cEPCs in the peripheral blood serve as a reserve for EC maintenance, and contribute up to 25% of ECs in newly formed vessels of ischemic lesions [25, 27–32]. In addition, estrogen preserves ischemic heart function during myocardial infarction by augmenting the mobilization and incorporation of bone marrow-derived EPCs into sites of neovascularization . In female reproductive organs, EPCs appear to play a crucial role in vascularization of the uterine endometrium at the site of embryo implantation and placentation [33–35]. During the menstrual cycle, the number of cEPCs in peripheral blood exhibits a cyclic change, whereby it is markedly increased during the luteal and menstrual phase, and decreased during the follicular phase. The number of cEPCs has been shown to decrease over the course of pregnancy . Since EPCs were also found in the corpus luteum and uterine endometrium, the estrogen-mediated increase in EPCs is thought to be involved in periodic neovascularization during the menstrual cycle [28, 37, 38].
The differences in EPC proliferation and cEPC concentration between the menstrual and luteal phases were the most important findings of this study. We demonstrated that EPCs from menstrual phase did not proliferate after seven days in culture; however, there was a dose-dependent increase in proliferation after cells were treated with E2β or P4. On the other hand, proliferation was increased in EPCs from luteal phase after seven days in culture; however, the proliferation was not influenced by addition of E2β or P4. Therefore, we hypothesize that luteal EPCs may have already reached maximum stimulation in vivo and could not be activated furtherer.
Decreased proliferation of EPCs from menstrual phase suggests that this observed effect was due to decreased concentration of E2β or P4 during menstruation. Therefore, the increase in proliferation was a sex steroid-mediated dose-dependent increase. Since the expression of ERα and ERβ in EPCs was different between the menstrual and luteal phases, E2β-induced neovascularization may be mediated by both receptors, and the differential expression of ERα and ERβ indicates that they have different effects on neovascularization. ERα mRNA expression levels in EPCs from menstrual phase was higher than those from luteal phase as shown in Figure 6. On the other hand, the ERβ mRNA expression level in EPCs from luteal phase was higher than ERα expression. Since estrogen-responsive element-dependent gene transcription activities are severely impaired in EPCs obtained from ERα-knockout mice, vascular growth is down-regulated in ERα-knockout EPCs [39, 40], and epithelial cell proliferation can be reduced through the activation of ERβ , the increased expression of ERα may at least partially explain the increase in E2β-induced proliferation of EPCs derived from subjects in the menstrual phase. However, the roles of ERα and ERβ in EPC biology during the menstrual cycle will require further elucidation.
The cEPC concentration was lowest in the follicular phase, and EPC proliferation in the cultures was not different compared to the other cultures. It has been well established that the serum concentration of E2 and VEGF is very low during the early follicular phase [12, 41]. Therefore, it is possible that decreased VEGF and E2 expression during the follicular phase could reduce the recruitment of cells from bone marrow into blood, since VEGF can induce the recruitment of EPCs from bone marrow . These observations suggest that the initial priming of the ovarian hormone is required for EPC proliferation during the menstrual cycle.
Estrogen also stimulates extensive placental neovascularization through the up-regulation of angiogenic factors in the uterus during pregnancy , and the number of cEPCs peaks from luteal phase through the 1st trimester . This finding is consistent with the fact that vasculogenesis peaks during embryogenesis [35, 45]. On the other hand, estrogen is reduced after menopause, which leads to the depletion of EPCs , and estrogen replacement therapy can delay the onset of senescence in bone marrow-derived EPCs . Therefore, estrogen is critical in promoting angiogenesis and vasculogenesis in female reproductive organs.