Saudi Cultural Missions Theses & Dissertations
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Item Restricted Role of IDO1 in the Spiral Artery remodeling during pregnancy(Drexel University, 2024-07) Ghaban, Dema; Muller, AlexanderDuring gestation, the trophoectoderm, the outer layer of the blastocyst, engages with the uterine wall to form the placenta, a temporary organ at the maternal-fetal vascular interface. To increase blood flow through the placenta, uterine spiral arteries transform into large, dilated, low-resistance vessels in which the endothelial layer has been replaced by infiltrating extravillous trophoblasts that invade the uterine myometrium. While it has been widely assumed that these invasive trophoblasts drive the spiral artery remodeling (SAR) process, careful temporal analysis has established that SAR initiation occurs prior to trophoblast arrival. Rather, it is decidual immune cells that are implicated in causing the disorganization of vascular cells and separation of smooth muscle layers characteristic of early-stage SAR. Mouse studies have shown that uterine natural killer (uNK) cells, which expand rapidly in the decidua at mid-gestation, are required for SAR to proceed normally and that this activity is dependent on the inflammatory cytokine interferon-gamma (IFNγ). It therefore has been clearly established that inflammation plays a fundamental role in this complex, physiological process, but thus far a detailed understanding of the specific factors involved downstream of IFNγ has remained elusive. Addressing the unresolved role of inflammation in the reshaping the vasculature at this key stage of gestation has been the core focus of my research project. To better understand how the inflammatory microenvironment established by decidual immune cells promotes SAR, it is important to have an assay that quantifiably assesses the remodeling process. To this end, I describe the development of a method whereby sections of mid-gestation mouse embryos are prepared for comparative assessment of SAR initiation based on confocal microscopy-based immunofluorescence quantification of staining for smooth muscle alpha actin (SMA) within individual arterial vessels (Chapter 2). This technique provides a reproducible and objectively quantifiable methodology for monitoring this initial stage of the SAR process for any perturbations that might result from modifications to the inflammatory microenvironment. Trophoblasts must next migrate to the spiral arteries for SAR to proceed, and I further describe the use methods for assessing trophoblast invasiveness as a later aspect of the SAR process using both in vitro human cell and in vivo mouse-based experimental models (Chapter 3). The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) is an IFNγ-inducible protein that is highly expressed in the placenta at mid-gestation. Through its well-established capacity to promote T cell tolerance, IDO1 has gained widespread recognition as a mechanism for protecting hemi-allogeneic fetus from maternal immunity. More recently, our laboratory has determined that a specific myeloid subpopulation that protects neovasculature from IFNγ due to IDO1 mediated activation of the integrated stress response through general control non-derepressible 2 (GCN2) that results in elevated production of interleukin 6 (IL6). Research studies have reported that IDO1 deficiency can induce symptoms akin to preeclampsia in both mice and humans. While these findings suggest a role of IDO1 in decidual vascularization during pregnancy, this has yet to be explored. Altered expression of IL6 and its receptor in decidua have likewise been reported in cases of sporadic miscarriage and in vitro assays have identified IL6 as a potential factor driving trophoblast invasion. Based on our findings in settings of pathological neovascularization, I have investigated the hypothesis that the initiation of SAR similarly runs through IFNγ/IDO1/IL6. In line with previous reports, vessel wall SMA remained significantly elevated in mice lacking IFNγ, and consistent with our hypothesis lack of either IDO1 or GCN2 resulted in a similar retention of SMA. However, in contrast to IDO1 or GCN2 deficiency which only partially attenuated SMA loss, IL6 deficiency blocked SMA loss almost completely, suggesting that IDO1 is not the sole input driving IL6 in this context. IFNγ expression was unaffected in mice lacking IDO1, GCN2 or IL6 despite the defects in vessel wall remodeling observed in these mice while IL6 expression was impaired in mice lacking IFNγ, IDO1 or GCN2 consistent with IFNγ being an upstream driver and IL6 a downstream effector in the SAR process. In addition to these effects on the vessel wall, IL6 was confirmed to promote trophoblast migration in vitro and loss of IFNγ, IDO1, GCN2 or IL6 all compromised trophoblast invasiveness in vivo. Thus, two key features of the SAR process, vessel wall remodeling and trophoblast invasion are subject to control by this inflammatory pathway. These data provide important elucidation of mechanism by which inflammation controls SAR an suggest potential pathways for interventional therapy to mitigate the effects when this process goes awry.9 0