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Hypertension before and during early pregnancy has been associated with an increased risk of gestational diabetes mellitus (GDM) in retrospective analyses. We aimed to investigate the prospective blood pressure trackings in a population-based cohort of pregnant women, who were stratified according to their metabolic status in early third trimester.We recorded blood pressure longitudinally during pregnancy in 1230 women from the Odense Child Cohort, Denmark. Fasting glucose and insulin were measured at gestational weeks 28-30. Metabolic status was evaluated according to the WHO 2013 threshold for GDM (GDM-WHO: fasting plasma glucose ≥5.1 mmol/l), insulin and homeostatic model assessment of insulin resistance (HOMA-IR). Relationships between metabolic status in third trimester and blood pressure trajectories were evaluated with adjusted linear mixed models. Trajectory was defined as blood pressure records in pregnancy per 4 weeks interval.Prevalence of GDM-WHO was 40% (498/1230). GDM-WHO was associated with 1.46 (0.22-2.70) mmHg higher SBP and 1.04 (0.07-2.01) mmHg higher DBP trajectories in the overall cohort. The associations were driven by differences in the overweight group, with 3.14 (1.05-5.25) mmHg higher SBP and 1.94 (0.42-3.47) mmHg higher DBP per 4 weeks in women with GDM-WHO compared with women without GDM-WHO. GDM-WHO was not associated with blood pressure in women with normal weight. Blood pressure trajectories were elevated across quartiles of insulin resistance.GDM-WHO is associated with higher blood pressure in pregnancy, and there appears to be a stronger effect in overweight women.

Supplemental Digital Content is available in the text., One driving factor for developing preeclampsia—a pregnancy disorder, often associated with poor spiral artery (SpA)-remodeling and fetal growth restriction—is the anti-angiogenic sFLT1 (soluble fms-like tyrosine kinase-1), which is found to be highly upregulated in preeclampsia patients. The sFLT1-mediated endothelial dysfunction is a common theory for the manifestation of maternal preeclampsia symptoms. However, the influence of sFLT1 on SpA-remodeling and the link between placental and maternal preeclampsia symptoms is less understood. To dissect the hsFLT1 (human sFLT1) effects on maternal and/or fetoplacental physiology in preeclampsia, sFLT1-transgenic mice with systemic hsFLT1 overexpression from midgestation onwards were used. SpA-remodeling was analyzed on histological and molecular level in placental/mesometrial triangle tissues. Maternal kidney and aorta morphology was investigated, combined with blood pressure measurements via telemetry. hsFLT1 overexpression resulted in maternal hypertension, aortic wall thickening, and elastin breakdown. Furthermore, maternal kidneys showed glomerular endotheliosis, podocyte damage, and proteinuria. preeclampsia symptoms were combined with fetal growth restriction already at the end of the second trimester and SpA-remodeling was strongly impaired as shown by persisted vascular smooth muscle cells. This phenotype was associated with shallow trophoblast invasion, delayed presence of uterine natural killer cells, and altered lymphatic angiogenesis. Overall, this study showed that circulating maternal hsFLT1 is sufficient to induce typical maternal preeclampsia-like symptoms in mice and impair the SpA-remodeling independent from the fetoplacental compartment, revealing new insights into the interaction between the placental and maternal contribution of preeclampsia.

SummaryPre-eclampsia (PE) is a syndrome that affects multiple organ systems and is the most severe hypertensive disorder in pregnancy. It frequently leads to preterm delivery, maternal and fetal morbidity and mortality and life-long complications1. We currently lack efficient screening tools2, 3and early therapies4, 5to address PE. To investigate the early stages of early onset PE, and identify candidate markers and pathways, we performed spatio-temporal multi-omics profiling of human PE placentae and healthy controls and validated targets in early gestation in a longitudinal clinical cohort. We used a single-nuclei RNA-seq approach combined with spatial proteo- and transcriptomics and mechanisticin vitrosignalling analyses to bridge the gap from late pregnancy disease to early pregnancy pathomechanisms. We discovered a key disruption in villous trophoblast differentiation, which is driven by the increase of transcriptional coactivator p300, that ultimately ends with a senescence-associated secretory phenotype (SASP) of trophoblasts. We found a significant increase in the senescence marker activin A in preeclamptic maternal serum in early gestation, before the development of clinical symptoms, indicating a translation of the placental syndrome to the maternal side. Our work describes a new disease progression, starting with a disturbed transition in villous trophoblast differentiation. Our study identifies potential pathophysiology-relevant biomarkers for the early diagnosis of the disease as well as possible targets for interventions, which would be crucial steps toward protecting the mother and child from gestational mortality and morbidity and an increased risk of cardiovascular disease later in life.

Several studies show an association of maternal diabetes during pregnancy with adverse offspring metabolic health. Other studies, however, suggest that this effect might be biased by obesity, which is independently associated with offspring metabolic disease and often coexistent to maternal diabetes. We performed a prospective study in a rat model to test the hypothesis that the burden of a diabetic pregnancy without obesity deteriorates metabolic health in male offspring. We generated maternal type 2 diabetes before conception that persisted during pregnancy by knockdown of the insulin receptor in small hairpin RNA–expressing transgenic rats. Male WT (wild type) offspring were followed up until adulthood and metabolically challenged by high-fat diet. Blood glucose was measured continuously via a telemetry device. Glucose and insulin tolerance tests were performed, and body composition was analyzed. Weight gain and glucose levels during adolescence and adulthood were similar in male offspring of diabetic and control pregnancies. Body weight and fat mass after high-fat diet, as well as glucose and insulin tolerance tests, were unaltered between male adult offspring of both groups. Glycemic control consisting of up to 49 000 individual glucose measures was comparable between both groups. Intrauterine exposure to maternal hyperglycemia and hyperinsulinemia without obesity had no impact on male offspring metabolic health in our model. We conclude that the intrauterine exposure itself does not represent a mechanism for fetal programming of diabetes and obesity in our model. Other maternal metabolic parameters during pregnancy, such as obesity, might impact long-term offspring metabolic health.

