D. Foster1,2, C. D. Marshall1,2, R. C. Ransom2, A. Manjunath3, G. Gulati3, M. S. Hu2, C. C. Chan3, W. T. Leavitt2, A. L. Moore2, L. A. Barnes2, M. Murphy2, M. T. Longaker1,2,3 1Stanford University,Department Of Surgery,Stanford, CA, USA 2Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine,Stanford, CA, USA 3Stanford University,Institute For Stem Cell Biology And Regenerative Medicine,Stanford, CA, USA
Introduction: After surgical interventions or secondary to fibrotic disease, intra-abdominal adhesions can form. The presence of adhesions makes further surgery challenging and can have other negative consequences such as infertility, bowel obstructions and chronic pain. Adhesions are thought to form by fibroblast collagen deposition, similar to cutaneous scar formation. The mechanisms underlying adhesion formation, including origin of the fibroblasts involved or signaling process governing this phenomenon, however, remain poorly characterized. Currently, there is no effective therapy to prevent or treat adhesive disease.
Methods: Intra-abdominal adhesions were established between the bowel and the peritoneal lining of the abdominal sidewall in PDGFRα-GFP mice, in which PDGFRα (platelet derived growth factor receptor alpha), a pan-fibroblast marker, is tagged with green fluorescent protein (GFP) [Fig. 1A]. Sham-surgery PDGFRα-GFP mice were used for comparison. Once formed, the adhesion, abdominal wall and bowel wall tissues were extracted and digested with collagenase. FACS sorting and quantitative PCR confirmed fibroblast identity via expression of known fibroblast genes. Bulk RNA sequencing was conducted on sorted fibroblasts and gene expression was compared between the adhesion and sham-surgery cohorts. For the next aim of this project, human abdominal adhesion tissue is being harvested from ileostomy takedown patients. All experiments were approved by Stanford University’s IRB or APLAC, as applicable.
Results: FACS sorting of mouse adhesion tissue showed consistent expression of fibroblast genes including PDGFRα, Vim (vimentin), and Col1a2 (encodes for collagen 1). This was confirmed with quantitative PCR. RNA sequencing showed significantly higher expression of relevant fibrosis-associated genes in both male and female mice from the adhesion cohort including Acta2, Tnc, and Col24a1, compared to sham [Fig. 1B]. Acta2 encodes for smooth muscle α-2-actin (αSMA) and is expressed by myofibroblasts and smooth muscle cells. Tnc encodes for tenascin C, an extracellular matrix protein, and Col24a1 is a member of the collagen gene family involved in type 1 collagen regulation. Human adhesion tissue will be sorted and sequenced, and gene expression will be analyzed and compared with mouse results. The results of our human adhesion tissue experiments are forthcoming.
Conclusion: Identification of this gene expression pattern in adhesions presents the opportunity for possible therapeutic targets. Comparison of our results from mouse experiments with cellular activity and gene expression patterns in human tissue will bring us closer to developing a potential therapy to combat adhesive disease.
