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

• 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
• 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

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（由应用提供）：根据美国癌症学会（Cancers Facts＆Figures，2012年），估计男性和女性的骨盆器官肿瘤分别占美国诊断出的新肿瘤的48％和21％。虽然辐照是治疗这些恶性肿瘤的关键疗法，但剂量受到发展辐射膀胱炎的可能性的限制。膀胱炎的急性期包括尿路上皮细胞（UC）渗透性屏障和传入神经的敏感性导致的炎症和膀胱过度活动。血管内皮细胞损伤，缺血，胶原蛋白沉积和改善的膀胱依从性，可以在6-12个月内出现慢性期。 在第一个赠款期间，我们使用了一氧化氮（no••），亚硝酸盐（NO2-）和过氧亚硝酸盐（ono2-） 微传感器以及CA2+成像，以确定辐射会诱导Ca2+的影响和释放，从而激活NO•合酶（NOS）。 NO•与细胞色素氧化物结合，导致超氧化物（*O2-）和ono2-抑制线粒体呼吸。这导致线粒体，细胞色素C释放和凋亡的肿胀和破裂。我们为NOS拮抗剂和自由基清除剂开发了线粒体靶向策略（美国专利11/565,779），以及小鼠模型，其中膀胱或下降结肠通过小切口撤回并有选择性地汇总。当在膀胱之前插入术时，这些药物是放射保护的，但不是结肠辐射（10 Gy; 1 Gy = 100 rad）。另一方面， 拮抗剂的胆碱内给药阻断了由于结肠照射而导致的交叉敏化和膀胱炎。这表明骨盆器官交叉敏化参与辐射膀胱炎的发展，并且必须系统地给予药物以提供多器官保护。在此提案中，我们将研究骨盆器官交叉敏化的机制，CA2+影响和释放的通道负责NOS激活以及新的治疗剂的益处，以预防或治疗辐射膀胱炎。 我们有三个特定的目标，这些目标将在控制和瞬态接收器电位上脉络蛋白 1和Vanilloid 1（TRPA1 - / - 和TRPV1 - / - ）敲除小鼠使用UC的光学映射，背根神经节（DRG）神经元和脊髓和脊髓中的传入神经末端。细胞和组织将用电压或Ca2+敏感的染料和/或使用伪行病毒标记的神经（PRV369和PRV823）染色，表达一般编码的CA2+指示剂GCAMP4。 TRPA1 - / - 和TRPV1 - / - 小鼠将用于确定这些通道在辐射诱导的损伤中的作用，而HPLC-MS将用于鉴定辐射诱导的亲电脂肪酸衍生物（EFAD）参与TRPA1和TRPV1通道激活。硝基脂肪酸（FA-NO2）和脑衍生的神经营养因子和神经生长因子抗体（BDNF-AB和NGF-AB）的治疗益处将进行测试。 我们的三个具体目标和相关的假设是： 1确定有助于辐射膀胱炎的膀胱 - 彩色传入交叉敏化的机制。假设：由于周围或脊髓周围或收敛性传入神经的交叉敏化，膀胱炎可以从结肠辐射发展。 2确定TRPA1和TRPV1在辐照诱导的尿路上皮损伤和传入的感觉中的作用。假设：辐射会产生激活TRPA1和TRPV1离子通道的EFAD，从而导致UC损伤和传入的感觉。 3确定新药物预防或治疗放疗的治疗益处。假设：FA-NO2通过使TRPA1和TRPV1通道脱敏以及BDNF-AB和NGF-AB受到辐射保护，通过抑制膀胱和脊髓的神经生长和敏感性，是治疗的。