SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 69A

Scientific Abstracts

O-037
Targeted Nanoparticles for Delivery of sFlt-1e15a-Specific siRNA
to the Placental Syncytiotrophoblast; a Novel Therapy for PreEclampsia? Chloe Logue,1 Susan Greenwood,1 Stephen Tong,2 Natalie
Hannan,2 Lynda K Harris*.1 1University of Manchester, Manchester,
United Kingdom; 2University of Melbourne, Melbourne, Australia.
INTRODUCTION: Preeclampsia is characterised by placental release
of elevated levels of soluble fms-like tyrosine kinase isoform e15a
(sFlt-1e15a) from the syncytiotrophoblast. sFlt-1e15a, an alternatively
spliced mRNA encoding a truncated, non-signalling fragment of vascular
endothelial growth factor receptor-1 (VEGFR1), antagonises the actions
of VEGF and placental growth factor (PlGF), contributing to maternal
systemic endothelial dysfunction. To reduce the risks associated with
systemic drug delivery and to create a novel therapy for pre-eclampsia,
targeted liposomes decorated with the placental homing peptide iRGD,
and containing sFlt-1e15a-specific small interfering RNA (siRNA) were
prepared.
METHODS: Liposomes were synthesised using the lipid film method
and decorated with fluorescent homing peptides; siRNA was encapsulated
via passive loading. Explants from normal, human term placentas were
incubated with liposomes containing either non-targeting or sFlt-1e15aspecific siRNA for up to 72h. Fluorescence microscopy was used to
assess liposome uptake and siRNA delivery. RT-qPCR and ELISA were
used to quantify sFlt-1e15a mRNA and protein secretion, respectively.
The rates of cytotrophoblast proliferation and apoptosis were examined
by immunostaining for Ki67 and M30; staining intensity was quantified
using Histoquest software. Non-parametric data (n>3) were expressed
as medians and analysed using Friedman's test with Dunn's multiple
comparisons.
RESULTS: Liposomes decorated with the targeting peptide iRGD
were capable of delivering payloads of fluorescent siRNA to the
syncytiotrophoblast layer, where VEGFR1 is expressed; however, sFlt1e15a mRNA expression and secreted protein levels were not significantly
decreased when compared to explants treated with targeted liposomes
containing a scrambled siRNA sequence. Cytotrophoblast turnover was
unchanged by any treatment.
CONCLUSION: This study demonstrates that a novel nanoparticle
formulation can be exploited to selectively deliver siRNA to the
syncytiotrophoblast layer, without interfering with normal placental
function. Although siRNA transfection under these experimental
conditions did not significantly reduce s-Flt-1e15a mRNA or protein,
further optimisation could lead to a placenta-specific therapy for
preeclampsia, which may alleviate maternal symptoms and minimise any
potentially harmful off-target effects.

O-038
Lysosome Biogenesis and Exocytosis is Augmented in Preeclampsia.
Leonardo Ermini†, 2 Jonathan Ausman†, 2 Martin Post*, 1 Isabella
Caniggia*.2 1Peter Gilgan Center, The Hospital for Sick Children, Toronto,
ON, Canada; 2Sinai Health System, Toronto, ON, Canada.
INTRODUCTION: Lysosomes are essential to maintain cell homeostasis
by orchestrating cellular processes including endocytosis, exocytosis,
autophagy and cholesterol homeostasis. Evidence indicates that lysosomal
biogenesis/ function are tightly controlled by transcription factors
belonging to the MiTF family, including the transcription factor EB
(TFEB). In particular, TFEB coordinates lysosomal activity in response
to a variety of stress stimuli. Ceramide is a key bioactive sphingolipid
that regulates cell fate in several pathophysiological processes. We

