A prospective observational cohort study of covid-19 epidemiology and vaccine seroconversion in South Western Sydney, Australia, during the 2021-2022 pandemic period.

BACKGROUND: It is known that COVID-19 disproportionally adversely affects the immunocompromised, including kidney transplant recipients (KTR), as compared to the general population. Risk factors for adverse outcomes and vaccine seroconversion patterns are not fully understood. Australia was uniquely positioned to reduce initial case numbers during the 2021-2022 pandemic period due to its relative isolation and several significant public health interventions. South-Western Sydney Local Heath District was one of the predominant regions affected. METHODS: A single centre, prospective cohort study of prevalent renal transplant recipients was conducted between 25th July 2021 and 1st May 2022. Baseline characteristics, COVID-19 vaccination status, COVID-19 diagnosis and outcomes were determined from the electronic medical record, Australian vaccination register and Australian and New Zealand Dialysis and Transplant Registry. Assessment of vaccine-induced seroconversion was assessed with ELISA in a subpopulation. Analysis was performed using SPSS v.28. RESULTS: We identified 444 prevalent transplant recipients (60% male, 50% diabetic, median age 58 years (Interquartile range (IQR)21.0) and eGFR 56 ml/min/1.73m2 (IQR 21.9). COVID-19 was identified in 32% (n = 142) of patients, of which 38% (n = 54) required hospitalisation and 7% (n = 10) died. At least one COVID-19 vaccination was received by 95% (n = 423) with 17 (4%) patients remaining unvaccinated throughout the study period. Seroconversion after 2 and 3 doses of vaccine was 22% and 48% respectively. Increased COVID-19 related deaths were associated with older age (aOR 1.1, 95% CI 1.004-1.192, p = 0.040), smoking exposure (aOR 8.2, 05% CI 1.020-65.649, p = 0.048) and respiratory disease (aOR 14.2, 95%CI:1.825-110.930, p = 0.011) on multi-variable regression analysis. Receipt of three doses of vaccination was protective against acquiring COVID-19 (aOR 0.48, 95% CI 0.287-0.796, p = 0.005) and death (aOR 0.6, 95% CI: 0.007-0.523, p = 0.011), but not against hospitalisation (p = 0.32). Seroconversion was protective for acquiring COVID-19 on multi-variable regression independent of vaccination dose (aOR 0.1, 95%CI: 0.0025-0.523, p = 0.011). CONCLUSIONS: COVID-19 was associated with a high mortality rate. Older age, respiratory disease and prior smoking exposure may be risk factors for increased mortality. Vaccination of 3 doses is protective against acquiring COVID-19 and death, however not hospitalisation. Antibody response is protective for acquiring COVID-19, however seroconversion rates are low.

Prevalence of long term metabolic, cardiovascular, cerebrovascular and renal disease in patients with hypertensive disorders in pregnancy remote from pregnancy (POMCH).

BACKGROUND: Hypertensive disorders of pregnancy (HDP) are associated with an increased risk of long-term cardiovascular, cerebrovascular, and adverse renal outcomes. Biomarkers including soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor (PlGF) are predictive of the development of preeclampsia. Their long-term value in predicting which women will develop cardiovascular complications remote from pregnancy is not yet established. OBJECTIVES: To determine the prevalence and incidence of Cardio-renal-metabolic outcomes at 10 years follow-up in a cohort of women screened for suspected preeclampsia from 2008 to 2009 and assess the relationship between pregnancy biomarkers and long-term outcomes. STUDY DESIGN: A retrospective cohort study of 117 women. Outcomes were assessed by auditing medical records. The primary outcome was the prevalence of cardiovascular, cerebrovascular, metabolic and renal outcomes at 10 years remote from the diagnosis of HDP. The secondary outcome was to assess the relationship of the remote from pregnancy outcomes to biomarkers (sFlt1, PlGF, soluble endoglin (sEng) and neutrophil gelatinase associated lipocalin (NGAL)) taken at the time of pregnancy comparing the results of those with adverse outcomes compared to those without. RESULTS: There was a 12.7% prevalence of cardiovascular and cerebrovascular disease, 44.4% prevalence of hypertension, 20.6% prevalence of chronic kidney disease and 17.5% prevalence of diabetes. Women who developed preeclampsia had an increased prevalence and incidence of adverse outcomes compared to women without preeclampsia. There was a weak relationship between sENg measured at the time of clinical suspicion of preeclampsia and the adverse outcomes 144 (139-146) months remote from pregnancy. CONCLUSIONS: Biomarkers taken at the time of pregnancy did not accurately predict the long-term adverse cardiometabolic outcomes.