Preeclampsia, with the hallmark features of new-onset hypertension and proteinuria after 20 weeks of gestation, is a major cause of fetal and maternal morbidity and mortality. Studies have demonstrated a role for the renin-angiotensin system (RAS) in its pathogenesis; however, small-molecule RAS blockers are contraindicated because of fetal toxicity. We evaluated whether siRNA targeting maternal hepatic angiotensinogen (Agt) could ameliorate symptoms of preeclampsia without adverse placental or fetal effects in 2 rodent models. The first model used a cross of females expressing human Agt with males expressing human renin, resulting in upregulation of the circulating and uteroplacental RAS. The second model induced ischemia/reperfusion injury and subsequent local and systemic inflammation by surgically reducing placental blood flow midgestation (reduced uterine perfusion pressure [RUPP]). These models featured hypertension, proteinuria, and fetal growth restriction, with altered biomarkers. siRNA treatment ameliorated the preeclamptic phenotype in both models, reduced blood pressure, and improved intrauterine growth restriction, with no observed deleterious effects on the fetus. Treatment also improved the angiogenic balance and proteinuria in the transgenic model, and it reduced angiotensin receptor activating antibodies in both. Thus, an RNAi therapeutic targeting Agt ameliorated the clinical sequelae and improved fetal outcomes in 2 rodent models of preeclampsia.

Contains fulltext : 225455.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Preeclampsia is associated with an elevated risk of cardiovascular disease later in life. Women with a history of preeclampsia are at risk of developing hypertension as well as ischemic heart disease. Identification of women at the highest risk is important to initiate preventive strategies. We investigated whether high-sensitivity cardiac troponin I (hs-cTnI) levels are associated with a history of early-onset preeclampsia, and with hypertension in these high-risk women. METHODS: Approximately 9-10 years after pregnancy, hs-cTnI levels were measured for 339 women of the Preeclampsia Risk Evaluation in FEMales cohort, consisting of 177 women with a history of early-onset preeclampsia and 162 women with a previous uncomplicated index pregnancy. Associations were analyzed using several statistical tests and linear regression analysis. RESULTS: The median hs-cTnI levels (IQR) were 2.50 ng/l (2.30) in women with a history of early-onset preeclampsia and 2.35 ng/l (2.50) in women without a history of preeclampsia, P = 0.53. Among women with a history of early-onset preeclampsia, the hs-cTnI levels were higher in women who were hypertensive compared with their normotensive counterparts (medians 2.60 versus 2.30; P = 0.03). In addition, blood pressure levels increased with increasing hs-cTnI levels. CONCLUSION: We did not find a difference in hs-cTnI levels between women with and without a history of early-onset preeclampsia. Nonetheless, hs-cTnI levels were statistically significantly higher in current hypertensive women with a history of preeclampsia compared with their normotensive counterparts. Therefore, hs-cTnI levels might improve risk prediction for women at the highest risk of cardiovascular disease.

The placenta is a temporary organ with a unique structure and function to ensure healthy fetal development. Placental dysfunction is involved in pre-eclampsia (PE), fetal growth restriction, preterm birth, and gestational diabetes mellitus (GDM). A diabetic state affects maternal and fetal health and may lead to functional alterations of placental metabolism, inflammation, hypoxia, and weight, amplifying the fetal stress. The placental molecular adaptations to the diabetic environment and the adaptive spatio–temporal consequences to elevated glucose or insulin are largely unknown (2). We aimed to identify gene expression signatures related to the diabetic placental pathology of placentas from women with diabetes mellitus. Human placenta samples (n = 77) consisting of healthy controls, women with either gestational diabetes mellitus (GDM), type 1 or type 2 diabetes, and women with GDM, type 1 or type 2 diabetes and superimposed PE were collected. Interestingly, gene expression differences quantified by total RNA sequencing were mainly driven by fetal sex rather than clinical diagnosis. Association of the principal components with a full set of clinical patient data identified fetal sex as the single main explanatory variable. Accordingly, placentas complicated by type 1 and type 2 diabetes showed only few differentially expressed genes, while possible effects of GDM and diabetic pregnancy complicated by PE were not identifiable in this cohort. We conclude that fetal sex has a prominent effect on the placental transcriptome, dominating and confounding gene expression signatures resulting from diabetes mellitus in settings of well-controlled diabetic disease. Our results support the notion of placenta as a sexual dimorphic organ. Keywords: diabetes mellitus; human; placenta; pregnancy; RNA sequencing

Preeclampsia is a life-threatening disorder associated with long-term cardiovascular risk ([1][1]). We investigated cardiac alterations in post-preeclamptic women and control subjects using cardiovascular magnetic resonance (CMR) imaging. Diffuse injury of the myocardium was assessed by parametric

Echocardiography is the most commonly applied technique for non-invasive assessment of cardiac function in small animals. Manual tracing of endocardial borders is time consuming and varies with operator experience. Therefore, we aimed to evaluate a novel automated two-dimensional software algorithm (Auto2DE) for small animals and compare it to the standard use of manual 2D-echocardiographic assessment (2DE). We hypothesized that novel Auto2DE will provide rapid and robust data sets, which are in agreement with manually assessed data of animals. 2DE and Auto2DE were carried out using a high-resolution imaging-system for small animals. First, validation cohorts of mouse and rat cine loops were used to compare Auto2DE against 2DE. These data were stratified for image quality by a blinded expert in small animal imaging. Second, we evaluated 2DE and Auto2DE in four mouse models and four rat models with different cardiac pathologies. Automated assessment of LV function by 2DE was faster than conventional 2DE analysis and independent of operator experience levels. The accuracy of Auto2DE-assessed data in healthy mice was dependent on cine loop quality, with excellent agreement between Auto2DE and 2DE in cine loops with adequate quality. Auto2DE allowed for valid detection of impaired cardiac function in animal models with pronounced cardiac phenotypes, but yielded poor performance in diabetic animal models independent of image quality. Auto2DE represents a novel automated analysis tool for rapid assessment of LV function, which is suitable for data acquisition in studies with good and very good echocardiographic image quality, but presents systematic problems in specific pathologies. Electronic supplementary material The online version of this article (10.1186/s12947-019-0156-0) contains supplementary material, which is available to authorized users.

