The Inhibitory Effect of Noscapine on the In Vitro Cathepsin G-Induced Collagen Expression in Equine Endometrium.

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 PGF2α Pathway in the Mare Endometrium.

Endometrosis, a fibrotic disease of mare endometrium, impairs uterine function. Prostaglandins (PG), despite modulating reproductive physiological functions, may also cause local pathological collagen deposition (fibrogenesis). We have previously shown that neutrophil extracellular traps (NETs) may also favor mare endometrosis. The aim of this study was to investigate the effect of enzymes present in NETs on PGF2α-pathway activation. Kenney and Doig's type I/IIA and IIB/III mare endometria, from follicular phase (FLP) and mid-luteal (MLP) phase, were cultured in vitro in the presence of NETs enzymes (elastase, cathepsin-G or myeloperoxidase). Production of PGF2α (EIA) and transcription (qPCR) of its synthases (PTGS2, AKR1C3) and receptor (PTGFR) genes were evaluated. PGF2α and PTGFR were influenced by endometrial category and estrous cycle phase. In FLP endometrium, NETs enzymes induced both high PGF2α production and/or PTGFR transcription. In MLP type I/IIA tissues, down-regulation of PTGFR transcripts occurred. However, in MLP type IIB/III endometrium, high levels of PTGFR transcripts were induced by NETs enzymes. As PGF2α-pathway activation facilitates fibrogenesis in other tissues, PGF2α may be involved in endometrosis pathogenesis. In the mare, the endocrine microenvironment of healthy and pathological endometrium might modulate the PGF2α pathway, as well as fibrosis outcome on endometrium challenged by NETs enzymes.

Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct.

The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (FGF1, FGF2, VEGF) and their receptors-FGFR1, FGFR2, KDR, respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares' oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns.

Myeloperoxidase Inhibition Decreases the Expression of Collagen and Metallopeptidase in Mare Endometria under In Vitro Conditions.

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.

Collagens and DNA methyltransferases in mare endometrosis.

Inflammation and fibroproliferative diseases may be modulated by epigenetic changes. Therefore, we suggest that epigenetic mechanisms could be involved in equine endometrosis pathogenesis. DNA methylation is one of the methods to evaluate epigenetics, through the transcription of methyltransferases (DNMT1, DNMT3A, DNMT3B). The correlation between DNMTs and collagen (COL) transcripts was assessed for the different Kenney and Doig's (Current Therapy in Theriogenology. Philadelphia: WB Saunders; 1986) endometrium categories. Endometrial biopsies were randomly collected from cyclic mares. Histological classification (category I, n = 13; II A, n = 17; II B, n = 12; and III, n = 7) and evaluation of COL1A2, COL3A1 and DNMTs transcripts by qPCR, were performed. Data were analysed by one-way analysis of variance (ANOVA), Kruskal-Wallis test and Pearson correlation. As mares aged, there was an increase in endometrium fibrosis (p < .01), and in DNMT1 mRNA (p < .001). Considering DNMT3B transcripts for each category, there was an increase with fibrosis (p < .05). No changes were observed for DNMT1 and DNMT3A transcripts. However, DNMT3A mRNA levels were the highest in all categories (p < .01). In category I endometrium, a positive correlation was observed for transcripts of all DNMTs in both COLs (p < .01). In category IIA, this correlation was also maintained for all DNMTs transcripts in COL1A2 (p < .05), but only for DNMT3B in COL3A1 (p < .05). In category IIB, there was a positive correlation between DNMT3B and COL3A1 (p < .05). In category III, a positive correlation was only observed between DNMT3B and COL3A1 (p < .05). Our results suggest that there is a disturbance in COLs and DNMTs correlation during fibrosis.

Impairment of the antifibrotic prostaglandin E2 pathway may influence neutrophil extracellular traps-induced fibrosis in the mare endometrium.

Prostaglandin E2 (PGE2) has contradictory effects in many organs. It may have proinflammatory, anti-inflammatory, or anti-fibrotic roles, depending on the type of receptors to which it binds. By signaling through its receptors EP2 and EP4, PGE2 mediates anti-inflammatory and anti-fibrotic actions. In spite of chronic endometrial fibrosis (endometrosis) being a major cause of mare infertility, its pathogenesis is not fully understood. We have shown that contact of mare endometrium in vitro with neutrophil extracellular traps (NETs) proteases favors endometrial collagen type I production. Therefore, we investigated the involvement of the PGE2 pathway in collagen deposition in mare endometrium, challenged in vitro with proteases present in NETs. Mare endometria (Kenney and Doig categories I/IIA and IIB/III), obtained in the follicular phase (FLP) and mid-luteal phase (MLP), were incubated for 24 h with components found in NETs (elastase, cathepsin-G, and myeloperoxidase). Secretion of PGE2 and transcripts for specific PGE synthase (PGES) and PGE2 receptors (EP2 and EP4) were evaluated. Impaired PGE2 production and low EP2 transcript abundance depended on the endometrial category and estrous cycle phase. Impairment of PGE2 and/or EP2 might play a role in FLP (category IIB/III) and MLP (I/IIA) endometrial fibrogenesis because of the reduction in its antifibrotic capacity. In conclusion, priming of the endometrium with endogenous ovarian steroids might inhibit the antifibrotic PGE2 pathway either in healthy or pathologic tissues with collagen formation after NETs proteases action.

Elastase inhibition affects collagen transcription and prostaglandin secretion in mare endometrium during the estrous cycle.

