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Mare endometrium : physiological and pathological involvement of hormones and neutrophil extracellular traps
Publication . Crisóstomo, Maria Rosa Rebordão Cordeiro Simões; Dias, Graça Maria Leitão Ferreira
Two reproductive topics in mares were addressed in this thesis. The aims of the studies were
to evaluate: (i) the effect of chronic oxytocin administration to mid-luteal phase mares on
luteal maintenance and its cellular and molecular mechanisms at endometrial level; (ii) the
capacity of equine neutrophils to produce neutrophil extracellular traps (NETs) in vitro when
stimulated with bacteria obtained from mares with endometritis, and to determine if NETs
release also occurred in vivo in mares with endometritis; (iii) the in vitro effects of some
NETs components on mare endometrial fibrogenic capacity and to determine if they could
depend on endometrial inflammatory lesions or estrous cycle phases; and (iv) the involvement
of PGF2α and PGE2 pathways in collagen deposition on mare endometrium, challenged with
NETs proteases. In the first study, luteal maintenance occurred in 67% of oxytocin treated
mares, which may be related to oxytocin and progesterone (PGR) receptors spatial expression
in endometrium. Reduction of endometrial estrogen receptor 2 (ESR2) may be responsible for
the maintenance of PGR in luminal and glandular epithelium and may attenuate ESR1
endometrial transcriptional activity. Equine neutrophils were able to release NETs in the
presence of bacteria that cause mare endometritis, and might be a complementary mechanism
to fight endometritis. By in vitro studies with NETs proteases, increased collagen type I
(COL1) production characteristic of fibrosis was observed, although endometrial response to
each NETs protease depended on estrous cycle and/or endometrial category. Also, NETs
proteases were linked to fibrogenesis, by increased synthesis of PGF2a and/or PGF2a
receptor transcripts and impaired PGE2 or PGE2 receptor 2 transcripts associated to increased
COL1. These effects were influenced by endometrium type and estrous cycle phases. Injury
induced-changes on PG mediators by NETs components may instigate PGF2α or PGE2 vias,
as additional pathways in mare endometrial fibrogenesis.
The In vitro inhibitory effect of sivelestat on elastase induced collagen and metallopeptidase expression in equine endometrium
Publication . Amaral, Ana; Fernandes, Carina; Rebordão, Maria Rosa; Szóstek-Mioduchowska, Anna; Lukasik, Karolina; Gawronska-Kozak, Barbara; Gama, Luis; Skarzynski, Dariusz J.; Ferreira-Dias, Graça
Abstract: Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease
found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium.
Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate
the e ects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and
gelatinolytic activity, as well as the potential inhibitory e ect of sivelestat on ELA-induced COL1 in
equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were
treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and
MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and
MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in
MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP
(48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h)
and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory e ect was detected in MMP9 transcripts
in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be
implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease
ELA-induced COL1 deposition and hinder endometrosis development.
Myeloperoxidase inhibition decreases the expression of collagen and metallopeptidase in mare endometria under in vitro conditions
Publication . Amaral, Ana; Fernandes, Carina; Rebordão, Maria Rosa; Szostek-Mioduchowska, Anna; Lukasik, Karolina; Pinto-Bravo, Pedro; Gama, Luis; Skarzynski, Dariusz Jan; Ferreira-Dias, Graça
ABSTRACT - Neutrophils can originate neutrophil extracellular traps (NETs). Myeloperoxidase (MPO) is
a peroxidase found in NETs associated to equine endometrosis and can be inhibited by 4-aminobenzoic
acid hydrazide (ABAH). Metallopeptidases (MMPs) participate in extracellular matrix stability and
fibrosis development. The objectives of this in vitro work were to investigate, in explants of mare’s
endometrium, (i) the ABAH capacity to inhibit MPO-induced collagen type I (COL1) expression;
and (ii) the action of MPO and ABAH on the expression and gelatinolytic activity of MMP-2/-9.
Explants retrieved from the endometrium of mares in follicular or mid-luteal phases were treated
with MPO, ABAH, or their combination, for 24 or 48 h. The qPCR analysis measured the transcription
of COL1A2, MMP2, and MMP9. Western blot and zymography were performed to evaluate COL1
protein relative abundance and gelatinolytic activity of MMP-2/-9, respectively. Myeloperoxidase
elevated COL1 relative protein abundance at both treatment times in follicular phase (p < 0.05). The
capacity of ABAH to inhibit MPO-induced COL1 was detected in follicular phase at 48 h (p < 0.05).
