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Placental insufficiency in the rat results in intrauterine growth restriction and development of hypertension in pre-pubertal male and female growth restricted offspring. However, after puberty only male growth restricted offspring remain hypertensive whereas female growth restricted offspring stabilize their blood pressure to levels comparable to adult female control. Since female rats reach their maximum levels of estrogen at puberty we hypothesize that estrogen may be a factor involved in the stabilization of blood pressure in adult female growth restricted offspring. At ten weeks of age female control and growth restricted offspring underwent ovariectomy or sham surgery and insertion of a telemetry probe. Mean arterial pressure was similar at sixteen weeks of age between control (123±4 mmHg) and growth restricted offspring (122±2 mmHg); however ovariectomy led to a significant increase in blood pressure in growth restricted offspring (140±2 mmHg, P<0.05 vs. intact counterpart) with no significant effect in control (124±1 mmHg). Estrogen replacement by subcutaneous mini-pellet initiated at fourteen weeks of age in a subset of ovariectomized-control and -growth restricted offspring reversed the effect of ovariectomy on blood pressure in growth restricted offspring at sixteen weeks of age (111±3 mmHg, P<0.05 vs. ovariectomized counterpart); renin angiotensin system blockade also abolished ovariectomy-induced hypertension in female growth restricted offspring (106±2 mmHg, P<0.05 vs. ovariectomized counterpart). Therefore, sex differences are observed in this model of fetal programmed hypertension, and results from this study suggest that estrogen contributes to normalization of blood pressure in adult female growth restricted offspring.
Hypertension shows a clear age-related sex dimorphism. Nearly one in three adult Americans have hypertension. A higher percentage of men than women have hypertension until age 45, the percentage is similar from ages 45 to 54, and becomes higher for women after that.1 Thus, the risk of hypertension increases in women after the onset of menopause and continues to rise with age.1-4 As a result, after menopause a greater percentage of women have hypertension than age-matched men.1, 5, 6 Epidemiological evidence suggests a regulatory role for estrogens in maintaining vascular function and structure.7-9 Loss of ovarian function results in estrogen deficiency and increased risk for development of cardiovascular diseases (CVD) such as hypertension in postmenopausal women and women with ovarian surgical ablation.7-10 In animal models of hypertension in which female rats are normotensive relative to their hypertensive male counterparts, ovariectomy induces hypertension.11-14 Therefore, it appears that while the ovaries are functional women have a lower risk of CVD than men, an observation supported by experimental studies.
Alterations in the fetal environment during a critical period of fetal development result in fetal adaptive changes that lead to long term consequences such as increased risk for development of hypertension and CVD later in life,15-19 an observation supported by numerous animal models.19-22 Sex differences are reported in different animal models of fetal programming; male offspring develop vascular dysfunction and hypertension, whereas female offspring appear to be protected.23-26 Therefore, a role for sex hormones is suggested in modulating cardiovascular responses to an adverse fetal environment.
In the model of fetal programming induced by placental insufficiency during late gestation in the rat both male and female intrauterine growth restricted (IUGR) offspring develop hypertension at pre-pubertal ages; however, only male IUGR offspring remain hypertensive in adulthood whereas female IUGR offspring stabilize their blood pressure after puberty.22 Therefore, sex hormones may contribute to sex differences in blood pressure in adult IUGR offspring. We previously reported an important role for testosterone and the renal renin angiotensin system (RAS) in the maintenance of established hypertension in adult or post-pubertal male IUGR offspring.27 Based on the fact that stabilization of blood pressure in female IUGR offspring is coincident with post-puberty, or the age at which female rats reach maximum levels of estrogen for this strain,28 we hypothesize that estrogen may protect against increases in blood pressure in post-pubertal female IUGR. Additionally, based on renal RAS involvement in adult male IUGR hypertension27, and experimental studies whereby estrogen modulates the renal RAS,29-32 we hypothesize that modulation of the RAS by estrogen may contribute to blood pressure regulation in female IUGR offspring. Thus, the purpose of this study was to determine if estrogen protects against increases in blood pressure in adult female IUGR offspring, and to determine whether modulation of the RAS may serve as a mechanism by which estrogen regulates blood pressure in female IUGR offspring.
