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

2008; Elsevier BV; Volume: 174; Issue: 1 Linguagem: Inglês

10.2353/ajpath.2009.080212

1525-2191

David D. Rahn, Jesús F. Acevedo, Shayzreen M. Roshanravan, Patrick W. Keller, Elaine C. Davis, Lihua Y. Marmorstein, R. Ann Word,

Pelvic and Acetabular Injuries

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. 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. Pelvic organ prolapse is a common pelvic floor disorder with substantial financial, social, and psychological implications.1Weber AM Richter HE Pelvic organ prolapse.Obstet Gynecol. 2005; 106: 615-634Crossref PubMed Scopus (189) Google Scholar, 2Jelovsek JE Barber MD Paraiso MFR Walters MD Functional bowel and anorectal disorders in patients with pelvic organ prolapse and incontinence.Am J Obstet Gynecol. 2005; 193: 2105-2111Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 3Woodman PJ Swift SE O'Boyle AL Valley MT Bland DR Kahn MA Schaffer JI Prevalence of severe pelvic organ prolapse in relation to job description and socioeconomic status: a multicenter cross-sectional study.Int Urogynecol J. 2006; 17: 340-345Crossref PubMed Scopus (73) Google Scholar More than 11% of women will ultimately require surgical correction of prolapse or incontinence in their lifetimes.4Olsen AL Smith VJ Bergstrom JO Colling JC Clark AL Epidemiology of surgically managed pelvic organ prolapse and urinary incontinence.Obstet Gynecol. 1997; 89: 501-506Crossref PubMed Scopus (2700) Google Scholar Despite the prevalence of prolapse, the true pathophysiology of the disease is not well understood although epidemiological studies indicate that aging and vaginal parity are major risk factors.1Weber AM Richter HE Pelvic organ prolapse.Obstet Gynecol. 2005; 106: 615-634Crossref PubMed Scopus (189) Google Scholar, 5Bump RC Norton PA Epidemiology and natural history of pelvic floor dysfunction.Obstet Gynecol Clin North Am. 1998; 25: 723-746Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar Connective tissues of the pelvic floor are important for support of the pelvic viscera. Elastic fibers, which are abundant in connective tissues of the vaginal wall, confer resilience to stretching and expansive forces.6Starcher B Percival S Elastin turnover in the rat uterus.Connect Tissue Res. 1985; 13: 207-215Crossref PubMed Scopus (37) Google Scholar, 7Kielty CM Sherratt MJ Shuttleworth CA Elastic fibres.J Cell Sci. 2002; 115: 2817-2828Crossref PubMed Google Scholar, 8Rahn DD Ruff MD Brown S Tibbals HF Word RA Biomechanical properties of the mouse vagina: changes seen in pregnancy and with elastinopathy.Am J Obstet Gynecol. 2008; 198 (590e6): 590e1Abstract Full Text Full Text PDF Scopus (108) Google Scholar The phenotype of pelvic organ prolapse in animals with defects in elastic fiber assembly and synthesis suggests that elastic fiber homeostasis pathways are involved in maintaining pelvic organ support. Elastic fiber synthesis and assembly is a complex process that requires tropoelastin monomers to be cross-linked in the extracellular matrix to form a growing elastic fiber. Microfibrils are the scaffold on which tropoelastin is deposited before it is cross-linked by one or more of the copper-requiring lysyl oxidases.9Sato F Wachi H Ishida M Nonaka R Onoue S Urban Z Starcher BC Seyama Y Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation.J Mol Biol. 2007; 369: 841-851Crossref PubMed Scopus (61) Google Scholar Fibulin-5 is a protein thought to participate in this elastic fiber building process by binding lysyl oxidase-like 1 (LOXL1) and possibly targeting it to fiber assembly sites in the extracellular matrix.10Nakamura T Lozano PR Ikeda Y Iwanaga Y Hinek A Minamisawa S Cheng CF Kobuke K Dalton N Takada Y Tashiro K Ross Jr, J Honjo T Chien KR Fibulin-5/DANCE is essential for elastogenesis in vivo.Nature. 