Hum. Reprod. Advance Access originally published online on November 25, 2005
Human Reproduction 2006 21(3):651-656; doi:10.1093/humrep/dei399
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Aortic function is compromised in a rat model of polycystic ovary syndrome
1 Ultrasound Department and 2 Department of Histopathology, North Middlesex Hospital, London, 3 Department of Surgery, 4 Department of Biochemistry and Molecular Biology, 5 Department of Chemical Pathology and 6 Academic Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, University College London
7 To whom correspondence should be addressed at: Academic Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, The Royal Free Hospital, Pond Street, London NW3 2PF, UK. E-mail: p.hardiman{at}medsch.ucl.ac.uk
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Abstract |
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BACKGROUND: Arterial mechanical parameters are modified in women with polycystic ovary syndrome (PCOS), before and during pregnancy. This study tested the hypothesis that aortic mechanics and endothelial function are modified in the mifepristone-treated rat model of PCOS. METHODS: Female rats injected daily with mifepristone or vehicle for 7–9 days were assessed by ultrasound to allow estimation of aortic stiffness index and compliance. The influence of acetylcholine (ACh) and sodium nitroprusside (SNP) on dissected phenylephrine-contracted aortic rings was assessed. RESULTS: Aortic compliance was reduced by 67% in mifepristone-treated rats versus controls (P < 0.05), while stiffness index was increased 2.3-fold (P < 0.02). ACh-induced dilation was less in aortic rings from mifepristone-treated rats (P = 0.022) and was less sensitive to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (P < 0.001), while SNP-induced dilation was greater (P = 0.001). CONCLUSIONS: Aortic mechanics in vivo and endothelial function in vitro were consistently perturbed in mifepristone-treated rats. Aortic ring behaviour suggested that NO release was depressed or degradation elevated, with a compensatory increase in NO sensitivity and/or activation of a non-NO-mediated relaxation mechanism. The mifepristone-treated rat is a valid model for investigation of the vascular deficits seen in PCOS.
Key words: animal model/polycystic ovaries/ultrasound/vascular system
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Introduction |
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Polycystic ovary syndrome (PCOS), characterized by infertility, oligo/amenorrhoea and hyperandrogenism, affects 5–10% of women of reproductive age. PCOS is also associated with metabolic abnormalities central to metabolic syndrome, such as hyperinsulinaemia, insulin resistance, impaired glucose tolerance and dyslipidaemia (Reaven, 2002
; Isomaa, 2003), and with type II non-insulin-dependent diabetes mellitus (NIDDM) (Talbott et al., 1995). NIDDM is an independent risk factor for atherosclerosis (Kannel and McGee, 1979) and many of the metabolic abnormalities are predictors for cardiovascular disease (Conway et al., 1992; Reaven, 2002). An increased prevalence of cardiovascular disease is expected in women with PCOS and we have demonstrated increased vascular stiffness (Lakhani et al., 2002) and intima–media thickness in the carotid and femoral artery (Lakhani et al., 2003) in young women with PCOS, suggesting atherosclerosis and underlying endothelial dysfunction (Balletshofer et al., 2003; Plutzky, 2003). Indeed, evidence of endothelial dysfunction in PCOS has been observed in vivo in the femoral artery (Paradisi et al., 2003) and the microvasculature (Kelly et al., 2003; Lakhani et al., 2005), though not in the brachial artery (Mather et al., 2000). Recently we noted increased stiffness and decreased compliance in the carotid arteries of pregnant women with PCOS in the first trimester and beyond (Hu et al., 2004). Decreased arterial compliance in the first trimester is associated with a history of pre-eclampsia, the most common serious complication of pregnancy (Spaanderman et al., 2000; Redman and Sargent, 2005), while a history of pre-eclampsia, obesity and hyperinsulinaemia are associated with further pre-eclampsic episodes (Duckitt and Harrington, 2005). Thus women with PCOS are at increased risk of hypertensive disease during pregnancy, and their babies of morbidity and mortality related to pre-term delivery and intrauterine growth restriction (de Vries et al., 1998).
The mechanisms by which PCOS modifies vascular function are poorly understood and require urgent investigation. An animal model of PCOS would allow in vivo analysis of arterial function and in vitro investigation of endothelial function. In the rat, daily injections of the antiprogestin mifepristone (also known as RU486) for 
1 week induce features characteristic of PCOS in humans, for example ovulatory failure, persistent vaginal cornification, and enlarged ovaries containing atretic follicles and follicular cysts (Sánchez-Criado et al., 1993; Ruiz et al., 1996) as well as increased serum LH, testosterone and estradiol, and decreased prolactin (Sánchez-Criado et al., 1992, 1993; Ruiz et al., 1996, 1997). Serum insulin-like growth factor 1 levels are also increased (Ruiz et al., 1997) and the response of such rats to therapies used in the treatment of PCOS is similar to that in humans (Ruiz et al., 1996). The mifepristone treated rat is a ‘fundamentally adequate’ model of PCOS (Ruiz et al., 1996) with which to investigate the effect of PCOS-like endocrinological perturbations on other physiological parameters in the short term (1–2 weeks).
