ERECTILE DYSFUNCTION AND NITRIC OXIDE SYNTHASE IN AGING

描述(由申请人提供)：勃起功能障碍(ED)在美国影响着超过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合成一氧化氮(NO)不足，以及由于平滑肌细胞(SMC)丢失而导致海绵体(CC)顺应性受损。对PDE5抑制剂的反应失败可能与这些因素有关。这项建议的总体目标是扩展我们先前的赠款发现，用于治疗衰老相关的ED，其基础是：a)增加阴茎神经元型一氧化氮合酶(PnNOS)的含量和活性，在性刺激时释放更多的NO；以及b)降低CC中诱导型一氧化氮合酶(INOS)和活性氧物种(ROS)的水平，以减少过氧亚硝酸盐的形成和SMC的凋亡。我们建议：1.目的：提高阴茎内PnNOS基因治疗的持续时间，方法是：a)使用新型辅助依赖性(HD)腺病毒(ADV)和组织特异性启动子引导β-半乳糖苷酶的表达，刺激成年大鼠阴茎中基因的持续表达；b)确定混合ADV/腺相关病毒(ADV-AAV)是否比HD-ADV更有效；c)比较电穿孔和小型泵的构建，并进行安全性测试；d)应用所选择的载体或细胞在老年CC中表达PnNOS和相关基因，以纠正海绵体神经电刺激所致的勃起反应受损；目的2：研究可能影响CC勃起控制的PnNOS活性调节因子：a)通过激活nNOS相关的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，调节其活性和勃起功能；目的3：通过调节CC内NO/ROS的比率，通过a)确定是否存在SMC复制以及这种复制是否随着年龄的增加而减少，并与iNOS、ROS、过氧亚硝酸盐和细胞凋亡的增加相关，以纠正与衰老有关的SMC丢失；B)降低NO(来自iNOS)和ROS水平，以逆转SMC的丢失并改善衰老相关的ED；以及c)确定iNOS基因敲除小鼠中iNOS的表达是否导致SMC的年龄相关丢失的减少。

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.