2002; 415: 171-175Crossref PubMed Scopus (529) Google Scholar, 11Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar, 12Yanagisawa H Davis EC Starcher BC Ouchi T Yanagisawa M Richardson JA Olson EN Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo.Nature. 2002; 415: 168-171Crossref PubMed Scopus (498) Google Scholar Mice with null mutations in genes encoding either fibulin-5 (Fbln5−/−)10Nakamura T Lozano PR Ikeda Y Iwanaga Y Hinek A Minamisawa S Cheng CF Kobuke K Dalton N Takada Y Tashiro K Ross Jr, J Honjo T Chien KR Fibulin-5/DANCE is essential for elastogenesis in vivo.Nature. 2002; 415: 171-175Crossref PubMed Scopus (529) Google Scholar, 12Yanagisawa H Davis EC Starcher BC Ouchi T Yanagisawa M Richardson JA Olson EN Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo.Nature. 2002; 415: 168-171Crossref PubMed Scopus (498) Google Scholar or LOXL1 (Loxl1−/−)11Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar develop elastinopathies including emphysematous lungs; defective elastic laminae of the great vessels; and loose, stretchy skin. Both mouse models also develop pelvic organ prolapse remarkably similar to that seen in primates, ie, descent of the vagina, cervix, and bladder herniating through the pelvic floor musculature. Previously, we reported that aging alone, regardless of parity, results in pelvic organ prolapse in more than 90% of Fbln5−/− mice.13Wieslander CK Acevedo JF Drewes PG Yanagisawa HK Word RA Pelvic organ prolapse severity increases with age in fibulin-5 knockout mice.Int Urogynecol J. 2006; 17: S371-S372Google Scholar Liu and colleagues14Liu X Zhao Y Pawlyk B Damaser M Li T Failure of elastic fiber homeostasis leads to pelvic floor disorders.Am J Pathol. 2006; 168: 519-528Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar reported that all parous Loxl1−/− mice developed prolapse after their first or second litters and that ∼50% of nulliparous animals ultimately progressed to prolapse after more than 1 year of age. The phenotype of these mouse models has led us to predict that other matrix proteins involved in elastic fiber assembly or degradation may be involved in the pathophysiology of pelvic organ prolapse. The fibulin family of proteins has seven known members characterized by tandem repeats of calcium binding-epidermal growth factor-like motifs and a C-terminal fibulin module. Repeating calcium binding-epidermal growth factor motifs are thought to be involved in protein-protein interactions, and biochemical analyses have revealed many interacting binding partners for fibulin-1 and fibulin-2 including fibronectin, proteoglycans, and basement membrane proteins.15Kobayashi N Kostka G Garbe JH Keene DR Bächinger HP Hanisch FG Markova D Tsuda T Timpl R Chu ML Sasaki T A comparative analysis of the fibulin protein family: biochemical characterization, binding interactions, and tissue localization.J Biol Chem. 2007; 282: 11805-11816Crossref PubMed Scopus (195) Google Scholar Fibulins are divided into two subgroups by similar size and binding preferences: fibulins-1, -2, -6 and fibulins-3, -4, -5, and -7.15Kobayashi N Kostka G Garbe JH Keene DR Bächinger HP Hanisch FG Markova D Tsuda T Timpl R Chu ML Sasaki T A comparative analysis of the fibulin protein family: biochemical characterization, binding interactions, and tissue localization.J Biol Chem. 2007; 282: 11805-11816Crossref PubMed Scopus (195) Google Scholar, 16de Vega S Iwamoto T Nakamura T Hozumi K McKnight DA Fisher LW Fukumoto S Yamada Y TM14 is a new member of the fibulin family (fibulin-7) that interacts with extracellular matrix molecules and is active for cell binding.J Biol Chem. 2007; 282: 30878-30888Crossref PubMed Scopus (77) Google Scholar Most have been shown to bind tropoelastin and are believed to contribute to elastic fiber assembly. There may be some redundancy in their roles, however, because absence of fibulin-1 or -2 does not appear to have effects on elastic fiber homeostasis in vivo.17Sicot FX Tsuda T Markova D Klement JF Arita M Zhang RZ Pan TC Mecham RP Birk DE Chu ML Fibulin-2 is dispensable for mouse development and elastic fiber formation.Mol Cell Biol. 2008; 28: 1061-1067Crossref PubMed Scopus (53) Google Scholar To date, only fibulin-4 and -5 have been found to be essential in elastic fiber assembly. Fbln4−/− mice die during or immediately after birth because of rupture of aortic aneurysms.10Nakamura T Lozano PR Ikeda Y Iwanaga Y Hinek A Minamisawa S Cheng CF Kobuke K Dalton N Takada Y Tashiro K Ross Jr, J Honjo T Chien KR Fibulin-5/DANCE is essential for elastogenesis in vivo.Nature. 2002; 415: 171-175Crossref PubMed Scopus (529) Google Scholar, 12Yanagisawa H Davis EC Starcher BC Ouchi T Yanagisawa M Richardson JA Olson EN Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo.Nature. 2002; 415: 168-171Crossref PubMed Scopus (498) Google Scholar, 18McLaughlin PJ Chen Q Horiguchi M Starcher BC Stanton JB Broekelmann TJ Marmorstein AD McKay B Mecham R Nakamura T Marmorstein LY Targeted disruption of fibulin-4 abolishes elastogenesis and causes perinatal lethality in mice.Mol Cell Biol. 2006; 26: 1700-1709Crossref PubMed Scopus (172) Google Scholar Fbln5−/− mice are born with elastic fiber defects in the lungs, skin, and aorta, but do not develop prolapse until 12 weeks of age.13Wieslander CK Acevedo JF Drewes PG Yanagisawa HK Word RA Pelvic organ prolapse severity increases with age in fibulin-5 knockout mice.Int Urogynecol J. 2006; 17: S371-S372Google Scholar The role of fibulin-3 (also known as EFEMP1, MBP1, H411, or UPH1) in elastic fiber assembly or pelvic organ prolapse is unknown. Among the seven known fibulins, fibulin-3 shares highest homology with fibulin-4 and -5.19Argraves WS Greene LM Cooley MA Gallagher WM Fibulins: physiological and disease perspectives.EMBO Rep. 2003; 4: 1127-1131Crossref PubMed Scopus (253) Google Scholar, 20Gallagher WM Currid CA Whelan LC Fibulins and cancer: friend or foe?.Trends Mol Med. 2005; 11: 336-340Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 21Chu ML Tsuda T Fibulins in development and heritable disease.Birth Defects Res C Embryo Today. 2004; 72: 25-36Crossref PubMed Scopus (68) Google Scholar, 22Markova D Zou Y Ringpfeil F Sasaki T Kostka G Timpl R Uitto J Chu ML Genetic heterogeneity of cutis laxa: a heterozygous tandem duplication within the fibulin-5 (FBLN5) gene.Am J Hum Genet. 2003; 72: 998-1004Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Recently, McLaughlin and colleagues23McLaughlin PJ Bakall B Choi J Liu Z Sasaki T Davis EC Marmorstein AD Marmorstein LY Lack of fibulin-3 causes early aging and herniation, but not macular degeneration in mice.Hum Mol Genet. 2007; 16: 3059-3070Crossref PubMed Scopus (118) Google Scholar reported that fibulin-3 has a specific effect on the integrity of elastic fibers in fascial connective tissues. Fbln3−/− mice develop early aging and herniation of the abdominal wall that increases progressively with age.23McLaughlin PJ Bakall B Choi J Liu Z Sasaki T Davis EC Marmorstein AD Marmorstein LY Lack of fibulin-3 causes early aging and herniation, but not macular degeneration in mice.Hum Mol Genet. 