We have used this model to test the hypothesis that arterial mechanics and endothelial function are modified in PCOS due to the underlying endocrine dysfunction, since it allowed in vivo assessment of the mechanical properties of the aorta followed by precise in vitro assessment of endothelial and vascular smooth muscle function.
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All chemicals were obtained from Sigma–Aldrich Chemicals, Gillingham, UK, unless otherwise stated. Animal procedures were performed in compliance with the Animals (Scientific Procedures) Act 1986.
Adult female Sprague–Dawley rats (aged 12–14 weeks, body weight 242 ± 4.5 g) were bred and housed locally at 22°C under a 14 h on/10 h off light cycle, with free access to food and water. Rats showing at least two consecutive 4–5 day estrous cycles were used, as assessed by vaginal smear. The treated group comprised eight rats injected s.c. daily with mifepristone in olive oil (2 mg/0.1 ml olive oil/100 g body weight/day), beginning on day 1 of the estrous cycle (Sánchez-Criado et al., 1993; Ruiz et al., 1996). Six control rats were injected with olive oil alone (0.1 ml/100 g body weight/day). After 7–9 days, anaesthesia was induced with Hypnorm (0.5 ml/kg i.m.; Jannsen Animal Healthcare Ltd, Bucks, UK) and diazepam (2.5 mg/kg i.p.; Dumex Ltd, Herts, UK), and maintained using isoflurane (Baxter Healthcare Ltd, Norfolk, UK), nitrous oxide and oxygen through a standard circuit (Yang et al., 2003). Body temperature was monitored with a rectal probe and maintained with an electric mat. Arterial oxygen saturation and heart rate were monitored non-invasively with a Biox 3740 pulse oximeter (Ohmeda Inc, Louisville, Colorado, USA), to ensure that a consistent level of anaesthesia was maintained.
When rats were stably anaesthetized, the abdomen was shaved to facilitate ultrasound aortic measurements. Real-time B- and M-mode images of the aortic wall were recorded using a 7.5 MHz linear probe (Pie Medical Systems, Maastricht, Netherlands), in the sagittal plane at 90° to the long axis, with signal output to a high resolution, echo-locked wall tracking system (Wall Track; Pie Medical Systems, Maastricht, The Netherlands). This system allowed measurement of vessel wall movement over time by automatically tracking assigned points representing the anterior and posterior aortic walls.
After ultrasound, a midline laparotomy was performed. Aortic systolic and diastolic blood pressures (SBP and DBP respectively) were measured by intra-aortic probe (Datex Engstron Light Monitor; Instrumentarium Corp, Helsinki, Finland) and aortic blood flow with a HT 207 flowmeter (Transonic Systems Inc., New York, USA). Trunk blood was obtained from the vena cava and serum prepared by centrifugation and stored at –20°C. Animals were killed by exsanguination and the thoracic aortae dissected and cleaned of adherent tissue. Aortae were then washed several times and kept in Krebs buffer (NaCl 118.6 mmol/l; KCl 2.8 mmol/l; CaCl2 2.5 mmol/l; MgSO4 1.2 mmol/l; NaHCO3 25.1 mmol/l; KH2PO4 1.2 mmol/l; glucose 5.5 mmol/l, gassed with 95% O2/5% CO2), until in vitro function was assessed. The right ovary was also dissected, fixed in formalin and embedded in paraffin wax.
