Raloxifene⁎ increases proliferation of human endothelial cells in association with increased gene expression of cyclins A and B1  Pilar J. Oviedo, B.Sc., Carlos Hermenegildo, M.D., Ph.D., Juan J. Tarín, Ph.D., Antonio Cano, M.D., Ph.D.  Fertility and Sterility  Volume 88, Issue 2, Pages 326-332 (August 2007) DOI: 10.1016/j.fertnstert.2006.11.185 Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Proliferation of HUVECs in response to either E2 or raloxifene (MTT assay). Quiescent HUVECs were exposed to increasing doses of either E2 or raloxifene (0.1–10 nM), and allowed to proliferate for up to 5 days. To investigate the role of ERs, cells were coexposed to 1 μM ICI 182780. The culture medium was then discarded, and the MTT protocol was followed. Data are the ratio of formazan produced by treated cells to that produced by control cells, and are the mean ± SEM of quadruplicate determinations from 7–13 different experiments performed in cells from three different cultures. *P<.05 versus control values, †P<.05 versus same dose of E2 without ICI 182780, and ‡P<.05 versus ICI 182780 alone. Oviedo. Raloxifene and HUVEC proliferation. Fertil Steril 2007. Fertility and Sterility 2007 88, 326-332DOI: (10.1016/j.fertnstert.2006.11.185) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Proliferation of HUVECs in response to either E2 or raloxifene (BrdU and XTT assays). Quiescent HUVECs were exposed to 10 nM of either E2 or raloxifene, with or without 1 μM ICI 182780. Data are expressed as the ratio of either BrdU incorporated (A) or formazan produced (B) by treated cells to that incorporated or produced by control cells, and are given mean ± SEM of quadruplicate determinations from 4–10 different experiments performed in cells from four different cultures. *P<.05 versus control values, and †P<.05 versus E2 alone. Oviedo. Raloxifene and HUVEC proliferation. Fertil Steril 2007. Fertility and Sterility 2007 88, 326-332DOI: (10.1016/j.fertnstert.2006.11.185) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Estrogen receptor expression in cultured human ECs. Quiescent HUVECs were exposed to either E2 or raloxifene (10 nM) alone, or were associated with 1 μM ICI 182780 for 24 hours. Then cells were collected in lysis buffer, protein was measured, and equal amounts of protein (range, 70–130 μg) were subjected to 8.5% acrylamide gel electrophoresis and immunoblotted with specific anti-ERα (A) or anti-ERβ (B) antibodies. Relative levels of ERα and ERβ were assessed by densitometry of bands of 66 kd (ERα) or 56-kd (ERβ). Data are expressed as the mean ± SEM of 5–8 different experiments performed in cells from three different cultures. *P<.05 versus control values, and †P<.05 versus raloxifene alone and versus ICI 182780 alone. Oviedo. Raloxifene and HUVEC proliferation. Fertil Steril 2007. Fertility and Sterility 2007 88, 326-332DOI: (10.1016/j.fertnstert.2006.11.185) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Cyclins A and B1 and p27Kip1 protein expression in cultured human ECs. Quiescent HUVEC were exposed to 1 nM either raloxifene (A) or E2 (B) for different periods of time (0–48 hours). Then cells were collected in lysis buffer, protein was measured, and equal amounts of protein (range, 45–60 μg) were subjected to 15% acrylamide gel electrophoresis and immunoblotted with specific anti-cyclin A, or anti-cyclin B1, or anti-p27 antibodies. Examples of 55–60-kd bands (cyclins A and B1) or 27-kd bands (p27) are shown. Oviedo. Raloxifene and HUVEC proliferation. Fertil Steril 2007. Fertility and Sterility 2007 88, 326-332DOI: (10.1016/j.fertnstert.2006.11.185) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Modulation of relative expression of cyclins A and B1 mRNA in HUVECs by E2 or raloxifene. Quiescent HUVECs were treated with 1–10 nM of either E2 or raloxifene alone, or were associated with ICI 182780 (1 μM) for 24 hours. The mRNA was extracted and reverse-transcribed (1 μg total RNA). The relative gene expressions of cyclins A and B1, and of GAPDH (housekeeping gene), were quantified by using QRT-PCR. Data are expressed as the fold response over control values ± SEM of 7–17 duplicate determinations, corresponding to five different experiments performed in cells from five different primary cultures. *P<.05 versus control values, †P<.05 versus 1 nM E2 plus ICI 182780, ‡P<.05 vs. ICI 182780 alone, and §P<.005 versus 1 nM of the same compound. Oviedo. Raloxifene and HUVEC proliferation. Fertil Steril 2007. Fertility and Sterility 2007 88, 326-332DOI: (10.1016/j.fertnstert.2006.11.185) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions