Table 1.
Demographic profile of the study population.
Fig 1.
Characteristics of ectopic ovarian endometriosis.
Hematoxylin and Eosin staining of ectopic endometriosis show presence of (A,D) endometrial glands (black arrow) and stroma(black asterisk), (B,E) hemorrhage and iron deposition (red asterisk) and (C,F) aberrant blood vessel formation(yellow arrow). The dashed box indicates the magnified area in the lower panel.
Fig 2.
Involvement of angiogenesis in ovarian endometriosis.
(A) Immunofluorescence for Von Willebrand factor (red flourescence) in early and late stages (n = 3 samples/stage) of ovarian endometriosis. Nucleus was stained with DAPI (blue flourescence). (B) Immunohistochemistry for cycloxygenase-2 in different stages (n = 3 samples/stage) of endometriosis. (C-D) Quantification of VEGF receptor-2(n = 14 samples/stage) and VEGF expressions (n = 12 samples/stage) in ectopic samples of endometriosis. Means ± SEM. p < 0.05 was accepted as level of significance; *** p < 0.001; ** p < 0.01; * p < 0.05; NS p > 0.05.
Fig 3.
Involvement of MMP-2 activity in ovarian endometriosis.
(A,B) Status of MMP-2 activity in eutopic endometrium (n = 15 samples/group) between women with and without endometriosis. (C,D) Serum MMP-2 status in different stages of ovarian endometriosis patients. (E-G) Status of MMP-2 activity in ectopic ovarian endometriotic tissues. (F,G) Representative zymography profile for pro and active MMP-2 in ectopic ovarian endometriosis. Number of patients for stage I (n = 19), stage II (n = 14), stage III (n = 24), stage IV (n = 31). Means ± SEM. p<0.05 was accepted as level of significance; *** p < 0.001; ** p < 0.01; * p < 0.05; NS p > 0.05.
Fig 4.
Status of TIMP-2 and MT1MMP expressions in ovarian endometriosis ectopic samples.
Representative western blots (A) and profiles for expressions of TIMP-2 (n = 14 samples/stage) and MT1MMP (n = 12 samples/stage) in ectopic ovarian endometriosis(B,C). Means ± SEM. p<0.05 was accepted as level of significance; *** p < 0.001; ** p < 0.01; * p < 0.05; NS p > 0.05.
Fig 5.
Effects of prostaglandin E2 on MMP-2 activity in endothelial cells.
(A,B) HUVEC cells were treated with PGE2 (0.1–1μM) for 6hr and gelatin zymography was performed with cell supernatant. (C,D) Whole cell extract (40μg/lane) were immunoblotted for VEGF, pAKT, AKT, COX-2. Experiment was repeated at least three times independently. *** p < 0.001; ** p < 0.01; * p < 0.05; NS p > 0.05.
Fig 6.
Inhibition of prostaglandinE2 attenuates tube formation along with MMP-2 activity in endothelial cells.
(A,B) HUVEC cells were inoculated on matrigel in the presence of PGE2 (1μM) with or without AKT1/2 kinase inhibitor (5μM), or only COX-2 inhibitor (5μM) and tube formation was monitored. (C,D) Gelatin zymography was performed to assess MMP-2 activity from cell supernatant. Experiments were repeated for at least three times independently. *** p < 0.001; ** p < 0.01; * p < 0.05; NS p > 0.05.
Fig 7.
Role of MMP-2 on cellular invasion and angiogenesis.
(A,B) Migration and (C,D) invasion assay was performed on MDAMB-231 cells with or without inhibitors (MMP-2i, 12.5μM or MMPi, 12.5μM). (E,F) Tube formation using HUVEC cells was performed on matrigel in the presence or absence of inhibitors (MMP-2i, 5μM or MMPi, 5μM). (G,H) Angiogenesis assay (chick chorioallantoic membrane assay) was performed using human endometriosis extract (500μg/implant) with or without MMP inhibitors (MMP-2i, 25μM or MMPi, 25μM). Dashed area signifies zone of quantification. Experiments were repeated for at least three times independently. *** p < 0.001; ** p < 0.01; * p < 0.05; NS p > 0.05.
Fig 8.
Effect for inhibition of MMP-2 and PGE2 in mouse model of endometriosis.
Mouse model of endometriosis were developed by inoculation of endometrial cells into peritoneum of the recipient mice as described in the ‘material and methods’. MMP-2i (20mg/kg) and COX-2i (40mg/kg) were administrated every alternate day for 10days. Mice (n = 4 for each group) were sacrificed on day 10 from the day of endometriosis induction. (A) Histogram for numbers of developed lesions in mice peritoneum. Inset shows the developed lesions. (B) Gelatin zymography was performed for MMP-2 activities of endometriosis. (C) Western blot was performed with the whole tissue lysate for mouse model of endometriosis. Means ± SEM. p < 0.05 was accepted as level of significance; * p < 0.05.
Fig 9.
PGE2-mediated MMP-2 activity promotes angiogenesis in endometriosis.
Endometriosis is an inflammatory, proliferative disease with increased PGE2 responses. The production of PGE2 from arachidonic acid (A.A.) is regulated by cycloxygenase-2 (COX-2). The present study found that the COX-2-PGE2-pAKT axis is involved in regulation of MMP-2 activity in endothelial cells which in turn promote angiogenesis in endometriosis.