[Show abstract][Hide abstract] ABSTRACT: Objectives:
Lysyl oxidase-like 1 knockout (Loxl1) mice demonstrate deficient elastin homeostasis associated with pelvic organ prolapse (POP). To further investigate the pathophysiology of POP in these animals, a genetically matched homozygous positive (Loxl1) or wild-type strain is needed. This study sought to create and validate genetically matched Loxl1 and Loxl1 strains.
Female Loxl1 mice were backcrossed with male wild-type mice. The resultant heterozygous mice were bred to produce Loxl1 and Loxl1 mice, whose genotype was confirmed by polymerase chain reaction (PCR). Multiparous female Loxl1 (n = 7) and Loxl1 (n = 9) mice were assessed for POP weekly for 12 weeks after their first vaginal delivery. Pelvic organ prolapse was compared between groups using a Kaplan-Meier survival curve with P of less than 0.05 indicating a significant difference. Vaginal connective tissue histologic finding was assessed qualitatively and quantitatively.
There were no significant differences between the groups in age or parity. Of the 7 Loxl1 mice, 4 developed prolapse by 8 weeks and 6 by 12 weeks postpartum. No Loxl1 mouse prolapsed. Loxl1 mice had significantly larger vaginas as determined by area within the lumen and total cross-sectional tissue area. Striated muscle fibers of the urethra in Loxl1 mice were less organized, shorter, and thinner than in Loxl1 mice.
Genetically matched Loxl1 and Loxl1 strains can be reliably created by a backcross method and differentiate in their prolapse phenotype. Loxl1 mice demonstrate pathology primarily characterized by enlargement of the vagina. Further studies are needed to elucidate the cause of this finding.
Journal of Pelvic Medicine and Surgery 09/2014; 20(5):287-292. DOI:10.1097/SPV.0000000000000104 · 1.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Human childbirth simulated by vaginal distention is known to increase the expression of chemokines and receptors involved in stem cell homing and tissue repair. We hypothesized that pregnancy and parturition in rats contributes to the expression of chemokines and receptors after vaginal distention.
Materials and methods:
We used 72 age matched female Lewis rats, including virgin rats with and without vaginal distention, and delivered rats with and without vaginal distention. Each rat was sacrificed immediately, or 3 or 7 days after vaginal distention and/or parturition, and the urethra was harvested. Relative expression of chemokines and receptors was determined by real-time polymerase chain reaction. Mixed models were used with the Bonferroni correction for multiple comparisons.
Vaginal distention up-regulated urethral expression of CCL7 immediately after injury in virgin and postpartum rats. Hypoxia inducible factor-1α and vascular endothelial growth factor were up-regulated only in virgin rats immediately after vaginal distention. CD191 expression was immediately up-regulated in postpartum rats without vaginal distention compared to virgin rats without vaginal distention. CD195 was up-regulated in virgin rats 3 days after vaginal distention compared to virgin rats without vaginal distention. CD193 and CXCR4 showed delayed up-regulation in virgin rats 7 days after vaginal distention. CXCL12 was up-regulated in virgin rats 3 days after vaginal distention compared to immediately after vaginal distention. Interleukin-8 and CD192 showed no differential expression.
Vaginal distention results in up-regulation of the chemokines and receptors expressed during tissue injury, which may facilitate the spontaneous functional recovery previously noted. Pregnancy and delivery up-regulated CD191 and attenuated the expression of hypoxia inducible factor-1α and vascular endothelial growth factor in the setting of vaginal distention, likely by decreasing hypoxia.
The Journal of urology 09/2012; 189(4). DOI:10.1016/j.juro.2012.09.096 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies of the urothelium, the specialized epithelial lining of the urinary bladder, are critical for understanding diseases affecting the lower urinary tract, including interstitial cystitis, urinary tract infections and cancer. However, our understanding of urothelial pathophysiology has been hampered by a lack of appropriate model systems. Here, we describe the isolation and characterization of a non-transformed urothelial cell line (TRT-HU1), originally explanted from normal tissue and immortalized with hTERT, the catalytic subunit of telomerase. We demonstrate responsiveness of the cells to anti-proliferative factor (APF), a glycopeptide implicated in the pathogenesis of interstitial cystitis. TRT-HU1 carries a deletion on the short arm of chromosome 9, an early genetic lesion in development of bladder cancer. TRT-HU1 urothelial cells displayed growth and migration characteristics similar to the low-grade papilloma cell line RT4. In contrast, we observed marked differences in both phenotype and gene expression profiles between TRT-HU1 and the highly malignant T24 cell line. Together, these findings provide the first demonstration of a non-transformed, continuous urothelial cell line that responds to APF. This cell line will be valuable for studies of both benign and malignant urothelial cell biology.
