A potencial função do nodal na endometrose da égua

Dissertação de Mestrado Integrado em Medicina VeterináriaAlguns membros da superfamília do TGFβ, tais como o Nodal e o TGFβ1, têm um papel importante na reprodução da égua, sendo que a sua disfunção pode contribuir para patologias uterinas. A endometrose é uma doença degenerativa em que o endométrio normal vai sendo substituído por tecido fibrótico. O objectivo deste estudo consistiu em avaliar: (i) como o Nodal pode influenciar o nível de mRNA dos recetores da PGE2 (EP2; EP4), do TGFβ1 (ALK5; TGFRII), e os próprios (ALK4; ALK7), e ainda, a secreção de prostaglandinas (PGs; PGE2; PGF2α) no endométrio equino; e (ii) como a fase do ciclo éstrico e grau de endometrose pode influenciar estas vias. Endométrios da fase folicular (FF; n=6) e da fase lútea (FL; n=6) foram classificados de acordo com o sistema de Kenney e Doig em categorias I e IIA (n=7), ou IIB e III (n=5). Os explantes foram incubados (24h; 37ºC, 5% CO2) com TNFα, ocitocina ou Nodal (0.1;1;10 ng/mL). A expressão de mRNA foi avaliada por qRT-PCR e a medição de prostaglandinas por ELISA. Em endométrios de categoria I/IIA, o Nodal inibiu a expressão génica de EP2, EP4 e ALK4 e estimulou a de TGFRII, em ambas as fases do ciclo éstrico; e estimulou também os níveis de mRNA de ALK5 e ALK7, apenas na FL. Em endométrios de categoria IIB/III, o Nodal estimulou os níveis de mRNA de EP2, EP4 e ALK5, na FF, e de ALK4 e ALK7, na FL, embora tenha inibido TGFRII e ALK4, na FF, e EP2, EP4, ALK5 e TGFRII na FL. O Nodal na concentração testada mais baixa (0.1ng/mL) estimulou a produção de PGE2 na FF e FL, enquanto que numa concentração superior a inibiu na FF (1ng/mL). A produção de PGF2α foi estimulada na FL com Nodal (0.1 e 10ng/mL). Concluindo, o Nodal parece estar envolvido na endometrose da égua, por afetar negativamente a sinalização da PGE2 anti-fibrótica e positivamente a da citocina pró-fibrótica TGFβ1 e a produção de PGF2α.ABSTRACT - THE POTENCIAL ROLE OF NODAL IN MAR ENDOMETROSIS - Members of TGFβ superfamily, as Nodal and TGFβ1, have an important role in mare´s reproduction, and as such, their dysfunction may contribute for uterine pathologies. Endometrosis is a degenerative process with a switch of normal endometrium to fibrotic tissue. The aim of the study was to assess: (i) how Nodal may influence the receptors of PGE2 (EP2; EP4), TGFβ1 (ALK5; TGFRII), and its own (ALK4; ALK7) mRNA level and prostaglandin (PG) secretion in equine endometrium; and (ii) estrous cycle and endometrosis influence on these vias. Endometria from follicular (FP; n=6) and mid luteal phases (MLP; n=6) were classified in Kenney and Doig´s categories (cat) I and IIA (n=7), or IIB and III (n=5). Endometrium explants were incubated (24h; 37ºC, 5% CO2) with TNF, oxytocin or Nodal (0.1, 1; 10ng/mL). The mRNA expression was assessed by qRT-PCR and ELISA was used for PG measurement. In cat I/IIA endometria, Nodal down-regulated EP2, EP4 and ALK4 mRNA expression and up-regulated TGFRII in both FP and MLP; and ALK5 and ALK7 only in MLP. In cat IIB/III, Nodal up-regulated mRNA levels of EP2, EP4 and ALK5 in FP, and ALK4 and ALK7 in MLP, whereas it inhibited TGFRII and ALK4 in FP, and EP2, EP4, ALK5 and TGFRII in MLP. Nodal (0.1ng/mL) stimulated PGE2 production in both FF and FL, while at a higher concentration (1ng/mL) it decreased PGE2 in FP. The production of PGF2α increased in MLP with Nodal stimulation (at 0.1 and 10ng/mL). In conclusion, Nodal may be involved in endometrosis in the mare, by impairment of anti-fibrotic PGE2 and pro-fibrotic TGFβ1 signaling pathways and increasing PGF2α production.N/

The inhibition of Cathepsin G on endometrial explants with endometrosis in the mare

Research Areas: Veterinary SciencesAlthough 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.info:eu-repo/semantics/publishedVersio