All experimental procedures were in accordance with National Institutes of Health guidelines with approval by the Animal Care and Use Committee at the University of Mississippi Medical Center. Rats were housed in a temperature-controlled room (23°C) with a 12:12-hour light/dark cycle with food and water available ad libitum. Timed pregnant Sprague Dawley rats were purchased from Harlan Inc (Indianapolis, IN). At day 14 of gestation rats destined for reduced uterine perfusion were clipped as described below. All dams were allowed to deliver at term with offspring birth weight recorded within 12 hours of birth. At this time the number of pups in the control and reduced uterine perfusion litter were trimmed with a size of 8 pups per dam to ensure equal nutrient access for all offspring. Animals were weighed twice weekly. Pupswere weaned at 3 weeks of age. Female offspring from 13 control pregnant and 16 reduced uterine perfusion pregnant litters were randomly assigned into 4 groups: control-intact (n=14), control ovariectomized (control-OVX) (n=20), IUGR-intact (n=14), and IUGR-OVX (n=23). Implantation of telemetry probes and initiation of either ovariectomy (OVX) or sham OVX were performed at 10 weeks of age. The angiotensin converting enzyme (ACE) inhibitor, enalapril (40mg/kg/day, P.O.), was administered in a randomly selected subset of intact and ovariectomized animals: control-intact + Enalapril (n=7), IUGR-intact + Enalapril (n=7), control-OVX + Enalapril (n=7), IUGR-OVX + Enalapril (n=7). Estradiol (E2) replacement was initiated in a randomly selected subset of ovariectomized animals: control-OVX + E2 (n=6), IUGR-OVX + E2 (n=8). Animals that did not receive enalapril or estradiol were used as untreated controls for each group. Plasma for measurement of plasma renin activity and plasma renin substrate was collected from intact and ovariectomized animals following decapitation to prevent activation of the RAS. Serum for measurement of estradiol levels was collected while animals were under anesthesia from the abdominal aorta to prevent hemolytic phenomenon in the samples. Estradiol levels were measured from different sets of animals at 4, 6, 8, 10, 12, and 16 weeks of age to characterize the onset of puberty and stabilization of estradiol in control and IUGR offspring.(n= 6-8 per group).
Reduced utero-placental perfusion, as previously described22 was utilized for induction of IUGR. Briefly, rats undergoing surgical procedures were anesthetized with 2% isoflurane. At day 14 of gestation, a silver clip (0.203-mm ID) was placed around the lower abdominal aorta above the iliac bifurcation. Since compensation of blood flow occurs through an adaptive increase in ovarian blood flow, a silver clip was slipped around both branches of the ovarian artery (0.100-mm ID). Pregnant rats used for the control group were not exposed to surgical procedures. Based on previous observations, no differences have been noted between offspring from pregnant rats undergoing a sham operation and offspring from pregnant rats not exposed to surgical procedures. (Alexander BT, unpublished data, 2003).
Animals were anesthetized with 2% isoflurane and a flexible catheter attached to a radio transmitter (Data Sciences, Minneapolis, MN) was inserted in the abdominal aorta just below the renal arteries as previously described.27 The transmitter was secured to the abdominal muscle and remained in the abdominal cavity for the duration of the experiment. After surgery, rats were housed in individual cages positioned over an RLA-3000 radio-telemetry receiver. Rats received food and water ad libitum. Blood pressure measurements obtained with a 10-second sampling period were recorded every 10 minutes, 24 hours a day in unrestricted animals, initiated at 12 weeks of age until the end of experiments at 16 weeks of age. Mean arterial pressure (MAP) expressed in results was obtained weekly from an average of the first 3 days of the week for each rat.
All rats undergoing surgical procedures were anesthetized with 2% isoflurane. Ovariectomy was performed at 10 weeks of age. The skin was prepared for aseptic surgery followed by a ventral mid-line incision. The abdominal musculature and peritoneum were incised and the ovaries visualized. Ovarian vessels were tied off and the ovaries were removed (OVX group). The sham operation involved a ventral midline incision. The abdominal musculature and peritoneum were incised and the ovaries visualized, but not removed (intact group). The abdomen was closed in two layers, muscular and skin.
The ACE inhibitor, enalapril (40mg/kg/day, P.O.), was administered in the drinking water from 14 weeks of age until the end of the experiment at 16 weeks of age.
17β estradiol valerate mini pellets (1.5mg for 21 days release) were used for continuous release of hormone (Innovative Research of America, Sarasota, FL) at a dose chosen to mimic the normal estradiol levels observed in adult Sprague-Dawley female control animals (25±2 ng/dL). This dose characterized the average estradiol level of synchronized adult female control and IUGR rats housed in the same cage and represented a combination of the different stages of estrous cycle as confirmed by cytology characteristic of vaginal smearing.