We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular-FP (n = 8) and mid luteal-MLP (n = 7) phases explants were cultured for 24-48 hr with medium alone (Control), ELA (0.5 µg/ml,1 µg/ml), sivelestat - ELA inhibitor (INH,10 µg/ml), or ELA (0.5 µg/ml,1 µg/ml) + INH (10 µg/ml). COL1 gene transcription was done by qRT-PCR and PGE2 and PGF2 α determination in culture medium by EIA. In FP, at 24 hr, ELA0.5 increased COL1 transcription (p < 0.001) but its inhibition (ELA0.5 + INH10) decreased COL1 transcription (p < 0.01) and PGF2 α production (p < 0.05). Also, ELA0.5 + INH10 or ELA1 + INH10 raised PGE2 production (p < 0.01). At 48 hr, ELA1 increased COL1 transcription (p < 0.01) and PGF2 α production (p < 0.001), but its inhibition (ELA1 + INH10) decreased these actions (p < 0.01; p < 0.05, respectively). Besides, ELA1 + INH10 incubation increased PGE2 (p < 0.05). PGF2 α also augmented with ELA0.5 (p < 0.001), but lowered with ELA0.5 + INH10 (p < 0.01). In MLP, ELA0.5 up-regulated COL1 transcription (24 hr, p < 0.01; 48 hr, p < 0.001), but ELA0.5 + INH10 decreased it (24 hr, p < 0.05; 48 hr, p < 0.001). At 48 hr, incubation with ELA1 also increased COL1 transcription and PGF2 α production (p < 0.05), but PGF2 α production decreased with ELA1 + INH10 incubation (p < 0.05). PGE2 production was higher in ELA1 + INH10 incubation (p < 0.05). Therefore, ELA inhibition may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti-fibrotic PGE2 and inhibiting pro-fibrotic PGF2 α.

Constituents of neutrophil extracellular traps induce in vitro collagen formation in mare endometrium.

Neutrophil extracellular traps (NETs) are DNA complexes carrying nuclear and cytoplasmic proteins, such as elastase (ELA), cathepsin-G (CAT) and myeloperoxidase (MPO). Mare endometrosis is a chronic degenerative process characterized by excessive collagen in endometrium. While NETs fight bacteria that cause endometritis, they may trigger endometrial fibrogenesis. The aim was to evaluate the in vitro effect of some NETs components on mare endometrial fibrogenesis and determine its relationship with histopathology or estrous cycle. Endometrial explants were incubated with NETs components (ELA, CAT, MPO or oxytocin). Collagen type I (COL1) protein and type I and III (COL3) gene transcription were evaluated in follicular and mid-luteal phases endometria (Kenney and Doig type I/IIA and IIB/III). Increased COL1 occurred with all NETs proteins, although endometrial response to each NETs protease depended on estrous cycle and/or endometrial category. Since ELA enhanced COL1 production, NETs persistence might be linked to endometrosis. Estrous cycle influenced COL1 protein concentration and COL3 transcripts, suggesting that follicular phase may favor endometrial collagen production. However, luteal phase endometria with moderate or severe lesions may be also susceptible to fibrotic effects of NETs constituents. These data propose that NETs involvement in chronic endometritis in mares may act as putative endometrial fibrogenic mediators.

Endometrial prostaglandin synthases, ovarian steroids, and oxytocin receptors in mares with oxytocin-induced luteal maintenance.

Oxytocin (OXT) has been used to prolong the luteal phase in mares, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of chronic exogenous OXT administration to mid-luteal phase mares on luteal maintenance. Also, endometrial expression of prostaglandin endoperoxide synthase 2 (PTGS2), prostaglandin F2α, E2 and I2 synthases (AKR1C3, PTGES, and PTGIS), oxytocin receptor (OXTR), progesterone receptor (PGR), and estrogen receptors 1 (ESR1) and 2 (ESR2) were assessed in mares experiencing luteal maintenance 2 weeks after chronic exogenous OXT administration. Control mares (n = 5; C group) received 6 mL of saline im, whereas OXT (60 units/mare) was administered im (n = 6; OXT group), every 12 hours, on days 7 to 14 postovulation. After endometrial biopsy in groups C (Day 10) and OXT (Day 24), luteolysis occurred within 3 or 6 days, respectively. Luteal maintenance took place in 4 of 6 (67%) of OXT-treated mares. Progesterone in C group was the highest on biopsy day (P < 0.05). In OXT mares, PTGS2, ESR1 (P < 0.05), PTGES, PTGIS, PGR, and ESR2 (P < 0.01) gene transcription decreased, whereas OXTR increased (P < 0.05) in comparison with the C group. In OXT-treated mares, endometrial ESR2 protein expression decreased (P < 0.05), but OXTR increased (P < 0.05) compared with control animals. In both experimental groups, PTGS2 was mainly immunolocalized in surface epithelium, whereas AKR1C3, PTGES, PTGIS, and PGR were in surface and glandular epithelia. ESR1 and ESR2 were found in glandular epithelium and OXTR in stromal cells. High immunolabeling for PTGES, PTGIS, PGR, and OXTR and low for ESR2 was detected in endometrium of OXT-group mares with extended diestrus. Prolonged luteal function associated with chronic OXT treatment may be related to different spatial expression of OXTR and PGR in the endometrium. The observed reduction of endometrial ESR2 may be responsible for the maintenance of PGR in luminal and glandular epithelium. Also, ESR2 may attenuate the transcriptional activity of ESR1 in mare endometrium. This study offers new knowledge on the endometrial expression of ovarian steroids and OXT receptors in OXT pharmacologically induced luteal maintenance in the mare.