Florian Herse

Dysregulation of the Circulating and Tissue-Based Renin-Angiotensin System in Preeclampsia

The renin–angiotensin system (RAS) participates in preeclampsia; however, the relative contributions from the circulating RAS and the tissue-based, uteroplacental RAS are unknown. We hypothesized that the tissue-based uteroplacental RAS is dysregulated in preeclampsia. We performed microarray and gene expression studies and confirmed the findings on the protein level by immunohistochemistry in ureteroplacental units from 10 preeclamptic women and 10 women with uneventful pregnancies. All of the women were delivered by cesarean section. We also analyzed plasma renin activity and circulating agonistic angiotensin II type 1 (AT1) receptor autoantibodies. In preeclampsia, we found that the angiotensin II AT1 receptor gene was 5-fold upregulated in decidua (maternal origin). We also found AT1 autoantibodies in preeclamptic women and in their offspring by neonatal cardiomyocyte bioassay compared with women with normal pregnancies and their infants (mother: 17.5±2.2 versus 0.05±0.4; fetus: 14.5±1.8 versus 0.5±0.5 &Dgr;bpm). Gene expressions for renin (35.0-fold), angiotensin-converting enzyme (2.9-fold), and angiotensinogen (8.9-fold) were higher in decidua than placenta (fetal origin) in both control and preeclamptic women, whereas the AT1 receptor was expressed 10-fold higher in placenta than in decidua in both groups. Our findings elucidate the ureteroplacental unit RAS in preeclamptic and normal pregnancies. We found that, in preeclampsia, the AT1 receptor expression is particularly high in decidua, combined with pregnancy-specific tissue RAS involving decidual angiotensin II production and AT1 autoantibodies. We also showed that AT1 autoantibodies cross the ureteroplacental barrier. These components could participate in the pathophysiology of preeclampsia.

Autoantibodies to the Angiotensin Type I Receptor in Response to Placental Ischemia and Tumor Necrosis Factor α in Pregnant Rats

Circulating factors, such as agonistic autoantibodies to the angiotensin II type 1 (AT1) receptor (AT1-AAs), and inflammatory cytokines, including tumor necrosis factor &agr; (TNF-&agr;), are suggested to be important links between placental ischemia and hypertension in preeclamptic women. The purpose of this study was to determine the role of placental ischemia and TNF-&agr; in stimulating the AT1-AA and the importance of AT1 receptor activation in mediating hypertension during reductions in uterine perfusion pressure (RUPP) and chronic TNF-&agr; excess in pregnant rats. Increased mean arterial pressure in RUPP pregnant rats (122±1 mm Hg RUPP versus 101±1 mm Hg normal pregnant [NP]; P<0.001) was associated with increased circulating TNF-&agr; (RUPP 48±13 pg/mL versus N 8±1 pg/mL; P<0.05) and AT1-AA (RUPP 15.3±1.6 U versus NP 0.6±0.3 U; P<0.001). Moreover, TNF-&agr;–induced hypertension (97±2 to 112±2 mm Hg; P<0.05) in pregnant rats was associated with AT1-AA production (TNF-&agr; rats 9.2±2.3 U versus NP rats 1.0±0.8 U; P<0.05). To determine the importance of AT1 receptor activation in mediating hypertension in RUPP– and TNF-&agr;–treated rats, we administered an AT1 receptor antagonist to RUPP–, TNF-&agr;–treated, and NP rats. Blood pressure responses were attenuated in RUPP rats (&Dgr; 32 mm Hg versus &Dgr; 20 mm Hg, NP; P<0.001), as well as in TNF-&agr;–treated rats (&Dgr; 10 mm Hg versus &Dgr; 5 mm Hg, NP; P<0.05). Collectively, these data indicate that placental ischemia and TNF-&agr; are important stimuli of AT1-AA, and activation of the AT1 receptor appears to, in part, mediate hypertension produced by RUPP and TNF-&agr; in pregnant rats.