Supplemental Digital Content is available in the text., Background: Arterial hypertension and its organ sequelae show characteristics of T cell–mediated inflammatory diseases. Experimental anti-inflammatory therapies have been shown to ameliorate hypertensive end-organ damage. Recently, the CANTOS study (Canakinumab Antiinflammatory Thrombosis Outcome Study) targeting interleukin-1β demonstrated that anti-inflammatory therapy reduces cardiovascular risk. The gut microbiome plays a pivotal role in immune homeostasis and cardiovascular health. Short-chain fatty acids (SCFAs) are produced from dietary fiber by gut bacteria and affect host immune homeostasis. Here, we investigated effects of the SCFA propionate in 2 different mouse models of hypertensive cardiovascular damage. Methods: To investigate the effect of SCFAs on hypertensive cardiac damage and atherosclerosis, wild-type NMRI or apolipoprotein E knockout–deficient mice received propionate (200 mmol/L) or control in the drinking water. To induce hypertension, wild-type NMRI mice were infused with angiotensin II (1.44 mg·kg–1·d–1 subcutaneous) for 14 days. To accelerate the development of atherosclerosis, apolipoprotein E knockout mice were infused with angiotensin II (0.72 mg·kg–1·d–1 subcutaneous) for 28 days. Cardiac damage and atherosclerosis were assessed using histology, echocardiography, in vivo electrophysiology, immunofluorescence, and flow cytometry. Blood pressure was measured by radiotelemetry. Regulatory T cell depletion using PC61 antibody was used to examine the mode of action of propionate. Results: Propionate significantly attenuated cardiac hypertrophy, fibrosis, vascular dysfunction, and hypertension in both models. Susceptibility to cardiac ventricular arrhythmias was significantly reduced in propionate-treated angiotensin II–infused wild-type NMRI mice. Aortic atherosclerotic lesion area was significantly decreased in propionate-treated apolipoprotein E knockout–deficient mice. Systemic inflammation was mitigated by propionate treatment, quantified as a reduction in splenic effector memory T cell frequencies and splenic T helper 17 cells in both models, and a decrease in local cardiac immune cell infiltration in wild-type NMRI mice. Cardioprotective effects of propionate were abrogated in regulatory T cell–depleted angiotensin II–infused mice, suggesting the effect is regulatory T cell–dependent. Conclusions: Our data emphasize an immune-modulatory role of SCFAs and their importance for cardiovascular health. The data suggest that lifestyle modifications leading to augmented SCFA production could be a beneficial nonpharmacological preventive strategy for patients with hypertensive cardiovascular disease.

Preeclampsia is associated with an increased cardiovascular risk later in life. Anti-GPCR autoantibodies have been shown to contribute to the development of cardiovascular disease. We investigated whether anti-GPCR autoantibodies are elevated in women with a history of early-onset preeclampsia 8-11 years postpartum, and whether they correlate with clinical outcomes. We investigated data from the Preeclampsia Risk EValuation in FEMales cohort, a retrospective matched case-control study. Anti AT1R-, beta1AR-, ETAR-, PAR1- and CXCR3- autoantibodies were determined in 485 samples by using commercially available ELISA. Women with the lowest combined levels of autoantibodies and a history of early preeclampsia had significantly higher SBP, DBP and MAP (all p

Objective: Hypertension before and during early pregnancy has been associated with an increased risk of gestational diabetes mellitus (GDM) in retrospective analyses. We investigated the prospective blood pressure patterns in a population-based cohort of pregnant women, who were stratified according to their metabolic status in early 3rd trimester. Research Design and Methods: We recorded blood pressure longitudinally during pregnancy in 1,230 women from the Odense Child Cohort, Denmark. Fasting glucose and insulin were measured at gestational weeks 28-30. Metabolic status was evaluated according to the World Health Organization (WHO) 2013 threshold for GDM (GDM-WHO: fasting plasma glucose ≥5.1 mmol/l), insulin and homeostatic model assessment of insulin resistance (HOMA-IR). Relationships between metabolic status in 3rd trimester and blood pressure trajectories throughout pregnancy were evaluated with adjusted linear mixed models. Results: GDM-WHO prevalence was high, 40% (498/1,230). Significant associations between GDM-WHO and blood pressure were seen only in overweight women, with 2.48 (1.08; 3.88) mmHg higher systolic and 1.58 (0.57; 2.58) mmHg higher diastolic trajectories in women with GDM-WHO compared with differences of -0.38 (-1.51; 0.75) and 0.03 (-0.83; 0.89) mmHg in lean women with GDM-WHO. The associations were independent of progression to gestational hypertension. Blood pressure trajectories in pregnancy were elevated across insulin and HOMA-IR quartiles. Conclusions: GDM-WHO was associated with moderate elevations of blood pressure troughout pregnancy. The relationships were independent of progression to overt gestational hypertension, but largely modified by overweight status. Disclosure A. Birukov: None. R. Dechend: None. M. S. Andersen: None. D. Glintborg: None. M. B. Schulze: None. T. K. Jensen: None. L. B. Andersen: None. K. Kräker: None. E. Polemiti: None. B. L. Jensen: None. J. S. Joergensen: None. Funding German Ministry of Education and Research; State of Brandenburg (82DZD00302)

Reduced expression of the plasma membrane citrate transporter INDY (acronym I’m Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target., Deletion of mIndy reduces blood pressure and heart rate by attenuating catecholamine biosynthesis and recapitulates beneficial cardiovascular and metabolic responses to caloric restriction.