S-glutathionylation of the Na+-K+ Pump: A Novel Redox Mechanism in Preeclampsia.

CONTEXT: Reduced Na+-K+ pump activity is widely reported in preeclampsia and may be caused by a reversible oxidative modification that is a novel pathological feature of preeclampsia. OBJECTIVE: This work aims to determine whether ß 1 subunit (GSS-ß 1) protein glutathionylation of the Na+-K + pump occurs in preeclampsia. METHODS: The GSS-ß1 of the Na+-K+ pump and its subunit expression in human placentas were compared between women with healthy pregnancies and women with preeclampsia. Human placental samples of pregnant women with preeclampsia (n = 11, mean gestational age 36.5 weeks) were used to examine the GSS-ß 1 of the Na+-K+ pump, compared to healthy pregnancies (n = 11, mean gestational age 39 weeks).The potential pathogenetic role of GSS-ß 1-mediated Na+-K+ pump dysfunction in preeclampsia was investigated. RESULTS: Protein expression of the ß 1 subunit was unchanged in placentas from women with preeclampsia vs those with normotensive pregnancies. Preeclamptic placentas had a significantly increased GSS-ß 1 of the Na+-K+ pump compared to those from healthy pregnancies, and this was linked to a decrease in α 1/ß 1 subunit coimmunoprecipitation. The cytosolic p47phox nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase subunit and its coimmunoprecipitation with the α 1 Na+-K+ pump subunit was increased in preeclamptic placentas, thus implicating NADPH oxidase-dependent pump inhibition. CONCLUSIONS: The high level of ß 1 pump subunit glutathionylation provides new insights into the mechanism of Na+-K+ pump dysfunction in preeclampsia.

The 15-Epilipoxin-A4 Pathway with Prophylactic Aspirin in Preventing Preeclampsia: A Longitudinal Cohort Study.

INTRODUCTION: The benefit of aspirin in preventing preeclampsia is increasingly recognized; however, its mechanism of action remains unclear. Nonobstetric studies have described an anti-inflammatory effect of aspirin through the 15-epilipoxin-A4 pathway (aspirin-triggered lipoxin [ATL]). However, the anti-inflammatory mechanism of aspirin in the prevention of preeclampsia remains unknown. OBJECTIVE/HYPOTHESIS: To examine (1) the difference in longitudinal endogenous lipoxin-A4 (En-Lipoxin-A4) concentration in low-risk (LR) and high-risk (HR) pregnancies, and (2) the effect of aspirin on endogenous ATL concentration and the associated effect on cytokine profile of HR women. METHODS: Plasma from 220 HR women was collected at 12, 16, 20, 24, 28, 32, and 36 weeks of gestation. Adherence to aspirin was biochemically verified. Plasma En-Lipoxin-A4 and ATL concentrations were analyzed using liquid chromatography mass spectrometry, and cytokines, interleukin (IL)-10, tumor necrosis factor-α, interferon-γ, IL-8, and IL-1ß, with the high-sensitivity multibead Luminex® assay. RESULTS: HR women have up to 70% lower plasma concentration of En-Lipoxin-A4 (P < 0.001) than LR women. HR women with adequate aspirin adherence (HR-AA) (n = 82) had higher plasma concentration of ATL (P < .001), lower concentration of IL-8 from 16 to 36 weeks of gestation (P < .001), and increased IL-10 concentration from 16 to 28 weeks of gestation (P = .03) compared with high-risk women who were not on aspirin (HR-NA). HR-AA who did not develop preeclampsia had higher plasma En-lipoxin-A4 (P < .001), ATL (P = .02), and IL-10 concentrations (P < .001) with lower IL-8 concentration (P = .004) than HR women who developed preeclampsia. DISCUSSION: Plasma concentration of En-Lipoxin-A4 is lower in HR women than in LR controls. Adequate adherence with aspirin results in an increase in ATL and IL-10 with reduced IL-8 plasma concentration. This study suggests a potential anti-inflammatory role of aspirin through the ATL pathway with prophylactic aspirin in HR pregnant women.

Calcium deficient placental growth restriction is mediated by an increase in non-invasive integrin α5 and ß4 phenotype.

