The expression of placental soluble fms-like tyrosine kinase 1 in mouse placenta varies significantly across different litters from normal pregnant mice.

OBJECTIVE: Gene expression studies often pool tissues from multiple placentas when using animal models of preeclampsia without accounting for the potential confounders of litter origin or pup sex. We aimed to determine whether placental gene expression differs based on sex or litter. METHODS: We examined the differential expression of soluble fms-like tyrosine kinase 1 (Flt-1) using 35 pups from six normal pregnant mice. RESULTS: Expression of sFlt-1 (p = 0.003) was significantly different between litters but not between sexes (p = 0.17). CONCLUSIONS: These findings highlight the importance of adequate sampling from multiple litters in expression studies when using animal models in clinical research.

OS010. Use of retinal imaging to characterise physiological vascularchanges throughout pregnancy.

INTRODUCTION: Visualisation of the microcirculation through retinal imaging can provide information on the health of systemic vasculature. Characterisation of the retinal vasculature throughout pregnancy using retinal imaging is a novel approach to examine physiological changes to the cardiovascular system, and may be useful to predict early pathophysiological signs of adverse maternal outcomes. OBJECTIVES: To characterise the retinal vascular and blood pressure (BP) changes that occur throughout a healthy pregnancy. METHODS: Data was collected from women recruited at 13±2 weeks of gestation from Royal Prince Alfred Hospital, a major tertiary referral hospital in Sydney, Australia. Retinal images centred on the optic disc and BP readings were collected throughout pregnancy. Postnatal data was collected from medical records, and women with hypertensive disorders of pregnancy and gestational diabetes mellitus were excluded. This left a final group of 19 women. Retinal images from 13±2, 19±2, 29±2 and 38±2 weeks gestation were graded using semi-automated retinal vascular calibre measurement (IVAN) software and the central retinal arteriolar equivalent (CRAE), and central retinal venular equivalent (CRVE). BP data was collected at the same time points as the retinal images. Analysis of data was performed using paired t-tests and repeated measures analysis of variance (ANOVA). Women with missing data points were excluded from the analysis at the relevant time points. RESULTS: Over the course of pregnancy, there was a significant dilatation of retinal arterioles between 13±2 and 19±2weeks (from 166.4 to 172.7µm, SE: 3.7µm, n=19, p=0.01), corresponding to a significant fall in diastolic BP during this time (from 64.6 to 60.2mmHg, SE: 1.5mmHg, p=0.01). No significant changes in venular diameter or systolic BP were noted. Between 19±2 and 29±2weeks (n=4), no significant changes to retinal arteriolar or venular diameter were seen although there were significant increases in both systolic and diastolic BP (SBP: from 100.3 to 109.9mmHg, SE: 1.9mmHg, p=0.01; DBP: from 59.3 to 64.6mmHg, SE: 6.9mmHg, p=0.01). Between 29±2 and 38±2weeks (n=3), no significant changes in retinal arteriolar, and venular diameter or BP were observed. CONCLUSION: An increase in retinal arteriolar diameter between 13±2 and 19±2 weeks gestation was observed, which corresponded to a decrease in both systolic and diastolic BP. However, between 19±2 and 29±2 weeks there was no change in vasculature, even though there was a significant increase in BP. By characterising the changes to retinal vessels that occur throughout a healthy pregnancy, we can further our understanding of the response of the systemic vasculature to pregnancy, which may provide clues to early vascular disease of pregnancies.

OS016. Retinal vascular changes in hypertensive disorders of pregnancy.