2007; 16: 3059-3070Crossref PubMed Scopus (118) Google Scholar In this study, we report the potential role of fibulin-3 in pelvic organ support by characterizing the gross and ultrastructural changes in the vaginal muscularis of mice deficient in fibulin-3 (Fbln3−/−). Further, studies were conducted to lend insight into potential mechanisms by which the lack of fibulin-3 led to pelvic organ prolapse. All mice were studied and euthanized in accordance with the standards of humane animal care described by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, using protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center. Animals were housed under a 12-hour dark/light cycle at 22°C. Fbln3−/− mice and wild-type (WT, n = 81) mice were the same strain (C57BL/6x129Sv/J). At sacrifice, after disarticulation of the pubic symphysis, uterine horns together with the bladder, cervix, and vagina were dissected down to the perineal skin. The vaginal dissection included the entire vaginal muscularis and the connective tissue suspending the vaginal wall to the pubocaudalis. Using microinstruments and a dissection microscope, the uterus was removed at the utero-cervico junction. Perineal skin was removed and the bladder and urethra dissected from the anterior vaginal wall. Wet weight of the vagina was determined by subtracting the weight of the dissected cervix from that of the cervicovaginal complex. The intact vaginal wall (muscularis and epithelium) was snap-frozen in liquid N2 and stored at −80°C. A validated mouse pelvic organ quantification (MOPQ) examination was used to serially measure the degree of vaginal, perineal, and rectal prolapse in 227 Fbln3−/− and 81 WT mice weekly for up to 75 weeks of age. Animals less than 11 weeks (n = 197) were not measured because prolapse was not observed before this age. The MOPQ was conducted with one investigator holding the animal by the scruff of the neck, which resulted in a prolonged, reflex valsalva (evidenced by defecation), whereas the other investigator performed the measurements using a caliper with the precision of 0.01 mm. Six assessments were performed in each animal: i) stage of perineal bulge; ii) magnitude of perineal bulge; iii) cervical descent; iv) anal prolapse; v) perineal body length, and vi) vaginal diameter.13Wieslander CK Acevedo JF Drewes PG Yanagisawa HK Word RA Pelvic organ prolapse severity increases with age in fibulin-5 knockout mice.Int Urogynecol J. 2006; 17: S371-S372Google Scholar Perineal bulge (0, none; 1, detectable but small; 2, moderate size bulge; 3, huge; 4, vagina coming out), cervical descent (0, not visible; 1, visible on straining inside the introitus; 2, at the level of the introitus; 3, outside the introitus), and anal prolapse (0, none; 1, present but mild; 2, severe) were measured on an ordinal scale. Magnitude of perineal bulge was measured in mm from the point of insertion of inner thigh to maximal edge of perineum. Perineal body was measured in mm from the posterior fourchette to mid-anus. Vaginal diameter was measured in mm from the anterior to posterior vaginal walls at the level of the introitus. WT, Fbln3−/−, and Fbln5−/− mice were anesthetized and perfused with 10% neutral buffered formalin (pH 7.4). Thereafter, the female urogenital tract was dissected en bloc. Serial transverse sections (5 μm) were obtained in 100-μm increments throughout the specimen, stained with hematoxylin and eosin or Hart's stain, and analyzed with an Eclipse E1000N microscope (Nikon, Tokyo, Japan). For transmission electron microscopy, anesthetized animals were perfused first with ice-cold phosphate-buffered saline and then with 3% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.4). Full thickness rings of vaginal wall from WT and Fbln3−/− mice were then dissected and fixed in fresh fixative overnight. Samples were then sequentially treated with 1% osmium tetroxide, 2% tannic acid, and 2% uranyl acetate before dehydration and Epon embedding. Thin sections (60 nm) were placed on formvar-coated grids and counterstained with 7% methanolic uranyl acetate followed by lead citrate. Sections were viewed using a Tecnai 12 (FEI