Roles of Nitric Oxide and Superoxide in Cystitis

一氧化氮和超氧化物在膀胱炎中的作用

基本信息

批准号：
9043860
负责人：
Anthony John Kanai
金额：
$ 33.55万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9043860
关键词：
ANK1 gene Accounting Acute Aftercare American Cancer Society Antibodies Apoptosis Binding Bladder Brain-Derived Neurotrophic Factor Catheterization Cells Chronic Chronic Phase Coagulation Process Collagen Colon Cystitis Deposition Descending colon Development Diagnosis Dose Dyes Dysuria Electrocoagulation Fatty Acids Free Radical Scavengers Frequencies Grant Growth Health Hematuria High Pressure Liquid Chromatography Hyperbaric Oxygenation Image Incontinence Inflammation Intracolonic Ion Channel Irrigation Ischemia Knockout Mice Label Lasers Legal patent Life Malignant Neoplasms Maps Methods Mitochondria Mus Muscarinic Antagonists Nerve Nerve Growth Factors Neurons New Agents Nitric Oxide Nitric Oxide Synthase Nitrites Optics Organ Pelvis Pentosan Polysulfate Permeability Peroxonitrite Phase Prevalence Prevention Radiation Radiation therapy Radioprotection Respiration Role Rupture Secondary to Spinal Cord Spinal Ganglia Staining method Stains Suid Herpesvirus 1 Superoxides Surgical incisions Swelling TRPV1 gene Testing Therapeutic Therapeutic Agents Tissues United States Urethra Urinary Retention Urothelial Cell Urothelium Vanilloid Vascular Endothelial Cell Woman acute symptom afferent nerve cell injury cytochrome c cytochrome c oxidase irradiation men microsensor mouse model novel therapeutics prevent receptor tumor voltage

项目摘要

DESCRIPTION (provided by applicant): Pelvic organ tumors in men and women are estimated to account for 48% and 21%, respectively, of new tumors diagnosed in the United States in 2012 according to the American Cancer Society (Cancer Facts & Figures, 2012). While irradiation is a key therapy for treating these malignancies, the dose is limited by the potential for developing radiation cystitis. The acute phase of cystitis includes inflammation and bladder overactivity resulting from disruption of the urothelial cell (UC) permeability barrier and sensitization of afferent nerves. A chronic phase can develop in 6-12 months with vascular endothelial cell damage, ischemia, collagen deposition and decreased bladder compliance. During the first grant period, we used nitric oxide (NO•), nitrite (NO2-) and peroxynitrite (ONO2-) microsensors along with Ca2+ imaging to determine that irradiation induces Ca2+ influx and release in UC that activate NO• synthase (NOS). NO• binds to cytochrome oxidase, resulting in superoxide (*O2-) and ONO2- which inhibits mitochondrial respiration. This leads to swelling and rupture of the mitochondria, cytochrome c release and apoptosis. We developed a mitochondrial targeting strategy (U.S. Patent 11/565,779) for NOS antagonists and free radical scavengers, and mouse models where the bladder or descending colon is withdrawn through a small incision and selectively irradiated. These agents were radioprotective when intravesically instilled prior to bladder, but not colonic, irradiation (10 Gy; 1 Gy=100 rads). On the other hand, intracolonic administration of the antagonists blocked cross-sensitization and cystitis due to colonic irradiation. This suggests that pelvic organ cross-sensitization is involved in the development of radiation cystitis and that agents must be given systemically to afford multiple organ protection. In this proposal, we will investigate the mechanism for pelvic organ cross-sensitization, the channels involved in Ca2+ influx and release responsible for NOS activation and the benefits of new therapeutic agents to prevent or treat radiation cystitis. We have three specific aims that will be tested in control and transient receptor potential ankyrin 1 and vanilloid 1 (TRPA1-/- and TRPV1-/-) knockout mice using optical mapping of UC, dorsal root ganglia (DRG) neurons and afferent nerve terminals in the bladder wall and spinal cord. Cells and tissues will be stained with voltage- or Ca2+-sensitive dyes and/or afferent nerves labeled using pseudorabies virus (PRV369 and PRV823) expressing the genetically encoded Ca2+ indicator, GCaMP4. TRPA1-/- and TRPV1-/- mice will be used to determine the roles of these channels in irradiation-induced damage and HPLC-MS will be used to identify irradiation-induced electrophilic fatty acid derivatives (EFAD) involved in TRPA1 and TRPV1 channel activation. The therapeutic benefits of nitro-fatty acids (FA-NO2) and brain-derived neurotrophic factor- and nerve growth factor-antibodies (BDNF-AB and NGF-AB) will be tested. Our three specific aims and associated hypotheses are: 1 Determine the mechanism of bladder-colon afferent cross-sensitization that contributes to radiation cystitis. Hypothesis: Cystitis can develop from colonic irradiation due to cross-sensitization of divergent afferent nerves in the periphery or convergent ones in the spinal cord. 2 Determine the roles of TRPA1 and TRPV1 in irradiation-induced urothelial damage and afferent sensitization. Hypothesis: Irradiation produces EFAD that activate TRPA1 and TRPV1 ion channels resulting in UC damage and afferent sensitization. 3 Determine the therapeutic benefits of new agents for the prevention or treatment of radiation cystitis. Hypothesis: FA-NO2 are radioprotective by desensitizing TRPA1 and TRPV1 channels and BDNF-AB and NGF-AB are therapeutic by inhibiting nerve growth and sensitization in bladder and spinal cord.

