Preexisting hypertension and pregnancy-induced hypertension reveal molecular differences in placental proteome in rodents.

Preexisting or new onset of hypertension affects pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. In certain cases, it also leads to long-term maternal cardiovascular complications. The placenta is a key player in the pathogenesis of complicated hypertensive pregnancies, however the pathomechanisms leading to an abnormal placenta are poorly understood. In this study, we compared the placental proteome of two pregnant hypertensive models with their corresponding normotensive controls: a preexisting hypertension pregnancy model (stroke-prone spontaneously hypertensive rats; SHRSP) versus Wistar-Kyoto and the transgenic RAS activated gestational hypertension model (transgenic for human angiotensinogen Sprague-Dawley rats; SD-PE) versus Sprague-Dawley rats, respectively. Label-free proteomics using nano LC-MS/MS was performed for identification and quantification of proteins. Between the two models, we found widespread differences in the expression of placental proteins including those related to hypertension, inflammation, and trophoblast invasion, whereas pathways such as regulation of serine endopeptidase activity, tissue injury response, coagulation, and complement activation were enriched in both models. We present for the first time the placental proteome of SHRSP and SD-PE and provide insight into the molecular make-up of models of hypertensive pregnancy. Our study informs future research into specific preeclampsia and chronic hypertension pregnancy mechanisms and translation of rodent data to the clinic.

HLA gene expression is altered in whole blood and placenta from women who later developed preeclampsia.

Preeclampsia is a multisystem disease that significantly contributes to maternal and fetal morbidity and mortality. In this study, we used a non-biased microarray approach to identify dysregulated genes in maternal whole blood samples which may be associated with the development of preeclampsia. Whole blood samples were obtained at 28 wk of gestation from 5 women who later developed preeclampsia (cases) and 10 matched women with normotensive pregnancies (controls). Placenta samples were obtained from an independent cohort of 19 women with preeclampsia matched with 19 women with normotensive pregnancies. We studied gene expression profiles using Illumina microarray in blood and validated changes in gene expression in whole blood and placenta tissue by qPCR. We found a transcriptional profile differentiating cases from controls; 336 genes were significantly dysregulated in blood from women who developed preeclampsia. Functional annotation of microarray results indicated that most of the genes found to be dysregulated were involved in inflammatory pathways. While general trends were preserved, only HLA-A was validated in whole blood samples from cases using qPCR (2.30- ± 0.9-fold change) whereas in placental tissue HLA-DRB1 expression was found to be significantly increased in samples from women with preeclampsia (5.88- ± 2.24-fold change). We have identified that HLA-A is upregulated in the circulation of women who went on to develop preeclampsia. In placenta of women with preeclampsia we identified that HLA-DRB1 is upregulated. Our data provide further evidence for involvement of the HLA gene family in the pathogenesis of preeclampsia.

Resistin Mediates Sex-Dependent Effects of Perivascular Adipose Tissue on Vascular Function in the Shrsp.

Premenopausal women are relatively protected from developing hypertension compared to men. Perivascular adipose tissue (PVAT) has been shown to mediate vasoactive effects; however, a sex-dependent difference in PVAT function in the setting of hypertension has not yet been explored. We investigated the effect of PVAT on resistance vessel biology in male and female 16 week old stroke prone spontaneously hypertensive rats (SHRSP). This preclinical model of hypertension exhibits a sex-dependent difference in the development of hypertension similar to humans. Wire myography was used to assess vascular function in third-order mesenteric arteries. KATP channel-mediated vasorelaxation by cromakalim was significantly impaired in vessels from SHRSP males + PVAT relative to females (maximum relaxation: male + PVAT 46.9 ± 3.9% vs. female + PVAT 97.3 ± 2.7%). A cross-over study assessing the function of male PVAT on female vessels confirmed the reduced vasorelaxation response to cromakalim associated with male PVAT (maximum relaxation: female + PVATfemale 90.6 ± 1.4% vs. female + PVATmale 65.8 ± 3.5%). In order to explore the sex-dependent differences in PVAT at a molecular level, an adipokine array and subsequent western blot validation identified resistin expression to be increased approximately 2-fold in PVAT from male SHRSP vessels. Further wire myography experiments showed that pre-incubation with resistin (40 ng/ml) significantly impaired the ability of female + PVAT vessels to relax in response to cromakalim (maximum relaxation: female + PVAT 97.3 ± 0.9% vs. female + PVAT + resistin[40ng/ml] 36.8 ± 2.3%). These findings indicate a novel role for resistin in mediating sex-dependent vascular function in hypertension through a KATP channel-mediated mechanism.

