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。目前还没有令人满意的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，而且还没有很好的了解。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。在以前发表的研究中，我们已经证明了通过基因转移在大鼠语体中过度表达阿片吗啡基因会导致异常勃起状态，并且在镰状细胞病的动物模型(伯克利镰状细胞(BERK)小鼠)中，在异常勃起(在这些动物中经常发生)之前，小鼠的组织中阿片吗啡基因的表达水平升高。我们最近的工作发现，阿片吗啡在低氧条件下上调，阿片吗啡可以调节HIF1a和a2br的表达，这两个基因以前被认为与平滑肌松弛通路和勃起有关。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。正是这些“松弛”通路的过度激活可能导致异常勃起。了解镰状细胞病引起的躯体组织中阿片吗啡激活之间的关系可能会导致开发预后标记物和新的药理学策略来预防或治疗与镰状细胞病相关的异常勃起。为了验证这一假说，我们提出了以下三个具体目标：在第一个特定的AM中，我们将使用体外模型来确定低氧对人和小鼠异常勃起发生或调节平滑肌松弛通路的阿片吗啡和其他相关基因表达的影响。我们将确定这些途径中的哪些是由阿片吗啡直接调节的。我们还将使用小鼠全身缺氧的模型来确定类似的反应机制是否在体内起作用。在第二个特定目的中，我们将使用镰状细胞病的Berk小鼠模型来确定阿片吗啡表达上调是否是异常勃起发生之前的早期事件，以及类似的途径是否像低氧模型中那样被激活。我们将通过对小鼠异常勃起的化学诱导来区分这些途径中的哪些对异常勃起的生理条件是“起因的”而不是“反应的”。在患者中，我们将比较镰状细胞和正常人血液中阿片吗啡的循环水平，并确定血液中阿片吗啡水平是否与发生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吗啡在勃起功能中发挥作用。我们将使用体内基因转移在小鼠体内过度表达阿片吗啡基因，并确定这是否会导致类似异常勃起的情况。我们将产生有条件的小鼠阿片吗啡基因敲除，并确定阿片吗啡基因敲除后对勃起功能的影响。我们还将把阿片吗啡基因敲除小鼠与伯克镰状细胞小鼠进行杂交，以确定阿片奥啡的表达是否影响镰状细胞小鼠异常勃起的发展。我们的研究可能会为诊断、预后、预防和治疗与镰状细胞疾病相关的异常勃起确定新的药理靶点。

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.