Changes in transcription profile in pelvic organ fibroblasts in response to stretch

Failure of ligamentous support of the genital tract to resist intra-abdominal pressure is a plausible underlying mechanism for the development of pelvic organ prolapse, but the nature of molecular response of pelvic tissue support remains unknown. We hypothesized that the expression of genes coding for proteins involved in maintaining the cellular and extracellular integrity would be altered in cases of pelvic organ prolapse. Therefore, cDNA microarrays were used to examine the difference in transcriptional profile in RNA of primary culture fibroblasts subjected to mechanical stretch and those that remained static. Keywords: Changes in transcription profile in pelvic organ fibroblasts in response to stretch Source of fibroblasts: Three tissue samples were collected from uterine specimens of abdominal hysterectomy performed on premenopausal Caucasian women with no history of uterine prolapse. The supra-cervical area was cut (5 mm thick slices) with a scalpel to obtain the medial ends of the cardinal ligament. Every patient had to sign a consent form preoperatively, allowing the use of tissues removed at surgery for research purposes. The local ethics committee has approved this research program. Primary culture: The specimens were placed immediately in a cold Hanks’ Balanced Salt Solution, HBSS, (Gibco, Paisley, Scotland, UK) and transferred to the tissue culture laboratory for immediate processing. They were minced into one mm3 cubes, treated with 1% Collagenase type I (Gibco, Paisley, Scotland, UK) in HBSS, and incubated at 37oC for 2h with gentle rotation. The supernatant, containing the fibroblasts, was washed with HBSS and Medium 199 Earle’s MOD Salts (Gibco, Paisley, Scotland, UK) to which 1% antibiotic/antimycotic solution (10 000 U penicillin, 10 mg streptomycin, 25 μg Amphotericin B per ml, Sigma-Aldrich, Dorset, UK) and 15% heat-inactivated fetal bovine serum (HyClone, Logan, Utah, USA) were added. The cell suspension was spun down twice to remove collagenase. The pellets were re-suspended in growth medium, plated in 25 cm2 tissue culture flasks with Nunclon surface (Nalge Nunc International Corporation, Hereford, UK) and incubated at 37oC in humidified air with 5% CO2. The medium was changed on alternate days and confluent fibroblasts cells were passaged into a 75 cm2 flask (Nalge Nunc International Corporation, Hereford, UK) using 0.1% trypsin-EDTA (Gibco, Paisley, Scotland, UK) for 10 min at 37oC. Stretch experiments: Preparation of cells for treatment: Fibroblasts from the 4th to 6th passages were seeded (5x104 cells/well) into 6-well BioFlex® flexible plates with Collagen-I coated elastomer membrane bottoms (FlexCell International Corporation, McKeesport, Pennsylvania, USA), and allowed to grow. They were serum-starved for 48h, by using 1% charcoal/dextran treated heat-inactivated fetal bovine serum (HyClone, Logan, Utah, USA) added to the tissue culture medium to achieve quiescence, and then they were cultured with 15% of the same serum added to the tissue culture medium. Chronic cyclical mechanical stretch: The Flexercell Stress Unit (FlexCell International Corporation, McKeesport, Pennsylvania, USA) provides the equipment for the application of stretch to cultured cells in vitro. The plates are housed within a manifold and subjected to a pre-calibrated computer-controlled vacuum stretch (Hsieh et al., 2000, Stanley et al., 2000). The cells were exposed to stretch amplitude of (21%)-relaxation (0%) cycles of 12h each, at a frequency of 1 Hz, for 96h, at 37oC in humidified air with 5% CO2. The corresponding BioFlex® static plate was kept in the same incubator. Changes in gene expression were analyzed using three cDNA arrays, consisting of pairs of arrays forward labeled with Cy5 and Cy3 dyes, hybridizing RNA from stretched fibroblasts against controls. All controls were pooled. Microarray hybridization and analysis were performed as previously described (Turton et al., 2001). Changes in gene expression were analysed hybridizing RNA from treated cells against pooled controls. In this study Cy3 was added to control samples and Cy5 to treated samples and was referred to as forward labelling