Hypertension: Focus on autoimmunity and oxidative stress.

Understanding the causal role of the immune and inflammatory responses in hypertension has led to questions regarding the links between hypertension and autoimmunity. Immune pathology in primary hypertension mimics several autoimmune mechanisms observed in the pathogenesis of systemic lupus erythematosus, psoriasis, systemic sclerosis, rheumatoid arthritis and periodontitis. More importantly, the prevalence of hypertension in patients with these autoimmune diseases is significantly increased, when compared to control populations. Clinical and epidemiological evidence is reviewed along with possible mechanisms linking hypertension and autoimmunity. Inflammation and oxidative stress are linked in a self-perpetuating cycle that significantly contributes to the vascular dysfunction and renal damage associated with hypertension. T cell, B cell, macrophage and NK cell infiltration into these organs is essential for this pathology. Effector cytokines such as IFN-γ, TNF-α and IL-17 affect Na+/H+ exchangers in the kidney. In blood vessels, they lead to endothelial dysfunction and loss of nitric oxide bioavailability and cause vasoconstriction. Both renal and vascular effects are, in part, mediated through induction of reactive oxygen species-producing enzymes such as superoxide anion generating NADPH oxidases and dysfunction of anti-oxidant systems. These mechanisms have recently become important therapeutic targets of novel therapies focused on scavenging oxidative (isolevuglandin) modification of neo-antigenic peptides. Effects of classical immune targeted therapies focused on immunosuppression and anti-cytokine treatments are also reviewed.

Natural killer cells in placentation and cancer: Implications for hypertension during pregnancy.

Hypertension during pregnancy is the most common medical condition encountered during gestation. Despite this, knowledge of the mechanisms that underlie the disease and the development of new therapies are limited. Hypertension during pregnancy and some forms of cancer confer an increased risk to the development of cardiovascular disease later in life; one mechanism which may link these conditions is the involvement of natural killer (NK) cells. Whilst immunology and immunotherapy are well-developed areas in oncology; the complex mechanisms of the immune system in health and disease at the maternal-fetal interface are less well-defined. Natural killer (NK) cells have emerged as key immune cells involved in physiology and pathology of pregnancy. These small lymphocytes are present in the decidua (the uterine-specific uNK cells) and are distinct from peripheral NK cells. The uNK cell population plays a vital role in mediating trophoblast invasion and affecting decidual vascular remodelling whereas the role of the peripheral NK cell population during pregnancy is less well-defined. This review will give an overview of NK cell biology followed by a discussion of the current evidence for the role of uterine and peripheral NK cells at the maternal-fetal interface in health and disease. Furthermore, examples of NK cell research from cancer biology will be employed to inform future directions of research. By combining this knowledge from oncology where the field of immunotherapy has now matured into clinical trials; it is hopeful that new mechanisms can be elucidated to generate targets for similar therapeutic strategies for women with hypertensive pregnancies where interventions are needed.

Polymerization-Incompetent Uromodulin in the Pregnant Stroke-Prone Spontaneously Hypertensive Rat.

The kidney is centrally involved in blood pressure regulation and undergoes extensive changes during pregnancy. Hypertension during pregnancy may result in an altered urinary peptidome that could be used to indicate new targets of therapeutic or diagnostic interest. The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of maternal chronic hypertension. Capillary electrophoresis-mass spectrometry was conducted to interrogate the urinary peptidome in SHRSP and the control Wistar-Kyoto strain at three time points: prepregnancy and gestational days 12 and 18. The comparison within and between the Wistar-Kyoto and SHRSP peptidome at all time points detected 123 differentially expressed peptides (fold change >1.5; P<0.05). Sequencing of these peptides identified fragments of collagen α-chains, albumin, prothrombin, actin, serpin A3K, proepidermal growth factor, and uromodulin. Uromodulin peptides showed a pregnancy-specific alteration in SHRSP with a 7.8-fold (P<0.01) and 8.8-fold (P<0.05) increase at gestational days 12 and 18, respectively, relative to the Wistar-Kyoto. Further investigation revealed that these peptides belonged to the polymerization-inhibitory region of uromodulin. Two forms of uromodulin (polymerization competent and polymerization incompetent) were found in urine from both Wistar-Kyoto and SHRSP, where the polymerization-incompetent form was increased in a pregnancy-specific manner in SHRSP. Nifedipine-treated pregnant SHRSP showed only polymerization-competent uromodulin, indicating that calcium may be mechanistically involved in uromodulin polymerization. This study highlights, for the first time, a potential role of uromodulin and its polymerization in hypertensive pregnancy.