Aims/hypothesis The impact of diabetic pregnancy has been investigated extensively regarding offspring metabolism; however, little is known about the influence on the heart. We aimed to characterise the effects of a diabetic pregnancy on male adult offspring cardiac health after feeding a high-fat diet in an established transgenic rat model. Methods We applied our rat model for maternal type 2 diabetes characterised by maternal insulin resistance with hyperglycaemia and hyperinsulinaemia. Diabetes was induced preconceptionally via doxycycline-induced knock down of the insulin receptor in transgenic rats. Male wild-type offspring of diabetic and normoglycaemic pregnancies were raised by foster mothers, followed up into adulthood and subgroups were challenged by a high-fat diet. Cardiac phenotype was assessed by innovative speckle tracking echocardiography, circulating factors, immunohistochemistry and gene expression in the heart. Results When feeding normal chow, we did not observe differences in cardiac function, gene expression and plasma brain natriuretic peptide between adult diabetic or normoglycaemic offspring. Interestingly, when being fed a high-fat diet, adult offspring of diabetic pregnancy demonstrated decreased global longitudinal (−14.82 ± 0.59 vs −16.60 ± 0.48%) and circumferential strain (−23.40 ± 0.57 vs −26.74 ± 0.34%), increased relative wall thickness (0.53 ± 0.06 vs 0.37 ± 0.02), altered cardiac gene expression, enlarged cardiomyocytes (106.60 ± 4.14 vs 87.94 ± 1.67 μm), an accumulation of immune cells in the heart (10.27 ± 0.30 vs 6.48 ± 0.48 per fov) and higher plasma brain natriuretic peptide levels (0.50 ± 0.12 vs 0.12 ± 0.03 ng/ml) compared with normoglycaemic offspring on a high-fat diet. Blood pressure, urinary albumin, blood glucose and body weight were unaltered between groups on a high-fat diet. Conclusions/interpretation Diabetic pregnancy in rats induces cardiac dysfunction, left ventricular hypertrophy and altered proinflammatory status in adult offspring only after a high-fat diet. A diabetic pregnancy itself was not sufficient to impair myocardial function and gene expression in male offspring later in life. This suggests that a postnatal high-fat diet is important for the development of cardiac dysfunction in rat offspring after diabetic pregnancy. Our data provide evidence that a diabetic pregnancy is a novel cardiac risk factor that becomes relevant when other challenges, such as a high-fat diet, are present. Graphical abstract

Pregnancy-induced hypertension is a severe pregnancy complication, increasing risk of long-term cardiovascular disease in mothers and offspring. We hypothesized that maternal blood pressure in pregnancy associated with offspring blood pressure; that the associations were sex-specific; and that maternal circulating placental angiogenic markers (PlGF [placental growth factor] and sFlt-1 [soluble fms-like tyrosine kinase-1]) mediated this relationship. We analyzed data from 2434 women and 2217 children from the Odense Child Cohort, a prospective Danish cohort study. Offspring blood pressure trajectory from 4 months to 5 years was highly associated to maternal first, second, and third trimester blood pressure, and mean blood pressure in pregnancy, independent of maternal and offspring covariates. There were offspring sex-specific associations: Girls from mothers in the highest quartile of first and third trimester blood pressure had significantly higher systolic blood pressure at 5 years than the rest of the cohort (mean difference±SEM: 1.81±0.59 and 2.11±0.59 mm Hg, respectively, all P P

Bone protection and metabolism are directly linked to estrogen levels, but exercise is also considered to have bone protective effects. Reduced estrogen levels lead to a variety of disorders, for example, bone loss and reduced movement drive. The objective of this study was to investigate the effects of estrogen on individual voluntary exercise motivation and bone protection. We investigated sham operated, ovariectomized, and ovariectomized with estrogen supplemented Wistar rats (20 weeks old) either with or without access to exercise wheels. We selected an experimental approach where we could monitor the individual exercise of group-housed rats with ad libitum access to a running wheel with the help of a subcutaneous chip. In vivo and ex vivo microcomputed tomography analyses of the tibia were performed at two-week intervals from week 0 to week 6. Furthermore, tibial trabecular structure was evaluated based on histomorphometric analyses. We observed a significant bone protective effect of E2. For exercise performance, a substantially high intra-group variability was observed, especially in the E2 group. We presume that dominant behavior occurs within the group-housed rats resulting in a hierarchical access to the running wheel and a high variability of distance run. Exercise did not prevent ovariectomy-induced bone loss. However, lack of estrogen within the ovariectomized rats led to a drastically reduced activity prevented by estrogen supplementation. Our findings are important for future studies working with group-housed rats and exercise. The reason for the high intra-group variability in exercise needs to be investigated in future studies.

Preeclampsia is associated with increased cardiovascular long-term risk; however, the underlying functional and structural mechanisms are unknown. We investigated maternal cardiac alterations after preeclampsia. Female rats harboring the human angiotensinogen gene [TGR(hAogen)L1623] develop a preeclamptic phenotype with hypertension and albuminuria during pregnancy when mated with male rats bearing the human renin gene [TGR(hRen)L10J] but behave physiologically normal before and after pregnancy. Furthermore, rats were treated with pravastatin. We tested the hypothesis that statins are a potential therapeutic intervention to reduce cardiovascular alterations due to simulated preeclamptic pregnancy. Although hypertension persists for only 8 days in pregnancy, former preeclampsia rats exhibit significant cardiac hypertrophy 28 days after pregnancy observed in both speckle tracking echocardiography and histological staining. In addition, fibrosis and capillary rarefaction was evident. Pravastatin treatment ameliorated the remodeling and improved cardiac output postpartum. Preeclamptic pregnancy induces irreversible structural changes of cardiac hypertrophy and fibrosis, which can be moderated by pravastatin treatment. This pathological cardiac remodeling might be involved in increased cardiovascular risk in later life.

