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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Hypertension. 2014 May 19;64(2):405–414. doi: 10.1161/HYPERTENSIONAHA.114.03283

ELEVATED TESTOSTERONE LEVELS DURING RAT PREGNANCY CAUSE HYPERSENSITIVITY TO ANG II AND ATTENUATION OF ENDOTHELIUM-DEPENDENT VASODILATION IN UTERINE ARTERIES

Vijayakumar Chinnathambi 1, Chellakkan S Blesson 2, Kathleen L Vincent 1, George R Saade 1, Gary D Hankins 1, Chandra Yallampalli 2, Kunju Sathishkumar 1,*
PMCID: PMC4096063  NIHMSID: NIHMS588761  PMID: 24842922

Abstract

Elevated testosterone levels increase maternal blood pressure and decrease uterine blood flow in pregnancy, resulting in abnormal perinatal outcomes. We tested whether elevated testosterone alters uterine artery adaptations during pregnancy, and if these alterations depend upon endothelium-derived factors such as nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostacyclin, or endothelium-independent mechanisms such as angiotensin II. Pregnant Sprague-Dawley rats were injected with vehicle (n=20) or testosterone propionate (0.5mg/Kg/day from gestation-day 15-19;n=20). Plasma testosterone levels increased 2-fold in testosterone-injected rats compared to controls. Elevated testosterone significantly decreased placental and pup weights compared to controls. In endothelium-intact uterine arteries, contractile responses to thromboxane, phenylephrine and angiotensin II were greater in testosterone-treated rats compared to controls. In endothelium-denuded arteries, contractile responses to angiotensin II (pD2=9.1±0.04 vs. 8.7±0.04 in controls,p<0.05) but not thromboxane and phenylephrine were greater in testosterone-treated rats. Angiotensin II type-1b receptor expression was increased while angiotensin II type-2 receptor was decreased in testosterone-exposed arteries. In endothelium-denuded arteries, relaxations to sodium nitroprusside were unaffected. Endothelium-dependent relaxation to acetylcholine was significantly lower in arteries from testosterone-treated dams (Emax=51.80%±6.9% vs. 91.98%±1.4% in controls,p<0.05). Assessment of endothelial factors showed NO-, EDHF- and prostacyclin-mediated relaxations were blunted in testosterone-treated dams. Endothelial NO-synthase, small conductance calcium-activated potassium channel-3 and prostacyclin receptor expressions were significantly decreased in arteries from testosterone-treated dams. Hypoxia-inducible factor-1α, Ankrd37 and Egln were significantly increased in testosterone-exposed placentae. These results suggest that elevated maternal testosterone impairs uterine vascular function, which may lead to an increased vascular resistance and a decrease in uterine blood flow.

Keywords: Uterine artery, vasodilation, vasoconstriction, EDHF, prostacyclin, NO, placental hypoxia, fetal growth

INTRODUCTION

Elevated maternal testosterone (T) levels have long been associated with adverse pregnancy outcomes, both for the mother, her fetus, and the newborn. Increased maternal T produces at least two major potential effects: direct actions of T on the fetus and indirect actions on the maternal-fetal unit, which result in intrauterine growth retardation and chronic conditions in adult life, such as hypertension, dyslipidemia, and diabetes mellitus.1-6 Numerous studies have demonstrated that T directly causes fetal damage.7-11 On the other hand, the adverse effects of T on fetal growth could be from indirect action of T on the maternal-fetal unit and the uteroplacental circulation. Studies in humans have shown that elevated T as observed in polycystic ovary syndrome (PCOS) pregnancies is associated with impaired decidual trophoblast invasion, increased uterine artery resistance index and reduced blood flow, resulting in an increase in abnormal perinatal outcomes.12,13 However, the mechanisms underlying the T-induced reduction of uterine blood flow in pregnancy are not fully understood.

