Reductions in angiotensin II type 2 receptor-mediated vasodilation contribute to increased angiotensin II vasoconstrictor sensitivity in women with preeclampsia history Open Access

Preeclampsia is defined as new onset hypertension in pregnancy accompanied by end organ dysfunction [1]. Although the clinical preeclampsia symptoms resolve following delivery, women with a history of preeclampsia (hxPE) have a significantly greater risk of cardiovascular disease (CVD) morbidity and mortality than women who had normotensive pregnancy [2-4]. Vascular studies reveal that women with a hxPE have subclinical vascular dysfunction that likely contributes to the elevated CVD risk [5-8]. Given that CVD is the leading cause of death among women worldwide, healthy women with a hxPE represent a high-risk cohort that requires early mechanism-specific intervention to prevent or delay CVD development.

One putative mechanism underlying vascular dysfunction after preeclampsia is dysregulated renin-angiotensin-aldosterone system (RAAS) signaling, including an increased vasoconstrictor sensitivity to angiotensin (ang) II. Ang II binds to ang II type 1 receptors (AT₁R) on vascular smooth muscle to cause vasoconstriction or to ang II type 2 receptors (AT₂R) on the endothelium to induce vasodilation [9]. Women with a hxPE have greater vasoconstrictor sensitivity to ang II, including an exaggerated pressor response [10,11] and increased microvascular constriction [12-14] compared with healthy postpartum controls. This increased vascular sensitivity is found in the absence of differences in circulating ang II [11,15], although AT₁R agonistic autoantibody (AT1-AA) concentrations, which are elevated during preeclampsia and contribute to a pro-constricted and anti-angiogenic milieu during preeclampsia [16,17], are also elevated postpartum [18-20]. Our group has recently demonstrated that exaggerated ang II-mediated constriction can be reduced in the microvasculature with local or systemic pharmacological inhibition of AT₁R [13,14], or local activation of the counterregulatory RAAS with ang 1–7 [12] or AT₂R agonism [15], suggesting that approaches that counteract excessive AT₁R activation may be feasible strategies to improve vascular function in women who had preeclampsia prior to the onset of chronic CVD.

AT₂R is an endogenous counterbalance to AT₁R-mediated responses, and the imbalance of the constrictor AT₁R and dilatory AT₂R RAAS axes plays a prominent role in the pathophysiology of preeclampsia. Specifically, the up-regulation of AT₂R present during healthy pregnancy [21,22] is absent during preeclampsia [23,24] and likely contributes to exaggerated AT₁R-mediated vasoconstriction postpartum [11]. We have recently demonstrated that women who had preeclampsia have reduced AT₂R-mediated dilation that contributes to persistent microvascular endothelial dysfunction after preeclampsia [15]; however, no studies have mechanistically examined the role of AT₂R in exaggerated ang II-mediated constriction in women with a hxPE.

The purpose of the present study was to determine the extent to which changes in AT₂R signaling contribute to exaggerated activation of the vasoconstrictor RAAS axis via AT₁R in otherwise healthy women with a hxPE compared with matched control women with a history of healthy pregnancy (hxHC). Our overarching hypothesis is that the balance of AT₁R:AT₂R is altered to favor ang II-mediated vasoconstriction in women who had a pregnancy complicated by preeclampsia. Using the cutaneous circulation as a model of global microvascular function [25,26], we hypothesized that hxPE would have reduced AT₂R-mediated dilation compared with hxHC, and that local AT₁R inhibition would improve AT₂R-mediated dilation in hxPE. Additionally, we hypothesized that exaggerated ang II-mediated constriction in hxPE is a result of reductions in AT₂R signaling, such that local AT₂R inhibition would increase ang II-mediated constriction in hxHC but have no effect in hxPE.

The data that support the findings of the present study are available from the corresponding author upon reasonable request. Written and verbal informed consent were obtained before study enrollment in accordance with the World Medical Association Declaration of Helsinki. All experimental protocols were approved by the University of Iowa Institutional Review Board (IRB no. 202309383) and a U.S. Food and Drug Administration (IND no. 124294) was obtained for the use of the pharmacological agents with intradermal microdialysis.

Twenty-four healthy normotensive women who were within 5 years postpartum were enrolled. This included 12 women who had preeclampsia in their most recent pregnancy, diagnosed as new onset hypertension and multi-system organ dysfunction after the 20th weeks of pregnancy by their obstetrician and confirmed by medical chart review [1] (hxPE), and 12 women who had a hxHC.