Aortic function was assessed in vitro by measuring acetylcholine (ACh)-induced dilation in phenylephrine (PE)-contracted aortic rings, since this process requires functional interaction between the endothelium and smooth muscle. Aortae were washed several times in fresh Krebs buffer and cut into 2 mm wide rings, which were mounted on two hooks in a 7 ml organ bath containing Krebs buffer at 37°C, gassed with 95% O2/5% CO2. Isometric tension was measured with a transducer (Grass Instruments, Quincy, MA, USA) and digitized using a multi-channel recording system (Linton Instrumentation, Diss, Norfolk, UK) with AcqAcknowledge ACK100W software (Biopac Systems Inc., Goleta, CA, USA), which allowed simultaneous testing of six rings per aorta. Rings were tensioned to 
1 g, equilibrated for 60 min, and contracted with 3 µmol/l PE. The dilatory response to cumulative doses of ACh (1 nmol/l–100 µmol/l) was then measured. The PE–ACh treatment was repeated, then again after 100 µmol/l N G-nitro-L-arginine methyl ester (L-NAME) was added to three of the six aortic rings. A final treatment cycle used the nitric oxide (NO) donor sodium nitroprusside (SNP; 1 nmol/l–100 µmol/l) instead of ACh, L-NAME being added to the same three rings (Li and Forstermann 2000). Aortic tension was expressed as percentage relaxation, such that the tension induced by 3 µmol/l PE was defined as 0% relaxation, and the tension prior to PE treatment was defined as 100% relaxation.
Serum LH, FSH and insulin were assayed in duplicate by enzyme-linked immunoassay using Biotrak kits (APBiotech, Amersham, UK), with coefficients of variation (CV) of 7.6, 11.4 and 13.8% respectively. Serum testosterone was determined using an enzyme-linked immunosorbent assay kit (Roche Diagnostics, Welwyn Garden City, Herts, UK) with an intra-assay CV of 11.7%. Ovarian morphology was assessed in 6 µm sections cut from the wax-embedded ovary, to determine the presence of features consistent with previous studies of mifepristone-treated rats (Sánchez-Criado et al., 1993; Ruiz et al., 1996).
The aortic diametrical compliance (C) and stiffness index (
) were calculated using the mean aortic wall movement over at least three cardiac cycles and the aortic blood pressure and flow estimates taken immediately after laparotomy (Lakhani et al., 2002). Statistical significance (P < 0.05) was tested by analysis of variance (ANOVA) with post hoc analysis by Fisher’s protected least significant difference (PLSD) test. All data are expressed as mean ± SEM.
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Treatment of female rats with mifepristone for 7–9 days had no significant effect on body weight, SBP, DBP or mean arterial pressure (Table I). Endocrine disturbances were apparent (Table I), as previously noted (Sánchez-Criado et al., 1993
; Ruiz et al., 1996). Mifepristone-treated rats exhibited increased serum LH and testosterone. Serum insulin also tended to be elevated, but not significantly due to hypervariable insulin concentrations in the treated rats (Table I). Mifepristone treatment had no effect on serum FSH. Mifepristone-treated ovaries exhibited evidence of an increase in the abundance of follicular cysts and of arrested follicular growth (Figure 1), as previously reported (Sánchez-Criado et al., 1993; Ruiz et al., 1996).
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CV for ultrasound estimates of aortic parameters were in nearly all cases <10%, indicating that measurements were made reproducibly. In mifepristone-treated animals, the mean aortic diameter and blood flow were unaffected; however, aortic compliance was reduced by 67% while the stiffness index was increased 2.3-fold, relative to controls (Table II).
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In vitro organ bath assessment of endothelial and smooth muscle function was performed to determine whether the changes in aortic compliance and stiffness were related to dysfunction in these tissues. The PE-induced contraction (post 3 µ mol/l PE tension–resting tension) was decreased in the mifepristone-treated animals compared to the controls (349 ± 34 and 781 ± 126 mg respectively, P < 0.001). ACh induced a concentration-dependent relaxation in PE-contracted aortic rings from mifepristone-treated and control animals (P < 0.001, ACh effect by two-way ANOVA; Figure 2); however, relaxation was less in mifepristone-treated animals compared to controls (P = 0.022, mifepristone treatment effect by two-way ANOVA), notably at 0.1 µmol/l and 1.0 µmol/l ACh (Figure 2). This difference was not reflected by changes in the maximal relaxation (76 versus 81% at 100 µmol/l ACh, mifepristone-treated versus control) or in ED50 (i.e. –log10 of the concentration producing a half-maximal response), which was 7.9 (95% CI, 7.8–8.0) in mifepristone-treated rat aortic rings and 7.8 (95% CI 7.7–8.1) in controls.
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ACh-stimulated aortic relaxation is endothelium dependent, and thought to be due to activation of endothelial nitric oxide synthase (eNOS), producing nitric oxide (NO), which diffuses to the underlying smooth muscle causing relaxation. Indeed L-NAME, which inhibits 80% of eNOS activity, considerably impaired ACh-induced relaxation in aortic rings from normal and mifepristone-treated animals (P < 0.001, L-NAME treatment effect in both the mifepristone-treated and control groups by 2-way ANOVA; Figure 2). The inhibitory effect of L-NAME on ACh-induced relaxation was, however, less in aortic rings from the mifepristone-treated rats than in controls (P < 0.001, effect of mifepristone treatment on ACh induced/L-NAME antagonized relaxation by two-way ANOVA), notably at ACh concentrations of 1 µmol/l (Figure 2). This difference was reflected by a change in the maximal relaxation produced by 100 µmol/l ACh after 100 µmol/l L-NAME blockade (35% versus 16%, P < 0.005, mifepristone-treated versus control), but not in ED50 {7.1 [95% confidence interval (CI), 6.5–7.5] versus 7.1 (95% CI 6.5–7.6), mifepristone-treated versus control}.
