The key novel findings observed from this study are: 1) UAECs express CYP1A1, CYP1A2, CYP1B1, CYP3A4, and COMT; 2) E2β, 2-OHE2, 4-OHE2, and 4-ME2 stimulate P-UAEC, but not NP-UAEC, proliferation; and 3) E2β-induced cell proliferative responses are mediated primarily via ER-β, whereas E2β metabolites-induced proliferative responses are independent of ER-α and ER-β.
UAECs constitutively express enzymes that may metabolize E2
β to its hydroxy-(CYP1A1, CYP1A2, CYP3A4, CYP1B1) and subsequently methoxy- (COMT) metabolites. Consistent with our findings, are reports showing that CYP450s and COMT are expressed in aortic, coronary artery and umbilical vein ECs.11, 16, 17,18
However, this is the first characterization of the localized intracellular expression of CYP450s in endothelial cells. Our data also confirm findings that COMT is primarily an intracellular cytosolic enzyme.19
Although little is known about the intracellular localization of these enzymes in endothelial cells, it is possible that intracellular compartmentalization is associated with enzymatic function.
The physiologic plasma E2
β concentration in women ranges from 0.1-2.2 nmol/L and increase dramatically during pregnancy.5
We demonstrate that a physiologic concentration of E2
β stimulates P-UAEC, but not NP-UAEC proliferation. The P-UAEC proliferative responses are similar to estrogenic stimulation of HUVECs and retinal microvascular ECs.8,13,20
β promotes murine endometrial endothelial proliferation in vivo
These current data are also consistent with our previous findings23
β increases P-UAEC [H3
]-thymidine incorporation and tube formation; however maximum P-UAEC responses to E2
β were seen at 1 nmol/L23
and not 0.1 nmol/L; NP-UAECs were not evaluated. These results demonstrate that P-UAEC proliferative responses are induced by gestational programming at the level of endothelial cell signaling, supporting reports that pregnancy-induced programming in P-UAECs leads to increased responsiveness to agonists and these effects are retained in cultured primary cell lines.15
Furthermore, the complete lack of mitogenic response of NP-UAECs may be specific to E2
β since NP-UAECs show proliferation in response to ATP,VEGF, bFGF, and high (≥5%) serum.15, 24, 25,26
The mechanistic significance of pregnancy-induced estrogenic programming on physiologic angiogenesis in P-UAECs remains to be elucidated.
Plasma catecholestrogens levels in pregnancy are 10-fold greater than in nonpregnant women.27
Our finding that low levels of 2-OHE2
stimulate P-UAEC, but not NP-UAEC proliferation, supports the proposal that CYP450s- and COMT-derived metabolites of E2
β may play roles in the regulation of uterine angiogenesis during pregnancy. Low concentrations of 2-OHE2
stimulate HUVEC proliferation and direct uterine arterial infusion of 2-OHE2
in nonpregnant sheep causes vasodilatation, whereas 4-OHE2
interacts directly with calcium channels to locally increase blood flow in gilts.13,28,29
These findings suggest that catecholestradiols play roles in pregnancy-induced vascular adaptations.
-methylation of catecholestradiols produces less potent and antiproliferative metabolites of E2
Interestingly, we demonstrate that 4-ME2
stimulated P-UAEC, but not NP-UAEC proliferation, consistent with observations that low 4-ME2
concentrations stimulate HUVEC proliferation.13
was not mitogenic on UAECs supporting numerous reports of its antiproliferative effects.30,31,32
The reason for divergent proliferative patterns between 2-ME2
is unclear. However, 2-ME2
disrupts tubulin polymeration and induces cell-cycle arrest in the mitotic phase in endothelial and smooth muscle cells.33,34,35
Thus, differences in association of 2-ME2
with regulators of mitosis, may likely account for their divergent responses.
Demonstrating a role for ER-α and ER-β, ICI abrogated E2
β-induced P-UAEC proliferation, supporting previous observations that ICI blocks E2
β-induced P-UAEC [H3
β-induced VEGF-mediated proliferation.9
Antagonism of ER-β with PHTPP abrogated E2
β-induced P-UAEC proliferation and ER-β activation with DPN-induced proliferation demonstrating an ER-β only effect. However, although activation of ER-α with PPT did not alter P-UAEC proliferation, PPT stimulates proliferation of human myometrial microvascular endothelial cells.36
Therefore, the differences in P-UAECs proliferation in response to DPN, PPT, or E2
β may be due to their distinct differences in affinity for ERs in association with the complex nature of ER-ligand complexes.37,38
Nevertheless, PHTPP inhibition of E2
β- and DPN-induced P-UAEC proliferation validates that these E2
β effects are solely ER-β mediated and independent of ER-α. Equally importantly, NP-UAECs, P-UAECs, and P-UAECs treated with E2
β express similar levels of ER-β (Figure S1
, please see http://hyper.ahajournals.org
.) demonstrating that the ER-β-mediated E2
β effects are not dependent on ER-β expression levels, but rather on other gestational-programming factors at the level of P-UAECs signaling.
β metabolites possess little affinity for classical ERs10,39
and unlike E2
β, the effects of its metabolites on P-UAECs proliferation are not mediated via ER-α or ER-β. These results also indirectly suggest that CYP450s and COMT expressed in the UAECs do not possess high enough enzymatic activity under these conditions to metabolize E2
β. However, E2
β metabolites may induce proliferative effects via other estrogen associated receptors like GPR30 found in endothelial cells.40,41
Nonetheless, the exact mechanism of action of estrogen metabolites on uterine vascular ECs remains to be determined.