Participants were recruited via: public advertisement in Iowa City, IA and surrounding communities; the University of Iowa and University of Iowa Health Care Medical Centers mass email; and by contacting eligible participants who had previously consented to being contacted for future research studies within our laboratory. Eight hxPE and 1 hxHC participated in a previous study in our laboratory [15]. All participants completed a medical screening that included a physical examination, collection of health history, urine pregnancy test, and fasted blood chemistry and lipid profile (University of Iowa Diagnostic Laboratories, Iowa City, IA). All participants were 18–45 years of age, premenopausal, and self-reported to be physically active. Exclusion criteria included current or past cardiovascular, renal, and/or metabolic disease, current use of anti-hypertensive or cholesterol-lowering medications or use of these medications within 2 months of study enrollment, current tobacco/e-cigarette use, history of gestational hypertension or gestational diabetes in any pregnancy, or currently pregnant. Race and ethnicity were self-reported by all participants.

Participants completed one experimental visit and were asked to fast for 8 hours, withhold caffeine for 12 hours, and refrain from strenuous physical activity and alcohol for 24 hours prior. This is a standardized approach utilized by our laboratory and others to control for external influences on vascular function [14,15,27,28]. All experiments were scheduled in the morning, with a start time between 7:00 and 10:00 am local time. Following 5 minutes of local ice application to anesthetize the skin, four intradermal microdialysis fibers (CMA 31 Linear Microdialysis Probe, CMA Microdialysis, Holliston, MA) separated by ≥4 cm were aseptically placed in the left ventral forearm for the local delivery of pharmacological agents. Pharmacological agents were weighed just prior to use, mixed with lactated Ringer’s solution, sterilized with syringe microfilters (Acrodisc; Pall, Ann Arbor, MI), wrapped in foil to prevent photodegradation, and perfused through each fiber at a rate of 2 μL/min (Bee Hive controller and Baby Bee microinfusion pumps; Bioanalytical Systems). Laser-Doppler flowmetry probes were placed in local heaters (Moor Instruments, Wilmington, DE) set to thermoneutral (33°C) directly over each microdialysis site to continuously measure cutaneous red cell flux within the tissue (~1 mm³) treated by the microdialysis perfusates. Automated brachial blood pressure and heart rate (SureSigns VS2+, Philips Healthcare, Andover, MA) were measured every 5 minutes throughout the protocol. Blood pressure was measured in the contralateral (right) arm at heart level. Participants were instrumented in a semi-recumbent position and remained in this position for the duration of the visit. Following an initial hyperemia-resolution period (~60 minutes), baseline measurements were collected (~10 minutes), and two separate dose–response protocols commenced as described below.

Compound 21 dose–response: Two microdialysis fibers were randomly selected to receive either lactated Ringer’s (control) or 43 µmol/L losartan (United States Pharmacopeial) for the inhibition of AT₁R [13,15,29]. Following baseline measurements, ascending concentrations of compound 21 (C21; AT₂R agonist, 10^-14–10^-8 M; Sigma-Aldrich) mixed with each site-specific treatment were perfused sequentially for 5 min each to ensure a plateau [15,29]. Following C21 doses, 28 mM sodium nitroprusside (SNP; United States Pharmacopeial) was perfused at 4 μL/min and local heaters were increased to 43°C until a maximal blood flow plateau on each site was obtained (~20 minutes).

Ang II dose–response: Two microdialysis fibers were randomly assigned for the local delivery of lactated Ringer’s (control) or 1 µmol/L PD-123319 (Tocris, Ellisville, MO) for inhibition of AT₂R [30,31]. After baseline was achieved, both sites received ascending concentrations of ang II (10^-20–10^-4 mol/L; Tocris), perfused sequentially for 5 min each, mixed with the site-specific treatment [12,13].

On the day of the experiment, blood samples were collected via venipuncture for the analysis of serum AT1-AA. Sera samples were isolated and frozen (–80°C) until analysis. Circulating AT1-AA was measured using a cardiomyocyte bioassay as previously described in Refs. [32-35]. Total IgG fraction was isolated from serum using a Protein G HP Column (Cytiva, Marlborough, MA) following manufacturer’s instructions. Neonatal ventricular cardiomyocytes were isolated and cultured as previously described in Booz and Baker [36]. Cells were loaded with calcium-sensitive Fluo-4 AM dye (Fisher, Waltham, MA), and the cell beating rate was measured by kinetic fluorescent microscopy. Baseline beating rate was calculated, and sample IgG fractions were applied to the cells. A duplicate of each sample was premixed with specific AT1-AA inhibitory peptide ‘n7AAc’ to confirm specific action of AT1-AA. The change in the beats per minute (ΔBPM) was calculated for each sample and indicates circulating AT1-AA activity.