描述（由申请人提供）：根据美国癌症协会的数据，2012 年美国诊断出的新肿瘤中，男性和女性盆腔器官肿瘤分别占 48% 和 21%（癌症事实与数字，2012 年）虽然放射治疗是治疗这些恶性肿瘤的关键疗法，但剂量受到发生放射性膀胱炎的可能性的限制。膀胱炎的急性期包括炎症和膀胱过度活动。尿路上皮细胞 (UC) 通透性屏障破坏和传入神经敏化可在 6-12 个月内形成慢性期，伴有血管内皮细胞损伤、缺血、胶原沉积和膀胱顺应性降低。在第一个资助期间，我们使用一氧化氮 (NO•)、亚硝酸盐 (NO2-) 和过氧亚硝酸盐 (ONO2-) 微传感器与 Ca2+ 成像一起确定辐射会诱导 UC 中 Ca2+ 流入和释放，从而激活 NO• 合酶 (NOS) 与细胞色素氧化酶结合，产生超氧化物 (*O2-) 和 ONO2-，从而抑制线粒体呼吸。我们开发了一种线粒体靶向策略（美国专利）。 11/565,779）用于 NOS 拮抗剂和自由基清除剂，以及通过小切口取出膀胱或降结肠并选择性照射的小鼠模型，这些药物在膀胱照射（但不是结肠照射）（10 Gy）之前进行膀胱内滴注时具有辐射防护作用。 ; 1 Gy=100 拉德）。结肠内给予拮抗剂可阻止结肠照射引起的交叉致敏和膀胱炎，这表明盆腔器官交叉致敏与放射性膀胱炎的发展有关，并且必须全身给予药物以提供多器官保护。将研究盆腔器官交叉致敏的机制、负责 NOS 激活的 Ca2+ 流入和释放的通道以及新治疗药物预防或治疗放射性膀胱炎的益处。我们有三个具体目标，将在控制和瞬时受体电位锚蛋白中进行测试使用光学图谱对 UC、背根神经节 (DRG) 神经元以及膀胱壁和脊髓中的传入神经末梢细胞和组织进行电压染色。 -或使用表达基因编码的Ca2+指示剂的伪狂犬病病毒（PRV369和PRV823）标记的Ca2+敏感染料和/或传入神经， GCaMP4-/- 和 TRPV1-/- 小鼠将用于确定这些通道在辐射引起的损伤中的作用，HPLC-MS 将用于鉴定参与 TRPA1 和 TRPA1 的辐射诱导的亲电脂肪酸衍生物 (EFAD)。 TRPV1 通道激活。硝基脂肪酸 (FA-NO2) 和脑源性神经营养因子和神经生长因子抗体 (BDNF-AB 和NGF-AB）将进行测试。我们的三个具体目标和相关假设是： 1 确定导致放射性膀胱炎的膀胱-结肠传入交叉敏化的机制假设：由于周围发散传入神经或脊髓中会聚传入神经的交叉敏化，结肠照射可导致膀胱炎。 2 确定 TRPA1 和 TRPV1 在辐射引起的尿路上皮损伤和传入敏化中的作用假设：辐射产生 EFAD，激活 TRPA1 和 TRPV1 离子通道，导致 UC 损伤和传入敏化。 3 确定新药物预防或治疗放射性膀胱炎的治疗效果假设：FA-NO2 通过使 TRPA1 和 TRPV1 通道脱敏而具有辐射防护作用，BDNF-AB 和 NGF-AB 通过抑制膀胱和脊髓的神经生长和敏化而具有治疗作用。绳索。