Patrick W. Keller

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.

Failure of Pelvic Organ Support in Mice Deficient In Fibulin-3

Fibulin-5 is crucial for normal elastic fiber synthesis in the vaginal wall; more than 90% of fibulin-5-knockout mice develop pelvic organ prolapse by 20 weeks of age. In contrast, fibulin-1 and -2 deficiencies do not result in similar pathologies, and fibulin-4-knockout mice die shortly after birth. EFEMP1 encodes fibulin-3, an extracellular matrix protein important in the maintenance of abdominal fascia. Herein, we evaluated the role of fibulin-3 in pelvic organ support. Pelvic organ support was impaired significantly in female Efemp1 knockout mice (Fbln3(-[supi]/-)), and overt vaginal, perineal, and rectal prolapse occurred in 26.9% of animals. Prolapse severity increased with age but not parity. Fibulin-5 was up-regulated in vaginal tissues from Fbln3(-[supi]/-) mice regardless of prolapse. Despite increased expression of fibulin-5 in the vaginal wall, pelvic organ support failure occurred in Fbln3(-[supi]/-) animals, suggesting that factors related to aging led to prolapse. Elastic fiber abnormalities in vaginal tissues from young Fbln3(-[supi]/-) mice progressed to severe elastic fiber disruption with age, and vaginal matrix metalloprotease activity was increased significantly in Fbln3(-[supi]/-) animals with prolapse compared with Fbln3(-[supi]/-) mice without prolapse. Overall, these results indicate that both fibulin-3 and -5 are important in maintaining pelvic organ support in mice. We suggest that increased vaginal protease activity and abnormal elastic fibers in the vaginal wall are important components in the pathogenesis of pelvic organ prolapse.

Neuraminidase-1 is required for the normal assembly of elastic fibers

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.

Recovery of the injured external anal sphincter after injection of local or intravenous mesenchymal stem cells

OBJECTIVES: To understand the endogenous process of wound healing after anal sphincter injury and to determine possible mechanisms by which mesenchymal stem cells (MSCs) exert their regenerative potential. METHODS: Virginal female rats (n=204) underwent anal sphincter laceration and repair. Thereafter, animals were randomly assigned to control injection, injection with intravenous MSCs, or direct injection of MSCs into the injured sphincter. Twenty uninjured animals served as baseline controls. Sphincters were analyzed for contractile function and parameters of wound healing 24 hours, 48 hours, 7 days, and 21 days after injury. RESULTS: Direct injection of MSCs into the injured anal sphincter resulted in improved contractile function 21 days after injury compared with controls. Although expression of both proinflammatory (cyclooxygenase-2 and interleukin-6) and anti-inflammatory (interleukin-10 and tumor necrosis factor-&agr;–stimulated gene-6) genes were increased dramatically and transiently after injury, MSCs did not alter this response. In contrast, transforming growth factor (TFG)-&bgr;1 (an important mediator of matrix deposition by mesenchymal cells) and lysyl oxidase (an enzyme important for synthesis of collagen and elastin) expression increased dramatically at earlier time points in the direct MSC injection group compared with controls. Increased expression of TFG-&bgr;1 and lysyl oxidase in directly injected sphincters was associated with increased collagen deposition and engraftment of MSCs in the sphincter. CONCLUSION: In this preclinical animal model, direct, but not intravenous, injection of MSCs into the injured anal sphincter at the time of repair resulted in improved contractile function of the sphincter after injury, increased matrix deposition in the external anal sphincter, and increased expression of TFG-&bgr;1 and lysyl oxidase in the acute phase after injury.

Effect of myogenic stem cells on contractile properties of the repaired and unrepaired transected external anal sphincter in an animal model

OBJECTIVE: To estimate the effect of myogenic stem cells on contractile function of the external anal sphincter after transection with or without repair in an animal model. METHODS: One hundred twenty virginal female rats were randomly assinged to repair (n=60) or no repair (n=60) after anal sphincter transection. Animals were further divided into two groups: 40-microliter injection at the transection site with either phosphate-buffered solution (control) or myogenic stem cells (3.2×106 cells). Animals were killed at 7, 21, or 90 days, and the anal sphincter complex dissected and analyzed for contractile function. RESULTS: Contractile function of the external anal sphincter was severely impaired 7 days after sphincter transection with or without repair. Twitch tension, maximal tetanic contraction, and maximal contractile force in response to electrical field stimulation improved significantly with time after sphincter repair. Injection of myogenic stem cells in the anal sphincter at the time of repair resulted in superior contractile function at both 7 days and 90 days compared with controls. Interestingly, contractile function of the nonrepaired external anal sphincter did not improve with time with or without myogenic stem cells. Indicators of denervation (fatigue and twitch or tetany ratios) did not change among groups. CONCLUSION: In this animal model, injection of myogenic stem cells at the time of external anal sphincter repair resulted in enhanced contractile function at 90 days compared with repair alone. Without repair, function of the external anal sphincter was not improved by stem cell therapy at any time point. These results suggest that addition of myogenic stem cells improves both acute and long-term function of the external anal sphincter after mechanical injury.