The gelatinolytic activity of activated MMP-2 augmented in mid-luteal phase at 24 h after MPO
treatment, but it was reduced with MPO+ABAH treatment. The activity of MMP-9 active form
augmented in MPO-treated explants. However, this effect was inhibited by ABAH in the follicular
phase at 48 h (p < 0.05). By inhibiting the pro-fibrotic effects of MPO, it might be possible to reduce
the development of endometrosis. Metallopeptidase-2 might be involved in an acute response to
MPO in the mid-luteal phase, while MMP-9 might be implicated in a prolonged exposition to MPO
in the follicular phase.
Noscapine acts as a protease inhibitor of in vitro elastase-induced collagen deposition in equine endometrium
Publication . Amaral, Ana; Fernandes, Carina; Szóstek-Mioduchowska, Anna; Rebordão, Maria Rosa; Skarzynski, Dariusz Jan; Ferreira-Dias, Graça
ABSTRACT - Endometrosis is a reproductive pathology that is responsible for mare infertility. Our
recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as
elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived
from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present
work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain
(COL1A2) mRNA and COL1 protein relative abundance induced by ELA in endometrial explants
of mares in the follicular or mid-luteal phases at 24 or 48 h of treatment. The COL1A2 mRNA was
evaluated by qPCR and COL1 protein relative abundance by Western blot. In equine endometrial
explants, ELA increased COL 1 expression, while NOSC inhibited it at both estrous cycle phases
and treatment times. These findings contribute to the future development of new endometrosis
treatment approaches. Noscapine could be a drug capable of preventing collagen synthesis in mare’s
endometrium and facilitate the therapeutic approach.
New insights on the inhibition of neutrophil extracellular traps enzymes in equine endometrium
Publication . Amaral, Ana Sofia Pires; Dias, Graça Maria Leitão Ferreira; Skarzynski, Dariusz Jan
Mares physiologically develop a post-breeding endometritis characterized by a fast arrival of neutrophils into the uterine lumen. These neutrophils besides releasing granules of proteolytic and cytotoxic enzymes, may also deliver to the extracellular environment their DNA, histones and enzymes forming neutrophil extracellular traps (NETs). Besides trapping and fighting pathogens, NETs persistence has been also associated to the development of pathological conditions, such as fibrosis. The enzymes found in NETs, such as elastase (ELA), cathepsin G (CAT) and myeloperoxidase (MPO) act as pro-fibrotic factors in equine endometrial fibrosis, by inducing collagen type I (COL1) accumulation. Matrix metallopeptidases (MMPs) are crucial for this extracellular matrix remodeling. Prostaglandins (PG)s E2 and F2α have been described as possessing anti or pro-fibrotic effects. Equine endometrial explants from follicular phase (FP) or mid-luteal phase (MLP) were treated in vitro with ELA, CAT or MPO and their specific inhibitors for 24 or 48h. This work aimed to evaluate the explants response to: (i) ELA inhibition by sivelestat sodium salt (SIV) on COL1A2 transcription and PGE2 and PGF2α secretion; (ii) ELA and SIV treatment on MMP-2 and MMP-9 activity, and the inhibitory effect of SIV on ELA-induced COL1; (iii) CAT and Cathepsin G inhibitor I (β-keto-phosphonic acid; INH) treatment on MMP-2 and MMP-9 activity, and the effect of INH on CAT-induced COL1 production; (iv) the inhibitory effect of 4-aminobenzoic acid hydrazide (ABAH) on MPO-induced COL1 and the effect of MPO and ABAH on MMP-2 and MMP-9 gelatinolytic activity. In FP, COL1A2 transcription decreased in SIV-treated group, simultaneously with reduced pro-fibrotic PGF2α and increased anti-fibrotic PGE2 production. In ELA- and SIV-treated explants, MMPs expression depended on estrous cycle phase and time of treatment. Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h). The effect of INH was observed on CAT-induced COL1 in both phases at 48h. The MMP-2 might be involved in an earlier response to CAT, while MMP-9 in a later response in FP. The inhibitory effect of ABAH on MPO-induced COL1 was detected in FP at 48h. Matrix metallopeptidase-2 appears to be involved in an acute response to MPO treatment in MLP and MMP-9 in FP in a prolonged MPO treatment. The use of specific inhibitors of ELA, CAT or MPO, might be the grounds for future development of specific drugs to be used as prophylaxis or therapy of endometrosis in the mare.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
3599-PPCDT
Funding Award Number
PTDC/CVT-REP/4202/2014