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勃起障碍影响了美国数千名男性和全球数百万名男性。目前还没有令人满意的医学治疗方法来预防这些患者的勃起功能障碍，部分原因是涉及勃起功能障碍发展的机制很复杂，尚未得到很好的理解。我们的研究小组已经确定，一个名为opiorphins的五肽家族在阴茎勃起症的发展中起着重要作用。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中过度表达opiorphin基因会导致阴茎勃起样状态，并且在镰状细胞病的动物模型（伯克利镰状细胞（BERK）小鼠）中，在阴茎勃起发展之前，小鼠体表组织中opiorphin基因的表达升高（这在这些动物中经常发生）。我们最近的工作发现，opiorphins在缺氧条件下上调，并且opiorphins可以调节hif1a和a2br的表达，这两个基因之前被认为与平滑肌松弛途径和阴茎勃起有关。这导致我们提出以下假设：镰状细胞病引起体表组织缺氧/缺血，导致opiorphin表达增加。然后，Opiorphin充当代偿性平滑肌“松弛”通路的主要调节器。正是这些“松弛”通路的过度激活导致了阴茎勃起。了解镰状细胞病导致的体表组织中opiorphin激活之间的关联，可能会导致预后标志物的发展和新的药物策略，以预防或治疗与镰状细胞病相关的勃起功能障碍。为了验证这一假设，我们提出了以下三个具体目标：在第一个具体目标中，我们将使用体外模型来确定缺氧对opiorphins和其他基因表达的影响，这些基因参与了人类和小鼠下体平滑肌细胞中阴茎勃起的发展或平滑肌松弛途径的调节。我们将确定哪些途径是由opiorphins直接调节的。我们还将使用小鼠下体缺氧模型来确定类似的反应机制是否在体内运作。在第二个特定目标中，我们将使用镰状细胞病的BERK小鼠模型来确定opiorphin表达的上调是否在勃起功能障碍发展之前的早期事件，以及是否与缺氧模型中相似的途径被激活。我们将通过小鼠阴茎勃起的化学诱导来区分这些途径中哪些是“导致”而不是“响应”阴茎勃起的生理条件。在患者中，我们将比较镰状细胞患者和正常男性的opiorphin循环水平，并确定opiorphin血液水平是否与发生阴茎勃起的风险相关。在第三个具体目标中，我们将使用小鼠模型直接证明opiorphin在勃起功能中发挥作用。我们将在小鼠体内使用体内基因转移来过度表达opiorphin基因，并确定这是否会导致类似priapic的情况。我们将有条件地敲除小鼠opiorphin基因，并确定敲除opiorphin基因时对勃起功能的影响。我们还将把opiorphin敲除小鼠与BERK镰状细胞小鼠杂交，以确定opiorphin表达是否影响镰状细胞小鼠阴茎勃起症的发展。我们的研究将潜在地为镰状细胞病相关阴茎勃起障碍的诊断、预后、预防和治疗确定新的药理学靶点。

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.