OBJECTIVES: Integrins are cell adhesion receptors that participated in endovascular invasion by cytotrophoblasts in preeclampsia. This study aimed to investigate the effect of calcium on cellular pathways influencing the trophoblast integration into endothelial cellular networks in vitro. STUDY DESIGN: Red fluorescent-labelled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. Green fluorescent-labelled HTR-8/SVneo trophoblast cells were then co-cultured with endothelial cells in different concentrations of calcium for 24 h. MAIN OUTCOME MEASURES: The calcium effects on HTR-8/SVneo cell integration were quantified by Image J. Quantitative PCR was performed to measure mRNA expression of integrins α1, α5, α6, ß1 and ß4. The concentrations of interleukin IL-6, matrix metalloproteinase-2 (MMP-2), MMP-9, PlGF and sFlt-1 in the conditioned medium were measured by ELISA while levels of cytokines IL-1ß, IL-8, IL-10, TNF-α and INF-γ were assessed by magnetic Luminex assays™. RESULTS: Both calcium depletion (0.4 mM) and low calcium (1.8 mM) groups demonstrated inhibited integration of trophoblast cells into endothelial cellular networks, compared with the normal calcium group (2.4 mM). The IL-6 production was reduced from conditioned media in both calcium depletion and low calcium groups. In calcium depletion group, mRNA expression of integrin α5 and ß4 in trophoblasts was increased while integrin α1 was decreased. CONCLUSIONS: The in vitro trophoblast cell integration into endothelial cellular networks could be modified by altering media calcium through integrin switch away from integrins α5 and ß4 and towards integrin α1 which may be required for healthy early trophoblast integration.

Effect of placental growth factor in models of experimental pre-eclampsia and trophoblast invasion.

Placental growth factor (PlGF) is decreased in early gestation of pregnant women who subsequently develop pre-eclampsia. In this study, pre-emptive treatment with PlGF to prevent pre-eclampsia was evaluated in an in vivo rodent model of experimental pre-eclampsia (EPE) induced by TNF-α and in an in vitro model of human first-trimester trophoblast invasion. Pregnant C57/BL6 mice were treated with recombinant mouse placental growth factor-2 (rmPlGF-2) 100 µg/kg/day IP from gestational day (gd) 10. Animals had EPE induced by continuous TNF-α infusion on gd 13 and were subject to either continuous blood pressure monitoring by radiotelemetry throughout pregnancy or live placenta T2 -weighted magnetic resonance imaging (MRI) to demonstrate placental function on gd 17. There was no difference in BP (P > .99), proteinuria (P = .9) or T2 values on MRI (P = .9) between control and rmPlGF-2-treated animals. On gd 13, animals treated with rmPlGF-2 demonstrated increased placenta PlGF (P = .01) and Toll-like receptor-3 (P = .03) mRNA expression as compared with controls. Fluorescent-labelled human uterine microvascular endothelial cells and HTR8/SVNeo cells were co-cultured on Matrigel™ and treated with recombinant human PlGF (rhPlGF) (10 ng/mL) and/or TNF-α (0.5 ng/mL). Trophoblast integration into endothelial networks was reduced by added TNF-α (P = .006), as was rhPlGF concentration in conditioned media (P < .0001). Cell integration was not ameliorated by addition of rhPlGF (P > .9). Although TNF-α-induced EPE was not reversed with pre-emptive rmPlGF-2, a further trial of pre-emptive rhPlGF in vivo is required to determine whether the absence of effect of rhPlGF demonstrated in vitro precludes PlGF as a preventative therapy for pre-eclampsia.

Adolescent Perinatal Outcomes in South West Sydney, Australia.

OBJECTIVE: To compare perinatal outcomes, blood pressures throughout pregnancy, rates of hypertensive disorders of pregnancy, preeclampsia, gestational diabetes mellitus, and immediate obstetric outcomes in adolescents younger than 20 years at delivery and those in the 20- to 34-year age group. PATIENTS AND METHODS: Questionnaires were administered to pregnant women at Campbelltown and Liverpool hospitals within South West Sydney, Australia, as part of a broader study of sleep-disordered breathing in pregnancy between February 1, 2009, and February 28, 2013. Data collected included demographic data, blood pressure readings, pregnancy complications, delivery type, and neonatal outcomes. Adolescents were compared with older women using Student t tests and χ2 statistics. RESULTS: A total of 103 adolescents were compared with 2291 women aged 20 to 34 years. Adolescents were more likely to be primiparous, had longer average gestations, and had lower pre-pregnancy body mass index. Adolescents had lower rates of cesarean section delivery and gestational diabetes mellitus. There was no significant difference in smoking rates, perinatal mortality rate, small for gestational age, intrauterine growth restriction, Apgar score of less than 7 at 5 minutes, admission to special care nursery, or hypertensive disorder of pregnancy rates. Adolescents had lower booking systolic and diastolic blood pressures, and their highest antenatal systolic blood pressures were lower. CONCLUSION: Adolescents have birth outcomes to similar to those of their older counterparts. Adolescents had lower booking blood pressures. This may have implications for the screening and diagnosis of hypertensive disorders of pregnancy in adolescents.

