Neutrophil extracellular traps and fibrotic cytokines: a challenge in endometrium fibrosis

Funder

Organizational Unit

Publications

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.

2021-01-20Doctoral thesis

Open access

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.

2020-05-16Journal article

Open access

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.

2021-05-19Journal article

Open access

The inhibitory effect of noscapine on the in vitro cathepsin G-induced collagen expression in equine endometrium

Publication . Amaral, Ana; Fernandes, Carina; Szostek-Mioduchowska, Anna; Lukasik, Karolina; Rebordão, Maria Rosa; Pinto-Bravo, Pedro; Skarzynski, Dariusz Jan; Ferreira-Dias, Graça

ABSTRACT - 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.

2021-10-19Journal article

Open access

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.

2021-01-16Journal article

Open access