Objectives: Preeclampsia (PE), the leading cause of maternal and neonatal morbidity and mortality, is associated with an increased long-term risk for cardiovascular disease (CVD). We aimed to characterize myocardium and investigate potential functional and structural cardiac alterations in asymptomatic women after PE using cardiovascular magnetic resonance (CMR) imaging. Methods and Results: We performed a post-pregnancy case-control study (clinicaltrials.gov ID: NCT03313063). In total, 22 cases and 23 controls were recruited. Participants were matched by age, BMI and parity. Hemodynamic and morphologic characteristics of the myocardium were assessed using established and innovative CMR technics as parametric mapping, late gadolinium enhancement (LGE) imaging and tissue-tracking. Increased left-atrial end-diastolic volume (LA-EDV normalized to BSA: 37.69±6.83 vs. 32.85±6.28, p=0.02) and stroke volume (LA-SV normalized to BSA: 22.57±5.83 vs 18.96±5.10, p=0.02) with a slight tendency to left-ventricular hypertrophy (LV-SV normalized to BSA: 47.28±7.03 vs. 51.58±6.32, p=0.04, LV remodeling index: 0.59±0.11 vs. 0.53±0.08, p=0.045) were found in the post-PE group. Associations between history of PE and changes in LA dimensions remained even after adjustment for BMI, age, parity, DBP, smoking status and years postpartum (adj. β-coefficient for LA-EDV [95% CI]: 9.14 [1.47; 16.81], adj. β-coefficient for LA-SV [95% CI]: 6.74 [0.77; 12.70]). Global circumferential (GCS) and radial (GRS) stains were significantly reduced in post-PE women (-18.28±2.53 vs. -19.82±2.12 and 30.72±6.99 vs. 35.09±6.73, respectively). We could not detect significant differences in T1 or T2 mapping between the groups or fibrosis in LGE imaging. Conclusion: Our findings confirm PE as a risk factor for CVD later in life. Changes in LV and LA dimensions were found as early as 2 years after PE pregnancy. These changes might point toward a manifest end-organ damage following PE in the future. We propose that post-PE women will notably benefit from early cardiovascular screenings.

The mineralocorticoid aldosterone increases in plasma in healthy pregnancy along with renin and angiotensin II and plays a key role in the physiological plasma volume expansion. In mice, aldosterone contributes to an optimal fetal development by enhancing PlGF (placental growth factor) expression and trophoblast cell proliferation. In preeclampsia, there is coincident suppression of aldosterone and impaired placental development. We hypothesized that aldosterone independently contributes to placental and birth weight in humans, and high dietary sodium and low potassium intakes affect this relationship adversely. We analyzed 24-hour urine collections and plasma samples from gestational week 29 in a subsample of 569 pregnant women from the Odense Child Cohort—a Danish population-based longitudinal cohort study. Plasma and urinary aldosterone were measured by ELISA, sodium and potassium excretions by flame photometer. Predictive values of aldosterone levels and sodium and potassium intakes were assessed by multiple and Cox regression analyses. Primary outcomes were placental weight and birth weight. Secondary outcome was preeclampsia. Urinary aldosterone excretion at gestational week 29 independently contributed to placental and birth weights (adjusted β-coefficients [95% CI], 24.50 [9.66–39.35] and 9.59 [4.57–14.61], respectively). Aldosterone levels were not associated to preeclampsia incidence. Salt intake >6 g/d was associated with development of preeclampsia (hazard ratio [95% CI], 5.68 [1.51–21.36]). At gestational week 29, urinary aldosterone excretion is an independent predictor of placental and birth weights. High salt intake is a risk factor for preeclampsia. In perspective, suppression of aldosterone in pregnancy has adverse trophic effects.

Diabetic pregnancy is correlated with increased risk of metabolic and neurological disorders in the offspring putatively mediated epigenetically. Little is known about epigenetic changes already present in fetuses of diabetic pregnancies. We aimed at characterizing the perinatal environment after preexisting maternal diabetes mellitus and at identifying relevant epigenetic changes in the fetus. We focused on the transcription factor Srebf2 (sterol regulatory element binding transcription factor 2), a master gene in regulation of cholesterol metabolism. We tested whether diabetic pregnancy induces epigenetic changes in the Srebf2 promoter and if they become manifest in altered Srebf2 gene expression. We worked with a transgenic rat model of type 2 diabetes mellitus (Tet29) in which the insulin receptor is knocked down by doxycycline-induced RNA interference. Doxycycline was administered preconceptionally to Tet29 and wild-type control rats. Only Tet29 doxycycline dams were hyperglycemic, hyperinsulinemic, and hyperlipidemic. Gene expression was analyzed with quantitative real-time reverse transcriptase polymerase chain reaction and CpG promoter methylation with pyrosequencing. Immunohistochemistry was performed on fetal brains. Fetuses from diabetic Tet29 dams were hyperglycemic and growth restricted at the end of pregnancy. They further displayed decreased liver and brain weight with concomitant decreased microglial activation in the hippocampus in comparison to fetuses of normoglycemic mothers. Importantly, diabetic pregnancy induced CpG hypermethylation of the Srebf2 promoter in the fetal liver and brain, which was associated with decreased Srebf2 gene expression. In conclusion, diabetic and hyperlipidemic pregnancy induces neurological, metabolic, and epigenetic alterations in the rat fetus. Srebf2 is a potential candidate mediating intrauterine environment-driven epigenetic changes and later diabetic offspring health.

Heart failure with preserved ejection fraction (HFpEF) can arise from cardiac and vascular remodeling processes following long-lasting hypertension. Efficacy of common HF therapeutics is unsatisfactory in HFpEF. Evidence suggests that stimulators of the nitric oxide–sensitive soluble guanylyl cyclase (NOsGC) could be of use here. We aimed to characterize the complex cardiovascular effects of NOsGC stimulation using NO-independent stimulator BAY 41-8543 in a double-transgenic rat (dTGR) model of HFpEF. We show a drastically improved survival rate of treated dTGR. We observed less cardiac fibrosis, macrophage infiltration, and gap junction remodeling in treated dTGR. Microarray analysis revealed that treatment of dTGR corrected the dysregulateion of cardiac genes associated with fibrosis, inflammation, apoptosis, oxidative stress, and ion channel function toward an expression profile similar to healthy controls. Treatment reduced systemic blood pressure levels and improved endothelium-dependent vasorelaxation of resistance vessels. Further comprehensive in vivo phenotyping showed an improved diastolic cardiac function, improved hemodynamics, and less susceptibility to ventricular arrhythmias. Short-term BAY 41-8543 application in isolated untreated transgenic hearts with structural remodeling significantly reduced the occurrence of ventricular arrhythmias, suggesting a direct nongenomic role of NOsGC stimulation on excitation. Thus, NOsGC stimulation was highly effective in improving several HFpEF facets in this animal model, underscoring its potential value for patients.

