Hypertens Pregnancy, 2014; 33(4): 375–388 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2014.911884
ORIGINAL ARTICLE
Hemodynamic responses to angiotensin-(1-7) in women in their third trimester of pregnancy Liliya M. Yamaleyeva,1 David C. Merrill,2,3* Thomas J. Ebert,4* Thomas L. Smith,1,5 Heather L. Mertz,2 and K. Bridget Brosnihan1 1
The Hypertension & Vascular Research Center, Wake Forest School of Medicine, Winston-Salem, NC, USA, 2 Department of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, NC, USA, 3 Aurora Health Care, Kenosha, WI, USA, 4 Department of Anesthesiology, Zablocki VA Medical Center and Medical College of Wisconsin, Milwaukee, WI, USA, and 5 Department of Orthopaedic Surgery, Wake Forest School of Medicine, WinstonSalem, NC, USA Background: To understand the role of Angiotensin-(1-7) (Ang-(1-7)) in vasculature of pregnant women, we compared cardiac output (CO), total peripheral resistance (TPR) and forearm blood flow (FBF) responses to Ang-(1-7) infusion between normotensive pregnant women in their third trimester and healthy age matched non-pregnant women. The responses of skin microcirculation to Ang-(1-7) were tested in preeclamptic, normotensive pregnant and non-pregnant women. Responses to Angiotensin II (Ang II) were also determined. Methods: Non-invasive methods for systemic (bioimpedance and VascuMAP), FBF (venous occlusion strain gauge plethysmography), and skin (laser Doppler) hemodynamics assessments were used. Results: Compared to non-pregnant women, systemic infusion of Ang-(1-7) (2000 pmol/min) resulted in a greater increase in CO (9.4 ± 6.4 versus 3.3 ± 2.1%, n = 9–10) in normotensive pregnant women. Brachial local infusion of Ang-(1-7) had no effect on FBF in either group. In non-pregnant and normotensive pregnant women, local Ang II induced a dose-dependent decrease in FBF and increase in forearm resistance at 50 and 100 pmol/min (p50.05 versus corresponding baseline, n = 7–10). Following iontophoretic application of 5 mmol/l dose of Ang-(1-7), the change in skin flow was higher in normotensive pregnant versus preeclamptic women (182.5 ± 93 versus 15.76 ± 19.46%, n = 14–15). Skin flow was lower in normotensive pregnant versus preeclamptic women (46.5 ± 48.7 versus 108.7 ± 49.1%, n = 14–15) following Ang II infusion at 1.0 pmol/min. Conclusion: In the third trimester of pregnancy, Ang-(1-7) induces alterations in CO and differentially regulates micro- and macro-circulations, depending on the dose. Dysregulation in skin vasculature may contribute to the development of vascular dysfunction and hypertension in preeclampsia. Keywords Angiotensin-(1-7), Noninvasive methods, Pregnancy, Regional (skin and brachial) blood flow.
*David C. Merrill and Thomas J. Ebert contributed equally as first author. Correspondence: K. Bridget Brosnihan, PhD, Hypertension and Vascular Research Center, Wake Forest School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA. Tel: 336-716-2795. Fax: 336-716-2456. E-mail: [email protected]
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INTRODUCTION The function of local vascular beds during pregnancy depends on systemic and local vasoactive factors. Components of the renin-angiotensin system (RAS) – such as renin, angiotensinogen, angiotensin II (Ang II), and angiotensin-(1-7) (Ang-(1-7)) – are elevated in the circulation of pregnant women (1). However, plasma Ang-(1-7) levels are reduced in women with preeclampsia in their third trimester (1). Since the RAS regulates cardiovascular function and body fluid and electrolyte balance, Ang-(1-7) and Ang II – two RAS peptides with opposing actions – may be involved in vascular adaptations in pregnancy. Other than pregnancy, Ang-(1-7) induces vasodilation and anti-proliferation, and Ang II elicits vasoconstriction, cell proliferation, and hypertrophy. The peripheral vasculature of pregnant women is resistant to pressor effects of Ang II, in part due to reduced sensitivity of arterial smooth muscle and down-regulation of AT1 receptors (2–4). However, the contribution of Ang-(1-7) to systemic or peripheral circulation in pregnant women is not known. Ang-(1-7) is expressed in vascular endothelial and smooth muscle cells (VSMC), and can influence the release of endothelium-derived vasodilatory factors or factors attenuating VSMC hypertrophy, suggesting that Ang-(1-7) can contribute to the vasodilatory state in pregnancy (5–7). Ang-(1-7) induces vascular effects via activation of the Mas/AT1-7 receptor (8). In the third trimester of pregnancy, Ang-(1-7) may act to reduce total peripheral resistance (TPR) and increase blood flow via systemic vasodilatory effects (1,9). In spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, Ang-(1-7) regulates CO and TPR without much change in blood pressure (10). In normal pregnant rats, Ang-(1-7) significantly dilates mesenteric arteries, an effect blocked by the Ang-(1-7) antagonist [D-Ala7]-Ang-(1-7) (9). To elucidate the contributions of Ang-(1-7) in vascular responses during pregnancy, we compared the influence of Ang-(1-7) on systemic hemodynamics and peripheral regional circulation (brachial artery) in normotensive pregnant and non-pregnant women. We also studied hypertensive women with preeclampsia, but only for assessments of skin (finger) microcirculation.