All data collection and analysis procedures were standardized prior to testing. Blood flow data were recorded at 40 Hz and stored for offline analysis (PowerLab and LabChart; AD Instruments, Sydney, Australia). Absolute cutaneous vascular conductance (CVC) was calculated (CVC = laser-Doppler flux/mean arterial pressure) and normalized to either a percentage of site-specific maximum (%max) for vasodilation data or site-specific baseline (%base) for vasoconstriction data. Total individual dilation was calculated as the area under the curve (AUC) from %max across all compound 21 doses between the control and losartan-treated sites. Because ang II can elicit vasodilation at low doses systemically [37] and in the microvasculature [31], net AUC was calculated as the difference between the area above and below baseline (net AUC = AUC above the baseline – AUC below the baseline) to assess individual responses [38] (GraphPad Prism 10.2.2, San Diego, CA). The relation between microvascular responses and circulating AT1-AA was evaluated using linear regression (GraphPad Prism).

Sample size was determined a priori by power analysis [repeated-measures analysis of variance (ANOVA); power = 0.80, α = 0.05]. Using previously published data with similar primary outcomes [13,15,31] and pilot data, we determined that n = 12/group would provide ≥80% power to detect meaningful physiological differences of 15% max for vasodilation sites and 20% base for vasoconstriction sites between groups and across treatment sites within group. Student’s unpaired t-tests were used to compare participant characteristics and AT1-AA activity. Paired t-tests were used to assess within-group changes in AUC. Dose–response CVC data were analyzed using a three-way repeated measures ANOVA (group*dose*pharmacological site; SAS 9.4, Cary, IN) with post hoc Tukey corrections applied for specific planned comparisons when appropriate. Values are mean ± standard error, and individual values are presented within each figure when appropriate.

Participant characteristics are presented in Table 1. There were no group differences in age, parity, time postpartum, body mass index, blood pressure, or blood chemistry. Four hxHC (2 oral pills, 1 intrauterine device, and 1 implant) and nine hxPE (2 oral pills, 5 intrauterine devices, and 2 implants) were using a form of hormonal contraceptive. Four hxPE and four hxHC reported antidepressant use at the time of the study visit. Of these eight, one hxPE and one hxHC reported concurrent use of an anti-anxiolytic. Five hxPE received treatment to manage preeclampsia during pregnancy, and six hxPE required pharmacological management acutely postpartum (Table 1). None of the participants were currently using anti-hypertensive medications at the time of the study visit. There were no group or site differences in baseline or maximal CVC (Table 2; all P>0.05).

Considering the role of AT₂R during healthy pregnancy and preeclampsia, we first assessed AT₂R-mediated dilation responses to the specific AT₂R agonist compound 21 in postpartum women. Microvascular AT₂R-mediated dilation (CVC, %max) responses to increasing doses of compound 21 were blunted in hxPE compared with hxHC (P=0.002, Figure 1A) as was the total vasodilation response (AUC; P<0.001, Figure 1B). In order to examine whether AT₁R-mediated constriction masks AT₂R-mediated responses in the microvasculature of hxPE, we acutely inhibited AT₁R with losartan during the assessment of AT₂R-mediated dilation. Local losartan treatment increased AT₂R-mediated dilation in hxPE (P<0.001 vs. control site) and AUC (P<0.001 vs. control site) to values similar to that observed in hxHC (P=0.68 hxPE losartan vs. hxHC control). However, in the hxHC group, losartan treatment attenuated AT₂R-mediated dilation (P<0.001) and AUC (P=0.005) compared with the control site.

To examine the role that AT1-AA may have in microvascular RAAS responses, we measured activity of circulating AT1-AA and assessed whether AT1-AA activity was associated with microvascular responses in a subset of our participants (n=10/group). Eight hxPE and four hxHC had detectable activity of circulating AT1-AA (∆bpm≥7.2). Circulating AT1-AA activity was higher in hxPE compared with hxHC (P=0.0145; Figure 2A). Furthermore, circulating AT1-AA activity was negatively associated with AT₂R-mediated dilation (compound 21 control site AUC; P=0.035, r²=0.22; Figure 2B) across groups.