Introduction: Aldosterone is involved in plasma volume expansion and its levels, along with the levels of other members of the renin-angiotensin-aldosterone system (RAAS), are increased in healthy pregnancy. Aldosterone appears to contribute to an optimal fetal development by enhancing placental growth factor (PlGF) expression and trophoblast cell proliferation. Hypothesis: Aldosterone independently contributes to placental and fetal growth. Methods: The project is based on data from the Odense Child Cohort, a prospective population-based study from Odense, Denmark, currently with 2500 active families. The participants were recruited between 2010 and 2012. To analyze plasma aldosterone levels and urinary aldosterone excretion (UAldoV), we used a subsample of 607 urine samples (24-h collections) from gestational week 28. Plasma aldosterone and UAldoV were determined by a commercially available ELISA. Predictive values of aldosterone were assessed by multiple regression analysis. Primary outcomes were placental weight (PW) and gestational age-adjusted birth weight Z-score (BW sds). Results: UAldoV, but not plasma aldosterone concentration, independently contributed to PW and BW sds (adjusted β coefficients ± SEM: 3.21 ± 1.51, p < 0.05 and 0.04 ± 0.01, p < 0.001, respectively). No significant differences in aldosterone excretion were found between women who later developed preeclampsia and the control group. Discussion: At 28 weeks of gestation, 24-h urinary aldosterone excretion was an independent predictor of placental and birth weights. In perspective, mineralocorticoid supplementation could be considered for pregnancies with high risk for IUGR in order to achieve elevated levels of mineralocorticoids and thus benefit placental and fetal development.

Diabetic pregnancy is correlated with increased risk of metabolic and neurological disorders in the offspring putatively mediated epigenetically. Little is known about epigenetic changes already present in fetuses of diabetic pregnancies. We aimed at characterizing the perinatal environment after preexisting maternal diabetes mellitus and at identifying relevant epigenetic changes in the fetus. We focused on the transcription factor

Preeclampsia is a pregnancy-related disorder characterized by hypertension and proteinuria, affecting about 5-8% of pregnancies. It is a major cause of fetal and maternal morbidity/mortality. The disease is most probably multifactorial and caused by several placental dysfunctions. As an important mediator, sFLT1 is induced in the placenta and in the serum of preeclamptic women, acting as a decoy receptor for placenta like growth factors (PLGF) and inducing an antiangiogenic balance. RNAi mediated gene specific silencing could represent a new therapeutic option in preeclampsia management. We tested sFLT1-specific small interfering RNA (siRNA) formulated with the liposomal siRNA delivery system AtuPLEX for the ability to ameliorate symptoms without deteriorating fetal health in an established rat model of preeclampsia. Transgenic rats expressing human angiotensinogen (hAGT) and renin (hREN) were crossed to produce a model of preeclampsia (PE rat) in the dams. Beginning on day 7 of gestation, transgenic hAGT dams were dosed intravenously with 2.8 mg/kg siRNA every third day through gestational day 19. Mean blood pressure was continuously recorded by radiotelemetry and 24 hour urine samples were collected in metabolic cages at day 17/18 of gestation. Rats were euthanized at day 21 of gestation. Biodistribution experiments showed that sFLT1 siRNA formulated with AtuPLEX delivers siRNA to the placenta but not to the embryo. In PE rats, our treatment was able to decrease sFLT1 mRNA expression in placenta, especially in the labyrinth layer. Circulating sFLT1 was also reduced by FLT1 siRNA treatment but did not show statistical significance. Silencing of sFLT1 did not prevent blood pressure increase in the last third of pregnancy in our preeclamptic rat model (159 ± 5 mmHg vehicle vs. 152 ± 2 mmHg sFLT1 siRNA). Proteinuria was not ameliorated (5712 ± 2038 μg/d vehicle vs. 3585 ± 1301 μg/d sFLT1 siRNA). In addition, sFLT1 siRNA had no influence on IUGR, measured by brain to liver weight ratio. AtuPLEX formulated siRNA specifically silenced maternal and placental sFLT1. The mode of delivery appears to be safe for fetal health. However, this sFLT1 knock down showed no protective effect on the preeclamptic phenotype in this Ang-II-based model.

8-Prenylnaringenin (8-PN) and naringenin (Nar) are phytoestrogens found in food items and nutritional supplements, while 6-(1.1-dimethylallyl)naringenin (6-DMAN) is a component of an African plant. Besides their assumed beneficial effects they may promote mammary and endometrial cancer. We therefore assessed their proliferative and estrogenic potential on the mammary gland in vitro and in vivo. In competitive estrogen receptor (ER) ligand binding assays 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα and had the strongest mitotic effect on MCF-7 cells among the test substances. In a three day exposure in young adult ovariectomized female rats 15 mg/kg 8-PN had the highest capacity to increase the number of terminal end buds (TEB) in the mammary gland and stimulated expression of proliferation markers in epithelial ductal cells, followed by 6-DMAN and Nar, but overall their capacity to stimulate proliferation was weak in comparison to 17β-Estradiol (E2).