The effect of acetyl salicylic acid (Aspirin) on trophoblast-endothelial interaction in vitro.

Early administration of low dose acetyl salicylic acid (Aspirin) in high risk women reduces the risk of early onset preeclampsia. This study aims to investigate the effect of aspirin on trophoblast integration and the its effect on angiogenic and invasive pathways in an in-vitro model of preeclampsia. Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on matrigel to form endothelial networks. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells were co-cultured with the endothelial networks with/without TNF-a (0.5ng/mL) and/or aspirin (0.1mM) for 24h. Fluorescent images were captured and quantified by Image J to examine the effects of TNF-a and aspirin on the trophoblast-endothelial integration. Conditioned media were collected to measure free VEGF, PlGF and sFlt-1 by ELISA and PGF1a by Enzyme immunoassay (EIA). Cells were retrieved to examine mRNA expression of angiogenic factors (VEGF, PlGF and sFlt-1), invasion markers (MMP-2 and TIMP-1), endothelial cell activation markers (E-selectin and VCAM), eNOS and cyclooxygenase (COX)-2 by quantitative PCR. Aspirin reversed the inhibitory effect of TNF-a on trophoblast cell integration into endothelial cellular networks. TNF-a increased PGF1a production (128±11%, p<0.05), whilst aspirin reversed the TNF-a effect on PGF1a production (19±4%, p<0.01). TNF-a decreased the mRNA expression of PlGF, eNOS, MMP-2 and TIMP-1, and stimulated COX2, E-selectin and VCAM mRNA expression. Aspirin did not reverse the TNF-a effect on these molecules. Aspirin improves trophoblast cell integration into endothelial cellular networks by inhibiting the effect of TNF-a via PGI2 with no significant effect on antiangiogenic, invasive or endothelial activation markers.

The Utility of Sonographic Assessment in Selecting Patients for Percutaneous Insertion of Peritoneal Dialysis Catheter.

BACKGROUND: Percutaneous insertion of peritoneal dialysis (PD) catheters by nephrologists is a safe and effective alternative to open surgical techniques. These patients are usually carefully selected due to anatomical considerations and medical comorbidities, with the current literature suggesting exclusion of patients with prior abdominal surgery. METHOD: We conducted a retrospective cohort study of pre-dialysis patients who attended a preprocedural clinic in a tertiary center over 6 years. Procedural complications and catheter survival were assessed. Chi-squared test and Kaplan-Meier survival analysis were undertaken. Inpatient assessments were excluded. RESULTS: A total of 217 patients were assessed, of whom 171 (78.8%) were accepted for percutaneous PD catheter insertion by a nephrologist. The key exclusion criteria were: (1) the clinical presence of abdominal hernia (p < 0.001), (2) ultrasound findings of skin to peritoneum depth of > 5.5 cm (p < 0.001) and (3) ultrasound findings of impaired visceral slide test (p < 0.001). Prior abdominal surgery was not a default exclusion criterion (p = 0.1), as 63 patients (37%) with prior abdominal surgery, average of 1.3 prior surgeries per patient, were assessed as appropriate for the percutaneous procedure. There was no difference in the procedural complication rate and catheter survival between patients with and without prior abdominal surgery. CONCLUSION: A comprehensive preprocedural assessment utilizing ultrasound permits an objective selection of patients for percutaneous insertion of PD catheters by nephrologists. This allowed for successful and safe percutaneous insertion of PD catheters in patients who may have otherwise been excluded, e.g., prior abdominal surgery, patients with large bilateral polycystic kidneys, and central obesity.

Antihypertensive methyldopa, labetalol, hydralazine, and clonidine reversed tumour necrosis factor-α inhibited endothelial nitric oxide synthase expression in endothelial-trophoblast cellular networks.

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor-α (TNF-α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose TNF-α (0.5 ng/mL) or TNF-α plus soluble fms-like tyrosine kinase-1 (sFlt-1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR-8/SVneo cells were co-cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF-α on eNOS mRNA expression. After pre-incubating endothelial cells with TNF-α and sFlt-1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF-α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF-α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF-α. The anti-angiogenic molecule sFlt-1 may antagonise the potential benefit of these medications by interfering with the NOS pathway.

