Opiorphin as a master regulator of pathways leading to priapism

描述（由申请人提供）：与镰状细胞病相关的阴茎异常勃起影响美国数千名男性和全球数百万男性。目前，还没有令人满意的药物治疗来预防这些患者的阴茎异常勃起的发作，部分原因是阴茎异常勃起的发生机制很复杂，而且还不清楚。我们的研究小组已经确定，一个名为阿片肽的五肽家族在阴茎异常勃起的发展中起作用。在先前发表的研究中，我们已经证明，通过基因转移在大鼠体内过表达阿片肽基因会导致阴茎异常勃起样状态，并且在镰状细胞病的动物模型（伯克利镰状细胞（BERK）小鼠）中，在阴茎异常勃起（在这些动物中经常发生）发生之前，在身体组织中小鼠阿片肽基因的表达升高。我们最近的工作发现，阿片肽在缺氧条件下上调，并且阿片肽可以调节hif 1a和a2 br的表达，这两个基因以前与平滑肌松弛途径和阴茎异常勃起有关。这使我们提出了以下假设：镰状细胞病导致身体组织缺氧/缺血，导致阿片肽表达增加。然后，阿片肽充当补偿性平滑肌“松弛剂”途径的主要调节剂。正是这些“松弛”途径的过度激活可能导致阴茎异常勃起。了解由于镰状细胞病而导致的身体组织中阿片肽激活之间的关联可能会导致预后标志物和新的药理学策略的发展，以预防或治疗与镰状细胞病相关的阴茎异常勃起。为了验证这一假设，我们提出了以下三个具体目标：在第一个具体的上午，我们将使用一个体外模型，以确定缺氧对阿片肽和其他基因的表达参与阴茎异常勃起的发展或调节平滑肌松弛剂途径在人类和小鼠的身体平滑肌细胞的影响。我们将确定这些途径中的哪些直接受阿片肽的调节。我们还将使用小鼠模型的身体缺氧，以确定是否类似的反应机制在体内运行。在第二个具体目标中，我们将使用镰状细胞病的BERK小鼠模型来确定阿片肽表达的上调是否是阴茎异常勃起发展之前的早期事件，以及是否与缺氧模型中类似的途径被激活。我们将区分哪些途径是“病因”，而不是“响应”的生理条件下，阴茎异常勃起，通过使用化学诱导小鼠阴茎异常勃起。在患者中，我们将比较镰状细胞和正常男性的阿片肽循环水平，并确定血液中的阿片肽水平是否与发生阴茎异常勃起的风险相关。在第三个具体目标中，我们将使用小鼠模型直接证明阿片肽在勃起功能中发挥作用。我们将使用脑内基因转移在小鼠中过度表达阿片肽基因，并确定这是否会导致阴茎异常勃起。我们将产生小鼠opiorphin基因的条件性敲除，并确定当opiorphin基因被敲除时对勃起功能的影响。我们还将使阿片肽敲除小鼠与BERK镰状细胞小鼠杂交，以确定阿片肽表达是否影响镰状细胞小鼠中阴茎异常勃起的发展。我们的研究将有可能确定新的药理学靶点，用于诊断、预后、预防和治疗与镰状细胞病相关的阴茎异常勃起。

DESCRIPTION (provided by applicant): Priapism associated with sickle cell disease affects several thousand men in the US and millions worldwide. At present there are no satisfactory medical treatments 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 plays a role in the development of priapism. In previously published studies we have demonstrated that over-expression of the opiorphin gene in the rat corpora by gene transfer results in a priapic-like state, and in an animal model of sickle cell disease (the Berkley sickle cell (BERK) mouse) there is elevated expression of the mouse opiorphin genes in corporal tissue prior to the development of priapism (which occurs regularly in these animals). Our recent work has discovered that opiorphins are up-regulated in hypoxic conditions, and that opiorphins can regulate hif1a and a2br expression, two genes that have previously been associated with smooth muscle relaxant pathways and priapism. 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. It is the excessive activation of these "relaxant" pathways that could then result in priapism. Understanding the association between opiorphin activation in corporal tissue as a result of sickle cell disease may lead to the development of prognostic markers and novel pharmacological strategies to prevent or treat priapism associated with sickle cell disease. In order to test this hypothesis we propose the following three specific aims: In the first specific am we will use an in vitro model to determine the effect of hypoxia on expression of opiorphins and other genes involved in the development of priapism or regulation of smooth muscle relaxant pathways in human and mouse corporal smooth muscle cells. We will determine which of these pathways are directly regulated by opiorphins. We will also use a mouse model of corporal hypoxia to determine if similar response mechanisms operate in vivo. In the second specific aim we will use a BERK mouse model of sickle cell disease to determine if up-regulation of opiorphins expression is an early event prior to the development of priapism, and if similar pathways are activated as in the hypoxic models. We will distinguish which of these pathways are "causative" rather than "responsive" to the physiological condition of priapism by using chemical induction of priapism in the mouse. In patients we will compare circulating levels of opiorphin in sickle cell and normal men, and determine if blood levels of opiorphin correlate with the risk of developing priapism. In the third specific aim we will use mouse models to directly demonstrate that opiorphins play a role in erectile function. We will use intracorporal gene transfer to over- express opiorphin genes in the mouse, and determine if this will result in a priapic-like condition. We will generate conditional knockouts of the mouse opiorphin genes, and determine the effect on erectile function when the opiorphin gene is knocked-out. We will also cross the opiorphin knockout mice with the BERK sickle cell mice to determine if opiorphin expression effects the development of priapism in sickle cell mice. Our research will potentially identify novel pharmacological targets for the diagnosis, prognosis, prevention and treatment of priapism associated with sickle cell disease.