Roles of Nitric Oxide and Superoxide in Cystitis

描述（由申请人提供）：根据美国癌症协会（Cancer Facts & Figures，2012），2012年在美国诊断的新肿瘤中，男性和女性的盆腔器官肿瘤估计分别占48%和21%。虽然放射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由尿路上皮细胞（UC）渗透性屏障破坏和传入神经致敏引起的炎症和膀胱过度活动。慢性期可在6-12个月内发展，伴随血管内皮细胞损伤、局部缺血、胶原沉积和膀胱顺应性降低。 在第一批研究期间，我们使用了一氧化氮（NO·）、亚硝酸盐（NO2-）和过氧亚硝酸盐（ONO 2-） 微传感器沿着与Ca 2+成像，以确定辐射诱导UC中激活NO·合成酶（NOS）的Ca 2+内流和释放。NO·与细胞色素氧化酶结合，产生超氧阴离子（*O2-）和ONO 2-，抑制线粒体呼吸。这导致线粒体肿胀和破裂，细胞色素c释放和细胞凋亡。我们开发了一种用于NOS拮抗剂和自由基清除剂的线粒体靶向策略（美国专利11/565，779），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（10戈伊; 1戈伊=100拉德）前膀胱内灌注时具有放射防护作用，但结肠照射除外。另一方面，在一项研究中， 拮抗剂的结肠内给药阻断了由于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生有关，必须全身给予药物以提供多器官保护。在这个提议中，我们将调查盆腔器官交叉致敏的机制，参与Ca 2+内流和释放的通道负责NOS激活和新的治疗药物的好处，以预防或治疗放射性膀胱炎。 我们有三个具体的目标，将在控制和瞬时受体电位锚蛋白测试 1和香草素1（TRPA 1-/-和TRPV 1-/-）敲除小鼠，使用膀胱壁和脊髓中UC、背根神经节（DRG）神经元和传入神经末梢的光学映射。细胞和组织将用电压或Ca 2+敏感性染料染色，和/或用表达遗传编码的Ca 2+指示剂GCaMP 4的伪狂犬病病毒（PRV 369和PRV 823）标记传入神经。TRPA 1-/-和TRPV 1-/-小鼠将用于确定这些通道在辐射诱导的损伤中的作用，HPLC-MS将用于鉴定参与TRPA 1和TRPV 1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）的治疗益处。 我们的三个具体目标和相关假设是： 1确定膀胱-结肠传入交叉致敏导致放射性膀胱炎的机制。 假设：膀胱炎可以从结肠照射由于交叉敏感的发散传入神经在外周或会聚的脊髓。 2研究TRPA 1和TRPV 1在放射性尿路上皮损伤和传入敏化中的作用。 假设：辐照产生EFAD，EFAD激活TRPA 1和TRPV 1离子通道，导致UC损伤和传入致敏。 3确定预防或治疗放射性膀胱炎的新药物的治疗益处。 假设：FA-NO2通过使TRPA 1和TRPV 1通道脱敏而具有辐射保护作用，BDNF-AB和NGF-AB通过抑制膀胱和脊髓中的神经生长和致敏而具有治疗作用。

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.