Background: Pregnant women have been recommended to take folic acid daily for preventing birth defects. However, we previously demonstrated that folic acid can inhibit angiogenesis. We propose that there should have some mechanisms allowing the fetus and the pregnant mother to escape from the folic acid-induced anti-angiogenesis. The present study was designed to study the effect of sex hormones on the folic acid-inhibited proliferation and migration of vascular endothelial cells. Methods: The protein levels and protein-protein interaction were examined by Western blot analysis and immunoprecipitation assay, respectively. The cell number was evaluated using MTT assay and cell migration was examined using wound healing assay. Results: Treatment with folic acid (0.1-10 micromolar) concentration-dependently inhibited endothelial cell proliferation and migration. Co-treatment with folic acid and sex hormones (estradiol, progesterone or androgen receptor agonist) together abolished the folic acid-induced proliferation and migration inhibition in vascular endothelial cells. However, the inhibitory effects caused by sex hormones on folic acid-induced anti-proliferation and anti-migration in endothelial cells were prevented by pre-treatment with their individual receptor antagonists (an estrogen receptor beta (ER beta) antagonist, PHTPP, a progesterone receptor antagonist, Org 31710, or an androgen receptor antagonist, hydroxyflutamide), respectively. Using immunoprecipitation technique, we demonstrated that cSrc, the key molecule involved in folic acid-and sex hormones-regulated endothelial cell proliferation and migration, forms a complex with estrogen receptor, progesterone receptor and androgen receptor in endothelial cells. Conclusions: These data suggest that sex hormones can interfere with the folic acid's action on vascular endothelial cell behaviors.
Folic acid (FA) has been suggested as an important daily dietary supplement, especially for women during pregnancy need to add a lot of FA to prevent some of the embryonic development of the lesions. Our previous studies have indicated that FA has anti‐angiogenic effect. However, there is no relevant studies indicated that pregnant women ingestion of FA induce lesions due to anti‐angiogenesis. Therefore, we proposed that estrogen might be able to antagonize the folic acid‐induced anti‐angiogenesis activity, and this hypothesis has been proved. However, previous studies showed endothelial cells also express androgen receptors (AR). Accordingly, this study was aimed to investigate whether androgen could influence the FA‐induced anti‐angiogenesis. We conducted in vitro study to investigate whether androgen has any impact on proliferation and migration. The preliminarydata showed that treatment with androgen agonist, R1881 or FA alone could inhibit cell proliferation and migration. However, co‐treatment will abolish the FA‐induced inhibitions on cell proliferation and migration. Moreover, we previously found that FA inhibits proliferation by increasing the levels of p21 and p27 proteins, and reduced the cell migration activity by inhibiting the RhoA activity. Thus, we examined the androgen on the signaling pathway involved in cell proliferation and migration. Our results showed R1881 could reduce the cSrc and up‐regulations of p21 and p27. Taken together, the results of this study suggest that activation of AR could abolish the FA‐induced inhibitions on endothelial cell proliferation and migration.
The effect of androgen on angiogenesis has been documented. However, its molecular mechanisms underlying has not been well illustrated. Here, we conducted both in vitro migration assay and proliferation assay to investigate whether androgen receptor activation have any impacts on the angiogenesis. Treatment with an androgen receptor (AR) agonist, metribolone (R1881) at a range of concentrations (0.05-5 nM) or dihydrotestosterone (DHT) at a range of concentrations (0.5-2 nM) caused concentration-dependent inhibition of proliferation and migration in human umbilical venous endothelial cells (HUVEC). Blockade of the AR activity by pre-treatment with HF (5 nM), an AR antagonist, or knockdown of AR expression using the lenti-virus shRNA technique abolished the R1881-induced proliferation and migration inhibition in HUVEC, suggesting that AR receptor activation can inhibit endothelial cell proliferation and migration. To further delineate the signaling pathway involved in the AR activation-induced proliferation inhibition, our data indicate that R1881 inhibited proliferation in vascular endothelial cells through activating the AR/cSrc/AKT/p38/ERK/NFκB signaling pathway, which in turn up-regulated the expression of p53, p21 and p27 protein, and finally reduced endothelial cell proliferation. To investigate signaling pathway involved in the AR activation-induced migration inhibition, our data showed that R1881 can reduce the membrane translocation of RhoA and Rac-1, suggesting that inhibition of the RhoA and Rac-1 activity might be involved in the R1881-inhibited endothelial cell migration. Over-expression of RhoA prevented the R1881-inhibited endothelial cell migration and this effect was abolished by pre-treatment with Y27623, a ROCK inhibitor, confirming that inhibiting RhoA activity participated in the R1881-inhibited endothelial cell migration. Using the zebrafish and Matrigel angiogenesis models, we also demonstrated that R1881 inhibited angiogenesis through the AR-mediated pathway in vivo.
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