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）损失引起的海绵体（CC）顺应性受损。PDE5抑制剂无效可能与这些因素有关。总的来说。本提案的目标是将我们之前的研究成果扩展到治疗与衰老相关的ED的方法：a)增加阴茎神经元一氧化氮合酶（PnNOS）在CC中的含量和活性，在性刺激下释放更多的NO；b)降低CC中诱导的NOS （iNOS）和活性氧（ROS）水平，减少过氧亚硝酸盐的形成和SMC的凋亡。我们提出：目的1：通过：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，提高PnNOS cDNA海绵体内基因治疗的持续时间；b)确定混合型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输送构建物的方法，并进行安全性测试；d)应用选定的载体或细胞在老年CC中表达PnNOS及相关基因，以纠正海海绵神经电刺激导致的勃起反应受损；目的2：表征可能影响CC勃起控制的PnNOS活性调节剂，通过：a)通过激活nnos相关的NMDA受体刺激PnNOS活性和勃起功能；b)发现调节PnNOS活性和勃起功能的新型PnNOS结合蛋白因子；目的3：通过控制CC内NO/ROS比率来纠正与衰老相关的SMC损失，a)确定SMC是否存在复制，是否随着年龄的增长而减少，以及与iNOS、ROS、过氧亚硝酸盐和凋亡的增加相关；b)降低NO（来自iNOS）和ROS水平，以逆转SMC的损失并改善衰老相关的ED；c)确定iNOS敲除小鼠中iNOS表达的消除是否导致SMC年龄相关性损失的减少。

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.