Effect of oxygen on the expression of renin-angiotensin system components in a human trophoblast cell line

During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin-angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development

Decidualisation of human endometrial stromal cells is associated with increased expression and secretion of prorenin

Background: In pregnancy, the decidualised endometrium expresses high levels of prorenin and other genes of the renin-angiotensin system (RAS) pathway. In this study we aimed to determined if the RAS was present in endometrial stromal cells and if decidualisation upregulated the expression of prorenin, the prorenin receptor ((P)RR) and associated RAS pathways. Immortalised human endometrial stromal cells (HESCs) can be stimulated to decidualise by combined treatment with medroxyprogesterone acetate (MPA), 17ß-estradiol (E₂) and cAMP (MPA-mix) or with 5-aza-2'-deoxycytidine (AZA), a global demethylating agent. Methods: HESCs were incubated for 10days with one of the following treatments: vehicle, MPA-mix, a combination of medroxyprogesterone acetate (MPA) and estradiol-17ß alone, or AZA. Messenger RNA abundance and protein levels of prorenin (REN), the (P)RR (ATP6AP2), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time PCR and ELISA's, respectively. Promyelocytic zinc finger (PLZF) and phospho-inositol-3 kinase (PIK3R1) mRNA abundances were also measured. Results: HESCs expressed the prorenin receptor (ATP6AP2), REN, AGT, ACE and low levels of AGTR1. MPA-mix and AZA stimulated expression of REN. Prorenin protein secretion was increased in MPA-mix treated HESCs. E₂ + MPA had no effect on any RAS genes. MPA-mix treatment was associated with increased VEGF (VEGFA) and PAI-1 (SERPINE1) mRNA and VEGF protein. Conclusions: An endometrial prorenin receptor/renin angiotensin system is activated by decidualisation. Since (P)RR is abundant, the increase in prorenin secretion could have stimulated VEGF A and SERPINE1 expression via Ang II, as both ACE and AGTR1 are present, or by Ang II independent pathways. Activation of the RAS in human endometrium with decidualisation, through stimulation of VEGF expression and secretion, could be critical in establishing an adequate blood supply to the developing maternal placental vascular bed

Expression of renin–angiotensin system (RAS) components in endometrial cancer

A dysfunctional endometrial renin–angiotensin system (RAS) could aid the growth and spread of endometrial cancer. To determine if the RAS is altered in endometrial cancer, we measured RAS gene expression and protein levels in 30 human formalin-fixed, paraffin-embedded (FFPE) endometrioid carcinomas and their adjacent endometrium. All components of the RAS were expressed in most tumours and in adjacent endometrium; mRNA levels of (pro)renin receptor (ATP6AP2), angiotensin II type 1 receptor (AGTR1), angiotensin-converting enzyme (ACE1) and angiotensin-converting enzyme 2 (ACE2) mRNA levels were greater in tumour tissue than adjacent non-cancerous endometrium (P = 0.023, 0.008, 0.004 and 0.046, respectively). Prorenin, ATP6AP2, AGTR1, AGTR2 and ACE2 proteins were abundantly expressed in both cancerous and adjacent non-cancerous endometrium. Staining was most intense in cancerous glandular epithelium. One potential target of the endometrial RAS, transforming growth factor beta-1 (TGFB1), which is essential for epithelial-to-mesenchymal transition, was also upregulated in endometrial cancer tissue (P = 0.001). Interestingly, TGFB1 was strongly correlated with RAS expression and was upregulated in tumour tissue. This study is the first to characterise the mRNA and protein expression of all RAS components in cancerous and adjacent non-cancerous endometrium. The greater expression of ATP6AP2, AGTR1 and ACE1, key elements of the pro-angiogenic/proliferative arm of the RAS, suggests that the RAS plays a role in the growth and spread of endometrial cancer. Therefore, existing drugs that inhibit the RAS and which are used to treat hypertension may have potential as treatments for endometrial cancer

The Gomeroi gaaynggal cohort: a preliminary study of the maternal determinants of pregnancy outcomes in Indigenous Australian women

The life expectancy of Indigenous Australians is amongst the lowest of any population group within developed nations and chronic diseases collectively account for over 80% of the gap in life expectancy between Indigenous and non-Indigenous Australians. The Gomeroi gaaynggal cohort is a prospective, longitudinal maternal-infant cohort established to examine the origins of chronic disease in Indigenous Australians. This study aimed to determine the major antenatal factors associated with adverse birth outcomes (preterm delivery, low birth weight) and other pregnancy-related complications (gestational diabetes and hypertensive disorders of pregnancy) in Indigenous Australian women. Pregnant women who identified as Indigenous Australians or pregnant non-Indigenous women giving birth to an Indigenous infant were eligible to participate in the cohort (n=227). Physical measurements and biological sample collection (including blood and urine) were undertaken up to 3 times in pregnancy. Median weight and BMI of the cohort was 80.7 kg and 30.3 kg/m² at enrolment (median 23 weeks gestation). 43% reported smoking cigarettes during pregnancy. Of the 158 women in whom pregnancy outcomes were known, 43% had an uncomplicated pregnancy, 13.9% delivered preterm, 14.6% delivered a small-for-gestational age infant, 10% developed a hypertensive disorder of pregnancy, and 6.3% developed gestational diabetes. In addition, many women showed evidence of underlying renal dysfunction (proteinuria or albuminuria). The ratio of male to female offspring in this cohort was 1.38. Eighty-seven percent of preterm infants were male, as were 83.3% of babies from women with gestational hypertension. This skewed sex distribution was far higher than for those who had a healthy pregnancy outcome (59%). This study demonstrates that key factors including maternal obesity, exposure to cigarette smoke and underlying renal impairment, influence pregnancy outcome. Preliminary findings from this study also suggest that more male babies are born early and from complicated pregnancies in this Indigenous cohort