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- Myeloperoxidase inhibition decreases the expression of collagen and metallopeptidase in mare endometria under in vitro conditionsPublication . Amaral, Ana; Fernandes, Carina; Rebordão, Maria Rosa; Szostek-Mioduchowska, Anna; Lukasik, Karolina; Pinto-Bravo, Pedro; Gama, Luis; Skarzynski, Dariusz Jan; Ferreira-Dias, GraçaABSTRACT - 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.
- Inhibition of Myeloperoxidase Pro-Fibrotic Effect by Noscapine in Equine EndometriumPublication . Amaral, Ana; Cebola, Nélio; Zóstek-Mioduchowska, Anna; Rebordão, Maria Rosa; Kordowitzki, Paweł; Skarzynski, Dariusz; Ferreira-Dias, GraçaMyeloperoxidase 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.
- Noscapine acts as a protease inhibitor of in vitro elastase-induced collagen deposition in equine endometriumPublication . Amaral, Ana; Fernandes, Carina; Szóstek-Mioduchowska, Anna; Rebordão, Maria Rosa; Skarzynski, Dariusz Jan; Ferreira-Dias, GraçaABSTRACT - 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.
- The inhibition of Cathepsin G on endometrial explants with endometrosis in the marePublication . Amaral, Ana; Fernandes, Carina; Faes Morazzo, Sofia; Rebordão, Maria Rosa; Szóstek-Mioduchowska, Anna; Lukasik, Karolina; Gawronska-Kozak, Barbara; Gama, Luis; Jan Skarzynski, Dariusz; Ferreira-Dias, GraçaAlthough proteases found in neutrophil extracellular traps (NETs) have antimicrobial properties, they also stimulate collagen type 1 (COL1) production by the mare endometrium, contributing for the development of endometrosis. Cathepsin G (CAT), a protease present in NETs, is inhibited by specific inhibitors, such as cathepsin G inhibitor I (INH; beta-keto-phosphonic acid). Matrix metallopeptidases (MMPs) are proteases involved in the equilibrium of the extracellular matrix. The objective of this study was to investigate the effect of CAT and INH (a selective CAT inhibitor) on the expression of MMP-2 and MMP-9 and on gelatinolytic activity. In addition, the putative inhibitory effect of INH on CAT-induced COL1 production in mare endometrium was assessed. Endometrial explants retrieved from mares in follicular phase or midluteal phase were treated for 24 or 48 h with CAT, inhibitor alone, or both treatments. In explants, transcripts (quantitative polymerase chain reaction) of COL1A2, MMP2, and MMP9, as well as the relative abundance of COL1 protein (Western blot), and activity of MMP-2 and MMP-9 (zymography) were evaluated. The protease CAT induced COL1 expression in explants, at both estrous cycle phases and treatment times. The inhibitory effect of INH was observed on COL1A2 transcripts in follicular phase at 24-h treatment, and in midluteal phase at 48 h (P < 0.05), and on the relative abundance of COL protein in follicular phase and midluteal phase explants, at 48 h (P < 0.001). Our study suggests that MMP-2 might also be involved in an earlier response to CAT, and MMP-9 in a later response, mainly in the follicular phase. While the use of INH reduced CAT-induced COL1 endometrial expression, MMPs might be involved in the fibrogenic response to CAT. Therefore, in mare endometrium, the use of INH may be a future potential therapeutic means to reduce CAT-induced COL1 formation and to hamper endometrosis establishment.
- The In vitro inhibitory effect of sivelestat on elastase induced collagen and metallopeptidase expression in equine endometriumPublication . Amaral, Ana; Fernandes, Carina; Rebordão, Maria Rosa; Szóstek-Mioduchowska, Anna; Lukasik, Karolina; Gawronska-Kozak, Barbara; Gama, Luis; Skarzynski, Dariusz J.; Ferreira-Dias, GraçaAbstract: 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.
- The inhibitory effect of noscapine on the in vitro cathepsin G-induced collagen expression in equine endometriumPublication . Amaral, Ana; Fernandes, Carina; Szostek-Mioduchowska, Anna; Lukasik, Karolina; Rebordão, Maria Rosa; Pinto-Bravo, Pedro; Skarzynski, Dariusz Jan; Ferreira-Dias, GraçaABSTRACT - 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.
- Enzymes present in neutrophil extracellular traps may stimulate the fibrogenic PGF(2 alpha) pathway in the mare endometriumPublication . Rebordão, Maria Rosa; Amaral, Ana; Fernandes, Carina; Silva, E; Lukasik, Karolina; Szóstek-Mioduchowska, Anna; Bravo, Pedro; Galvão, António; Skarzynski, Dariusz J.; Ferreira-Dias, GraçaEndometrosis 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.