To determine smooth muscle function independent of endothelial NO, aortic dilation due to smooth muscle relaxation was assessed using cumulative doses of SNP, a non-endothelium-dependent NO donor. As for ACh, SNP induced a concentration-dependent (P < 0.001, SNP effect by two-way ANOVA) relaxation in aortic rings from mifepristone-treated and control animals (Figure 3). Relaxation was, however, greater in mifepristone-treated animals compared to controls (P = 0.001, mifepristone treatment effect by two-way ANOVA), notably at 0.01 µmol/l and 0.1 µmol/l SNP (Figure 3). This was reflected by a change in the ED50 for SNP from 7.4 (95% CI 7.1–7.6) in mifepristone-treated rat aortic rings to 8.1 (95% CI, 7.9–8.2) in controls, but not in the maximal relaxation produced by 0.1 mmol/l SNP (103 versus 105%, mifepristone-treated versus control).
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Discussion |
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These results demonstrate increased stiffness index and decreased compliance in the aorta in vivo in this rat model of PCOS, replicating the changes seen in PCOS women, albeit in other arteries (Lakhani et al., 2002,
2003; Kelly et al., 2003). The altered arterial mechanics have important implications in women with PCOS if they conceive, as the reduced arterial compliance persists in the first trimester and beyond (Hu et al., 2004) and is associated with a history and further risk of pre-eclampsia (Spaanderman et al., 2000; Duckitt and Harrington, 2005). These findings are compatible with reports of an association between PCOS and hypertensive disorders of pregnancy (de Vries et al., 1998), a hypothesis we are currently investigating.
ACh-stimulated endothelium-dependent relaxation was attenuated in aortic rings from mifepristone-treated rats, whereas endothelium-independent SNP relaxation was exaggerated. L-NAME partially inhibited ACh relaxation, indicating it to be NO-mediated. Residual relaxation may be due to non-NO-mediated mechanisms or because L-NAME inhibits only 80% of eNOS activity (Li and Forstermann, 2000). L-NAME inhibition was reduced in aortic rings from mifepristone-treated rats, perhaps because a non-NO-mediated relaxation mechanism was more active. The mifepristone-related changes in aortic stiffness and compliance in vivo were consistent with the effects on aortic dilation noted in vitro. The endothelium synthesizes many vasodilators, NO being prominent in the rat aorta, with prostacyclin having a lesser role and endothelium-derived hyperpolarizing factor (EDHF) being more active in smaller arteries (Palmer et al., 1987; Shimokowa et al., 1996; Ge and He, 2000). This study suggests that aortic NO synthesis is impaired, and/or NO degradation enhanced, in mifepristone-treated animals. In partial compensation, aortic smooth muscle is more sensitive to NO, and non-NO-mediated ACh-stimulated vasodilation is more active, perhaps based on EDHF or prostacyclin.
Changes in endothelial behaviour and aortic mechanics may result from the endocrine effects of mifepristone injection. Serum estradiol is elevated in this model (Sánchez-Criado et al., 1993; Ruiz et al., 1996) but this should enhance, not diminish, vasodilation since estradiol stimulates endothelial NOS expression and NO synthesis (Goetz et al., 1994; Andersen et al., 1999), and ACh-induced aortic relaxation (Huang et al., 1998; Teoh et al., 2000). The elevated serum testosterone in mifepristone-treated rats could diminish vasodilatory responses to ACh, since in PCOS women, leg blood flow response to methacholine is negatively correlated with serum testosterone (Steinberg et al., 1996; Paradisi et al., 2003). The causal link is perhaps with insulin resistance, as noted in women with NIDDM, due to its effect on arterial smooth muscle NO sensitivity and degradation (Williams et al., 1996). In the present study, mifepristone had no effect on serum insulin and it enhanced aortic ring SNP sensitivity; nevertheless NO degradation might be increased. Testosterone stimulates aortic ring prostanoid synthesis and dilation in vitro (Selles et al., 2002; Tep-areenan et al., 2003). This cannot explain the diminished ACh dilation in the present study, but might underlie the non-NO-mediated vasodilatory pathway. The aortic effects of testosterone need further investigation, in androgen-treated ovariectomized female rats for example, before firm conclusions are made.
