Metallopeptidades 2 and 9 genes epigenetically modulate equine endometrial fibrosis

Abstract

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, <i>n</i> = 6; category IIA, <i>n</i> = 6; category IIB, <i>n</i> = 6 and category III, <i>n</i> = 6). Transcription of <i>COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1</i>, and <i>TIMP2</i> genes and DNA methylation pattern by pyrosequencing of <i>COL1A1, MMP2, MMP9, TIMP1</i> genes were evaluated. Both <i>MMP2</i> and <i>MMP9</i> transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (<i>P</i> < 0.05). <i>TIMP1</i> transcripts were higher in category III, when compared to category I endometrium (<i>P</i> < 0.05). No differences were found for <i>COL1A1, COL1A2, COL3A1</i> and <i>TIMP2</i> transcripts between endometrial categories. There were higher methylation levels of (i) <i>COL1A1</i> in category IIB (<i>P</i> < 0.05) and III (<i>P</i> < 0.01), when compared to category I; (ii) <i>MMP2</i> in category III, when compared to category I (<i>P</i> < 0.001) and IIA (<i>P</i> < 0.05); and (iii) <i>MMP9</i> in category III, when compared to category I and IIA (<i>P</i> < 0.05). No differences in <i>TIMP1</i> methylation levels were observed between endometrial categories. The hypermethylation of <i>MMP2</i> and <i>MMP9</i>, but not of <i>COL1A1</i> 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 (<i>MMP2</i> and <i>MMP9</i>) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.