Glycosaminoglycans For IC Pathophysiology and Prognosis

描述（由申请人提供）：尽管患者倡导团体做出了巨大的努力，但我们对间质性膀胱炎（IC）的病因，诊断和治疗的了解仍然不足。由于管腔糖胺聚糖（GAG）层保护膀胱尿路上皮免受尿液中有害物质的侵害，因此尿路上皮GAG的改变可能与IC的病理生理有关。经验证的O’leary - sant问卷和临床指标判断，重症IC患者尿中总（即非硫酸化和硫酸化）gag和透明质酸（HA）水平升高。这些患者的尿液还含有一种或多种独特的GAG物种和高分子量的HA物种。尿总GAG水平和GAG谱似乎是监测疾病严重程度的准确标志物，无论患者正在接受何种治疗。与正常尿路上皮细胞相比，IC尿路上皮培养物分泌更高水平的基质金属蛋白酶(MMPs)-2和-9，提示MMPs与该疾病之间存在关联。本研究旨在探讨IC特异性GAGs、高分子量HA和MMPs在IC病理生理中的作用，以及gag样物质如何缓解症状。此外，在一项多中心试验中评估尿总GAG和HA水平、GAG和HA谱和尿MMP水平在IC患者随访中的有效性。为了鉴定IC特异性GAGs及其细胞来源，这些GAGs将从IC患者的尿液和原代尿路上皮培养条件培养基（CM）中通过顺序液相色谱、gag降解酶和高效液相色谱（Aim 1）纯化。通过对不同疾病严重程度的IC患者尿液、组织提取物和尿路上皮CM中的GAG水平和GAG特征进行两两比较，将评估GAGs定性和定量改变的细胞基础。IC特异性GAGs参与IC病理生理和gag样物质引起症状缓解，将通过cDNA微阵列分析分别在IC特异性GAGs和聚硫酸戊聚糖处理的正常和IC尿路上皮细胞中基因表达的变化来评估（目的2）。了解它们与IC、HA水平的关系；将分析不同疾病严重程度IC患者的尿液、组织提取物和尿路上皮CM中的HA谱和MMP水平。用高分子量HA治疗后正常尿路上皮基因表达的变化将通过cDNA微阵列分析进行评估，并与IC尿路上皮细胞中的基因表达进行比较，以揭示HA在IC病理生理中的作用（目的3）。在一项多中心试验中，将评估总GAG水平、GAG和HA谱以及MMP水平在IC患者随访中的有效性及其用于监测治疗反应的用途（Aim 4）。本研究将揭示尿路上皮GAGs（包括HA）和MMPs在1C病理生理中的功能和诊断潜力。此外，它可能产生一种或多种测试，可用于IC患者的随访和监测治疗反应。

DESCRIPTION (provided by applicant): Despite superb efforts of patient advocacy groups, our insight into the etiology, diagnosis and treatment for interstitial cystitis (IC) is still inadequate. Since the luminal glycosaminoglycan (GAG) layer protects the bladder urothelium from noxious substances in urine, alterations in urothelial GAGs may be associated with IC pathophysiology. Urinary levels of total (i.e., non-sulfated and sulfated) GAGs and hyaluronic acid (HA) are elevated in IC patients with severe disease, as judged by the validated O'Leary-Sant questionnaire and clinical index. These patients' urine also contain one or more unique GAG species and a high molecular mass HA species. Urinary total GAG levels and GAG profile appear to be accurate markers for monitoring disease severity, regardless of the type of treatment the patients are undergoing. IC urothelial cultures secrete higher levels of matrix metalloproteinases (MMPs)-2 and -9, when compared with normal urothelial cells, suggesting an association between MMPs and this disease. This proposal is designed to investigate the involvement of IC specific GAGs, high molecular mass HA and MMPs in the pathophysiology of IC and how GAG-like substances may bring about symptom relief. Furthermore, to evaluate in a multi-center trial the usefulness of total urinary GAG and HA levels, GAG and HA profiles and urinary MMP levels in the follow-up of IC patients. To identify IC-specific GAGs and their cellular source, these GAGs will be purified from IC patients' urine and primary urothelial culture conditioned media (CM), by sequential liquid chromatographies, digestion with GAG-degrading enzymes and HPLC (Aim 1). The cellular basis of qualitative and quantitative alterations in GAGs, will be evaluated by performing a pair-wise comparison of GAG levels and GAG profile in urine, tissue extracts and urothelial CM from IC patients with varying degrees of disease severity. The involvement of IC-specific GAGs in IC pathophysiology and of GAG-like substances in causing symptom relief, will be evaluated by cDNA microarray analysis of changes in gene expression in normal and IC urothelial cells treated with IC-specific GAGs and pentosan polysulfate, respectively (Aim 2). To understand their association with IC, HA levels; HA profile and MMP levels will be analyzed in urine, tissue extracts and urothelial CM from IC patients with varying degrees of disease severity. Changes in normal urothelial gene expression following treatment with high molecular mass HA will be evaluated by cDNA microarray analysis, and compared with gene expression in IC urothelial cells, to reveal the involvement of HA in IC pathophysiology (Aim 3). In a multi-center trial, the usefulness of total GAG levels, GAG and HA profile and MMP levels in IC patient follow-up and their use for monitoring treatment response will be evaluated (Aim 4). The proposed study will reveal the function and diagnostic potential of urothelial GAGs (including HA) and MMPs in 1C pathophysiology. Furthermore, it might yield a test or a combination of tests that can be used in the follow-up of IC patients and for monitoring 9 treatment responses.