During pregnancy, the development of uteroplacental circulation with low vascular tone accommodates a more than 20-fold increase in uterine blood flow in near-term pregnant sheep and in humans, which ensures normal placental perfusion and fetal development.14,15 The adaptation of uterine artery contraction and relaxation mechanisms to pregnancy is complex. In addition to growth and remodeling of the uterine vasculature, decreased uterine artery resistance is accomplished by significantly blunted vascular contractility16,17 and increased endothelial nitric oxide (NO),18-20 prostacyclin (PGI2),21-24 and endothelium-derived hyperpolarizing factor (EDHF) 25,26 synthesis/release with enhanced endothelium-dependent relaxation of the uterine artery.27,28 The effects of T on vascular contractions are controversial and vary in different species, sex, vascular bed, and experimental conditions. T can inhibit NO,5,29-32 PGI2,33 and EDHF34 production and/or function and decrease endothelium-dependent vasorelaxation.32 In addition, both vasoconstriction and relaxation induced by T have been reported previously.35 Recent studies in rat models also suggest an important role for androgens in inducing key features of preeclampsia, including elevated mean arterial pressure, proteinuria, systemic endothelial dysfunction and reduced fetal weight.4,32 It is also demonstrated that increased T levels are associated with impaired placental development with significant decrease in endovascular trophoblast invasion13 and nutrient transport capacity.4 However, in spite of the well-documented effects of elevated maternal T in placental development and the available circumstantial evidence of its association with increased uterine artery resistance and pulsatility index36,37 and decreased uterine blood flow in PCOS pregnancy,12 the effect of T on uterine artery contractility has not been studied. Herein, we present evidence in an in vivo pregnancy rat model system that elevated T, at concentrations similar to that observed in clinical conditions, promotes selective hyperresponsiveness to angiotensin II (Ang II) and impairs endothelium-dependent NO-, EDHF- and PGI2-mediated relaxation in uterine arteries associated with placental hypoxia.

MATERIALS AND METHODS

All procedures were in accordance with National Institutes of Health guidelines (NIH Publication No. 85–23, revised 1996) with approval by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch at Galveston. On gestational day (GD) 13, dams were randomly divided into 2 groups. Dams in the treatment group were subcutaneously injected with T propionate (0.5 mg/Kg/day, n=20) for 5 days from GD 15–19. The control group received vehicle (sesame oil, n=20). This dose and duration of exposure is commonly used to mimic the two fold increase in plasma T levels observed in preeclamptic women and PCOS pregnancies.1,2,4,5,32 At GD20, animals were sacrificed, a portion of the uterine arteries were separated for vascular reactivity studies and the remaining arteries, and placenta were quickly frozen for RNA/protein isolation. Fetal weight and crown-rump length and placental weight and diameter were measured. Some dams were allowed to deliver at term and the birth weight of pups was recorded. An expanded Methods section is available in the online-only Data Supplement, which includes, animals, arterial segment preparation, vascular contractile and relaxation responses, RNA isolation and Quantitative real-time PCR, Western blotting and statistical analysis

RESULTS

Elevated T decreased placental weights and birth weight of pups

The length of gestation and mean litter size were not significantly affected by T treatment (supplement Table S2). Fetal weight and crown-rump length, placental weight and birth weight of pups were significantly reduced in the T-treated group compared with controls (supplement Table S2), consistent with previous reports.1,4,5,32 The plasma T levels in T-treated dams were 2.2 ±0.23 ng/mL compared to 1.0±0.25 ng/mL in vehicle-treated control dams (n=7 in each group; P < 0.05).

Contractile response to Ang II was selectively increased in endothelium-denuded uterine arteries of T rats

Figure 1 shows the effect of elevated T exposure on U46619-, phenylephrine (PE)- and Ang II– induced concentration-dependent contractions of endothelium–intact and –denuded uterine arteries. As shown in Table 1, in endothelium–intact arteries, the maximal response and the pD2 values of U46619- and PE- and Ang II–induced contractions were significantly increased in T rats compared to controls (n=5 to 8 in each group; P < 0.05). Removal of the endothelium significantly enhanced U46619-, PE- and Ang II–induced contraction to a greater extent in control than in T rats (Fig. 1 and Table 1; n=7 to 8 in each group; P < 0.05). The U46619- and PE-induced contractions in endothelium–denuded arteries of T rats were not significantly different from controls (Fig. 1A and B and Table 1; n=5 to 8 in each group). In contrast, in endothelium–denuded arteries, there remains a significant increase in Ang II–induced contraction in T-treated rats, as compared with that of controls (Fig. 1C and Table 1; n= 8 in each group; P < 0.05). These data indicate that T selectively increases Ang II induced contraction in endothelium-denuded uterine arteries.