69A

have recently reported that placental and maternal circulating ceramide
levels are elevated in pregnancies complicated by preeclampsia leading
to increased trophoblast cell death. However, to date, the relevance of
ceramide in lysosomal biogenesis remains to be established. Hence
we examined the role of ceramide in regulating lysosomal function in
preeclampsia.
METHODS: Placentae from preeclamptic (PE; n=8) and normotensive
pre-term and term control pregnancies, (PTC n=5; TC, n=5) were
processed for TEM to quantify lysosomes. Levels of TFEB and LAMP1, lysosomal markers, in PE and PTC lysates were analyzed by western
blotting (WB). Syncytial microvillus membranes (MVM) from PTC and
PE placentae were isolated by differential centrifugations and analysed
for LAMP-1 expression by WB. Tandem mass spectrometry (MS/MS)
was employed to examine CERs levels in isolated MVM from PE and
PTC. Human choriocarcinoma JEG3 cells were treated with synthetic
C16 CER (20μM), and TFEB and LAMP1 content was analyzed by WB.
Immunofluorescence analysis (IF) was used to assess the subcellular
distribution of TFEB and LAMP-1 in JEG3 cells following C16 CER
exposure.
RESULTS: TEM revealed a significant increase in the number of
lysosomes in the syncytium of PE relative to controls, and this was
primarily due to a striking abundance in heterolysosomes (originated from
lysosome fusion with other cytoplasmic vesicles as autophagolysosomes).
WB analysis showed increased content of TFEB and LAMP-1 in lysates
from PE placentae vs. controls. Accelerated fusion of lysosome with the
plasma membrane in PE, indicative of active lysosomal exocytosis, was
further confirmed by high LAMP-1 levels at the apical MVM. Notably,
MS/MS analysis revealed increased CER 18:0 in MVM from PE compared
to PTC. Treatment of JEG3 cells with C16 CER induced an increase in
the number of lysosomes, and triggered TFEB nuclear translocation. This
associated with augmented LAMP-1 and lysotracker staining, indicating
increased lysosome exocytosis.
CONCLUSION: Our data demonstrate that exuberant lysosome
biogenesis and exocytosis in PE placentae may be due to ceramide-induced
expression and translocation of TFEB. (Funded by CIHR and NIH).

O-039
Circulating mRNA Coding Adrenomedullin is Decreased in Women
Destined to Develop Pre-Eclampsia and May Be of Placental and/or
Endothelial Origin. Carole-Anne Whigham†,1,2 Teresa MacDonald,1,2
Sue Walker,1,2 Natalie Hannan,1,2 Ping Canon,1,2 Tuong Vi Nguyen,1,2
Stephen Tong,1,2 Tu'uhevaha Kaitu'u-Lino.1,2 1Mercy Hospital for Women,
Melbourne, Australia; 2University of Melbourne, Melbourne, Australia.
INTRODUCTION: Pre-eclampsia (PE) is characterised by elevated
release of anti-angiogenic factors, hypertension, maternal endothelial
dysfunction and end organ injury. Adrenomedullin (ADM) is a proangiogenic peptide. It is highly expressed in placenta and endothelial cells.
Our study objectives were to examine whether circulating ADM mRNA is
differentially expressed in 1) women with PE, 2) those destined to develop
PE; and 3) to examine the possible tissue origins of the ADM mRNA.
METHODS: We performed a case control study, selected from a
prospective collection of 2000 participants. We measured circulating
ADM mRNA (using qPCR) at 28 and 36 weeks' gestation among 37
mothers destined to develop PE and 194 normal pregnancies. We measured
circulating ADM mRNA from women with established preterm PE
(n=34) vs gestation matched controls (n=23). To determine if the origin
of circulating ADM mRNA may be placental or endothelial, we measured
ADM mRNA expression in 1)a cohort of placentas <34 weeks gestation
(55 from preterm PE vs 31 preterm placentas without PE) 2) human
umbilical vein endothelial cells (HUVECs) after provoking dysfunction
with either trophoblast conditioned media or TNFα.
RESULTS: Circulating levels of ADM mRNA were very significantly
reduced at both 28 and 36 weeks gestation (p=0.005 at 28 weeks,
p=<0.0001 at 36 weeks) among those destined to develop PE later,
compared to controls. As a predictive test, the area under the curve was
0.64 at 28 weeks and 0.72 at 36 weeks. In established PE, circulating ADM
mRNA was also significantly reduced by ~40% (p=0.01). In PE placentas
there was a non-significant decrease in ADM mRNA expression (p=0.09),

Thursday Orals

GA were observed for K (p=0.02) and Dp (p=0.001) indicating increased
heterogeneity and microcirculatory perfusion. The DDC, Dk and fp did
not correlate with GA.
CONCLUSION: Metrics derived from multiple b-value DWI data
reflect placental perfusion and microstructural changes with gestational
age. This novel imaging method may lead to more accurate diagnosis
of placental perfusion disorders and improved understanding of fetal
growth abnormalities.
*Figure(s) will be available online.

Reproductive Sciences Vol. 25, Supplement 1, March 2018

Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018