Given that women who had preeclampsia have increased vascular sensitivity to ang II, we next examined the role that AT₂R contributes to this response. hxPE had an exaggerated vasoconstriction (CVC, %base) response to ang II compared with hxHC (P<0.001, Figure 3A), and the net vasoconstriction response (AUC) to ang II was greater in the control site in hxPE compared with hxHC (P<0.001, Figure 3B). Local AT₂R blockade with PD-123319 potentiated ang II-mediated vasoconstriction in hxHC (P<0.001 vs. control site) but had no effect in hxPE (P=0.19) such that there was no difference in PD-123319 treated sites between groups (P=0.77) (Figure 3A). Local AT₂R inhibition increased the net vasoconstrictor response in hxHC (P<0.001 vs. control site) but had no effect in hxPE (P=0.16) (Figure 3B). There were no group differences in net vasoconstriction in the presence of local AT₂R inhibition (P=0.47). No significant association was found between circulating AT1-AA and ang-II-mediated constriction (ang II control site AUC; all P>0.3; data not shown).

The primary findings of the present study are as follows: (1) healthy women with a hxPE have attenuated AT₂R-mediated vasodilation compared with matched women who had a healthy pregnancy, (2) acute inhibition of AT₁R with losartan augmented AT₂R-mediated vasodilation in women with a hxPE, and (3) exaggerated ang II vasoconstrictor sensitivity present in women after preeclampsia is driven, in part, by a reduction in AT₂R-mediated responses. We also found that women with a hxPE have greater circulating AT1-AA activity than women who had normotensive pregnancy and that there was a negative association between AT1-AA activity and AT₂R-mediated dilation across groups. Together, these findings suggest that despite the absence of clinical CVD risk factors, healthy women with a hxPE have alterations in the balance of AT₁R- and AT₂R-mediated vascular responses that contribute to increased ang II-mediated constriction. This imbalance may contribute to the accelerated progression of CVD following preeclampsia.

Despite the remission of clinical preeclampsia symptoms after delivery, women with a hxPE develop chronic CVD at a younger age (~10 years earlier), with greater frequency, and have higher CVD mortality than women who had a healthy pregnancy [3,4,39,40]. Under physiological conditions, AT₂R functions not only as an endogenous counterbalance to AT₁R but also as an independent effector [41,42]. We recently found that AT₂R-mediated dilation is reduced in otherwise healthy women with a hxPE and that this reduction contributes to endothelial dysfunction in these women [15]. In the current study, we extended these findings and examined the extent to which alterations in the vascular balance of AT₁R:AT₂R responses contribute to reduced AT₂R-mediated dilation in otherwise healthy women with a hxPE. Previous reports demonstrate that in physiological states where the ratio of AT₁R:AT₂R expression favors AT₁R-mediated responses, such as in male rodents [43] and humans [29], AT₂R-mediated dilation is blunted or even completely masked by tonic AT₁R activation [44-46]. Considering that women with preeclampsia have increased AT₁R expression in placental vessels and vascular endothelial cells during pregnancy [23,24,47] and have increased AT₁R expression in skin biopsy samples after preeclampsia [13], we hypothesized that exaggerated AT₁R-mediated constriction attenuates AT₂R-mediated dilation after preeclampsia. Indeed, we found that local AT₁R inhibition with losartan increased AT₂R-mediated dilation in hxPE to a magnitude similar to that seen in the control site in hxHC. Given that AT₂R activation can reduce AT₁R expression and function [23,48,49], these data support our overarching hypothesis that increased vasoconstrictor sensitivity to ang II after preeclampsia is mediated by alterations in both AT₂R and AT₁R signaling. Interestingly, local losartan treatment unexpectedly caused a significant reduction in AT₂R-mediated vasodilation in hxHC. This finding may be evidence of compound 21 binding to AT₂R on vascular smooth muscle to produce inflammatory responses in the absence of vascular dysfunction [50,51]. However, examining the role of downstream AT₂R signaling was not the focus of the present study and requires further investigation.