METHODS The protocol for these experiments was reviewed and approved by the Wake Forest School of Medicine and Zablocki VA Medical Center Institutional Review Boards. Consent forms were signed by all participants at the time of their enrollment in the study. All pregnant subjects were in their third trimester of pregnancy, and free of any known pre-existing cardiovascular, endocrine, or connective tissue disease. Study 1: Ang-(1-7) Contribution to Systemic Hemodynamics (Blood Pressure, Cardiac Output, Heart Rate, and Total Peripheral Resistance) and Forearm Blood Flow (FBF) in Normotensive Pregnant and Non-Pregnant Women Group 1 (n = 7) was composed of non-pregnant, normotensive women who were not taking oral contraceptives; the stage of the menstrual cycle was not determined. Group 2 (n = 13) was composed of nulliparous, normotensive
Ang-(1-7) and vascular responses in pregnancy Table 1. Systemic infusion study: patient population.
Number of subjects Race (percent of Caucasian/African American, American Indian and Hispanic) Age (years) Body mass index (kg/m2) Gestation age at the time of study (wks)
Non-pregnant
Normotensive pregnant
7 85.7/14.3
13 54/46
24.6 ± 3.1 21.9 ± 1.1
21.8 ± 0.8 29.3 ± 1.4* 37.4 ± 0.3
*p50.05 versus non-pregnant.
pregnant women. The non-pregnant and normotensive pregnant subjects are described in Table 1. All subjects were given systemic (intravenous) infusions of Ang-(1-7) in supine position with a 15 elevation from the back to the head. Three doses of Ang-(1-7) were administered (1000, 2000, and 3000 pmol/min) with each infusion dose lasting 30 min. Arterial pressures were measured using a radial artery catheter and VascuMAP (Carolina Medical, King, NC). The VascuMAP was placed on the arm contralateral to the infusion. CO and TPR were measured by bioimpedance using the BioZ device (CardioDynamics International Corporation, San Diego, CA). The advantage of the BioZ System is its ability to assess accurately the changes in perfusion/flux in response to a drug or maneuver. The procedure has been validated against the thermal dilution and Fick methods in animal studies and in adult critically ill surgical patients (11,12). CO data were expressed as a percent change relative to control (saline infusion). Blood samples were obtained for measurement of Ang-(1-7) before Ang-(1-7) infusion and at the end of each dosage (a total of four samples). FBF was measured by bilateral venous occlusion strain gauge plethysmography. Pediatric cuffs were placed around both wrists and inflated to 200 mmHg during each blood flow measurement. Strain gauges were placed around the forearms 5 cm distal to the olecranon. Study 2: Ang-(1-7) and Forearm (brachial) Blood Flow in Normotensive Pregnant and Non-Pregnant Women Group 1 (n = 7–13) was composed of non-pregnant, normotensive women. Group 2 (n = 5–10) was composed of nulliparous, normotensive pregnant women (Table 2) as characterized in the Study 1. All subjects were studied in the supine position. FBF was measured by bilateral venous occlusion strain gauge plethysmography as described in Study 1. Blood pressure cuffs placed around the upper arm were inflated to 40 mmHg and deflated every 15 s. All drugs were infused via a catheter (27 gauge) inserted into the brachial artery of the non-dominant arm under local anesthesia with 1% lidocaine. Baseline FBF was measured at least 60 min after the cannulation. Each subject received Ang II or Ang-(1-7) intra-arterially with measurement of FBF in the order indicated and with increasing doses. Angiotensin II was given at low (1, 5, 10, 20 pmol/min) and high (5, 25, 50, 100 pmol/min) doses. Ang-(1-7) was given at low (1, 5, 10, 50 pmol/min) and high (10, 50, 100, 500 pmol/min) doses. We used three different doses of Ang-(1-7), previously reported to have vasodilatory effects
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L. M. Yamaleyeva et al. Table 2. Forearm blood flow (FBF) study: patient population.
