Induction of Cellular Senescence in Rat Vaginal Fibroblasts and Treatment With Senolytics: An in Vitro Model for the Study of Pelvic Organ Prolapse.

OBJECTIVE: The objective of this study was to develop an in vitro model of cellular senescence using rat vaginal fibroblasts and determine the effects of treatment with senolytics. METHODS: Rat vaginal tissue biopsies were collected. Primary vaginal fibroblasts were isolated and characterized by immunofluorescence. To induce cellular senescence, fibroblasts were treated with etoposide at 3, 10, and 20 mM for 24 hours, followed by treatment with the senolytics dasatinib (1 mM) and/or quercetin (20 mM). After treatment, RNA was extracted and the expression of selected genes was quantified. Immunostaining of senescence markers was also performed. RESULTS: Fibroblasts were confirmed by positive immunostaining for α-smooth muscle actin and vimentin, and negative immunostaining for pan-cytokeratin. Treatment with etoposide resulted in a dose-dependent increase in expression of the senescence-associated secretory phenotype markers MMP-7, MMP-9, and IL-b1 (P < 0.05) compared with controls. Immunostaining showed increased expression of γ-H2A and p21 after treatment with etoposide. Cells treated with dasatinib and quercetin after etoposide treatment had decreased expression of p21, MMP-7, MMP-9, and IL-1b compared with cells treated only with etoposide (P < 0.05). CONCLUSIONS: Upregulation of senescence-associated factors provided evidence that senescence can be induced in vaginal fibroblasts in vitro. Furthermore, treatment with the senolytics dasatinib and quercetin abrogated the senescence phenotype induced by etoposide in rat vaginal fibroblasts. Our findings provide a novel model for the study and development of new therapies targeting the disordered extracellular matrix associated with pelvic organ prolapse.

Effect of single-dose ibuprofen on follicular fluid levels of interleukins in poor responders undergoing in vitro fertilization.

Premature follicular rupture during in vitro fertilization (IVF) is a well-known culprit for cycle cancellation. We sought to evaluate whether a single oral dose of ibuprofen will have an effect on the follicular fluid (FF) levels of inflammatory markers involved in ovulation. This is a prospective within-subjects study following nine patients undergoing IVF. Every patient underwent a first cycle of minimal stimulation IVF followed by a second cycle using the same stimulation protocol, except one oral dose of ibuprofen 800 mg was administered 15-18 h post-trigger injection. FF was obtained during oocyte retrievals of both cycles and analyzed for levels of selected inflammatory markers. A total of 27 cytokines and 9 matrix metalloproteinases (MMPs) were tested. Results demonstrate significantly decreased levels of interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor (G-CSF), eotaxin, MMP3, MMP7, MMP12, and MMP13 in FF of cycles where ibuprofen was administered. However, other cytokines levels, such IL-1 and vascular endothelial growth factor (VEGF), were similar with or without ibuprofen. Levels of MMPs described to be involved in ovulation, namely MMP-2 and MMP-9, were either undetectable or unchanged by ibuprofen, respectively. In conclusion, our data show that one dose of ibuprofen administered orally the day after trigger injection revealed a significant impact on the FF inflammatory milieu. Abbreviations: IVF: in vitro fertilization; MMP: matrix metalloproteinase; IL: interleukins; FF: follicular fluid; VEGF: vascular endothelial growth factor; NSAIDS: non-steroidal anti-inflammatories; POR: poor ovarian response; AMH: anti-Mullerian hormone; TAFC: total antral follicle count; HMG: human menopausal gonadotropin; hCG: human chorionic gonadotropin; COX: cyclooxygenase enzymes; PGH2: prostaglandin H2; RANTES: regulated on activation, normal T expressed and secreted; NF-κb: nuclear factor kappa-light-chain-enhancer of activated B cells.

Thrombin Alters Human Endometrial Stromal Cell Differentiation During Decidualization.

