Progress 01/15/12 to 01/14/15
Outputs
Target Audience: Scientific and medical community with an interest in placental function and improving maternal/child health. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate and graduate students have received training in animal user health and safety, general laboratory safety, animal husbandry, tissue collection and processing, DNA extraction, RNA extraction, paraffin embedding of tissues, histology techniques, cell culture methodology, data analysis, presentation of data (oral and poster)and manuscript preparation. How have the results been disseminated to communities of interest? Through publications and presentations at EB 2015 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Maternal choline supplementation modulates fetal growth patterns andimproves placental functioning in amouse model of placental insufficiency We investigated the effects of maternal choline supplementation (MCS) in a mouse model of placental insufficiency. Dlx3 +/- female mice were assigned to control, 2X or 4X choline intake during gestation and were sacrificed at embryonic days E10.5, E12.5, E15.5, and E18.5. Data were analyzed using linear mixed models with dam as a random factor. At E10.5 the 4x choline treatment increasedembryo weight by 70% in wildtype (WT) pups (P<.001) and by 60% in heterozygous (HET) pups (P=.013) compared to controls. Similarly, placental efficiency (ratio of embryo weight to placental weight) was increased by 44% in HET pups (P=.044) on 4X choline. By E12.5, neither 2X or 4X choline group pups weighed more than controls and at E18.5, WT (P=.024) and HET (P=.024) 4X pups showed 10% lower weight as compared to controls. To gain insights into the molecular mechanisms through which MCS improves fetal growth in early pregnancy, we examined placental expression of angiogenic and inflammatory genes. MCS decreased endoglin expression among female placental. A trend towards decreased Vegf expression (by 28%) was detected in 4X vs. Ctrl (P=.098). When stratified by sex, the trend remained only in females (P=.119). No effects of choline were seen on Pgf expression. Expression of NFkB was also decreased by MCS but statistical significance was not achieved. No effects of choline were seen on Tnfα (tumor necrosis factor α) and Il-1β. In sum, maternal choline supplementation accelerates fetal growth and placental efficiency in early gestation in Dlx3+/-, followed by a possible compensatory slowing of growth towards late gestation. MCS significantly reduced expression of endoglin at E10.5, perhaps due to improved placental efficiency requiring less expression of angiogenic factors. The effect of MCS on markers of placental function appears to vary by sex. Future studies should employ larger sample sizes to account for these sex-specific effects. Maternal Choline Supplementation During Normal MousePregnancy Reduces Placental Inflammation and Improves Placental Development. Placentas fromNon-Swiss Albino (NSA) outbred mice receiving 1, 2 or 4 times the recommended choline intake were collected at gestational day (E) 10.5, 12.5, 15.5 or 18.5. QPCR was conducted to assess the expression of inflammatory cytokines, angiogenic factors and nutrient transporters. Data were analyzed using a mixed linear model. A higher choline intake signficantly reduced IL-1β expression in female placentasatE12.5andE18.5, and in male placenta of small litters (n=5-8) at E18.5,In male placenta,extra choline significantly decreasedTNF-alpha expressionatE10.5, andat E18.5(small litter size only; n=5-8).Choline treatment also increased placental VEGF alpha expression infemales at E15.5 and 18.5, and signficantly improved placental labyrinth vasculature.In males, extra choline enhanced nutrient transporter (Snat 1) and significantly improved maternal decidua vasculature. In sum, a higher maternal choline intake during normal pregnancy improves biomarkers of placenta development and function in a manner dependent on gestational-age, sexand litter size. Choline insufficiency induces inflammation and placental dysfunction in a human trophoblast cell culture model Immortalized HTR-8/SVneo trophoblasts were cultured in different choline concentrations (8, 13 and 28 [control] μM) for 96-h and markers of angiogenesis, inflammation, apoptosis, and blood vessel formation were examined. Choline insufficiency altered the angiogenic profile, impaired in vitro angiogenesis, increased inflammation, induced apoptosis, increased oxidative stress, and yielded greater levels of protein kinase C (PKC) isoforms δ and ε possibly through increases in the PKC activators 1-stearoyl-2-arachidonoyl-sn-glycerol and 1-stearoyl-2-docosahexaenoyl-sn-glycerol. Notably, the addition of a PKC inhibitor normalized angiogenesis and apoptosis, and partially rescued the aberrant gene expression profile. Together these results suggest that choline inadequacy may contribute to placental dysfunction and the development of disorders related to placental insufficiency by activating PKC.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jiang X, Jones S, Andrew BY, Ganti A, Malysheva OV, Giallourou N, Brannon PM, Roberson MS, Caudill MA. Choline inadequacy impairs trophoblast function and vascularization in cultured human placental trophoblasts. J Cell Physiol. 2014;229:1016-27.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Julia H King, Sze Ting Kwan, Jian Yan, Vladislav G Fomin, Mark S Roberson and Marie A Caudill.Maternal Choline Supplementation Modulates Fetal Growth Patterns in a Mouse Model of Placental. Abstract 4646, Experimental Biology 2015