Number of subjects Race (percent of Caucasian/ African American, American Indian and Hispanic) Age (years) Body mass index (kg/m2) Gestation age at the time of study (wks)
Non-pregnant low dose
Non-pregnant high dose
Normotensive pregnant low dose
Normotensive pregnant high dose
13 77/23
7 100/0
5 40/60
10 50/50
25.8 ± 7.7 22.9 ± 3.7
20.7 ± 1.9 23.8 ± 3.7
21.5 ± 5.9 30.1 ± 2.9
22.6 ± 3.9 33.5 ± 6.4
35.3 ± 1.9
37.2 ± 0.9
in non-pregnant women or men (13,14). Each dose was given for 10 min with a 30-min washout period between each agent. The doses of drugs were selected to produce local changes in the infused arm only. Systemic arterial pressure was measured by VascuMAP in the contralateral arm. Since venous occlusion plethysmography provides only a relative assessment of blood flow, the data were expressed as a ratio of measurements recorded in infused – to a noninfused (control) arm to reduce the possibility of small changes in blood pressure or sympathetic nervous system affecting the responses to vasoactive substances (15). Study 3: The Contribution of Ang-(1-7) to Skin Microcirculation Group 1 (n = 14) was composed of non-pregnant women and Group 2 (n = 15) was composed of normotensive pregnant women according to the same criteria as described in the Study 1. Group 3 (n = 15) was composed of nulliparous preeclamptic women, matched to Group 2 by gestational age. Preeclampsia was defined by the onset of pregnancy-induced hypertension (blood pressure 140/90 mmHg) and proteinuria (300 mg/24 h or 2+ or more protein levels on random sample collection) (Table 3). Skin blood flow was determined by laser Doppler and Ang II and Ang-(1-7) were delivered using iontophoresis, a drug delivery method based on the stimulation of charged ions migration across the skin (16). The iontophoresis laser Doppler unit (PeriIont Micropharmacology System, Perimed AB, Stockholm, Sweden) allows for direct measurements of skin blood flow over the iontophoresized area (17,18). Since this method does not provide absolute perfusion values but arbitrary perfusion units that represent blood ‘‘flux’’ (F) changes, the data are shown as percent change determined by the following equation: Fpost Fpre Percentage change in flux ¼ 100: Fpre biologic zero
Ang-(1-7) and vascular responses in pregnancy Table 3. Skin microcirculation study: patient population.
Number of subjects Race (percent of Caucasian/African American, American Indian and Hispanic) Age (years) Body mass index (kg/m2) SBP (mmHg; baseline) DBP (mmHg; baseline) Proteinuria (mg) Gestation age at the time of study (wks) Gestational age at delivery (wks) Birthweight, g Birth centile Baby gender (female-to-male ratio)
Nonpregnant
Normotensive pregnant
Preeclamptic
14 63.3/35.7
15 66. 7/33.3
15 71.4/28.6
21.5 ± 5.9 26.3 ± 1.8 108.9 ± 3.4 65.8 ± 0.4
20.7 ± 1.9 28.5 ± 1.6 105.4 ± 2.4 65.7 ± 1.7 Negligible 30.9 ± 1.2 36.8 ± 1.2 2750 ± 249.4 47.7 ± 0.1 10/5
24.3 ± 1.7 36.6 ± 1.9*y 142.6 ± 1.9*y 86.2 ± 3.1*y 507.3 ± 214.4y 31.5 ± 0.8 32.9 ± 0.9y 2031 ± 241.0y 39.3 ± 0.1 5/10
*p50.05 versus non-pregnant; yp50.05 versus pregnant.
All participants were examined in the supine position. Subjects were warmed with a blanket until the finger temperature was 30 ± 2 C. Skin (finger) blood flow responses to each of the following iontophoretically applied agents were studied: (a) Ang II (0.5, 1.0 and 2.0 mmol/l), (b) Ang-(1-7) (1.0, 5.0 and 20.0 mmol/l), and (c) physiological saline (control). Drugs were administered in the order and dose indicated. Iontophoresis (200 mA) was performed for 2 min with each agent at each concentration. Skin blood flow was measured at the same site as the iontophoresis, with a 15-min interval between each drug and dose. Angiotensin-(1-7) Radioimmunoassay Blood (14 ml) was taken in a cocktail of inhibitors (19) and plasma was extracted using Sep-Pak columns. Ang-(1-7) was measured by an in-house radioimmunoassay, as previously described (19). Statistical Analysis Statistical analyses of data were performed using GraphPad Prism IV (San Diego, CA) plotting and statistical software. Comparisons of demographic indices between normotensive pregnant and preeclamptic women in the systemic infusion study were performed using unpaired t-tests. The comparisons of demographic parameters among non-pregnant, normotensive pregnant, and preeclamptic women in the skin flow study were performed using one-way analysis of variance (ANOVA) with Tukey posttests. Hemodynamic responses were evaluated using two-way ANOVA with Bonferroni post-hoc tests. Dose-dependent responses in each group were evaluated using one-way ANOVA with repeated measures test for matched observations. All measurements were expressed as the mean ± standard error of the mean (SEM). The criterion for statistical significance was p50.05.
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RESULTS Study 1 In the systemic hemodynamics study, most non-pregnant and pregnant women were Caucasians, but there were more African American or Hispanic women in the normotensive pregnant group compared to non-pregnant women group (Table 1). Systemic infusion of Ang-(1-7) at 2000 pmol/min resulted in a greater change in CO (pregnant: 9.4 ± 6.4 versus non-pregnant: 3.3 ± 2.1%, p50.05) and less change in TPR (pregnant: 12.9 ± 5.5 versus non-pregnant: 1.7 ± 3.1%) in pregnant women compared to non-pregnant women (Figure 1). There were no differences between groups in response to the systemic infusion of Ang-(1-7) at 1000 or 3000 pmol/min. The change in FBF was greater in
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Figure 1. Responses to systemic Ang-(1-7) infusion in non-pregnant and normotensive pregnant women. Data are mean ± SEM; *p50.05 versus non-pregnant women, n = 7–13. FBF = forearm blood flow.