Figure 1.

Figure 1

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T exposure enhances uterine artery responses to contractile agonists. Contractile responses were taken in endothelium-intact and –denuded uterine arteries to cumulative additions of (A) thromboxane agonist- U46619, (B) phenylephrine (PE), and (C) Ang II. (D) Ang II dose response in endothelium-intact arteries in absence or presence of losartan and PD123319. Values are given as means±SEM of 5 to 8 rats in each group.

Table 1.

The Emax and pD2 of concentration response curves induced contractile agonists in uterine arteries of control and T groups

Contractile agonists Control T
pD2 Emax pD2 Emax
U46619 – Endo + 6.62±0.01 80.5±0.99 7.01±0.02* 90.4±1.35*
U46619 – Endo - 7.09±0.01† 103.7±1.18† 7.10±0.01 101.8±1.37†
PE– Endo + 5.5±0.02 106.8±5.32 5.8±0.02* 119.9±1.77*
PE– Endo - 5.9±0.04† 125.3±5.65† 5.9±0.02 129.3±1.46†
Ang II– Endo + 8.3±0.05 45.5±3.31 8.7±0.02* 82.1±3.61*
Ang II– Endo - 8.7±0.04† 102.9±5.03† 9.1±0.04*† 135.0±14.80*†
PD + Ang II– Endo + 8.6±0.07‡ 87.9±4.60‡ 8.8±0.04 105.4±5.13*‡
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Values are expressed as mean±SEM of 10 to 16 uterine arterial rings from 5 to 8 rats in each group. pD2 refers to –log EC50 (mol/l) and Emax is presented as percent of 80 mM KCl contraction. Endo+ indicates endothelium intact. Endo- indicates endothelium denuded. PD indicates PD123319 (AT2 receptor antagonist).

*

p<0.05 compared to Control group.

†

p<0.05 compared to respective endothelium intact groups.

‡

p<0.05 compared to Ang II– Endo +.

Losartan and PD123319 on Ang II–induced contractions

To determine the receptor subtype through which Ang II mediated vascular contractions, uterine arterial rings were pretreated with losartan or PD123319. Losartan completely blocked Ang II– induced contractions of the endothelium–intact and –denuded arteries from both control and T-treated rats (Figure 1D and supplement Figure S3; n=5 to 8 in each group). PD123319 significantly enhanced Ang II–induced contractions in endothelium-intact arteries of both control and T rats, however, the magnitude of increase was greater in the arteries of controls than in T rats (Figure 1D and Table 1; P < 0.05; n=5 to 8 in each group). PD123319 did not significantly affect Ang II–induced contractions in endothelium-denuded arteries from control and T rats (supplement Figure S3).

Uterine arterial expression of Ang II receptors in T rats is altered with increased Ang II type-1b receptor and decreased Ang II type-2 receptor levels

To determine whether Ang II receptors expression in the uterine arteries correlated with alteration of Ang II contractile responses in T rats, mRNA and protein levels of Ang II receptors were determined with quantitative RT-PCR and Western blot analyses. As shown in Figure 2A, T rats had no significant changes in Ang II type-1a receptor (AT1aR) mRNA expression, but significantly increased Ang II type-1b receptor (AT1bR) mRNA expression by 3-fold in uterine arteries compared to controls (n=6 in each group; P < 0.05). In contrast, Ang II type-2 receptor (AT2R) was significantly decreased by 50% in uterine arteries of T rats (n=6) compared to controls (n=6; P<0.05; Figure 2A). Thus presence of elevated T levels significantly increased the AT1bR/AT2R ratio in uterine arteries by 6-fold compared to controls (n=6; P < 0.05; Figure 2A).

Figure 2.

Figure 2

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T exposure alters the mRNA and protein expression of Ang II receptors in uterine arteries. (A) T exposure increases AT1bR and decreases AT2R mRNA in uterine arteries. Real-time reverse transcriptase PCR was used to assess vascular AT1aR, AT1bR and AT2R mRNA expression. Quantitation of vascular AT1aR, AT1bR and AT2R expression was normalized relative to β-actin levels. (B) T exposure decreases AT2R protein expression in the uterine arteries. Protein were isolated from uterine arteries and probed for AT1R and AT2R. Representative Western blots for AT1R, AT2R and β-actin are shown at top; blot density obtained from densitometric scanning of AT1R and AT2R normalized to β-actin is shown at bottom. Values are given as means±SEM of 6 rats in each group. *P≤0.05 vs control.

