Restoration of Fecal Continence in Aging IAS

描述(由申请者提供)：对与年龄相关的肛门内括约肌(IAS)功能下降的机制或病理生理学知之甚少。IAS平滑肌的机械效率降低导致括约肌关闭压力降低，从而极大地导致大便失禁，这种情况在老年人中不成比例地普遍存在。组织工程学的最新进展为我们提供了一个良好的模拟体内功能的体外模型来研究衰老对IAS平滑肌分子机制的影响。我们首次从分离的人类间质动脉平滑肌细胞中获得了生物工程三维(3-D)环。这些环产生张力，以剂量依赖的方式对乙酰胆碱产生反应，并在外源性松弛介质8-溴-cAMP的作用下松弛。人IAS和人环状结肠平滑肌细胞(CSMC)的初步结果表明：1)RhoA、磷酸化PKC1(S657)和HSP27仅在静息状态下IAS细胞的富含小窝蛋白的脂筏微域中表达，而不是CSMC；2)与CSMC相比，人IAS细胞中HSP27、RhoA、PKC1和磷酸化CPI-17的表达更高。与成年大鼠IAS环相比，老年大鼠IAS细胞环表现出收缩反应(最大力产生5N和峰值时间反应)降低，这与HSP27磷酸化降低有关。HSP27的磷酸化程度降低会影响肌动蛋白细胞骨架的稳定性，导致小窝形成障碍。在陈旧的IAS平滑肌细胞中过表达磷酸化的HSP27显示PKC1和HSP27与小窝蛋白-1的相关性增加，并恢复了由这些细胞生物工程构建的IAS环产生的力的大小和收缩峰值的时间。这项拨款建议的具体目的是：1)从人、成年和老年大鼠的IAS细胞中分离出体外生物工程的IAS建立三维生理模型，并研究衰老对紧张性IAS收缩的分子机制的影响；2)研究磷酸化的HSP27在IAS平滑肌功能随年龄下降中的作用；以及3)研究由转Phomimic-HSP27基因的IAS细胞生物工程得到的IAS环生理收缩功能的恢复。

DESCRIPTION (provided by applicant): Little is known about the mechanisms or pathophysiology responsible for age-related decline of internal anal sphincter (IAS) function. Decreased mechanical efficiency of smooth muscle of the IAS results in decreased closure pressure of the sphincter, thus greatly contributing to fecal incontinence which is disproportionately prevalent in the elderly. Recent advances in tissue engineering provide us with an excellent in vitro model mimicking in vivo function to study the effects of aging on the molecular mechanisms of the IAS smooth muscle. We have for the first time, bioengineered three- dimensional (3-D) rings from isolated smooth muscle cells from human IAS. These rings developed tone, responded to acetylcholine in a dose-dependent manner and relaxed upon the exogenous addition of the relaxant mediator 8-Br-cAMP. Preliminary results from Human IAS and Human circular colonic smooth muscle cells (CSMC) indicate: 1) Sequestration of RhoA, phospho-PKC1 (S657) and HSP27 only in the caveolin-rich lipid raft microdomains of IAS cells at rest and not CSMC; 2) a greater expression of HSP27, RhoA, PKC1 and phospho CPI-17 in Human IAS cells vs. CSMC. Rings from old Rat IAS cells showed decreased contractile response (maximal force generation 5N and time-to-peak response) when compared to IAS rings from adult rats that correlated with decreased HSP27 phosphorylation. Reduced phosphorylation of HSP27 affects actin cytoskeleton stability leading to disturbed caveolae formation. Overexpression of phosphomimic-HSP27 in old IAS smooth muscle cells exhibited increased association of PKC1 and HSP27 with caveolin-1, and also reinstated the magnitude of force generated and the time-to-peak contraction in IAS rings bioengineered from these cells. The specific aims of this grant proposal are: 1) Develop a 3-D physiological model of the IAS bioengineered in vitro from cells isolated from the IAS of human and of adult and aged rats, and examine the effect of aging on the molecular mechanisms of tonic IAS smooth muscle contraction 2) Study the role of phosphorylated-HSP27 in age-related decline of IAS smooth muscle function, and 3) Examine the reinstatement of physiological contractile function in 3-D IAS rings bioengineered from IAS smooth muscle cells transfected with phosphomimic-HSP27 cDNA.