Co., Hillsboro, OR) transmission electron microscope at 120 kV and images were digitally captured. Rabbit polyclonal antibody BSYN5128 was raised against mouse fibulin-3 polypeptide (78IVNNEHPQQETQPAAEASS95; Biosynthesis Inc., Lewisville, TX). Purified immunoglobulin-γ (IgG) and affinity-purified antibodies were prepared. Formalin-fixed, paraffin-embedded tissues from WT and Fbln3−/− animals were sectioned at 5 μm. After drying, slides were deparaffinized in xylene and rehydrated in graded alcohols to distilled water. Endogenous peroxidase activity was quenched for 10 minutes at room temperature, using 0.3% H2O2 with 0.1% sodium azide. Slides were subjected to steam-heat epitope retrieval in ethylenediaminetetraacetic acid buffer (1 mmol/L, pH 8.0) for 30 minutes. After rinsing in phosphate-buffered saline (PBS), slides were incubated in primary antibody (BSYN5128) for 30 minutes at 25°C using gentle orbital rotation. Negative control specimens were processed simultaneously in an identical manner, with the exception that PBS was used in place of primary antibody. After another rinse in PBS, slides were incubated with appropriate horseradish peroxidase-conjugated polymer (PowerVision reagent; ImmunoVision Technologies Co., Daly City, CA) for 30 minutes at 25°C. Finally, the slides were immersed for 5 minutes in 25°C diaminobenzidine (Invitrogen, Carlsbad, CA), enhanced with 0.5% copper sulfate in PBS for 5 minutes at 25°C, counterstained in hematoxylin, dehydrated in graded alcohols, cleared in xylene, and covered with a coverslip. Vaginal tissues were thawed on ice, minced, washed in PBS, and homogenized in MMP2 assay buffer (EnzoLyte 520 MMP-2 assay kit; Anaspec, San Jose, CA) containing 0.1% Triton-X 100 (95× volume:tissue wet weight). Thereafter, homogenates were centrifuged at 10,000 × g for 15 minutes at 4°C and supernatants used for determination of protease activity. Protein concentrations were determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Samples (5 μg per lane) were applied to gelatin polyacrylamide minigels (Invitrogen) (10%) in standard sodium dodecyl sulfate loading buffer containing 0.1% sodium dodecyl sulfate with no β-mercaptoethanol, and the samples were not boiled before loading. Gels were run at room temperature at 125 V. After electrophoresis, gels were soaked in renaturing buffer (2.7% Triton X-100 in distilled water) in a shaker for 30 minutes with one change after 30 minutes to remove sodium dodecyl sulfate. Next, gels were soaked in assay buffer (50 mmol/L Tris, 200 mmol/L NaCl, 10 mmol/L CaCl2, 0.05% Brij 35, pH 7.5) for 4 to 16 hours at 37°C and then stained with Coomassie Brilliant Blue-R 250 in 50% methanol and 10% acetic acid followed by destaining with 25% methanol and 7% acetic acid. Clear zones of lysis against a dark background indicated enzyme activity, and purified enzymes were used as positive controls (Anaspec). Conditions of zymography and analysis were quantitative because enzyme activity was linear with time of incubation and protein loading, and samples for each experiment were applied to the same gel to avoid intergel variation. Areas of lysis were quantified using a Fuji LAS 3000 image analysis system (Fujifilm Life Science, Stamford, CT). Side-by-side immunoblots for α-tubulin protein were conducted to ensure equal protein loading of the gel. MMP activity was expressed as relative units per unit α-tubulin. Relative amounts of fibulin-5 and tropoelastin in the vaginal wall from five virginal WT mice, six Fbln3−/− mice without prolapse, and five Fbln3−/− mice with prolapse were determined using immunoblot analysis as described previously using 10 μg of urea-extracted protein per lane.24Drewes PG Yanagisawa H Starcher B Hornstra IK Csiszar K Marinis SI Keller P Word RA Pelvic organ prolapse in fibulin-5 knockout mice: pregnancy changes in elastic fiber homeostasis in mouse vagina.Am J Pathol. 