Date of Award

12-1-2018

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Torry, Donald

Abstract

ABSTRACT Preeclampsia (PE) is a one of the more common pregnancy complications that affects 5-8% of pregnancies worldwide and produces significant morbidity and mortality for mother and fetus. Shallow trophoblast invasion and insufficient maternal spiral artery remodeling early in gestation is believed to lead to a relatively hypoxic placenta with inflammatory and trophoblast endoplasmic reticulum (ER) stress. These stresses cause an imbalance in trophoblast expression of angiogenic/anti-angiogenic molecules with decreased placental growth factor (PGF) and increased soluble fms like tyrosine kinase-1 receptor (sFlt-1) production. The decrease in trophoblast PGF seems to be mediated at the transcriptional level while increased expression of sFlt-1 is mediated by alternative splicing. Two variants known to be elevated in PE are the sFlt-1i13 and sFlt-1e15a isoforms. Both share the first 13 exons of Flt-1. A read through into intron 13 and utilization of an alternative poly (A) signal sequence produces the sFlt-1i13 variants protein, while sFlt-1e15a results from alternative splicing of exon 14 to exon 15a, rather than exon 15, and utilization of an alternative poly (A) signal sequence. This angiogenic imbalance contributes to the clinical manifestations of PE later in pregnancy including maternal hypertension and proteinuria. Currently, there are no pharmacological options available for the prevention of PE and the only way to reverse PE symptoms is by delivery. The overall goal of my project was (1) to investigate potential therapeutic mechanisms that could be used to relieve the maternal symptoms of PE by correcting the angiogenic imbalance, and (2) to gain a better understanding of the alternative splicing mechanisms responsible for producing sFlt-1 gene expression in human trophoblast. PE shares some similar pathophysiology and risk factors with cardiovascular diseases. This has prompted use of statins as a potential therapy for PE. However, existing preclinical investigations for statin use has been mostly restricted to PE animal models without elucidating the cell types that respond to statin treatment. Therefore, we sought to determine the effect of statins on angiogenic gene expression in cells that are in direct contact with maternal blood during pregnancy and could contribute to PE: primary trophoblast and endothelial cells. Placental tissue and isolated cells were cultured under hypoxic stress (1% O2) as a model for the hypoxic environment noted in PE. We compared the effectiveness of two types of statins (hydrophilic vs hydrophobic) on angiogenic and anti-angiogenic gene expression from the human tissues and cells. Human placenta villus explants, umbilical vein endothelial cells (HUVECs), and cytotrophoblast were isolated from normal term placentae and cultured under low oxygen tension (1% O2) with serial concentrations of statins. Expression of proangiogenic genes (VEGF and PGF) and the prominent anti-angiogenic sFlt-1 isoforms (sFlt-1i13 & sFlt-1e15a) were analyzed. In villus explants, hypoxia (1% O2) tended to alter angiogenic gene expression in their predicted fashion, by increasing VEGF mRNA (hypoxia marker), decreasing PGF mRNA, and increasing both sFlt-1i13 and sFlt-1e15a mRNA expression. However, the changes in gene expression were quite variable and statistically not significant. Hypoxia significantly increased both sFlt-1i13 and sFlt-1e15a mRNA and protein expression in primary trophoblast but had limited effects on expression in HUVECs. Hypoxia significantly decreased PGF mRNA and protein expression in primary trophoblast, yet significantly increased PGF mRNA and protein expression in HUVECs. Concentrations of pravastatin or simvastatin used had limited effects on altering PGF mRNA and protein expression in any of the cell types. In primary trophoblast, lower concentrations of pravastatin (100/500 µg/ml) had no significant effects on sFlt-1i13 or sFlt-1e15a mRNA expression while higher concentrations (1000 µg/ml) significantly decreased sFlt-1i13 and tended to decrease sFlt-1e15a mRNA expression. Secreted sFlt-1 protein from trophoblast decreased with increasing concentrations of pravastatin. Similarly, simvastatin had limited effects and