24 research outputs found
Mare endometrium : physiological and pathological involvement of hormones and neutrophil extracellular traps
Tese de Doutoramento em Ciências Veterinárias, Especialidade de Ciências Biológicas e Biomédicas.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.RESUMO - Endométrio da égua: envolvimento fisiológico e patológico de hormonas e de redes extracelulares de neutrófilos - Nesta tese foram abordados dois temas reprodutivos em éguas. Os objectivos destes estudos
consistiram na avaliação: (i) da administração crónica de ocitocina na manutenção do corpo
lúteo em éguas na fase lútea média, e os mecanismos celulares e moleculares no endométrio;
(ii) da capacidade dos neutrófilos equinos para produzirem redes extracelulares de neutrófilos
(NETs) in vitro e in vivo quando estimulados com bactérias de éguas com endometrite; (iii)
dos efeitos in vitro de componentes das NETs na fibrogénese do endométrio equino e sua
associação com lesões inflamatórias no endométrio ou com o ciclo éstrico; e (iv) do
envolvimento das vias da PGF2α e PGE2 na deposição de colagénio no endométrio da égua,
incubado com proteases das NETs. No primeiro estudo, o prolongamento da função lútea em
67% das éguas tratadas com ocitocina, parece estar relacionado com alteração da expressão
espacial dos receptores de ocitocina e progesterona (PGR) no endométrio. A diminuição do
receptor 2 dos estrogénios (ESR2) pode ser responsável pela manutenção do PGR no epitélio
luminal e glandular do endométrio e atenuar a transcrição do ESR1. Os neutrófilos equinos
libertaram NETs na presença de bactérias causadoras de endometrite podendo ser um
mecanismo complementar no combate à endometrite. As proteases das NETs in vitro
aumentaram a produção de colagénio do tipo I (COL1), característico de fibrose, no
endométrio, embora a resposta a cada protease dependesse do ciclo éstrico e/ou da categoria
do endométrio. O aumento do COL1 associado ao incremento de PGF2α e/ou da transcrição
do seu receptor e à diminuição da PGE2 e/ou do seu receptor 2, parecem implicar as
prostaglandinas e as proteases das NETs na fibrogénese no endométrio equino. Alterações nos
mediadores das prostaglandinas, induzidas pelos componentes das NETs, podem instigar as
vias da PGF2a ou da PGE2, como mecanismos adicionais de fibrose do endométrio da égua.N/
Cytokines and neutrophil extracellular traps in the equine endometrium: friends or foes?
Articles in International JournalsCytokines may play a dual role in the reproductive tract – either involved in physiologic processes or mediating inflammation and other
pathologic processes. Physiologic secretory and angiogenic function in the equine endometrium appears to be regulated by cytokines
TNFa, FasL, IFNg through their receptors. These receptors are present in glandular epithelium, and stroma cells and their mRNA expression
changes throughout the estrous cycle. Besides, interleukins (IL-1a and IL-1b) and their receptors mRNA expression vary according to
various degrees of endometrium inflammation (endometritis) and fibrosis (endometrosis). A novel paradigm in innate immunity and neutrophils
(PMN) hyperactivation is PMN ability to cast out their DNA in response to infectious stimuli. These PMN extracellular traps (NETs)
bind and kill pathogens, at the infection site. The intriguing dilemma is that even though NETs may function as a first line of defense, they
also release molecules that may contribute to tissue damage. Thus, we postulate that PMN present in the endometrium at estrus, mating
or infection, might form NETs, and release nucleic and cytoplasmic proteins with immunomodulatory properties. Equine PMN stimulated in
vitro showed NETs formation capacity when in contact with some bacteria strains obtained from mares with endometritis, such as Streptococcus
zooepidemicus, Escherichia coli and Staphylococus capitis. In this regard, even though NETs and cytokines function as an effective
antimicrobial first line of defense or modulate physiologic endometrial function, respectively (friends), they may also be involved in endometrial
fibrosis pathogenesis and endometrial secretory function impairment, due to enhanced NETs formation and/or a decrease on NETs
degradation (foes)
Noscapine acts as a protease inhibitor of in vitro elastase-induced collagen deposition in equine endometrium
Research Areas: Biochemistry & Molecular Biology ; ChemistryABSTRACT - 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.info:eu-repo/semantics/publishedVersio
The inhibitory effect of noscapine on the in vitro cathepsin G-induced collagen expression in equine endometrium
Research Areas: Life Sciences & Biomedicine ; MicrobiologyABSTRACT - Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium
in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine
endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with
anti-neoplastic and anti-fibrotic properties) would reduce COL1A2 transcription (evaluated by qPCR)
and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicular and mid-luteal phases treated for 24 or 48 h. The explants treated with
CAT increased COL1 expression. Noscapine decreased COL1A2 transcription at both estrous cycle
phases, but COL1 relative protein only at the follicular phase, both induced by CAT. Additionally,
the noscapine anti-fibrotic action was found to be more effective in the follicular phase. The CAT
treatment caused more fibrosis at the longest period of treatment, while noscapine acted better at the
shortest time of treatment. Our results showed that noscapine could act as an anti-fibrotic drug in
equine endometrosis by inhibiting CAT in vitro. Noscapine offers a new promising therapeutic tool
for treating fibrosis as a single non-selective agent to be considered in the future.info:eu-repo/semantics/publishedVersio