The effects of mifepristone were unlikely to have been due to its antagonism of progesterone or glucocorticoid action (Philibert, 1984) in the rat aorta. Progesterone has vasodilatory activity in rat aorta (Glusa et al., 1997; Mukerji et al., 2000; Zhang et al., 2002) but it is not mediated via the nuclear progesterone receptor and thus should be mifepristone insensitive (Glusa et al., 1997; Selles et al., 2002; Zhang et al., 2002). Mifepristone can also have had minimal acute effect in vitro, as the aortic rings were repeatedly washed (Zhang et al., 2002). A long-term mifepristone effect on aortic gene expression was also unlikely, since 8 days of progesterone injection in ovarectomized rats had no effect on aortic ring vasodilation (Sampaio-Moura and Marcondes, 2001). Finally, glucocorticoids inhibit prostacyclin and NO synthesis in rat aorta (Jeremy and Dandona, 1986; Wallerath et al., 1999), thus mifepristone blockade of glucocorticoid action promotes aortic ring vasodilation in vitro (Selles et al., 2002), in contrast to the present results.
Hyperlipidaemia impairs endothelial function (Shimokowa et al., 1989), but the effect of hyperglycaemia is controversial (Poston and Taylor, 1995; Oltman et al., 1997). Thus changes in aortic function in this study may be the result of metabolic modifications related to mifepristone injection. This model is, however, poorly characterized at the metabolic level and it is difficult to address the possible involvement of metabolic dysfunction at this time.
Mifepristone-injected female rats displayed disrupted ovarian morphology and elevated serum LH and testosterone, as previously seen in this model and in human PCOS women (Ruiz et al., 1996, 1997; Sánchez-Criado et al., 1992, 1993). They provide a valid model of PCOS (Ruiz et al., 1996) in which vascular function can be investigated in greater detail than in humans, using in vitro analysis. Findings in this model will assist the investigation of the association between PCOS and hypertensive disorders of pregnancy. An impaired contractile response to PE was noted in aortic rings from mifepristone-treated rats. This may be related to their elevated serum testosterone, since testosterone inhibits PE-mediated aortic ring contraction by enhancing endothelial NO release and inhibiting smooth muscle calcium influx (Costarella et al., 1996; Crews and Khalil, 1999). ACh-induced relaxation was calculated as a percentage, 0% relaxation being the PE-contracted tension and 100% relaxation being the resting tension prior to PE contraction. The differences in ACh-induced relaxation in aortic rings from mifepristone-treated and control rats should not therefore be related to the differences in PE-induced tension. A further experimental caveat is that aortic parameters were measured under anaesthesia, albeit under conditions standardized to remove variability due to its depth and the use of nitrous oxide, which is vasoactive. Thus differences in aortic parameters were mifepristone-related, but their degree may have been limited by anaesthesia. Aortic blood flow was unchanged whereas SPB tended to be elevated (P = 0.1), as seen in human PCOS women (Conway et al., 1992; Talbott et al., 1995; Elting et al., 2001). Aortic blood flow and pressure may be more disturbed in conscious rats. Another caveat is that the mechanical calculations relied on pre-laparotomy ultrasound and post-laparotomy blood pressure measurements; blood pressure may have been modified by laparotomy. However, consistent anaesthesia was achieved in all rats, thus variability in the stress of laparotomy was thought to be minimal.
This is the first demonstration that aortic mechanics are perturbed in vivo in the mifepristone-treated rat model of PCOS, in a manner comparable with arteries from human PCOS women (Talbott et al., 1995; Lakhani et al., 2002; Kelly et al., 2003; Lakhani et al., 2003). Aortic ring behaviour is modified in vitro in a manner suggestive of depressed endothelial NO release or elevated NO degradation. In compensation, smooth muscle sensitivity to NO is elevated, and a non-NO-mediated, ACh-stimulated relaxation mechanism appears activated, perhaps due to increased prostacyclin or EDHF. Perturbations in in vitro behaviour are compatible with the in vivo changes in mechanical properties, and may result from the endocrine perturbations in this model, perhaps in serum testosterone. Endocrine changes in women with PCOS may therefore cause their disturbed arterial properties. Further studies are necessary to characterize the endocrinological and metabolic changes in the mifepristone rat model and to clarify the mechanisms underlying the altered vascular behaviour.
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Submitted on June 10, 2005; resubmitted on September 8, 2005; accepted on October 12, 2005.
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