Peter A. Binkley

Induction of endometrial epithelial cell invasion and c-fms expression by transforming growth factor beta

Transforming growth factor beta 1 (TGF-beta1) levels are increased in the peritoneal fluid of endometriosis patients, and endometrial cells express TGF-beta signaling components; however, little is known regarding the role of TGF-beta in endometriosis. Our objective was to examine the effects of TGF-beta1 on (i) the expression of macrophage colony-stimulating factor receptor encoded by the c-fms gene, (ii) transmesothelial invasiveness of endometrial cells, (iii) cellular proliferation and (iv) attachment to peritoneal mesothelial cells (PMCs). Effects of TGF-beta1 on c-fms mRNA expression were determined by real-time RT-PCR and c-fms cell-surface expression by flow cytometry. Effects of TGF-beta1 on the invasiveness of the immortalized endometrial epithelial cell (EEC) line EM42 and primary EECs were examined using a three-dimensional in vitro system modeling the peritoneum. Cellular proliferation and attachment to PMCs were also examined using established techniques. TGF-beta1 had little or no effect on cellular proliferation and endometrial cell attachment to PMCs. TGF-beta1 significantly induced the expression of c-fms mRNA and c-fms cell-surface expression. TGF-beta1 enhanced transmesothelial invasion by EM42 cells and EECs. Antagonists of TGF-beta1 signaling significantly inhibited both the induction of c-fms expression and cellular invasiveness, suggesting that additional studies are warranted to assess the therapeutic potential of TGF-beta antagonists in endometriosis.

Peroxisome-proliferator activator receptor-gamma activation decreases attachment of endometrial cells to peritoneal mesothelial cells in an in vitro model of the early endometriotic lesion

The aim of this study was to investigate whether peroxisome proliferator-activated receptor (PPAR)-gamma activation has an effect on the attachment of endometrial cells to peritoneal mesothelial cells in a well-established in vitro model of the early endometriotic lesion. The endometrial epithelial cell line EM42 and mesothelial cell line LP9 were used for this study. EM42 cells, LP9 cells or both were treated with the PPAR-gamma agonist ciglitazone (CTZ) at varying concentrations (10, 20 and 40 microM) x 48 h with subsequent co-culture of EM42 and LP9 cells. The rate of EM42 attachment and invasion through LP9 cells was then assessed and compared with control (EM42 and LP9 cells co-cultured without prior treatment with CTZ). Next, attachment of CTZ-treated and untreated EM42 cells to hyaluronic acid (HA), a cell adhesion molecule (CAM) on peritoneal mesothelial cells, were assessed. Although there was no difference in EM42 attachment when LP9 cells alone were treated with CTZ, treatment of EM42 cells with 40 microM CTZ decreased EM42 attachment to LP9 cells by 27% (P < 0.01). Treatment of both EM42 and LP9 cells with 40 microM CTZ decreased EM42 attachment to LP9 by 37% (P < 0.01). Treatment of EM42 cells with 40 microM CTZ decreased attachment to HA by 66% (P = 0.056). CTZ did not decrease invasion of EM42 cells through the LP9 monolayer. CTZ may inhibit EM42 cell proliferation. In conclusion, CTZ significantly decreased EM42 attachment to LP9 cells and HA in an in vitro model of the early endometriotic lesion.

Inhibition of CD44 N- and O-linked Glycosylation Decreases Endometrial Cell Lines Attachment to Peritoneal Mesothelial Cells

The attachment of endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) to peritoneal mesothelial cells (PMCs) with and without inhibition of N- and O-linked glycosylation, the viability of EECs and ESCs, and the expression of CD44 surface density were evaluated. Inhibition of CD44 N- and O-linked glycosylation by using tunicamycin and/or B-GalNAc statistically significantly inhibited endometrial cell attachment to peritoneal mesothelial cells, suggesting a role in establishment of early endometriotic lesions.

Colony-stimulating factor-1 exerts direct effects on the proliferation and invasiveness of endometrial epithelial cells

Although macrophage colony-stimulating factor (CSF-1) has been suggested to play a role in maintaining the chronic inflammatory response in endometriosis, our data suggest that CSF-1 may also play a role in early endometriosis lesion formation. We have shown that CSF-1, in an autocrine fashion, has a direct effect on endometrial epithelial cell proliferation and attachment to peritoneal mesothelial cells, early steps in endometriosis lesion formation on the peritoneum.

Raf-1, a potential therapeutic target, mediates early steps in endometriosis lesion development by endometrial epithelial and stromal cells.

Endometriosis is a hormone-sensitive gynecological disorder characterized by the benign growth of endometrial-like tissue in the pelvic cavity. Endometriotic lesions composed of endometrial stromal cells (ESC) and glandular epithelial cells (EEC) are thought to arise from menstrual endometrial tissue reaching the pelvic cavity via retrograde menstruation. The cause of endometriotic lesion formation is still not clear. Recent evidence suggest that cytokines may play a role in the early development of endometriosis lesions. Because cytokines and growth factors signal via the v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase pathway, we have examined the role of Raf-1 in early steps of endometriosis lesion formation, specifically attachment of endometrial cells to peritoneal mesothelial cells (PMC) and invasion of endometrial cells through PMC (trans-mesothelial invasion). Raf-1 antagonist GW5074 decreased attachment to PMC and trans-mesothelial invasion by primary EEC and ESC. Raf-1 also mediated TGFβ-induced trans-mesothelial invasion by the established, low-invasive EEC line EM42. TGFβ treatment of EEC resulted in Raf-1 phosphorylation at S338 and phosphorylation of ERK, suggesting that TGFβ activates Raf-1 signaling in these cells. GW5074 had little effect on ESC proliferation but inhibited EEC growth significantly under reduced serum conditions. Antagonizing Raf-1 activity and expression via GW5074 and specific Raf-1 small interfering RNA, respectively, did not alter EEC resistance to growth inhibition by TGFβ. Raf-1 inhibition blocked induction of EEC growth by epidermal growth factor. Our data suggest that Raf-1 may mediate pathologic steps involved in early endometriosis lesion formation and may be a mediator of TGFβ and epidermal growth factor actions in endometriosis.