There are no commercial antibodies available that can individually detect AT1aR and AT1bR, hence we used an antibody that detects both isoforms. As shown in Figure 2B, T rats showed a trend of increase in AT1R protein expression in the uterine arteries compared to controls (n=6; P = 0.06; Figure 2B). However, AT2R protein was significantly decreased in uterine arteries of T rats compared to controls (n=6 in each group; P < 0.05; Figure 2B). The AT1R/AT2R ratio in uterine arteries was significantly higher by 2.1-fold in T rats compared with controls (n=6; P< 0.05; Figure 2B).

Endothelium-dependent, but not endothelium-independent, relaxation in uterine arteries is impaired in T rats

We next determined the effect of T exposure on endothelium-dependent relaxations induced by acetylcholine (ACh) in uterine arteries. As shown in Figure 3A, ACh produced a concentration-dependent relaxation in uterine arteries from both control and T rats. T exposure resulted in a significant decrease in the maximal relaxation induced by ACh (Control: 91.98% ± 1.4% and T rats: 51.80% ± 6.9%; n=9 in each group; P<0.05).

Figure 3.

Figure 3

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T exposure impaired endothelium-dependent vascular relaxation in uterine arteries. Arterial rings were precontracted with PE (10−6 M) and examined for relaxation to ACh. Values are given as means±SEM of 9 rats in each group. *P≤0.05 vs control.

To determine the potential effect of T exposure on endothelium-independent relaxations, sodium nitroprusside (SNP)-induced relaxations of the uterine arteries were also examined in the present study. SNP-induced relaxations of the uterine arteries were not significantly different between control and T rats (supplementary Figure S4; n=9 in each group). These data indicate that T impairs endothelium-dependent, but not endothelium-independent, relaxation in uterine arteries.

NO-, EDHF- and PGI2-mediated endothelium-dependent relaxation is decreased in uterine arteries of T rats

Because endothelium-dependent relaxation is mediated by NO, EDHF and PGI2, we next examined the effect of elevated T on these 3 distinct relaxation pathways. The NO-component of relaxation was significantly reduced by 33% in the T rats (Emax = 48.64 ± 3.73%, n=8, P≤0.05) compared with controls (Emax = 72.56 ± 11.51%, n=8; Figure 4A). The EDHF-component of ACh-induced relaxation was significantly reduced by 84% in the T rats (Emax = 9.72% ± 8.76%, n=8; P≤0.05) compared with controls (Emax = 62.33% ± 3.70%, n=7) (Figure 4B). The maximal PGI2-mediated relaxation was 50.18 ± 8.53% (n=8; P< 0.05) in control rats and this was completely abolished in the T rats (-3.18 ± 1.73 %; n=8; Figure 4C). Blockade of all three pathways completely abolished ACh-induced relaxation (n=7; Figure 4D).These data suggest that presence of elevated T levels in pregnant rats impair all 3 endothelial relaxation pathways.

Figure 4.

Figure 4

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Prenatal T exposure selectively impairs endothelium-dependent NO-, EDHF-, and PGI2-mediated vascular relaxation in uterine arteries. Arterial rings were pretreated with specific blockers, contracted with PE, and examined for relaxation to ACh. (A) NO-mediated endothelium-dependent relaxation. ACh relaxation was taken in presence of PGI2 blocker indomethacin (10−5 M) and EDHF blocker (charybdotoxin (CTx) plus apamin, each 10-7 M). (B) EDHF-mediated endothelium-dependent relaxation. ACh relaxation was taken in presence of NOS inhibitor L-NAME (10-4 M) and with PGI2 blocker indomethacin. (C) PGI2-mediated endothelium-dependent relaxation. ACh relaxation was taken in presence of EDHF blocker and NOS inhibitor. (D) ACh relaxation in presence of blockers for all 3 pathways. ACh relaxation was taken in presence of EDHF, PGI2 and NOS inhibitor. Values are means ± SEM (n = 7 to 8 rats, 2 vessel segments/rat). *P < 0.05 compared with control group.

