The central hypothesis of the original proposal was: Epithelial cell protection by endogenous PGE2 is an important component of the physiologic response to intestinal injury and is mediated by decreased apoptosis and escape from the cell cycle arrest. The proposed Specific Aims are a continuation of the old Specific Aims which have been amended to accommodate the progress made during the current funding period. The new amended Specific Aims continue to test the central hypothesis of the original proposal. The old Specific Aims were: 1. To define the mechanism by which PGE2 induces an early release from cell cycle arrest in intestinal epithelial cells subjected to radiation injury.
This Specific Aim has been eliminated. 2. To define the mechanisms by which DSS induces cell cycle arrest and by which PGE2 rescues this arrest. Progress has been made on this Specific Aim and we propose to continue this Specific Aim largely as before. In particular, as shown in Ref 4, we have developed preliminary evidence that DSS is taken up into macrophages by a TLR dependent pathway. We will define the mechanism for this uptake and define the signaling mechanisms by which this uptake results in PGE2 production (Ref 4). 3. To define the mechanisms for the increased radiation induced apoptosis and MyD88'''mice and the rescue by dimethyl PGE2. This is the Specific Aim which we have made the most progress (Ref 2, 3, 4, 5, 8). We have determined that the source of the PGE2 that protects the epithelial cells during injury is COX-2 expressing mesenchymal stem cells. Moreover we have found that in response to injury these cells migrate to an area adjacent to the epithelial stem cells. We also determined that TLR signaling is involved in this migration but the nature of this involvement is not fully defined. During the upcoming funding period we propose to extend these studies: a. We plan to identify the cells responding to TLR activation. The most likely scenario is that TLR activation of either epithelial cells at the base of the crypts or macrophages surrounding the crypts results in a generation of chemotactic factor that causes the migration of the COX-2 mesenchymal stem cells. To address this issue we have bred MyD88 and Villin-Cre mice to generate mice which lack MyD88 only in intestinal epithelial cells, and we have bred MyD88 and lyso-Cre* mice to generate mice that lack MyD88 only in myeloid cells, (neutrophils, monocytes and macrophages). We propose to treat these mice with intraperitoneal hyaluronic acid and quantify mesenchymal stem cell migration in the small intestine. Similarly we will treat these mice with DSS and quantify mesenchymal stem cell migration. This study should allow us to determine if the TLR activation that is related to the migration of mesenchymal stem cells occurs in epithelial cells or in myeloid cells. b. We propose to identify the chemotactic factor that mediates the migration of the COX-2 expressing mesenchymal stem cells to the area surrounding the intestinal or colonic crypt. The experiment described above should allow us to determine if the source of the chemotactic factor is epithelial cell or macrophages surrounding the intestinal crypt. To address this issue we will treat wild type mice with intraperitoneal hyaluronic acid and then, using laser capture micro dissection and microarray analysis we will compare the mRNA of either epithelial cells or macrophages surrounding the crypt base from hyaluronic acid treated and control animals. This microarray analysis should generate a series of candidate molecules as potential chemotactic agents for mediating the migration of the mesenchymal stem cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK033165-30
Application #
8668027
Study Section
Special Emphasis Panel (NSS)
Program Officer
Hamilton, Frank A
Project Start
1984-04-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
30
Fiscal Year
2014
Total Cost
$376,200
Indirect Cost
$128,700
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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