The protective effect of apolipoprotein in models of trophoblast invasion and preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy. It is associated with abnormal placentation via poor placental invasion of the uterine vasculature by trophoblast cells, leading to poor placental perfusion, oxidative stress, and inflammation, all of which are implicated in its pathogenesis. A dyslipidemia characterized by low plasma levels of high-density lipoproteins (HDL) and elevated triglycerides has been described in preeclampsia. Apolipoprotein A-I (apoA-I), a constituent of HDL is an anti-inflammatory agent. This study investigated whether apoA-I protects against hypertension and adverse placental changes in a proinflammatory cytokine (TNF-α)-induced model of preeclampsia. Further, this study investigated whether apoA-I protects against the inhibitory effect of TNF-α in a human in vitro model of trophoblast invasion. Administration of apoA-I to pregnant mice before infusion with TNF-α resulted in a significant reduction in the cytokine-induced increase in systolic blood pressure. MRI measurement of T2 relaxation, a parameter that is tissue specific and sensitive to physiological changes within tissues, showed a reversal of TNF-α-induced placental changes. Preincubation of endothelial cells with apoA-I protected against the TNF-α-induced inhibition of HTR-8/SVneo (trophoblast) cell integration into endothelial (UtMVEC) networks. These data suggest that a healthy lipid profile may affect pregnancy outcomes by priming endothelial cells in preparation for trophoblast invasion.

A longitudinal analysis of angiotensin II type 1 receptor antibody and angiogenic markers in pregnancy.

BACKGROUND: Preeclampsia can be caused by shallow trophoblast invasion and results in endothelial dysfunction. Angiotensin II type 1 receptor antibodies may have a role in both processes. Other angiogenic markers (placental growth factor, soluble fms-like tyrosine kinase-1, and soluble endoglin) have been shown to alter before clinically evident preeclampsia. OBJECTIVE: The aim of this study is to assess the longitudinal changes and utility of biomarker angiotensin II type 1 receptor antibodies and angiogenic markers in hypertensive disorders of pregnancy, gestational hypertension, and preeclampsia. STUDY DESIGN: A longitudinal prospective cohort observational study of angiogenic markers and a secondary retrospective case-control study of angiotensin II type 1 receptor antibody changes were conducted. The studies were conducted in a large tertiary metropolitan teaching hospital (Sydney, Australia). Sequential recruitment of women with a singleton pregnancy (N = 351) was undertaken. Plasma concentrations of angiotensin II type 1 receptor antibodies, placental growth factor, soluble fms-like tyrosine kinase-1, and soluble endoglin were measured using validated enzyme-linked immunosorbent assays at 12, 18, 28, 36, and 40 weeks' gestation and 6 weeks' postpartum. Clinical, demographic, and pregnancy data were prospectively collected. Pregnancy outcomes were classified as normotensive, gestational hypertension, or preeclampsia. Analyses were carried out using software and significance set at P < .05. RESULTS: In all, 351 women were recruited, 17 developed gestational hypertension, and 18 developed preeclampsia. Women with preeclampsia at baseline were heavier (P = .015), were taller (P = .046), and had higher systolic (P = .029) and diastolic (P = .006) blood pressure. The preeclampsia group had higher soluble fms-like tyrosine kinase-1 from ≥28 weeks (P = .003) and lower placental growth factor from 18 weeks (P = .004). Soluble endoglin and angiotensin II type 1 receptor antibodies did not vary over time or between groups. Angiotensin II type 1 receptor antibody (12 weeks) was positively correlated with serum pregnancy associated plasma protein A (P = .008) and human chorionic gonadotrophin (P = .04). CONCLUSION: Angiogenic markers vary longitudinally during pregnancy and placental growth factor and soluble fms-like tyrosine kinase-1 have a role for predicting and diagnosing preeclampsia later in disease. Our data show that angiotensin II type 1 receptor antibodies are not sensitive for disease and hence not useful as a biomarker. Larger studies are required to describe the role and functionality of angiotensin II type 1 receptor antibodies in preeclampsia.

The incidence of IgG4-positive plasma cells staining TIN in patients with biopsy-proven tubulointerstitial nephritis.

