to relax to baseline, and equilibrated for an additional hour. The vessels used achieved a mean relaxation of 70%.
Cumulative contraction curves to NA (1 nM to 30 µM) and ET-1 (1 pM to 0.3 µM) were performed for all three patient groups.
Drugs and solutions
All drugs were obtained from Sigma (Poole, UK) and were made up in distilled H2O. PSS had the following composition: 118 mM NaCl, 25 mM NaHCO3, 4.5 mM KCl, 1.0 mM KH2PO4, 2.5 mM CaCl2, 1.0 mM MgSO4, 6.0 mM C6H12O6, 0.023 mM EDTA, and 0.03 mM ascorbic acid.
Statistical analysis
Contractile responses are expressed as a percentage of each individual vessel’s maximum potassium contraction, allowing for standardization of responses between vessels. Data are expressed as the mean ± SEM. The number of experiments is represented as n(N), where n is the number of vessels, and N is the number of patients. Maximum contraction and sensitivity (pD2, negative log of the concentration required to produce 50% of the maximum response) were compared using t test, with P < 0.05 being accepted as statistically significant.
Results
As there were no differences in vessel responses between the
smokers and nonsmokers or between indications for cesarean section, all
results were combined. There was also no significant difference in the
maximum responses to K+ or NA among the three
groups of women. These results are summarized in Table 2.
|
]. No difference in the maximum
response was seen between the diabetic and nondiabetic pregnant women
[maximum response (percent K+): nondiabetic
pregnant, 142.44 ± 12.37%, n = 9(9); diabetic pregnant,
138.29 ± 6.80%, n = 8(7)].
|
]. No
difference in sensitivity was seen between the healthy pregnant and
nonpregnant women (pD2: nondiabetic pregnant,
5.51 ± 0.13, n = 9(9); nonpregnant, 5.32 ± 0.24,
n = 6(5)].
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Our main findings in this study were a significant increase in the maximum vasoconstriction response to ET-1 in pregnant compared with nonpregnant women and a significant reduction in sensitivity to ET-1 in pregnant women with type 1 diabetes compared with healthy pregnant women.
Endogenous ET-1 appears to contribute to the regulation of basal vascular tone, systemic blood pressure, placental blood flow, and normal fetal development (8, 9, 10, 11, 12). Plasma ET-1 levels have been shown to be significantly elevated in conditions associated with vascular dysfunction, such as diabetes mellitus, preeclampsia, and chronic cardiac failure (13, 17, 18, 19). In this study supraphysiological concentrations of ET-1 were used to demonstrate the pharmacological effects of the peptide in the different patient groups, which give an indication of the maximum response that can be achieved by the tissue and may not reflect the effects found at physiological concentrations.
As there were no differences in responses to either potassium or NA, the results of this study are specific to ET-1. This suggests that any alteration in the ET-1 response is more likely to be secondary to changes in receptor expression and not the second messenger pathway within the smooth muscle (20). We have shown a significant increase in the maximum vasoconstrictor response to ET-1 in pregnancy, reflecting the ability of the vessels to respond to the peptide, suggesting that the normal vascular changes in pregnancy may be partly modified by ET-1. The nonpregnant women recruited into the study were slightly older than the pregnant women. However, this is unlikely to significantly alter vascular function, as the age difference was less than 10 yr, and all of the nonpregnant women were premenopausal, with no increased incidence in smoking.
In contrast, our findings in pregnant diabetic women show a different type of effect, with an attenuation of vascular tissue sensitivity to ET-1, but no change in the maximum vasoconstrictor response. This implies that vessels obtained from pregnant diabetic women require a higher concentration of ET-1 to produce a similar response, which may reflect vascular dysfunction. However, the diabetic women in our study achieved good glycemic control, as shown by the mean hemoglobin A1c level, the fetal weight at delivery, and the delivery of the infants at term (i.e. >37 completed wk). It is possible that the reduction in sensitivity we observed in pregnant diabetic women may be secondary to a down-regulation of ETA receptor signaling in response to a rise in plasma ET-1 levels. Wolff et al. (21) demonstrated that resistance vessels pretreated with the ETA receptor antagonist, BQ-123, showed a similar shift in the response curve to the right and a reduction in sensitivity to ET-1. Previous studies also demonstrated changes in vascular tissue sensitivity to other endogenous vasoconstrictor peptides during pregnancy. Gant et al. (22) showed that healthy pregnant women were remarkably resistant to the pressor effects of infused angiotensin II, and that the likely explanation for this reduction in sensitivity was the increased plasma angiotensin II concentrations seen during pregnancy (23).
The absence of a group of young nonpregnant type 1 diabetic women does not allow the effect of pregnancy on ET-1 responses in diabetic women to be studied. However, there would be significant difficulty in recruiting a sufficient number of young diabetic women undergoing laparotomy, and it would have been necessary to obtain the vessels from gluteal biopsies.
In a recent study by McIntyre et al. (20), vascular responses to ET-1 were studied in a mixed sample of male and female type 1 diabetic and nondiabetic subjects. They reported an increase in ET-1 sensitivity with no change in maximum response in the diabetic group and suggested that these findings may be due to either a down-regulation of ETB1 receptors or an up-regulation of ETA/ETB2 receptors. Another possibility would be the increased production of ET-1 resulting from a reduction in tissue sensitivity due to vascular damage. However, this type of response is much less well understood. The greater importance of the vasoconstrictor pathway suggests that the elevated plasma ET-1 concentrations seen in diabetics is more likely to result in a down-regulation of ETA/ETB2 receptors and to be observed as a reduction in tissue sensitivity to ET-1. Unfortunately, ET-1 levels were not measured in this study.
Insulin has been shown to increase ET-1 production, receptor synthesis, and gene expression (24, 25). Wolff et al. (13) reported constant plasma ET-1 levels throughout pregnancy similar to those in nonpregnant women, but significantly elevated levels in diabetic pregnant women. If insulin increases ET-1 production, one would expect elevated plasma ET-1 levels in healthy pregnant women who are physiologically insulin resistant and hyperinsulinemic. Therefore, the raised ET-1 levels seen in diabetics are unlikely to be due to exogenous insulin administration, but, rather, are caused by diabetes per se. The role of glucose in the vascular effects of ET-1 is unclear. Studies have reported both increased and decreased ET-1 release in endothelial cell cultures after glucose administration (26, 27).
Conclusions
This study has shown that pregnancy is associated with a greater maximum response to ET-1, whereas diabetes reduces ET-1 sensitivity in the peripheral vasculature. These important observations suggest that diabetes may significantly alter vessel sensitivity to circulating peptides and result in an increased disposition to developing microvascular disease.
Acknowledgments
We thank the patients and staff of the Western Infirmary, Glasgow Royal Maternity and Queen Mother’s Hospitals, for their help with this study, and Fiona Johnston for technical assistance.
Footnotes
This work was supported by the Wellcome Trust and the Glasgow Royal Infirmary Research Endowment Fund.
Abbreviations: ET-1, Endothelin-1; NA, noradrenaline; PSS, physiological saline solution.
Received March 28, 2001.
Accepted July 3, 2001.
References
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  K. Sharma, L. Deelman, M. Madesh, B. Kurz, E. Ciccone, S. Siva, T. Hu, Y. Zhu, L. Wang, R. Henning, et al. Involvement of transforming growth factor-{beta} in regulation of calcium transients in diabetic vascular smooth muscle cells Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1258 - F1270. [Abstract] [Full Text]
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Healthy pregnant (n = 9); 
, healthy nonpregnant (n = 5).
*, P < 0.05.