INTRODUCTION: Hypertensive disorders of pregnancy (HDP) are characterised by vascular dysfunction. Retinal vascular imaging is a novel, non-invasive way to characterise early microvascular changes in pregnancy, and as a result has the potential to be used to predict the onset of HDP. OBJECTIVES: To characterise retinal vascular changes that occur in HDP, and compare these changes to those in healthy pregnancies. METHODS: Women were recruited at 13±2 weeks of gestation from Royal Prince Alfred Hospital, a major metropolitan tertiary referral hospital in Sydney, Australia. Retinal images centred on the optic disc and blood pressure (BP) readings were collected at 13±2, 19±2, 29±2 and 38±2 weeks gestation. Retinal images were graded using semi-automated retinal vascular calibre measurement software (IVAN) and the central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE) were calculated. Within and between subject repeat measures analysis was performed on images from each trimester, using paired t-tests and repeated measures analysis of variance (ANOVA). Multiple linear regressions were used to model the average arteriole diameter adjusted for age, tobacco consumption and body mass index (BMI). All tests were two-sided using a 5% level of significance. A clinical diagnosis of HDP was obtained from postnatal medical record data. Women with missing data points were excluded from the analysis at that time point. RESULTS: Of the 39 women included in the study, 6 (15%) were diagnosed with HDP. In the HDP cohort, repeated measures ANOVA revealed no significant changes in arteriolar or venular diameter measurements throughout pregnancy. Paired t-tests indicated no significant differences in any of the outcome measures between HDP and healthy pregnancies at 13±2 (n=36) and 19±2 (n=39)weeks. At 29±2weeks (n=39), there was a significantly smaller venular diameter in HDP pregnancies (220.4±6.9µm vs 239.1± 5.4µm in healthy pregnancies, p=0.03). At 38±2weeks (n=39), arteriolar diameter was significantly smaller in HDP pregnancies (148.6±6.0µm vs 164.1±4.6µm in healthy pregnancies, p=0.04). Similar results persisted following adjustments for cardiovascular risk factors (age, tobacco use and BMI). CONCLUSION: Significant differences in the retinal vasculature develop in HDP as compared to healthy pregnancies. These differences appear at29±2weeks gestation and persist throughout the rest of the pregnancy. Retinal vascular imaging is a promising tool for the detection of the early microvascular changes in HDP, prior to diagnosis.

OS055. Sex-dependent differences in expression of FLT-1 variants andJMJD6 in mouse placenta.

INTRODUCTION: There is evidence for fetal sex-dependent differences in the way in which preeclamptic pregnancies proceed, and in maternal and neonatal outcomes. Mouse models are common in the study of preeclampsia and pooled tissue from multiple placentae is often used to obtain samples for expression studies. Potential concerns regarding this practice are the sex-dependent differences in placental expression of candidate factors. One biomolecule of interest is soluble fms-like tyrosine kinase 1 (sFLT-1) which is a known marker of preeclampsia and commonly used to determine the severity of the induced preeclampsia-like syndrome in rodent models. OBJECTIVES: It was the aim of this preliminary study to determine whether variation exists in the expression of different genes in murine placenta based on pup sex in C57BL/6JArc mice. A novel gene, Jumonji domain-containing protein 6 (Jmjd6) that may prove to have a role in the pathogenesis of preeclampsia, mFLT-1 and sFLT-1 were selected as targets. METHODS: Seventeen pups were retrieved from three normal pregnant female mice euthanized via cervical dislocation (CD) on day 17.5 or 18.5. Tails and corresponding placentas were collected from the pups, snap frozen in liquid nitrogen and stored at -80°C. Tails were used to accurately determine pup sex via PCR amplification of sex chromosome-specific sequences and revealed the presence of 3 females and 14 males. Quantitative PCR was used to determine the relative expression of the FLT-1 and Jmjd6 transcripts in each placenta. The placenta collected from the first pup of the first pregnant female served as the reference sample and transcript expression in the remaining samples was expressed relative to this sample. General linear modelling using linear regression with categorical variables was used to evaluate the difference in transcript expression between the sexes and Pearson's correlation coefficient used to examine relationships between variables. RESULTS: Pup sex was found to have a significant effect on the relative expression of sFLT-1 after controlling for litter, pup weight and gestational age (p=0.013), with 1.5 times more expression in the placentas of female pups. The expression of sFLT-1 was highly correlated with mFLT-1 (r=0.690,p=0.002). The relative expression of Jmjd6 was not significantly different in male and female placentas and sFlt-1 was not correlated with Jmjd6. CONCLUSION: This is the first study to demonstrate a link between fetal sex and placental sFLT-1 expression in mice, finding increased levels of this gene in the placentas of female pups. It is possible that in normal pregnancies, female placentas produce more sFLT-1 which acts to condition them and offer some protection during the sFLT-1 spike seen in preeclampsia. The findings of this study also highlight a possible need to consider sex as a variable in placental expression studies using mice to ensure the accuracy of results.

