ERECTILE DYSFUNCTION AND NITRIC OXIDE SYNTHASE IN AGING

描述（由申请人提供）：勃起功能障碍（艾德）影响美国超过1500万男性及其伴侣。衰老是艾德的主要危险因素，可能是由于阴茎神经中一氧化氮（NO）合成不足和平滑肌细胞（SMC）损失引起的海绵体（CC）顺应性受损。对PDE 5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先前的研究成果扩展到衰老相关艾德的治疗方法，该方法基于：a）增加CC中阴茎神经元型一氧化氮合酶（PnNOS）的含量和活性，以在性刺激时释放更多的NO;和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：通过以下方法改善用PnNOS cDNA进行阴茎海绵体内基因治疗的持续时间：a）使用新的辅助依赖性（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续性; B）确定杂合AdV/腺相关病毒（AdV-AAV）是否比HD-AdV更有效; c）比较用于构建体递送的电穿孔和微型泵，并进行安全性测试，以及d）应用所选择的载体或细胞以在老年CC中表达PnNOS和相关基因，以校正对海绵体神经的电刺激的受损的勃起反应;通过以下方式表征可能影响CC中勃起控制的PnNOS活性调节剂：a）通过激活nNOS相关的NMDA受体刺激PnNOS活性和勃起功能;和B）发现调节其活性和勃起功能的新的PnNOS结合蛋白因子;目的3：通过操作CC内的NO/ROS比率来校正SMC的老化相关损失，通过a）确定是否存在SMC复制以及其是否随老化而减少，并与iNOS、ROS、过氧亚硝酸盐和凋亡的增加相关联; B）降低NO（来自iNOS）和ROS水平以逆转SMC的损失并改善衰老相关的艾德;和c）确定在iNOS敲除小鼠中iNOS表达的消除是否导致SMC的衰老相关损失的减少。

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.