Opiorphin as a master regulator of pathways leading to priapism

 描述(由申请者提供)：与镰状细胞疾病相关的异常勃起在美国影响数千名男性，在全球影响数百万人。目前还没有药物治疗来预防这些患者的异常勃起，部分原因是异常勃起的发生机制复杂，而且还不清楚。我们的研究小组已经证实，一种名为阿片吗啡的五肽家族在异常勃起的发展过程中发挥了作用。我们最新的工作发现，阿片吗啡在体部平滑组织中对缺氧的反应是上调的，然后阿片吗啡调节起作用的基因的表达，这些基因作用于平滑肌“松弛”途径。这导致我们提出了以下假设：镰状细胞病导致体部组织缺氧/缺血，从而导致阿片吗啡表达增加。然后，阿片吗啡作为代偿性平滑肌“松弛”通路的主调节器。这些“松弛”通路在躯体组织中的过度激活通常被认为是导致异常勃起的原因。我们的假设将在三个具体目标上得到检验。在特定的目标1中，我们将确定阿片吗啡的表达是否在体外的小鼠和人类细胞中因缺氧而增加，以及阿片吗啡表达的上调是否反过来激活了参与体部平滑肌松弛的通路。我们将证实，体外对低氧的反应与体内低氧小鼠模型的松弛通路的类似变化相关，如果在该动物模型中，器官浴研究中的体肌条高度松弛。在具体目标2中，我们将重点放在镰状细胞病的小鼠模型上，并确定躯体组织中阿片吗啡表达上调是否是镰状细胞病的早期事件。我们将在镰状细胞小鼠的躯体组织中确定是否与缺氧模型(在特定目标1中确定)一样，参与松弛通路的一组相似的基因被激活。我们将把松弛通路中涉及的基因的变化与勃起倾向的增加和体部平滑肌组织的高度松弛联系起来。此外，我们还将确定化学诱导的异常勃起小鼠模型中基因表达的变化。对blbbistatin小鼠模型的分析将区分哪些基因发生了“次级”改变(即。对异常勃起的反应)，而不是异常勃起的“起因”。在一项临床研究中，我们将比较镰状细胞和非镰状细胞患者血液中阿片吗啡的表达，并确定阿片吗啡水平是否在异常勃起危机期间增加或预测。在特定的目标3将开发阿片吗啡超过 表达或击倒小鼠模型，直接证明阿片吗啡在异常勃起/勃起功能中发挥作用。这些研究将利用爱因斯坦的乔尔·弗里德曼斯博士(Co-I)开发的一种新型给药系统--一种携带阿片吗啡siRNA的顺磁性纳米颗粒，它可以靶向阴茎。我们将使用这个系统来特异性地下调阿片吗啡在镰状细胞小鼠体内组织中的表达，并确定阿片吗啡表达下调是否会导致勃起倾向降低或平滑肌松弛通路活性降低。如果有证据证实，在这种动物模型中，阿片吗啡的击倒减少了异常勃起的类活动，这将为支持这一方法或其他针对阿片吗啡的策略，以预防镰状细胞患者的异常勃起提供“原则证据”。

 DESCRIPTION (provided by applicant): Priapism associated with sickle cell disease affects several thousand men in the US and millions worldwide. At present there is no pharmacological treatment to prevent the onset of priapism in these patients, in part because the mechanisms involved in the development of priapism are complex and not well understood. Our group has established that a family of pentapeptides called opiorphins play a role in the development of priapism. Our most recent work has discovered that opiorphins are up-regulated in corporal smooth tissue in response to hypoxia and that opiorphins then regulate expression of genes that function in smooth muscle "relaxant" pathways. This has led us to propose the following hypothesis: Sickle cell disease causes hypoxia/ischemia in corporal tissue which results in increased opiorphin expression. Opiorphin then acts as a master regulator of compensatory smooth muscle "relaxant" pathways. The excessive activation of these "relaxant" pathways in corporal tissue is generally considered to lead to priapism. Our hypothesis will be tested in three Specific Aims. In Specific Aim 1 we will determine if opiorphin expression is increased in response to hypoxia in mouse and human cells in vitro and if up-regulation of opiorphin expression in turn activates pathways involved in corporal smooth muscle relaxation. We will confirm that the in vitro response to hypoxia correlates to similar changes in relaxant pathways in an in vivo corporal mouse model of hypoxia and if in this animal model there is heightened relaxation of corporal smooth muscle strips in organ bath studies. In Specific Aim 2 we will focus on a mouse model of sickle cell disease and determine if up-regulation of opiorphin expression in corporal tissue is an early event in sickle cell disease. We will determine in the sickle cell mouse corporal tissue if a similar set of genes involved in relaxant pathways are activated as in models of hypoxia (identified in Specific Aim 1). We will correlate changes in the genes involved in relaxant pathways with increased erectile tendencies and heighted relaxation of corporal smooth muscle tissue. In addition, we will determine changes in gene expression in a chemically-(blebbistatin)-induced mouse model of priapism. Analysis of the blebbistatin mouse model will distinguish genes that are changed "secondary" (ie. in response to priapism) rather than "causative" of priapism. In a clinical study we will compare the expression of opiorphin in the blood of sickle cell and non-sickle cell patients, and determine if opiorphin levels are increased during, or predictive of, a priapic-crisis. In Specific Aim 3 will develop opiorphin over expression or knock-down mouse models to directly demonstrate that opiorphins play a role in priapism/erectile function. These studies will utilize a novel delivery system developed by Dr. Joel Friedmans (Co-I) at Einstein- a paramagnetic nanoparticle carrying opiorphin-siRNA which can be targeted to the penis. We will use this system to specifically knock-down expression of opiorphin in the corporal tissue of sickle cell mice and determine if knock-down of opiorphin expression results in decreased erectile tendencies or decreased activity of smooth muscle relaxant pathways. If evidence confirms that knock-down of opiorphin reduces priapic-like activity in this animal model it would provide "proof-of-principle" to support this approach, or other strategies targeting opiorphin, to prevent priapism in sickle cell patients.