Diabetes Erectile dysfunction and apoptosis of cavernous tissues

描述(申请人提供)：本提案的主要目的是调查海绵体组织的氧化应激和细胞凋亡是否是勃起功能障碍的主要途径。我们还将研究抑制氧化应激和细胞凋亡是否可以恢复勃起功能。这个项目的基本原理是，勃起功能障碍在美国影响约3000万男性，在全球范围内影响超过2亿男性。根据我们的初步数据和以前的出版物，我们假设海绵体组织的凋亡是勃起功能障碍的下游事件。我们进一步假设抗氧化剂或bcl2基因治疗可以恢复动物模型的勃起功能。我们将通过以下实验来检验这些假说：具体目的1.检验糖尿病引起的勃起功能障碍是由于海绵体组织的凋亡所致的假说。在这一特定的目标下，我们将对糖尿病大鼠的勃起功能进行分析，然后分析细胞凋亡指数、抗凋亡基因、促凋亡基因、细胞色素c、超氧化物歧化酶和平滑肌分化标志物。我们还将研究抗氧化剂(Tempoll)是否能恢复糖尿病大鼠的勃起功能。我们将使用以下技术：RT-PCR、实时RT-PCR、原位杂交、免疫组织化学、电刺激海绵体神经进行压力测量、基因传递、组织学、光镜和电子显微镜。具体目的#2.验证胰岛素治疗可以通过恢复糖尿病大鼠海绵体组织中的抗凋亡因子来恢复勃起功能的假说。在这个特定的目标下，我们将用胰岛素治疗糖尿病大鼠，然后研究是否通过恢复特定目的#1中描述的抗凋亡通路来恢复勃起功能。特定目的#3.验证Bcl2抗凋亡基因治疗可以恢复糖尿病大鼠勃起功能障碍的功能、细胞和分子机制的假设。在这一特定目标下，将使用腺相关病毒载体将Bcl2基因导入糖尿病大鼠的海绵体组织。我们将进行以下实验：(A)Bcl2基因治疗对糖尿病大鼠阴茎勃起的影响的功能评估；(B)Bcl2基因治疗对特定目的1中所述的氧化应激途径和细胞凋亡途径的影响；(C)Bcl2基因治疗对糖尿病大鼠阴茎平滑肌标志物表达的影响。这一建议是新颖的，因为糖尿病引起的勃起功能障碍中的氧化应激和细胞凋亡途径从未被研究过。这些实验的完成将为勃起功能障碍提供新的分子机制，并有助于指导我们开发治疗勃起功能障碍的新疗法。

DESCRIPTION (provided by applicant): The main goal of this proposal is to investigate whether oxidative stress and apoptosis of cavernous tissues are major pathways in erectile dysfunction. We will also investigate whether inhibition of oxidative stress and apoptosis can restore erectile function. The rationale for this project is that erectile dysfunction affects about 30 million men in the United States and more than 200 million men worldwide. Based on our preliminary data and prior publications, we hypothesize that apoptosis of cavernous tissues are down stream events in erectile dysfunction. We further hypothesize that anti-oxidative agent or Bcl2 gene therapy can restore erectile function in animal model. We will test these hypotheses through the following experiments: Specific Aim# 1. To test the hypothesis that diabetes-induced erectile dysfunction is due to apoptosis of cavernous tissues. Under this specific aim, we will analyze erectile function in diabetic rats and then analyze apoptotic index, anti-apoptotic genes, pro-apoptotic genes, cytochrome c, superoxide dismutase and smooth muscle differentiation markers. We will also investigate whether an anti-oxidative agent (TEMPOL) can restore erectile function in diabetic rats. We will use the following techniques: RT-PCR, real-time RT-PCR, in situ hybridization, immunohistochemistry, electro-stimulation of cavernous nerve for pressure measurements, gene delivery, histology, light and electron microscopy. Specific Aim # 2. To test the hypothesis that insulin treatment can restore erectile function through restoration of anti-apoptotic factors in cavernous tissues of diabetic rats. Under this specific aim, we will treat diabetic rats with insulin and then investigate whether erectile function is restored through restoration of anti-apoptotic pathways described under specific aim # 1. Specific Aim # 3. To test the hypothesis that gene therapy with the Bcl2 anti-apoptotic gene can restore functional, cellular and molecular mechanisms of erectile dysfunction in diabetic rats. Under this specific aim, the Bcl2 gene will be transfected into the cavernous tissue of diabetic rats using an adeno-associated virus vector. We will do the following experiments: (a) Functional assessment of the effects of Bcl2 gene therapy on penile erection in diabetic rats, (b) Analysis of the effects of Bcl2 gene therapy on the oxidative stress pathways and the apoptotic pathways described under specific aim 1. (c) Analysis of the effects of Bcl2 gene therapy on the expression of smooth muscle markers in diabetic rat penis. This proposal is novel because oxidative stress and apoptotic pathways in diabetes-induced erectile dysfunction have never been investigated. Accomplishment of these experiments will provide novel molecular mechanisms of erectile dysfunction and can help guide us towards the development of new therapies for the treatment of erectile dysfunction.