Endothelial NO synthase, small conductance calcium-activated potassium channel-3, and PGI2 receptor expression is decreased in uterine arteries of T rats

To determine whether endothelial NO synthase (eNOS) expression in the uterine arteries correlated with decrease of NO-mediated relaxations in T rats, eNOS expression and activity status was determined. There was a significant decrease in eNOS mRNA (Figure 5A) and protein levels (Figure 5B) in uterine arteries from T rats (n=6; P≤0.05) compared with controls (n=6). Examination of phosphorylation status of eNOS, as an indicator activity state showed phosphorylation at Ser1177 was significantly lower in the uterine arteries of T rats compared with controls (Fig. 5B; P≤0.05; n= 5 in each group).

Figure 5.

Figure 5

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T exposure decreases eNOS mRNA and protein expression in uterine arteries. (A) T exposure decreases eNOS mRNA in uterine arteries. Real-time reverse transcriptase PCR was used to assess vascular eNOS mRNA expression. Quantitation of vascular eNOS expression was normalized relative to β-actin levels. (B) T exposure decreases eNOS protein expression and its phosphorylation at Ser1177 in uterine arteries. Protein were isolated from uterine arteries and probed for total eNOS and Ser1177-phosphorylated eNOS. Representative Western blots for eNOS, Ser1177-phosphorylated eNOS and β-actin are shown at top; blot density obtained from densitometric scanning of eNOS and Ser1177-phosphorylated eNOS normalized to β-actin is shown at bottom. Values are given as means±SEM of 5 to 6 rats in each group. *P≤0.05 vs control.

Small conductance calcium-activated potassium channel-3 (SK3) and intermediate conductance calcium-activated potassium channel-1 (IK1) are a major source of EDHF generation in the vasculature. We determined whether the expression of SK3 and IK1 in the uterine arteries correlated with decrease of EDHF-mediated relaxations in T rats. There was a significant decrease in SK3 mRNA levels in uterine arteries from T rats (n=6; P≤0.05) compared with controls (n=6; Figure 6; left panel). However, the mRNA levels of IK1, was not significantly different between control and T rats (n=6 in each group; Figure 6). Our attempt to determine the SK3 and IK1 protein expression was less conclusive with many bands at inappropriate molecular weights similar to previous reports.38

Figure 6.

Figure 6

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T exposure decreases SK3 channel mRNA expression in uterine arteries. Real-time reverse transcriptase PCR was used to assess vascular SK3 and IK1 mRNA expression. Quantitation of vascular SK3 and IK1 components was normalized relative to β-actin levels. Values are given as means±SEM of 6 rats in each group. *P≤0.05 vs control.

Studies show that PGI2 is synthesized through cyclooxygenase-2 (Cox-2) and prostacyclin synthase (Pgis) and acts on PGI2 receptor (IPR) to cause vasodilation. The expression levels of Cox-2, Pgis and IPR in uterine arteries was determined to find if they correlated with decrease of PGI2-mediated relaxations in T rats. There was no significant difference in the mRNA and protein levels of Cox-2 and Pgis but the IPR expression (n=6; P≤0.05) was significantly reduced in uterine arteries of T rats compared to controls (n=6; Figure 7A and B).

Figure 7.

Figure 7

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T exposure decreases prostacyclin (IP) receptor mRNA and protein expression in uterine arteries. (A) mRNA expression. Real-time reverse transcriptase PCR was used to assess vascular Cox-2, Pgis and IP receptor mRNA expression. Quantitation of vascular Cox-2, Pgis and IP receptor was normalized relative to β-actin levels. (B) Protein expression. Protein were isolated from uterine arteries and probed for Cox-2, Pgis and IP receptor Representative Western blots for Cox-2, Pgis, IP receptor and β-actin are shown at top; blot density obtained from densitometric scanning of Cox-2, Pgis and IP receptor normalized to β-actin is shown at bottom. Values are given as means±SEM of 6 rats in each group. *P≤0.05 vs control.

