Endothelial dysfunction in myometrial arteries of women with gestational diabetes

diabetes research and clinical practice 89 (2010) 134–140

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Endothelial dysfunction in myometrial arteries of women with gestational diabetes Haiju H. Chirayath *, Mark Wareing, Michael J. Taggart, Philip N. Baker The Maternal and Foetal Health Research Centre, St. Mary’s Hospital, Manchester M13 9WL, United Kingdom

article info

abstract

Article history:

Aims: This study directly examined endothelial function of myometrial arteries in the

Received 31 January 2010

uterus of women with gestational diabetes mellitus (GDM), healthy pregnant and non-

Accepted 29 March 2010

pregnant women. It also examined whether endothelial function was affected by the

Published on line 22 April 2010

changes in glucose concentration (from 2 to 12 mmol/L) that is seen in poorly controlled diabetes.

Keywords:

Methods: Ex vivo myometrial arteries from the uterus of women with GDM (N = 16) as well as

Diabetes

healthy pregnant (N = 16) and non-pregnant women (N = 15) were mounted on a wire

Gestational diabetes

myograph. The effect of changing glucose concentrations from 5 mmol/L to 2, 8 and

Endothelial dysfunction

12 mmol/L glucose for 30 min was studied.

Glucose

Results: Myometrial arteries from women with GDM had significantly impaired endothe-

Uterus

lium-dependent relaxation compared to those from both healthy pregnant and non-preg-

Endothelium

nant women (two-way ANOVA, p < 0.001). Changes in glucose concentration had no effect on constriction or endothelium-dependent relaxation in any group studied (two-way ANOVA, p > 0.05). Conclusions: Fluctuations of glucose concentrations between 2 and 12 mmol/L do not affect endothelial function. Endothelial dysfunction is present in the myometrial arteries of women with GDM, an inherent defect which may be responsible for some of the complications of GDM. # 2010 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

Gestational diabetes mellitus (GDM) is associated with increased morbidity and mortality for both mother and foetus [1,2]. It has been demonstrated that patients with poorly controlled GDM have an adverse pregnancy outcome [3,4]; poor control being characterised by both hyper- and hypoglycaemia. However, the precise pathophysiology linking poorly controlled diabetes to adverse pregnancy outcome remains largely unknown. Various studies have demonstrated that endothelial dysfunction is associated with diabetic complica-

tions, although the results vary between vascular beds [5]. The myometrial arteries in the body of the uterus form a crucial conduit for the blood supply from maternal circulation to the growing foetus [6]. It represents the terminal maternal vascular bed prior to the start of foetal circulation in the placental vessels [7]. We examined whether myometrial arteries in women with GDM was associated with aberrant endothelial function, as this may be a cause of some of the complications seen in this condition. We also investigated whether the changes in glucose concentration seen in diabetes are associated with alterations in endothelium-

* Corresponding author at: Diabetes Department, Milton Keynes Hospital, Milton Keynes MK6 5LD, United Kingdom. Tel.: +44 1908 660033; fax: +44 1908 669348. E-mail address: [email protected] (H.H. Chirayath). 0168-8227/$ – see front matter # 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2010.03.022

diabetes research and clinical practice 89 (2010) 134–140

dependent relaxation. This is the first report of endothelial function in the myometrial arteries of women with GDM. A novel feature of this study was that endothelial dysfunction was investigated directly in ex vivo vessels rather than using surrogate markers for endothelial dysfunction.

2.

Materials and methods

The study was approved by the Central Manchester Local Research Ethics Committee and it conformed to the guidelines of the Declaration of Helsinki [8]. Informed written consent was obtained from all participants. BMI was calculated at the time of the first antenatal booking, typically in the first two months of pregnancy. All patients had an elective Caesarean section at term. The two most common indications for Caesarean section were breech delivery and previous Caesarean section in healthy women, with macrosomia being an additional indication in women with GDM. None of the women were in labour at the time of section and pre-eclampsia had been excluded in all patients (BP less than 140/90 mm Hg with no proteinuria).

2.1.

Subjects

2.1.1.

