Intraperitoneal adhesions have been a major and expensive complication of abdominal surgeries and lead to a significant fraction of postsurgical morbidities, small bowel obstructions, female infertility, and chronic pain. Despite its prevalence not much is known about the molecular and cellular mechanisms that underlie adhesion formation. Studies have implicated the surface mesothelium, and suggest either its interaction with fibrin matrices or its denudation as causes for adhesion formation. Because much is still unknown about adhesion pathogenesis, few effective preventative or postoperative treatments exist. We have shown that the peritoneal surface mesothelium activates and reacts to local insults such as abrasion and ischemia, leading to hypertrophy and proliferation, culminating in adhesion formation. We have begun to investigate this response for its role as a mediator of adhesion pathogenesis. Our initial experiments showed we can selectively label and purify mouse mesothelium using surface marker stains PDPN+LYVE-1-CD31-CD45-. Immunofluorescence imaging showed substantial mesothelial thickening in mice ~2h after initial insult and adhesion formation as early as 72h after. We purified surface mesothelium using FACS and performed RNA sequencing on mesothelium with no injury and 6h, 12h, and 24h after injury.
Aim 1 proposes to a) investigate the cellular dynamics of adhesion formation using a Cre dependent multi-color fluorescent reporter, and b) to perform parallel sorting and RNA sequencing analysis on other adhesion forming models. Our multi-color reporter will be crossed with a global Cre under the actin promoter, and a mesothelium specific Cre allowing us to address two fundamental questions regarding adhesion formation: 1) does only the mesothelium give rise to peritoneal adhesions? 2) is the surface mesothelium homogenous, or do there exist subsets within the mesothelium that specifically give rise to adhesions? Though we have a list of targets we believe are key mediators of adhesion formation, further analysis of our RNA seq data in conjunction with new analyses of other adhesion forming models will refine this list and provide insight towards a global mechanism.
Aim 2 proposes to use an in vivo system we have recently raised interrogate our molecular targets. We have developed a technique in which we electroporate the peritoneal wall of mice and observe robust transfection of plasmid containing a cDNA cassette or an shRNA system. We propose to use this system to provide transient upregulation or knock down of genes we find to be highly involved in adhesion formation. We expect that electroporating in these genes after initial adhesion-forming insults will prevent its pathogenesis. We are pursuing a parallel approach in which we will electroporate in plasmids containing single guide RNAs into a Cas9 knock-in mouse to achieve permanent knockout and expect similar results. Finally, we have accumulated a list of surface markers highly upregulated following adhesion induction, and are testing blocking antibodies and expect to prevent adhesion formation. These studies have the potential to establish a new understanding of peritoneal adhesions and a mechanism to facilitate discovery of new therapies.

Public Health Relevance

Intraperitoneal adhesions are an important complication of abdominal surgeries and are significant cause of surgical morbidities, small bowel obstructions, and female infertility. This proposal aims to demonstrate the major pathways and key regulators involved in adhesion formation. It also describes systems to interrogate these targets and which will provide insight into the mechanism of the disease and potential therapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30DK108561-02
Application #
9208053
Study Section
Special Emphasis Panel (ZDK1-GRB-2 (O1)L)
Program Officer
Densmore, Christine L
Project Start
2016-01-01
Project End
2018-12-31
Budget Start
2017-01-01
Budget End
2017-12-31
Support Year
2
Fiscal Year
2017
Total Cost
$35,971
Indirect Cost
Name
Stanford University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304