Restoration of Fecal Continence in Aging IAS

描述（由申请人提供）：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的平滑肌的机械效率降低导致括约肌的闭合压力降低，从而极大地促进老年人中不成比例地普遍存在的大便失禁。组织工程学的最新进展为我们提供了一个很好的体外模拟体内功能的模型来研究衰老对IAS平滑肌的分子机制的影响。我们第一次用生物工程技术从人IAS分离的平滑肌细胞中制造出三维环.这些环开发的音调，响应乙酰胆碱的剂量依赖性的方式和放松后，外源性添加的松弛介质8-Br-cAMP。来自人IAS和人结肠平滑肌细胞（CSMC）的初步结果表明：1）RhoA、磷酸化-PKC 1（S657）和HSP 27仅在静止时的IAS细胞而非CSMC的富含小窝蛋白的脂筏微结构域中被隔离; 2）与CSMC相比，人IAS细胞中HSP 27、RhoA、PKC 1和磷酸化CPI-17的表达更高。与来自成年大鼠的IAS环相比，来自老年大鼠IAS细胞的环显示出降低的收缩反应（最大力产生5 N和到达峰值反应的时间），这与降低的HSP 27磷酸化相关。HSP 27磷酸化的减少影响肌动蛋白细胞骨架的稳定性，导致小窝形成紊乱。在老年IAS平滑肌细胞中磷酸化HSP 27的过表达表现出PKC 1和HSP 27与小窝蛋白-1的关联增加，并且还恢复了从这些细胞生物工程化的IAS环中产生的力的大小和达到峰值收缩的时间。这项赠款建议的具体目标是：1）从分离自人、成年和老年大鼠的IAS的细胞中建立IAS生物工程体外的三维生理模型，并检查衰老对强直性IAS平滑肌收缩的分子机制的影响2）研究磷酸化HSP 27在IAS平滑肌功能与年龄相关的衰退中的作用，和3）检查由用磷酸化模拟-HSP 27 cDNA转染的IAS平滑肌细胞生物工程化的3-D IAS环中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.