did not significantly decrease sFlt-1i13 or sFlt-1e15a expression in hypoxic primary trophoblast. Both pravastatin and simvastatin significantly down-regulated sFlt-1i13 and sFlt-1e15a mRNA expression and sFlt-1 protein production in HUVECs. To overcome the effects of statin treatments on sFlt-1 expression, primary HUVECs were treated with farnesyl pyrophosphate ammonium salt (FPP), an intermediate in the cholesterol synthesis pathway. FPP partially restored sFlt-1i13 and sFlt-1e15a mRNA expression. Our data support that the angiogenic imbalance seen in PE can be medicated by hypoxia, and that statin could be a promising medication to limit PE symptoms. The effect of statins may be more evident on endothelial cells than on trophoblast, and the reduction in sFlt-1 expression by statins seems to be partially mediated through the cholesterol synthesis pathway in endothelial cells. The antiangiogenic protein, sFlt-1, plays a central role in the pathophysiology of PE. Excessive amounts of the sFlt-1 receptor in maternal circulation leads to maternal endothelial cell dysfunction and subsequent clinical symptoms of PE. However, the mechanism governing sFlt-1 mRNA expression in trophoblast remains unclear. Jumonji C domain containing gene 6 (JMJD6) has been shown to be involved in splicing of sFlt-1i13 in endothelial cells, although with conflicting outcomes as to whether it increases or decreases alternative splicing of sFlt-1i13. It is unknown if JMJD6 functions to regulate splicing in human primary trophoblast. Therefore, we assessed whether JMJD6 expression is altered in primary trophoblast under hypoxia or ER stress and its ability to regulate alternative splicing of sFlt-1. Human cytotrophoblast were isolated from normal term placentae and were cultured in the presence or absence of ER stress inducer (tunicamycin) or at 1% O2 to simulate trophoblast stressors during PE. Expression of JMJD6, C/EBP homologous protein (CHOP), and sFlt-1 (sFlt-1i13, and sFlt-1e15a) variants were analyzed. Hypoxic stress significantly increases JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression. ER stress also tended to increase JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression in primary trophoblast. Collectively, our results show that low oxygen tension (1% O2) or ER stress increase JMJD6 mRNA expression which may contribute to increased sFlt-1i13 and sFlt-1e15a variant expression in primary trophoblast. Similarly, JMJD6 knock down with siRNA tends to slightly decrease sFlt-1i13 and sFlt-1e15a mRNA expression in primary trophoblast. JMJD6 overexpression in HTR-8 cells (choriocarcinoma) tended to increase sFlt-1i13 and sFlt-1e15a mRNA expression; however, results using HTR-8 were inconsistent due to extremely low expression of endogenous Flt-1 mRNA. To overcome this, a Flt-1 minigene plasmid was transfected into HTR-8 cell line. Under 1%O2 these cells increased expression of the sFlt-1i13 isoform. To more directly confirm effects of JMJD6 and hypoxia on sFlt-1 expression, HEK293 and JEG3 stable clones harboring the Flt-1 minigene were generated. Preliminary results from selected single colony isolates show that several stable clones express the Flt-1 minigene products. HEK293 and JEG3 stable clones harboring the Flt-1 minigene, HEK293-Flt1#5 and JEG3-Flt1#5 respectively, were cultured at 1%O2 for 48 or 72 hours. Hypoxic stress had no significant on altering sFlt-1 variant production or JMJD6 mRNA expression in HEK293-Flt1#5 cells. However, hypoxic JEG3-Flt1#5 cells significantly increased sFlt-1i13 isoform mRNA expression (˜6 fold) and mFlt-1 mRNA expression (2.5 Fold) and also increased JMJD6 mRNA expression (1.8 Fold). In summary, these data suggest a role for statins as a potential therapeutic approach for the prevention and treatment of PE by decreasing systemic sFlt-1 expression in endothelial cells. This effect seems most significant in endothelial cells. If substantiated by clinical studies, use of statins would offer an affordable and easily accessible therapy to lessen PE symptoms. Moreover, our preliminary data suggest a potential involvement of JMJD6 in splicing process of sFlt-1i13. Confirming of JMJD6 role in splicing of Flt-1 may provide therapeutic strategies to treat Flt-1 associated disorder.

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