Circulating AT1-AA activity is elevated and contributes to vascular dysfunction during preeclampsia [17], and circulating AT1-AA remains elevated in some women up to 8 years, following a pregnancy complicated by preeclampsia [18-20]. AT1-AA binds to AT₁R to elicit AT₁R-mediated responses that are blocked by AT₁R inhibitors, and AT₁R antagonism improves postpartum outcomes in preeclamptic-like rats [52]. In the present study, we found that women with a hxPE had higher activity of circulating AT1-AA than matched control women with a history of normotensive pregnancy. Interestingly, AT1-AA activity was not exclusive to hxPE in our study. AT1-AAs have been detected in patient populations, including hypertension [53,54] and renal-allograft rejection [55], as well as in healthy, normotensive adults [53,54] and in women with a history of healthy pregnancy [19,20]. It currently remains unclear why some apparently healthy adults have detectable circulating AT1-AA activity. We also found that circulating AT1-AA was negatively associated with AT₂R-mediated dilation, demonstrating that there is a functional role for circulating AT1-AA in reduced microvascular AT₂R-mediated responses. Furthermore, this association suggests that AT1-AA contributes to a pro-constrictor RAAS balance by masking AT₂R-mediated responses and increasing ang II vasoconstrictor sensitivity after preeclampsia. However, similar to van der Graaf et al. [20], who demonstrated that the presence of AT1-AA was not correlated with the pressor response to exogenous ang II perfusion in women with a hxPE, we did not find a significant relation between circulating AT1-AA and ang II-mediated constriction in our cohort. It is important to note that AT1-AA non-competitively binds to an allosteric site on AT₁R that independently activates AT₁R and increases the sensitivity for ang II to bind [34,56]. Although ang II has greater affinity for AT₂R than AT₁R [57], it remains unclear whether or how AT1-AA may affect vascular AT₁R sensitivity to ang II. While future work specifically examining the effects of AT1-AA on AT₂R-mediated vascular responses in women who had preeclampsia is warranted, our data suggest that AT1-AA tonically activating AT₁R to increase ang II vasoconstrictor sensitivity may be one mechanism contributing to AT₂R dysfunction after preeclampsia.

Although systemic RAAS activity increases during healthy pregnancy, there is a concomitant decrease in vascular ang II sensitivity, resulting in a reduced pressor response to ang II [58]. Conversely, women with preeclampsia have an exaggerated pressor response to ang II [58,59] despite no elevation, or even a decrease [60], in circulating RAAS components during pregnancy. This vascular phenotype likely remains after delivery, as women who had preeclampsia demonstrate an exaggerated pressor response to ang II [10,11] even in the absence of increased systemic RAAS activity [11,15,61,62]. We have demonstrated that increased vascular sensitivity to ang II is present in the cutaneous microvasculature and contributes to reduced endothelium-dependent dilation in these women [12-15,63,64]. Furthermore, we have shown that endothelial function can be improved with local or systemic pharmacological AT₁R inhibition [13,14], local ang 1–7 treatment [12], or local AT₂R agonism with compound 21 [15], suggesting that blocking exaggerated AT₁R-mediated constriction and activating the counterregulatory RAAS are potential mechanistic approaches to improve endothelial function in these high-risk women before the development of overt CVD. Interestingly, one study has demonstrated that women who had preeclampsia have a significant relation between the ratio of AT₁R:AT₂R expression in the skin and the change in blood pressure during systemic ang II infusion [11]. The authors report that this relation was absent in women who had a healthy pregnancy and never pregnant normotensive women, suggesting that differences in the vascular tissue RAAS mediate exaggerated responsiveness to ang II following preeclampsia. In agreement, our data extend these findings and demonstrate that exaggerated ang II-mediated microvascular constriction in women who had preeclampsia is due, in part, to a reduction in counterregulatory AT₂R-mediated dilation. We found that acute AT₂R inhibition shifted the ang II vasoconstrictor curve downward and increased constriction in hxHC but had no effect on the constriction response in hxPE. Taken together, our data suggest that reductions in AT₂R signaling contribute to alterations in the RAAS balance that favors AT₁R-mediated constriction after preeclampsia.

Recent data suggest that preeclampsia may have two distinct phenotypes, often termed ‘early-onset’ (diagnosed before 34 weeks gestation) and ‘late-onset’ (diagnosed at or after 34 weeks gestation) preeclampsia [65]. Early-onset preeclampsia is associated with a greater lifetime risk of adverse vascular outcomes compared with late-onset preeclampsia [66,67]. We did not detect differences in microvascular function when we stratified our data by early- vs. late-onset preeclampsia; however, we are underpowered to make this comparison. Future work is required to determine if differences in microvascular function exist between early- and late-onset preeclampsia subtypes in the years following pregnancy. Similarly, it is likely that the time since the index pregnancy may influence vascular function. Women with a hxPE have premature development of hypertension andCVD, resulting in an increased use of blood pressure- and lipid-lowering medication at a younger age [68]. Therefore, we choose to enroll women up to 5 years postpartum, prior to the manifestation and pharmacological management of clinical disease. Although recent findings suggest that pre-clinical vascular dysfunction in women who had preeclampsia may be reversible at any point up to the development of clinical disease [69], future work examining microvascular function beyond 5 years postpartum in women who did and did not have preeclampsia is warranted.

