Choline as a Modulator of Inflammation and Placental Function

Non-Technical Summary:<br/>
Placental insufficiency contributes to several adverse pregnancy outcomes including preeclampsia and intrauterine growth restriction (IUGR). Although the etiology of placental insufficiency is incompletely understood, the disorder is characterized by insufficient invasion of trophoblast cells into the maternal arteries along with oxidative stress and placental production of pro-inflammatory cytokines. Preliminary data from our research group suggest that supplementing the maternal diet of healthy third trimester women with choline, an essential micronutrient, beneficially alters placental markers of inflammation and angiogenesis. We have also identified a transcription factor, distal-less homeobox 3 (Dlx3), which is required for normal placental development and function. Loss of a single allele (Dlx3 +/- genotype) in mouse pups results in placental insufficiency and an IUGR phenotype. To investigate whether maternal choline intake can improve placental insufficiency, and to clarify the molecular mechanisms involved, the maternal diet of Dlx3 +/- mice will be supplemented with choline: 4 2, or 1 times normal intake. Placental and circulating inflammatory and angiogenic factors as well as placental morphological parameters will be quantified at embryonic days 9.5-15.5. Fetal weights and crown rump measurements will also be obtained in the +/+ and +/- pups. These proof-of-principle experiments are expected to show the beneficial effects of the bioactive micronutrient, choline, on inflammation, angiogenic balance, placental function and fetal well-being. As such, data generated from these studies would justify the need for clinical studies in human study participants that seek to explore the use of choline as a prophylaxis for disorders related to placental dysfunction and more broadly diseases with an inflammatory component.
<P>
Approach:<br/>
Distal-less homeobox 3(Dlx3)is a transcription factor that is required for normal placental development and function. Loss of a single allele (Dlx3 +/- genotype) in mouse pups results in placental insufficiency and an intrauterine growth restriction (IUGR) phenotype. To investigate whether maternal choline intake can ameliorate placental insufficiency, and to clarify the molecular mechanisms involved, the maternal diet of Dlx3 +/- mice will be supplemented with choline: 4 2, or 1 times normal intake. Placental and circulating inflammatory and angiogenic factors as well as placental morphological parameters will be quantified at embryonic days 9.5-15.5. Fetal weights and crown rump measurements will also be obtained in the +/+ and +/- pups. In parallel, we will use human immortalized placental trophoblast cells (HTR-8/SVneo)to directly assess the impact of varied choline exposure on human trophoblast function. Three concentrations of choline will be added to cell culture: 0 ?M, 100 ?M, or 500 ?M; and the cells will be cultured at 2%, 8% or 20% oxygen. After culture, cells and culture medium will be obtained for the following analyses: (i) cell viability; (ii) choline metabolites; (iii) cellular expression of pro- and anti-inflammatory genes as well as angiogenic peptides; (iv) secreted inflammatory cytokines and angiogenic peptides; (v) DNA methylation of growth and stress related genes; (vi) cell apoptosis; and (vii) cellular oxidative stress. Data generated from this study will be used to determine whether a higher maternal intake of the micronutrient choline can beneficially influence placental function and fetal well-being. This information is particularly important given that the majority of U.S. women are not consuming choline intakes at recommended intake levels and that prenatal vitamins do not contain choline. The findings of this study will also advance understanding of the molecular mechanisms through which choline is influencing placental function. Collectively, these data may provide a strong rationale for women of childbearing age to increase their dietary intake of choline and serve as an impetus for developing or promoting foods enriched with choline. These results can also scientifically inform the development of a choline intervention study in pregnant women with placental insufficiency.
<P>
Progress:<br/>
2012/01 TO 2013/01<br/>
OUTPUTS: Mouse Project: All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committees at Cornell University. Female Dlx3 +/- mice (n~75 of the 120 target) and female Dlx3 +/+ mice (n~75 of the 120 target) were assigned to either the control diet (CD; n=40), the choline supplemented diet at 2 times normal intake (CS-A; n=40) or the choline supplement group at 4 times normal intake (CS-B; n=40). Dams were sacrificed at E10.5 (n=30), E12.5 (n=30), E15.5 (n=30), and E18.5 (n=30) and tissues (e.g., maternal liver, placenta and pups) were collected, weighed (placenta and pups), processed and stored. Embryo morphology, crown rump measurements, and Dlx3 genotyping for these animals have also been performed. Trophoblast Cell Culture Project: HTR-8/SVneo cells were cultured for 96-h in total choline concentrations of 8, 13, 18, 28, 48 or 108 uM at 20% oxygen or total concentrations of 13, 18, 28, 48 at 1% oxygen. Cell counts and viability were measured and total RNA extracted. For the 8, 13 and 28 uM choline treatment groups at 20% oxygen, cellular mRNA abundance of the angiogenic factors VEGFA, sFLT1, PGF, ENG, KDR, MMP14 and the proinflammatory factors NFKB1, RELA, IL6, IL1B were measured along with the cellular concentrations of choline metabolites. Intracellular levels of reactive oxygen species, oxidative DNA damage, and apoptosis were also examined. For the 13, 18, 28 and 48 choline treatment groups at 1% oxygen, cellular mRNA abundance of the angiogenic factors VEGF, PGF, sFLT1 and the proinflammatory panel (NFKB1, RELA, IL6, IL1B) were examined.
<br/>PARTICIPANTS: Individuals who worked on this project include Marie Caudill (PI), Mark Roberson (Co-I), Patsy Brannon (Co-I), Julia King (pre-doctoral graduate student), Sze Ting (Cecilia) Kwan (pre-doctoral graduate student), Xinyin Jiang (pre-doctoral graduate student), Jian Yan (pre-doctoral graduate student), Steven Henick (undergraduate research assistant), and Hallie Klein (undergraduate research assistant). Training in animal user health and safety, general laboratory safety, animal husbandry, tissue collection and processing, and cell culture techniques were provided.
<br/>TARGET AUDIENCES: Pregnant women and their health care providers.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<P>
IMPACT: Outcomes: Preliminary data from the mouse model of placental insufficiency suggest that supplementing the maternal diet with extra choline improves fetal growth among the female Dlx3 +/+ and +/- mice. Data from the human trophoblast cell culture model cultured under normal oxygen levels (20%) show graded adverse effects of choline insufficiency on angiogenic factors (VEGF), inflammatory markers (ILB1), apoptosis, oxidative stress, DNA integrity, angiogenesis and protein kinase C signaling. Notably, the addition of a PKC inhibitor rescued the impaired angiogenesis, attenuated apoptosis, and partially normalized the gene expression profile. Under conditions of oxygen deprivation (i.e., 1%), choline treatment did not affect the angiogenic or inflammatory markers. In conclusion, choline availability plays an important role in trophoblast development and placental vasculature. The influence of choline on the trophoblast functioning may be partially mediated by PKC signaling.