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Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion, migration, and invasion of human endometriotic cells
P-374 Wednesday, October 27, 2010 THE ROLE OF MACROPHAGES IN THE DEVELOPMENT OF THE ADHESION PHENOTYPE. J. White, Z. Jiang, M. Diamond, G. Saed. OB/GYN, Wayne State University, Detroit, MI. OBJECTIVE: Hypoxia exposure promotes the development of the adhesion phenotype in normal peritoneal fibroblasts (NPF) which is characterized, in part, by increased expression of TGF-b1, VEGF, and type I collagen. In this study we sought to determine whether macrophages, exposed to hypoxia, release factors that stimulate normal peritoneal fibroblasts to acquire the adhesion phenotype. DESIGN: University Research Laboratory. MATERIALS AND METHODS: Media retrieved from macrophages cultured under normal (20% O2) and hypoxic conditions (2% O2) were utilized to culture NPF. Total RNA from each group was extracted and complimentary DNA (cDNA) was synthesized. Aliquots of cDNA were subjected to real-time reverse transcriptase polymerase chain reaction to determine mRNA levels for TGF-b1, VEGF, and Col-I. Western Blot analysis will confirm mRNA levels. RESULTS: NPF cultured under hypoxic conditions resulted in a significant increase in TGF-b1 (from 69.13 to 126.02 pg TGFb1/ug RNA; 31% increase in protein; p < .05), VEGF (from 0.40 to 1.14 pg VEGF/ug RNA; 31% increase in protein; p < .05;), and Col-I (from 1.16 to 2.63 pg Col-I/ug RNA; p < .05) as compared to normoxic conditions. NPF cultured utilizing media obtained from macrophages cultured under hypoxic conditions resulted in a significant increase in TGF-b1 (from 108.08 pg TGFb1/ug RNA to 141.83 pg TGFb1/ug RNA; 32% increase in protein; p < .05), VEGF (0.43 pg VEGF/ug RNA to 0.84 pg VEGF/ug RNA; 27% increase in protein; p < .05), and Col-I (2.36 pg Col-I/ug RNA to 2.83 pg Col-I/ug RNA; p < .05) when compared to media retrieved from macrophages cultured under normoxic conditions. CONCLUSION: Human macrophages, cultured under hypoxic conditions, release factors that induce the expression of the adhesion phenotype in normal peritoneal fibroblasts. This highlights an important role for human macrophages during peritoneal wound-healing. Supported by: In part by grant number 1RO1 GM069941-01A3, National Institutes of Health, Bethesda, Maryland to Ghassan M. Saed.
P-375 Wednesday, October 27, 2010 SELECTIVE INHIBITION OF PROSTAGLANDIN E2 RECEPTORS EP2 AND EP4 INHIBITS ADHESION, MIGRATION, AND INVASION OF HUMAN ENDOMETRIOTIC CELLS. J. A. Arosh, J. Lee, S. K. Banu. Integrative Biosciences, Texas A&M University, College Station, TX; Integrative Biosciences, Texas A&M University, College Station, TX; Integrative Biosciences, Texas A&M University, College Station, TX. OBJECTIVE: Prostaglandin E2 plays an important role in the pathogenesis of endometriosis. The objective of the present study was to determine functional interaction between PGE2 signaling and adhesion, migration, and invasion of human endometriotic epithelial and stromal cells and to unravel the underlying molecular and cellular mechanisms. DESIGN: Human immortalized endometriotic epithelial and stromal cells were treated with EP2 and EP4 receptor inhibitors, and their effects on adhesion, migration, and invasion of cells were studied and the underlined molecular mechanisms were uncovered. MATERIALS AND METHODS: Adhesion of endometriotic cells with ECM substrate and invasion of endometriotic ells into matrigel were analyzed in vitro. Set of integrins, MMPs and TIMPS proteins were analyzed by western blot. RESULTS: Results of the present study indicated that selective inhibition of EP2 and EP4-mediated PGE2 signaling: (i) suppressed expression and/or activity specific integrin receptors a2b1, avb3, a5b1, and a3b1 and thereby decreased adhesion of human endometriotic epithelial and stromal cells to extracellular matrix substrates collagen I, collagen IV, vitronectin, fibronectin, and laminins; and (ii) suppressed expression and/or activity of MMP1, MPP2, MMP3, MMP7 and MMP9 and increased expression of TIMP1, TIMP2, TIMP3, and TIMP4 and thereby decreased migration and invasion of human endometriotic epithelial and stromal cells. CONCLUSION: These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonestrogen or nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age
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Abstracts
women. Further, our results suggest potential opportunities for translational studies that could lead to preclinical and clinical-phase trials. Supported by: Program development award from Texas A&M University.
