R. Ann Word

Cervical remodeling during pregnancy and parturition: molecular characterization of the softening phase in mice

Cervical remodeling during pregnancy and parturition is a single progressive process that can be loosely divided into four overlapping phases termed softening, ripening, dilation/labor, and post partum repair. Elucidating the molecular mechanisms that facilitate all phases of cervical remodeling is critical for an understanding of parturition and for identifying processes that are misregulated in preterm labor, a significant cause of perinatal morbidity. In the present study, biomechanical measurements indicate that softening was initiated between gestation days 10 and 12 of mouse pregnancy, and in contrast to cervical ripening on day 18, the softened cervix maintains tissue strength. Although preceded by increased collagen solubility, cervical softening is not characterized by significant increases in cell proliferation, tissue hydration or changes in the distribution of inflammatory cells. Gene expression studies reveal a potentially important role of cervical epithelia during softening and ripening in maintenance of an immunomucosal barrier that protects the stromal compartment during matrix remodeling. Expression of two genes involved in repair and protection of the epithelial permeability barrier in the gut (trefoil factor 1) and skin (serine protease inhibitor Kazal type 5) were increased during softening and/or ripening. Another gene whose function remains to be elucidated, purkinje cell protein 4, declines in expression as remodeling progressed. Collectively, these results indicate that cervical softening during pregnancy is a unique phase of the tissue remodeling process characterized by increased collagen solubility, maintenance of tissue strength, and upregulation of genes involved in mucosal protection.

Human Myometrial Gene Expression Before and During Parturition

Abstract Identification of temporal and spatial changes in myometrial gene expression during parturition may further the understanding of the coordinated regulation of myometrial contractions during parturition. The objective of this study was to compare the gene expression profiles of human fundal myometrium from pregnant women before and after the onset of labor using a functional genomics approach, and to further characterize the spatial and temporal expression patterns of three genes believed to be important in parturition. Fundal myometrial mRNA was isolated from five women in labor and five women not in labor, and analyzed using human UniGEM-V microarrays with 9182 cDNA elements. Real-time polymerase chain reaction using myometrial RNA from pregnant women in labor or not in labor was used to examine mRNA levels for three of the genes; namely, prostaglandin-endoperoxide synthase 2 (PTGS2), calgranulin B (S100A9), and oxytocin receptor (OXTR). The spatial expression pattern of these genes throughout the pregnant uterus before and after labor was also determined. Immunolocalization of cyclooxygenase-2 (also known as PTGS2) and S100A9 within the uterine cervix and myometrium were analyzed by immunohistochemistry. Few genes were differentially expressed in fundal myometrial tissues at term with the onset of labor. However, there appears to be a subset of genes important in the parturition cascade. The cellular properties of S100A9, its spatial localization, and dramatic increase in cervix and myometrium of women in labor suggest that this protein may be very important in the initiation or propagation of human labor.

Telomerase Immortalization of Human Myometrial Cells

Abstract Several strategies have been described for the primary culture of human myometrial cells. However, primary cultures of myometrial cells have a limited life span, making continual tissue acquisition and cell isolation necessary. Recent studies have demonstrated that cell culture life span is related to chromosomal telomere length, and cellular senescence results from progressive telomere shortening and the lack of telomerase expression. Transfection of cells with expression vectors containing the human telomerase reverse transcriptase (hTERT) maintains telomere length and effectively gives normal cells an unlimited life span in culture. In addition, hTERT extends the life span of cultured cells far beyond normal senescence without causing neoplastic transformation. In the present study, we developed a cell line from hTERT-infected myometrial cells (hTERT-HM). Cells were isolated from myometrial tissue obtained from women undergoing hysterectomy, and retroviral infection was used to express the catalytic subunit of telomerase in myometrial cells. Cells expressing hTERT have been in continuous culture for >10 mo, whereas the control culture senesced after approximately 2 mo. Telomerase activity was monitored in cells with a polymerase chain reaction-based telomerase activity assay. Telomerase-expressing cells contained mRNA for α smooth muscle actin, smoothelin, oxytocin receptor, and estrogen receptor α, but the estrogen receptor β receptor was lost. Immunoblotting analysis identified the expression of calponin, caldesmon, α smooth muscle actin, and oxytocin receptor. Although estrogen receptor expression was below the level of detection with immunoblotting, transfection experiments performed with reporter constructs driven by estrogen response elements demonstrated estrogen responsiveness in the hTERT-HM. In addition, treatment of hTERT-HM with oxytocin caused a concentration-dependent increase in intracellular calcium levels, confirming the presence of functional oxytocin receptors. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in primary cultures of smooth muscle cells. The expression of various smooth muscle/myometrium cell markers suggests that these cells may be an appropriate model system to study certain aspects of human myometrial function.