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice.

Previous studies have shown endometrial cell (EC) CD44 and peritoneal mesothelial cell (PMC)-associated hyaluronan (hyaluronic acid [HA]) are involved in the attachment of endometrial stroma and epithelial cells to peritoneal mesothelium. Here we assess the CD44–HA interaction in the formation of the early endometriotic lesion using CD44–/– (knockout) mice. Using an established murine model and crossover technique, endometrial tissue from donor mice (wild type [WT] and CD44–/–) was used to induce endometriosis in recipient mice (WT and CD44–/–). Endometriotic lesions were visualized by fluorescent microscopy and confirmed by hematoxylin and eosin staining. Early endometriotic lesions were decreased when CD44–/– endometrium was placed in WT recipients and when WT endometrium was placed in CD44–/– recipients (P = .002). Early endometriotic lesions were also significantly decreased when both peritoneal and endometrial tissues lacked CD44 expression (P < .01). These studies demonstrate that both EC and PMC CD44 play a role in the development of early endometriotic lesion.

Increased expression of macrophage colony–stimulating factor and its receptor in patients with endometriosis

OBJECTIVE To investigate the expression and regulation of colony-stimulating factor 1 (CSF-1) and its receptor, C-FMS, in endometriosis. DESIGN In vivo and vitro study. SETTING University-based academic medical center. PATIENT(S) Reproductive-age women undergoing surgery for benign conditions. INTERVENTION(S) Peritoneal and endometrial tissue samples were obtained. MAIN OUTCOME MEASURE(S) CSF-1 and C-FMS expression. RESULT(S) Significantly higher CSF-1 levels were found in peritoneal fluid of patients with endometriosis compared with control subjects. Ectopic endometriotic tissue had 3.5-fold and 1.7-fold increases in CSF-1 and C-FMS expression, respectively, compared with eutopic tissue. Coculture of endometrial cells from either established cell lines or patient samples with peritoneal mesothelial cells (PMCs) led to increased expression of CSF-1 and C-FMS. A higher but nonsignificant increase in levels of C-FMS and CSF-1 was found in cocultures of endometrial epithelial cells from patients with endometriosis compared with those without endometriosis. CONCLUSION(S) Increased CSF-1 levels may contribute to endometriosis lesion formation and progression. Elevation in CSF-1 after coculture of endometrial cells with PMCs suggests that endometrial tissue may be a source of peritoneal CSF-1. Increased C-FMS expression in endometrial cells from women with endometriosis cocultured with PMCs suggests that endometrial tissue involved in lesion formation is highly responsive to CSF-1 signaling.

Imatinib decreases endometrial stromal cell transmesothial migration and proliferation in the extracellular matrix of modeled peritoneum

OBJECTIVE To characterize imatinibs effect on endometrial stromal cell (ESC) attachment, proliferation, and invasion in modeled peritoneum. DESIGN In vitro study. SETTING Academic medical center. PATIENT(S) Twelve normally cycling women. INTERVENTION(S) Imatinib treatment in ESCs from women without endometriosis. MAIN OUTCOME MEASURE(S) Rate of ESC attachment, proliferation, and invasion. RESULT(S) Imatinib treatment at 10 μM had no effect on ESC attachment. Treatment with 0.5 μM, 2 μM, and 10 μM of imatinib reduced ESC proliferation by 30%, 72%, and 76%, respectively. The 0.1 μM dose of imatinib had no effect on proliferation. Treatment with 5 μM and 10 μM of imatinib reduced ESC invasion by 30% and 73%, respectively. The 2 μM dose had no effect on invasion. CONCLUSION(S) Imatinib treatment reduces ESC proliferation and invasion in modeled peritoneum without altering attachment. Imatinib may have a therapeutic role in endometriosis treatment.

A Combination of a GnRH Antagonist and Agonist for Fertility Preservation in an Adolescent Female Murine Model

Recent studies have suggested that GnRH agonists (GnRHags) protect ovarian function following chemotherapy. Here, we study the effect of a combination of GnRH antagonist (GnRHan) and GnRHag for gonadal protection from gonadotoxic chemotherapy in adolescent female rats. Cycling Sprague Dawley rats were treated at adolescent age. Thirty female rats were randomized to 5 treatment groups (n = 6/group): (1) placebo, (2) cyclophosphamide (CPA) alone, (3) GnRHan followed by GnRHag with placebo, (4) GnRHan followed by GnRHag with CPA, and (5) GnRHag with CPA. The main outcome measure was live birth rate (LBR), and secondary measures included rat weight, ovarian volume, and follicles. Group 2 had decreased LBR compared to all other groups. Group 4 and 5 had LBR similar to placebo. There was no difference in the ovarian volume. The CPA-alone group had decreased number of antral follicles compared to control. These studies demonstrate that the combination of GnRHan and GnRHag and GnRHag alone preserved fertility in female adolescent rats following gonadotoxic chemotherapy treatment. The addition of a GnRHan to a GnRHag does not confer a greater protective effect.