Restoration of Fecal Continence in Aging IAS

主要研究者/项目负责人（最后，第一，中间）：Bitar，Khalil，N 研究及相关其他项目信息 1. * 是否涉及人类受试者？m是l否 1.a.如果对人类受试者为是 IRB审查是否待定？m是m否 IRB批准日期： 豁免编号：1 2 3 4 5 6 人类受试者保证编号 2. * 是否使用脊椎动物？l是m否 2.a.如果是，脊椎动物 IACUC审查是否待定？m是l否 IACUC批准日期：2006年11月29日 动物福利保证编号A3114-01 3. * 是否为专有/特许信息m是l否 包括在申请？ 4.a.*本项目是否对m有实际或潜在影响是l否 环境？ 4.b.如果是，请解释： 4.c.如果该项目对环境有实际或潜在影响，是否已批准豁免或进行环境评估（EA），或 环境影响报告书（EIS）？m是m否 4.d.如果是，请解释： 5.a.*该项目是否涉及美国以外的活动或m是l否 与国际合作伙伴？ 5.b.如果是，请指明国家： 5.c.可选说明： 6. * Project Summary/Abstract 7519-PROJECTABSTRACT.pdf Mime Type：application/pdf 7. * Project Narrative 8426-PROJECT_NARRATIVE.pdf Mime Type：application/pdf 8.参考书目和参考文献引用5924-REFERENCESCITED. pdf Mime Type：application/pdf 9. Facilities & Other Resources 3457-RESOURCES.pdf Mime Type：application/pdf 10. Equipment 7977-MAJOR_EQUMENT.pdf Mime Type：application/pdf 跟踪编号：其他信息第5页OMB编号：4040-0001 到期日期：2008年4月30日 主要研究者/项目负责人（最后，第一，中间）：Bitar，Khalil，N 摘要 关于与年龄相关的衰老的机制或病理生理学知之甚少。 肛门内括约肌（IAS）功能下降。机械效率降低， IAS的平滑肌导致括约肌的闭合压力降低，因此 这极大地导致了大便失禁， 老人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，以研究衰老对分子生物学的影响。 IAS平滑肌的机制。我们第一次用生物工程技术制造了三个- 来自人IAS的分离的平滑肌细胞的三维（3-D）环。这些环 发达国家的语气，响应乙酰胆碱在剂量依赖性的方式和放松 在外源性添加松弛介质8-Br-cAMP后。初步结果 来自人IAS和人环形结肠平滑肌细胞（CSMC）的结果表明：1） RhoA、磷酸化-PKC <$（S657）和HSP 27仅在富含小窝蛋白的脂质中的螯合 静止时IAS细胞而非CSMC的筏状微区; 2）HSP 27的更高表达， RhoA、PKC和磷酸CPI-17在人IAS细胞与CSMC中的表达。老老鼠的戒指 IAS细胞显示收缩反应降低（最大力产生<$N和 达到峰值反应的时间）与成年大鼠的IAS环相比， 降低HSP 27磷酸化。HSP 27磷酸化水平降低影响肌动蛋白 细胞骨架的稳定性导致小窝的形成受到干扰。过表达 在老年IAS平滑肌细胞中，磷酸化HSP 27的表达增加， PKC β和HSP 27与小窝蛋白-1，也恢复了所产生的力的大小 以及从这些细胞生物工程化的IAS环中达到峰值收缩的时间。的 这项拨款的具体目标是：1）开发IAS的三维生理模型 从分离自人、成人和老年人IAS的细胞进行体外生物工程化 大鼠，并检查老化对强直性IAS平滑的分子机制的影响 2）研究磷酸化热休克蛋白27在增龄性肌肉收缩功能下降中的作用。 IAS平滑肌功能，以及3）检查生理性 从IAS平滑肌细胞生物工程化的3-D IAS环的收缩功能 用磷酸化模拟HSP 27 cDNA转染。 项目描述第6页

Principal Investigator/Program Director (Last, first, middle): Bitar, Khalil, N RESEARCH & RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? l Yes m No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes l No IACUC Approval Date: 11-29-2006 Animal Welfare Assurance Number A3114-01 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 7519-PROJECTABSTRACT.pdf Mime Type: application/pdf 7. * Project Narrative 8426-PROJECT_NARRATIVE.pdf Mime Type: application/pdf 8. Bibliography & References Cited 5924-REFERENCESCITED.pdf Mime Type: application/pdf 9. Facilities & Other Resources 3457-RESOURCES.pdf Mime Type: application/pdf 10. Equipment 7977-MAJOR_EQUIPMENT.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Bitar, Khalil, N ABSTRACT 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-PKC¿ (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, PKC¿ and phospho CPI-17 in Human IAS cells vs. CSMC. Rings from old Rat IAS cells showed decreased contractile response (maximal force generation ¿N 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 PKC¿ 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. Project Description Page 6