Leiomyoma cells do not properly sense or respond to mechanical cues

O-155 Tuesday, October 20, 2009 5:30 PM LEIOMYOMA CELLS DO NOT PROPERLY SENSE OR RESPOND TO MECHANICAL CUES. C. M. Owen, J. M. Norian, C. Korecki, J. Taboas, R. S. Tuan, J. H. Segars. Clinical Research Training Program, Office of Clinical Research Training and Medical Education, NIH, Bethesda, MD; Program in Reproductive and Adult Endocrinology, NICHD, NIH, Bethesda, MD; Cartilage Biology and Orthopedics Branch, NIAMS, NIH, Bethesda, MD. OBJECTIVE: Leiomyoma cells reside in an extracellular matrix (ECM) of increased mechanical stress that they create (Rogers et al. 2008), although it is unclear why this transformation occurs. Normal cells respond to increased mechanical stress with activation of Rho signaling, which leads to re-organization of the cell cytoskeleton. Here we tested whether leiomyoma cells appropriately sense ambient mechanical cues. DESIGN: Experimental Investigation. MATERIALS AND METHODS: Immortalized leiomyoma and myometrial cells were cultured on pronectin-coated, flexible silicone substrates (Flex Cell Intl) and levels of active RhoAwere quantified at baseline and after 2 hours of 8.6% uni-axial cyclic strain at 1 Hertz. Next, cells were treated with Y-27632, a selective inhibitor of the Rho-associated kinase (ROCK), then exposed to 18 hours of the same cyclic strain before the cytoskeleton was assessed by cytoimmunofluorescence. To quantify their response to substrate stiffness, cells were cultured on collagen-coated polyacrylamide gels with stiffness ranging from 30 to 140.5 kPa. Cells were then treated with Calcein AM and fluorescent images were obtained at 24 hours. RESULTS: Two hours of cyclic strain resulted in a 1.5-fold increased RhoA activation in myometrial cells, while levels in leiomyoma cells were increased 2-fold at baseline and showed a reduced response to load compared with myometrial cells. Inhibition of ROCK resulted in morphologic changes in myometrial cells, while results with leiomyoma cells were consistent with an attenuated response. Leiomyoma cells exhibited a reduced ability to respond to increasing substrate stiffness compared with myometrial cells. CONCLUSIONS: These data indicate that leiomyoma cells do not sense or respond appropriately to mechanical cues. Altered mechanosensing by leiomyoma cells may play a role in the aberrant structure of the ECM of leiomyoma. Supported by: HD Z01-008737-07, AR Z01-41131, and CRTP a publicprivate partnership supported by NIH and Pfizer Inc via a grant to the NIH Foundation from Pfizer.

O-156 Tuesday, October 20, 2009 5:45 PM STEROIDOGENIC ACUTE REGULATORY PROTEIN (STAR) FACILITATES DE NOVO SYNTHESIS OF PROGESTERONE BY HUMAN LEIOMYOMA (LEIO) AND MYOMETRIUM (MYO) CELLS. E. E. Marsh, D. S. Palm, J. B. Parker, C. R. Parker, S. E. Bulun. Obstetrics and Gynecology, Feinberg School of Medicine - Northwestern University, Chicago, IL; Obstetrics and Gynecology, University of Alabama Medical School, Birmingham, AL; Obstetrics and Gynecology, Feinberg School of Medicine - Northwestern University, Chicago, IL. OBJECTIVE: To determine the ability of human LEIO to synthesize progesterone de novo. DESIGN: Tissue and cell culture based experiments. MATERIALS AND METHODS: LEIO and MYO tissues were collected from subjects undergoing hysterectomy. Real time RT-PCR was performed for StAR, CYP11A, and 3b-HSD on cDNA created from tissue. Cells plated from the tissue were serum starved overnight and then treated with either low density lipoprotein (LDL), 22(R)-hydroxycholesterol (22RC) , or vehicle for 48 hours. Cells were then collected for protein. The cell culture media was collected for progesterone enzyme immunoassay (EIA). RESULTS: Real time RT-PCR revealed that StAR, CYP11A1 and 3bHSD mRNA were present in LEIO and MYO (n¼6). Western blots of whole tissue protein revealed that StAR was present in 8 out of 10 LEIO, and 3bHSD and CYP11A were present in 7 out of 7 samples. Western blots of matched MYO and LEIO pairs revealed no differences in the protein levels of CYP11A or 3b-HSD. StAR protein levels were elevated in LEIO in half of the matched pairs (n¼6) . EIA of cell culture media showed no progesterone in any of the plates treated with vehicle alone. There was progesterone present in the media of cells treated with 22RC with significantly higher levels seen in the LEIO media than the MYO media (56.8 pg/ml versus 31.4 pg/ml; p¼0.017). CONCLUSIONS: LEIO and MYO express StAR, CYP11A1, and 3b-HSD mRNA and protein in vivo and in vitro and are capable of the de novo

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synthesis of progesterone. While mRNA and protein levels of sex steroid synthesis enzymes are not consistently elevated in LEIO versus MYO, significantly higher levels of progesterone were synthesized by LEIO cells. Understanding the clinical significance of this autocrine production of progesterone in LEIO is the goal of future studies. Supported by: This work was supported by the RWJ Foundation and the Feinberg School of Medicine, Northwestern University.