Vaginal bleeding and subchorionic hematomas are associated with increased risk of both early and late pregnancy loss. Thrombin generation may play a pivotal role in the development of these complications. To determine the effects of thrombin on human endometrial stromal cells (hESCs), cells were treated with thrombin at baseline or during decidualization with cyclic adenosine monophosphate (cAMP)+medroxyprogesterone acetate (MPA). Next-generation RNA sequencing revealed that markers of decidualization (IGF-1, IGFBP-1, and prolactin [PRL]) were induced after the initiation of decidualization, whereas thrombin suppressed insulin-like growth factor ( IGF)-1, Insulin-like growth factor binding protein ( IGFBP)-1, and PRL gene expression at baseline and during decidualization. These effects were mediated through protease activated receptor (PAR)-1- and PAR-1-independent pathways. Thrombin decreased the secretion of a key marker of decidualization (PRL), altered the morphological transformation of decidualizing hESCs, and activated genes involved in matrix degradation and proinflammatory chemokines ( Interleukin-8 and Interleukin-6). Genes encoding factors important for matrix stability ( Col1α1, LOX) were suppressed. We suggest that intrauterine bleeding and generation of thrombin accentuates leukocyte extravasation and endometrial inflammation, impairs decidualization, and endometrial support of early pregnancy.

Effect of Body Weight on Metabolic Hormones and Fatty Acid Metabolism in Follicular Fluid of Women Undergoing In Vitro Fertilization: A Pilot Study.

INTRODUCTION: Obesity is an epidemic affecting more than one-third of adults in the United States. Obese women experience decreased fertility, explained in part by oocyte quality. Since follicular fluid (FF) provides an important microenvironment for oocyte growth, we sought to evaluate the effect of increased body weight on FF levels of 11 metabolic hormones and fatty acid metabolism. METHODS: The FF was collected from 25 women (10 normal weight, 10 overweight, and 5 obese) with diminished ovarian reserve undergoing in vitro fertilization (IVF) following a minimal stimulation protocol. Hormone levels were determined by multiplex immunoassay using the MAGPIX (Luminex, Austin, Texas) instrument. Fatty acid metabolites were determined using gas and ultra-high pressure liquid chromatography coupled with mass spectrometry. RESULTS: Levels of hormones related to glucose and energy homeostasis and regulation of fat stores (insulin, glucagon, glucagon-like peptide-1, C-peptide, and leptin) were increased significantly in FF from obese women compared to FF from nonobese(normal weight and overweight) women. Interestingly, FF levels of branched-chain amino acids (BCAA) isoleucine, leucine, and valine as well as uric acid, isocaproic acid, butanoic acid, tyrosine, threonine, glycine, and methionine correlated positively with body mass index. CONCLUSION: This pilot study demonstrates significant alterations in the FF milieu of obese women undergoing IVF, which may contribute to the decreased fecundity of obese women. Although the impact of this environment on oocyte and embryo development is not fully realized, these changes may also lead to imprinting at the genomic level and long-term sequelae on offspring.

Dysregulation of fibulin-5 and matrix metalloproteases in epithelial ovarian cancer.

Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility. Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC). FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary. Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression. FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC. We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5. MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC. Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors. This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment. Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells. Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

Prostaglandin dehydrogenase is a target for successful induction of cervical ripening.

The cervix represents a formidable structural barrier for successful induction of labor. Approximately 10% of pregnancies undergo induction of cervical ripening and labor with prostaglandin (PG) E2 or PGE analogs, often requiring many hours of hospitalization and monitoring. On the other hand, preterm cervical ripening in the second trimester predicts preterm birth. The regulatory mechanisms of this paradoxical function of the cervix are unknown. Here, we show that PGE2 uses cell-specific EP2 receptor-mediated increases in Ca2+ to dephosphorylate and translocate histone deacetylase 4 (HDAC4) to the nucleus for repression of 15-hydroxy prostaglandin dehydrogenase (15-PGDH). The crucial role of 15-PGDH in cervical ripening was confirmed in vivo. Although PGE2 or 15-PGDH inhibitor alone did not alter gestational length, treatment with 15-PGDH inhibitor + PGE2 or metabolism-resistant dimethyl-PGE2 resulted in preterm cervical ripening and delivery in mice. The ability of PGE2 to selectively autoamplify its own synthesis in stromal cells by signaling transcriptional repression of 15-PGDH elucidates long sought-after molecular mechanisms that govern PG action in the cervix. This report details unique mechanisms of action in the cervix and serves as a catalyst for (i) the use of 15-PGDH inhibitors to initiate or amplify low-dose PGE2-mediated cervical ripening or (ii) EP2 receptor antagonists, HDAC4 inhibitors, and 15-PGDH activators to prevent preterm cervical ripening and preterm birth.