Serum estradiol levels were determined with a commercially available radioimmunoassay kit (Ultra-Sensitive Estradiol RIA DSL-4800).
PRA was measured by radioimmunoassay (RIA) using a modification of the method by Haber 33with Angiotensin I (AI) standards, tracer, and antibody from National Bureau of Standards, NEN, and Arnel, respectively.
PRS was measured by RIA, as previously described. 34
Total RNA was utilized for quantification of the mRNA by Real-Time PCR. Kidneys were removed, quick frozen in liquid nitrogen, and stored at −80°C. Each kidney was first ground using liquid nitrogen chilled mortar and pestle and total RNA was isolated using a guanidine thiocyanide, acid phenol:chloroform procedure (ToTALLY RNA kit, Ambion, Austin, TX). All RNA isolates were treated with DNAse (DNA-free kit, Ambion, Austin, TX) to remove DNA. Total RNA (2 μg) was reverse transcribed using a modified MMLV-derived reverse transcriptase and a unique blend of oligo (dT) and random hexamer primers (iScriptTMcDNA Synthesis Kit, BIO-RAD). The resulting cDNA (1μl) was amplified by real-time PCR using SYBR Green (iQTM SYBR Green Supermix, BIO-RAD) as fluorophore in an iCycler real-time thermal cycler (BIO-RAD). ACE and ACE2 mRNA expression were assessed using RT2 PCR Primer Set for Rat ACE and ACE2 (SuperArray Bioscience Corporation). Results were calculated using the 2−ΔΔCT method and expressed in folds increase/decrease of the gene of interest in IUGR vs. control rats. All reactions were performed in triplicates and β-actin was used as an internal control (RT-PCR Primer and Control Set, Invitrogen).
GB-STAT version 7 for MS Windows was used for all statistical analyses. For comparison made between groups, ANOVA, with adjustments for multiple comparisons (MANOVA) was used. For two groups comparisons T- test was used. A value of P<0.05 was considered statistically significant.
Weight at birth was significantly reduced in female IUGR offspring from reduced uterine perfusion dams as compared to female control offspring from control dams (Table 1). At 16 weeks of age body weight did not differ upon comparison of female IUGR to female control offspring (Table 1.). Therefore, female IUGR offspring exhibited catch-up growth as differences in body weight were normalized by 16 weeks of age. Body weight did not differ upon comparison in any group (Table 1.). Thus, neither ovariectomy nor ACE inhibition affected body weight in control or IUGR offspring by 16 weeks of age.
As previously reported by our group22 and now confirmed by telemetry, MAP did not differ in adult female IUGR offspring as compared to adult female control offspring (Figure 1). Ovariectomy induced hypertension in adult IUGR-OVX offspring (mean increase of 18 mmHg,) (Figure 1), yet had no significant effect on blood pressure in adult control-OVX (mean increase of 1 mmHg). Estradiol replacement for 2 weeks initiated at 14 weeks of age in post-pubertal offspring significantly reduced blood pressure by 16 weeks of age in control-OVX offspring (mean decrease of 20 mmHg; P<0.05 vs. control-OVX) and IUGR-OVX offspring (mean decrease of 29 mmHg; P<0.05 vs. IUGR-OVX) (Figure 2). However, estradiol replacement abolished the significant difference in MAP observed upon comparison of control-OVX and IUGR-OVX offspring normalizing blood pressure in IUGR-OVX offspring to values comparables to control-OVX.
The ACE inhibitor, enalapril, initiated at 14 weeks of age decreased blood pressure in IUGR-OVX and control-OVX rats (Figure 3). However, the depressor response to ACE inhibition was greater in IUGR-OVX rats (mean decrease of 35 mmHg; P<0.01, vs. untreated IUGR-OVX) as compared to control-OVX rats (mean decrease of 5 mmHg; P<0.05, vs. untreated control-OVX) (Figure 3). ACE inhibition led to a significant decrease in blood pressure in intact-IUGR (mean decrease of 9 mmHg), but not intact-control offspring (mean decrease of 3 mmHg) as compared to their intact untreated counterparts. MAP did not significantly differ upon comparison of treated intact–control and treated OVX-control offspring. Additionally, ACE inhibition abolished the significant difference in MAP observed upon comparison of untreated OVX-IUGR and untreated OVX-control offspring.