Agonistic angiotensin II type 1 receptor autoantibodies in postpartum women with a history of preeclampsia

Activating angiotensin II type 1 autoantibodies (AT1-AAs) develop in women with preeclampsia and may contribute to the disorder. Insulin resistance and serum concentrations of the antiangiogenic soluble fms-like tyrosine kinase 1 (sFlt-1) are also increased in women with preeclampsia compared with normal pregnancy. sFlt-1 and insulin resistance decrease substantially after delivery; however, significant group differences persist postpartum. Women who have had preeclampsia are at increased cardiovascular risk later in life. We measured AT1-AAs in groups of women with previous preeclampsia (n=29) and previous normal pregnancies (n=35) 18±9 months after the first completed pregnancy. These women had had sFlt-1, insulin resistance homeostasis model assessment score, and related cardiovascular risk factors measured. Activating antibodies were detected by the chronotropic response of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists (losartan and prazosin). AT1-AAs were detected in 17.2% of women with previous preeclampsia versus 2.9% of women with previous uncomplicated pregnancies (P<0.05). In contrast, there was no difference in the prevalence of autoantibodies against the α1-adrenoceptor (10% of previous preeclamptic versus 14% of previous normal pregnant). Women with activating autoantibodies had significantly increased sFlt-1, reduced free vascular endothelial growth factor, and higher insulin resistance homeostasis model assessment values compared with autoantibody-negative women. These data suggest that, as with sFlt-1 and insulin resistance, the AT1-AA does not regress completely after delivery and, secondarily, that correlations exist among these variables. The impact of AT1-AA after preeclampsia, especially in the context of cardiovascular risk, remains to be determined.

Angiotensin II type 1 receptor antibodies and increased angiotensin II sensitivity in pregnant rats.

Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT1) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. We generated and purified activating antibodies against the AT1 receptor (AT1-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT1 from patients with preeclampsia. We then purified AT1-AB using affinity chromatography with the AFHYESQ peptide. We were able to detect AT1-AB both by ELISA and a functional bioassay. We then passively transferred AT1-AB into pregnant rats, alone or combined with Ang II. AT1-AB activated protein kinase C-&agr; and extracellular-related kinase 1/2. Passive transfer of AT1-AB alone or Ang II (435 ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT1-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1&agr; was upregulated by Ang II plus AT1-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT1-AB. We show that AT1-AB induces Ang II sensitivity. Our mechanistic study supports the existence of an “autoimmune-activating receptor” that could contribute to Ang II sensitivity and possible to preeclampsia.

Dietary n-3 Polyunsaturated Fatty Acids and Direct Renin Inhibition Improve Electrical Remodeling in a Model of High Human Renin Hypertension

We compared the effect n-3 polyunsaturated fatty acids (PUFAs) with direct renin inhibition on electrophysiological remodeling in angiotensin II-induced cardiac injury. We treated double-transgenic rats expressing the human renin and angiotensinogen genes (dTGRs) from week 4 to 7 with n-3 PUFA ethyl-esters (Omacor; 25-g/kg diet) or a direct renin inhibitor (aliskiren; 3 mg/kg per day). Sprague-Dawley rats were controls. We performed electrocardiographic, magnetocardiographic, and programmed electrical stimulation. Dietary n-3 PUFAs increased the cardiac content of eicosapentaenoic and docosahexaenoic acid. At week 7, mortality in dTGRs was 31%, whereas none of the n-3 PUFA- or aliskiren-treated dTGRs died. Systolic blood pressure was modestly reduced in n-3 PUFA-treated (180±3 mm Hg) compared with dTGRs (208±5 mm Hg). Aliskiren-treated dTGRs and Sprague-Dawley rats were normotensive (110±3 and 119±6 mm Hg, respectively). Both n-3 PUFA-treated and untreated dTGRs showed cardiac hypertrophy and increased atrial natriuretic peptide levels. Prolonged QRS and QTc intervals and increased T-wave dispersion in dTGRs were reduced by n-3 PUFAs or aliskiren. Both treatments reduced arrhythmia induction from 75% in dTGRs to 17% versus 0% in Sprague-Dawley rats. Macrophage infiltration and fibrosis were reduced by n-3 PUFAs and aliskiren. Connexin 43, a mediator of intermyocyte conduction, was redistributed to the lateral cell membranes in dTGRs. n-3 PUFAs and aliskiren restored normal localization to the intercalated disks. Thus, n-3 PUFAs and aliskiren improved electrical remodeling, arrhythmia induction, and connexin 43 expression, despite a 70-mm Hg difference in blood pressure and the development of cardiac hypertrophy.