Systematic Review of Micro-RNA Expression in Pre-Eclampsia Identifies a Number of Common Pathways Associated with the Disease.

BACKGROUND: Pre-eclampsia (PE) is a complex, multi-systemic condition of pregnancy which greatly impacts maternal and perinatal morbidity and mortality. MicroRNAs (miRs) are differentially expressed in PE and may be important in helping to understand the condition and its pathogenesis. METHODS: Case-control studies investigating expression of miRs in PE were collected through a systematic literature search. Data was extracted and compared from 58 studies to identify the most promising miRs associated with PE pathogenesis and identify areas of methodology which could account for often conflicting results. RESULTS: Some of the most frequently differentially expressed miRs in PE include miR-210, miR-223 and miR-126/126* which associate strongly with the etiological domains of hypoxia, immunology and angiogenesis. Members of the miR-515 family belonging to the imprinted chromosome 19 miR cluster with putative roles in trophoblast invasion were also found to be differentially expressed. Certain miRs appear to associate with more severe forms of PE such as miR-210 and the immune-related miR-181a and miR-15 families. Patterns of miR expression may help pinpoint key pathways (e.g. IL-6/miR-223/STAT3) and aid in untangling the heterogeneous nature of PE. The detectable presence of many PE-associated miRs in antenatal circulatory samples suggests their usefulness as predictive biomarkers. Further progress in ascertaining the clinical value of miRs and in understanding how they might contribute to pathogenesis is predicated upon resolving current methodological challenges in studies. These include differences in diagnostic criteria, cohort characteristics, sampling technique, RNA isolation and platform-dependent variation in miR profiling. CONCLUSION: Reviewing studies of PE-associated miRs has revealed their potential as informants of underlying target genes and pathways relating to PE pathogenesis. However, the incongruity in results across current studies hampers their capacity to be useful biomarkers of the condition.

Abnormal uterine artery remodelling in the stroke prone spontaneously hypertensive rat.

INTRODUCTION: The stroke prone spontaneously hypertensive rat (SHRSP) is an established model of human cardiovascular risk. We sought to characterise the uteroplacental vascular response to pregnancy in this model and determine whether this is affected by the pre-existing maternal hypertension. METHODS: Doppler ultrasound and myography were utilised to assess uterine artery functional and structural changes pre-pregnancy and at gestational day 18 in SHRSP (untreated and nifedipine treated) and in the normotensive Wistar-Kyoto (WKY) rat. Maternal adaptations to pregnancy were also assessed along with histology and expression of genes involved in oxidative stress in the placenta. RESULTS: SHRSP uterine arteries had a pulsatile blood flow and were significantly smaller (70906 ± 3903 µm(2) vs. 95656 ± 8524 µm(2) cross-sectional area; p < 0.01), had a significant increase in contractile response (57.3 ± 10.5 kPa vs 27.7 ± 1.9 kPa; p < 0.01) and exhibited impaired endothelium-dependent vasorelaxation (58.0 ± 5.9% vs 13.9 ± 4.6%; p < 0.01) compared to WKY. Despite significant blood pressure lowering, nifedipine did not improve uterine artery remodelling, function or blood flow in SHRSP. Maternal plasma sFLT-1/PlGF ratio (5.3 ± 0.3 vs 4.6 ± 0.1; p < 0.01) and the urinary albumin/creatinine ratio (1.9 ± 0.2 vs 0.6 ± 0.1; p < 0.01) was increased in SHRSP vs WKY. The SHRSP placenta had a significant reduction in glycogen cell content and an increase in Hif1α, Sod1 and Vegf. DISCUSSION: We conclude that the SHRSP exhibits a number of promising characteristics as a model of spontaneous deficient uteroplacental remodelling that adversely affect pregnancy outcome, independent of pre-existing hypertension.

Role of Tumor Necrosis Factor-α and Natural Killer Cells in Uterine Artery Function and Pregnancy Outcome in the Stroke-Prone Spontaneously Hypertensive Rat.