Expression of hypoxia responsive genes are increased in placentas of T rats

Enhanced uterine artery contraction and reduced relaxation mechanisms may decrease uterine arterial blood flow, which would be anticipated to decrease oxygen delivery to the placenta. We, therefore, predicted that the placentas in T rats would be more hypoxic in vivo. To test this, we examined the expression of hypoxia-inducible factor-1α (HIF-1α, a key transcription factor for response to low oxygen), and other hypoxia responsive genes, Ankrd 37 and Egln. The mRNA and protein levels of HIF-1α were significantly increased in placentae of T rats (n=6; P≤0.05) compared to control placentae (n=6; Figure 8A). Consistently, the measurement of hypoxia responsive genes, Ankrd 37 and Egln also showed a significant increase in their mRNA levels in placentae of T rats (n=6; P≤0.05) compared to control placentae (n=6; Figure 8B).

Figure 8.

Figure 8

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T exposure increases hypoxia responsive gene expression in uterine arteries. (A) T exposure increases Hif-1α mRNA and protein expression in uterine arteries. Real-time reverse transcriptase PCR was used to assess vascular Hif-1α mRNA expression. Quantitation of vascular Hif-1α was normalized relative to β-actin levels. Protein were isolated from uterine arteries and probed for Hif-1α. Representative Western blots for Hif-1α and β-actin are shown at top; blot density obtained from densitometric scanning of Hif-1α normalized to β-actin is shown at bottom. (B) T exposure increases mRNA expression of hypoxia responsive genes, Ankrd 37 and Egln in uterine arteries. Real-time reverse transcriptase PCR was used to assess vascular Ankrd 37 and Egln mRNA expression. Quantitation of vascular Ankrd 37 and Egln was normalized relative to β-actin levels. Values are given as means±SEM of 6 rats in each group. *P≤0.05 vs control.

DISCUSSION

The major findings of the present study are that, in pregnant rat uterine arteries, a 2-fold increase in plasma T levels, similar to that observed in compromised pregnancies: 1) did not affect thromboxane- and α1-adrenoceptor-mediated contractions but concentration-dependently increased Ang II induced contractions with associated increase in AT1bR expression and decrease in AT2R expression; and 2) did not affect endothelium-independent relaxations mediated by SNP whereas attenuated the ACh-induced, endothelium-dependent NO-, EDHF- and PGI2-mediated relaxations with correlated decreases in eNOS, SK3 channel and IPR receptor expression, respectively. The enhanced uterine artery contraction and reduced relaxation mechanisms may decrease uterine arterial blood flow that may lead to decreased oxygen delivery to the placenta as indicated by increase in expression of hypoxia responsive genes in placentae.

In the present study, U46619-, PE-, and Ang II–induced contractions of endothelium-intact uterine arteries were significantly increased in T rats, suggesting an increased arterial sensitivity to contractile agonists. Removal of the endothelium potentiated U46619- and PE-induced contractions to a greater extent in control rats than in T rats, and there was no significant difference in U46619- and PE-induced contractions in endothelium-denuded arteries from control and T rats. This finding indicates that the enhanced U46619- and PE-induced contractions of uterine arteries in T rats are primarily due to the loss of the endothelial function, rather than increased U46619- and PE-induced contractions per se. In contrast, T treatment produced enhanced Ang II–induced contractions in the absence or presence of endothelium. This suggests that the enhanced arterial sensitivity to Ang II is primarily because of increased Ang II– induced contractions, per se, rather than the loss of the endothelium-mediated relaxation component. Thus, it is likely that the effect T on vascular smooth muscle contractile responses is agonist specific. In agreement with our finding, T is shown to selectively enhance the pressor effects of Ang II in male rats.39 These findings suggest that T-mediated uterine vascular smooth muscle dysfunction occurs at the agonist-specific receptor level rather than at common intracellular signaling pathways.

