Figure 1.
Diagrammatic representation of experimental endometriosis induction in mice and histological representation of ectopic endometriotic implant.
A) Diagram depicts donor mice (and genotype) from which endometrial tissue was dissected and separated into myometrium and endometrium (stromal and epithelial components), transfer of 10 equal size (1 mm3) endometrial fragments to recipient mice and subsequent assessment of establishment of ectopic “endometriotic implants.” B) histological representation of “endometriotic implant” with red boxed area enlarged in C) white arrow indicates epithelial component and stromal compartment and underlying peritoneum are labeled. Notice the stromal and peritoneal contact at the site of implant attachment. White scale bar = 50 um.
Figure 2.
Development of experimental endometriosis is associated with the level of implant miR-451 expression.
Experimental endometriosis was induced and development assessed as described under “Materials and Methods”. A. The number of implants of each genotype that developed in wild-type host mice (N = 8/genotype). Data are presented as the mean ± SEM and were analyzed by one-way ANOVA followed by post-hoc analysis. Different letters indicate statistical significance (P<0.05). B. Elevated miR-451 expression is associated with wild-type “implant” tissue that develops ectopically. Uterine fragments were obtained from wild-type mice at the time (0 h) of PMSG administration or 44 h later (the time of endometrial fragment harvest). miR-451 and miR-144 expression was determined by qRT-PCR. Different letters indicate statistical significance (P<0.05) between groups (by unpaired t-test). ND indicates that miR-144 levels were not detectable by qRT-PCR.
Figure 3.
Development of experimental endometriosis is dependent upon implant miR-451 expression and not host expression.
A. Endometriosis was induced in wild-type and miR-451 null mice using donor tissue from both wild-type and miR-451 null mice and the number of implants which developed was assessed as described under “Materials and Methods.” Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means as determined by one-way ANOVA followed by post-hoc analysis (N = 6 per group). B. miR-451 wild-type and not miR-451 deficient implants develop in the same wild-type host. Endometriosis was induced as described under “Materials and Methods” in wild-type host mice using both miR-451 wild-type (expressing EGFP) and miR-451 deficient (non-EGFP) endometrial fragments and implant establishment was assessed. Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means as determined by unpaired t-test (N = 6 per group).
Figure 4.
CyDye switch, two dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of wild-type and miR-451 deficient endometrial fragment proteomes.
Endometrial fragments were obtained from wild-type (N = 6) and miR-451 deficient mice (N = 6) as described under “Materials and Methods”. Wild-type samples were labeled with Cy3 (green) and miR-451 deficient samples with Cy5 (red). Samples were then mixed and separated on analytical 2-D DIGE. The resulting gel was scanned and the merged image is shown where red proteins represent proteins whose expression is higher in the miR-451 deficient tissue and green proteins represent proteins whose expression is higher in the wild-type tissue. Circled and numbered spots represent proteins which were most differentially expressed of which only those indicated by white (up-regulated) or yellow (down-regulated) arrows are reported in Table 1. Red arrow indicates protein #5 which was identified as fibrinogen, alpha polypeptide isoform 2 precursor.
Table 1.
Most significantly modulated proteins in uterine fragments from miR-451 deficient mice compared to wild-type counterparts.
Figure 5.
Fibrinogen transcript, protein and active plasmin expression in endometrial fragments of wild-type and miR-451 deficient mice.
Fibrinogen alpha chain (Fga) transcript (A) and protein (B) were analyzed by qRT-PCR and Western blot expression, respectively. Data are representative of 5 observations per endpoint per genotype (N = 5). Fga mRNA levels were not normally distributed and were analyzed using Mann-Whitney tests. Bar graph data are displayed as the mean ± SEM. In B), black arrow indicates molecular weight (95 kDa) of fibrinogen alpha chain while white arrows indicate major Fga fragments of approximately 42 kDa and 34 kDa. C) Plasmin activity was determined in uterine fragments from wild-type and miR-451 null mice as described under “Materials and Methods.” Data are displaed as the mean ± SEM and are representative of 6 separate observations per genotype (N = 6). P values are indicated in each figure and data were analyzed by unpaired t-tests in B and C. As Fga mRNA data (A) were not normally distributed, data was analyzed using the non-parametric t-test (Mann-Whitney).
Figure 6.
Fibrinogen alpha polypeptide isoform 2 precursor contains multiple cell adhesion motifs which modulate cell adhesion in vitro.
A. Protein sequence of mouse fibrinogen alpha polypeptide isoform 2 precursor. Three RGD sequences were detected in the protein sequence and are indicated in red underlined text. B. Pre-treatment of the immortalized human endometrial stromal cell line, t-HESC with cyclic RGD peptide inhibits in vitro cell spreading and survival. T-HESC cells were treated and cell adhesion, spreading and survival assessed as described under “Materials and Methods.” Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means within each substrate as determined by one-way ANOVA followed by post-hoc analysis (N = 4 separate experiments). C. Pre-treatment with RGD cyclic peptide does not induce cell death. Data are displayed as the mean ± SEM and are representative of 4 separate experiments (N = 4 separate experiments). Means are not significantly different among the different doses of RGD peptide.
Figure 7.
RGD cycle peptide inhibits development of experimental endometriosis.
Experimental endometriosis was induced in wild-type mice with wild-type endometrial fragments which were pre-treated with either vehicle (Veh) or RGD peptide (RGD) and development of ectopic lesions was assessed as described under “Materials and Methods.” Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means between treatments as determined by unpaired t-tests (N = 8/group). B Chi square analysis of the proportion of mice which received implant tissue pre-treated with either vehicle (Veh) or RGD cyclic peptide (RGD).