Ang-(1-7) and vascular responses in pregnancy
pregnant women after systemic administration of Ang-(1-7) at 2000 pmol/min (pregnant: 12.7 ± 7.5 versus non-pregnant: 13.8 ± 5.1%, p50.01, n = 9–10) and at 3000 pmol/min (pregnant: 21.2 ± 11.6 versus non-pregnant: 20.3 ± 6.2%, p50.01, n = 9–10) but not at 1000 pmol/min. There were no significant changes in blood pressure, heart rate or stroke volume. Plasma levels of Ang-(1-7) were significantly elevated following the infusion of 1000 pmol/min (69.9 ± 5.6 pmol/ml), 2000 pmol/min (122.5 ± 9.9 pmol/ml) and 3000 pmol/min (127.3 ± 11.8 pmol/ml) of the peptide in comparison to pretreatment state (21 ± 3.6 pmol/ml). Plasma Ang-(1-7) levels were measured in normotensive pregnant women; p50.05; n = 15 in each group. In addition, the plasma levels of Ang-(1-7) were increased in a dose-dependent manner such as the peptide levels detected following 2000 and 3000 pmol/min infusion were significantly higher compared to the levels of the peptide detected following 1000 pmol/min infusion (p50.05; n = 15 in each group). Study 2 In the forearm (brachial) circulation study, most non-pregnant women were Caucasians. However, there were more participants of African American, American Indian or Hispanic heritage among the normotensive pregnant women compared to non-pregnant women (Table 2). Local infusion of Ang-(1-7) (Figure 2) had no effect on forearm flow, brachial resistance, or mean blood pressure at any dose. Ang II induced a dose-dependent decrease in FBF at 25, 50 and 100 pmol/min (2.1 fold at 25 pmol/min, 2.5 fold at 50 pmol/min and 2.7 fold at 100 pmol/min; all versus baseline in non-pregnant women, p50.05) and an increase in resistance in non-pregnant women at 50 and 100 pmol/min (2.9 fold at 50 pmol/min and 3.1 fold at 100 pmol/min, p50.05 versus baseline in non-pregnant women). In normotensive pregnant women, FBF was lower at 50 and at 100 pmol/min (1.65-fold at 50 pmol/min and 2.1-fold at 100 pmol/min, all versus baseline in pregnant women, p50.05) and resistance was higher at 50 and 100 pmol/min following Ang II administration (2.1-fold at 50 pmol/min and 2-fold at 100 pmol/min; all versus baseline in pregnant women, p50.05) (Figure 3). These differences were not observed in response to Ang II infusion at low doses. Mean blood pressure did not change in any group in response to brachial infusion of Ang II, regardless of dose. Study 3 Participants in the skin microcirculation experiment were predominantly Caucasian. As expected, resting systolic and diastolic blood pressures and proteinuria were greater in preeclamptic women versus normotensive pregnant and non-pregnant women (Table 3). Body mass index was greater in preeclamptic subjects compared to normotensive pregnant or to non-pregnant women. On the average, preeclamptic women had shorter pregnancies and smaller babies. There were more boys born to preeclamptic mothers in this cohort of women. Changes in cutaneous flow were significantly higher in normotensive pregnant women versus preeclamptic women in response to 5 mmol/l of Ang-(17) (pregnant: 182.5 ± 93 versus preeclamptic: 15.76 ± 19.46%, p50.05) (Figure 4A), but not in response to Ang-(1-7) at either 1 or 20 mmol/l dose. Preeclamptic women lost the vasodilatory response to 5 mmol/l. Skin flow was
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Figure 2. Responses of forearm (brachial) circulation to local infusion of Ang-(1-7) in non-pregnant and normotensive pregnant women. Data are mean ± SEM, n = 5–13 (low dose), n = 7–10 (high dose).
reduced in normotensive pregnant women compared to preeclamptic women in response to Ang II (1.0 mmol/l) (Figure 4B). There were no differences in skin flow among studied groups in response to Ang II at either 0.5 or 2.0 mmol/l dose (Figure 4B).
DISCUSSION In this study, systemic infusion of Ang-(1-7) resulted in greater increase in the change of CO and lower decrease in the change of TPR in normotensive pregnant women compared to non-pregnant women. In contrast, in our forearm circulation experiments, Ang-(1-7) had no effect on the change of FBF
Ang-(1-7) and vascular responses in pregnancy
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Figure 3. Responses of forearm (brachial) circulation to local infusion of Ang II in non-pregnant and normotensive pregnant women. Data are mean ± SEM, n = 5–13 (low dose), n = 7–10 (high dose); *p50.05 versus baseline in non-pregnant women; #p50.05 versus baseline in pregnant women; ^p50.05 versus 5 pmol/min.