Healthy group

Pregnant: 16 healthy pregnant women (with no known disease) who had uncomplicated singleton pregnancies and who were not on any medication were selected. Non-pregnant: 15 healthy non-pregnant women (with no known disease) and who were not on any medication were selected. These patients were undergoing a hysterectomy, the common indications for this being menorrhagia or fibroids.

2.1.2.

GDM group

16 patients with GDM, who had no other co-existing disease, were not on medication (except insulin) and who had uncomplicated singleton pregnancies were chosen. GDM was diagnosed in pregnancy by an oral glucose tolerance test, which was normally performed at 24–28 weeks of gestation. All the patients with GDM fulfilled the WHO criteria for having gestational diabetes [9] i.e., glucose intolerance was first recognized during pregnancy and on a 75 g oral glucose tolerance test the fasting blood glucose was 7 mmol/L (126 mg/dL) and/or 2 h 7.8 mmol/L (140 mg/dL). Of the 16 patients, 8 had a normal post-natal glucose tolerance test and 8 patients, although requested to attend, abstained from the test as they had normal postnatal blood glucose readings. 11 patients were on insulin and 5 patients controlled their diabetes by dietary measures alone.

2.2.

Sample collection

Myometrial biopsies from the uterus were obtained from the upper lip of the transverse lower segment Caesarean section incision and from the same anatomic site in hysterectomy specimens. This tissue was immediately immersed in ice-cold modified Krebs’s solution (in mmol/L: NaCl 136.9, KCl 2.7, CaCl2 1.8, MgSO4 0.6, NaHCO3 11.9, KH2PO4 0.5 and glucose

135

11.5). Small myometrial arteries were carefully dissected out from the biopsy tissue.

2.3.

Wire myography

Segments of arteries approximately 2 mm long were mounted on a wire myograph (Multi Myograph System 610 M, Danish Myo Technology A/S, Aarhus, Denmark) and their lengths measured using a calibrated microscope eyepiece on a stereomicroscope. Two 40 mm steel wires were inserted into the lumen of an artery and the ends of the wire were secured to a set of steel jaws (one side attached to a strain gauge, the other to a movable micrometer) in a tissue bath. Initially, the bath contained 7 ml of physiological saline solution (PSS) with 5 mmol/L glucose (inmmol/L: 127.76 NaCl, 25 NaHCO3, 4.69 KCl, 2.4 MgSO4, 1.6 CaCl2, 1.18 KH2PO4, 5.0 glucose, 0.034 EDTA and pH 7.4), warmed to 37 8C and aerated with an air mixture containing 21% oxygen (British Oxygen Company, Surrey, UK).

2.4.

Normalization

The classical normalization procedure was used to set the resting vessel diameter for experimentation [10]. Briefly, vessels were stretched in small increments and allowed to equilibrate for 2–10 min between stretches. Passive tension was recorded and the vessel was stretched until the passive tension had reached the limit of 13.3 kPa (100 mm Hg). Vessels were then normalized to an internal circumference calculated as 90% of this limit for the remainder of the experiment, as it has been previously demonstrated that a maximal force is generated at this setting [11].

2.5.

Drugs

Arteries were constricted using 60 mmol/L potassium in physiological saline solution (KPSS, in mmol/L: 72.45 NaCl, 25 NaHCO3, 60 KCl, 2.4 MgSO4, 1.6 CaCl2, 1.18 KH2PO4, 0.034 EDTA and 2, 5, 8 or 12 glucose). After constriction had reached a plateau, incremental doses (10 10–10 6 M) of the endothelium-specific vasorelaxant bradykinin (Sigma–Aldrich, UK) were added at 2 min intervals.

2.6.

Experiment protocol

Myometrial arteries were obtained from both normal pregnant individuals and patients with GDM. For each experiment, arteries were all initially incubated in individual chambers at a glucose level of 5 mmol/L. Two constriction–relaxation curves were performed at this concentration and the data averaged. Vessels were then exposed to 2, 5 (control), 8 or 12 mmol/L glucose for 30 min. To avoid methodological error, vessels were randomly allocated to a particular glucose level. After 30 min exposure to the new glucose concentration, two further constriction–relaxation curves were performed and the data averaged.