2007; 170: 578-589Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar All mice were matched for age and estrus cycle (metestrus), except in some mice with advanced prolapse that were not cycling. Protein extracts from Fbln5−/− mice served as negative controls for fibulin-5. Quantitative PCR was used to determine the relative levels of Timp1, Timp2, and Timp3 in vaginal tissues as described previously.25Wieslander CK Marinis SI Drewes PG Keller PW Acevedo JF Word RA Regulation of elastolytic proteases in the mouse vagina during pregnancy, parturition, and puerperium.Biol Reprod. 2008; 78: 521-528Crossref PubMed Scopus (32) Google Scholar Primer sequences for amplifications were chosen using published cDNA sequences and chosen such that the resulting amplicons would cross an exon junction thereby eliminating the potential of false-positive signals from genomic DNA contamination. Primer sets for Timp3 (BC014713) were 5′-CACGGAAGCCTCTGAAAGTCTT-3′(513–534) and 5′-CATACACGCGCCCTGTCA-3′(592–575). SYBR Green was used for amplicon detection, and gene expression was normalized to that of the housekeeping gene β2-microglobulin (B2M). All assays included positive, negative, and no template controls. Elastic fiber content was assessed by radioimmunoassay for desmosine, an amino acid crosslink found only in elastin. After urea extraction, pelleted tissue extracts were hydrolyzed in 6 N HCl at 100°C for 24 hours. An aliquot was evaporated again to dryness and redissolved in 100 μl of H2O, vortexed, microfuged, and assayed for desmosine as previously described.26Starcher B Conrad M A role for neutrophil elastase in the progression of solar elastosis.Connect Tissue Res. 1995; 31: 133-140Crossref PubMed Scopus (107) Google Scholar Differences in continuous variables of MOPQ scores, protease activities, and amounts of fibulin-5 and tropoelastin for Fbln3−/− mice with and without prolapse versus WT mice were assessed using analysis of variance with Student-Newman-Keuls method for pair-wise multiple comparisons. To determine whether age or parity were independent predictors of prolapse, a multiple logistic regression analysis was used with pelvic organ prolapse as the dependent variable and age and parity as independent variables. Vaginal weight comparisons between knockout and WT groups were made with the Student's t-test. Statistical software used was SigmaStat version 2.03 (Jandel Scientific, San Rafael, CA). P values ≤0.05 were considered statistically significant. During the course of this investigation, overt pelvic organ prolapse was noted in a significant number of Fbln3−/− female mice (Figure 1A). Grossly, the phenotype of prolapse in Fbln3−/− knockout animals resembled that of Fbln5−/− and Loxl1−/− animals.14Liu X Zhao Y Pawlyk B Damaser M Li T Failure of elastic fiber homeostasis leads to pelvic floor disorders.Am J Pathol. 2006; 168: 519-528Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 24Drewes PG Yanagisawa H Starcher B Hornstra IK Csiszar K Marinis SI Keller P Word RA Pelvic organ prolapse in fibulin-5 knockout mice: pregnancy changes in elastic fiber homeostasis in mouse vagina.Am J Pathol. 