SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 154A

154A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

blood flow velocity (CBFV) in the bilateral middle cerebral arteries and
finger plethysmography to record mean arterial pressure (MAP). We
calculated DCA capacity using mean velocity index (Mx), a validated
time-domain correlation method, with higher Mx indicating a higher
degree of correlation between CBFV and MAP and thus poorer DCA. A
cutoff of 0.3 was set as the threshold for abnormal DCA, based on prior
studies showing this threshold to be associated with poor neurological
outcomes in patients with traumatic brain injury. Overall mean Mx was
compared between groups using 2-sample t-test.
RESULTS: We performed 49 DCA assessments in 17 postpartum women
(9 with PEC, 8 normotensive), none of whom developed neurological
complications. Mean Mx throughout the postpartum period was elevated
in women with and without PEC (mean PEC Mx 0.42, versus mean
normotensive Mx 0.32, p=0.23), indicating impaired DCA. In the PEC
group, more severe DCA dysfunction was seen on postpartum day 2
(mean Mx 0.57 vs 0.32, p=0.17), but the difference did not reach statistical
significance.
*Figure(s) will be available online.
CONCLUSION: Dynamic cerebral autoregulation was impaired in
postpartum women with and without PEC. Women with PEC may have
more severe impairment of DCA in the immediate postpartum period, but
due to small sample size in this feasibility study, we were underpowered
to detect a statistical difference. More research is needed to better
characterize DCA dysfunction in this population, and to determine if
the degree of DCA dysfunction predicts postpartum cerebrovascular
complications.

T-134
The Role of VEGFR-1 and VEGFR-2 in Mediating Human
Fetoplacental Endothelial Cell Angiogenesis. Shuhan Ji†, Yingchun Li*,
Emily J Su*. University of Colorado Denver, Aurora, CO, United States.
INTRODUCTION: Proper fetoplacental blood flow is imperative for
a healthy pregnancy outcome, and fetoplacental angiogenesis plays
a vital role. Vascular endothelial growth factor A (VEGFA) is one of
many major regulators of angiogenesis. It primarily binds to two related
receptor tyrosine kinases, vascular endothelial growth factor receptor-1
(VEGFR-1; Flt1) and vascular endothelial growth factor receptor-2
(VEGFR-2; KDR/Flk-1). In most vascular beds, VEGFR-2 appears to
be a main mediator of angiogenesis. However, the role of the VEGFRs
within the human placenta remain unknown. Thus, our objective was
to determine the role of VEGFR-1 and VEGFR-2 on primarily isolated
human fetoplacental endothelial cell (EC) characteristics of angiogenesis,
and we hypothesized that EC migration is mediated primarily through
VEGFR-1 while proliferation is regulated through VEGFR-2.
METHODS: Human fetoplacental ECs were isolated/cultured from
placentas of full-term, uncomplicated, singleton pregnancies after
scheduled Cesarean section. Cells were subjected to RNA interference
of either VEGFR-1 and -2 followed by MTT assays, evaluation of
downstream gene expression, wound scratch, and tube formation assays.
ECs were serum starved after RNA interference and treated with VEGFA
(60 ng/ml), then subjected to western blot and qPCR. All experiments were
performed in triplicate utilizing ECs from at least three separate subjects.
Images were analyzed by ImageJ software. One-way ANOVA with Tukey
post-hoc testing was utilized for statistical analysis.
RESULTS: Significant knock-down of VEGFR-1 and -2 was confirmed
by qPCR and WB (p<0.001). To further verify appropriate RNA
interference, we found that VEGFR-2 inhibition resulted in diminished
phosphorylation of Akt and eNOS, both of which are downstream of
VEGFR-2. In contrast, VEGFR-1 silencing did not affect Akt and eNOS
phosphorylation. VEGFR-1 RNA interference had significant effect on
inhibition angiopoietin 2 (Ang2) and junctional adhesional molecule C
9 (JAM3) expression at 0 hrs (p<0.05) and 24 hrs (p<0.01) as compared
to control siRNA treatment and siVEGFR-2. Knock-down of VEGFR-2
significantly decreased metabolic activity of ECs (p<0.01), with no effect
via siVEGFR-1 transfection (p=0.7859). There was no difference in
apoptosis regardless of VEGFR-1 or -2 inhibition. Surprisingly, VEGFR-2

Scientific Abstracts

effects on presumed EC proliferation had no effect on cell migration. In
contrast, knock-down of VEGFR-1 demonstrated significantly impaired
wound scratch closure (p<0.01) and tube formation (p<0.05).
CONCLUSION: Human fetoplacental EC migration is mediated
primarily by VEGFR-1 while proliferation is regulated through VEGFR-2.