or resistance, whereas high doses of Ang II induced a dose-dependent decrease in FBF and increased resistance in normotensive pregnant and non-pregnant women. We also found that the change in skin blood flow in response to Ang(1-7) (5 mmol/l) was greater in normotensive pregnant women and had little or no effect in preeclamptic women. These findings suggest a possible role for Ang-(1-7) in the regulation of systemic and local skin hemodynamics in the third trimester of normotensive pregnancy. Here, we demonstrate for the first time that systemic infusion of a single dose of Ang-(1-7) in the third trimester of pregnancy increases CO and decreases TPR without altering blood pressure. Changes in stroke volume probably led to the increase in CO in pregnant women. However, our study was
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ORIGINAL ARTICLE
Hemodynamic responses to angiotensin-(1-7) in women in their third trimester of pregnancy Liliya M. Yamaleyeva,1 David C. Merrill,2,3* Thomas J. Ebert,4* Thomas L. Smith,1,5 Heather L. Mertz,2 and K. Bridget Brosnihan1 1
The Hypertension & Vascular Research Center, Wake Forest School of Medicine, Winston-Salem, NC, USA, 2 Department of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, NC, USA, 3 Aurora Health Care, Kenosha, WI, USA, 4 Department of Anesthesiology, Zablocki VA Medical Center and Medical College of Wisconsin, Milwaukee, WI, USA, and 5 Department of Orthopaedic Surgery, Wake Forest School of Medicine, WinstonSalem, NC, USA Background: To understand the role of Angiotensin-(1-7) (Ang-(1-7)) in vasculature of pregnant women, we compared cardiac output (CO), total peripheral resistance (TPR) and forearm blood flow (FBF) responses to Ang-(1-7) infusion between normotensive pregnant women in their third trimester and healthy age matched non-pregnant women. The responses of skin microcirculation to Ang-(1-7) were tested in preeclamptic, normotensive pregnant and non-pregnant women. Responses to Angiotensin II (Ang II) were also determined. Methods: Non-invasive methods for systemic (bioimpedance and VascuMAP), FBF (venous occlusion strain gauge plethysmography), and skin (laser Doppler) hemodynamics assessments were used. Results: Compared to non-pregnant women, systemic infusion of Ang-(1-7) (2000 pmol/min) resulted in a greater increase in CO (9.4 ± 6.4 versus 3.3 ± 2.1%, n = 9–10) in normotensive pregnant women. Brachial local infusion of Ang-(1-7) had no effect on FBF in either group. In non-pregnant and normotensive pregnant women, local Ang II induced a dose-dependent decrease in FBF and increase in forearm resistance at 50 and 100 pmol/min (p50.05 versus corresponding baseline, n = 7–10). Following iontophoretic application of 5 mmol/l dose of Ang-(1-7), the change in skin flow was higher in normotensive pregnant versus preeclamptic women (182.5 ± 93 versus 15.76 ± 19.46%, n = 14–15). Skin flow was lower in normotensive pregnant versus preeclamptic women (46.5 ± 48.7 versus 108.7 ± 49.1%, n = 14–15) following Ang II infusion at 1.0 pmol/min. Conclusion: In the third trimester of pregnancy, Ang-(1-7) induces alterations in CO and differentially regulates micro- and macro-circulations, depending on the dose. Dysregulation in skin vasculature may contribute to the development of vascular dysfunction and hypertension in preeclampsia. Keywords Angiotensin-(1-7), Noninvasive methods, Pregnancy, Regional (skin and brachial) blood flow.
*David C. Merrill and Thomas J. Ebert contributed equally as first author. Correspondence: K. Bridget Brosnihan, PhD, Hypertension and Vascular Research Center, Wake Forest School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA. Tel: 336-716-2795. Fax: 336-716-2456. E-mail: [email protected]
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INTRODUCTION The function of local vascular beds during pregnancy depends on systemic and local vasoactive factors. Components of the renin-angiotensin system (RAS) – such as renin, angiotensinogen, angiotensin II (Ang II), and angiotensin-(1-7) (Ang-(1-7)) – are elevated in the circulation of pregnant women (1). However, plasma Ang-(1-7) levels are reduced in women with preeclampsia in their third trimester (1). Since the RAS regulates cardiovascular function and body fluid and electrolyte balance, Ang-(1-7) and Ang II – two RAS peptides with opposing actions – may be involved in vascular adaptations in pregnancy. Other than pregnancy, Ang-(1-7) induces vasodilation and anti-proliferation, and Ang II elicits vasoconstriction, cell proliferation, and hypertrophy. The peripheral vasculature of pregnant women is resistant to pressor effects of Ang II, in part due to reduced sensitivity of arterial smooth muscle and down-regulation of AT1 receptors (2–4). However, the contribution of Ang-(1-7) to systemic or peripheral circulation in pregnant women is not known. Ang-(1-7) is expressed in vascular endothelial and smooth muscle cells (VSMC), and can influence the release of endothelium-derived vasodilatory factors or factors attenuating VSMC hypertrophy, suggesting that Ang-(1-7) can contribute to the vasodilatory state in pregnancy (5–7). Ang-(1-7) induces vascular effects via activation of the Mas/AT1-7 receptor (8). In the third trimester of pregnancy, Ang-(1-7) may act to reduce total peripheral resistance (TPR) and increase blood flow via systemic vasodilatory effects (1,9). In spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, Ang-(1-7) regulates CO and TPR without much change in blood pressure (10). In normal pregnant rats, Ang-(1-7) significantly dilates mesenteric arteries, an effect blocked by the Ang-(1-7) antagonist [D-Ala7]-Ang-(1-7) (9). To elucidate the contributions of Ang-(1-7) in vascular responses during pregnancy, we compared the influence of Ang-(1-7) on systemic hemodynamics and peripheral regional circulation (brachial artery) in normotensive pregnant and non-pregnant women. We also studied hypertensive women with preeclampsia, but only for assessments of skin (finger) microcirculation.