AIM: IgG4 disease is rare. However, IgG4 tubulointerstitial nephritis (TIN) is the most common renal manifestation. IgG4 disease is usually associated with elevated serum IgG4 levels and other organ involvement, low-density renal lesions on enhanced CT imaging and immune activation. The incidence of IgG4-TIN may be underestimated, as staining for IgG4 is not routine. This study sought to describe the prevalence of previously undiagnosed IgG4-TIN. Due to the complexity of the diagnosis, we only attempt to look at IgG4-positive plasma cell TIN as a potential indication for IgG4 renal disease. METHODS: A retrospective review of native renal biopsies performed between 2002 and 2012 with a primary diagnosis of TIN was selected. Samples for which interstitial nephritis was secondary to a glomerular disease were excluded. The tissues were stained for IgG4 and scored by two blinded observers. Demographic and follow-up details were collected. This study was approved by the local ethics committee. RESULTS: 82 cases of interstitial nephritis from a total of 1238 renal biopsies (2002-2012) were available after staining for further assessment. 12 samples demonstrated staining consistent with the criteria for IgG4-positive plasma cell TIN, of which 3 had mildly positive staining, 7 moderately positive staining and 2 had markedly positive staining. There were no statistically significant differences in the baseline characteristics between the positive and negative staining groups. CONCLUSIONS: A number of cases of IgG4-positive plasma cell TIN were observed histologically that had been previously diagnosed as non-specific chronic TIN. IgG4-positive plasma cell TIN made up 1% of all renal biopsies performed over 10 years and 13% of all biopsies demonstrating TIN not related to glomerular disease. IgG4 staining should be considered routinely in biopsies demonstrating primary TIN.

Placental Growth Factor Reduces Blood Pressure in a Uteroplacental Ischemia Model of Preeclampsia in Nonhuman Primates.

An imbalance in the angiogenesis axis during pregnancy manifests as clinical preeclampsia because of endothelial dysfunction. Circulating soluble fms-like tyrosine kinase 1 (sFLT-1) increases and placental growth factor (PlGF) reduces before and during disease. We investigated the clinical and biochemical effects of replenishing the reduced circulating PlGF with recombinant human PlGF (rhPlGF) and thus restoring the angiogenic balance. Hypertensive proteinuria was induced in a nonhuman primate (Papio hamadryas) by uterine artery ligation at 136 days gestation (of a 182-day pregnancy). Two weeks after uteroplacental ischemia, rhPlGF (rhPlGF, n=3) or normal saline (control, n=4) was administered by subcutaneous injection (100 µg/kg per day) for 5 days. Blood pressure was monitored by intra-arterial radiotelemetry and sFLT-1 and PlGF by ELISA. Uteroplacental ischemia resulted in experimental preeclampsia evidenced by increased blood pressure, proteinuria, and endotheliosis on renal biopsy and elevated sFLT-1. PlGF significantly reduced after uteroplacental ischemia. rhPlGF reduced systolic blood pressure in the treated group (-5.2±0.8 mm Hg; from 132.6±6.6 mm Hg to 124.1±7.6 mm Hg) compared with an increase in systolic blood pressure in controls (6.5±3 mm Hg; from 131.3±1.5 mm Hg to 138.6±1.5 mm Hg). Proteinuria reduced in the treated group (-72.7±55.7 mg/mmol) but increased in the control group. Circulating levels of total sFLT-1 were not affected by the administration of PlGF; however, a reduction in placental sFLT-1 mRNA expression was demonstrated. There was no significant difference between the weights or lengths of the neonates in the rhPlGF or control group; however, this study was not designed to assess fetal safety or outcomes. Increasing circulating PlGF by the administration of rhPlGF improves clinical parameters in a primate animal model of experimental preeclampsia.

DNA methylation profiles in preeclampsia and healthy control placentas.

Preeclampsia is a hypertensive disorder of pregnancy that affects 3-5% of all pregnancies. There is evidence to suggest that epigenetic mechanisms, such as DNA methylation, play a role in placental development and function. This study compared DNA methylation profiles of placentas from preeclampsia-affected pregnancies with placentas from healthy pregnancies to identify gene-specific changes in DNA methylation that may contribute to the development of preeclampsia. The methylation status of eight placental biopsies taken from preeclampsia-affected and 16 healthy pregnancies was analyzed using the Illumina Infinium Methylation 450 BeadChip array. Bisulfite pyrosequencing was used to confirm regions found to be differentially methylated between preeclampsia and healthy placentas. A total of 303 differentially methylated regions, 214 hypermethylated and 89 hypomethylated, between preeclampsia cases and controls were identified, after adjusting for gestational age (adjusted P < 0.05). Functional annotation found cell adhesion, wingless type MMTV Integration Site family member 2 (Wnt) signaling pathway, and regulation of transcription were significantly enriched in these gene regions. Hypermethylation of WNT2, sperm equatorial segment protein (SPESP1), NADPH oxidase 5 (NOX5), and activated leukocyte cell adhesion molecule (ALCAM) in preeclampsia placentas was confirmed with pyrosequencing. This study found differences in methylation in gene regions involved in cell signaling (WNT2), fertilization and implantation (SPESP1), reactive oxygen species signaling (NOX5), and cell adhesion (ALCAM). These results build on recently published studies that have reported significant differences in DNA methylation in preeclampsia placentas.