[Show abstract][Hide abstract] ABSTRACT: : Lysyl oxidase like-1 (LOXL1) knockout mice have abnormal elastic fiber homeostasis and frequently develop pelvic floor dysfunction after pregnancy and delivery. The objective of this study was to test the hypothesis that tissue changes associated with vaginal delivery lead to pelvic floor dysfunction as a result of abnormal elastic fiber homeostasis.
: Female LOXL1 knockout mice delivered either spontaneously or by cesarean delivery. Mice were assessed weekly for pelvic organ prolapse (POP). At 12 weeks postpartum, lower urinary tract function was assessed by cystometry and leak-point pressure testing. Urethrovaginal cross-sections were analyzed using a histologic grading scale to assess elastin fiber disorganization.
: A total of 39 mice delivered by spontaneous vaginal delivery and 36 by cesarean delivery. Twelve weeks after spontaneous vaginal delivery or cesarean delivery, 23 (59%) and 11 (31%) mice had developed POP, respectively. The mean time to develop POP was 7.2 weeks after spontaneous vaginal delivery and 10.5 weeks after cesarean delivery (log rank, P = 0.0008). The Cox proportional hazard ratio was 0.55 (95% confidence interval, 0.38-0.79). Mice with POP had increased frequency of bladder contractions not associated with voiding during cystometry (P = 0.02). POP, but not mode of delivery, was associated with increased elastic fiber disorganization.
: Cesarean delivery delays the development of POP in LOXL1 knockout mice. POP is associated with increased bladder contraction frequency and increased elastic fiber disorganization in the urethra and vagina. The mechanisms underlying these findings warrant further investigation.
Journal of Pelvic Medicine and Surgery 01/2010; 16(1):21-30. DOI:10.1097/SPV.0b013e3181d00035 · 1.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaginal distension (VD) in outbred rats has been shown to decrease urethral resistance, as well as increase the expression of the stem cell-homing chemokine, monocyte chemotactic factor 3 (MCP-3), but not stromal derived factor 1 (SDF-1). The aim of this study was to determine if similar responses are induced by VD in an inbred rat strain.
Forty female Lewis rats underwent VD or sham VD followed by leak point pressure (LPP) testing 4 or 10 days later. Ten additional rats served as controls. The urethra and vagina were then dissected for histology. To examine chemokine expression, eight additional rats underwent VD with organs harvested immediately or 1 day after the procedure for reverse transcriptase polymerase chain reaction (RT-PCR) of MCP-3 and SDF-1. Four age-matched rats served as controls.
Four days after VD, LPP was significantly lower in VD rats (14.3 +/- 1.6 cm H(2)O) than controls (18.7 +/- 1.3 cm H(2)O). Ten days after VD, LPP in both VD (19.7 +/- 2.6 cm H(2)O) and sham (18.4 +/- 1.3 cm H(2)O) groups was not significantly different from controls. Urethral histology demonstrated marked disruption and atrophy of smooth and striated muscle in VD rats compared to shams and controls. RT-PCR yielded a 25-fold significant increase in expression of urethral MCP-3 immediately following VD. SDF-1 was significantly decreased in the urethra and vagina immediately after VD and in the bladder 24 hr after VD.
VD in Lewis rats produces functional, histological and molecular results similar to that of outbred rats. This model could be utilized in future studies investigating cellular transplant methods of improving urethral function.
Neurourology and Urodynamics 04/2009; 28(4):356-61. DOI:10.1002/nau.20644 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the effect of the duration of vaginal distention on the differential expression of stem cell homing, tissue repair cytokines and cytokine receptors to identify the factors most important for recovery from injury.
A total of 20, 10-week-old virgin Sprague-Dawley rats were divided into 4 groups, including 1, 4 and 6-hour vaginal distention, and anesthetized sham operation. The vagina, bladder, urethra and rectum were harvested immediately after vaginal distention. Real-time polymerase chain reaction was used to determine the relative expression of cytokines and receptors of interest. Mixed models analysis was used to determine associations between expression levels and vaginal distention duration.