METHODS The protocol for these experiments was reviewed and approved by the Wake Forest School of Medicine and Zablocki VA Medical Center Institutional Review Boards. Consent forms were signed by all participants at the time of their enrollment in the study. All pregnant subjects were in their third trimester of pregnancy, and free of any known pre-existing cardiovascular, endocrine, or connective tissue disease. Study 1: Ang-(1-7) Contribution to Systemic Hemodynamics (Blood Pressure, Cardiac Output, Heart Rate, and Total Peripheral Resistance) and Forearm Blood Flow (FBF) in Normotensive Pregnant and Non-Pregnant Women Group 1 (n = 7) was composed of non-pregnant, normotensive women who were not taking oral contraceptives; the stage of the menstrual cycle was not determined. Group 2 (n = 13) was composed of nulliparous, normotensive
Ang-(1-7) and vascular responses in pregnancy Table 1. Systemic infusion study: patient population.
Number of subjects Race (percent of Caucasian/African American, American Indian and Hispanic) Age (years) Body mass index (kg/m2) Gestation age at the time of study (wks)
Non-pregnant
Normotensive pregnant
7 85.7/14.3
13 54/46
24.6 ± 3.1 21.9 ± 1.1
21.8 ± 0.8 29.3 ± 1.4* 37.4 ± 0.3
*p50.05 versus non-pregnant.
pregnant women. The non-pregnant and normotensive pregnant subjects are described in Table 1. All subjects were given systemic (intravenous) infusions of Ang-(1-7) in supine position with a 15 elevation from the back to the head. Three doses of Ang-(1-7) were administered (1000, 2000, and 3000 pmol/min) with each infusion dose lasting 30 min. Arterial pressures were measured using a radial artery catheter and VascuMAP (Carolina Medical, King, NC). The VascuMAP was placed on the arm contralateral to the infusion. CO and TPR were measured by bioimpedance using the BioZ device (CardioDynamics International Corporation, San Diego, CA). The advantage of the BioZ System is its ability to assess accurately the changes in perfusion/flux in response to a drug or maneuver. The procedure has been validated against the thermal dilution and Fick methods in animal studies and in adult critically ill surgical patients (11,12). CO data were expressed as a percent change relative to control (saline infusion). Blood samples were obtained for measurement of Ang-(1-7) before Ang-(1-7) infusion and at the end of each dosage (a total of four samples). FBF was measured by bilateral venous occlusion strain gauge plethysmography. Pediatric cuffs were placed around both wrists and inflated to 200 mmHg during each blood flow measurement. Strain gauges were placed around the forearms 5 cm distal to the olecranon. Study 2: Ang-(1-7) and Forearm (brachial) Blood Flow in Normotensive Pregnant and Non-Pregnant Women Group 1 (n = 7–13) was composed of non-pregnant, normotensive women. Group 2 (n = 5–10) was composed of nulliparous, normotensive pregnant women (Table 2) as characterized in the Study 1. All subjects were studied in the supine position. FBF was measured by bilateral venous occlusion strain gauge plethysmography as described in Study 1. Blood pressure cuffs placed around the upper arm were inflated to 40 mmHg and deflated every 15 s. All drugs were infused via a catheter (27 gauge) inserted into the brachial artery of the non-dominant arm under local anesthesia with 1% lidocaine. Baseline FBF was measured at least 60 min after the cannulation. Each subject received Ang II or Ang-(1-7) intra-arterially with measurement of FBF in the order indicated and with increasing doses. Angiotensin II was given at low (1, 5, 10, 20 pmol/min) and high (5, 25, 50, 100 pmol/min) doses. Ang-(1-7) was given at low (1, 5, 10, 50 pmol/min) and high (10, 50, 100, 500 pmol/min) doses. We used three different doses of Ang-(1-7), previously reported to have vasodilatory effects
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Number of subjects Race (percent of Caucasian/ African American, American Indian and Hispanic) Age (years) Body mass index (kg/m2) Gestation age at the time of study (wks)
Non-pregnant low dose
Non-pregnant high dose
Normotensive pregnant low dose
Normotensive pregnant high dose
13 77/23
7 100/0
5 40/60
10 50/50
25.8 ± 7.7 22.9 ± 3.7
20.7 ± 1.9 23.8 ± 3.7
21.5 ± 5.9 30.1 ± 2.9
22.6 ± 3.9 33.5 ± 6.4
35.3 ± 1.9
37.2 ± 0.9
in non-pregnant women or men (13,14). Each dose was given for 10 min with a 30-min washout period between each agent. The doses of drugs were selected to produce local changes in the infused arm only. Systemic arterial pressure was measured by VascuMAP in the contralateral arm. Since venous occlusion plethysmography provides only a relative assessment of blood flow, the data were expressed as a ratio of measurements recorded in infused – to a noninfused (control) arm to reduce the possibility of small changes in blood pressure or sympathetic nervous system affecting the responses to vasoactive substances (15). Study 3: The Contribution of Ang-(1-7) to Skin Microcirculation Group 1 (n = 14) was composed of non-pregnant women and Group 2 (n = 15) was composed of normotensive pregnant women according to the same criteria as described in the Study 1. Group 3 (n = 15) was composed of nulliparous preeclamptic women, matched to Group 2 by gestational age. Preeclampsia was defined by the onset of pregnancy-induced hypertension (blood pressure 140/90 mmHg) and proteinuria (300 mg/24 h or 2+ or more protein levels on random sample collection) (Table 3). Skin blood flow was determined by laser Doppler and Ang II and Ang-(1-7) were delivered using iontophoresis, a drug delivery method based on the stimulation of charged ions migration across the skin (16). The iontophoresis laser Doppler unit (PeriIont Micropharmacology System, Perimed AB, Stockholm, Sweden) allows for direct measurements of skin blood flow over the iontophoresized area (17,18). Since this method does not provide absolute perfusion values but arbitrary perfusion units that represent blood ‘‘flux’’ (F) changes, the data are shown as percent change determined by the following equation: Fpost Fpre Percentage change in flux ¼ 100: Fpre biologic zero
Ang-(1-7) and vascular responses in pregnancy Table 3. Skin microcirculation study: patient population.
