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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK071614-03
Application #
7759595
Study Section
Clinical and Integrative Gastrointestinal Pathobiology Study Section (CIGP)
Program Officer
Hamilton, Frank A
Project Start
2008-02-01
Project End
2013-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
3
Fiscal Year
2010
Total Cost
$313,910
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pediatrics
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Bohl, Jaime L; Zakhem, Elie; Bitar, Khalil N (2017) Successful Treatment of Passive Fecal Incontinence in an Animal Model Using Engineered Biosphincters: A 3-Month Follow-Up Study. Stem Cells Transl Med 6:1795-1802
Rego, Stephen L; Zakhem, Elie; Orlando, Giuseppe et al. (2016) Bioengineering functional human sphincteric and non-sphincteric gastrointestinal smooth muscle constructs. Methods 99:128-34
Bitar, Khalil N; Zakhem, Elie (2016) Bioengineering the gut: future prospects of regenerative medicine. Nat Rev Gastroenterol Hepatol 13:543-56
Rego, Stephen Lee; Zakhem, Elie; Orlando, Giuseppe et al. (2016) Bioengineered Human Pyloric Sphincters Using Autologous Smooth Muscle and Neural Progenitor Cells. Tissue Eng Part A 22:151-60
Zakhem, Elie; Elbahrawy, Mostafa; Orlando, Giuseppe et al. (2015) Successful implantation of an engineered tubular neuromuscular tissue composed of human cells and chitosan scaffold. Surgery 158:1598-608
Zakhem, Elie; Rego, Stephen L; Raghavan, Shreya et al. (2015) The appendix as a viable source of neural progenitor cells to functionally innervate bioengineered gastrointestinal smooth muscle tissues. Stem Cells Transl Med 4:548-54
Zakhem, Elie; Raghavan, Shreya; Bitar, Khalil N (2014) Neo-innervation of a bioengineered intestinal smooth muscle construct around chitosan scaffold. Biomaterials 35:1882-9
Raghavan, Shreya; Bitar, Khalil N (2014) The influence of extracellular matrix composition on the differentiation of neuronal subtypes in tissue engineered innervated intestinal smooth muscle sheets. Biomaterials 35:7429-40
Bitar, Khalil N; Zakhem, Elie (2014) Design strategies of biodegradable scaffolds for tissue regeneration. Biomed Eng Comput Biol 6:13-20
Gilmont, Robert R; Raghavan, Shreya; Somara, Sita et al. (2014) Bioengineering of physiologically functional intrinsically innervated human internal anal sphincter constructs. Tissue Eng Part A 20:1603-11

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