2.7.

Data analysis

The data obtained as above was processed using the Myodata 2.02 program (Myonic Software, National Instruments

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diabetes research and clinical practice 89 (2010) 134–140

Table 1 – Characteristics of study participants. Parameters Age (years) BMI (kg/m2) Max. systolic BP (mm Hg) Max. diastolic BP (mm Hg) Gravidity Parity Antenatal blood glucose (low reading, mmol/L) Antenatal blood glucose (high reading, mmol/L) Mean antenatal HbA1C (%) Mother’s blood glucose at delivery (mmol/L) Baby’s blood glucose at delivery (mmol/L) Gestational age at delivery (weeks) Birth weight (g)

GDM (N = 16)

Healthy pregnant women (N = 16)

33.7  1.1 34.4  1.9 131  4 83  3 3  0.2 1.9  0.2 4.2  0.2 9.7  0.7 6.6  0.2 4.9  0.4 2.2  0.3 38.5  0.2 4039  143

31.6  1.0 26.6  0.8 121  2 72  2 2.3  0.2 0.9  0.1 – – – – – 39  0.2 3600  97

Corporation, USA). A paired comparison was made of the endothelium-dependent relaxation before and after the change in glucose levels. The degree of relaxation obtained by each incremental dose of bradykinin was expressed as a percentage of maximum constriction (which was taken as 100%); variation in the numerical value of tension developed in different vessels was thus eliminated. The active tension produced by the vessels was measured in mN/mm to eliminate variations arising due to different vessel lengths. Arteries with an active tension of less than 0.4 mN/mm were excluded from the analysis due to poor constriction. Student’s t test was used to analyse the constriction of arteries and patient details. Repeated measures ANOVA was used for comparing endothelium-dependent relaxation at different glucose levels. Results were then analysed and graphically plotted using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, USA). For all tests, a significant difference was taken as p < 0.05 and if such a difference was detected, Bonferroni post hoc tests were performed. Mean values  standard error of mean have been calculated.

3.

Results

3.1.

Clinical characteristics

The clinical characteristics of women with GDM, healthy pregnant women and healthy non-pregnant women are given in Table 1. There was no significant difference between healthy pregnant women and women with GDM in maternal age, gestational age or birth weight (unpaired t test, p > 0.05). However, women with GDM had a significantly higher BMI than healthy pregnant women (34.4  1.9 vs 26.6  0.8: unpaired t test, p < 0.05). The average HbA1c for each woman during the course of her pregnancy was calculated. The mean HbA1c of all women (6.6  0.2%) was only slightly above the upper limit of normal (6.5%). Pre-prandial antenatal blood glucose levels ranged between 4.2  0.2 and 9.7  0.7 mmol/L. These values represent the mean value of the lowest reading and the mean value of the highest reading in each patient. 11 patients with GDM were on human insulin, and the others were controlled through diet modification only.

3.2.

Healthy non-pregnant women (N = 15) 43  2.4 34.4  3.4 120  9 77  5 2  0.4 1.7  0.4 – – – – – – –

Comparison of constriction of arteries

The maximum constriction of myometrial arteries, expressed as active effective pressure, was compared between normal individuals and patients with GDM. No significant difference was noted in the active effective pressure between the two groups (unpaired t test, p > 0.05).

3.3.

The effect of acute changes in glucose concentration

The constriction as well as the endothelium-dependent relaxation of arteries at 5 mmol/L was compared to that after 30 min of incubation at 2, 8 and 12 mmol/L glucose, maintaining a control vessel at 5 mmol/L glucose. Acute changes in glucose concentration had no significant effect either on constriction (paired t test, p > 0.05) or endothelium-dependent relaxation (repeated measures ANOVA, p > 0.05: Fig. 1) in arteries from women with GDM. Similar results were obtained in arteries from healthy pregnant and non-pregnant women.