2007; 170: 578-589Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Specifically, the perineal bulge was comprised of the bladder, vaginal vault, and descended uterine horns caudal to the pubic ligament (Figure 1C). Rectal prolapse and hernias were observed in the flank and inguinal areas (Figure 1B). Vaginal weight was increased in knockout animals compared with WT (mean ± SD): 77.6 ± 18.3 mg versus 44.2 ± 7.3 mg, respectively (P < 0.001). Prolapsed Fbln3−/− vaginas were thin and patulous (Figure 1D). Like Fbln5−/− mice, the connective tissue paravaginal and apical attachments (eg, the uterosacral ligaments) were either absent or attenuated in Fbln3−/− mice with prolapse. Rectal prolapse was a common finding among Fbln3−/− animals with concomitant perineal prolapse (18 of 53 or 34%) (Figure 1B). In contrast, rectal prolapse occurred in only 4 of 74 (5.4%) Fbln5−/− mice. To study the effect of fibulin-3 on subtle increases (and waxing and waning) in perineal bulge, rectal prolapse, and cervical descent, a longitudinal observation of pelvic organ support was conducted in WT and Fbln3−/− mice. Knockout (n = 227) and C57BL/6 WT (n = 81) mice were examined weekly with MOPQ scores from birth; the oldest of which were greater than 1 year of age. The effects of pregnancy, parturition, and aging were noted. Continuous variable measurements of the MOPQ (magnitude of perineal bulge, perineal body length, and vaginal diameter) of WT and knockout animals with and without gross evidence of pelvic organ prolapse were compared and validated by examiners blinded to genotype (Figure 2). All parameters were increased significantly in knockout relative to WT animals (all P < 0.001, Figure 2), suggesting that pelvic organ support is abnormal in Fbln3−/− mice even in the absence of obvious prolapse. In agreement with our previous study involving WT mice of C3BL/6 strain,13Wieslander CK Acevedo JF Drewes PG Yanagisawa HK Word RA Pelvic organ prolapse severity increases with age in fibulin-5 knockout mice.Int Urogynecol J. 2006; 17: S371-S372Google Scholar, 25Wieslander CK Marinis SI Drewes PG Keller PW Acevedo JF Word RA Regulation of elastolytic proteases in the mouse vagina during pregnancy, parturition, and puerperium.Biol Reprod. 2008; 78: 521-528Crossref PubMed Scopus (32) Google Scholar WT C57BL/6x129Sv mice did not develop perineal prolapse. In contrast, 53 of 197 knockout animals ≥11 weeks of age (26.9%) developed at least stage 1 perineal bulge. For these animals with prolapse, the average age at time of diagnosis was 37 weeks (range, 11.3 to 70.9 weeks). The majority of knockout animals (89%) developed prolapse after 20 weeks of age (n = 47, Figure 3). Of these older knockout mice with prolapse, 18 (34%) were parous and 35 (66%) were virginal. Using multivariate logistic regression analysis including age and parity as independent variables, prolapse was significantly related to age (P < 0.001; Figure 3, A–C) but not parity. As described by McLaughlin and colleagues,23McLaughlin PJ Bakall B Choi J Liu Z Sasaki T Davis EC Marmorstein AD Marmorstein LY Lack of fibulin-3 causes early aging and herniation, but not macular degeneration in mice.Hum Mol Genet. 2007; 16: 3059-3070Crossref PubMed Scopus (118) Google Scholar Fbln3−/− mice developed hernias in either one or both flanks or inguinal area. These hernias were distinct from the perineal bulge of pelvic organ prolapse. Herein, we found that of 129 animals older than 40 weeks of age, 53 (53.5%) developed hernias, and of the mice with pelvic prolapse, 32% had concomitant hernias. Using multivariate logistic regression analysis, hernias were associated with mouse age (P < 0.001) but not parity. Interestingly, the background strain altered the prevalence of hernias in Fbln3−/− mice.23McLaughlin PJ Bakall B Choi J Liu Z Sasaki T Davis EC Marmorstein AD Marmorstein LY Lack of fibulin-3 causes early aging and herniation, but not macular degeneration in mice.Hum Mol Genet. 2007; 16: 3059-3070Crossref PubMed Scopus (118) Google Scholar To determine whether strain may alter the prevalence of prolapse, Fbln3−/− mice on a BALB/c background were examined for 18 months. Of 36 animals younger than 1 year of age, none developed prolapse. Older BALB/c Fbln3−/− mice (>1 year of age), however, developed severe sta