Introduction Preeclampsia complicates about 5% of all pregnancies worldwide and is the major cause of maternal, fetal and neonatal morbidity and mortality in the western world. Long-term morbidity has also been observed in former preeclamptic mothers, with a substantial increased risk for coronary heart disease. Recent studies demonstrate that women after preterm PE show echocardiographic signs of AHA classification stage B (asymptomatic heart failure) 2 years postpartum although they were normotensive. Objectives The main objective is to detect where and what kind of changes during preeclamptic pregnancy occur. Materials and methods We established a transgenic rat model for preeclampsia, featuring hypertension, proteinuria and intrauterine growth restriction (IUGR) where a female rat harbouring the human angiotensinogen gene is mated with a male transgenic for the human renin. For evaluation we used echocardiography, immunostaining and mRNA expression levels. Results Echocardiographic data show a decreasing ejection fraction and decreasing stroke volume in c ombination with a reduced left ventricular diameter during diastole stage at the end of pregnancy in the preeclamptic rat. The pathology is visible already at day 18, five days after blood pressure rises. In addition, speckle tracking analysis allows description of myocardial deformation normalized to its original shape (strain) and the speed at which this occurs (strain rate). In all dimensions (longitudinal, circumferential and radial) in long as well as in short axis significant reduced global function of strain and strain rate was observed in the heart of preeclamptic rats compared to controls. In the posterior part of the left ventricle we could detect a regional decrease of strain rate which was not found in septal part. Furthermore, an increased wall thickness and increased perivascular fibrosis was measured in this impaired part of the heart (posterior wall). Conclusion These results demonstrate alterations based on a pathological pregnancy leading to a change in structure and function of the left ventricle heart. Future studies will show how long these changes will remain postpartum and how big the impact on later cardiovascular health is.

Introduction Placental macrophages regulate villous trophoblast differentiation and activity. Disturbance of this well-balanced immune-regulation and a pro-inflammatory cytokine milieu can lead to dysfunctional placentas and preeclampsia. Cluster of differentiation 74 (CD74) downregulation in placental macrophages leads to altered macrophage-trophoblast interaction, a pro-inflammatory status and is involved in preeclampsia. Oxylipins, metabolites derived from polyunsaturated fatty acids (PUFAs), are implicated in the development of preeclampsia. Hypothesis Disturbed pro-inflammatory cytokine milieu activates a dysregulated PUFA metabolism enzyme expression pattern leading to disturbed oxylipin levels that can be detected in circulation of preeclamptic mothers. Methods A trophoblast-derived cell line (SGHPL-4) and first trimester villous explants were stimulated by the pro-inflammatory cytokines TNFa, CCL5 and MCP-1. Furthermore, we analyzed placental expression pattern of cytochrome P450 epoxygenases, cyclooxygenases and lipoxygenases by microarray and qRT-PCR from human cohorts and analyzed serum from preeclamptic and uneventful pregnancies by mass spectrometry. Results Microarray studies of preeclamptic placentas revealed a distinguished expression pattern of PUFA metabolism enzymes. Results could be confirmed on two different cohorts (qRT-PCR). Stimulation of SGHPL-4 and villous explants by the pro-inflammatory cytokines TNFa, CCL5 and MCP-1 resulted in similar expression pattern. Oxylipins were dysregulated in the circulation of preeclamptic women compared to uneventful pregnancies atgestational week 28 and week 9–12. Composition of oxylipins at week 9–12 in women that later develop preeclampsia showed a good ROC predictive performance with an AUC = 0.8. Conclusion The pro-inflammatory cytokine milieu induced by the disturbed macrophage-trophoblast interaction induces a distinguished PUFA metabolism leading to dysregulated oxylipins that can predict preeclampsia before onset of clinical symptoms.

Introduction Preeclampsia is a common and devastating pregnancy disorder, featuring hypertension and proteinuria. Studies have demonstrated that dysregulation of Renin-Angiotensin-System (RAS) is involved in the pathogenesis of the disease; however, treatment with a RAS-blocker is contraindicated due to fetal toxicity. Objective RNA interference (RNAi) is a potent means of gene-specific silencing. We sought to demonstrate maternal-specific RAS blockade by targeting maternal hepatic angiotensinogen (AGT) using small interfering RNA (siRNA). In this study we tested the ability of AGT-targeting siRNA to ameliorate symptoms of preeclampsia in two rat models, without inducing a placental pathology or affecting fetal health. Methods Two animal models of preeclampsia were used. The first model (transgenic) acts by upregulation of the circulating and uteroplacental Renin-Angiotensin-System (RAS). The second model is a surgical model that induces ischemia/reperfusion injury and subsequent local and systemic inflammation restriction (RUPP). Beginning on day 3 of gestation, transgenic rats were dosed subcutaneously with 10 mg/kg siRNA every third day through gestation day 15. In RUPP rats, siRNA was subcutaneously injected once (10 mg/kg) on day 12 of gestation. Results The major finding is that RNAi therapeutics targeting maternal hepatic AGT ameliorated the preeclamptic phenotype in both models. We were able to selectively reduce maternal RAS signaling while preserving the fetal RAS. In the transgenic model, silencing of hAGT leads to a reduction of blood pressure and urinary albumin excretion. Moreover, we improved intrauterine growth retardation, indicating an improved fetal development. The RUPP model confirmed the principal findings in a model that is not explicitly driven by the RAS. Discussion Investigational RNAi therapeutics targeting AGT ameliorated the clinical sequelae of preeclampsia in a transgenic rat model and improved the outcome of the fetus. We conclude that maternal specific RAS blockade improves maternal symptoms without deteriorating fetal health in rodent models.