Number of subjects Race (percent of Caucasian/African American, American Indian and Hispanic) Age (years) Body mass index (kg/m2) SBP (mmHg; baseline) DBP (mmHg; baseline) Proteinuria (mg) Gestation age at the time of study (wks) Gestational age at delivery (wks) Birthweight, g Birth centile Baby gender (female-to-male ratio)
Nonpregnant
Normotensive pregnant
Preeclamptic
14 63.3/35.7
15 66. 7/33.3
15 71.4/28.6
21.5 ± 5.9 26.3 ± 1.8 108.9 ± 3.4 65.8 ± 0.4
20.7 ± 1.9 28.5 ± 1.6 105.4 ± 2.4 65.7 ± 1.7 Negligible 30.9 ± 1.2 36.8 ± 1.2 2750 ± 249.4 47.7 ± 0.1 10/5
24.3 ± 1.7 36.6 ± 1.9*y 142.6 ± 1.9*y 86.2 ± 3.1*y 507.3 ± 214.4y 31.5 ± 0.8 32.9 ± 0.9y 2031 ± 241.0y 39.3 ± 0.1 5/10
*p50.05 versus non-pregnant; yp50.05 versus pregnant.
All participants were examined in the supine position. Subjects were warmed with a blanket until the finger temperature was 30 ± 2 C. Skin (finger) blood flow responses to each of the following iontophoretically applied agents were studied: (a) Ang II (0.5, 1.0 and 2.0 mmol/l), (b) Ang-(1-7) (1.0, 5.0 and 20.0 mmol/l), and (c) physiological saline (control). Drugs were administered in the order and dose indicated. Iontophoresis (200 mA) was performed for 2 min with each agent at each concentration. Skin blood flow was measured at the same site as the iontophoresis, with a 15-min interval between each drug and dose. Angiotensin-(1-7) Radioimmunoassay Blood (14 ml) was taken in a cocktail of inhibitors (19) and plasma was extracted using Sep-Pak columns. Ang-(1-7) was measured by an in-house radioimmunoassay, as previously described (19). Statistical Analysis Statistical analyses of data were performed using GraphPad Prism IV (San Diego, CA) plotting and statistical software. Comparisons of demographic indices between normotensive pregnant and preeclamptic women in the systemic infusion study were performed using unpaired t-tests. The comparisons of demographic parameters among non-pregnant, normotensive pregnant, and preeclamptic women in the skin flow study were performed using one-way analysis of variance (ANOVA) with Tukey posttests. Hemodynamic responses were evaluated using two-way ANOVA with Bonferroni post-hoc tests. Dose-dependent responses in each group were evaluated using one-way ANOVA with repeated measures test for matched observations. All measurements were expressed as the mean ± standard error of the mean (SEM). The criterion for statistical significance was p50.05.
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RESULTS Study 1 In the systemic hemodynamics study, most non-pregnant and pregnant women were Caucasians, but there were more African American or Hispanic women in the normotensive pregnant group compared to non-pregnant women group (Table 1). Systemic infusion of Ang-(1-7) at 2000 pmol/min resulted in a greater change in CO (pregnant: 9.4 ± 6.4 versus non-pregnant: 3.3 ± 2.1%, p50.05) and less change in TPR (pregnant: 12.9 ± 5.5 versus non-pregnant: 1.7 ± 3.1%) in pregnant women compared to non-pregnant women (Figure 1). There were no differences between groups in response to the systemic infusion of Ang-(1-7) at 1000 or 3000 pmol/min. The change in FBF was greater in
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Figure 1. Responses to systemic Ang-(1-7) infusion in non-pregnant and normotensive pregnant women. Data are mean ± SEM; *p50.05 versus non-pregnant women, n = 7–13. FBF = forearm blood flow.