Our approach did not measure circulating RAAS components in our participants. We and others have demonstrated that systemic RAAS components and their activity are not different in postpartum women who did or did not have preeclampsia [11,15,61,62], suggesting that alterations in ang II receptor sensitivity within the vasculature are the likely mechanism underlying enhanced ang II constrictor sensitivity in women with a hxPE. Another limitation is that we did not control for menstrual cycle phase or contraceptive use, nor did we assess circulating hormone concentrations on the day of the experimental visit. Previous reports suggest that microvascular function is not influenced by menstrual cycle phase [70]. However, the RAAS can be influenced by chronic sex hormone exposure, specifically by differences in estradiol [71-73] compared with testosterone [74,75]. It is also possible that differences in synthetic hormone status based on contraceptive use may have influenced our results. Nine women with a hxPE were utilizing a form of hormonal contraceptive compared with four hxHC women. As such, variations in the number and type of contraceptives (progesterone alone vs. progesterone + estradiol) within each group may have contributed to the variability in our data. Future work designed to specifically assess these outcomes is required to examine how differences in ovarian hormone status and type of contraceptive use may influence mechanisms mediating microvascular responses.

We utilized the human cutaneous microvascular bed to examine mechanisms of microvascular function and dysfunction in vivo. There is a significant relation between microvascular function measured in the skin and that measured invasively in the coronary and renal microvasculature [26,76], and attenuations in microvascular responses are a direct predictor of CVD morbidity and mortality [77]. Given that systemic microvascular dysfunction drives the clinical presentation of preeclampsia [78,79], it is likely that subclinical dysfunction persists in the maternal microvasculature following delivery [5]. In support of this hypothesis, our group and others have consistently demonstrated that women who had preeclampsia have reductions in microvascular function [8,14,15,63,64,80-82]. We have shown that blocking the increased vasoconstrictor sensitivity to ang II present in the cutaneous microvasculature of women with a hxPE can improve microvascular function [12-14] and that reductions in AT₂R-mediated responses contribute to microvascular endothelial dysfunction after preeclampsia [15]. In the current study, we extended those findings and demonstrated that reductions in AT₂R-mediated responses contribute to exaggerated AT₁R-mediated microvascular constriction after preeclampsia (Figure 4). Collectively, our data indicate that ang II vasoconstrictor sensitivity is present in the microvasculature of women who had preeclampsia and may be mediated, in part, by subclinical alterations in AT₂R signaling.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

The authors have no competing interests to disclose.

This project was supported in part by the National Institutes of Health Clinical and Translational Science Award (UM1TR004403) and T32 (HL007344 to K.S.S.), the University of Iowa Graduate College (fellowship to K.S.S.), and the Mississippi Center for Excellence in Perinatal Research (P20GM121334).

Research design, data collection, analysis and interpretation, manuscript preparation: K.S.S.; Research design, analysis and interpretation, manuscript preparation: N.C.; Research design, analysis and interpretation, manuscript preparation: D.I.J.; Funding and research design, data collection, analysis and interpretation, manuscript preparation: A.E.S. All authors approved the final version of the manuscript.

Written and verbal informed consent were obtained before study enrollment in accordance with the World Medical Association Declaration of Helsinki. All experimental protocols were approved by the University of Iowa Institutional Review Board (IRB no. 202309383) and a U.S. Food and Drug Administration (IND no. 124,294) was obtained for the use of the pharmacological agents with intradermal microdialysis.

The authors thank the University of Mississippi Medical Center’s Pharmacology Clinical Research Core and the Mississippi Center for Excellence in Perinatal Research for AT1-AA bioassay analysis. We would also like to thank Claire Goebel for their assistance with data processing and our participants for contributing their time and effort to the completion of this project.

AT1-AAs

agonistic autoantibodies

AT1R

ang II type 1 receptor

AT2R

ang II type 2 receptor

AUC

area under the curve

BPM

beats per minute

C21

compound 21

CVC

cutaneous vascular conductance

CVD

cardiovascular disease

RAAS

renin-angiotensin-aldosterone system

ang II

angiotensin II

hxHC

history of healthy pregnancy

hxPE

history of preeclampsia