Roles of Nitric Oxide and Superoxide in Cystitis

描述(申请人提供)：根据美国癌症协会的数据，男性和女性的盆腔器官肿瘤分别占2012年美国新诊断肿瘤的48%和21%(癌症事实和数字，2012年)。虽然放射治疗是治疗这些恶性肿瘤的关键疗法，但剂量受到发展为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，原因是尿路上皮细胞(UC)通透性屏障的破坏和传入神经的敏化。慢性期可在6-12个月内发展，伴有血管内皮细胞损伤、缺血、胶原沉积和膀胱顺应性下降。 在第一次赠款期间，我们使用了一氧化氮(NO·)、亚硝酸盐(NO2-)和过氧亚硝酸盐(ONO2-) 微传感器和钙离子成像确定照射诱导UC内钙离子内流和释放，激活一氧化氮合酶(NOS)。NO·与细胞色素氧化酶结合，产生超氧化物(*O2-)和抑制线粒体呼吸的ONO2-。这会导致线粒体肿胀和破裂，细胞色素c释放和细胞凋亡。我们开发了一种线粒体靶向策略(美国专利11/565,779)，适用于一氧化氮合酶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前膀胱内滴注，而不是结肠照射(10Gy1Gy100rad)时，具有辐射防护作用。另一方面， 结肠内注射拮抗剂可阻止交叉敏感化和因结肠照射引起的膀胱炎。这表明盆腔器官交叉敏化参与了放射性膀胱炎的发生，必须系统地给予药物以提供多器官保护。在这个方案中，我们将探讨盆腔器官交叉敏化的机制，参与钙离子内流和释放的通道，以及参与NOS激活的通道，以及新的治疗药物在预防或治疗放射性膀胱炎方面的好处。 我们有三个特定的目标，将在对照和瞬时受体潜在强直蛋白中进行测试 1和香草素1(TRPA1-/-和TRPV1-/-)基因敲除小鼠，对UC、背根神经节(DRG)神经元和膀胱壁和脊髓的传入神经末梢进行光学定位。细胞和组织将被电压或钙敏感染料染色和/或用伪狂犬病病毒(PRV369和PRV823)标记的传入神经表达遗传编码的钙指示剂GCaMP4。TRPA1-/-和TRPV1-/-小鼠将被用来确定这些通道在辐射损伤中的作用，而高效液相-质谱仪将被用来鉴定参与TRPA1和TRPV1通道激活的辐射诱导的亲电脂肪酸衍生物(EFAD)。将测试硝基脂肪酸(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)的治疗效果。 我们的三个具体目标和相关假设是： 1确定引起放射性膀胱炎的膀胱-结肠传入交叉敏化的机制。假设：由于周围发散的传入神经或脊髓的汇聚传入神经的交叉敏感化，结肠照射可导致膀胱炎。 2确定TRPA1和TRPV1在辐射诱导的尿路上皮损伤和传入增敏中的作用。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确定预防或治疗放射性膀胱炎的新药物的治疗效益。假设：FA-NO2通过脱敏TRPA1和TRPV1通道具有放射防护作用，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。

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.