O-157 Tuesday, October 20, 2009 6:00 PM ANGIOTENSION EXPRESSION AND ITS EFFECTS ON RHO SIGNALING MAY EXPLAIN THE LINK BETWEEN HYPERTENSION AND FIBROIDS. J. M. Norian, C. M. Owen, C. X. Guo, W. H. Catherino, M. Malik, J. H. Segars. Program in Reproductive and Adult Endocrinology, NICHD, NIH, Bethesda, MD; Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, MD. OBJECTIVE: A key feature of uterine fibroids is an excess amount of fibrotic extracellular matrix accompanied by altered mechanical homeostasis and by altered Rho signaling. Uterine fibroids are associated with hypertension and there is increasing evidence that angiotensin II (AngII), a major regulator of blood pressure and cardiovascular homeostasis, is involved in tissue remodeling and fibrosis of the lung and heart. We sought to examine whether AngII altered the Rho-signaling pathway in fibroids. DESIGN: Experimental investigation. MATERIALS AND METHODS: Western blot analysis of whole tissue lysates from fibroid and myometrial surgical samples (n¼6) were probed for the AngII receptor type 1 (ATR1). Immortalized uterine fibroid and myometrial cells (Malik et al. 2008) were cultured to 60% confluency in 10% Fetal Bovine Serum DMEM/F12. FBS was then reduced to 0.5% for 16 hours and cell lines were then treated for 30 minutes with AngII or an ATR1-blocker Losartan or for 3 minutes with a known RhoA activator lysophospatidic acid (LPA). Active RhoA levels were quantified using the G-LISA method. RESULTS: ATR1 was over-expressed 3-fold (3.5 þ/-1.3 integrated density) in fibroid tissue extracts relative to matched myometrium. Untreated uterine fibroid cells had 1.6-fold higher levels of active RhoA relative to untreated myometrial cells. Fibroid cells stimulated with AngII demonstrated increased expression of active RhoA (2.6-fold) but treated myometrial cells remained unchanged. As an internal control, LPA increased active RhoA in myometrial cells (6.2-fold) more than fibroid cells (5.4-fold). Pretreatment with Losartan decreased the active RhoA expression by 35% in fibroid cells. CONCLUSIONS: These data suggest that angiotensin signaling is altered in uterine fibroids. ATR1 expression was increased in fibroid tissue compared to matched myometrium. The positive relationship between angiotensin stimulation and active Rho expression may have relevance to the association of uterine fibroids and hypertensive disease. Supported by: HD ZO1-008737-07.

IMAGING IN REPRODUCTIVE MEDICINE SPECIAL INTEREST GROUP O-158 Tuesday, October 20, 2009 4:15 PM TUBAL PATENCY EVALUATION: FEASIBILITY OF NEW AUTOMATED THREE DIMENSIONAL SONOGRAPHIC METHOD. C. Exacoustos, A. Di Giovanni, B. Szabolcs, C. Gabardi, E. Zupi, D. Arduini. Department of Surgery, Obstetrics and Gynecology Unit, Universita` of Tor Vergata, Rome, Italy; GE Healthcare, Zipf, Austria. OBJECTIVE: To evaluate the feasibility of a new automated three dimensional contrast-enhanced ultrasound technology called ‘‘Coded Contrast Imaging’’ (3D CCI-GE Healthcare) in the evaluation of tubal patency and visualization of tubal course. DESIGN: Patients undergoing transvaginal hystero-salpingo-contrast-sonography (TVS HyCoSy) with automated 3D CCI were prospectively evaluated. MATERIALS AND METHODS: Sixtytwo patients of reproductive age underwent automated 3D CCI HyCoSy. The CCI allows the detection of an ultrasound contrast agent in the tube by discriminating the harmonic response of contrast bubbles from the echoes of other tissues. 3D TVS allows the visualization of tubal course. In order to evaluate the feasibility of this method we considered first the tubal patency and the 3D visualization of

Vol. 92., No. 3, Supplement, September 2009