T-135
Localization of the Insulin-Sensitive GLUT4 Transporter and the
Insulin Receptor in the Human Placenta across Gestation. Laura
B James-Allan†, Theresa L Powell*, Thomas Jansson*. University of
Colorado, Denver, CO, United States.
INTRODUCTION: Glucose is transported across the human
placenta mediated by glucose transporters (GLUTs), expressed in the
syncytiotrophoblast (ST). The ST consists of two polarized plasma
membranes: the fetal facing basal plasma membrane (BM) and the
maternal facing microvillus plasma membrane (MVM). GLUT1 is
believed to be the main glucose transporter in the human placenta and
is expressed in both MVM and BM with a 3-fold higher expression in
the MVM. Therefore, the BM has been suggested to be the rate-limiting
step in transplacental glucose transport. The subcellular localization of
the insulin-sensitive GLUT4 transporter and the insulin receptor (IR) in
the human placenta across gestation remains to be fully established. We
tested the hypothesis that the GLUT4 transporter is expressed in both ST
membranes and that expression increases across gestation and that the
insulin receptor is predominantly localized in the MVM.
METHODS: Placental tissue was collected from healthy women who
underwent elective termination of pregnancy at 8-22 weeks of gestation
and from normal term pregnancies. ST MVM and BM were isolated using
homogenization, magnesium precipitation and differential centrifugation.
Mean MVM enrichment of alkaline phosphatase activity, an established
MVM marker, was 12.3 ± 1.5 and 12.6 ±1.0 (mean ± SEM) in early
gestation and term, respectively. Mean BM enrichment, determined by
ferroportin protein expression, was 10.3 ± 1.1 in early pregnancy and
33.0 ± 3.9 at term. Protein expression of GLUT4 and IRβ was determined
by western blot.
RESULTS: GLUT4 protein expression was exclusively expressed in
the BM in both early gestation and term placentas, which was confirmed
using two different antibodies. In the BM, GLUT4 protein expression
significantly increased across gestation (n=19, p<0.05). The protein
expression of IRβ was ~10-fold higher in MVM than in BM in both
early gestation (n=6, p<0.01) and term samples (n=6, p<0.0001). MVM
IRβ protein expression increased significantly across gestation (n=6,
p<0.05). BM GLUT4 expression was not affected by maternal obesity
in early pregnancy.
CONCLUSION: We show for the first time that insulin-sensitive GLUT4
is exclusively expressed in the fetal-facing BM of the ST and expression
increases across gestation. In contrast, the IR is predominantly expressed
in the MVM with increasing expression across gestation. These findings
are consistent with a model of maternal insulin modulating the transport
of glucose to the fetus by regulating GLUT4 expression in the BM.

T-136
Sex- and Gestational-Related Differences in Hypoxia-Induced
Mitochondrial Function in the Guinea Pig Placenta. Hong Song,2
Bhanu Telugu,1 Loren P Thompson*.2 1University of Maryland College
Park, College Park, MD, United States; 2University of Maryland SOM,
Baltimore, MD, United States.
INTRODUCTION: Chronic hypoxia is one of the most significant
clinical challenges during pregnancy and generates placenta dysfunction
via oxidative stress. Hypoxia-induced changes in placental development
and function may be mediated by underlying changes in mitochondrial
function. This study measured the effect of chronic hypoxia on indices
of mitochondrial (mito) function in the guinea pig (GP) placenta during
mid and late term gestation between males and females.
METHODS: Pregnant GPs were exposed to either normoxia (NMX) or
hypoxia (HPX; 10.5%O2) at 25d gestation until term (65d). NMX and
HPX GPs were anesthetized and fetuses and placentas (N=6 NMX/HPX
males, N=6 NMX/HPX females) were excised at either midterm (40d) or
Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018