P-376 Wednesday, October 27, 2010 LABELED RED BLOOD CELL SCINTIGRAPHY IN THE NON-INVASIVE DIAGNOSTICS OF ENDOMETRIOSIS. F. Demirel, G. Koca, K. Demirel, H. Aydogmus, M. Korkmaz, B. Gokmen. Obstetrics&Gynecology, Ministry of Health Ankara Training and Research Hospital, Ankara, Turkey; Nuclear Medicine, Ministry of Health Ankara Training and Research Hospital, Ankara, Turkey. OBJECTIVE: Our aim in this study was to show the activated implants with labeled red cell scintigraphy in patients with recurrent endometriosis and compare the results with pelvic MRI results. DESIGN: Endometriosis is a hormone-dependent clinically benign disease associated with pelvic pain and infertility. Although, laparoscopy and histopathology are the gold standarts for diagnosis of endometriosis and evaluation of the implants, it is also investigated that active implants can be shown by scintigraphy. MATERIALS AND METHODS: Thirty patients, who were diagnosed as recurrence by clinical and laboratory terms and 10 healthy volunteer (control group) patients were included in the study. Patients who had operated with diagnostic and/or therapeutic, laparascopy or laparotomy and diagnosed histopathologically as endometriosis, were examined in our study. Between the second and fifth days of menstruation when the endometriotic lesions were highly activated, radionuclide imaging was performed by red blood cellTc-99m scintigraphy in both early&late phases and compared with pelvic MRI findings. The results were statistically assessed. RESULTS: The patients mean age was 33.33 8.05 years. Compared to the control group we found a total of pathological accumulation of radioactivity with marked red blood cell scintigraphy in 27 patients out of 30 (90%). The pathological accumulation observed in 26/30 (86.7%) patients, there was definite increased focal accumulation in scintigraphy and in one patient, however was modarate focal accumulation. In 22/27 patients (81.5%) there was increased radioactivity accumulation in late phase of radionuclide images where there was findings of endometriosis in MRI. CONCLUSION: Imaging of endometriosis regions with labeled RBC scan can be used as an alternative diagnostic procedure for the patients with recurrent endometriosis.
P-377 Wednesday, October 27, 2010 EFFECT OF RESVERATROL ON THE INDUCTION OF ENDOMETRIOSIS IN A RAT MODEL. M. Oktem, P. Ozcan, O. Erdem, C. Karakaya, H. Guner, O. Karabacak. Obstetrics & Gynecology, Gazi University School of Medicine, C ¸ ankaya, Ankara, Turkey; Pathology, Gazi University School of Medicine, C ¸ ankaya, Ankara, Turkey. OBJECTIVE: Resveratrol is a natural polyphenol which has anti-angiogenic, anti-oxidant and anti-inflammatory effects.We aimed to assess the effect of resveratrol in a rat endometriosis model. DESIGN: Prospective randomized animal study. MATERIALS AND METHODS: Endometriosis was induced surgically in 24 rats.3 weeks after the initial surgery, the rats underwent laparotomy to determine the size of endometrial implants. Two rats died during the procedures. The remaining rats were randomized into three groups. Group I (Resveratrol group, seven rats) was given 60 mg/kg/d resveratrol via orogastric tube. Group II (GnRH agonist group, eight rats) was given a single dose of 1 mg/kg s.c. leuprolide acetate. Group III served as control group (seven rats). All rats received the treatment for 21 days. After treatments, the rats were euthanized and a third laparotomy was performed and the surface areas of the implants were calculated. Also before and after medications, peritoneal fluids were collected to measure VEGF and MCP-1 levels. The implants were then excised and examined histopathologically. RESULTS: All rats developed similar areas of implants at the second laparotomy. After treatment, the mean areas of implants were smaller in the resveratrol and leuprolide acetate groups than those in the control group. The mean areas of implants decreased from 44.1 5 to 7.1 2.9 mm2 in the resveratrol group (P <.05). The mean histopathological score of the implants at the end of the treatment was lower in the resveratrol group when compared with the control group (1.2 1.2 vs. 2.5 0.5, P <.05). Peritoneal level of
Vol. 94., No. 4, Supplement, September 2010
P-375 Wednesday, October 27, 2010 SELECTIVE INHIBITION OF PROSTAGLANDIN E2 RECEPTORS EP2 AND EP4 INHIBITS ADHESION, MIGRATION, AND INVASION OF HUMAN ENDOMETRIOTIC CELLS. J. A. Arosh, J. Lee, S. K. Banu. Integrative Biosciences, Texas A&M University, College Station, TX; Integrative Biosciences, Texas A&M University, College Station, TX; Integrative Biosciences, Texas A&M University, College Station, TX. OBJECTIVE: Prostaglandin E2 plays an important role in the pathogenesis of endometriosis. The objective of the present study was to determine functional interaction between PGE2 signaling and adhesion, migration, and invasion of human endometriotic epithelial and stromal cells and to unravel the underlying molecular and cellular mechanisms. DESIGN: Human immortalized endometriotic epithelial and stromal cells were treated with EP2 and EP4 receptor inhibitors, and their effects on adhesion, migration, and invasion of cells were studied and the underlined molecular mechanisms were uncovered. MATERIALS AND METHODS: Adhesion of endometriotic cells with ECM substrate and invasion of endometriotic ells into matrigel were analyzed in vitro. Set of integrins, MMPs and TIMPS proteins were analyzed by western blot. RESULTS: Results of the present study indicated that selective inhibition of EP2 and EP4-mediated PGE2 signaling: (i) suppressed expression and/or activity specific integrin receptors a2b1, avb3, a5b1, and a3b1 and thereby decreased adhesion of human endometriotic epithelial and stromal cells to extracellular matrix substrates collagen I, collagen IV, vitronectin, fibronectin, and laminins; and (ii) suppressed expression and/or activity of MMP1, MPP2, MMP3, MMP7 and MMP9 and increased expression of TIMP1, TIMP2, TIMP3, and TIMP4 and thereby decreased migration and invasion of human endometriotic epithelial and stromal cells. CONCLUSION: These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonestrogen or nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age
S202
Abstracts
women. Further, our results suggest potential opportunities for translational studies that could lead to preclinical and clinical-phase trials. Supported by: Program development award from Texas A&M University.
