Expression and Localization of Anti-Angiogenic Factors in the Endometrium of Macaques During Menstruation.

Many women suffer from unwanted heavy menstrual bleeding. Macaques are menstruating nonhuman primates that provide the most physiologically relevant model for preclinical studies of heavy menstrual bleeding. Angiogenic factors including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) are up-regulated in the primate endometrium during early menstruation. This could stimulate new blood vessel growth at the menstrual interface and exacerbate bleeding. Paradoxically, endothelial cell proliferation is minimal during menses and peaks after menses concludes on cycle day 8 of the proliferative phase. We hypothesize that anti-angiogenic factors could play a role in suppression of angiogenesis during menstruation. Our goal in this study was to assess the expression of anti-angiogenic factors in the menstrual endometrium. Quantitative reverse transcription polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were used to quantify and localize expression of anti-angiogenic factors including: thrombospondin-1 (THBS-1), vasohibin1 (VASH-1), and plasminogen activator inhibitor-1 (PAI-1) in endometrium of rhesus macaques. Archived samples of endometrium were obtained from ovariectomized macaques that were sequentially treated with estrogen and progesterone (P) to induce artificial menstrual cycles. Endometria from menstrual cycle days 0 (the day of P withdrawal), 1, 2, 3, 4, 6, 7 (n=4/time point), and 14 (n=7) were used in the analysis. RNA samples were analyzed via multiplex qPCR on an ABI 7900HT FAST Real-Time PCR System. Primer/probe sets for THBS-1, VASH-1, and PAI-1 were based on the predicted Macaca mulatta sequences XM_001093770, XM_001100694, and XM_002803227, respectively. qPCR determinations were normalized to the expression of S10, a ribosomal transcript. IHC was conducted with monoclonal primary antibodies to THBS-1 (Abcam), VASH-1 (Lifespan Biosciences), and PAI-1 (Abcam). qPCR revealed that THBS-1 and PAI-1 increased significantly, greater than 10 fold (P<0.01), by day 2 of the menstrual phase of the cycle and declined to baseline by day 6, the final day of frank menstruation. In contrast, VASH-1 levels remained unchanged during the menstrual phase of the cycle. Immunostaining revealed that the increase of THBS-1 was exclusively localized to the endometrial stroma of the sloughing endometrium, the endometrial zone that expresses matrix metalloproteinases and VEGF during menses. VASH-1 and PAI-1 were localized to endothelial cells and leukocytic macrophages throughout the endometrial stroma. These data indicate that a delicate balance of previously reported angiogenic factors and newly identified anti-angiogenic factors develops in the premenstrual and menstruating endometrium. Coexpression of anti-angiogenic factors during menses could prevent overgrowth of small vessels in the menstrual zones. Dysregulation of anti-angiogenic factors during the menstrual phase of the cycle could play a role in menorrhagia. Supported by NIH P51 RR00163.