Women with chronic hypertension are at increased risk of maternal and fetal morbidity and mortality. We have previously characterized the stroke-prone spontaneously hypertensive rat (SHRSP) as a model of deficient uterine artery function and adverse pregnancy outcome compared with the control Wistar-Kyoto. The activation of the immune system plays a role in hypertension and adverse pregnancy outcome. Therefore, we investigated the role of tumor necrosis factor-α in the SHRSP phenotype in an intervention study using etanercept (0.8 mg/kg SC) at gestational days 0, 6, 12, and 18 in pregnant SHRSP compared with vehicle-treated controls (n=6). Etanercept treatment significantly lowered systolic blood pressure after gestational day 12 and increased litter size in SHRSP. At gestational day 18, etanercept improved the function of uterine arteries from pregnant SHRSP normalizing the contractile response and increasing endothelium-dependent relaxation, resulting in increased pregnancy-dependent diastolic blood flow in the uterine arteries. We identified that the source of excess tumor necrosis factor-α in the SHRSP was a pregnancy-dependent increase in peripheral and placental CD3- CD161+ natural killer cells. Etanercept treatment also had effects on placental CD161+ cells by reducing the expression of CD161 receptor, which was associated with a decrease in cytotoxic granzyme B expression. Etanercept treatment improves maternal blood pressure, pregnancy outcome, and uterine artery function in SHRSP by antagonizing signaling from excess tumor necrosis factor-α production and the reduction of granzyme B expression in CD161+ natural killer cells in SHRSP.

Differential expression of microRNA-206 and its target genes in preeclampsia.

OBJECTIVES: Preeclampsia is a multisystem disease that significantly contributes to maternal and foetal morbidity and mortality. In this study, we used a nonbiased microarray approach to identify novel circulating miRNAs in maternal plasma that may be associated with preeclampsia. METHODS: Plasma samples were obtained at 16 and 28 weeks of gestation from 18 women who later developed preeclampsia (cases) and 18 matched women with normotensive pregnancies (controls). We studied miRNA expression profiles in plasma and subsequently confirmed miRNA and target gene expression in placenta samples. Placental samples were obtained from an independent cohort of 19 women with preeclampsia matched with 19 women with normotensive pregnancies. RESULTS: From the microarray, we identified one miRNA that was significantly differentially expressed between cases and controls at 16 weeks of gestation and six miRNAs that were significantly differentially expressed at 28 weeks. Following qPCR validation, only one miR-206 was found to be significantly increased in 28-week samples in women who later developed preeclampsia (1.4-fold change ±â€Š0.2). The trend for increase in miR-206 expression was mirrored within placental tissue from women with preeclampsia. In parallel, IGF-1, a target gene of miR-206, was also found to be downregulated (0.41 ±â€Š0.04) in placental tissue from women with preeclampsia. miR-206 expression was also detectable in myometrium tissue and trophoblast cell lines. CONCLUSION: Our pilot study has identified miRNA-206 as a novel factor upregulated in preeclampsia within the maternal circulation and in placental tissue.

Hypertension due to antiangiogenic cancer therapy with vascular endothelial growth factor inhibitors: understanding and managing a new syndrome.

Novel antiangiogenic cancer therapies, particularly agents that block vascular endothelial growth factor (VEGF) signalling, have improved outcomes in patients with cancers and are now used as first-line therapies for some tumours. However, with VEGF inhibitors (VEGFIs) are new complications, particularly hypertension. VEGFI-induced hypertension is a dose-dependent phenomenon due to on-target effects rather than off-target effects. Increased blood pressure occurs in almost 100% of patients who take VEGFIs, with a subset who develop severe hypertension. Molecular mechanisms underlying VEGFI-induced hypertension are unclear, but endothelial dysfunction and increased vascular resistance, due to impaired nitric oxide signalling, reduced prostacyclin production, endothelin-1 (ET-1) upregulation, oxidative stress, and rarefaction have been implicated. Treatment of hypertension should be aimed at reducing the risk of short-term morbidity associated with hypertension while maintaining effective dosing of antiangiogenic therapy for optimal cancer treatment. Although specific guidelines are not yet available for the management of VEGFI-induced hypertension, angiotensin-converting enzyme inhibitors and dihydropyridine calcium channel blockers are commonly used. Severe hypertension might require reduction of VEGFI dosing, or in some cases, interruption of treatment. As more potent VEGFIs are developed and as more cancer patients are treated with VEGFIs, the burden of hypertension toxicity will increase. This will be further compounded as the use of antiangiogenic drugs broadens to include older patients and those with pre-existing cardiovascular disease. Here we focus on VEGF as a target for antiangiogenesis and how this affects increased blood pressure. Putative mechanisms underlying VEGFI-induced hypertension are highlighted and therapeutic strategies to manage such hypertension are discussed.