Placental Regulation of Inflammation and Hypoxia after TNF-α Infusion in Mice.

PROBLEM: Increased levels of inflammatory cytokines are demonstrated in the serum of women with pre-eclampsia. TNF-α infusion in animal models induces proteinuric hypertension similar to human pre-eclampsia. The effect of TNF-α on regulation of the immune and hypoxic pathways in the developing placenta and their relationship with experimental pre-eclampsia remains unexamined. METHOD OF STUDY: TNF-α was infused into pregnant mice, and the effects on maternal hypertension, proteinuria, circulating levels of sFlt-1 and corresponding placental changes in molecules responding to inflammation (TLR-3 and TLR-4) and hypoxia (HIF-1α) were examined. RESULTS: TNF-α infusion resulted in maternal hypertension and proteinuria. Molecular changes in the placenta involved upregulation of TLR-3, TLR-4 and HIF-1α. Serum levels of sFlt-1 were high in pregnant animals, but not further upregulated by TNF-α infusion. CONCLUSION: A role for maladaptive regulation of TLR and HIF-1α induced by an imbalance in inflammatory cytokines is implicated in the pathogenesis of pre-eclampsia.

Nicotinic acetylcholine receptors (nAChR) are increased in the pre-eclamptic placenta.

OBJECTIVE: The role of the nicotinic acetylcholine receptors (nAChR) in pre-eclampsia is unknown. Given that ACh levels are affected in pre-eclampsia, it has been suggested that compensatory changes in nAChR expression may ensue. This study aimed to determine the effects of pre-eclampsia on the mRNA and protein expression of 12 mammalian nAChR subunits. METHODS: Placentas were collected from healthy term pregnancies (n = 8) and pregnancies complicated by pre-eclampsia (n = 7), both being non-cigarette smoke exposed to rule out any role of nicotine. Using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), 12 subunits (α2, α3, α4, α5, α6, α7, α9, ß1, ß2, ß4, δ, and γ) were able to be studied at the mRNA level, while at the protein level using Western blotting, nine subunits (α2, α3, α4, α5, α7, α9, ß1, ß2, and γ) were studied. RESULTS: At the mRNA level, pre-eclamptic placentas showed an increase in α2 (p = 0.003), α9 (p < 0.001), ß1 (p = 0.03) and ß2 (p = 0.02) subunit expression, while at the protein level, α7 (p = 0.004), α9 (p = 0.02), and δ (p = 0.003) subunits were increased compared to controls. CONCLUSION: Certain nAChR subunits are increased in the pre-eclamptic placenta. Given the absence of cigarette smoking, the changes in expression are hypothesised to be due to the hypoxic environment resulting from the pathophysiology of pre-eclampsia, which subsequently affects endogenous ACh levels, yielding compensatory increases in α2, α7, α9, ß1, ß2, and δ nAChR subunits.

Variability in mRNA expression of fms-like tyrosine kinase-1 variants in normal and preeclamptic placenta.