Serum estradiol levels did not differ upon comparison of adult female control and adult female IUGR offspring at 16 weeks of age (Figure 4a). Estradiol levels were measured at 4, 6, 8, 10, 12, and 16 weeks of age in separate set of animals. Estradiol levels oscillated from 4 to 6 weeks of age between 0.7 to 0.9 ng/dl and no significant differences were observed upon comparison of IUGR to control offspring at these ages. However, estradiol levels were increased at 8 weeks of age, a value that correlates with puberty for this rat strain28 and were maintained at this level up to 16 weeks of age, or the end of the study, with no significant differences observed upon comparison of female IUGR to control offspring (Figure 4a). At puberty, recognized by vaginal opening, daily vaginal smearing was performed to identify the stage of estrous cycle at the time of estradiol measurement. Although estradiol levels may represent a combination of the different stages of estrous cycle, bi-weekly estradiol levels reflected synchronized cycles in female rats housed in the same cage. Estradiol levels were significantly decreased at 6 weeks after ovariectomy in both control and IUGR offspring upon comparison to their intact counterparts (Figure 4b). E2 replacement reinstated estradiol levels to levels comparable to intact animals (Figure 4b).
No significant differences in PRA or PRS were observed upon comparison of control and IUGR rats at 16 weeks of age (PRA: 4±1 vs. 5±1 nmoles AI/L/hr, and PRS: 25±2 vs. 27±6 nmoles AI/ml/hr; control vs. IUGR, respectively). Ovariectomy had no effect on either PRA or PRS levels in control or IUGR rats (PRA: 4±1 vs. 3±1 nmoles AI/L/Hr, and PRS: 27±1 vs. 23±2 nmoles AI/ml/hr; control vs. IUGR, respectively).
Renal ACE2 mRNA expression was significantly increased in adult female intact IUGR offspring, upon comparison to adult female control offspring (Figure 5). Ovariectomy had no effect on renal ACE mRNA expression in either control or IUGR offspring. However, renal ACE2 mRNA expression was significantly decreased by ovariectomy in IUGR-OVX rats (Figure 5); ovariectomy had no effect on renal ACE2 mRNA expression in control-OVX rats.
Hypertension induced by placental insufficiency exhibits sex-specific differences in adult IUGR offspring.22, 27 Hypertension in female IUGR offspring from reduced uterine perfusion dams returns to normotensive values in adulthood while male IUGR offspring remain hypertensive.22, 27 We recently reported that testosterone contributes to the maintenance of established hypertension in post-pubertal male IGUR offspring.27 This study demonstrates that estrogen protects against increases in blood pressure in post-pubertal female IUGR offspring. Importantly, this study also demonstrates a potential role for the RAS as an underlying mechanism in mediating hypertension induced by ovariectomy in adult female IUGR offspring.
Normalization of blood pressure in post-pubertal female IUGR offspring occurred in conjunction with attainment of adult female estradiol levels for this strain.28 Furthermore, ovariectomy led to development of significant elevations in MAP in adult female IUGR offspring with no effect in adult female control offspring. Thus, the possibility that estradiol plays a protective role in the normalization of arterial pressure was strongly suggested. However, no difference is estradiol levels were observed upon comparison of adult female control and adult female IUGR offspring. To clarify the importance of estradiol on blood pressure control in female IUGR, estradiol replacement in ovariectomized rats was initiated to return estrogen levels to that observed in adult female rats of this strain.28 Estrogen replacement abolished hypertension induced by ovariectomy in adult female IUGR offspring and normalized blood pressure to levels observed in the ovariectomized control plus estrogen replacement group suggesting that estrogen does provide a protective status in adult female IUGR offspring. However, the protective role of estradiol may not be directly related to the level of estradiol per se, but to the effect of estradiol on other systems controlling blood pressure in adult female IUGR offspring.