Prevalence of Agonistic Autoantibodies Against the Angiotensin II Type 1 Receptor and Soluble fms-Like Tyrosine Kinase 1 in a Gestational Age–Matched Case Study

We showed earlier that activating autoantibodies against the angiotensin II type 1 (AT1) receptor (AT1-AA) circulate in preeclamptic women. They may be involved in the pathogenesis of preeclampsia. Protein alignment suggests that the binding site for AT1-AAs is highly homologous to the capsid protein VP2 of parvovirus B19. We performed a prospective, nested, case-control study of 30 gestational age–matched women with preeclampsia and 30 normotensive pregnant women. We measured AT1-AA, soluble fms-like tyrosine kinase 1 (sFlt-1), and serum immunoglobulin G against parvovirus B19 proteins. AT1-AAs were present in 70% of preeclamptic patients and absent in 80% of controls. Prediction by AT1-AA was improved in late-onset preeclampsia. The discrimination for sFlt-1 was 96%. We did not find an interaction between sFlt-1 and AT1-AA. A human monoclonal immunoglobulin G antibody against parvovirus B19 VP2-protein showed a positive reaction in the AT1-AA bioassay, which could be blocked by an AT1 receptor blocker, as well as by the epitope amino acid sequence. Immunoglobulin G against parvovirus B19 proteins was similarly distributed between preeclamptic patients and controls and had no significant importance. We detected significantly more AT1-AA in women with an immune response corresponding with parvovirus B19 infection corresponding with a distant viral infection associated with virus elimination. We concluded that AT1-AAs were common in patients with preeclampsia in a prospective case-control study, although sFlt-1 was a superior biomarker. AT1-AA may represent a better marker for late disease, whereas sFlt1 is a better marker for early onset disease.

Hypertension in response to placental ischemia during pregnancy: Role of B lymphocytes

Preeclampsia is associated with innate inflammatory response resulting in elevated tumor necrosis factor-&agr;, agonistic autoantibodies to the angiotensin II type I receptor, and activation of endothelin 1 (ET-1). This study was designed to determine the role of B-cell depletion, resulting in agonistic autoantibodies to the angiotensin II type I receptor suppression to mediate hypertension via activation of ET-1 in the placental ischemic reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. To achieve this goal we examined the effect of RUPP on mean arterial pressure and ET-1 in the presence and absence of chronically infused rituximab (R; 250 mg/kg), a B-lymphocyte–suppressive agent used clinically to treat autoimmune diseases. Mean arterial pressure was 103±1 mm Hg in normal pregnant (NP) rats; 103±3 mm Hg in NP+R versus 133±2 mm Hg in RUPP rats, and 118±2 mm Hg in RUPP+R rats (P<0.001 vs RUPP controls). B lymphocytes decreased from 6.0±0.5% gated cells in RUPP to 3.7±0.8% gated cells in RUPP+R rats. Importantly, agonistic autoantibodies to the angiotensin II type I receptor decreased from 18±1 bpm in RUPP rats to 10±1 bpm in RUPP+R rats. ET-1 decreased 1.5-fold in kidneys and 4-fold in the placenta (P<0.01) of RUPP+R versus RUPP rats. Media ET-1 excretion from endothelial cells exposed to serum from NP, RUPP, NP+R, or RUPP+R rats was determined. ET-1 from endothelial cells treated with NP serum was 53+13 pg/mg and increased to 75+10 pg/mg with RUPP serum. In contrast, ET-1 secretion decreased in response to B-cell–depleted RUPP serum to 50±8 pg/mg and was unchanged in response to NP+R sera (46±12 pg/mg). These data demonstrate the important roles that B-lymphocyte activation and agonistic autoantibodies to the angiotensin II type I receptors play in the pathophysiology of hypertension in response to placental ischemia.

