Collagenolysis and Elastolysis in Urinary Incontinence

描述（由申请人提供）：尿失禁在美国是一个主要的健康问题，导致尿失禁和/或脱垂的女性终身手术风险为11%。分子病理生理学知之甚少，抑制了医学预防或治疗这一日益增长的残疾领域的发展。胶原蛋白和弹性蛋白是骨盆结构的主要支撑成分。因此，胶原酶、弹性酶及其抑制剂的表达控制了细胞外基质的蛋白水解，并可能对泌尿功能产生负面影响。我们假设应激性尿失禁（SUI）是由于骨盆胶原蛋白和弹性蛋白代谢异常引起的，这是由于基质金属蛋白酶（MMPs）和弹性蛋白酶及其各自的蛋白质抑制剂、金属蛋白酶组织抑制剂（TIMPs）和a -1抗胰蛋白酶的差异表达所致。首先，我们建议使用定量竞争反转录聚合酶链反应（QC-RT-PCR）、Western blot、免疫组织化学分析和酶谱分析，研究在月经周期卵泡期和黄体期分离的SUI女性和大陆女性阴道壁组织中蛋白水解酶及其抑制剂的表达。其次，我们将比较失禁和失禁绝经后妇女阴道组织样本中MMPs和TIMPs以及中性粒细胞弹性蛋白酶和A-1抗胰蛋白酶的表达，以确定雌激素剥夺对RNA和蛋白质表达以及胶原和弹性蛋白水解活性的影响。此外，我们将使用微阵列分析比较失禁和失禁妇女之间基因表达的广泛模式。第三，为了评估生长因子在泌尿功能障碍病因学中的作用，我们将检查松弛素和转化生长因子- b （TGF-B）对来自绝经前失禁和失禁妇女的培养成纤维细胞胶原溶解和弹性溶解的调节。由于泌尿功能障碍随着女性年龄的增长而发展，我们的第四个目标是检查17-B雌二醇和黄体酮在阴道成纤维细胞培养中改变MMPs/TIMPs比例和总弹性酶活性/A-1抗胰蛋白酶体外表达的能力。三分之二的尿失禁负担由妇女承担，患病率为14-41%。我们的目标是确定与生殖事件和衰老相关的特定病理生理变化，这些变化是SUI发展的基础，作为确定治疗干预潜在目标的第一步。

DESCRIPTION (provided by applicant): Urinary incontinence is a major health problem in the United States, resulting in an 11% lifetime risk of surgery for women with incontinence and/or prolapse. The molecular pathophysiology is poorly understood, inhibiting the development of medical prophylaxis or therapy for this growing area of disability. Collagen and elastin are major supporting elements of pelvic structures. Thus, expression of collagenases, elastases and their inhibitors controls proteolysis of the extracellular matrix and may negatively impact urinary function. We hypothesize that stress urinary incontinence (SUI) results from abnormal pelvic collagen and elastin metabolism as a function of differential expression of the matrix metalloproteinases (MMPs) and elastases and their respective protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) and A-1 anti-trypsin. First, we propose to investigate expression of both the proteolytic enzymes and their inhibitors in vaginal wall tissue isolated from women with SUI and continent women during the follicular and luteal phases of the menstrual cycle using quantitative-competitive reverse transcription polymerase chain reaction (QC-RT-PCR), Western blot, immunohistochemical analysis, and zymography. Second, we will compare the expression of MMPs and TIMPs as well as neutrophil elastase and A-1 anti-trypsin in vaginal tissue samples from continent and incontinent postmenopausal women to determine the effect of estrogen deprivation on RNA and protein expression as well as on collagen and elastin proteolytic activity. In addition, we will compare broad patterns of gene expression between continent and incontinent women using microarray analysis. Third, to assess the role of growth factors in the etiology of urinary dysfunction, we will examine relaxin and transforming growth factor-B (TGF-B) modulation of both collagenolysis and elastolysis in cultured fibroblasts derived from premenopausal continent and incontinent women. Because urinary dysfunction develops as women age, our fourth goal is to examine the ability of 17-B estradiol and progesterone to alter in vitro expression of the ratio of MMPs/TIMPs and total elastase activity/A-1 anti-trypsin in vaginal fibroblast cultures. Two-thirds of the burden of urinary incontinence is borne by women with prevalence rates of 14-41%. Our goal is to identify specific pathophysiologic changes related to reproductive events and aging which underlie the development of SUI as a first step to identifying potential targets for therapeutic intervention.