Regulation of Elastolytic Proteases in the Mouse Vagina During Pregnancy, Parturition, and Puerperium

Abstract Recent evidence indicates that failure of elastic fiber assembly and synthesis is involved in the pathophysiology of pelvic organ prolapse in mice. It has been long been hypothesized that parturition-induced activation of proteases in the vaginal wall and its supportive tissues may contribute to pelvic organ prolapse in women. In this investigation, we determined the expression of matrix metalloproteases with elastase activity (matrix metalloproteinase [MMP] 2, MMP9, and MMP12) and their inhibitors in the vaginal wall of nonpregnant, pregnant, and postpartum mice. Data obtained using mRNA levels and enzyme activity measurements indicate that MMP2, MMP9, and 21- to 24-kDa caseinolytic serine proteases are regulated in vaginal tissues from pregnant and postpartum mice. Although suppressed during pregnancy and the early postpartum time period, MMP2 and MMP9 enzyme activities are increased after 48 h, a time when mRNA levels of protease inhibitors (tissue inhibitor of MMP2 [Timp2], cystatin C [Cst3], and alpha-1 antitrypsin [Serpina1]) are decreased. We conclude that recovery of the vaginal wall from pregnancy and parturition requires increased elastic fiber assembly and synthesis to counteract the marked increase in elastolytic activity of the postpartum vagina.

Fetal Fibronectin Signaling Induces Matrix Metalloproteases and Cyclooxygenase-2 (COX-2) in Amnion Cells and Preterm Birth in Mice

Background: The function of fetal fibronectin (fFN) in the pathogenesis of preterm labor is not known. Results: fFN activates MMP-1, MMP-9, and COX-2 in mesenchymal cells and causes preterm labor in mice. Conclusion: fFN is biologically active and plays a significant role in the pathogenesis of preterm labor. Significance: Signaling of fFN in fetal membranes is important in the pathophysiology of premature preterm rupture of the membranes. Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E2 synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes.

Interleukin-1β induces expression of neuropeptide Y in primary astrocyte cultures in a cytokine-specific manner: induction in human but not rat astrocytes

Previous studies have demonstrated that astrocyte cultures express neuropeptide Y (NPY) in a regulated manner, namely, phorbol ester leads to an increase in proNPY-mRNA and NPY production. In this respect, the behavior of astrocytes derived from the human fetal or rat neonatal brain is similar (Regul. Pept. 75 (1998) 293). Since astrocytes can be exposed to high levels of IL-1beta, we addressed the question: Does IL-1beta regulate NPY expression by the astrocytes? Primary astrocytes derived from the human fetal or rat neonatal cortex were cultured in serum-free medium. IL-1beta, but not IL-6 or TNF-alpha, led to an increase in NPY production dose-dependently. IL-1beta action manifested in the human but not in the rat astrocytes and it was completely abolished by IL-1 receptor antagonist. The responsiveness to IL-1beta did not diminish upon sub-culture of the astrocytes (five passages). In addition, IL-1beta led to an increase in the abundance of proNPY-mRNA, which was preceded by a rapid and transient increase in cFos-mRNA and a rapid and sustained increase in JunB-mRNA. In contrast to cFos/JunB, IL-1beta did not alter the abundance of cJun-mRNA. In summary, we demonstrate that IL-1beta induction of NPY expression in astrocytes is species- and cytokine-specific and that IL-1 receptor is involved. Moreover, induction of NPY expression is preceded by a rapid increase in the expression of two transcription factors (cFos, JunB) that have been previously (Oncogene 9 (1994) 2369; J. Neurochem. 70 (1998) 1887) implicated in transcriptional regulation of the human NPY gene.

Effect of Thrombin on Human Amnion Mesenchymal Cells, Mouse Fetal Membranes, and Preterm Birth

Background: Bleeding during pregnancy is a risk factor for premature rupture of the fetal membranes. Results: Thrombin causes preterm birth in mice and activates PAR-1 and TLR4 to increase MMPs and COX-2. Conclusion: Thrombin acts through multiple mechanisms to increase MMPs and PGE2 in amnion. Significance: Thrombin plays a pivotal role in the pathogenesis of preterm labor and rupture of the membranes. Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.

Estrogen Alters Remodeling of the Vaginal Wall after Surgical Injury in Guinea Pigs

ABSTRACT Loss of pelvic organ support (i.e., pelvic organ prolapse) is common in menopausal women. Surgical reconstruction of pelvic organ prolapse is plagued with high failure rates. The objective of this study was to determine the effects of estrogen on biomechanical properties, lysyl oxidase (LOX), collagen content, and histomorphology of the vagina with or without surgical injury. Nulliparous ovariectomized guinea pigs were treated systemically with either 50 μg/kg/day estradiol (E2,) or vehicle. After 2 wk, vaginal surgery was performed, and animals were treated with either beta-aminopropionitrile (BAPN, an irreversible LOX inhibitor), or vehicle to determine the role of LOX in recovery of the vaginal wall from injury with or without E2. Estradiol resulted in (i) significant growth, increased smooth muscle, and increased thickness of the vagina, (ii) increased distensibility without compromise of maximal force at failure, and (iii) increased total and cross-linked collagen. In the absence of E2, BAPN resulted in decreased collagen and vaginal wall strength in the area of the injury. In contrast, in E2-treated animals, increased distensibility, maximal forces, and total collagen were maintained despite BAPN. Interestingly, LOX mRNA was induced dramatically (9.5-fold) in the injured vagina with or without E2 at 4 days. By 21 days, however, LOX levels declined to near baseline in E2-deprived animals. LOX mRNA levels remained strikingly elevated (12-fold) at 21 days in the estrogenized vagina. The results suggest that prolonged E2 induced increases in LOX, and collagen cross-links may act to sustain a matrix environment that optimizes long-term surgical wound healing in the vagina.