SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 290A

290A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

upregulated in lesions whereas several oxidative stress-related enzymes
and factors were expressed at a lower level in lesions. Moreover, some
novel upregulated proteins were identified. RNAseq revealed large
differential expression of 44 genes in ectopic samples confirming ROS
detoxification, GSH conjugation and iron/heme overload pathways. We
found in addition pathways such as protein repair, which had previously
not been identified through the proteomic dataset.
CONCLUSION: Using combined proteomic and transcriptomic
approaches, we identified novel targets, including upregulated prooxidative enzymes as well as downregulated protective factors supporting
the observation of increased oxidative protein modification in ectopic
lesions.

S-114
The Medium-Chain Free Fatty Acid Receptor GPR84 in Endometriosis
Associated Pain. Frank Sacher*,1 Fernando Martinez*,2 Thomas M
Zollner*,1 Oliver M Fischer*,1 Manman Guo†,2 C Bafligil†,2 SG Dakin†,2
Jens Nagel*,1 Krina Zondervan*,2 Christian Becker*,2 Udo Opermann*.2
1
Bayer AG, Berlin, Germany; 2University of Oxford, Oxford, United
Kingdom.
INTRODUCTION: The G-protein coupled receptor GPR84 binds and
becomes activated by saturated medium-chain free fatty acids, is expressed
on pro-inflammatory macrophages and pharmacological inhibition leads
to reduction of inflammatory markers.
METHODS: First, we study GPR84 expression and medium-chain free
fatty acids levels in eutopic endometrium and endometriotic lesions.
Furthermore, we study the effect of GPR84 agonists and antagonist in
various in vitro inflammatory models on cytokine secretion. Finally,
we characterized the link between GRP84 and inflammatory pain in
experimental models including an endometrial explant and an in vivo
CFA inflammatory pain model.
RESULTS: Our results indicate that GPR84 is expressed in endometriotic
lesions and that the endogenous agonists, the medium-chain free fatty acids,
are more abundant in lesions than in eutopic endometrium. Furthermore,
the receptor is induced by pro-inflammatory stimuli such as LPS and
IFNgama and regulates production of inflammatory mediators such as
TNFalpha and IL-6 in peripheral blood monocytes. Inhibition of GPR84
with the antagonist CMPD139 reduces pro-inflammatory cytokines and
growth factors in inflammatory macrophage and endometrial tissue slice
assays. Pharmacological GPR84 inhibition in an in vivo inflammatory
pain model leads to significant reduction of pain scores.
CONCLUSION: GPR84 is a pro-inflammatory receptor and inhibition
reduces cytokine levels and pain score in inflammatory in vitro and in
vivo models suggesting therapeutic benefit of modulating GPR84 ligand
binding in inflammatory conditions such as endometriosis.

S-115
Vitamin D Reverses Uterine Fibroids Associated DNA Damage.
Mohamed Ali†,1,2 Qiwei Yang,1 Lauren Prusinski,1 Sara M Shahin,2
Nagwa A Sabri,2 Ayman Al-Hendy*.1 1Augusta University, Augusta, GA,
United States; 2Clinical Pharmacy Department, Faculty of Pharmacy,
ASU, Cairo, Egypt.
INTRODUCTION: Uterine fibroids (UFs) are more prevalent in African
American (AA) women which is related in part to their vitamin D
deficiency, yet its exact preventive mechanism hasn't been fully revealed.
Literature showed the chemopreventive effect of vitamin D especially in
breast cancer. We have recently shown DNA repair defect in UF. Thus,
we hypothesize that vitamin D3 deficiency exacerbates a DNA damage
response (DDR) defect in UFs, enhancing tumor progression, which may
be rescued by vitamin D treatment.
METHODS: Study was approved by Augusta University IRB. Primary
cells were isolated from post-hysterectomy UF and adjacent myometrium
(MM) tissues of 3 AA patients. The protein and RNA expressions of DNA
damage sensor γH2AX and DNA repair related effectors ATM, RAD50,
RAD51, BRCA1, NBS1 and MRE11 were examined using Western blot
(WB) and RT-qPCR respectively. To determine the role of vitamin D
signaling on DNA damage repair system, uterine smooth muscle cells
(UTSM) were transduced with lentiviruses expressing control or vitamin