Ang-(1-7) and vascular responses in pregnancy
pregnant women after systemic administration of Ang-(1-7) at 2000 pmol/min (pregnant: 12.7 ± 7.5 versus non-pregnant: 13.8 ± 5.1%, p50.01, n = 9–10) and at 3000 pmol/min (pregnant: 21.2 ± 11.6 versus non-pregnant: 20.3 ± 6.2%, p50.01, n = 9–10) but not at 1000 pmol/min. There were no significant changes in blood pressure, heart rate or stroke volume. Plasma levels of Ang-(1-7) were significantly elevated following the infusion of 1000 pmol/min (69.9 ± 5.6 pmol/ml), 2000 pmol/min (122.5 ± 9.9 pmol/ml) and 3000 pmol/min (127.3 ± 11.8 pmol/ml) of the peptide in comparison to pretreatment state (21 ± 3.6 pmol/ml). Plasma Ang-(1-7) levels were measured in normotensive pregnant women; p50.05; n = 15 in each group. In addition, the plasma levels of Ang-(1-7) were increased in a dose-dependent manner such as the peptide levels detected following 2000 and 3000 pmol/min infusion were significantly higher compared to the levels of the peptide detected following 1000 pmol/min infusion (p50.05; n = 15 in each group). Study 2 In the forearm (brachial) circulation study, most non-pregnant women were Caucasians. However, there were more participants of African American, American Indian or Hispanic heritage among the normotensive pregnant women compared to non-pregnant women (Table 2). Local infusion of Ang-(1-7) (Figure 2) had no effect on forearm flow, brachial resistance, or mean blood pressure at any dose. Ang II induced a dose-dependent decrease in FBF at 25, 50 and 100 pmol/min (2.1 fold at 25 pmol/min, 2.5 fold at 50 pmol/min and 2.7 fold at 100 pmol/min; all versus baseline in non-pregnant women, p50.05) and an increase in resistance in non-pregnant women at 50 and 100 pmol/min (2.9 fold at 50 pmol/min and 3.1 fold at 100 pmol/min, p50.05 versus baseline in non-pregnant women). In normotensive pregnant women, FBF was lower at 50 and at 100 pmol/min (1.65-fold at 50 pmol/min and 2.1-fold at 100 pmol/min, all versus baseline in pregnant women, p50.05) and resistance was higher at 50 and 100 pmol/min following Ang II administration (2.1-fold at 50 pmol/min and 2-fold at 100 pmol/min; all versus baseline in pregnant women, p50.05) (Figure 3). These differences were not observed in response to Ang II infusion at low doses. Mean blood pressure did not change in any group in response to brachial infusion of Ang II, regardless of dose. Study 3 Participants in the skin microcirculation experiment were predominantly Caucasian. As expected, resting systolic and diastolic blood pressures and proteinuria were greater in preeclamptic women versus normotensive pregnant and non-pregnant women (Table 3). Body mass index was greater in preeclamptic subjects compared to normotensive pregnant or to non-pregnant women. On the average, preeclamptic women had shorter pregnancies and smaller babies. There were more boys born to preeclamptic mothers in this cohort of women. Changes in cutaneous flow were significantly higher in normotensive pregnant women versus preeclamptic women in response to 5 mmol/l of Ang-(17) (pregnant: 182.5 ± 93 versus preeclamptic: 15.76 ± 19.46%, p50.05) (Figure 4A), but not in response to Ang-(1-7) at either 1 or 20 mmol/l dose. Preeclamptic women lost the vasodilatory response to 5 mmol/l. Skin flow was
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Figure 2. Responses of forearm (brachial) circulation to local infusion of Ang-(1-7) in non-pregnant and normotensive pregnant women. Data are mean ± SEM, n = 5–13 (low dose), n = 7–10 (high dose).
reduced in normotensive pregnant women compared to preeclamptic women in response to Ang II (1.0 mmol/l) (Figure 4B). There were no differences in skin flow among studied groups in response to Ang II at either 0.5 or 2.0 mmol/l dose (Figure 4B).
DISCUSSION In this study, systemic infusion of Ang-(1-7) resulted in greater increase in the change of CO and lower decrease in the change of TPR in normotensive pregnant women compared to non-pregnant women. In contrast, in our forearm circulation experiments, Ang-(1-7) had no effect on the change of FBF
Ang-(1-7) and vascular responses in pregnancy
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Figure 3. Responses of forearm (brachial) circulation to local infusion of Ang II in non-pregnant and normotensive pregnant women. Data are mean ± SEM, n = 5–13 (low dose), n = 7–10 (high dose); *p50.05 versus baseline in non-pregnant women; #p50.05 versus baseline in pregnant women; ^p50.05 versus 5 pmol/min.
or resistance, whereas high doses of Ang II induced a dose-dependent decrease in FBF and increased resistance in normotensive pregnant and non-pregnant women. We also found that the change in skin blood flow in response to Ang(1-7) (5 mmol/l) was greater in normotensive pregnant women and had little or no effect in preeclamptic women. These findings suggest a possible role for Ang-(1-7) in the regulation of systemic and local skin hemodynamics in the third trimester of normotensive pregnancy. Here, we demonstrate for the first time that systemic infusion of a single dose of Ang-(1-7) in the third trimester of pregnancy increases CO and decreases TPR without altering blood pressure. Changes in stroke volume probably led to the increase in CO in pregnant women. However, our study was
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