Jesus F. Acevedo

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.

EFFECT OF VAGINAL DISTENTION ON ELASTIC FIBER SYNTHESIS AND MATRIX DEGRADATION IN THE VAGINAL WALL: POTENTIAL ROLE IN THE PATHOGENESIS OF PELVIC ORGAN PROLAPSE

Matrix metalloprotease (MMP) activity is increased in the postpartum vagina of wild-type (WT) animals. This degradative activity is also accompanied by a burst in elastic fiber synthesis and assembly. The mechanisms that precipitate these changes are unclear. The goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. Vaginal distention with a balloon simulating parturition resulted in increased MMP-2 and MMP-9 activity in the vaginal wall of nonpregnant and pregnant animals. This was accompanied by visible fragmented and disrupted elastic fibers in the vaginal wall. In nonpregnant animals, the abundant amounts of tropoelastin and fibulin-5 in the vagina were not increased further by distention. In contrast, in pregnant animals, the suppressed levels of both proteins were increased 3-fold after vaginal distention. Distention performed in fibulin-5-deficient (Fbln5(-/-)) mice with defective elastic fiber synthesis and assembly induced accelerated pelvic organ prolapse, which never recovered. We conclude that, in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. Distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. The finding that distention results in accelerated pelvic organ prolapse in Fbln5(-/-) animals, but not in WT, indicates that elastic fiber synthesis is crucial for recovery of the vaginal wall from distention-induced increases in vaginal protease activity.

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.

Quantification of Pelvic Organ Prolapse in Mice: Vaginal Protease Activity Precedes Increased MOPQ Scores in Fibulin 5 Knockout Mice

Abstract Two mouse models of pelvic organ prolapse have been generated recently, both of which have null mutations in genes involved in elastic fiber synthesis and assembly (fibulin 5 and lysyl oxidase-like 1). Interestingly, although these mice exhibit elastinopathies early in life, pelvic organ prolapse does not develop until later in life. In this investigation we developed and validated a tool to quantify the severity of pelvic organ prolapse in mice, and we used this tool prospectively to study the role of fibulin 5, aging, and vaginal proteases in the development of pelvic organ prolapse. The results indicate that >90% of Fbln5−/− mice develop prolapse by 6 mo of age, even in the absence of vaginal delivery, and that increased vaginal protease activity precedes the development of prolapse.

Effect of Vaginal or Systemic Estrogen on Dynamics of Collagen Assembly in the Rat Vaginal Wall

ABSTRACT The objective of this study was to compare the effects of systemic and local estrogen treatment on collagen assembly and biomechanical properties of the vaginal wall. Ovariectomized nulliparous rats were treated with estradiol or conjugated equine estrogens (CEEs) either systemically, vaginal CEE, or vaginal placebo cream for 4 wk. Low-dose local CEE treatment resulted in increased vaginal epithelial thickness and significant vaginal growth without uterine hyperplasia. Furthermore, vaginal wall distensibility increased without compromise of maximal force at failure. Systemic estradiol resulted in modest increases in collagen type I with no change in collagen type III mRNA. Low-dose vaginal treatment, however, resulted in dramatic increases in both collagen subtypes whereas moderate and high dose local therapies were less effective. Consistent with the mRNA results, low-dose vaginal estrogen resulted in increased total and cross-linked collagen content. The inverse relationship between vaginal dose and collagen expression may be explained in part by progressive downregulation of estrogen receptor-alpha mRNA with increasing estrogen dose. We conclude that, in this menopausal rat model, local estrogen treatment increased total and cross-linked collagen content and markedly stimulated collagen mRNA expression in an inverse dose-effect relationship. High-dose vaginal estrogen resulted in downregulation of estrogen receptor-alpha and loss of estrogen-induced increases in vaginal collagen. These results may have important clinical implications regarding the use of local vaginal estrogen therapy and its role as an adjunctive treatment in women with loss of vaginal support.

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.

Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1β by human fetal membranes in a two-compartment culture system.

INTRODUCTION Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1β mediated by E. coli infection. METHODS Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 10⁴, 10⁵, and 10⁶ colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1β were assessed using the enzyme-linked immunosorbent assay (ELISA) method. RESULTS After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1β secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70. DISCUSSION Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1β.

Thermosensitive hydrogels deliver bioactive protein to the vaginal wall.

The pathophysiology and natural history of pelvic organ prolapse (POP) are poorly understood. Consequently, our approaches to treatment of POP are limited. Alterations in the extracellular matrix components of pelvic support ligaments and vaginal tissue, including collagen and elastin, have been associated with the development of POP in animals and women. Prior studies have shown the protease MMP-9, a key player of ECM degradation, is upregulated in vaginal tissues from both mice and women with POP. On the other hand, fibulin-5, an elastogenic organizer, has been found to inhibit MMP-9 in the vaginal wall. Hence, we hypothesized that prolonged release of fibulin-5 may delay progression of POP. To test the hypothesis, oligo (ethylene glycol)-based thermosensitive hydrogels were fabricated, characterized and then used to deliver fibulin-5 to the vaginal wall and inhibit MMP-9 activity. The results indicate that hydrogels are cell and tissue compatible. The hydrogels also prolong the ½ life of fibulin-5 in cultured vaginal fibroblasts and in the vaginal wall in vivo. Finally, fibulin-5-containing hydrogels resulted in incorporation of fibulin-5 into the vaginal matrix and inhibition of MMP-9 for several weeks after injection. These results support the idea of fibulin-5 releasing hydrogel being developed as a new treatment for POP.