Fig. 1 – Endothelium-dependent relaxation in myometrial arteries from women with GDM at 5 and 12 mmol/L glucose concentrations. Two-way ANOVA, p > 0.05.

diabetes research and clinical practice 89 (2010) 134–140

Fig. 2 – Comparison of endothelium-dependent relaxation in myometrial arteries from healthy pregnant women and women with GDM. Two-way ANOVA, p < 0.001.

3.4. Comparison of endothelium-dependent relaxation in myometrial arteries from patients with gestational diabetes, healthy pregnant women and healthy non-pregnant women At the normoglycemic level (5 mmol/L glucose), endotheliumdependent relaxation was significantly impaired in arteries from patients with GDM compared to healthy pregnant women (two-way ANOVA, p < 0.001: Fig. 2). Similar impaired relaxation of vessels was also noticed in GDM compared to healthy non-pregnant women (two-way ANOVA, p < 0.001: Fig. 3), whereas endothelium-dependent relaxation was similar in healthy pregnant and non-pregnant women (twoway ANOVA: p > 0.05: Fig. 4).

4.

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Fig. 3 – Comparison of endothelium-dependent relaxation in myometrial arteries from healthy non-pregnant women and women with GDM. Two-way ANOVA, p < 0.001.

women with GDM [12]. Reduced nitric oxide synthesis has been demonstrated in the placenta of pregnant women with GDM, which may cause increased vascular resistance [13]. In patients with diabetes, vascular smooth muscle may also be less sensitive to the vasorelaxant effects of nitric oxide [14].

Discussion

The main finding of this study is that endothelium-dependent relaxation is impaired in the myometrial vascular bed in women with GDM compared to both healthy pregnant and non-pregnant women. At 5 mmol/L glucose, myometrial arteries from women with GDM exhibited impaired endothelium-dependent relaxation compared to those from healthy women. Impairment in these arteries was present even though these patients did not have poorly controlled diabetes, as evidenced by their glucose levels during pregnancy and their near-normal HbA1c. This signifies that factors independent of diabetes duration and glycemic control are important in the pathogenesis of endothelial dysfunction. Although the precise cause of endothelial dysfunction in GDM is uncertain, possible factors may include insulin resistance and reduced production of nitric oxide which is an endothelium-derived vasodilator. Insulin resistance has been noted even in lean

Fig. 4 – Comparison of endothelium-dependent relaxation in myometrial arteries from healthy pregnant women and healthy non-pregnant women. Two-way ANOVA, p > 0.05.

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Gestational diabetes is associated with a higher rate of preeclampsia [15]. The aberrant endothelial function reported in pre-eclampsia [16] may form a link between these two conditions. Increased insulin resistance has also been noted in pre-eclampsia [17], further strengthening the association with GDM. Although the precise cause of pre-eclampsia remains unknown, low nitric oxide levels have been demonstrated to be involved in its aetiology [18,19], which is yet another common feature it shares with pregnancies complicated by GDM. Myometrial arteries influence blood flow to the foetus and changes in arterial tone may be associated with aberrant blood supply to the growing foetus. As flow is proportional to the fourth power of radius [20], even small changes in vessel calibre may cause significant changes in blood flow to the foetus. However, it is uncertain whether the endothelial dysfunction demonstrated in this study leads to impaired blood flow in vivo. Zaidi et al. have demonstrated that an absence of subendometrial blood flow results in a poor success rate during in vitro fertilization [21]. As GDM is associated with impaired fertility [22], aberrant uterine blood supply may be a factor in the pathogenesis of this complication. Hyperglycemia has been associated with impaired endothelium-dependent relaxation in various arterial beds such as the forearm [23]. This study has demonstrated that rises and falls in glucose concentration (for 30 min) had no effect on either vessel constriction or endothelium-dependent relaxation in any of the three groups studied. Studies to date have examined the effect of glucose levels on endothelial function for long time periods of up to 24 h [24], and at very high levels such as 44 mmol/L [25]: neither scenario is seen clinically. The mechanisms of endothelium-dependent vasodilatation have been demonstrated to vary according to the size of the vessel and its anatomical location [5,26,27]. Therefore, extrapolating results drawn from one vessel bed, or indeed a specific type of diabetes may give rise to erroneous conclusions. For example, the study of endothelial dysfunction in pregnancies of women with Type 1 diabetes has led to conflicting results with impaired [28] and preserved endothelial function [29]. These studies differed both in the vascular bed studied as well as vessel calibre (brachial artery and subcutaneous arteries, respectively). It is therefore important to study clinically relevant models of endothelial dysfunction in physiologically important vascular beds. Hence for this study it was decided to study myometrial arteries using physiologically relevant glucose levels (2, 5, 8 and 12 mmol/L) that a poorly controlled diabetic patient commonly encounters on a day-to-day basis for a clinically relevant time period of 30 min [30]. The time frame of 30 min was chosen as it has been demonstrated by Buhling et al. (using the continuous glucose monitoring system) to be representative of the acute fluctuations of blood glucose that are seen in women with GDM [30]. However, it is possible that this time frame is insufficient to allow the production of free radicals that may be responsible for reducing the bio-availability of nitric oxide, a factor that has been postulated to be associated with endothelial dysfunction in diabetes [31]. The effect of hypoglycaemia on endothelial function is unknown and a novel finding of this study was that low glucose concentrations did not affect endothelium-dependent relaxation.