Epidemiological studies of hormonal replacement therapy are controversial as to whether estradiol provides a protective status in post-menopausal women. 35-39 However, estradiol is associated with protective cardio-renal effects in many animal models of hypertension11, 12, 40, 41 and the deleterious effects of ovariectomy such as induced hypertension, renal injury, or endothelial dysfunction are reversed with estradiol therapy.12, 13, 41 The cardio-renal protective effect of estradiol appears to be complex and includes a wide range of regulatory system involvement with a role for the RAS strongly suggested in both human and animal studies.30, 42-46
We previously reported that ACE inhibition abolishes hypertension in adult male IUGR offspring.27 Furthermore, activation of the renal RAS, but not peripheral RAS, is associated with hypertension in adult male IUGR 47suggesting a role for the renal RAS in adult male IUGR hypertension. To investigate whether the RAS contributes to hypertension induced by ovariectomy in adult female IUGR offspring, we examined the effect of RAS blockade utilizing the ACE inhibitor, enalapril. Enalapril abolished hypertension induced by ovariectomy in adult female IUGR offspring suggesting the RAS plays a critical role in mediating hypertension induced by ovariectomy in female IUGR offspring. Thus, ovariectomy leads to development of hypertension in adult female IUGR offspring; an effect reversed by RAS blockade.
One potential mechanism for the protective status mediated by estradiol in adult IUGR female offspring may involve modulation of the renal RAS by estrogen. ACE- and ACE2-dependent pathways generate peptides, angiotensin II (ANG II) and ANG (1-7), respectively, which are critical for blood pressure regulation.48, 49 ANG (1-7) acts as a negative regulator of the vasoconstrictor effects of ANG II, thus suggesting ACE2 provides a counter-regulatory balance to ACE. 13, 50, 51 ACE, ACE2, and the Angiotensin type 1 receptor (AT1R) are components of the RAS associated with the cardio-renal protective effects of estradiol. 31, 52-55 Modulation of the RAS by estradiol involves alterations in the vasoconstrictor-vasodilator actions of the RAS by influencing the ACE and ACE2 pathways.31, 32, 51, 53-56 Various animal models have been used to investigate participation of RAS in the regulatory effects of estradiol.32, 46, 52, 56-58 Although the exact mechanism(s) remains unclear, it is suggested that when ACE activity is greater than ACE2, vasoconstriction due to Ang II will predominate and hypertension will be induced.48 Since estrogen is shown to regulate renal ACE message in the rat,56 we determined whether renal ACE and ACE2 message expression were altered in IUGR offspring relative to control offspring, and whether renal ACE and ACE2 message expression were altered in response to ovariectomy. Significant elevations in renal ACE2 mRNA expression were observed in intact adult female IUGR offspring upon comparison to intact adult female control offspring. Ovariectomy induced a significant decrease in renal mRNA expression of ACE2 in adult female IUGR offspring with no effect in adult female control. Ovariectomy had no effect on renal ACE mRNA expression in adult female IUGR offspring. Thus, loss of ovarian function may decrease the vasodilator effect provided by the ACE2 pathway leading to increases in blood pressure in post-pubertal female IUGR offspring. Ovariectomy did not affect blood pressure or renal ACE and ACE2 mRNA expression in control animals. Therefore, the abnormal response to loss of ovarian function on blood pressure regulation in adult female IUGR offspring may reflect permanent alterations in the regulatory systems important in the long-term control of arterial pressure regulation that develop as a consequence of fetal adapted changes to placental insufficiency.
Hypertension is present in pre-pubertal female IUGR offspring; however, estrogen protects against hypertension in post-pubertal female IUGR offspring. Modulation of the RAS, in particular the ACE2 pathway, by estrogen may be one mechanism critical to normalization of blood pressure in adult female IUGR offspring. However, other factors may be modulated by estrogen and mechanism(s) mediating ovariectomy-induced elevations in arterial pressure in adult female IUGR offspring may also involve oxidative stress. Investigation into the regulation of these factors by estrogen in this model of IUGR will be a very exciting opportunity for future investigations.
The influence of the fetal environment is a novel factor in the etiology of hypertension. Various animal models of programmed hypertension have been used to study the mechanisms underlying the pathophysiology of this condition, and the regulatory systems involved in these mechanisms. Placental insufficiency results in IUGR offspring that reveal sex-specific differences in the development of programmed hypertension. Adult female IUGR offspring appear protected while the ovaries remain intact and the RAS with participation of the ACE2 counterbalance pathway may be involved. Thus, mechanisms common to sex differences in this model of programmed hypertension may enlighten the complex mechanisms underlying the sex differences in human hypertension.
Sources of Funding
This work was supported by NIH grant HL074927.
Norma B Ojeda: No disclosures
Daniela Grigore: No disclosures
Elliott B Robertson: No disclosures
Barbara T Alexander:
Research Grant: NIH grant HL074927., Amount: >= $10,000