Progesterone modulates integrin {alpha}2 (ITGA2) and {alpha}11 (ITGA11) in the pregnant cervix.

OBJECTIVE: Fibrillar collagen in the cervical extracellular matrix (ECM) is the predominant component providing mechanical support. Cellular integrins contribute to structural integrity by cross-linking ECM components. We investigated the expression of collagen-binding integrins in the normal rat gestation and after treatment with mifepristone to determine whether integrin modulation is involved in changes in tissue resistance. STUDY DESIGN: Cervical tissue was harvested from nonpregnant and timed pregnant Sprague-Dawley rats. Normal gestational expression was evaluated in nonpregnant and timed pregnant tissue on days 12, 16, 18, 20, 21 and 22. Progesterone inhibition was induced with 3 mg mifepristone administered on day 15. Primary rat cervical stromal (RCS) cell cultures were generated from nonpregnant rats using tissue explants. The effects of progesterone environment on RCS cells were evaluated in the presence and absence of various inhibitors. Protein expression and signaling pathways were evaluated by Western blot. RESULTS: Integrin α2 (ITGA2) expression increased over gestation, peaking at the end of gestation (analysis of variance [ANOVA] P < .01). Integrin α11 (ITGA11) expression increased through mid-gestation, peaking on day 18 and decreasing through day 22 (ANOVA P < .001). Progesterone increased the expression of ITGA11 and phosphorylated focal adhesion kinase ([pFAK] P < .002). Mifepristone blocked these effects in vitro. Mifepristone increased ITGA2 and phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) in vivo and in vitro. Mifepristone-induced upregulation of ITGA2 was abrogated by inhibition of ERK1/2. CONCLUSION: Progesterone/progesterone withdrawal is involved in regulating the expression of collagen-binding integrins. These changes differ among the collagen-binding integrins. Mitogen-activated protein kinase (MAPK) signaling is involved in regulating some of these integrins.

The role of transforming growth factor beta in cervical remodeling within the rat cervix.

OBJECTIVE: Transforming growth factor beta (TGFbeta) plays a central role in extracellular matrix remodeling. We hypothesized that TGFbeta signaling is involved in cervical remodeling. This study evaluated patterns within this signaling pathway. STUDY DESIGN: The cervices of nonpregnant and timed pregnant rats were obtained. Messenger ribonucleic acid (mRNA) expression of TGFbeta1, TGFbeta receptor 1 (TbetaR1), TbetaR2, and TbetaR3 was evaluated. Four animals were euthanized for each time point. Western blotting was performed for protein expression. Phosphorylated mothers against decapentaplegic (Smad)-2 and -3 phosphorylation was assessed to evaluate TGFbeta activation. RESULTS: TGFbeta1 mRNA increased through day 21 and declined on day 22 (analysis of variance, P = .001). TbetaR1 expression was unchanged. TbetaR2 and TbetaR3 mRNA expression was similar to TGFbeta1. TbetaR3 protein expression was similar to mRNA. Smad2 phosphorylation paralleled changes in TbetaR3. CONCLUSION: Components of the TGFbeta signaling pathway increase during pregnancy along with Smad2 activation. The decline on day 22 correlates with a transition to the ripening phase supporting a role in cervical remodeling.

Androgen-regulated cervical ripening: a structural, biomechanical, and molecular analysis.

OBJECTIVE: Androgens regulate biomechanical responses in load-bearing tissues. Evidence suggests that androgens may play a role in the cervix. We hypothesized that androgens directly regulate cervical remodeling by altering both collagen structure and proteoglycan composition. STUDY DESIGN: Cervical resistance was evaluated using the cervical creep method after the administration of intravaginal dihydrotestosterone or oral flutamide. Microstructural changes in collagen were evaluated by transmission electron microscopy and polarized light birefringence. Proteoglycan expression was evaluated by reverse transcription-polymerase chain reaction for the core proteins (decorin, biglycan, fibromodulin, aggrecan, versican) and fluorophore-assisted carbohydrate analysis. RESULTS: Dihydrotestosterone decreased cervical resistance, whereas flutamide inhibited the decline in cervical resistance, compared with vehicle controls. Flutamide was associated with higher levels of organized collagen and increased aggrecan expression with a greater proportion of chondroitin/dermatan sulfate glycosaminoglycans. Flutamide inhibited the increase in hyaluronan. CONCLUSION: Androgens appear to play a role in regulating cervical resistance by altering proteoglycan content. Structural analysis indicates that flutamide may alter collagen fibril organization and/or structure.

Prostaglandin E2-regulated cervical ripening: analysis of proteoglycan expression in the rat cervix.

OBJECTIVE: Prostaglandins reduce cervical resistance by reorganizing collagen fibrils. Proteoglycans are involved in collagen fibril organization and structure. We evaluated the changes in proteoglycan composition induced by prostaglandin E(2) (PGE(2)). STUDY DESIGN: Prostaglandins were administered intravaginally to induce cervical ripening in timed pregnant Sprague-Dawley rats. Changes in proteoglycan messenger ribonucleic acid (mRNA) expression were measured using reverse transcription (RT-PCR) for core protein. Fluorophore assisted carbohydrate gel electrophoresis (FACE) was used to evaluate proteoglycan glycosaminoglycan composition along with size exclusion high-performance liquid chromatography (HPLC). RESULTS: No change in core protein mRNA expression was detected after PGE(2) treatment. Total glycosaminoglycan (GAG) decreased more than 20% after PGE(2) (P = .02). FACE demonstrated a shift in disaccharide subunit composition after PGE(2), with a decrease in 4-sulfated disaccharides (P = .02). HPLC confirmed a decrease in total GAG (P = .04). CONCLUSION: Although there was no change in core protein mRNA expression, alterations in GAG composition was detected after PGE(2). The decrease in sulfated GAG could decrease electrostatic interactions that would weaken interfibrillar interactions. These findings would be consistent with a decline in cervical resistance.

The use of streptolysin o for the treatment of scars, adhesions and fibrosis: initial investigations using murine models of scleroderma.

Diseases and conditions involving the deposition of excessive amounts of collagen include scleroderma, fibrosis, and scar and surgical adhesion formation. Diseases such as scleroderma may result from acute and chronic inflammation, disturbances in the normal parenchymal area, and activation of fibroblasts. ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is being developed for the treatment of such collagen-related disorders. At sublytic concentrations in vitro, ML-05 was shown to activate CD44 expression. This may modulate production of collagen, hyaluronate, and their associated enzymes to allow a restoration of normal extracellular matrices within tissues. More importantly, ML-05 appeared to decrease skin collagen levels in two in vivo models of collagen disorders, the tight skin mouse (Tsk) model of scleroderma, and the bleomycin-induced mouse skin fibrosis model. In the Tsk model, levels of hydroxyproline (a measure of total collagen) decreased by 25% in the Tsk+ML-05 treatment group relative to the Tsk+saline control group over a 3-month period. In the bleomycin-induced skin fibrosis study, hydroxyproline levels decreased from 15-22% over a 6-week period in a bleomycin-induced ML-05 treatment group (relative to levels in a bleomycin-induced, untreated control group). Hydroxyproline levels in samples from this treatment group were only slightly greater than levels in an uninduced control group at 8 weeks. Thus, ML-05 treatment appeared to reduce collagen levels in two separate mouse skin fibrosis models, one genetically based and the other chemically induced.