The association between obesity and endothelial dysfunction is unclear. Obesity has been demonstrated to cause impaired endothelial function [32], However a study in monozygotic twins has shown that endothelial dysfunction was not correlated with obesity, but with the accompanying metabolic abnormality of adiponectin deficiency [33]. Two studies examining endothelial function in obese women with previous GDM have not found obesity to be associated with altered endothelial function. In a study by Anastasiou et al., it had been demonstrated that flow-mediated dilatation was equally decreased in both obese and non-obese women with GDM [34]. Bergholm et al. have demonstrated that moderate weight loss and therapeutic lowering of LDL cholesterol were associated with improvement of endothelial function in obese women with previous GDM; whereas a similar improvement was not seen with weight loss alone [35]. In our study it is interesting to note that in the myometrial vascular bed, endothelial function was similar in healthy obese (non-pregnant) and non-obese (pregnant) women. However arteries from women with GDM had impaired endothelium-dependent relaxation compared to both nonobese healthy pregnant women and obese non-pregnant women. In this study, despite demonstrating impaired endothelial function in myometrial arteries, the fetal birth weight of women with GDM was higher than normal. The aetiology of large babies in GDM is multi-factorial but has not been associated with uterine blood supply. Hardy et al. have demonstrated that macrosomia was not related to gestational age, ethnic group or insulin treatment but was associated with non-pregnant weight, weight gain during pregnancy and high fasting plasma glucose levels, among which non-pregnant weight was the strongest predictor of macrosomia [36]. These findings have been confirmed by many other studies [37–39]. Obesity may therefore be of more importance in the pathogenesis of macrosomia than myometrial artery blood flow. Furthermore, it has been shown that decreased uterine blood flow in the diabetic pregnant rat does not modify the augmented glucose transfer to the foetus [40]. It is therefore possible that impaired endothelial function in myometrial arteries does not influence the aetiology of macrosomia, which may be attributed to the high BMI of the patients with GDM in this study. In summary, this study on the blood vessels of women with GDM has demonstrated that aberrant glucose concentrations for short periods had no effect on vessel constriction or endothelium-dependent relaxation. Endothelial dysfunction was demonstrated in the myometrial vascular bed of women with GDM, which was present despite adequate glycemic control and was independent of the effect of obesity. This finding may explain some of the complications of GDM such as increased infertility and pre-eclampsia, although further studies are required to clarify this further.

Acknowledgements This study was supported by a research grant from the Novo Nordisk UK Research Foundation. The authors would like to thank all the patients and medical staff involved in this study

diabetes research and clinical practice 89 (2010) 134–140

at St. Mary’s Hospital, Hope Hospital and North Manchester Hospital.

Conflict of interests

[17] [18]

The authors declare that they have no conflict of interest. [19]

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