Ang II mediates its physiological effects mainly through 2 seven-transmembrane G-protein– coupled receptors, the AT1R and AT2R. The activation of AT1R, especially AT1bR (but not AT1aR) mediates vasoconstriction.40-42 The inhibition of Ang II–induced arterial contractions by losartan in both control and T rats indicated a primary role of AT1R in vasoconstrictions. In contrast to AT1R, the activation of AT2R induces vasodilation.43 Although most adult blood vessels express predominantly AT1R, pregnant uterine arteries contain predominantly AT2R (>70%).44,45 These AT2 receptors in pregnant uterine arteries are known to counteract AT1-mediated contractions in vitro46 and in vivo.47-51 The present findings that PD123319 did not affect Ang II-induced contractions in endothelium-denuded arteries but potentiated Ang II-induced contractions to a greater extent in endothelium-intact uterine arteries from controls than in T rats suggesting that endothelial AT2R plays a primary role in modulating Ang II contractions and that the function of AT2R is impaired in T rats. Consistently, elevated T levels significantly increased AT1bR expressions and decreased AT2R expression in the uterine arteries of pregnant rats suggesting that the increased AT1bR/AT2R ratio may contribute to the exaggerated vasoconstriction to Ang II.

The present study shows that elevated T levels attenuate ACh-induced relaxations in the uterine artery. To determine whether the impaired relaxations of the uterine artery in T-treated vessels were not related to a downstream signal of the endothelium, we examined the endothelium-independent relaxation of the uterine artery by SNP. The finding that SNP-induced relaxations of the uterine arteries were not significantly changed in T rats suggests that the elevated T causes an inhibition of endothelial function.

Studies have shown that endothelium-dependent vasodilation is primarily mediated by NO, EDHF and PGI2. Consistently, our studies show that combined presence of inhibitors of all 3 endothelial factors completely abolishes endothelium-dependent ACh induced vasodilation in pregnant rat uterine arteries. The present finding that elevated T levels decrease NO-, EDHF- and PGI2 mediated relaxation in uterine arteries of T rats suggests that T blunts all 3 endothelial pathways. Elevated levels of T had a greater impact on the contribution of PGI2 and EDHF as compared to NO. In contrast, previous studies have shown that elevated T levels in pregnant rats selectively impairs only NO-mediated vasodilation in mesenteric arteries.32 This discrepancy may be due to the differences in vascular bed. Indeed, major differences have been reported between mesenteric and uterine vascular adaptations during pregnancy.52

The finding of significant decreases in eNOS protein levels and its phosphorylation status at Ser1177 in uterine arteries of T rats in the present study, suggests that decreased NO-mediated relaxations of the uterine arteries after T treatment results primarily from a decreases in eNOS protein levels and its activity state. These findings agree well with previous studies that showed a reduction in eNOS expression/activity in rat mesenteric artery and in human umbilical vein endothelial cells exposed to T.32,53 Indeed, previous studies have shown that elevated T levels in pregnant rats significantly decreased endothelial-dependent NO-mediated relaxations in the mesenteric arteries.32 In addition, elevated T levels significantly decreased plasma NOx levels32 further emphasizing that T decreases the function of the eNOS enzyme.

The present finding of decreased EDHF mediated relaxation in uterine arteries of T rats correlates well with the decreased expression of SK3 channels, which is important in initiation of endothelial cell hyperpolarization. These findings agree well with previous studies that showed a T-dependent reduction in SK3 expression in rat mesenteric artery.2 In support of the concept that SK3 channels are important for EDHF mediated vascular function, studies using transgenic mice (SK3T/T) show that SK3 channels exert a profound hyperpolarizing influence in resistance arteries and that suppression of SK3 channel expression causes decreased vasodilation and pronounced hypertension.54 A possible mechanism for decreased PGI2-mediated vasodilation is via inhibition of Cox or Pgis, which are involved in PGI2 production. Previous study show that T inhibits PGI2 production in cultured aortic vascular smooth muscle cells in vitro.33 Although we did not measure PGI2 production in this study, it is less likely that PGI2 production is altered in uterine arteries of T rats as similar expression levels of PGI2 biosynthetic enzymes were observed in T and control rats. However, the expression of IP receptor that mediates the vasodilatory effects of PGI2 was significantly reduced in uterine arteries of T rats suggesting that decreased IP receptor may contribute for reduced PGI2 mediated vasodilation.

The present study shows that placenta from T rats have significantly increased expression of HIF-1α and other hypoxia responsive genes. Since HIF-1α is a key transcription factor for response to low oxygen, the increased HIF-1α levels in T placentae suggests that the placenta may be underperfused and hypoxic. The enhanced vascular contraction and reduced vasodilation could contribute to underperfusion and persistent hypoxia. Poor placental perfusion and increased Hif-1α expression may explain for the observed decrease in placental nutrient transport from mother to fetus4 and fetal growth restriction in pregnant rats with elevated maternal T levels.4,5 Further studies that directly measures uterine blood flow and placentae oxygen status are needed.

