Restoration of Fecal Continence in Aging IAS

描述（由申请人提供）：关于内肛门括约肌（IAS）功能与年龄相关的衰退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闭合压力降低，从而极大地促进了老年人中不成比例地普遍存在的大便失禁。组织工程的最新进展为研究衰老对IAS平滑肌分子机制的影响提供了一种模拟体内功能的体外模型。我们首次从人类IAS分离的平滑肌细胞中获得生物工程三维（3-D）环。这些环形成音调，以剂量依赖的方式对乙酰胆碱作出反应，并在外源添加松弛介质8-Br-cAMP时松弛。在人IAS和人环状结肠平滑肌细胞（CSMC）的初步结果表明：1)RhoA、phospho-PKC1 （S657）和HSP27仅在静止状态下IAS细胞富含小泡蛋白的脂质筏微域中被隔离，而在CSMC中不被隔离；2)人IAS细胞中HSP27、RhoA、PKC1和磷酸化CPI-17的表达高于CSMC。与HSP27磷酸化降低的成年大鼠IAS细胞环相比，老年大鼠IAS细胞环的收缩反应（最大力产生5N和峰值时间反应）有所下降。HSP27磷酸化降低影响肌动蛋白细胞骨架稳定性，导致小泡形成紊乱。在老年IAS平滑肌细胞中，磷酸化-HSP27的过表达显示PKC1和HSP27与小窝蛋白-1的关联增加，并恢复了由这些细胞生物工程的IAS环产生的力的大小和峰前收缩的时间。这项拨款建议的具体目标是：1)利用人IAS及成年和老年大鼠IAS分离细胞建立IAS生物工程体外三维生理模型，探讨衰老对IAS强直性平滑肌收缩的分子机制影响。2)研究磷酸化hsp27在IAS平滑肌功能年龄相关性下降中的作用。3)用转染了hsp27 cDNA的IAS平滑肌细胞进行生物工程后，观察其生理收缩功能的恢复情况。

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.