Scientific Abstracts

D receptor (VDR) specific shRNAs. The expression of same 6 proteins
was examined. In addition, total RNA was tested against DNA damage
signaling pathway prime PCR array, expression of 11 DNA double strand
breaks repair related genes were validated using RT-qPCR including
BRCA1, BRCA2, CHECK1, CHECK2, RAD50, RAD51, RAD17, NBS1,
MRE11, EXO1 and MSH2. In addition, immortalized human leiomyoma
cells (HuLM) were treated with 100 nM 1, 25 dihydroxyvitamin D3 for
3 days, the RNA expression of same 11 genes was examined. Protein
lysates were tested for same 6 proteins by WB. Immunofluorescence
(IF) intensity was measured for γH2AX, ATM and RAD50. All in treated
and untreated cells.
RESULTS: The expression of DNA repair genes, ATM, RAD50, RAD51,
BRCA1, NBS1, MRE11 were significantly downregulated in UF primary
cells vs. MM at both protein and gene levels (P<.05). In addition, UF
primary cells exhibited a significant increase in γH2AX protein level
vs. MM (p=0.027). VDR silencing exhibited a significant reduction in
mRNA expression of BRCA1, BRCA2, CHECK1, CHECK2, RAD50,
RAD51, RAD17, NBS1, MRE11, EXO1 and MSH2, and protein levels of
ATM, RAD50, RAD51, BRCA1, NBS1, and MRE11 while increasing
γH2AX relative to the control (P<.05). Vitamin D treatment significantly
increased the mRNA expression of the same 11 DNA repair genes and
protein levels of ATM, RAD50, RAD51, BRCA1, NBS1, MRE11 and
decreased γH2AX protein. IF intensity of RAD50 and ATM increased,
while γH2AX decreased upon vitamin D treatment (P<.05).
CONCLUSION: Our data showed DNA repair impairment in
UFs. Vitamin D deficiency in UF patients is linked to DNA damage
exacerbation, suggesting that Vitamin D3 may restore the DDR in UFs and
reverse UF-associated DNA damage. Support: NIH R01HD089553-01,
R01ES028615-01.

S-116
A Novel Role for the Interaction of Myeloperoxidase and CD11b
in Leiomyoma Cells. Nicole M Fletcher†,2 Awoniyi O Awonuga,2 Ira
Memaj,2 Michael P Diamond,1 Ayman A Al-Hendy,1 Ghassan M Saed*.2
1
Augusta University, Augusta, GA, United States; 2Wayne State University,
Detroit, MI, United States.
INTRODUCTION: We have previously demonstrated that
myeloperoxidase (MPO), a marker of inflammation and oxidative stress,
was expressed in leiomyoma cells (DD) and not in normal myometrial
cells (USMC). The expression of MPO was surprising, as it was previously
known to be expressed solely in cells of myeloid origin. To investigate
the significance of MPO expression in DD cells, we sought to determine
the expression and function of β2 integrin (CD11b/CD18), a known
ligand for MPO.
METHODS: Matched pairs of DD and USMC (n=5) were utilized.
Cell viability was assessed after treatment with a CD11b antibody or
isotype control by the TACS MTT Cell Proliferation Assay. Expression
of CD11b mRNA and protein was determined by real-time RT-PCR,
western blot, and flow cytometry analysis. Unpaired t-tests were used to
compare groups for real-time RT-PCR while a one-way ANOVA followed
by Tukey's post hoc tests with Bonferroni correction was performed for
cytotoxicity comparisons.
RESULTS: CD11b expression was significantly higher in DD as
compared to USMC cells. As shown by real-time RT-PCR, CD11b mRNA
levels were 53.5% higher in DD (4.5 ± 0.8 fg/μg RNA) as compared to
USMC (2.1 ± 0.2 fg/μg RNA). CD11b protein levels were 48.9% ± 0.5
higher in DD as compared to USMC, as determined by Western Blot.
Flow cytometry analysis revealed that CD11b is expressed in 80% of
DD while only in 23% of USMC cells. Surprisingly, there was no CD18
detected in either cell line. Treatment with the CD11b antibody resulted
in an 26.6% ± 8.5 and 38.2% ± 10.2 increase in killing of USMC and a
28.8% ± 3.6 and 38.2% ± 5.0 increase in killing of DD cells at the 5 and
10 μg/ml doses, respectively, as compared to controls (p<0.05).
CONCLUSION: CD11b may serve as a potential localized therapeutic
target for leiomyoma. The lack of CD18 expression is surprising and may
indicate a novel role for the interaction of MPO and CD11b alone in the
pathogenesis of leiomyoma.

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