Vaginal estrogen: a dual-edged sword in postoperative healing of the vaginal wall.

OBJECTIVE: Reconstructive surgery for pelvic organ prolapse is plagued with high failure rates possibly due to impaired healing or regeneration of the vaginal wall. Here, we tested the hypothesis that postoperative administration of local estrogen, direct injection of mesenchymal stem cells (MSCs), or both lead to improved wound healing of the injured vagina in a menopausal rat model. METHODS: Ovariectomized rats underwent surgical injury to the posterior vaginal wall and were randomized to treatment with placebo (n = 41), estrogen cream (n = 47), direct injection of MSCs (n = 39), or both (n = 43). RESULTS: MSCs did not survive after injection and had no appreciable effects on healing of the vaginal wall. Acute postoperative administration of vaginal estrogen altered the response of the vaginal wall to injury with decreased stiffness, decreased collagen content, and decreased expression of transcripts for matrix components in the stromal compartment. Conversely, vaginal estrogen resulted in marked proliferation of the epithelial layer and increased expression of genes related to epithelial barrier function and protease inhibition. Transcripts for genes involved in chronic inflammation and adaptive immunity were also down-regulated in the estrogenized epithelium. CONCLUSIONS: Collectively, these data indicate that, in contrast to the reported positive effects of preoperative estrogen on the uninjured vagina, acute administration of postoperative vaginal estrogen has adverse effects on the early phase of healing of the stromal layer. In contrast, postoperative estrogen plays a positive role in healing of the vaginal epithelium after injury.

Nrf2 Activation Inhibits Effects of Thrombin in Human Amnion Cells and Thrombin-Induced Preterm Birth in Mice.

CONTEXT: Nrf2 is a key transcription factor that modulates cell defense mechanisms against endogenous and exogenous stress. Previously, we reported that thrombin increased matrix metalloproteinases and prostaglandin synthesis in human amnion mesenchymal cells. OBJECTIVE: We sought to determine whether activation of Nrf2 alters the effect of thrombin on prostaglandin synthesis, protease activation, and cytokine release in human amnion. Furthermore, we analyzed the effect of Nrf2 activation on thrombin-induced preterm labor in mice. DESIGN: Primary human amnion mesenchymal cells and pregnant mice were employed to investigate the effect of Nrf2 on thrombin-induced inflammation and preterm birth. SETTING: This was a laboratory-based study using cells and mice. RESULTS: As expected, thrombin increased cyclooxygenase-2, IL-1ß, IL-6, IL-8, and matrix metalloproteinase-1 in amnion mesenchymal cells. Preincubation with Nrf2 activators, diethyl maleate or 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), profoundly repressed thrombin-induced gene expression. In addition, Nrf2 activation inhibited thrombin-induced cyclooxygenase-2 protein levels and secretion of prostaglandin E2, IL-1ß, IL-6, IL-8, TNFα, and granulocyte-macrophage colony-stimulating factor in the media. Whereas vehicle and 15d-PGJ2 did not alter gestational length, all pregnant mice treated with thrombin delivered preterm. 15d-PGJ2 delayed thrombin-induced preterm birth significantly. CONCLUSIONS: The results indicate that Nrf2 activation represents a key stress response in amnion mesenchyme cells and in pregnant mice to mitigate the adverse proinflammatory effects of thrombin on the fetal membranes. We suggest, therefore, that pharmacological activation of Nrf2 may prevent the increased risk of preterm premature rupture of the membranes associated with thrombin activation that accompanies subchorionic hemorrhage or bleeding during pregnancy.

Estrogen alters remodeling of the vaginal wall after surgical injury in guinea pigs.

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.

Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.