Positive associations between vaginal distention duration and the urethral expression level were found for 1 of the receptors of monocyte chemotactic protein-3, CCR1 (p = 0.0001) as well as for monocyte chemotactic protein-3 (p = 0.025), CCR5 (p = 0.032) and hypoxia inducible factor-1alpha (p = 0.023). A positive relationship between vaginal distention duration and monocyte chemotactic protein-3 expression was also observed in rectal tissue (p = 0.035). Urethral expression of CCR2, another receptor for monocyte chemotactic protein-3, approached significance (p = 0.066). An inverse relationship between vaginal distention duration and interleukin-8 expression was found in the bladder (p = 0.0008). No association was noted between vaginal distention duration and the expression of stromal derived factor-1, CXCR4, CCR3 and vascular endothelial growth factor in any pelvic organs.
These data support a relationship between vaginal distention duration and the subsequent expression of monocyte chemotactic protein-3 and 1 of its associated receptors, CCR1, in the urethra immediately following vaginal distention. The increase in hypoxia-inducible factor1alpha expression in the urethra with prolonged vaginal distention suggests a limited role of tissue ischemia in the immediate response of pelvic organs to vaginal distention.
The Journal of urology 09/2008; 180(2):753-9. DOI:10.1016/j.juro.2008.03.182 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Female pelvic floor dysfunction (FPFD) is a complex group of conditions that include urinary incontinence and pelvic organ prolapse (POP). In humans, elastin homeostasis has been implicated in the pathophysiology of FPFD. Lysyl oxidase-like 1 knockout (LOXL1-KO) mice demonstrate abnormal elastic fiber homeostasis and develop FPFD after parturition. We compared the lower urogenital tract (LUT) anatomy and function in LOXL1-KO mice with and without POP. LUT anatomy was assessed in LOXL1-KO mice over 28 wk. Pelvic visceral anatomy in LOXL1-KO was evaluated with a 7-Tesla magnetic resonance imaging (MRI) scanner. LUT function was assessed using conscious cystometry and leak point pressure (LPP) testing. Quantitative histological analysis of elastic fibers was performed on external urethral sphincter (EUS) cross sections. By 25 wk of age, 50% of parous LOXL1-KO mice developed POP. LOXL1-KO mice with POP had greater variability in the size and location of the bladder on MRI compared with mice without POP. Parity and POP were associated with lower LPP. Elastin clusters were significantly increased in the EUS of LOXL1-KO mice with POP. Because parity triggers POP in LOXL1-KO mice, LOXL1-KO mice with POP have variable internal pelvic anatomy, and both parity and POP are associated with a decrease in LPP, we conclude that LOXL1 LUT anatomical and functional phenotype resembles FPFD in humans. The increase in elastin clusters in the urethra of LOXL1-KO mice with POP suggests that elastin disorganization may lead to functional abnormalities. We conclude that LOXL1 warrants further investigation in the pathphysiology of FPFD.
American journal of physiology. Renal physiology 06/2008; 295(2):F545-55. DOI:10.1152/ajprenal.00063.2008 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaginal delivery is a risk factor for stress urinary incontinence. Rat models of simulated childbirth demonstrated hypoxia of the urogenital organs as well as the development of stress urinary incontinence following vaginal distention. Stromal derived factor-1 and monocyte chemotactic protein-3 were identified as cytokines that are over expressed after myocardial ischemia and signal stem cell migration to ischemic sites in a rat cardiac model. Given the focal hypoxia observed with vaginal distention, we characterized stromal derived factor-1 and monocyte chemotactic protein-3 expression by pelvic organ tissues after vaginal distention.
A total of 16 female rats were randomized into 4 groups. Two groups underwent vaginal distention with harvest of pelvic tissues immediately or 24 hours after vaginal distention, a sham group underwent anesthesia only and a control group underwent no intervention. Reverse transcriptase-polymerase chain reaction was performed on RNA extracted from the urogenital organs.
Monocyte chemotactic protein-3 expression in the urethra was increased 20 and 6-fold immediately and 24 hours after vaginal distention, respectively. Monocyte chemotactic protein-3 was 8 and 4-fold increased in the vagina after vaginal distention. There was no difference in monocyte chemotactic protein-3 expression in the rectum or bladder in any group. Stromal derived factor-1 was significantly under expressed immediately after vaginal distention in all tissues.
Monocyte chemotactic protein-3 is significantly over expressed in rat urethral and vaginal tissues immediately following vaginal distention with above normal but decreasing expression 24 hours later. The association between monocyte chemotactic protein-3 over expression and targeted stem cell migration is under investigation. Successful characterization and control of such a repair mechanism in the lower urinary tract would introduce the potential for novel nonoperative treatments and/or preventive measures for stress urinary incontinence.
The Journal of Urology 05/2007; 177(4):1568-72. DOI:10.1016/j.juro.2006.11.047 · 4.47 Impact Factor