P-376 Wednesday, October 27, 2010 LABELED RED BLOOD CELL SCINTIGRAPHY IN THE NON-INVASIVE DIAGNOSTICS OF ENDOMETRIOSIS. F. Demirel, G. Koca, K. Demirel, H. Aydogmus, M. Korkmaz, B. Gokmen. Obstetrics&Gynecology, Ministry of Health Ankara Training and Research Hospital, Ankara, Turkey; Nuclear Medicine, Ministry of Health Ankara Training and Research Hospital, Ankara, Turkey. OBJECTIVE: Our aim in this study was to show the activated implants with labeled red cell scintigraphy in patients with recurrent endometriosis and compare the results with pelvic MRI results. DESIGN: Endometriosis is a hormone-dependent clinically benign disease associated with pelvic pain and infertility. Although, laparoscopy and histopathology are the gold standarts for diagnosis of endometriosis and evaluation of the implants, it is also investigated that active implants can be shown by scintigraphy. MATERIALS AND METHODS: Thirty patients, who were diagnosed as recurrence by clinical and laboratory terms and 10 healthy volunteer (control group) patients were included in the study. Patients who had operated with diagnostic and/or therapeutic, laparascopy or laparotomy and diagnosed histopathologically as endometriosis, were examined in our study. Between the second and fifth days of menstruation when the endometriotic lesions were highly activated, radionuclide imaging was performed by red blood cellTc-99m scintigraphy in both early&late phases and compared with pelvic MRI findings. The results were statistically assessed. RESULTS: The patients mean age was 33.33 8.05 years. Compared to the control group we found a total of pathological accumulation of radioactivity with marked red blood cell scintigraphy in 27 patients out of 30 (90%). The pathological accumulation observed in 26/30 (86.7%) patients, there was definite increased focal accumulation in scintigraphy and in one patient, however was modarate focal accumulation. In 22/27 patients (81.5%) there was increased radioactivity accumulation in late phase of radionuclide images where there was findings of endometriosis in MRI. CONCLUSION: Imaging of endometriosis regions with labeled RBC scan can be used as an alternative diagnostic procedure for the patients with recurrent endometriosis.
P-377 Wednesday, October 27, 2010 EFFECT OF RESVERATROL ON THE INDUCTION OF ENDOMETRIOSIS IN A RAT MODEL. M. Oktem, P. Ozcan, O. Erdem, C. Karakaya, H. Guner, O. Karabacak. Obstetrics & Gynecology, Gazi University School of Medicine, C ¸ ankaya, Ankara, Turkey; Pathology, Gazi University School of Medicine, C ¸ ankaya, Ankara, Turkey. OBJECTIVE: Resveratrol is a natural polyphenol which has anti-angiogenic, anti-oxidant and anti-inflammatory effects.We aimed to assess the effect of resveratrol in a rat endometriosis model. DESIGN: Prospective randomized animal study. MATERIALS AND METHODS: Endometriosis was induced surgically in 24 rats.3 weeks after the initial surgery, the rats underwent laparotomy to determine the size of endometrial implants. Two rats died during the procedures. The remaining rats were randomized into three groups. Group I (Resveratrol group, seven rats) was given 60 mg/kg/d resveratrol via orogastric tube. Group II (GnRH agonist group, eight rats) was given a single dose of 1 mg/kg s.c. leuprolide acetate. Group III served as control group (seven rats). All rats received the treatment for 21 days. After treatments, the rats were euthanized and a third laparotomy was performed and the surface areas of the implants were calculated. Also before and after medications, peritoneal fluids were collected to measure VEGF and MCP-1 levels. The implants were then excised and examined histopathologically. RESULTS: All rats developed similar areas of implants at the second laparotomy. After treatment, the mean areas of implants were smaller in the resveratrol and leuprolide acetate groups than those in the control group. The mean areas of implants decreased from 44.1 5 to 7.1 2.9 mm2 in the resveratrol group (P <.05). The mean histopathological score of the implants at the end of the treatment was lower in the resveratrol group when compared with the control group (1.2 1.2 vs. 2.5 0.5, P <.05). Peritoneal level of
Vol. 94., No. 4, Supplement, September 2010