The neuropilin 2 axis in smooth muscle contractility

 描述(申请人提供)：膀胱功能依赖于平滑肌(SM)收缩和松弛的循环，分别实现排尿和储存。膀胱SM收缩的强度或持续时间的损害是一种普遍但鲜为人知的临床症状，称为逼尿肌活动不足(DU)。DU是一种复杂的疾病，(I)由各种神经肌肉损伤引起，包括慢性膀胱出口梗阻、衰老和持续性糖尿病；(Ii)排尿效率降低；(Iii)可导致严重的泌尿系统并发症。无论病因如何，DU患者的正常膀胱收缩功能丧失是一个严重的临床问题，目前还不能从机制上加以理解。DU的药物治疗是有限的，主要包括副交感神经刺激药物，这些药物的疗效和副作用值得怀疑。对于DU患者来说，恢复膀胱收缩能力的更有效和更可耐受的治疗方案将是一个重大的治疗进步。然而，只有了解膀胱SM收缩的调节机制及其在DU中的失调，才能实现DU药物治疗的成功目标。我们最近发现逼尿肌是神经粘连蛋白2(Nrp2)的主要表达部位。膀胱平滑肌细胞暴露于神经粘连蛋白2配体信号素3F(SEMA3F)后，细胞骨架发生了深刻的变化，伴随着RhoA的抑制，肌球蛋白轻链磷酸化水平降低，细胞骨架僵硬程度降低。与此一致的是，在体内通过腺病毒转导强制表达SEMA3F导致了膀胱SM收缩能力的降低。相反，与非缺失对照相比，体内Nrp2或Sema3F基因敲除增强了膀胱SM的收缩能力。新的数据显示，在部分膀胱出口梗阻(PBOO)后经历失代偿的膀胱中，与未缺失的对照相比，Nrp2缺失恢复了SM的收缩能力。最后，对人类膀胱标本的新分析显示，逼尿肌收缩能力与NRP2的表达呈负相关。基于这些观察，我们假设SEMA3F-Neuropilin 2网络在活体内抑制了膀胱SM的收缩，而靶向这个轴可以恢复收缩 在DU条件下运行。这一假说将在以下特定目标下进行检验：(1)确定Sema3F-Nrp2介导的抑制膀胱SM收缩的潜在机制。(2)确定在pBOO模型中靶向Sema3F-Nrp2轴的功能后果。我们将使用新的Nrp2和Sema3F缺乏的小鼠模型、肌条张力测试、生化和组织学分析、膀胱计量学评估以及与生理相关的膀胱出口梗阻小鼠模型来实现我们的目标。在项目期结束时，我们预计已经确定了Sema3F-Nrp2轴抑制平滑肌收缩的机制(S)，Sema3F和Nrp2扰动对膀胱病理生理学的影响，以及如何利用Sema3F-Nrp2网络在治疗上恢复DU的收缩能力。

 DESCRIPTION (provided by applicant): Bladder function depends on cycles of smooth muscle (SM) contraction and relaxation to achieve voiding and storage, respectively. Impairment of the strength or duration of bladder SM contraction characterizes a prevalent but poorly understood clinical condition termed detrusor underactivity (DU). DU is a complex disorder that (i) arises from diverse neuromuscular insults, including chronic bladder outlet obstruction, aging and persistent diabetes; (ii) reduces voiding efficiency and (iii) can lead to significant urologic complications. Irrespective of etiology, the loss of normal bladder contractilty in DU is a serious clinical problem that is not understood in mechanistic terms. Pharmacotherapy for DU is limited, comprising primarily parasympathomimetic agents, which have questionable efficacy and adverse side effects. More effective and tolerable treatment options to restore bladder contractility would represent a major therapeutic advance for patients suffering from DU. However, the goal of successful pharmacologic treatment of DU will only be realized by understanding mechanisms regulating bladder SM contraction and their dysregulation in DU. We recently identified the detrusor smooth muscle as a major site of expression of neuropilin 2 (Nrp2). Exposure of bladder smooth muscle cells to the neuropilin 2 ligand semaphorin 3F (SEMA3F) evoked profound cytoskeletal changes, accompanied by inhibition of RhoA, decreased myosin light chain phosphorylation, and reduced cytoskeletal stiffness. Consistent with this, forced expression of SEMA3F via adenoviral transduction in vivo led to reduced bladder SM contractility. Conversely, knockout of either Nrp2 or Sema3F in vivo enhanced bladder SM contractility compared to non-deleted controls. New data show that in bladders undergoing decompensation following partial bladder outlet obstruction (pBOO), Nrp2 deletion restored SM contractility compared to non-deleted controls. Lastly, new analysis of human bladder specimens revealed an inverse correlation between detrusor contractility and NRP2 expression. Based on these observations, we hypothesize that the SEMA3F-neuropilin 2 network inhibits bladder SM contractility in vivo and that targeting this axis restores contractile function under conditions of DU. The hypothesis will be tested with the following Specific Aims: (1) Determine the mechanisms underlying Sema3F- Nrp2-mediated inhibition of bladder SM contractility. (2) Determine the functional consequences of targeting the Sema3F-Nrp2 axis in a model of pBOO. We will employ novel mouse models of Nrp2- and Sema3F- deficiency, tension testing in muscle strips, biochemical and histological analyses, cystometric evaluation and a physiologically relevant mouse model of bladder outlet obstruction to achieve our objectives. At the end of the project period we expect to have determined the mechanism(s) whereby the Sema3F-Nrp2 axis inhibits smooth muscle contractility, the consequences of Sema3F and Nrp2 perturbation for bladder pathophysiology and how the Sema3F-Nrp2 network may be exploited therapeutically to restore contractility in DU.