The In vitro inhibitory effect of sivelestat on elastase induced collagen and metallopeptidase expression in equine endometrium
Research Areas: Agriculture ; Veterinary Sciences ; ZoologyAbstract: 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.info:eu-repo/semantics/publishedVersio
Myeloperoxidase inhibition decreases the expression of collagen and metallopeptidase in mare endometria under in vitro conditions
Research Areas: Agriculture ; Veterinary Sciences ; ZoologyABSTRACT - 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.info:eu-repo/semantics/publishedVersio
Enzymes present in neutrophil extracellular traps may stimulate the fibrogenic PGF(2 alpha) pathway in the mare endometrium
Research Areas: AgricultureVeterinary SciencesEndometrosis is a fibrotic disease in mare endometrium whose pathological
mechanisms remain obscure. Prostaglandin (PG)F2α, despite modulating reproductive physiological
processes, may also provoke local pathological collagen deposition (fibrogenesis). Neutrophil extracellular traps (NETs) released during inflammation have been linked to fibrogenesis in several
tissues. We have previously shown that enzymes found in NETs increase in vitro collagen production
in mare endometrium. In this study, activation of PGF2α-pathway in equine endometrial explants
challenged in vitro by enzymes found in NETs is shown. Our results indicate that both endocrine
microenvironment (estrous cycle phase) and healthy or pathological conditions of endometrial tissues
play an important role in PGF2α-pathway activation. In the endometrium of the follicular phase,
we have observed both high production of PGF2α and/or PGF2α receptor gene transcription under
the action of enzymes found in NETs, both conditions associated with fibrogenesis in other tissues.
Nevertheless, transcription of the PGF2α receptor gene does not appear to be hormone-dependent,
albeit their levels seem to be dependent on endometrial category in the mid-luteal phase. This study
suggests that enzymes existing in NETs may instigate changes on PGF2α mediators, which may
become an additional mechanism of fibrogenesis in mare endometrium.info:eu-repo/semantics/publishedVersio
Metallopeptidades 2 and 9 genes epigenetically modulate equine endometrial fibrosis
Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium (P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB (P < 0.05) and III (P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I (P < 0.001) and IIA (P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA (P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9, but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.info:eu-repo/semantics/publishedVersio
Collagen Type III as a Possible Blood Biomarker of Fibrosis in Equine Endometrium
Research Areas:Agriculture ; Veterinary Sciences ; ZoologyCollagen pathological deposition in equine endometrium (endometrosis) is responsible for infertility. Kenney and Doig’s endometrial biopsy histopathological classification is the gold standard method for endometrosis evaluation, whereby blood biomarkers identification would be less invasive and could provide additional information regarding endometrosis diagnosis and fertility prognosis. This study aimed to identify blood biomarkers for endometrosis diagnosis (42 mares were used in experiment 1), and fertility assessment (50 mares were used in experiment 2). Reproductive examination, endometrial biopsy histopathological classification (Kenney and Doig) and blood collection were performed. Endometrium and serum collagen type I (COL1) and type III (COL3), and hydroxyproline concentrations were measured (ELISA). Serum COL3 cut-off value of 60.9 ng/mL allowed healthy endometria (category I) differentiation from endometria with degenerative/fibrotic lesions (categories IIA, IIB or III) with 100% specificity and 75.9% sensitivity. This cut-off value enabled category I + IIA differentiation from IIB + III (76% specificity, 81% sensitivity), and category III differentiation from others (65% specificity, 92.3% sensitivity). COL1 and hydroxyproline were not valid as blood biomarkers. Serum COL3 cut-off value of 146 ng/mL differentiated fertile from infertile mares (82.4% specificity, 55.6% sensitivity), and was not correlated with mares’ age. Only COL3 may prove useful as a diagnostic aid in mares with endometrial fibrosis and as a fertility indicator.info:eu-repo/semantics/publishedVersio
Inhibition of Myeloperoxidase Pro-Fibrotic Effect by Noscapine in Equine Endometrium
Myeloperoxidase is an enzyme released by neutrophils when neutrophil extracellular
traps (NETs) are formed. Besides myeloperoxidase activity against pathogens, it was also linked to many diseases, including inflammatory and fibrotic ones. Endometrosis is a fibrotic disease of the
mare endometrium, with a large impact on their fertility, where myeloperoxidase was shown to induce fibrosis. Noscapine is an alkaloid with a low toxicity, that has been studied as an anti-cancer drug, and more recently as an anti-fibrotic molecule. This work aims to evaluate noscapine inhibition
of collagen type 1 (COL1) induced by myeloperoxidase in equine endometrial explants from follicular and mid-luteal phases, at 24 and 48 h of treatment. The transcription of collagen type 1 alpha 2 chain (COL1A2), and COL1 protein relative abundance were evaluated by qPCR and Western
blot, respectively. The treatment with myeloperoxidase increased COL1A2 mRNA transcription and COL1 protein, whereas noscapine was able to reduce this effect with respect to COL1A2 mRNA transcription, in a time/estrous cycle phase-dependent manner (in explants from the follicular phase, at 24 h of treatment). Our study indicates that noscapine is a promising drug to be considered as an anti-fibrotic molecule to prevent endometrosis development, making noscapine a strong candidate to be applied in future endometrosis therapies