Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Sascha Drewlo

Abstract

The placenta in mammals forms the maternal fetal interface serving as the source of nutrition for the fetus throughout gestation. It comprises of two major trophoblast lineages: (i) the decidua invading extra-villous trophoblast (EVT) and (ii) the placenta residing villous trophoblast (VT). The EVT’s invade the maternal endometrium to establish pregnancy and secure blood-flow to the implantation site. The VT villous trophoblast forms the main maternal-fetal exchange surface and ensures nutrient and gas exchange to facilitate growth throughout pregnancy. Additionally, both lineages are involved in immunological functions such as maintaining allogenic tolerance and regulation of immune cell activation at the maternal fetal interface. They also express pattern recognition receptors like TLR (toll-like receptors) and NLR (Nod-like receptors) involved in pathogen recognition. Activation of these receptors leads to an inflammatory cascade aimed at elimination of the pathogen which in severe conditions can lead to preterm birth (PTB) and even fetal death. Proper development of trophoblast cells is thus crucial for placental function and hence for a successful pregnancy. Indeed, pregnancy disorders like pre-eclampsia (PE), intra – uterine growth disorders (IUGR) and preterm birth (PTB) have all been associated with abnormal trophoblast differentiation. Interestingly, elevated levels of systemic as well as placental inflammation is another feature commonly associated with these disorders. Localized inflammation (Chorioamnionitis) has been reported to be present in >85% spontaneous preterm births even in the absence of systemic inflammation. However, the potential effects of inflammatory pathways on placental function (and trophoblast differentiation) remain largely unexplored. Consequently, there is also less information available on molecular targets common to both these processes that can be used for development of therapeutic interventions.

The ligand activated transcription factor PPARγ has a known anti-inflammatory role and plays a crucial role in placental development. Abnormal levels of the receptor were also associated with disorders IUGR associated PE, GDM and even PTB. However, the potential role of PPARγ in regulation of the placental and systemic immune responses remains unexplored. Our preliminary studies in the mouse model of inflammation induced PTB showed that activation of PPARγ significantly reduced PTB and improved both placental and fetal weights. The current dissertation therefore aimed to evaluate PPARγ as the potential common link between inflammation and placental function. Based on our preliminary results, we hypothesized that PPARγ is an important modulator of placental immune responses and is as well involved in trophoblast function.

AIM 1: To determine the molecular mechanism of PPARγ mediated prevention of PTB in endotoxin induced PTB mouse model.

In our preliminary studies, we observed that treatment with Rosiglitazone (specific agonist for PPARγ) significantly reduced pre-term birth in the mouse model for endotoxin (bacterial lipopolysaccharide) mediated inflammatory preterm birth. The current aim was designed to evaluate the mechanism involved. Our results revealed that activation of PPARγ via Rosiglitazone had anti-inflammatory effects at both systemic and local levels. We also reported that the endotoxin (LPS) increases inflammation by upregulating its receptor TLR4 and contributes to oxidative stress by downregulating the anti-oxidant pathway. Rosiglitazone via PPARγ activation decreased the inflammatory cytokine levels in serum and downregulated activity of NF-κB pathway in macrophages at the maternal-fetal interface. It also upregulated expression of anti-oxidant pathway mediators NRF2 and HO-1 and reduced the expression of TLR4. We thus report for the first time, that PPARγ activation via Rosiglitazone prevents LPS induced preterm birth in mice by acting on inflammatory as well as anti-oxidative pathway.

AIM 2: To determine the effects of LPS exposure and PPARγ induction on human trophoblast physiology.

In our mice studies, we observed that endotoxin treatment caused reduction in placental weights suggesting inflammation mediated effects on mouse placenta. Since elevated inflammation is associated with approximately 30% of preterm deliveries in humans, we asked if inflammation had any effects on human placental function. Previous studies have focused on evaluating the inflammatory response of human placental trophoblast cells. However, the effects of inflammation on trophoblast cell differentiation and function remain comparatively unevaluated. Additionally, the potential anti-inflammatory effect of PPARγ in human placenta also remains unexplored. The current aim was designed to evaluate the effects of endotoxin exposure on the human trophoblast cell differentiation and function and the potential role of PPARγ in reversing these effects. Our results showed for the first time that inflammation alters trophoblast cell differentiation and function -- by downregulating expression of trophoblast differentiation proteins GCM1 and CG-β and reducing invasion -- which can be reversed by PPARγ activation.

AIM 3: To determine the role of PPARγ in differentiation of EVT and VT trophoblast lineages.

The data obtained in Aim 1 and Aim 2 highlighted the key role for anti-inflammatory activity of PPARγ in placental function and pregnancy in general. However, PPARγ is implicated to be involved in placental development via pathways beyond inflammation. Previous studies and results presented in chapter 4 suggested a key role for PPARγ in trophoblast differentiation via regulation of GCM1 – transcription factor crucial for differentiation towards both trophoblast lineages. The first part of Aim 3 therefore focused on validating the PPARγ – GCM1 molecular axis. Our results showed that PPARγ transcriptionally regulated GCM1 expression by binding to its specific binding site in the GCM1 promoter. The second part of Aim 3 was designed to further delineate the role of PPARγ in differentiation towards the VT and EVT lineages. A crucial initial step was to develop a model to simultaneously study both lineages. In the current study, we established the model and conducted preliminary experiments for determining the specific role of PPARγ in trophoblast lineage differentiation.

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