Introduction In preeclampsia, symptoms such as high blood pressure and albuminuria are caused by a state of anti-angiogenic and immune imbalance resulting in severe endothelial dysfunction. The evaluation of smaller vessels is a challenge, but clinically of increasing importance in understanding endothelial dysregulation. Objective We want to examine whether the increased risk for postpartum maternal cardiovascular disease after preeclamptic pregnancy is resultant from microvascular changes in connection with the structural remodeling processes. Methods We compared echocardiographic data from a human cohort with data from our transgenic animal model (hAGTxhRen) after preeclamptic pregnancy. In addition, we investigated cardiac changes in gene (qPCR) and protein expression levels (ELISA, IHC staining) in maternal rats, as well as alterations in microvascular 3D remodeling using Light Sheet Microscopy and Micro CT. Results We were able to show that the echocardiographic changes in our transgenic rat model are comparable to human data. In our rodent model we found maternal structural remodeling involving ventricular hypertrophy, myocardial fibrosis and inflammation at the end of pregnancy. The microvasculature and entire vascular network has been visualized so far only in cleared mouse brains and lymph nodes and partially in adult mouse hearts. Here, we present the 3D network of lectin-labeled blood vessels in cleared adult rat hearts with the analysis of cardiac small vessels with regard to branching points, vessel length and up to a diameter of 6 μm. Discussion Preeclampsia leads to a structural remodeling in maternal hearts as a presumed cause for weakened functionality. Studies are underway to quantify coronary microvascular pathology as a possible missing link between preeclampsia and higher risk for further cardiovascular disease.

Introduction Preeclampsia is a pregnancy-related disorder characterized by hypertension and excess protein excretion in the urine. It is the most common hypertensive disease of pregnancy and affects about 5–8% of pregnancies. The Renin-Angiotensin-System (RAS) is involved in the pathogenesis of the disease; however, treatment with a RAS-blocker is contraindicated due to fetal toxicity. Objective The rat angiotensinogen renin cross model has been described as a rodent model that recapitulates many of the changes observed in human preeclampsia. This experimental rat model of preeclampsia will be used to test the hypothesis that peptide antagonists targeting the RAS system impact both maternal and fetal outcomes. Methods Sprague-Dawley (SD) rats harboring the human angiotensinogen (hAGT) gene [SD-Tg(hAGT) L1623] were mated with male SD rats bearing the human renin (hREN) gene [SD-Tg(hRen) L10J] to produce a model of preeclampsia (PE) in the dams. These pregnant dams developed hypertension on day 13 of pregnancy (plug-recognition was assigned as day 1) and albuminuria. At day 15 of gestation when the animals were already hypertensive, a RAS peptide antagonist was administered via mini-osmotic pumps sc. Results The major finding of the study was that a peptide antagonist targeting RAS ameliorated the preeclamptic phenotype. In the transgenic model, treatment led to a reduction of blood pressure and urinary albumin excretion. The improvement of intrauterine growth retardation (IUGR) indicates an improved fetal development. Discussion RAS peptide antagonist ameliorates the preeclamptic phenotype of the rat model due to the reduction of blood pressure and albuminuria. Furthermore an improvement of the fetal outcome was observed by an amelioration of the IUGR.

Introduction Preeclampsia (PE) is associated with an increased cardiovascular risk in later life, especially in women after early-onset PE. We and others have shown that activating antibodies against angiotensin II receptor type 1 (AT1-) are present in women with PE, even after pregnancy. Further, regulatory (auto-) antibodies against G-coupled receptors (GPCR) have been shown to contribute to cardiovascular disease. Hypothesis We tested the hypothesis that regulatory autoantibodies’ titers are elevated in women with a history of early-onset PE and correlate to physiological parameters and clinical outcomes. Methods We investigated data from PREVFEM (Preeclampsia Risk EValuation in FEMales) retrospective matched case-control study, which was performed in Zwolle, The Netherlands, from 2008 until 2010. All women registered in the early PE database as well as an equal number of age-matched females without PE from the regular obstetric database in the same time period (1991–2007) were invited to participate in the PREVFEM study. Autoantibodies against AT1-, β1-adrenergic receptors, endothelin I receptor A (ETAR), protease-activated receptor 1 (PAR1) and C-X-C3 chemokine binding receptors (CXC3R) were determined in 609 samples (306 cases and 303 controls) by using commercially available ELISA (Celltrend, Luckenwalde, Germany). Results The titer levels of AT1-, β1-, ETAR-, PAR1- and CXC3R-autoantibodies were not significantly different between control and former PE groups 10 years after index pregnancy. Former PE women showed a significantly higher prevalence of hypertension (43% vs. 17%, p Discussion Regulatory autoantibodies against GPCR do not identify women at higher risk for cardiovascular disease 10 years after early-onset PE. The need to search for biomarkers identifying women at risk before cardiovascular symptoms is continuing.

Evaluation of stimulation of proliferation in female reproductive organs by phytoestrogens of reproductive tract organs is important to estimate their safety/risk profile if used as an alternative to hormone therapy. To assess the proliferative capacity of the flavanones 8-Prenylnaringenin (8-PN), 6-(1.1-dimethylallyl)naringenin (6-DMAN) and Naringenin (Nar) on the mammary gland we compared their estrogenic properties to 17β-Estradiol (E2) in three independent assays. We performed competitive estrogen receptor (ER) ligand binding assays, MCF-7 proliferation studies and a three day exposure experiment in young adult ovariectomized female rats. 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα (19.46 %) compared to ERβ (6.5 %). Among the tested flavanones 8-PN had the strongest mitotic effect on MCF-7 cells. In vivo 8-PN increased the number of terminal end buds (TEB) in the mammary gland and the protein expression of the proliferation markers Ki-67 and PCNA in epithelial ductal cells and further led to an increase of Amphiregulin (Areg) mRNA expression, endpoints correlated to mammary gland proliferation. 6-DMAN also possesses a higher relative binding affinity for the ERα than for the ERβ (1.46 %, 0.84 %) and stimulated the cell proliferation of MCF-7 cells. It enhanced Ki-67 protein expression and β-Casein mRNA expression in vivo. Nar displayed the weakest estrogenic properties and was the only tested compound with a higher affinity for the ERβ than the ERα (0.5 %, 0.08 %). It displayed proliferative effects on MCF-7 cells and slightly induced Ki-67 protein expression in vivo. Citation Format: Janina Helle, Oliver Zierau, Kristin Kräker, Annekathrin Keiler, Günter Vollmer, JoEllen Welsh, Georg Kretzschmar. Proliferative effects of the phytoestrogens 8-Prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and Naringenin in MCF-7 cells and the rat mammary gland. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2014-625