ERECTILE DYSFUNCTION AND NITRIC OXIDE SYNTHASE IN AGING

描述（由申请人提供）：在美国，勃起功能障碍 (ED) 影响着超过 1500 万男性及其伴侣。衰老是 ED 的主要危险因素，可能是由于阴茎神经中一氧化氮 (NO) 合成不足以及平滑肌细胞 (SMC) 损失导致海绵体 (CC) 顺应性受损。对 PDE5 抑制剂无反应可能与这些因素有关。该提案的总体目标是将我们之前的资助发现扩展到与衰老相关的 ED 的治疗方法，该方法基于：a) 增加 CC 中阴茎神经元一氧化氮合酶 (PnNOS) 的含量和活性，在性刺激时释放更多的 NO； b) 降低 CC 中诱导型 NOS (iNOS) 和活性氧 (ROS) 的水平，以减少过氧亚硝酸盐形成和 SMC 凋亡。我们建议： 目标 1：通过以下方式改善 PnNOS cDNA 海绵体内基因治疗的持续时间： a) 使用新型辅助依赖性 (HD) 腺病毒 (AdV) 和指导 β-半乳糖苷酶表达的组织特异性启动子刺激成年大鼠阴茎中基因表达的持久性； b)确定杂交AdV/腺相关病毒(AdV-AAV)是否比HD-AdV更有效； c)比较电穿孔和微型泵用于构建体递送，并进行安全测试，以及d)应用选定的载体或细胞在老化CC中表达PnNOS和相关基因，以纠正对海绵体神经电刺激受损的勃起反应；目标 2：通过以下方式表征可能影响 CC 勃起控制的 PnNOS 活性调节剂： a) 通过激活 nNOS 相关 NMDA 受体刺激 PnNOS 活性和勃起功能； b) 寻找新的 PnNOS 结合蛋白因子来调节其活性和勃起功能；目标 3：通过控制 CC 内的 NO/ROS 比率来纠正与衰老相关的 SMC 损失，方法是：a) 确定 SMC 复制是否存在以及复制是否随衰老而减少，并与 iNOS、ROS、过氧亚硝酸盐和细胞凋亡的增加相关联； b) 降低 NO（来自 iNOS）和 ROS 水平，以逆转 SMC 的损失并改善与衰老相关的 ED； c) 确定 iNOS 敲除小鼠中 iNOS 表达的消除是否会导致与年龄相关的 SMC 损失减少。

DESCRIPTION (provided by applicant): Erectile dysfunction (ED) affects over 15 million men and their partners in the USA. Aging is a major risk factor for ED, presumably due to an insufficient synthesis of nitric oxide (NO) in the penile nerves and an impaired compliance of the corpora cavernosa (CC) caused by a loss of smooth muscle cells (SMC). Failure to respond to PDE5 inhibitors may be related to these factors. The overall .goal of this proposal is to extend our previous grant findings towards a curative approach for aging-related ED based on: a) increasing the content and activity of penile neuronal nitric oxide synthase (PnNOS) in the CC, to release more NO upon sexual stimulation; and b) decreasing the levels of inducible NOS (iNOS) and reactive oxygen species (ROS) in the CC to reduce peroxynitrite formation and SMC apoptosis. We propose: Aim 1: to improve the duration of intracavernosal gene therapy with PnNOS cDNA, by: a) stimulating persistence of gene expression in the penis of adult rats using novel helper-dependent (HD) adenovirus (AdV) and tissue-specific promoters directing the expression of beta-galactosidase; b) determining whether hybrid AdV/adeno-associated virus (AdV-AAV) are more effective than HD-AdV; c) comparing electroporation and minipumps for construct delivery, and conducting safety tests, and d) applying the selected vectors or cells to express PnNOS and related genes in the aged CC, to correct the impaired erectile response to electrical stimulation of the cavernosal nerve; Aim 2: to characterize modulators of PnNOS activity that may affect the control of erection in the CC, by: a) stimulating PnNOS activity and erectile function through the activation of nNOS-associated NMDA receptors; and b) finding novel PnNOS binding protein factors modulating its activity and erectile function; Aim 3: to correct the aging-related loss of SMC by manipulating the NO/ROS ratio within the CC, by a) determining whether there is SMC replication and whether this is decreased with aging, and correlating with the increase in iNOS, ROS, peroxynitrite, and apoptosis; b) reducing NO (from iNOS) and ROS levels to reverse the loss of SMC and ameliorate aging-related ED; and c) determining whether obliteration of iNOS expression in the iNOS knock-out mouse leads to a reduction in the age-related loss of SMC.