Placental abnormalities and hypertension in pregnancy

  • Gabriele Bobek

Western Sydney University thesis: Doctoral thesis

Abstract

Preeclampsia is a significant and common complication of pregnancy, with characteristic signs of hypertension and proteinuria. Current theories postulate a role for altered placental perfusion as a consequence of abnormal placental development in the aetiology of preeclampsia. Animal models of human preeclampsia have shown that an imbalance of the inflammatory cytokine TNF-I leads to a similar maternal phenotype as seen with a surgical reduction of placental perfusion pressure. This suggests a role for the inflammatory response in generating the maternal signs of hypertension and proteinuria. Currently, there is no direct link showing that a cytokine imbalance (specifically increased TNF-) affects placental development in such a way as to result in altered blood flow. The ability to detect morphological changes and alterations in blood flow in experimental models of preeclampsia would provide a significant boost in our understanding of the syndrome. The aim of this study was to develop an "imbalance in pro-inflammatory cytokine (TNF-I)" experimental mouse model of preeclampsia and to utilize magnetic resonance imaging (MRI) for visualization of placental anatomy and for the analysis of changes in tissue morphology and function including blood flow and perfusion. Secondly, this study aimed to examine the relationship between; an imbalance in inflammatory cytokines; changes in placental markers involved in inflammation, hypoxia and pH homeostasis; and changes in blood flow in the aetiology of the maternal hypertensive response. Pregnant C57BL/6JArc mice were subject to either reduced utero-placental perfusion (RUPP), subcutaneous infusion of the inflammatory cytokine TNF-I, or control procedures. Blood pressure was measured by either tail cuff sphygmomanometry or by telemetry. Urine was collected to measure proteinuria and blood was collected to measure levels of the anti-angiogenic molecule soluble fms-like tyrosine kinase 1 (sFlt-1), a biomarker of the human disease. MRI images were acquired on anaesthetised mice on day 17 of gestation using a Bruker Avance 11.7 Tesla wide-bore spectrometer. Quantitative analysis of changes in T2 relaxation measurements were carried out by using Matlab™ to generate R2 (i.e., 1/T2) maps from the acquired T2 measurement data, with the T2 values being calculated from selected regions of interest. Additional high resolution MRI images were acquired on formalin fixed, Magnevist™ contrast agent infused placenta. Placentas were harvested on day 17 of pregnancy, either formalin fixed and paraffin embedded for histology or snap frozen for proteomics and genomics. Histology was performed on sections using either Haematoxylin and Eosin (H&E) or Periodic acid-Schiff (PAS) stains. Immunohistochemistry using secondary anti rabbit horse radish peroxidise linked polymer and visualising with DAB, or quantitative immunofluorescent histochemistry using Alexa 488 goat anti-rabbit IgG was performed using primary antibodies to Cytokeratin (trophoblast marker), HIF-1a (Hypoxia inducible transcription factor 1), CLIC-3 (chloride intracellular channel 3; Cl-/H+ co-transporter) and TLR-3 and TLR-4 (Toll-like receptor 3 and 4). Quantitative PCR (qPCR) was used to measure mRNA expression of mFlt-1, sFlt-1, hif-1, tlr-3, tlr-4, clic-3 and clic-4 in placental tissue. This thesis demonstrates that infusion of the inflammatory cytokine (TNF-I) is an experimental model for hypertension and proteinuria in murine pregnancy. Hypertension in the RUPP model was not definitively confirmed despite the proteinuria. No increase in sFlt-1 above the constitutively high levels of normal pregnancy was detected in the maternal serum of either model, suggesting sFlt-1 is not a reliable marker for disease in the mouse model. This thesis demonstrates that that morphologically distinct regions of the mouse placenta can be detected and quantified by MRI. Mapping of T2 relaxation times ,which are attenuated by both hypoxia (increased levels of deoxyhaemoglobin) and acidosis (increase in free protons), indicate contrast between regions which is is lost when blood flow ceases. Similar decrease in contrast is detected upon T2 mapping in the placentas of both the artificially reduced perfusion (RUPP) and imbalance of inflammatory cytokines (TNF-I) experimental models. Immunohistochemistry and qPCR detected increases in the presence of molecules involved in response to both inflammation (TLR-3 and TLR-4) and changes in oxygen (HIF-1I) and pH (CLIC-3) levels in placentas from animals subject to either TNF-I infusion or RUPP. These results demonstrate for the first time that morphological differences or abnormalities related to blood flow can be detected by T2 mapping in the placenta of mice subject to experimental models of preeclampsia and may be used to analyse changes quantitatively. This technology has the potential to be used when studying the dynamic changes in the placenta of pregnancies complicated by preeclampsia. Analysis of the MRI images suggests changes involve both increases in deoxyhaemoglobin (hypoxia) and decreases in intracellular pH (acidosis) and suggests that pH dependent mechanisms may be as equally important as hypoxia in the perturbed placenta. The results also indicate that the metabolic changes in the placenta in response to both decreased blood flow and TNF-I infusion involve upregulation of both TLR-3 and TLR-4 protein expression and upregulation of HIF-1I mRNA and protein. Alterations in expression and localisation of the H+/Cl- co transporter CLIC-3 was demonstrated in the placenta after TNF-I infusion, consistent with the metabolic change observed by MRI. Inflammation-driven changes in both oxygen and pH-dependent signalling pathways are thus implicated in alterations of the complex metabolic pathways of homeostasis and angiogenesis in the placenta that lead to the subsequent maternal hypertensive response.
Date of Award2015
Original languageEnglish

Keywords

  • preeclampsia
  • hypertension in pregnancy
  • pregnancy
  • complications
  • fetus
  • abnormalities
  • congential abnormalities

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