Fibulin-4 Deficiency Results in Ascending Aortic Aneurysms. A Potential Link Between Abnormal Smooth Muscle Cell Phenotype and Aneurysm Progression

Rationale: Loss of fibulin-4 during embryogenesis results in perinatal lethality because of aneurysm rupture, and defective elastic fiber assembly has been proposed as an underlying cause for the aneurysm phenotype. However, aneurysms are never seen in mice deficient for elastin, or for fibulin-5, which absence also leads to compromised elastic fibers. Objective: We sought to determine the mechanism of aneurysm development in the absence of fibulin-4 and establish the role of fibulin-4 in aortic development. Methods and Results: We generated germline and smooth muscle cell (SMC)-specific deletion of the fibulin-4 gene in mice (Fbln4GKO and Fbln4SMKO, respectively). Fbln4GKO and Fbln4SMKO aortic walls fail to fully differentiate, exhibiting reduced expression of SM-specific contractile genes and focal proliferation of SMCs accompanied by degenerative changes of the medial wall. Marked upregulation of extracellular signal-regulated kinase 1/2 signaling pathway was observed in the aneurysmal wall of Fbln4GKO and Fbln4SMKO mice and both mutants developed aneurysm predominantly in the ascending thoracic aorta. In vitro, Fbln4GKO SMCs exhibit an immature SMC phenotype with a marked reduction of SM-myosin heavy chain and increased proliferative capacity. Conclusions: The vascular phenotype in Fbln4 mutant mice is remarkably similar to a subset of human thoracic aortic aneurysms caused by mutations in SMC contractile genes. Our study provides a potential link between the intrinsic properties of SMCs and aneurysm progression in vivo and supports the dual role of fibulin-4 in the formation of elastic fibers as well as terminal differentiation and maturation of SMCs in the aortic wall.

Biomechanical Properties of The Vaginal Wall: Effect of Pregnancy, Elastic Fiber Deficiency, and Pelvic Organ Prolapse

OBJECTIVE The purpose of this study was to identify pregnancy-induced changes in biomechanical properties of the vaginal wall and to compare these with fibulin-5 knockout mice (Fbln5(-/-)) with and without prolapse. STUDY DESIGN Mid-vaginal segments of nonpregnant and late-pregnant wild-type mice, Fbln5(-/-) with prolapse mice and Fbln5(-/-) mice without prolapse were studied. Tissue length at failure, maximal strain, maximal stress, and tissue stiffness were determined. RESULTS Compared with nonpregnant mice, vaginas of pregnant and Fbln5(-/-) (with prolapse) mice exhibited decreased maximal stress, increased distensibility and strain, and decreased stiffness. Tissues from Fbln5(-/-) mice without prolapse were similar to nonpregnant wild-type animals. CONCLUSION Pregnancy confers remarkable changes in the vaginal wall that include increased distensibility and decreased stiffness and maximal stress. Elastinopathy alone is insufficient to cause significant changes in these properties, but prolapse confers additional alterations in distensibility and stiffness that are similar to those changes that have been observed in pregnancy. These changes may contribute to the poor durability of many restorative surgical procedures for prolapse.

Compartmentalization of type 117β-hydroxysteroid oxidoreductase in the human ovary

Abstract The steroid-metabolizing enzyme, type I 17β-hydroxysteroid oxidoreductase (17β-HSOR) also called 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a key role in ovarian synthesis of 17β-estradiol. This is the only enzyme in the steroid-metabolizing pathway which has not been localized in the human ovary by immunohistochemistry. In this study, using antibody directed against human placenta! cytosolic 17β-HSOR (type I), a single protein band with a relative molecular mass of approximately 34 kDa was demonstrated by Western analysis in both human luteinized granulosa cells and placental tissue. In placental tissue, immunoreactive type I 17β-HSOR was demonstrated within the syncytiotrophoblast using immunohistochemistry. In human ovary, immunoreactive type I 17β-HSOR was localized exclusively in granulosa cells of developing follicles, ranging from primary follicles with a single layer of cuboidal-shaped granulosa cells, preantral follicles with multiple layers of granulosa cells, and large antral follicles. No immunoreactivity was detected in spindle-shaped granulosa cells of primordial follicles, theca interna, theca externa or surrounding stroma. In the corpus luteum, type I 17β-HSOR immunoreactivity was localized solely in granulosa-lutein cells. For comparison, immunoreactive 3β-hydroxysteroid dehydrogenase (3β-HSD) was examined in the same tissues. Both theca interna and granulosa cells of preantral and antral follicles exhibited 3β-HSD staining. Primary follicles did not exhibit detectable 3β-HSD in either granulosa or theca cells. This study serves to demonstrate that in the human ovary, type I 17β-HSOR is compartmentalized in granulosa cells of the developing follicles and granulosa-lutein cells. In addition, the expression of type I 17β-HSOR appears to be present prior to that of 3β-HSD at the stage of primary follicle. With the strategic location of type I 17β-HSOR and the ability to aromatize theca-derived androstenedione to estrone, granulosa cells have the capacity to synthesize and maintain the high intrafollicular levels of 17β-estradiol, which are essential for normal follicular development.

Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium

Endothelin, a recently discovered sarafotoxin-like peptide secreted by endothelial cells, is a potent stimulator of vascular smooth muscle contraction. We found that the action of endothelin is not restricted to the vasculature; we demonstrated that endothelin causes an increase in the concentration of intracellular Ca++ and phosphorylation of the 20 kd light chain of myosin in human uterine smooth muscle cells in culture. In the absence of Ca++ in the buffer medium of myometrial cells, the effects of endothelin on intracellular Ca++ and myosin light chain phosphorylation are attenuated but not abolished. Endothelin also increases the frequency of contraction of human uterine smooth muscle (longitudinal and circular). The contractile effects of endothelin on myometrial strips are diminished in the presence of nifedipine. We conclude that (1) human myometrium is responsive to endothelin, (2) endothelin promotes contraction in myometrium by effecting an increase in intracellular Ca++ and thus an increase in myosin light chain phosphorylation, and (3) endothelin acts in myometrium by stimulating Ca++ influx as well as Ca++ release from intracellular stores.

Extracellular matrix proteases contribute to progression of pelvic organ prolapse in mice and humans

Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, fibulin-5 inhibited the β1 integrin-dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5RGE/RGE) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to fibulin-5 knockouts (Fbln5-/-), Fbln5RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with β-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5-/- mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre- and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women.

Estrogen and progesterone metabolism in the cervix during pregnancy and parturition.

CONTEXT Experimental and clinical studies in a variety of nonprimate species demonstrate that progesterone withdrawal leads to changes in gene expression that initiate parturition at term. Mice deficient in 5alpha-reductase type I fail to undergo cervical ripening at term despite the timely onset of luteolysis and progesterone withdrawal in blood. OBJECTIVE Our objective was to test the hypothesis that estrogen and progesterone metabolism is regulated in cervical tissues during pregnancy, even in species in which parturition is not characterized by progesterone withdrawal in blood. DESIGN Estradiol and progesterone metabolism was quantified in intact cervical tissues from nonpregnant and pregnant women at term before or after labor. SETTING The study was conducted at a university hospital. PATIENTS Tissues were obtained from five nonpregnant and 21 pregnant women (nine before labor and 12 in labor). MAIN OUTCOME MEASURES Enzyme activity measurements, Northern blot analysis, quantitative real-time RT-PCR, and immunohistochemistry were used to quantify steroid hormone metabolizing enzymes in cervical and myometrial tissues. RESULTS During pregnancy, 17beta-hydroxysteroid dehydrogenase type 2 was induced in glandular epithelial cells to catalyze the conversion of estradiol to estrone and stroma-derived 20alpha-hydroxyprogesterone to progesterone. During parturition, 17beta-hydroxysteroid dehydrogenase type 2 was down-regulated in endocervical cells, thereby creating a microenvironment favorable for cervical ripening. CONCLUSIONS Together, the data indicate that cervical ripening during parturition involves localized regulation of estrogen and progesterone metabolism through a complex relationship between cervical epithelium and stroma, and that steroid hormone metabolism in cervical tissues from pregnant women is unique from that in mice.

C-Reactive Protein Downregulates Endothelial NO Synthase and Attenuates Reendothelialization In Vivo in Mice

C-reactive protein (CRP) is an acute-phase reactant that is positively associated with cardiovascular disease risk and endothelial dysfunction. In cell culture, CRP decreases the expression of endothelial NO synthase (eNOS), which regulates diverse endothelial cell (EC) functions including migration. To determine whether CRP alters EC gene expression and phenotype in vivo, we studied CF1 transgenic mice expressing rabbit CRP (CF1-CRP) regulated by the phosphoenolpyruvate carboxykinase promoter such that levels could be altered by changing carbohydrate intake. Compared with CF1 controls with CRP of <1 &mgr;g/mL, carotid artery reendothelialization after perivascular electric injury was blunted in CF1-CRP mice, with CRP levels as low as 9 &mgr;g/mL. eNOS mRNA and enzyme abundance in carotid arteries was also blunted by CRP at 9 &mgr;g/mL in vivo, and ex vivo studies of isolated arteries showed that this occurs via direct action on the endothelium. The impaired reendothelialization with CRP was mimicked by NOS antagonism in CF1 mice; conversely, in cultured ECs CRP attenuation of migration was prevented by exogenous NO. Studies of EC transfected with human eNOS 5′ flanking sequence fused to luciferase indicated that CRP decreases eNOS gene transcription. Both mutagenesis and electrophoretic mobility shift assays further revealed that CRP-responsive elements reside within the first 79 bp of the eNOS promoter. Thus, CRP downregulates eNOS and attenuates reendothelialization in vivo in mice, and this action of CRP on eNOS is mediated at the level of gene transcription.