Extracellular matrix and microvascularization regulatory genes in the mare placenta

In the mare placenta, molecular mechanisms regulating extracellular matrix (ECM) collagen deposition and vascularization are not fully understood. This study evaluated: (i) gene transcription and expression of enzymes regulating collagen deposition (matrix metalloproteinases - MMP2, MMP9), and their inhibitors (TIMP1, TIMP2); (ii) gene transcription of vascular endothelial growth factor (VEGF-A) and its receptors VEGF receptor 1 and 2 (VEGFR1 and VEGFR2); and (iii) methylation pattern of these genes promoter regions (MMP2, TIMP1, TIMP2), in both placental horns (pregnant-PH and non-pregnant-NPH), of mares (n = 17). Gene transcription was analyzed by qPCR, protein expression by Western blotting, tissue protein localization by immunohistochemistry, and DNA methylation patterns by bisulfite pyrosequencing of CpG islands in gene promoter regions. Transcription of MMP2, TIMP1, and TIMP2 was upregulated in PH, whereas no differences were found for MMP9, VEGFA, VEGFR1, and VEGFR2. Increased transcription of MMP2, TIMP1 and TIMP2 genes in PH was associated with their hypomethylation, whereas its decrease in NPH was associated with hypermethylation. A positive correlation between MMP2 transcription and methylation was observed in PH, and a negative correlation for MMP2, TIMP1 and TIMP2 in NPH. An epigenetic mechanism might be involved in placenta gene regulation. Protein abundance of MMP-2, MMP-9, TIMP-1, and TIMP-2 was similar between horns. No differences were found in MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios, between horns. Proteins MMP-2, MMP-9, TIMP-1, and VEGF-A were localized in trophoblast cells, and TIMP-2 in chorioallantoic membrane stroma. This study suggests a regulatory role of epigenetics on specific modulatory ECM enzymes in the mare placenta at term.