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.

Androgens Upregulate Endometrial Epithelial Progesterone Receptor Expression: Potential Implications for Endometriosis.

BACKGROUND: Androgenic compounds have been implicated in induction of endometrial atrophy yet the mechanisms of androgen effects on human endometrium have not been well studied. We hypothesized that androgens may promote their endometrial effects via modulation of progesterone receptor (PR) expression. METHODS: Proliferative phase endometrial samples were collected at the time of hysterectomy. We evaluated the effect of the potent androgen 5α-dihydrotestosterone (DHT) on endometrial PR expression by treating human endometrial explants, endometrial stromal cells, and Ishikawa cells with DHT. Ishikawa cells were also treated with DHT ± the androgen receptor (AR) blocker flutamide. The PR-B, total PR messenger RNA (mRNA), and PR protein expression were assessed. Expression of cyclin D1 and D2 was checked as markers of cell proliferation. RESULTS: As expected, estradiol induced PR expression in isolated stromal cells, endometrial epithelial cells, and tissue explants. The DHT treatment also resulted in increased PR expression in endometrial explants and Ishikawa cells but not in stromal cells. Further, protein levels of both nuclear PR isoforms (PR-A and PR-B) were induced with the DHT treatment. Although flutamide treatment alone did not affect PR expression, flutamide diminished androgen-induced upregulation of PR in both endometrial explants and Ishikawa cells. Although estradiol induced both cyclin D1 and cyclin D2 mRNA, DHT did not induce these markers of cell proliferation. CONCLUSION: Androgens may mediate endometrial effects through upregulation of PR gene and protein expression. Endometrial PR upregulation by androgens is mediated, at least in part, through AR.

Effects of vaginal conjugated equine estrogens and ospemifene on the rat vaginal wall and lower urinary tract.

Although the positive effects of vaginal estrogens and the selective estrogen receptor modulator, ospemifene (OS), on the vaginal epithelium are well recognized, less is known regarding the effects of these therapies on the lower urinary tract or vaginal muscularis. Clinical evidence suggests that vaginally administered estrogen may improve overactive bladder-related symptoms. The objective of this study was to compare the effects of OS, vaginal conjugated equine estrogens (CEE), or both on the vaginal wall and lower urinary tract in a rat model of menopause. Contractile force of the bladder neck, dome, and external urethral sphincter at optimal field stimulation did not differ significantly among treatment groups. Pharmacologic responses to atropine, carbachol, and potassium chloride were similar among groups. Vaginal epithelial thickness and differentiation were differentially regulated by CEE or OS. Ospemifene altered epithelial differentiation pathways in vaginal epithelium in a unique way, and these effects were additive with local CEE. Unless contraindicated, the beneficial effects of vaginal CEE on the vaginal wall outweigh those of OS.

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.

Myogenic stem cell-laden hydrogel scaffold in wound healing of the disrupted external anal sphincter.

OBJECTIVE: To evaluate the effect of myogenic stem cell-laden hydrogel scaffold on contractile function and histomorphology of the external anal sphincter (EAS) after transection without repair. METHODS: Eighty female rats underwent anal sphincter transection without repair. After 2 weeks, animals were injected at the transection site with: nothing (non-repaired control, NRC group); a polyethylene glycol-based hydrogel matrix scaffold combined with phosphate-buffered saline (PBS/hydrogel group); a hydrogel matrix scaffold combined with myogenic stem cells (stem cell/hydrogel group): or type I collagen (collagen) group. 4 (n = 40) or 12 (n = 40) weeks later, the anal sphincter complexes were dissected out and analyzed for contractile function, disruption, and striated muscle volume. Time-matched unoperated controls (UOC) were utilized for each of the two time points (n = 20). RESULTS: After 4 weeks, maximal electrical field-stimulated (EFS) contractions were significantly decreased in all four non-repaired treatment groups compared with UOC. However, EFS-stimulated contractions, tetanic force generation, and twitch tension were improved in non-repaired EAS injected with stem cell/hydrogel group relative to the NRC, PBS/hydrogel, or collagen groups. NRC and sphincters injected with PBS/hydrogel deteriorated further by 12 weeks, while those receiving stem cell/hydrogel maintained improved contractile function at varying frequencies and voltages. Striated muscle volume increased from 4 to 12 weeks for PBS/hydrogel and stem cell/hydrogel animals. At 12 weeks, stem cell/hydrogel animals had greater sphincter striated muscle volumes compared with all other treatment groups. CONCLUSION: In this animal model, sustained improvement of contractile responses in non-repaired EAS treated with biogel scaffold and myogenic stem cells suggests that a biologically compatible matrix may facilitate stem cell survival, differentiation, or function leading to recovery of contractile function even after persistent disruption.

Effect of thrombin on human amnion mesenchymal cells, mouse fetal membranes, and preterm birth.

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.

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.

Fetal fibronectin signaling induces matrix metalloproteases and cyclooxygenase-2 (COX-2) in amnion cells and preterm birth in mice.

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 E(2) 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.

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-α-stimulated gene-6) genes were increased dramatically and transiently after injury, MSCs did not alter this response. In contrast, transforming growth factor (TFG)-ß1 (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-ß1 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-ß1 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 assigned 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 (3.2x10 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.

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.

Regulation of elastolytic proteases in the mouse vagina during pregnancy, parturition, and puerperium.

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.