Glycosaminoglycans For IC Pathophysiology and Prognosis

描述(由申请人提供)：尽管患者权益团体做出了卓越的努力，但我们对间质性膀胱炎(IC)的病因、诊断和治疗的洞察力仍然不足。由于腔内糖胺多聚糖(GAG)层保护膀胱尿路上皮免受尿液中有害物质的伤害，尿路上皮GAG的改变可能与IC的病理生理有关。根据有效的O‘Leary-Sant问卷和临床指数判断，重症IC患者尿中总(即非硫酸盐和硫酸盐)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物种和一种高分子质量的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培养的尿路上皮细胞分泌更高水平的基质金属蛋白酶(MMPs)-2和-9，提示MMPs与本病有关。本研究旨在探讨IC特异性GAG、大分子HA和MMPs在IC的病理生理过程中的作用，以及类GAG物质如何起到缓解症状的作用。此外，在一项多中心试验中，评估尿总GAG和HA水平、GAG和HA谱以及尿基质金属蛋白酶水平在IC患者随访中的有效性。为了鉴定IC特异性GAG及其细胞来源，将从IC患者的尿液和原代尿路上皮培养条件培养液(CM)中提纯这些GAG，方法是依次进行液相层析、GAG降解酶消化和高效液相色谱(AIM 1)。通过对疾病严重程度不同的IC患者尿液、组织提取物和尿路上皮CM中的GAG水平和GAG谱进行配对比较，将评估GAG定性和定量变化的细胞学基础。IC特异性GAG在IC病理生理学中的参与，以及GAG样物质在导致症状缓解中的作用，将通过分别用IC特异性GAG和多聚戊聚糖处理的正常和IC尿路上皮细胞中基因表达变化的基因芯片分析来评估(目标2)。为了了解它们与IC、HA水平的关系，将分析具有不同疾病严重程度的IC患者的尿液、组织提取液和尿路上皮CM中的HA概况和基质金属蛋白酶水平。用基因芯片技术分析高分子HA处理后正常尿路上皮细胞基因表达的变化，并与IC细胞的基因表达进行比较，以揭示HA参与IC的病理生理过程(AIM 3)。在一项多中心试验中，将评估总GAG水平、GAG和HA概况以及基质金属蛋白酶水平在IC患者随访中的有效性，以及它们在监测治疗反应方面的用途(目标4)。这项研究将揭示尿路上皮糖链(包括透明质酸)和基质金属蛋白酶在1C病理生理学中的功能和诊断潜力。此外，它可能会产生一项测试或一组测试的组合，可用于对IC患者的随访和监测9种治疗反应。

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.