Insufficiency
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Sze Ting Kwan, Julia King, Jian Yan, Hallie Klein, Vladislav Fomin, Mark Roberson, Marie Caudill. Maternal Choline Consumption During Pregnancy Modulates Cellular and Functional Aspects of Placental Development. Experimental Biology 2015; EXPERIMENTAL ANIMAL NUTRITION:ANIMAL MODELS OF FETAL
NUTRITION, PROGRAMMING AND NEONATAL DEVELOPMENT MINI-Symposium
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Progress 01/15/13 to 01/14/14
Outputs
Target Audience: Scientific community at-large with an interest in inflammation and placental functioning. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate and graduate students have received training in animal user health and safety, general laboratory safety, animal husbandry, tissue collection and processing, DNA extraction, RNA extraction, paraffin embedding of tissues, histology techniques, cell culture methodology, data analysis, and manuscript preparation. How have the results been disseminated to communities of interest? Through Publication What do you plan to do during the next reporting period to accomplish the goals? Placental gene expression assaysof inflammatory cytokines, angiogenic factors and nutrient transporters will be conducted at each embryonic timepoint (E10.5, 12.5, 15.5 and 18.5) alongwith measurements of circulating plasma concentrations ofthe inflammatory and angiogenic factors. In addition, placenta morphological parameters will be examined, statistical analyses conducted, and a manuscriptwith a description of study findings prepared.
Impacts
What was accomplished under these goals?
Mouse Study: The feeding and tissue collection phase of the mouse study was completed in December 2013. We achieved the target sample size of ~ 10 dams in each treatment group at each time point (i.e., n=7-11 per treatment group at E10.5, 12.5, 15.5 and 18.5). A preliminary analysis revealed improved fetal growth and placental functioning with maternal choline intakes at 2X or 4X the recommended intake level at one or more of these timepoints. Overall, these observations are consistent with the hypothesis that extra choline during pregnancy will improve placental development and functions. Placental Trophoblast Cell Culture Study: Immortalized HTR-8/SVneo trophoblasts were cultured in different choline concentrations (8, 13 and 28 [control] μM) for 96-h and markers of angiogenesis, inflammation, apoptosis, and blood vessel formation were examined. Choline insufficiency altered the angiogenic profile, impaired in vitro angiogenesis, increased inflammation, induced apoptosis, increased oxidative stress, and yielded greater levels of protein kinase C (PKC) isoforms δ and ε possibly through increases in the PKC activators 1-stearoyl-2-arachidonoyl-sn-glycerol and 1-stearoyl-2-docosahexaenoyl-sn-glycerol. Notably, the addition of a PKC inhibitor normalized angiogenesis and apoptosis, and partially rescued the aberrant gene expression profile. Together these results suggest that choline inadequacy may contribute to placental dysfunction and the development of disorders related to placental insufficiency by activating PKC. A manuscript containing these results has been accepted for publication.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Jiang X, Jones S, Andrew BY, Ganti A, Malysheva OV, Giallourou N, Brannon PM, Roberson MS, Caudill MA. Choline inadequacy impairs trophoblast function and vascularization in cultured human placental trophoblasts. J Cell Phys. In Press.
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Progress 01/15/12 to 01/14/13
Outputs
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. 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. TARGET AUDIENCES: Pregnant women and their health care providers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
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.
Publications
- Jiang X, Jones S, Ganti A, Andrew B, Brannon P, Roberson M, Caudill M. Choline affects placental vascular function and development in a dose response manner. Experimental Biology (Abstract). Boston Massachusetts. April 2013.
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