IL-17-mediated oxidative stress is an important stimulator of AT1-AA and hypertension during pregnancy

Preeclampsia is associated with autoimmune cells T(H)17, secreting interleukin-17, autoantibodies activating the angiotensin II type I receptor (AT1-AA), and placental oxidative stress (ROS). The objective of our study was to determine whether chronic IL-17 increases blood pressure by stimulating ROS and AT1-AAs during pregnancy. To answer this question four groups of rats were examined: normal pregnant (NP, n = 20), NP+IL-17 (n = 12), NP+tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (n = 7) (a superoxide dismutase mimetic that scavenges ROS), and NP+IL-17+tempol (n = 11). IL-17 (150 pg/day) was infused into NP rats while tempol was administered via the drinking water ad libitum. On day 19 blood pressure (MAP) was recorded, and plasma, urine, and tissue were collected for isolation of ROS detected by chemilluminescent technique. Urinary isoprostane was measured by ELISA. AT1-AAs were determined via cardiomyocyte assay and expressed as beats per minute. MAP increased from 98 ± 3 mmHg in NP to 123 ± 3 mmHg in IL-17-infused NP rats. Urinary isoprostane increased from 1,029 ± 1 in NP to 3,526 ± 2 pg·mg(-1)·day(-1) in IL-17-infused rats (P < 0.05). Placental ROS was 436 ± 4 RLU·ml(-1)·min(-1) (n = 4) in NP and 702 ± 5 (n = 5) RLU·ml(-1)·min(-1) in IL-17-treated rats. Importantly, AT1-AA increased from 0.41 ± 0.05 beats/min in NP rats (n = 8) to 18.4 ± 1 beats/min in IL-17 rats (n = 12). Administration of tempol attenuated the hypertension (101 ± 3 mmHg) ROS (459 ± 5 RLU·ml(-1)·min(-1)) and blunted AT1-AAs (7.3 ± 0.6 beats/min) in NP+IL-17+tempol-treated rats. Additionally, AT1 receptor blockade inhibited IL-17-induced hypertension and placental oxidative stress. MAP was 105 ± 5 mmHg and ROS was 418 ± 5 RLU·ml(-1)·min(-1) in NP+IL 17-treated with losartan. These data indicate that IL-17 causes placental oxidative stress, which serves as stimulus modulating AT1-AAs that may play an important role in mediating IL-17-induced hypertension during pregnancy.

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

Background Agonistic autoantibodies directed at the α1-adrenergic receptor (α1-AAB) have been described in patients with hypertension. We implied earlier that α1-AAB might have a mechanistic role and could represent a therapeutic target. Methodology/Principal Findings To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had α1-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α1-adrenergic receptor antibodies (α1-AB). Patient α1-AAB and rabbit α1-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human α1A-adrenergic receptor were incubated with patient α1-AAB and rabbit α1-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient α1-AAB and rabbit α1-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca2+ in cardiomyocytes and induce mesentery artery segment contraction. Conclusions/Significance Patient α1-AAB and rabbit α1-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α1-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

A recently evolved novel trophoblast-enriched secreted form of fms-like tyrosine kinase-1 variant is up-regulated in hypoxia and preeclampsia.

CONTEXT Recent published studies indicate a possible role for sFlt1 in the development of preeclampsia. OBJECTIVE The objective of the study was to investigate the expression and regulation of sFlt1-e15a, a recently described novel C-terminal variant isoform of sFlt1. DESIGN The studies included a computational comparative analysis of the genomic locus of sFlt1 across vertebrate species; an assessment of sFlt1 variants in human and rhesus cells and tissues; an analysis of sFlt1 variants transiently expressed in HeLa and COS-7 cells; an evaluation of the effect of hypoxia on sFlt1 expression in trophoblasts; and a comparison of placental sFlt1 expression between pregnancies complicated by preeclampsia and control pregnancies. RESULT AND CONCLUSIONS sFlt1-e15a emerged as an alternate transcript of Flt1 late in evolution with the insertion of an AluSq sequence into the primate genome after the emergence of the simian infraorder about 40 million years ago. sFlt1-e15a is particularly abundant in human placenta and trophoblasts and is also highly expressed in nonhuman primate placenta. The expressed protein has a C-terminal polyserine tail and, like reference sequence sFlt1 (sFlt1-i13), is glycosylated and secreted. Consistent with a role in placental pathophysiology, hypoxia stimulates sFlt1-e15a expression in isolated cytotrophoblasts and a trophoblast cell line, and differentiation into syncytiotrophoblasts further enhances the effect of hypoxia. Placental levels of sFlt1-e15a and sFlt1-i13 transcripts are significantly elevated in patients with preeclampsia compared with normal pregnancies. We speculate that sFlt1-e15a may contribute to the pathophysiology of preeclampsia.