BACKGROUND: Preeclampsia is a complication of pregnancy characterised by gestational hypertension and proteinuria and is a leading cause of morbidity and mortality in both mothers and infants. Certain anti-angiogenic factors have long been implicated in the pathogenesis of preeclampsia and the placental expression of factors such as soluble fms-like tyrosine kinase-1 (sFLT-1) are often reported in studies of normal and diseased placentae. Despite evidence showing significant differences in placental gene expression by collection site, many studies fail to provide sufficient details on sample selection and collection. FINDINGS: With ourselves and others investigating and reporting on the expression of FLT-1 variants and other genes in the placenta of normotensive and preeclamptic patients, we felt it prudent to examine the variation in expression of FLT-1 variants across human placenta. We examined the differential expression of FLT-1 variants in samples obtained from 12 sites on normal and preeclamptic placentae and found expression to be highly variable between sites. We therefore developed an algorithim to calculate the mean expression for any number of these sites collected and in any combination. The coefficient of variation for all combinations of sites was then used to determine the minimum number of sites required to reduce coefficient of variation to below an acceptable 10%. We found that 10 and 11 sites had to be sampled in the normal and preeclamptic placentae respectively to ensure a representative expression pattern for all FLT-1 variants for an individual placenta. CONCLUSIONS: These findings demonstrate significant variation in expression levels of several commonly investigated genes across sites in both normal and preeclamptic placenta. This highlights both the importance of adequate sampling of human placenta for expression studies and the effective communication of sample selection and collection methods, for data interpretation and to ensure the reproducibility and reliability of results and conclusions drawn.

Antihypertensive drugs methyldopa, labetalol, hydralazine, and clonidine improve trophoblast interaction with endothelial cellular networks in vitro.

OBJECTIVES: The interaction between trophoblasts and maternal endothelium is important for placental vascular modeling. Failure of uterine spiral artery transformation is linked to the etiopathology of preeclampsia. Antihypertensive medications used to control hypertension in early pregnancy can alter placental and circulating cytokines. This study investigated whether selected antihypertensive drugs can modulate the interaction between trophoblast and endothelial cells. METHODS: Human uterine myometrial microvascular endothelial cells were preincubated with (or without) low-dose tumor necrosis factor-α (TNF-α; 0.5 ng/ml) or TNF-α and soluble fms-like tyrosine kinase 1 (sFlt-1; 100 ng/ml). Red fluorescent-labeled endothelial cells were then cultured on Matrigel. After appearance of endothelial cellular networks, green fluorescent-labeled HTR-8/SVneo trophoblast cells were cocultured in the presence of pharmacological doses of methyldopa, labetalol, hydralazine, and clonidine. Images were captured after 24 h and drug effects on HTR-8/SVneo cell integration were quantified by Image Analysis software. The conditioned medium was collected to measure sFlt-1, vascular endothelial growth factor (VEGF), placental growth factor, interleukin-10, and interleukin-6 by ELISA. RESULTS: Methyldopa, labetalol, hydralazine, and clonidine increased trophoblast integration into TNF-α-preincubated endothelial cellular networks. In conditioned medium, sFlt-1 was reduced by methyldopa, hydralazine, and clonidine alone. VEGF was increased by methyldopa. A decrease in placental growth factor was seen by methyldopa and also in nontreated endothelial cell coculture of the other three drugs. CONCLUSION: Some antihypertensive drugs used in pregnancy may improve the cellular interaction between trophoblast and endothelial cells exposed to TNF-α. Methyldopa, hydralazine, and clonidine reduced sFlt-1 concentration in culture medium, whereas labetalol increased trophoblast integration independently of sFlt-1. Methyldopa increased VEGF concentration. Some pregnancy-related antihypertensives may affect placental vascularization.

Fetal-maternal alignment of regulatory T cells correlates with IL-10 and Bcl-2 upregulation in pregnancy.

Transplacental immune regulation refers to the concept that during pregnancy, significant cross-talk occurs between the maternal and fetal immune system with potential long-term effects for both the mother and child. In this study, we made the surprising observation that there is a strong correlation of peripheral blood regulatory T (Treg) cells between the mother and the fetus. In contrast, there is no significant Treg cell correlation between paternal fetal dyads (pairs), suggesting that the specific context of pregnancy, rather than the genetic parental similarity to the fetus, is responsible for this correlation. Gene microarray analysis of Treg cells identified a typical IL-10-dependent signature in maternal and fetal Treg cells. In addition, a direct correlation of serum IL-10 protein levels between maternal fetal dyads was observed. Furthermore, we show that maternal serum IL-10 levels correlate with serum estradiol and estriol, implicating hormonal involvement in this alignment. Interestingly, we show that Treg cells possess higher expression of IL-10 receptor α and that Treg cell IL-10 receptor α expression directly correlates with their Bcl-2 expression. Indeed, in vitro data in both humans and mice demonstrate that IL-10 upregulates Bcl-2 specifically in Treg cells but not non-Treg cells. Our results provide evidence for transplacental regulation of cellular immunity and suggest that IL-10 may influence Treg cell homeostasis through its effect on Treg cell Bcl-2 expression. These novel findings have important implications on immune tolerance in pregnancy and beyond in areas of autoimmunity, allergy, and transplantation.