Project description:Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impairs vascular smooth muscle cell (VSMC) and pericyte proliferation and/or migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and alpha smooth muscle actin (aSMA) double-immunohistochemistry assessed VSMC differentiation and proliferation in endometria from women pre- and postDepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA) or E2+MPA pellets. After treating cultured VSMCs with MPA or etonogestrel (ETO), whole genome profiling, proliferation and migration assays were performed. Endometrial vasculature of Depo-administered women displayed reduced M-DM-.SMA immunoreactivity and fewer PCNA (+) nuclei among aSMA (+) cells (P<0.008). Microarray analysis of VSMCs identified several MPA and ETO-altered transcripts regulated by STAT1 signaling (p<2.22x10-6), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduced VSMC proliferation and migration (p<0.001), recombinant CCL2 reversed this progestin inhibition, and, in turn, a STAT1 inhibitor abolished these CCL2 effects. Similarly, the endometria of MPA treated OVX-GPs displayed decreased aSMA staining and fewer PCNA (+) nuclei in VSMC (p<0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration. Total RNA (n=3) obtained from cultured TDCs incubated 6h with estradiol or estradio + medroxyprogesterone acetate with or without 1 ng/ml IL-1M-NM-2 for 6h.
Project description:Here we report the gene expression profile of in vitro cultured human endometrial stromal cells treated with siRNA targeting FOXO1 piror to eutherian differentiation media exposure. The eutherian differentiation media contains cyclic AMP (cAMP) analogue 8-Br-cAMP and the progesterone (P4) analogue medroxyprogesterone acetate (MPA).
Project description:Cells were cotreated with dihydrotestosterone, progesterone or medroxyprogesterone acetate and estrdiol to assess the combinatorial effects of hormone exposure in breast cancer cells
Project description:Analysis of long-acting progestin-only contraceptive (LAPC)-mediated differential gene expression in vascular smooth muscle cells (VSMCs). We tested the hypothesis that LAPCs influence expression of several common genes associated with differentiation, survival and migration of VMSCs. Results provide important information of several common and unique gene expression profiles in cultured VSMC treated with ETO, M or P. Among ETO and M responsive genes, up- or down-regulated common genes were determined and used further confirmation and in vitro functional analyses. Total RNA (n=3) obtained from cultured aortic VSMCs treated with vehicle only (CON) or ETO or M or P for 6h.
Project description:Analysis of long-acting progestin-only contraceptive (LAPC)-mediated differential gene expression in vascular smooth muscle cells (VSMCs). We tested the hypothesis that LAPCs influence expression of several common genes associated with differentiation, survival and migration of VMSCs. Results provide important information of several common and unique gene expression profiles in cultured VSMC treated with ETO, M or P. Among ETO and M responsive genes, up- or down-regulated common genes were determined and used further confirmation and in vitro functional analyses.
Project description:Cells were cotreated with estrogen and dihydrotestosterone, progesterone or medroxyprogesterone acetate to assess the combinatorial effects of progestogens or androgens with estrogen in T47D breast cancer cells
Project description:Comparison between the effects of progesterone (P4) and medroxyprogesterone acetate (MPA) combined to estradiol (E2) on gene expression in normal breast cells to provide insight on their possible different impact on breast cancer risk in vivo.
Project description:Analysis of differential gene expression in Human endometrial endothelial cells (HEECs) incubated with CMS derived from human endoemtrial stromal cells (HESCs) treated by LAPCs. We tested the hypothesis that paracrine factors sectreted from HESCs treated LAPCs, etonogestrol (ETO) or medorxyprogesterone acetate (M) influence several common genes associated with survival in HEECs. Thefore, whole genome analyses were performed in HEECs treated with HESC derived CMS obtained from vehicle (estrodial (E) as control) or progesterone (P) or ETO or M incubations under hypoxia (HX) and normoxia (NX). Results provide important information of several common and unique gene expression profiles in cultured HEECs treated with HESC CMS from P or ETO or M incubations. Among M and ETO responsive genes, up- or down-regulated survival related common genes were determined and used further confirmation and in vitro functional analyses.
Project description:Use of long-acting progestin-only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understand the mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+progesterone (P4) were analyzed by q-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depo-Provera (Depo) treated women (n=6) and ovariectomized guinea pigs (GPs; n=12) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67-gene group regulated by all three progestins, whereas a 235-gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as an upstream regulator of the 235 LAPCMPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both LAPCsMPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-Depo versus pre-Depo administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. LAPC MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting feeds back to inhibit PR- and GR-mediated transcription. The resultant PR- and/or GR-mediated functional withdrawal may contribute to associated endometrial inflammation, aberrant angiogenesis, and bleeding.
Project description:Analysis of differential gene expression in Human endometrial endothelial cells (HEECs) incubated with CMS derived from human endoemtrial stromal cells (HESCs) treated by LAPCs. We tested the hypothesis that paracrine factors sectreted from HESCs treated LAPCs, etonogestrol (ETO) or medorxyprogesterone acetate (M) influence several common genes associated with survival in HEECs. Thefore, whole genome analyses were performed in HEECs treated with HESC derived CMS obtained from vehicle (estrodial (E) as control) or progesterone (P) or ETO or M incubations under hypoxia (HX) and normoxia (NX). Results provide important information of several common and unique gene expression profiles in cultured HEECs treated with HESC CMS from P or ETO or M incubations. Among M and ETO responsive genes, up- or down-regulated survival related common genes were determined and used further confirmation and in vitro functional analyses. Total RNA (n=3) obtained from cultured HEECs incubated 6h with vehicle- or P4- or ETO- or MPA- treated NX or HX conditioned HESC-derived CMS.