PP001. Variability in MRNA expression of Jmjd6 and FLT-1 variants in normal and preeclamptic human placenta.

INTRODUCTION: Biomolecules such as soluble fms-like tyrosine kinase 1 (sFLT-1) have been implicated in the pathogenesis of preeclampsia with many studies reporting on their expression in human placenta. OBJECTIVES: This study aimed to determine whether variation exists in the expression of different genes in human placenta based on collection site. Expression of different FLT-1 variants including the primate-specific sFLT-1e15a and a novel gene, Jumonji domain-containing protein 6 (Jmjd6) that may prove to have a role in the pathogenesis of preeclampsia, was selected as targets. METHODS: Placental tissue was collected from one normotensive and one preeclamptic woman following caesarean section at 38 weeks. Twelve 1.5cm diameter×2mm thick samples were excised from various sites around the decidual surface. Quantitative PCR was used to determine the relative expression of the FLT-1 and Jmjd6 transcripts in the separate samples. Within a placenta, the first sample collected served as the reference and transcript expression in the remaining 11 samples was expressed relative to this sample. Between placentas, a pooled normal sample was used as a reference to determine the relative expression in preeclamptic compared to normal placental samples. One sample t -tests and coefficients of variation (CV) were used to explore the variation and Pearson's correlation coefficient was used to examine relationships. RESULTS: Within the normal placenta, significant variation was seen in the 12 collection sites for sFLT-1 e15a (CV=45.1% p=0.008) and Jmjd6 (CV=30.4% p=0.019). The CVs for sFLT-1 i13 and mFLT-1 were 25.6% and 23.7% respectively. Within the preeclamptic placenta, significant variation was seen in the expression of all FLT-1 variants; mFLT-1 (CV=66.9% p=0.023), sFLT-1 i13 (CV=64.8% p=0.033) and sFLT-1 e15a (CV=61.1% p=0.001) across different collection sites. Significant variation was also seen between preeclamptic placenta sites and a normotensive pool; mFLT-1 (CV=66.9% p=0.012), sFLT-1 e15a (CV=61.1% p=0.005) and Jmjd6(CV=65.2% p=0.029). Using cumulative moving means, the minimum number of samples required to obtain a zero difference in means for all transcripts in a data subset was 8 for the normal placenta and 6 for the preeclamptic placenta. Overall, the expression of Jmjd6 and all FLT-1 variants was increased in the samples from the preeclamptic placenta compared to normal. Expression of mFLT-1 was highly correlated with sFLT-1 i13 and sFLT-1 e15ain preeclamptic (r=0.808 p=0.001; r=0.841p=0.001) but not normal placenta, and Jmjd6 was not correlated with any transcript in either placenta. CONCLUSION: This study demonstrates significant variation in expression levels of several new and commonly investigated genes across sites in both normal and preeclamptic human placenta. These data show samples should be obtained from no less than 8 separate sites when pooling samples for expression analysis. Further, given that many studies examine relationships between different colocalised molecules, it may also be prudent to examine expression levels in each site separately to ensure that no relationships are missed.

PP033. High blood pressure in pregnancy: an indicator of future health outcomes.

INTRODUCTION: Hypertensive disorders of pregnancy (HDP) remain a leading cause of maternal and perinatal morbidity and mortality worldwide. In Australia approximately 10% of all pregnancies are affected by HDP. There is growing evidence that endothelial damage caused by HDP remains after pregnancy and has long term consequences on maternal health. OBJECTIVES: The aim of our research was to determine the association between HDP and risk of having high blood pressure in later life. METHODS: Self-reported data regarding a physician's diagnosis of HDP and of high blood pressure later in life were obtained from women recruited from the 45 and Up Study, Australia. Relative risks (converted from odds ratios) and 99% confidence intervals were estimated using logistic regression, adjusting for demographic and lifestyle characteristics. RESULTS: A total of 82,164 women were included in the study, of which 9,845 reported having HDP. Women who had HDP had a significantly increased risk of having high blood pressure later in life compared to women who did not have HDP (adjusted relative risk of 2.05, 99% CI 1.99-2.11, p<0.001). The results showed that women who had HDP develop high blood pressure 6.3 years (99% CI 5.85-6.66, p<0.001) earlier compared to women without HDP. CONCLUSION: Women who have HDP are at a greater risk of future onset of high blood pressure compared to women who have a healthy pregnancy. Women with HDP should be monitored closely in the years following pregnancy for early identification and intervention of high blood pressure.

Synthesis, cardiac electrophysiology, and beta-blocking activity of novel arylpiperazines with potential as class II/III antiarrhythmic agents.

A series of novel arylpiperazines have been prepared in an attempt to incorporate both class II (beta-receptor blocking) and class III antiarrhythmic properties in a single molecule. The key step in the preparation of the new compounds involves a regioselective heterocyclic ring formation. All but four compounds significantly prolonged action potential duration in canine cardiac Purkinje fibers (class III activity). All but one of the compounds demonstrated beta-receptor affinity in a competitive binding assay and three had beta 1-receptor selectivity. Compared to sotalol, a reference class II/III agent, arylpiperazine 7a (4-[(methylsulfonyl)amino]-N-[(4- phenylpiperazin-2-yl)methyl]benzamide) demonstrated beta 1-selectivity and was 1 order of magnitude more potent in the in vitro class III and the beta 1-receptor screens. Compound 7a was evaluated further and found to be effective in preventing programmed electrical stimulation-induced arrhythmias in conscious dogs (class III activity) and against epinephrine-induced arrhythmias in halothane anesthetized dogs (class II activity).

Synthesis of novel (aryloxy)propanolamines and related compounds possessing both class II and class III antiarrhythmic activity.

Several (aryloxy)propanolamines and related compounds (i.e. 5-13, 16-18, 20-24, 27-33, 35, 37-39, 41, and 42) were synthesized and investigated for their class III electrophysiological activity and class II (beta-blocking) effects with use of in vitro and in vivo models. Structure-activity relationships are discussed for a series of 30 compounds. A number of these compounds prolonged the action potential duration at 95% repolarization of isolated canine cardiac Purkinje fibers by 20% (C20APD95) at concentrations of less than 1.0 microM, with no significant effects on cardiac conduction. beta-Adrenergic receptor binding studies showed that some of these compounds were 2-20 times more potent for cardiac beta 1 receptors than for beta 2 receptors. In particular, compounds 32, 41, 1, and especially (S)-1 were found to be orally active class III agents in anesthetized mongrel dogs (1 or 3 mg/kg, id) and efficacious at suppressing programmed electrical stimulation induced arrhythmias in halothane-anesthetized dogs. The profile of these compounds was similar to that found for sotalol. Compound (S)-1, which was more potent than sotalol in the PES study and equieffective in the halothane/epinephrine dog model, is being investigated further as a combined class III/II antiarrhythmic agent.

Synthesis and cardiac electrophysiological activity of aryl-substituted derivatives of the class III antiarrhythmic agent sematilide. Potential class I/III agents.

Twelve novel derivatives of the selective class III antiarrhythmic agent sematilide were prepared in an attempt to incorporate both class I and class III electrophysiological properties into a single molecule. Electrophysiological activity was determined by standard microelectrode techniques in canine cardiac Purkinje fibers. Initial assessment of class I efficacy was carried out in a ouabain-induced arrhythmia model in guinea pigs. All of the compounds prolonged action potential duration in Purkinje fibers (class III activity), and three were active against ouabain-induced arrhythmias (class I activity). Selected compounds were evaluated further in dogs for efficacy against arrhythmias occurring 24 h following coronary ligation (automatic arrhythmias) and induced by using programmed electrical stimulation techniques (reentrant arrhythmias). The most effective compounds from the series are 3g and -j, which were effective in both canine models. Molecular modeling and structure-activity relationships are discussed.