Supplementary Material

1

Perspectives.

About 1 in every 12 babies in the United States and up to 15% of all pregnancies worldwide exhibit some degree of fetal growth restriction and are consequently at a higher risk of perinatal and childhood morbidity and mortality. Moreover, those growth restricted babies develop chronic conditions in adult life. Most pregnancy pathologies which cause fetal growth restriction are also presented with high androgen levels, such as preeclampsia, PCOS, congenital adrenal hyperplasia, maternal smoking or nicotine intake, caffeine intake, obesity or stress. Moreover, pregnant African-American women have higher serum T levels, with a greater frequency of low-birth-weight babies. Thus, it is of clinical significance to examine androgen's role in fetal growth restriction. The present study demonstrates for the first time that elevated T levels has adverse effects on vascular reactivity of the uterine artery in pregnancy with selective increase in Ang II-mediated contraction and decreased endothelium-dependent relaxations. Although it is not clear at present whether the increased vasoconstriction and inhibition of endothelium-dependent relaxation could be a major reason for the reduced uterine blood flow and fetal nutrient delivery observed with androgen exposure during pregnancy, these findings provides a potential mechanism. Strategies that target excessive androgen action in the uterine circulation could have important therapeutic potential in treatment of pregnancies complicated by fetal growth restriction.

NOVELTY AND SIGNIFICANCE.

What Is New?

  • In contrast to the well-studied beneficial roles of estrogen and progesterone in maternal cardiovascular adaptations to pregnancy, this study shows that elevated maternal plasma T, at levels similar to those observed in preeclampsia, PCOS mothers, and pregnant African-American women leads to enhanced vascular contraction and blunted endothelium-dependent relaxation in uterine arteries of pregnant rats.

  • Testosterone-mediated increases in uterine arterial contraction are mediated by selective hyperresponsiveness to Ang II which correlates with increased AT1bR expression and decreased in AT2R expression

  • Testosterone-mediated impairments in uterine arterial relaxation are mediated by decreases in the endothelium-dependent NO-, EDHF- and prostacyclin-components.

  • The enhanced uterine artery contraction and reduced relaxation mechanisms may decrease uterine arterial blood flow that may lead to decreased oxygen delivery to the placenta as indicated by increase in expression of hypoxia responsive genes in placentae.

What Is Relevant?

  • Most pregnancy pathologies, which cause fetal growth restriction, are also presented with high androgen levels, such as preeclampsia, PCOS, congenital adrenal hyperplasia, maternal smoking or nicotine intake, caffeine intake, obesity or stress. Moreover, pregnant African-American women have higher serum T levels, with a greater frequency of low-birth-weight babies. Thus, it is of clinical significance to examine androgen's role in fetal growth.

  • Numerous studies have demonstrated that T directly causes fetal damage. On the other hand, the adverse effects of T on fetal growth could be from indirect action of T on the maternal-fetal unit and the uteroplacental circulation.

  • Herein, we present evidence for the first time that elevated T levels has adverse effects on vascular reactivity of the uterine artery in pregnancy with selective increase in Ang II-mediated contraction and decreased endothelium-dependent relaxations.

  • The increased vasoconstriction and inhibition of endothelium dependent relaxation could be a major reason for the reduced uterine blood flow and fetal nutrient delivery observed with androgen exposure during pregnancy.

  • Strategies that target excessive androgen action in the uterine circulation could have important therapeutic potential in treatment of pregnancies complicated by fetal growth restriction.

Summary

  • This article is the first to show how elevated maternal T, at concentrations relevant to those observed in abnormal pregnancy conditions like preeclampsia, affects uterine artery function. Elevated T enhanced uterine artery vasoconstriction and blunted endothelium-dependent, which may lead to an increased vascular resistance and a decrease in uterine blood flow.

Acknowledgments

SOURCES OF FUNDING

Financial Support from the National Institute of Health (NIH) through grants HD069750 and HL119869 awarded to K. Sathishkumar and HL102866 and HL 58144 awarded to C. Yallampalli, is greatly appreciated.

Footnotes

CONFLICT OF INTEREST

None

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